Salmonella typhimurium

Infect Immun, 1998 Nov, 66(11), 5295 - 300
Inhibition of Salmonella typhimurium invasion by host cell expression of secreted bacterial invasion proteins; Carlson SA et al.; Pathogenic Salmonella species initiate infection of a host by inducing their own uptake into intestinal epithelial cells . An invasive phenotype is conferred to this pathogen by a number of proteins that are components of a type III secretion system . During the invasion process, the bacteria utilize this secretion system to release proteins that enter the host cell and apparently interact with unknown host cell components that induce alterations in the actin cytoskeleton . To investigate the role of secreted proteins as direct modulators of invasion, we have evaluated the ability of Salmonella typhimurium to enter mammalian cells that express portions of the Salmonella invasion proteins SipB and SipC . Plasma membrane localization of SipB and SipC was achieved by fusing carboxyl- and amino-terminal portions of each invasion protein to the intracellular carboxyl-terminal tail of a membrane-bound eukaryotic receptor . Expression of receptor chimeras possessing the carboxyl terminus of SipB or the amino terminus of SipC blocked Salmonella invasion, whereas expression of their chimeric counterparts had no effect on invasion . The effect on invasion was specific for Salmonella since the perturbation of uptake was not extended to other invasive bacterial species . These results suggest that Salmonella invasion can be competitively inhibited by preventing the intracellular effects of SipB or SipC . In addition, these experiments provide a model for examining interactions between bacterial invasion proteins and their host cell targets.

Zh Mikrobiol Epidemiol Immunobiol, 1998 Jul-Aug, (4), 19 - 23
{The design and study of an ogt deletion mutant of Salmonella typhi}; Satorov S et al.; The chromosome of S . typhi, similarly to those of Escherichia coli and Salmonella typhimurium, was found to contain ogt gene . Using Cmr gene, flanked by the corresponding sites of Salmonella chromosome, we managed to create S . typhi ogt deletion mutant strain . As shown in this study, ogt gene, together with giving protection against alkylating, methylating, ethylating and propylating agents, played an important role in protection against other mutagenic factors . In contrast to the wild strain, S . typhi ogt deletion mutant strain was found to be sensitive to UV radiation, pesticides and antimicrobial preparations.

Microbiology, 1998 Sep, 144 ( Pt 9), 2497 - 504
Hydrolysis of the soft amphiphilic antimicrobial agent tetradecyl betainate is retarded after binding to and killing Salmonella typhimurium; Ahlstrom B et al.; Hydrolysis of tetradecyl betainate (B14), a fast-acting microbicidal amphiphilic quaternary ammonium compound (QAC) being an ester of betaine and tetradecanol, occurred after binding to Salmonella typhimurium, resulting in release of the water-soluble betaine portion and retention of the lipophilic tetradecanol . The rate of the hydrolysis was significant but retarded in comparison to B14 in solution . As in free solution, the hydrolysis of substance bound to S . typhimurium was increased in an alkaline environment and by heat . At pH 6.0 and 20 degrees C the hydrolysis of bound ester was about 10% after 180 min, whereas at pH 9.0 and 50 degrees C it was about 50% after 60 min . These results are consistent with a model where amphiphilic QACs are inserted into the bacterial outer membrane (OM) with the quaternary ammonium head group facing outwards and the lipophilic portion, including the ester bond, being in the membrane lipid environment enough for retarding the hydrolysis . However, calculation of the mean concentration of B14 in the bacteria at MBC99 (minimum bactericidal concentration required to kill 99% of cells) showed a 7000-8000 times greater concentration than in the medium . At this concentration, when most B14 is considered to be bound to the OM, the available surface area for each molecule was only 2 A2 . This is only 6-7% of that required for close packing of the quaternary ammonium head group (30 A2), indicating that a three-dimensional, presumably continuous arrangement was formed . Since B14 is hydrolysed after its binding to bacteria with microbicidal effect, it may be used under conditions where stable QACs might be harmful to the close or the common environment.

Mol Microbiol, 1998 Sep, 29(6), 1471 - 80
The identification of exported proteins with gene fusions to invasin; Worley MJ et al.; Exported proteins are integral to understanding the biology of bacterial organisms . They have special significance in pathogenesis research because they can mediate critical interactions between pathogens and eukaryotic cell surfaces . Further, they frequently serve as targets for vaccines and diagnostic tests . The commonly used genetic assays for identifying exported proteins use fusions to alkaline phosphatase or beta-lactamase . These systems are not ideal for identifying outer membrane proteins because they identify a large number of inner membrane proteins as well . We addressed this problem by developing a gene fusion system that preferentially identifies proteins that contain cleavable signal sequences and are released from the inner membrane . This system selects fusions that restore outer membrane localization to an amino terminal-truncated Yersinia pseudotuberculosis invasin derivative . In the present study, a variety of Salmonella typhimurium proteins that localize beyond the inner membrane were identified with gene fusions to this invasin derivative . Previously undescribed proteins identified include ones that share homology with components of fimbrial operons, multiple drug resistance efflux pumps and a haemolysin . All of the positive clones analysed contain cleavable signal sequences . Moreover, over 40% of the genes identified encode putative outer membrane proteins . This system has several features that may make it especially useful in the study of genetically intractable organisms.

Mol Microbiol, 1998 Sep, 29(6), 1435 - 48
Gyrase and Topo IV modulate chromosome domain size in vivo; Staczek P et al.; In bacteria, DNA supercoil movement is restricted to subchromosomal regions or 'domains.' To elucidate the nature of domain boundaries, we analysed reaction kinetics for gammadelta site-specific resolution in six chromosomal intervals ranging in size from 14 to 90 kb . In stationary cultures of Salmonella typhimurium, resolution kinetics were rapid for both short and long intervals, suggesting that random stationary barriers occur with a 30% probability at approximately 80 kb intervals along DNA . To test the biochemical nature of domain barriers, a genetic screen was used to look for mutants with small domains . Rare temperature-sensitive alleles of DNA gyrase and Topo IV (the two essential type II topoisomerases) had more supercoil barriers than wild-type strains in all growth states . The most severe gyrase mutants were found to have twice as many barriers in growing cells as wild type throughout a 90 kb interval of the chromosome . We propose that knots and tangles in duplex DNA restrain supercoil diffusion in living bacteria.

Nippon Rinsho, 1998 Sep, 56(9), 2376 - 81
{Bleeding infectious enteritis}; Mitsuda T et al.; A recent trend on bleeding intestinal infections in Japan was described . Salmonella Enteritidis infection occupied over 42% of food-borne diseases in 1996 . Salmonella Enteritidis is the most popular infectious agent for food-borne outbreak in Japan . Salmonella Typhimurium DT104 which is widely spread in Europe and in U.S.A . is not common in Japan . We experienced large outbreak of foodbore EHEC/VTEC O157: H7 infections in 1996 . Since then, diagnostic and therapeutic studies on EHEC/VTEC infection and haemolytic uremic syndrome are promoted by the Government . HACCP may take an important roll for the prevention of large outbreaks of foodbore EHEC/VTEC infections.

Chem Res Toxicol, 1998 Oct, 11(10), 1195 - 200
Identification of a 2-phenylbenzotriazole (PBTA)-type mutagen, PBTA-2, in water from the Nishitakase River in Kyoto; Oguri A et al.; We previously isolated five mutagens, compounds I-V, in blue rayon-adsorbed materials from the Nishitakase River in Kyoto . The chemical structure of compound I, a major mutagen that accounted for 21% of the total mutagenicity, was determined to be 2-{2-(acetylamino)-4-{bis(2-methoxyethyl)amino}-5-methoxyphenyl}-5-am ino-7-bromo-4-chloro-2H-benzotriazole (PBTA-1) . Compound II was also a major mutagen and accounted for 17% of the total mutagenicity . In this study, a large quantity (1.2 mg) of compound II was isolated from adsorbate to 27 kg of blue cotton, and its UV, mass, and 1H NMR spectra were analyzed . On the basis of the spectral data, compound II was deduced to be the PBTA-1 analogue 2-{2-(acetylamino)-4-{N-(2-cyanoethyl)ethylamino}-5-methoxyphenyl}-5- amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-2) . As with PBTA-1, PBTA-2 was synthesized from an azo dye by reduction and chlorination . Since all of the spectra of PBTA-2 coincided with those of compound II obtained from river water, compound II was concluded to be PBTA-2 . PBTA-2 is a newly identified potent mutagen, which induces 93 000 and 3 200 000 revertants of Salmonella typhimurium TA98 and YG1024 per microgram, respectively, in the presence of S9 mix . Like PBTA-1, PBTA-2 may also be produced from an azo dye during industrial processes in dyeing factories and treatment at sewage plants.

Chem Res Toxicol, 1998 Oct, 11(10), 1137 - 44
1,3-Dichloropropene epoxides: intermediates in bioactivation of the promutagen 1,3-dichloropropene; Schneider M et al.; 1,3-Dichloropropene (1,3-D), a major soil fumigant nematicide, is genotoxic in many types of assays, leading to its classification as possibly carcinogenic in humans . This study tests in three steps the hypothesis that 1,3-D is a promutagen activated by epoxidation and further reaction of the 1,3-D-epoxides . Stereospecific epoxidation of 1,3-D (examined as the cis/trans mixture and as individual isomers) to the corresponding cis- and trans-1,3-D-epoxides is demonstrated here for the first time, both in vitro in a mouse liver microsome-NADPH system and in vivo in the liver of ip-treated mice, using GC/MS for product identification and quantitation . The cis epoxide is observed in higher yield than the trans epoxide, both in vitro and in vivo, and the cis isomer also reacts slower than the trans isomer with GSH alone or catalyzed by GSH S-transferase . cis- and trans-1,3-D-Epoxides are stable in acetone or chloroform but degrade completely in Me2SO exclusively to 2-chloroacrolein (30 min at 40 degrees C) . Epoxide decomposition is slower in pH 7.4 phosphate buffer (t1/2 = 116 and 64 min for cis and trans, respectively, at 41 degrees C) with a >99% yield of 3-chloro-2-hydroxypropanal (and its dimer) and <0.5% formation of 2-chloroacrolein (for which the t1/2 is 248 min at 41 degrees C) . Mutagenicity assays in Salmonella typhimurium TA100 (standard plate incorporation) establish high potencies of 37, 17, and 150 revertants/nmol for cis- and trans-1, 3-D-epoxides and 2-chloroacrolein, respectively . The mutagenicity of the epoxides is due either to their direct action or to a degradation product formed at physiological pH, i.e., 3-chloro-2-hydroxypropanal or its dehydrochlorination products . The candidate mutagens methylglyoxal and glycidaldehyde are not detected as breakdown products of 3-chloro-2-hydroxypropanal at pH 7.4 and also have low mutagenic activity in TA100 . It is therefore proposed that the penultimate and ultimate mutagens of 1,3-D metabolism are the corresponding epoxides and their direct hydrolysis product 3-chloro-2-hydroxypropanal, respectively.

Environ Mol Mutagen, 1998, 32(2), 192 - 6
Selection of agar for use in Salmonella typhimurium and Escherichia coli mutation assays; Majeska JB et al.; The spontaneous and induced revertant frequency of four Salmonella typhimurium strains (TA1535, TA1537, TA98, and TA100) and Escherichia coli {WP2 uvrA (pKM101)} was evaluated using Vogel Bonner minimal plates prepared with ten different agars . In addition to the Difco Bacto agar originally recommended by Ames, Difco Noble, granulated and Bitek agars; BD grade A, BBL granulated and purified agars; Oxoid purified and No . 1 agars; and GIBCO select agar were tested . Several of these agars have been reported as acceptable alternatives for these Salmonella strains, but comparable studies with E . coli have not been done . The bacteria were treated with DMSO or an appropriate positive control in the presence or absence of an Aroclor 1254-induced rat liver activation system . With the exception of Noble agar in the presence of S9, there was little difference among the responses of the Salmonella strains on any of the agars . However, with E . coli the responses include either a reduction or an increase in spontaneous revertants numbers as well as a reduction in absolute and relative induced revertant frequency . Difco Bacto agar appears to be the most consistent agar for use with these strains . As an alternative, only BBL purified agar resulted in consistent results for all of these strains under all testing conditions . These results emphasize the need to evaluate the components of the standard mutation assay when incorporating additional bacterial strains . Suboptimal responses related to the agar or other components could compromise the detection of weak mutagens.

Microb Comp Genomics, 1998, 3(3), 151 - 69
The CorA magnesium transporter gene family; Kehres DG et al.; The CorA transport system is the primary Mg2+ influx system of Salmonella typhimurium and Escherichia coli . The CorA protein has no homology to any other known family of proteins . It has an unusual membrane topology, with a large, soluble, highly charged periplasmic N-terminal domain with three transmembrane segments in a shorter, hydrophobic C-terminal domain . Previous phenotypic and molecular data had suggested that this transport system was widespread in the Bacteria . In this report we show that CorA is virtually ubiquitous in the Bacteria and Archaea, forming a distinct family of transport proteins . Genomic sequences to date have revealed at least 22 members of the CorA family in the Bacteria and the Archaea, with 6 more distant members in the yeasts . Only three of the smallest bacterial genomes lack a CorA homologue . Strikingly, phylogenetic analysis does not show clustering by related species or even within kingdom . Several species of Bacteria contain two or even three CorA paralogues . Within species, these paralogues are not closely related, however, and we suggest that they might have distinct transport functions . A multiple alignment suggests three extended consensus regions within the N-terminal soluble domain of CorA, which is predicted to be virtually all alpha-helical . A fourth consensus region includes the last 20 residues of the soluble domain and continues through the entire membrane domain . The first half of this last consensus domain may form an amphipathic alpha-helix that extends from the soluble domain into the first transmembrane segment . The degree of charge in the first transmembrane segment is quite variable, and we suggest that this transport family may include members with only two rather than three transmembrane segments . If so, this would place the N-terminal soluble domain on different sides of the membrane in different members of the family . We suggest that the CorA Mg2+ transport system forms the major Mg2+ uptake system in the Bacteria and Archaea but that some family members may have a function other than Mg2+ transport.

Comp Immunol Microbiol Infect Dis, 1998 Oct, 21(4), 327 - 36
Passive immunisation of neonatal lambs via colostrum and milk of ewes previously immunised with live attenuated Salmonella typhimurium protects neonatal lambs from experimental salmonellosis; Mukkur TK et al.; Lambs sucking non-immunised ewes or ewes immunised 4-5 weeks before lambing with live attenuated, aromatic-dependent (aroA) Salmonella typhimurium (strain CS 332) were challenged orally at either 2, 4 or 7 days of age with virulent S . typhimurium (strain CS 94) at doses ranging from 10(9) to 10(13) colony forming units . No lambs displayed signs of clinical salmonellosis and all survived challenge but those sucking immunised ewes had organisms of the challenge strain in their faeces for much shorter periods of time than lambs of the control ewes . High titres of specific antibodies were measured in colostrum and milk of immunised ewes in comparison with very low titres measured in samples from control ewes; these differences were reflected by the titres of antibodies in the sera of corresponding lambs . At 2 days after lambing, the major antibody isotype in the colostrum of immunised ewes and sera of their lambs was IgM whereas at 7 days IgG1 was the predominant isotype . While it was clear that vaccination of pregnant ewes with the live attenuated vaccination conferred protection against experimentally-induced salmonellosis in their lambs, considerable protection was observed in control lambs in spite of there being very low titres of antibodies in the mammary secretion of their dams . The latter observation could be related to the presence of contain non-antibody potent bactericidal factors previously described in colostrum and milk.

J Struct Biol, 1998, 122(3), 311 - 9
Architectural features of the Salmonella typhimurium flagellar motor switch revealed by disrupted C-rings; Khan S et al.; The three-dimensional surface topology of rapid-frozen Salmonella typhimurium flagellar hook basal body complexes was studied by stereo-examination of thin-film metal replicas . The complexes contained the extended cytoplasmic structure, composed of the switch complex proteins; FliG, FliM, and FliN . Distinct nanometer-scale element arrays, separated by grooves, defined the outer surface of the cytoplasmic (C-) ring . The number of array elements was comparable to previously determined FliG and FliM copy numbers in the basal body . In addition to basal body complexes lacking C-rings, complexes containing incomplete C-rings were identified . The incomplete C-rings had lost segments of the proximal array . Basal bodies with the distal C-ring array alone were not found . These findings are compatible with the spatial organization of the flagellar switch suggested by previous biochemical data .

Presse Med, 1998 May 23-30, 27(19), 909 - 10
{Cerebral abscess during a severe form of Salmonella typhimurium bacteremia in an immunocompetent patient}; Broux C et al.; BACKGROUND: Bacteremia rarely occurs in non-typhoid salmonella infections and the development of a brain abscess is exceptional . CASE REPORT: An immunocompetent patient developed severe Salmonella typhimurium bacteremia leading to septic shock and acute respiratory distress and acute renal failure . A brain abscess, which was not present on the initial brain tomodensitometry, developed and totally regressed after antibiotic therapy . DISCUSSION: We were unable to identify and factor favoring the development of salmonella bacteremia in this patient . There were no cerebral lesions on the initial brain tomodensitometry considered to be normal . To our knowledge, this is the first report of Salmonella typhimurium brain abscess in an immunocompetent subject.

Mol Microbiol, 1998 Sep, 29(5), 1191 - 202
Deletion analysis of MotA and MotB, components of the force-generating unit in the flagellar motor of Salmonella; Muramoto K et al.; MotA and MotB are cytoplasmic membrane proteins that form the force-generating unit of the flagellar motor in Salmonella typhimurium and many other bacteria . Many missense mutations in both proteins are known to cause slow motor rotation (slow-motile phenotype) or no rotation at all (non-motile or paralysed phenotype) . However, large stretches of sequence in the cytoplasmic regions of MotA and in the periplasmic region of MotB have failed to yield these types of mutations . In this study, we have investigated the effect of a series of 10-amino-acid deletions in these phenotypically silent regions . In the case of MotA, we found that only the C-terminal 5 amino acids were completely dispensable; an adjacent 10 amino acids were partially dispensable . In the cytoplasmic loop region of MotA, deletions made the protein unstable . For MotB, we found that two large segments of the periplasmic region were dispensable: the results with individual deletions showed that the first consisted of six deletions between the sole transmembrane span and the peptidoglycan binding motif, whereas the second consisted of four deletions at the C-terminus . We also found that deletions in the MotB cytoplasmic region at the N-terminus impaired motility but did not abolish it . Further investigations in MotB were carried out by combining dispensable deletion segments . The most extreme version of MotB that still retained some degree of function lacked a total of 99 amino acids in the periplasmic region, beginning immediately after the transmembrane span . These results indicate that the deleted regions in the MotA cytoplasmic loop region are essential for stability; they may or may not be directly involved in torque generation . Part of the MotA C-terminal cytoplasmic region is not essential for torque generation . MotB can be divided into three regions: an N-terminal region of about 30 amino acids in the cytoplasm, a transmembrane span and about 260 amino acids in the periplasm, including a peptidoglycan binding motif . In the periplasmic region, we suggest that the first of the two dispensable stretches in MotB may comprise part of a linker between the transmembrane span of MotB and its attachment point to the peptidoglycan layer, and that the length or specific sequence of much of that linker sequence is not critical . About 40 residues at the C-terminus are also unimportant.

Res Microbiol, 1998 May, 149(5), 309 - 18
Inhibition of Salmonella intracellular proliferation by non-phagocytic eucaryotic cells; Martinez-Moya M et al.; Salmonella typhimurium is an intracellular pathogen capable of proliferating within vacuolar compartments of non-phagocytic eucaryotic cells . This process has been shown to be essential for virulence in the mouse typhoid model (Leung and Finlay, Proc . Natl . Acad . Sci . USA, 88, 11470-11474, 1990) . Here we present evidence that certain non-phagocytic eucaryotic cell lines, such as 3T3 (mouse fibroblasts) and NRK (rat fibroblasts) cells, are not permissive for S . typhimurium intracellular proliferation . Moreover, viability of intracellular bacteria residing within both cell types notably decreases at late postinfection times (72 h) . These results clearly demonstrate that non-phagocytic eucaryotic cells are capable of destroying intracellular S . typhimurium . Experimentation with 3T3 and NRK cell lines might provide an appropriate in vitro model for identifying new bacterial and/or eucaryotic factors regulating Salmonella intracellular proliferation within vacuoles of the host eucaryotic cell.

J Food Prot, 1998 Sep, 61(9), 1124 - 8
Antibacterial effects of N-sulfonated and N-sulfobenzoyl chitosan and application to oyster preservation; Chen CS et al.; The antibacterial effects of sulfonated and sulfobenzoyl chitosans were evaluated and compared with that of 69% deacetylated chitosan (DD69 chitosan) . Minimal inhibitory concentrations of sulfonated chitosan (SC1, 0.63% sulfur content) against Shigella dysenteriae, Aeromonas hydrophila, Salmonella typhimurium, and Bacillus cereus were found to be lower than those of DD69 chitosan . A high sulfur content in sulfonated chitosan adversely influenced its antibacterial effect . Sulfobenzoyl chitosan (SBC) has excellent water solubility and an antibacterial effect comparable to that of SC1 . SBC at 1,000 and 2,000 ppm extended the shelf life of oysters at 5 degrees C by 4 days at the former or by 7 days at least at the latter concentration . The growth of coliforms and Pseudomonas, Aeromonas, and Vibrio species on oysters was retarded by the addition of DD69 chitosan or SBC.

J Bacteriol, 1998 Oct, 180(20), 5384 - 97
Flk couples flgM translation to flagellar ring assembly in Salmonella typhimurium; Karlinsey JE et al.; The hook-basal body (HBB) is a key intermediate structure in the flagellar assembly pathway in Salmonella typhimurium . The FlgM protein inhibits the flagellum-specific transcription factor sigma28 in the absence of the intact HBB structure and is secreted out of the cell following HBB completion . The flk gene encodes a positive regulator of the activity of FlgM at an assembly step just prior to HBB completion: at the point of assembly of the P- and L-rings . FlgM inhibition of sigma28-dependent class 3 flagellar gene transcription was relieved in P- and L-ring assembly mutants (flgA, flgH, and flgI) by introduction of a null mutation in the flk gene (J . E . Karlinsey et al., J . Bacteriol . 179:2389-2400, 1997) . In P- and L-ring mutant strains, recessive mutations in flk resulted in a reduction in intracellular FlgM levels to those seen in wild-type (Fla+) strains . The reduction in intracellular FlgM levels by mutations in the flk gene was concomitant with a 10-fold increase in transcription of the flgMN operon compared to that of the isogenic flk+ strain, while transcription of the flgAMN operon was unaffected . This was true for both direct measurement of the flgAMN and flgMN mRNA transcripts by RNase T2 protection assays and for lac operon fusions to either the flgAMN or flgMN promoter . Loss of Flk did not allow secretion of FlgM through basal-body structures lacking the P- and L-rings . Intracellular FlgM was stable to proteolysis, and turnover occurred primarily after export out of the cell . Loss of Flk did not result in increased FlgM turnover in either P- or L-ring mutant strains . With lacZ translational fusions to flgM, a null mutation in flk resulted in a significant reduction of flgM-lacZ mRNA translation, expressed from the class 3 flgMN promoter, in P- and L-ring mutant strains . No reduction in either flgAMN or flgMN mRNA stability was measured in the absence of Flk in Fla+, ring mutant, or HBB deletion strains . We conclude that the reduction in the intracellular FlgM levels by mutation in the flk gene is only at the level of flgM mRNA translation.

J Bacteriol, 1998 Oct, 180(20), 5299 - 305
Domain structure of the ATP-binding-cassette protein MalK of salmonella typhimurium as assessed by coexpressed half molecules and LacK'-'MalK chimeras; Schmees G et al.; ATP-binding-cassette (ABC) subunit MalK of the binding protein-dependent transport system for maltose of Salmonella typhimurium and Escherichia coli is crucial to the transport process but also exhibits a repressing activity on other genes of the maltose regulon . The latter function has been attributed to a carboxy-terminal extension by which MalK differs in length from a prototype ABC protein . In order to define the boundaries of putative functional domains of MalK, we have analyzed pairs of N- and C-terminally truncated MalK proteins of S . typhimurium . Coexpressed half molecules of about equal lengths (MalKN1: residues 1 to 179; MalKC1: residues 179 to 369) restored the transport activity of a malK strain and displayed substantial regulatory activity . The same regulatory activity was obtained when malKC1 was expressed separately . These results indicate that a covalent linkage is not absolutely essential for function and that the protein might be composed of two structurally distinct entities . To elucidate further the minimal structural requirements for the regulatory function of MalK, we have studied chimeric proteins that have C-terminal portions of MalK fused to the corresponding amino-terminal fragments of its close homolog LacK . Functional analyses revealed that a fusion containing only the C-terminal extension of MalK (Q263 to V369) is sufficient to display half-maximal regulatory activity . This activity increased with the lengths of the MalK portions present in the chimeras . Furthermore, the failure of two chimeras to support maltose transport suggests a structurally critical region between residues 243 and 264 . In the absence of a crystal structure, this work contributes to the understanding of the multiple functions of MalK.

Genes Dev, 1998 Oct 1, 12(19), 3123 - 36
The flagellar anti-sigma factor FlgM actively dissociates Salmonella typhimurium sigma28 RNA polymerase holoenzyme; Chadsey MS et al.; The anti-sigma factor FlgM of Salmonella typhimurium inhibits transcription of class 3 flagellar genes through a direct interaction with the flagellar-specific sigma factor, sigma28 . FlgM is believed to prevent RNA polymerase (RNAP) holoenzyme formation by sequestering free sigma28 . We have analyzed FlgM-mediated inhibition of sigma28 activity in vitro . FlgM is able to inhibit sigma28 activity even when sigma28 is first allowed to associate with core RNAP . Surface plasmon resonance (SPR) was used to evaluate the interaction between FlgM and both sigma28 and sigma28 holoenzyme (Esigma28) . The Kd of the sigma28-FlgM complex is approximately 2 x 10(-10) M; missense mutations in FlgM that cause a defect in sigma28 inhibition in vivo increase the Kd of this interaction by 4- to 10-fold . SPR measurements of Esigma28 dissociation in the presence of FlgM indicate that FlgM destabilizes Esigma28, presumably via an interaction with the sigma subunit . Our data provide the first direct evidence of an interaction between FlgM and Esigma28 . We propose that this secondary activity of FlgM, which we term holoenzyme destabilization, enhances the sensitivity of the cell to changes in FlgM levels during flagellar biogenesis.

Arch Toxicol, 1998 Jul-Aug, 72(8), 505 - 13
Correlation of 32P-postlabelling-detection of DNA adducts in mouse skin in vivo with the polycyclic aromatic compound content and mutagenicity in Salmonella typhimurium of a range of oil products; Booth ED et al.; The in vivo genotoxic activities in mouse skin of the dimethyl sulphoxide (DMSO) extracts of a range of oil products {residual aromatic extract; untreated heavy paraffinic distillate aromatic extract; mildly refined light naphthenic base oil; bitumen (vacuum residue); high viscosity index base oil obtained by catalytic hydrogenation} were evaluated by 32P-postlabelling DNA analysis . The results of quantitative 32P-postlabelling analyses of epidermal DNA from mice treated with the DMSO extracts showed linear relationships with the total polycyclic aromatic compound (PAC) contents, determined by the Institute of Petroleum method IP 346 and also the 3-6 ring PAC contents, measured by on-line liquid-liquid extraction using flow injection analysis . The 32P-postlabelling data also showed a linear relationship with the mutagenicity indices of these oil products determined in S . typhimurium TA98 using the modified Ames Salmonella microsome test . The in vivo genotoxicity of the DMSO extracts from the oil products was low, judged by 32P-postlabelling analysis of DNA adducts measured in epidermal DNA of treated mouse skin, and ranging from 2 to 723 attomole/microg DNA per mg oil product . The in vivo 32P-postlabelling data from this study are consistent with these materials expressing low genotoxicity in mouse skin in vivo . The DMSO extraction procedure coupled with 32P-postlabelling DNA analysis is useful for ranking the relative genotoxic potency in vivo of a wide range of oil products . In general the trend observed is similar to rankings based on physicochemical measurements of total PAC contents or 3 6 ring PAC contents of the oil products.

Anal Biochem, 1978 Nov, 91(1), 46 - 59
Improved radioisotopic assay for cytidine 5'-triphosphate synthetase (EC 6.3.4.2); Williams JC et al.; An improved radioassay for cytidine 5-triphosphate synthetase is reported which employs thin-layer chromatographic methods and provides a number of advantages over previously available techniques . (i) The method resolves the nucleotides and the degradation products generated during the time course of the enzymatic reaction by ascending chromatography employing polyethyleneimine cellulose plastic-backed sheets . (ii) Determinations of CTP formed and all nucleotide pairs generated during kinetic analysis of CTP synthetase are greatly simplified, further facilitating the detection of extraneous enzymatic activities . (iii) The sensitivity of the assay is enhanced and as little as 50 pmol of product formed was readily detected in supernatant fluids . This was made possible, in part, by the addition of NaF and phosphoenolpyruvate which together maintain the nucleotide triphosphates in the reaction mixture . (iv) A large number of samples can be handled at one time with highly reproducible results . The synthesis of CTP from UTP by enzyme preparations from rat liver, hepatomas, and Salmonella typhimurium LT2 was quantitated with this method.

J Mol Biol, 1998, 283(1), 121 - 33
Three-dimensional structure of O-acetylserine sulfhydrylase from Salmonella typhimurium; Burkhard P et al.; The last step in cysteine biosynthesis in enteric bacteria is catalyzed by the pyridoxal 5'-phosphate-dependent enzyme O-acetylserine sulfhydrylase . Here we report the crystal structure at 2.2 A resolution of the A-isozyme of O-acetylserine sulfhydrylase isolated from Salmonella typhimurium . O-acetylserine sulfhydrylase shares the same fold with tryptophan synthase-beta from Salmonella typhimurium but the sequence identity level is below 20% . There are some major structural differences: the loops providing the interface to the alpha-subunit in tryptophan synthase-beta and two surface helices of tryptophan synthase-beta are missing in O-acetylserine sulfhydrylase . The hydrophobic channel for indole transport from the alpha to the beta active site of tryptophan synthase-beta is, not unexpectedly, also absent in O-acetylserine sulfhydrylase . The dimer interface, on the other hand, is more or less conserved in the two enzymes . The active site cleft of O-acetylserine sulfhydrylase is wider and therefore more exposed to the solvent . A possible binding site for the substrate O-acetylserine is discussed .

Antimicrob Agents Chemother, 1998 Oct, 42(10), 2511 - 20
Intracellular delivery and antibacterial activity of gentamicin encapsulated in pH-sensitive liposomes; Lutwyche P et al.; Cell membranes are relatively impermeable to the antibiotic gentamicin, a factor that, along with the toxicity of gentamicin, precludes its use against many important intracellular bacterial infections . Liposomal encapsulation of this drug was used in order to achieve intracellular antibiotic delivery and therefore increase the drug's therapeutic activity against intracellular pathogens . Gentamicin encapsulation in several dipalmitoylphosphatidylcholine (DPPC) and pH-sensitive dioleoylphosphatidylethanolamine (DOPE)-based carrier systems was characterized . To systematically test the antibacterial efficacies of these formulations, a tissue culture assay system was developed wherein murine macrophage-like J774A.1 cells were infected with bacteria and were then treated with encapsulated drug . Of these formulations, DOPE-N-succinyl-DOPE and DOPE-N-glutaryl-DOPE (70:30;mol:mol) containing small amounts of polyethyleneglycol-ceramide showed appreciable antibacterial activities, killing greater than 75% of intracellular vacuole-resident wild-type Salmonella typhimurium compared to the level of killing of the control formulations . These formulations also efficiently eliminated intracellular infections caused by a recombinant hemolysin-expressing S . typhimurium strain and a Listeria monocytogenes strain, both of which escape the vacuole and reside in the cytoplasm . Control non-pH-sensitive liposomal formulations of gentamicin had poor antibacterial activities . A fluorescence resonance energy transfer assay indicated that the efficacious formulations undergo a pH-dependent lipid mixing and fusion event . Intracellular delivery of the fluorescent molecules encapsulated in these formulations was confirmed by confocal fluorescence microscopy and was shown to be dependent on endosomal acidification . This work shows that encapsulation of membrane-impermeative antibiotics in appropriately designed lipid-based delivery systems can enable their use in treating intracellular infections and details the development of a general assay for testing the intracellular delivery of encapsulated drug formulations.

Genetics, 1998 Oct, 150(2), 533 - 42
Mechanism and control of interspecies recombination in Escherichia coli . I . Mismatch repair, methylation, recombination and replication functions; Stambuk S et al.; A genetic analysis of interspecies recombination in Escherichia coli between the linear Hfr DNA from Salmonella typhimurium and the circular recipient chromosome reveals some fundamental aspects of recombination between related DNA sequences . The MutS and MutL mismatch binding proteins edit (prevent) homeologous recombination between these 16% diverged genomes by at least two distinct mechanisms . One is MutH independent and presumably acts by aborting the initiated recombination through the UvrD helicase activity . The RecBCD nuclease might contribute to this editing step, presumably by preventing reiterated initiations of recombination at a given locus . The other editing mechanism is MutH dependent, requires unmethylated GATC sequences, and probably corresponds to an incomplete long-patch mismatch repair process that does not depend on UvrD helicase activity . Insignificant effects of the Dam methylation of parental DNAs suggest that unmethylated GATC sequences involved in the MutH-dependent editing are newly synthesized in the course of recombination . This hypothetical, recombination-associated DNA synthesis involves PriA and RecF functions, which, therefore, determine the extent of MutH effect on interspecies recombination . Sequence divergence of recombining DNAs appears to limit the frequency, length, and stability of early heteroduplex intermediates, which can be stabilized, and the recombinants mature via the initiation of DNA replication.

Eur J Immunol, 1998 Sep, 28(9), 2913 - 20
Innate resistance to infection by intracellular bacterial pathogens differs in mice selected for maximal or minimal acute inflammatory response; Araujo LM et al.; The intensity of nonspecific immune reaction and the host resistance to facultative intracellular pathogens are found to be associated in lines of mice selected for maximal (AIRmax) or minimal (AIRmin) acute inflammatory reactivity . AIRmax are more resistant than AIRmin mice to Salmonella typhimurium and Listeria monocytogenes infection, the differences between lines in LD50 being > 1000 and 100 times, respectively . This difference was shown to be related to the initial bacterial containment at the infectious focus, and to the control of bacterial multiplication in the spleen during the 1st week after s . c . inoculation of the bacteria . Specific immune responses were not deeply affected by the selective process: antibody production and delayed-type hypersensitivity were both of similar intensity in AIRmax and AIRmin mice . The differential susceptibility to infection seems independent of the Nramp-1 locus polymorphism; therefore, these two lines represent a powerful model for investigating the role of other genetic loci regulating the nonspecific immunity effectors in the course of infectious diseases.

Can Vet J, 1998 Sep, 39(9), 559 - 65
Salmonella typhimurium DT104: a virulent and drug-resistant pathogen; Poppe C et al.; Salmonella typhimurium phage type (PT) or definitive type (DT) 104 is a virulent pathogen for humans and animals, particularly cattle . It has been isolated increasingly from humans and animals in the United Kingdom and several other European countries and, more recently, in the United States and Canada . Humans may acquire the infection from foods of animal origin contaminated with the infective organism . Farm families are particularly at risk of acquiring the infection by contact with infected animals or by drinking unpasteurized milk . The symptoms in cattle are watery to bloody diarrhea, a drop in milk production, pyrexia, anorexia, dehydration and depression . Infection may result in septicemic salmonellosis and, upon necropsy, a fibrinonecrotic enterocolitis may be observed . The infection occurs more commonly in the calving season than at other times . Feedlot cattle and pigs may also be affected . Prolonged carriage and shedding of the pathogen may occur . Symptoms in humans consist of diarrhea, fever, headache, nausea, abdominal pain, vomiting, and, less frequently, blood in the stool . Salmonella typhimurium DT104 strains are commonly resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline.

Yi Chuan Xue Bao, 1998 Apr, 25(2), 181 - 7
{Expression regulation of purine biosynthetic genes in Salmonella typhimurium . VI . Isolation and characterization of super-repressor mutants}; Tang H et al.; Starting from strain of Salmonella typhimurium purD::lac, 86 exponential cultures were mutagenized with NTG and white or light blue clones on E + ado + Xgal plate were selected as candidates of purRs mutant . Total 66 independent candidate strains were obtained . By assaying their beta-galactosidase activity under the repressed and derepressed conditions, determining their frequency of revertional mutation, Conducting transductional analysis of mutational site and dorminance test, 11 candidates strains were proved to be super-repressor mutants . These mutants are useful for studying the expression of purine biosynthetic gene and relationship between protein structure and function in general.

J Bacteriol, 1998 Oct, 180(19), 5231 - 4
Cell division inhibition in Salmonella typhimurium histidine-constitutive strains: an ftsI-like defect in the presence of wild-type penicillin-binding protein 3 levels; Cano DA et al.; Histidine-constitutive (Hisc) strains of Salmonella typhimurium undergo cell division inhibition in the presence of high concentrations of a metabolizable carbon source . Filaments formed by Hisc strains show constrictions and contain evenly spaced nucleoids, suggesting a defect in septum formation . Inhibitors of penicillin-binding protein 3 (PBP3) induce a filamentation pattern identical to that of Hisc strains . However, the Hisc septation defect is caused neither by reduced PBP3 synthesis nor by reduced PBP3 activity . Gross modifications of peptidoglycan composition are also ruled out . D-Cycloserine, an inhibitor of the soluble pathway producing peptidoglycan precursors, causes phenotypic suppression of filamentation, suggesting that the septation defect of Hisc strains may be caused by scarcity of PBP3 substrate.

Biochemistry, 1998 Sep 22, 37(38), 13359 - 69
Structure of Salmonella typhimurium nrdF ribonucleotide reductase in its oxidized and reduced forms; Eriksson M et al.; The first class Ib ribonucleotide reductase R2 structure, from Salmonella typhimurium, has been determined at 2.0 A resolution . The overall structure is similar to the Escherichia coli class Ia enzyme despite only 23% sequence identity . The most spectacular difference is the absence of the pleated sheet and adjacent parts present in the E . coli R2 structure; the heart-shaped structure loses its tip . From sequence comparisons, it appears that this feature is shared with all other class Ib enzymes and, in this respect, is more like the mammalian class Ia enzymes . Both the oxidized and reduced iron forms have been investigated . In the ferric iron center, both iron ions are octahedrally coordinated and bridged by one carboxylate and one oxide ion . The ferrous form has lost the bridging oxide ion but is bridged by two carboxylates . Accompanying the change in redox state, helix E changes its conformation from one covering the metal center in the oxidized form to a more open reduced form . A narrow channel is opened which may permit easier access of oxygen to the ferrous iron site and to efficiently generate the tyrosyl radical.

J Biol Chem, 1998 Oct 2, 273(40), 25745 - 50
The glucose transporter of Escherichia coli with circularly permuted domains is active in vivo and in vitro; Gutknecht R et al.; The bacterial phosphotransferase system (PTS) consists of two energy-coupling soluble proteins (enzyme I and HPr) and a large number of inner membrane transporters (enzymes II) that mediate concomitant phosphorylation and translocation of sugars and hexitols . The transporters consist of three functional units (IIA, IIB, IIC), which occur either as protein subunits or domains of a multidomain polypeptide . The membrane-spanning IIC domain contains the substrate binding site; IIA and IIB are phosphorylation domains that transfer phosphate from HPr to the transported sugar . The transporter complexes of the PTS are good examples for variation of design by modular assembly of domains and subunits . The domain order is IIC-IIB in the membrane subunit of the Escherichia coli glucose transporter (IICBGlc) and IIB-IIC in Salmonella typhimurium sucrose transporter (IIBCScr) . The phosphorylation domain of IICBGlc was translocated from the carboxyl-terminal to the amino-terminal end of the IIC domain, and the activity of the circularly permuted form was optimized by variation of the length and the composition of the interdomain linker . IIBapCGlc with an alanine-proline-rich interdomain linker has 70% of the control specific activity after purification and reconstitution into proteoliposomes . These results indicate that the amino-terminal end of IICBGlc must be on the cytoplasmic side of the inner membrane, that membrane insertion of the IIC domain is insensitive to the modification of its amino-terminal end, and that a domain swap as it could occur by a single DNA translocation event can rapidly lead to a functional protein . However, IIB could not be substituted for by glucokinase . Fusion proteins between the IIC domain and glucokinase do not transport and phosphorylate glucose in an ATP-dependent mechanism, although the IIC moiety displays transport activity upon complementation with soluble subclonal IIB, and the glucokinase moiety retains ATP-dependent nonvectorial kinase activity . This indicates that IIC and IIB are two cooperative units and not only sequentially acting upon a common substrate, and that translocation of glucose must be conformationally coupled to the phosphorylation/dephosphorylation cycle of IIB.

Vet Rec, 1998 Aug 8, 143(6), 155 - 8
Causes of death of wild birds of the family Fringillidae in Britain; Pennycott TW et al.; The provision of supplementary food for wild birds in gardens during the winter months is common in the UK, but it is possible that it may precipitate infectious diseases in the birds . This paper describes the results of postmortem examinations of 116 wild finches carried out over a period of four years . The two commonest causes of death in areas where high mortality had been reported were infections with the bacteria Salmonella typhimurium DT40 and Escherichia coli O86 . Coccidia of the genera Atoxoplasma or Isospora were found in several of the birds but were considered to be incidental . Megabacteria were also identified in some of the birds, for the first time in flocks of wild birds in the UK, but they were not considered to be significant.

Infect Immun, 1998 Oct, 66(10), 4624 - 32
Invasion by Salmonella typhimurium induces increased expression of the LMP, MECL, and PA28 proteasome genes and changes in the peptide repertoire of HLA-B27; Maksymowych WP et al.; We have analyzed proteasomal adaptation and associated changes in the B27-bound peptide repertoire in response to cellular invasion with Salmonella . The peptide repertoire of HLA-B27 complexes was analyzed by two different methods: (i) high-pressure liquid chromatography (HPLC) profiles of newly synthesized peptides eluted from B27 following metabolic labeling with arginine and (ii) reactivities with two B27 monoclonal antibodies, Ye-2 and B27.M2, sensitive to peptide-induced conformational changes . LMP, MECL, and PA28 expression was analyzed by reverse transcription-PCR (RT-PCR) of mRNA and by Western blot analysis for LMP2 . Invasion of HLA-B27-transfected HeLa cells by Salmonella typhimurium induced significant changes in the reactivities of HLA-B27 with these two antibodies, which was accompanied by significant quantitative and qualitative changes in the HPLC profile of peptides eluted from HLA-B27 . We also observed increases in the RT-PCR values for the LMP2, LMP7, and MECL proteasome subunit genes, as well as the proteasomal activator PA28alpha and -beta genes, and increased expression of the LMP2 protein by Western blotting . Upregulation of LMP2, but not LMP7, gene expression showed a close correlation with the changes in antibody reactivities observed upon bacterial invasion . We observed similar changes in reactivity with the Ye-2 or the B27.M2 antibody of lymphoblastoid cells upon gamma interferon treatment, which significantly correlated with the increased RT-PCR values for the LMP2 gene . This was accompanied by consistent HPLC profile changes for eluted peptides . Thus, Salmonella invasion leads to serologically recognizable changes in the B27-bound peptide repertoire, which may include peptides of host origin potentially through modulation of proteasome LMP2 subunit expression and, as a consequence, proteasomal activities.

Roum Arch Microbiol Immunol, 1997 Jul-Dec, 56(3-4), 127 - 38
Plasmid profile analysis and antibiotic resistance of Salmonella strains from clinical isolates in Cluj-Napoca; Fagarasan S et al.; Resistance patterns, plasmid profiles and the genetic resistance determinants were investigated in 38 isolates of Salmonella enterica serotype Typhimurium and 19 isolates of Salmonella enterica serovar Enteritidis derived from children hospitalized in two clinics in Cluj-Napoca, during the period of 1995-1997 . Incidence of plasmid and antibiotic resistance was very high in Salmonella typhimurium isolates . All strains were resistant to almost all antibiotics tested but susceptible to the third generation cephalosporines and fluoroquinolones . We identified three resistance patterns and six plasmid profiles . Each plasmid profile was characterized by the presence of two large plasmids of 150-180 Kbp . Approximately 60% of strains harbored three or four small plasmids of 1.3 to 9.5 Kbp . The plasmids of 8.5 Kbp encoded resistance to beta-lactam antibiotics and were non-conjugative . The other small plasmids were cryptic and also non-conjugative . Salmonella enteritidis isolates were susceptible to many antibiotics, except Tetracycline and Trimethoprim-Sulfamethoxazole . We identified three different resistance patterns but nine plasmid profiles . All plasmid profiles were characterized by the presence of a large plasmid (> 100 Kbp) . The number and the diversity of small plasmids were higher than in S . typhimurium strains . There was no parallelism between resistance and plasmid profile: for the same resistance pattern a number of two or three plasmid profiles were found . Our conclusions are that Salmonella typhimurium strains were multiresistant to antibiotics and that many genetically different strains of Salmonella typhimurium and Salmonella enteritidis were responsible for gastroenteritis in children from Cluj County . The increasing antibiotic resistance highlights the need for more refined methods in genetic and epidemiological characterization of bacteria involved in gastrointestinal infections.

Aust N Z J Public Health, 1998 Apr, 22(2), 243 - 6
A salmonellosis outbreak linked to internally contaminated pork meat; Delpech V et al.; In August 1995, we investigated an outbreak of salmonellosis among patrons who attended a church camp in southern Sydney . Of the 73 attendees interviewed, 22 reported a gastroenteritis illness within two days of the conclusion of the camp, with one attendee hospitalised . Two stool specimens, one from each of two attendees, were both positive for Salmonella typhimurium phage type 9 . A cohort study of 68 attendees established a statistically significant association between illness and the consumption of de-boned roast pork (estimated relative risk infinite, p = 0.03) and between illness and the degree of cooking of the pork meat (chi 2 for trend 5.8, p < 0.02) . The outbreak was most likely caused by consumption of roast pork that had been internally contaminated during the de-boning process . Meat and meat products that may be internally contaminated, such as de-boned meats, should be thoroughly cooked . Guidelines about minimum cooking temperatures of meats liable to internal contamination should be developed for commercial food handlers in Australia.

J Mol Biol, 1998 Oct 2, 282(4), 721 - 30
Regulation of UmuD cleavage: role of the amino-terminal tail; McDonald JP et al.; An essential step in SOS mutagenesis is the RecA-mediated posttranslational processing of UmuD-like proteins to the shorter, but mutagenically active, UmuD'-like proteins . Interestingly, the UmuD-like proteins undergo posttranslational processing at different rates . For example, although the Escherichia coli UmuD (UmuDEc) and the Salmonella typhimurium UmuD (UmuDSt) proteins are 73% identical, UmuDSt is processed in vivo at a significantly faster rate than the UmuDEc protein . Here, we report experiments aimed at investigating the molecular basis of these phenotypic differences . The faster rate of UmuDSt cleavage probably does not result solely from a better interaction with RecA, since we observed that, in vitro, UmuDSt undergoes RecA-independent autocatalytic processing about four-times faster than UmuDEc . By constructing chimeric UmuD proteins, we determined that the amino-terminal tail of the UmuD proteins proximal to the Cys24-Gly25 cleavage site is mainly responsible for the difference in UmuDSt and UmuDEc cleavage rates . Site-directed mutagenesis of the UmuDEc protein suggests that most of the enhanced cleavage observed with the UmuDSt protein can be attributed to the presence of a Pro23 residue, juxtaposed to the cleavage site in UmuDSt . Furthermore, this proline residue appears to result in a UmuD protein that is a much better substrate for intermolecular cleavage . These findings clearly implicate the N-terminal tail of the UmuD-like proteins as playing an important and unexpected regulatory function in the maturation of the mutagenically active UmuD'-like mutagenesis proteins .

FEMS Microbiol Lett, 1998 Aug 15, 165(2), 289 - 93
Two novel plasmid-mediated cefotaxime-hydrolyzing beta-lactamases (CTX-M-5 and CTX-M-6) from Salmonella typhimurium; Gazouli M et al.; Two novel plasmid-mediated beta-lactamases (CTX-M-5 and CTX-M-6) produced by Salmonella typhimurium clinical strains were characterized . The enzymes exhibited a pI of 8.4, hydrolyzed oxyimino-beta-lactams and were susceptible to mechanism-based beta-lactamase inhibitors . The respective bla genes were cloned and sequenced . The deduced amino acid sequences showed a high degree of homology with those of the previously described plasmid class A CTX-M-type enzymes and appeared related to the chromosomal beta-lactamases of Klebsiella oxytoca.

Biochem J, 1998 Oct 1, 335 ( Pt 1), 167 - 73
Cobalamin (vitamin B12) biosynthesis: functional characterization of the Bacillus megaterium cbi genes required to convert uroporphyrinogen III into cobyrinic acid a,c-diamide; Raux E et al.; The function of individual genes of the Bacillus megaterium cobI operon genes in cobalamin (vitamin B12) biosynthesis was investigated by their ability to complement defined Salmonella typhimurium cob mutants . This strategy confirmed the role of cbiA, -D, -F, -J, -L and cysGA . Furthermore the operon as a whole was used to restore corrin biosynthesis in Escherichia coli, which, although closely related to S . typhimurium, does not possess the CobI pathway . When the B . megaterium cob operon was cloned into a plasmid and transformed into an E . coli strain containing the S . typhimurium cbiP, it conferred upon the host strain the ability to make the cobyric acid de novo . However, cobyric acid synthesis was observed only when the strain was grown anaerobically . Derivatives of the corrin-producing E . coli strain were constructed in which genes of the B . megaterium cob operon had been inactivated . These strains were used to demonstrate that, whereas B . megaterium cbiD, -G and -X are essential for cobyric acid synthesis, the cbiW and -Y genes could be deleted without detriment to cobyric acid production in E . coli.

Int J Exp Pathol, 1998 Jun, 79(3), 183 - 92
Comparative histopathology in mouse typhoid among genetically diverse mice; Moncure CW et al.; Genetically resistant CBA and A/J mice and susceptible BALB/c and C57BL/6 mice were challenged with either an identical infective dose or a minimal lethal dose of Salmonella typhimurium . The histopathological progression of the disease was examined in tissue sections prepared by the JB-4 Plus resin embedding method and compared between the resistant and susceptible mice . In a fatal disease, the lesions in both animal hosts began with focal abscesses within the first three days post infection . Mononuclear cell infiltration started by day 4 and transformed the lesions into granulomata . Well-formed granulomata were evident by day 7 and persisted in sublethally infected resistant mice . Massive bacterial proliferation and extensive tissue degeneration marked the terminal stage of a lethal challenge . There were no distinguishable features that would identify the tissue response to infection in a resistant host from a susceptible one, except that the lesions in the sublethally infected resistant mice advanced slower and were discrete and self-limiting.

Mol Gen Genet, 1998 Jul, 259(1), 46 - 53
The sfiX, rfe and metN genes of Salmonella typhimurium and their involvement in the His(c) pleiotropic response; Mouslim C et al.; Two loci involved in the pleiotropic response of His(c) strains of Salmonella typhimurium (sfiX and sfiY) have been characterized at the molecular level . The sfiX gene (CS 44) has been identified as a homolog of the E . coli gene sanA, located downstream of the cytidine deaminase gene (cdd) . The cdd-sanA (or cdd-sfiX) operon shows a highly conserved structure in E . coli and Salmonella . Like its E . coli homolog, the sfiX gene of S . typhimurium is required for vancomycin resistance at high temperature . The dual effect of sfiX mutations (induction of vancomycin sensitivity and suppression of cell division inhibition) suggests a link between SfiX function and murein synthesis . The sfiY locus (CS 85), contains two genes arranged in a single transcriptional unit . The upstream gene is a homolog of the E . coli gene rfe; mutations in this gene suppress the cell division defect of His(c) strains . The suppressor effect of rfe mutations can be reproduced by tunicamycin, suggesting that suppression of filamentation results from an increase in the intracellular concentration of UDP-N-acetyl-D-glucosamine . The gene located downstream of rfe is also found in E . coli but its function is unknown . Insertions in rfe suppress the methionine requirement of His(c) strains of S . typhimurium by a polar effect on the downstream gene, tentatively designated metN . Complementation with a rfe+ clone indicates that the rfe gene is not involved in the methionine requirement of His(c) strains . Thus metN expression appears to cause methionine auxotrophy in a His(c) background.

J Biol Chem, 1998 Sep 25, 273(39), 25006 - 14
Detection of a conserved alpha-helix in the kinase-docking region of the aspartate receptor by cysteine and disulfide scanning; Bass RB et al.; The transmembrane aspartate receptor of Escherichia coli and Salmonella typhimurium propagates extracellular signals to the cytoplasm, where its cytoplasmic domain regulates the histidine kinase, CheA . Different signaling states of the cytoplasmic domain modulate the kinase autophosphorylation rate over at least a 100-fold range . Biochemical and genetic studies have implicated a specific region of the cytoplasmic domain, termed the signaling subdomain, as the region that transmits regulation from the receptor to the kinase . Here cysteine and disulfide scanning are applied to the N-terminal half of the signaling subdomain to probe its secondary structure, solvent exposure, and protein-protein interactions . The chemical reactivities of the scanned cysteines exhibit the characteristic periodicity of an alpha-helix with distinct solvent-exposed and buried faces . This helix, termed alpha7, ranges approximately from residue 355 through 386 . Activity measurements probing the effects of cysteine substitutions in vivo and in vitro reveal that both faces of helix alpha7 are critical for kinase activation, while the buried face is especially critical for kinase down-regulation . Disulfide scanning of the region suggests that helix alpha7 is not in direct contact with its symmetric partner (alpha7') from the other subunit; presently, the structural element that packs against the buried face of the helix remains unidentified . Finally, a novel approach termed "protein interactions by cysteine modification" indicates that the exposed C-terminal face of helix alpha7 provides an essential docking site for the kinase CheA or for the coupling protein CheW.

Food Chem Toxicol, 1998 Sep-Oct, 36(9-10), 811 - 7
Mutagenicity and DNA-damaging activity of decomposed products of food colours under UV irradiation; Ozaki A et al.; Five synthetic food colours Food Red Nos 3, 40 and 102 and Food Blue Nos 1 and 2, and their UV irradiated products were tested for mutagenic activity by means of the Ames test using Salmonella typhimurium strains TA98 and TA100 . Food colours were irradiated with UV light for 14 days . Food Red Nos 3, 40 and 102 and Food Blue No . 1 were non-mutagenic before and after irradiation . UV irradiated products of Food Blue No . 2 were mutagenic in TA98 with or without S-9 mix . The mutagenic activity increased with increasing irradiation period, reached maximum potency on day 6, and then decreased . Moreover, Food Blue No . 2 showed DNA-damaging activity after 14 days of irradiation in rec-assay using Bacillus subtilis strains H17 and M45 . The capillary electrophoresis was applied for the analysis of UV irradiated products of Food Blue No . 2 . The original peak of Food Blue No . 2 was decomposed into seven peaks after UV irradiation.

Biochem Biophys Res Commun, 1998 Sep 8, 250(1), 150 - 3
In vivo detection of mutations induced by aflatoxin B1 using human CYP3A7/HITEC hybrid mice; Yamada A et al.; CYP3A7-M10 mouse is a transgenic mouse carrying human CYP3A7 cDNA, in which CYP3A7 is expressed in the small intestine but not in the kidney . HITEC mouse is a transgenic mouse developed to detect mutagenic potency of various chemicals in vivo . The M10/HITEC mouse was established by crossmating of these two strains of mice . When a 9,000 x g supernatant fraction prepared from the small intestine was added to an incubation mixture for Ames test with Salmonella typhimurium TA98 strain to examine the mutagen-producing activity from aflatoxin B1 (AFB1), the mutagen-producing activities of the 9, 000 x g supernatant fraction from the small intestine was found to be 1.7-fold higher in the M10/HITEC mice than in HITEC mice . Such a difference in the capacity to activate AFB1 was not seen with the 9, 000 x g supernatant fraction from the kidney from both strains of mice . Male M10/HITEC mice of 8 weeks old were treated with a single i.p . injection of AFB1 ( 8 mg/kg body weight) . The mutation of the introduced rpsL gene in the genomic DNA from the small intestine and the kidney was analyzed . The mutation frequency in the small intestine of M10/HITEC mice was significantly higher (p<0.05) than that of HITEC mice, while the mutation frequency in both strains was similar in the kidney . These results provide the first evidence for the toxicological function of CYP3A7 in vivo .

Mutat Res, 1998 Sep 11, 417(2-3), 141 - 53
Evaluation of the mutagenicity and antimutagenicity of forty-two 3-substituted flavones in the Ames test; Beudot C et al.; The mutagenic and antimutagenic activities of forty-two synthetic flavones were assessed by the Ames test . The tested flavones included twenty-three 3-nitroflavones, eighteen 3-aminoflavones and the 3-chloroflavone . The mutagenicity was evaluated with Salmonella typhimurium TA100 and YG1042 (an overproducing nitroreductase and O-acetyltransferase TA100 strain) with and without metabolic activation (S9 mix) . The antimutagenicity of the non mutagenic derivatives was evaluated against 11 known reference mutagens . A total of 39 synthetic flavones were mutagenic . The mutagenic activities ranged from 0.1 rev/nmole (4'-chloro-6-methoxy-3-nitroflavone) to 6240 rev/nmole (4'-methoxy-3, 3'-diaminoflavone) . Two differences were found between the 3-amino and the 3-nitroflavones: (i) the mutagenicity of the 3-aminoflavones required the presence of the metabolic activation; (ii) the 3-amino derivatives were more mutagenic than their 3-nitro counterparts . Increased mutagenicity, as assessed with strain YG1042, was limited to 17/39 derivatives . The mutagenic activity was induced by the presence of the double bond at the 2,3-position for conjugation of the lone-pair electron with the carbonyl group on the 'C' ring . This mutagenicity was modulated by substituents at the 2'-position . Additional mutagenicity was brought by the aminoaromatic and nitroaromatic group reduction by bacterial nitroreductases and by the S9 mix; it was modulated by different substituents on the aromatic rings of the flavones . Three flavones: 3-chloroflavone (1C), 4'-hydroxy-3-nitroflavone (23N) and 2',3-diaminoflavone (2A) showed antimutagenic properties . Compound 1C was efficient against benzo(a)pyrene (BaP), 2-aminofluorene (2AF), 2-aminoanthracene (2AA), 4-nitroquinoline-1-oxide (4NQO) and 1-methyl-3'-nitro-1-nitrosoguanidine (MNNG) . Compound 23N inhibited the mutagenicity of BaP and MNNG . The antimutagenic activity of 2A was limited to MNNG .

Mutat Res, 1998 Sep 11, 417(2-3), 95 - 100
Genotoxicity of Farbasol and its component: 4-ethyltoluene; Janik-Spiechowicz E et al.; A combination of assays for gene mutations in Salmonella typhimurium TA97a, TA98, TA100 and TA102 strains with and without rat liver activation, and for micronucleus and sister chromatid exchange (SCE) in bone marrow cells of Imp:Balb/c mice was used to provide data on the mutagenic and genotoxic properties of the mixture of aromatic solvents, known under the trade name of Farbasol . In addition, 4-ethyltoluene (the main ethylmethylbenzenic component of Farbasol) was also tested for muta- and genotoxicity . The results revealed that neither Farbasol nor 4-ethyltoluene induced an increased reverse mutation in bacterial cells or the formation of micronucleated polychromatic erythrocytes in bone marrow . However, those compounds were found to be active as sister chromatid exchange (SCE) agents .

Mutat Res, 1998 Sep 11, 417(2-3), 75 - 84
Modulatory effects of melatonin on genotoxic response of reference mutagens in the Ames test and the comet assay; Musatov SA et al.; The effect of a potent endogenous antioxidant, the pineal gland indole melatonin (MLT) on the mutagenicity of twelve well-known mutagens and carcinogens has been investigated using two in vitro tests the Ames test and the single cell gel electrophoresis assay (SCGE assay or COMET assay) . The 12 mutagens used were 7, 12-dimethylbenz(a)anthracene (DMBA), benzo(a)pyrene (BP), 2-aminofluorene (AF), 1,2-dimethylhydrazine (DMH), bleomycin, cyclophosphamide (CP), 4-nitroquinoline-N-oxide (NQO), 2,4, 7-trinitro-9-fluorenone (TNF), 9-aminoacridine (AA), N-nitrosomethylurea (NMU), mitomycin C and sodium azide tested in the absence or in the presence of S9 mix . MLT alone turned out neither toxic nor mutagenic in the Ames test and revealed clastogenic activity at the highest concentration tested (100 microM) in the SCGE assay . In four Salmonella typhimurium tester strains TA 97, TA 98, TA 100 and TA 102 MLT significantly reduced the mutagenicity of chemicals which require S9 activation . In the SCGE assay performed on CHO cells, preincubation with MLT led to a strong inhibition of clastogenic activities of DMBA and CP, and in a lesser extent with BP and NMU . With mitomycin C, MLT exacerbated responses in both tests . The possible mechanisms of MLT's inhibitory action are discussed .

J Biol Chem, 1998 Sep 18, 273(38), 24575 - 82
Identification and characterization of a sialidase released by the salivary gland of the hematophagous insect Triatoma infestans; Amino R et al.; Sialidases (EC 3.2.1.18) are commonly found in viruses, bacteria, fungi, protozoa, and vertebrates, but not in invertebrates . We have previously reported the presence of a new sialidase activity in the gut of exclusively hematophagous insects of the Triatoma genus, which transmit Chagas' disease (Amino, R., Acosta, A., Morita, O . M., Chioccola, V . L . P., and Schenkman, S . (1995) Glycobiology 5, 625-631) . Here we show that this sialidase is present in the salivary gland of Triatoma infestans, and it is released with the saliva during the insect bite . The sialidase was purified to homogeneity (>5000 times) to a specific activity of more than 20 units/mg . It elutes from a gel filtration column with a volume corresponding to the size of 33 kDa, and it migrates as a single 26-kDa band in SDS-polyacrylamide gel electrophoresis, which is unusually smaller when compared with other known sialidases . T . infestans sialidase hydrolyzes preferentially alpha2-->3-linked sialic acids at pH 4-8, with maximal activity between pH 5.5 and 6.5, which is compatible with the optimal pH of secreted sialidases . The sialidase is competitively inhibited by 2-deoxy-2, 3-dehydro-N-acetyl-neuraminic acid (Ki = 0.075 mM) and differently from many sialidases, with exception of Salmonella typhimurium sialidase, it is inhibited competitively by HEPES (Ki = 15 mM) . The fact that T . infestans sialidase is released with the saliva and can hydrolyze sialyl-LewisX blood groups, which are the ligands for selectins, suggests that it might have a role in the blood feeding.

J Bacteriol, 1998 Sep, 180(18), 4775 - 80
Mutations in Salmonella pathogenicity island 2 (SPI2) genes affecting transcription of SPI1 genes and resistance to antimicrobial agents; Deiwick J et al.; The Salmonella typhimurium genome contains two pathogenicity islands (SPI) with genes encoding type III secretion systems for virulence proteins . SPI1 is required for the penetration of the epithelial layer of the intestine . SPI2 is important for the subsequent proliferation of bacteria in the spleens of infected hosts . Although most mutations in SPI2 lead to a strong reduction of virulence, they have different effects in vitro, with some mutants having significantly increased sensitivity to gentamicin and the antibacterial peptide polymyxin B . Previously we showed that certain mutations in SPI2 affect the ability of S . typhimurium to secrete SPI1 effector proteins and to invade cultured eukaryotic cells . In this study, we show that these SPI2 mutations affect the expression of the SPI1 invasion genes . Analysis of reporter fusions to various SPI1 genes reveals highly reduced expression of sipC, prgK, and hilA, the transcriptional activator of SPI1 genes . These observations indicate that the expression of one type III secretion system can be influenced dramatically by mutations in genes encoding a second type III secretion system in the same cell.

J Med Chem, 1998 Sep 10, 41(19), 3748 - 52
Anthracene-9,10-diones as potential anticancer agents: bacterial mutation studies of amido-substituted derivatives reveal an unexpected lack of mutagenicity; Venitt S et al.; Fifteen anthracene-9,10-dione ("anthraquinone") derivatives with (omega-aminoalkyl)carboxamido substituents at the 1-, 2-, 1,4-, or 2, 6-ring positions were tested for bacterial mutagenicity in reverse-mutation assays using Salmonella typhimurium frameshift strains TA1538, TA98, and TA97a, in the presence and absence of a metabolic activation system prepared from the livers of rats treated with Aroclor 1254 . Six of the compounds were also tested in S . typhimurium TA100 and Escherichia coli WP2uvrApKM101 strains, which carry mutations particularly sensitive to reversion by DNA base-pair substitution . Two structurally related compounds, mitoxantrone and bisantrene, were tested in parallel as positive controls . Mitoxantrone was mutagenic to S . typhimurium TA1538 and TA98, whereas bisantrene was weakly mutagenic to both these strains but strongly mutagenic toward the TA97a variant . By contrast, although they are also DNA-binding intercalators, none of the amide-functionalized anthracene-9,10-diones of the present study showed significant mutagenic activity in any of the bacterial strains examined . Further, neither substituent position nor systematic alterations in the nature of attached side chains appeared to induce mutagenicity with these agents, although other studies have shown that such structural factors markedly influence their cytotoxic potencies toward mammalian cells in vitro.

J Rheumatol, 1998 Sep, 25(9), 1765 - 71
HLA-B27 does not affect invasion of arthritogenic bacteria into human cells; Ortiz-Alvarez O et al.; OBJECTIVE: To investigate the effect of HLA-B27 expression on entry of Salmonella typhimurium and Yersinia enterocolitica into human cells . METHODS: We performed standard bacterial invasion assays with S . typhimurium and Y enterocolitica to analyze isogenic pairs of HeLa (epithelial), U937 (promonocyte), C1R (B lymphocyte), and Jurkat (T lymphocyte) human cell lines and their respective HLA-B27 transfectants . Invasion of peripheral blood derived T lymphocytes, monocytes, and B lymphocytes/dendritic cell fraction (corresponding to peripheral blood cells depleted of monocytes and T lymphocytes) from patients with ankylosing spondylitis and healthy donors was also analyzed . The percentage of internalized bacteria was quantified, and the differences between HLA-B27 positive and negative samples were compared . RESULTS: The percentages of intracellular S . typhimurium and Y enterocolitica in HeLa, U937, and C1R with or without B27 were not statistically different (independent t test) . We also found that the percentage of internalized bacteria did not differ significantly between HLA-B27 positive and negative samples in the different populations of peripheral blood derived cells . CONCLUSION: The presence of HLA-B27 on the surface of human cells does not alter the degree of bacterial invasion into either cultured human cell lines or peripheral blood derived human cells, and the influence of HLA-B27 expression on bacterial invasion should not be implicated in the pathogenesis of reactive arthritis related to Salmonella and Yersinia.

Indian J Exp Biol, 1998 Jun, 36(6), 588 - 92
Effect of gamma irradiation on chemical and biological properties of lipopolysaccharide from Salmonella typhimurium; Naidu MD et al.; Lipopolysaccharide (LPS) from S . typhimurium on exposure to gamma-radiation resulted in decrease in toxicity and was less mitogenic, Silver stained profiles of irradiated LPS on polyacrylamide gels revealed complete loss of its heteropolysaccharides which was confirmed further by analysing lipid A and LPS from Salmonella minnesota Re mutants on SDS-PAGE . Glucosamine and 2-keto 3-deoxy-octonate(Kdo) contents were significantly decreased on treatment . Lipid A obtained by removal of heteropolysaccharides from LPS was less toxic on exposure to gamma radiations.

Mutat Res, 1998 Aug 14, 416(3), 169 - 81
Comparisons of chemically-induced mutation among four bacterial strains, Salmonella typhimurium TA102 and TA2638, and Escherichia coli WP2/pKM101 and WP2 uvrA/pKM101: collaborative study III and evaluation of the usefulness of these strains; Watanabe K et al.; Over the past 5 years, a large collaborative study of chemically-induced mutation has been performed using the four bacterial strains Salmonella typhimurium TA102 and TA2638 and Escherichia coli WP2/pKM101 and WP2 uvrA/pKM101 in order to compare the specific spectrum of response to chemicals and to evaluate the usefulness (sensitivity) of each strain . Following the two collaborative studies to test the chemicals in category 1, chemicals previously judged as positive only in E . coli WP2 strains and derivatives of these chemicals, and category 2, oxidative agents or crosslinking agents, 22 compounds of category 3 consisting of 10 nonmutagenic carcinogens and another 12 chemicals were selected in this study . Twenty participating laboratories tested each compound in the same method as previous reports . In the group of nonmutagenic carcinogens, no chemical induced revertant colonies of any strain tested . In the group of other chemicals, response to the chemicals was similar in TA102 and WP2 uvrA/pKM101 . Overall, in the three collaborative studies, a total of 79 compounds were tested . No difference in qualitative response to the four strains was observed for 71% (56/79) of the test chemicals . The combination of strains providing the greatest number of positive responses was WP2 uvrA/pKM101 with TA102; 84% (66/79) of the test chemicals elicited the same qualitative response in these two strains . Therefore, it is suggested that WP2 uvrA/pKM101 and TA102 can be included as a part of the standard tester strains for detection of mutagenic activity of chemicals .

Mutat Res, 1998 Aug 14, 416(3), 149 - 57
Inhibitory effect of threo-9,10-dichlorostearic acid on the mutagenic activity of MeIQx, 2-AF and B{a}P in the Ames/Salmonella test; Vereskuns G et al.; The mutagenic activity of threo-9,10-dichlorostearic acid, one of the chlorinated fatty acids identified in fish lipids, was examined in the Ames/Salmonella test . No mutagenic activity was found on any of the Salmonella typhimurium strains TA 98, TA 100 and TA 102, either with or without S9 activation . On the other hand, dichlorostearic acid showed an inhibitory effect on the mutagenic activity of the indirectly-acting mutagens 2-amino-3, 8-dimethylimidazo{4,5-f}quinoxaline (MeIQx), 2-aminofluorene (2-AF) and benzo{a}pyrene (B{a}P) using strain TA 98 in the presence of S9 . However, no inhibition was observed when mixing MeIQx and S9 before the addition of dichlorostearic acid . Furthermore, dichlorostearic acid did not show any inhibitory effect on the mutagenic activity of the directly-acting mutagen 4-nitroquinoline-N-oxide (4NQO) using the tester strains TA 98 and TA 100 . We, therefore, suggest that dichlorostearic acid interacts with the enzymes of the S9 mix, thereby dose-dependently inhibiting the transformation of MeIQx, 2-AF and B{a}P into their active forms .

Mutat Res, 1998 Sep 1, 417(1), 31 - 7
Genotoxic activity of chlorinated butenoic acids in Salmonella typhimurium strains TA98, TA100 and TA104; Franzen R et al.; The mutagenic activities of several chlorinated butenoic acids, recently identified in chlorinated drinking waters, were determined by the Salmonella microsome assay . The Salmonella typhimurium tester strains TA98, TA100, and TA104 were used without S9 mix . The results from the investigation showed that (Z)-2-chloro-3-(dichloromethyl)-4-oxobutenoic acid (MX, in the open form) was the most potent mutagen of the compounds tested . However, a significant number of mutations was also induced by compounds with structural similarities to MX . In general, all the compounds, except the butenedioic acids, were mutagenic in the assays for both base-pair substitution strains (TA100, TA104) and for the frameshift strain TA98, with the highest mutagenic response observed in strain TA100 . When the aldehyde group of MX and of 2-chloro-3-(chloromethyl)-4-oxobutenoic acid (CMCF, in the open form) was replaced by a dichloromethyl group, the mutagenic response in strains TA98 and TA104 changed . We concluded that a frame-shift mutation occurred because of the replacement . The increase of the TA104 mutagenicity suggested that adenosine could be the target for these types of compounds . Further evidence for such possibility were the modified adenosine adducts we could identify for some chlorinated butenoic acids .

Appl Environ Microbiol, 1998 Sep, 64(9), 3458 - 63
Induction of acid resistance of Salmonella typhimurium by exposure to short-chain fatty acids; Kwon YM et al.; Exposure to short-chain fatty acids (SCFA) is one of the stress conditions Salmonella typhimurium encounters during its life cycle, because SCFA have been widely used as food preservatives and SCFA are also present at high concentrations in the gastrointestinal tracts of host animals . The effects of SCFA on the acid resistance of the organism were examined in an attempt to understand the potential role of SCFA in the pathogenesis of S . typhimurium . The percent survival of S . typhimurium at pH 3.0 was determined after exposure to SCFA for 1 h at pH 7.0 . The percent acid survival, which varied depending on the SCFA species and the concentration used, was 42 after exposure to 100 mM propionate at pH 7.0 under aerobic incubation conditions, while less than 1% could survive without exposure . The SCFA-induced acid resistance was markedly enhanced by anaerobiosis (64%), lowering pH conditions (138% at pH 5.0), or increasing incubation time (165% with 4 h) during exposure to propionic acid . When protein synthesis during exposure to propionate was blocked by chloramphenicol, the percent acid survival was less than 1, indicating that the protein synthesis induced by exposure to propionate is required for the induction of the acid resistance . The percent acid survival determined with the isogenic mutant strains defective in acid tolerance response revealed that AtrB protein is necessary for the full induction of acid resistance by exposure to propionate, while unexpectedly, inactivation of PhoP significantly increased acid resistance over that of the wild type (P < 0.05) . The results suggest that the virulence of S . typhimurium may be enhanced by increasing acid resistance upon exposure to SCFA during its life cycle and further enhanced by anaerobiosis, low pH, and prolonged exposure time.

Indian J Pathol Microbiol, 1995 Oct, 38(4), 365 - 8
In vitro susceptibility of multiple drug resistant Salmonella typhimurium to newer fluroquinolone derivatives; Shetty M et al.; A total of 105 strains of Salmonella typhimurium resistant to Ampicillin, Co-trimoxazole and Nalidixic acid were included in the present study . Among the new line of Fluroquinolones resistance to Ciprofloxacin (3.8%), Norflox (16.19%) and Ofloxacin (24.76%) was very low as compared to older antibiotics . All the 3 fluoroquinolones had MIC values less than 1 mcg/ml.

Mutat Res, 1998 Jul 17, 403(1-2), 223 - 7
Genotoxicity of ribo- and deoxyribonucleosides of 8-hydroxyguanine, 5-hydroxycytosine, and 2-hydroxyadenine: induction of SCE in human lymphocytes and mutagenicity in Salmonella typhimurium TA 100; Arashidani K et al.; The induction of SCE by ribo- and deoxyribonucleosides of 8-hydroxyguanine, 5-hydroxycytosine, and 2-hydroxyadenine was tested using human peripheral blood lymphocytes . All of these compounds caused an increase in SCE frequency . The potency of SCE induction was as follows: 5-OH-C, 5-OH-dC > 8.OH-G > 8-OH-dG > 2-OH-A, 2-OH-dA . These results suggest that the oxidized nucleosides are incorporated into DNA with different efficiencies (or are repaired with different efficiencies) and exhibit genotoxicity in human blood cells . Ribo- and deoxyribo-derivatives of 5-OH-Cyt and 2-OH-Ade also showed mutagenic activity in Salmonella typhimurium TA 100.

Mutat Res, 1998 Aug 7, 416(1-2), 125 - 8
Inhibition of PhIP mutagenicity by caffeine, lycopene, daidzein, and genistein; Weisburger JH et al.; The heterocyclic amine 2-amino-1-methyl-6-phenylimidazo{4,5-beta}pyridine NPhIP) is a major dietary component in individuals eating cooked meats or fish . This heterocyclic amine requires biochemical activation, mainly through cytochrome P4501A2, and can be detoxified chiefly by 4'hydroxylation through other cytochromes, and be in turn converted through phase 2 enzymes to readily excreted conjugates . The active form of PhIP is mutagenic in Salmonella typhimurium TA98 and is a useful substrate to study the possible chemoprotective action of phytochemicals . We found that black and green tea depressed the mutagenicity of PhIP in dose-related fashion, and decaffeinated tea was less powerful an inhibitor . This led to the study of caffeine, that displayed effective dose-related inhibition of the mutagenicity of PhIP . Other antioxidants such as lycopene, the active antioxidant from tomatoes, and daidzein and genistein from soy products, also had a dose-related inhibition of the mutagenicity of PhIP . We conclude that PhIP is a good substrate found in several human foods to determine the protective effect of phytochemicals from vegetables, and beverages.

Mutat Res, 1998 Aug 7, 416(1-2), 11 - 9
Antimutagenic activity of carotenoids in green peppers against some nitroarenes; Gonzaez de Mejia E et al.; In Mexico, as well as in Central and South American countries, the consumption of peppers (Capsicum annuum) has been tradition for thousands of years; the per capita dietary intake of peppers is about 40 g/day . Peppers are an important source of beta-carotene and vitamin A, which have antimutagenic and/or anticarcinogenic properties . In the present study, Salmonella typhimurium tester strain YG1024 in the plate-incorporation test was used to examine the antimutagenicity of carotenois extracted from five different types of Capsicum spp . ('Chilaca', 'Poblano', 'Serrano', 'Jalapeno' and 'Pimiento') which were chosen, based on their consumption and availability on the local market . Extracts from these peppers were tested against 1-6-dinitropyrene (1,6-DNP) and 1,8-dinitropyrene (1,8-DNP) mutagenicity . Dose-response mutagenicity curves of 1-NP; 1,6-DNP and 1,8-DNP were obtained . For the antimutagenicity studies, doses of 0.05 microgram/plate, 0.20 ng/plate and 0.06 ng/plate for 1-NP, 1,6-DPN and 1,8-DNP respectively were chosen, and the number of net revertants/plate were 1008 for 1-NP, 512 for 1,6-DNP, and 712 for 1,8-DPN . Trans-beta-carotene and the extracts were not toxic to the bacteria at the concentrations tested . The extracts obtained from the peppers showed more inhibition than pure trans-beta-carotene on 1-NP; 1,6-DNP and 1,8-DNP mutagenicity . Chilaca pepper extract required 0.36 g (34 nmol expressed as trans-beta-carotene equivalents) of fresh pepper to inhibit 94% on 1-NP mutagenicity, 78% on 1,6-DNP mutagenicity and 84% on 1,8-DNP mutagenicity . Bell pepper ('Pimiento') extract required 1.53 g (50 nmol expressed as trans-beta-carotene) to obtain 87%, 79% and 73% inhibition on 1-NP; 1,6-DNP and 1,8-DNP mutagenicity respectively . Since pure beta-carotene inhibited only approximately 50% the mutagenicity of nitroarenes, these results suggest that each one of the pepper extracts have more than one antimutagenic compound (e.g., beta-carotene and xanthophylls) and those functional nutrients apparently have a synergistic effect.

Int J Parasitol, 1998 Jul, 28(7), 1121 - 30
Progress in recombinant vaccine development against coccidiosis . A review and prospects into the next millennium; Vermeulen AN; The increasing problems encountered by the poultry industry, despite the extensive use of drugs, have emphasised the need for an immunological solution for the economic damage caused by the Eimeria parasite . Although immunity develops relatively fast following a natural infection, to induce protection by using parasite extracts or single antigens appears more difficult . Nevertheless, the development of a vaccine based on defined antigens seems the best solution in the long run . At the VIth International Coccidiosis Conference in 1993 the first promising results were reported from small-scale experiments using recombinant antigens . This review summarises the advances in this field of research from 1993 onwards . Although since then not many reports have been published about the effects of using recombinant . antigens as a vaccine against coccidiosis, a number of interesting new proteins which could be considered good targets for such a vaccine have been described and are referred to herein . Proteins involved in the process of invasion of the host cell by the extracellular parasite are regarded as key components in the developmental cycle of the parasite . These components possibly bind to receptors on the host cell . Interference with this process could be a target of the protective immune response . Progress has also been made in characterising the immune mechanisms activated by infection with the parasite . From experimental mouse models and from studies in chickens, a better insight has been obtained towards the involvement of CD4- and CD8-type T cells in, respectively, the inductor and the effector branch of the immune response, although not all questions have been answered . Several antigens have been selected using T-cell stimulation and cytokine assays and these are reviewed . In a third section, mostly unpublished results of our own experiments dealing with the use of live vectors to present defined antigens such as Ea1A and EaSC2, a parasite refractile body transhydrogenase and a lactate dehydrogenase, respectively, are summarised . Partial protection could be induced using Salmonella typhimurium as a carrier for these antigens, in that the oocyst output was reduced by up to 50% after challenge and weight gain could be improved by 5-10% over non-vaccinated challenged chickens, when tested in a floor-pen trial . Similar results were obtained when these antigens were presented by viral vectors such as Fowlpox virus or Herpes virus of turkey . These data seem to offer good prospects for the accomplishment of a safe and efficaceous vaccine based on recombinant DNA technology . These expectations are corroborated by recent breakthrough in transfection of related parasites such as Plasmodium and Toxoplasma gondii, and by the increasing amount of genomic information becoming available every day, the impact of which cannot even be estimated yet . These new technologies will allow us to solve the complex problems that we once created ourselves.

J Bacteriol, 1998 Sep, 180(17), 4757 - 9
The panE gene, encoding ketopantoate reductase, maps at 10 minutes and is allelic to apbA in Salmonella typhimurium; Frodyma ME et al.; In Salmonella typhimurium, precursors to the pyrimidine moiety of thiamine are synthesized de novo by the purine biosynthetic pathway or the alternative pyrimidine biosynthetic (APB) pathway . The apbA gene was the first locus defined as required for function of the APB pathway (D . M . Downs and L . Petersen, J . Bacteriol . 176:4858-4864, 1994) . Recent work showed the ApbA protein catalyzes the NADPH-specific reduction of ketopantoic acid to pantoic acid . This activity had previously been associated with the pantothenate biosynthetic gene panE . Although previous reports placed panE at 87 min on the Escherichia coli chromosome, we show herein that apbA and panE are allelic and map to 10 min on both the S . typhimurium and E . coli chromosomes . Results presented here suggest that the role of ApbA in thiamine synthesis is indirect since in vivo labeling studies showed that pantoic acid, the product of the ApbA-catalyzed reaction, is not a direct precursor to thiamine via the APB pathway.

J Bacteriol, 1998 Sep, 180(17), 4750 - 2
Gene transfer between related bacteria by electrotransformation: mapping Salmonella typhi genes in Salmonella typhimurium; Toro CS et al.; Transfer of newly isolated mutations into a fresh background is an essential step of genetic analysis and strain construction . Gene transfer is hampered in Salmonella typhi and in other pathogenic bacteria by the lack of a generalized transduction system . We show here that this problem can be partially circumvented by using electrotransformation as a means for delivering S . typhi DNA into suitable S . typhi or Salmonella typhimurium recipients . Transferred DNA can recombine with the homologous region in the host chromosome . In one application of the method, mutations isolated in S . typhi were genetically mapped in S . typhimurium.

J Bacteriol, 1998 Sep, 180(17), 4686 - 92
Multidrug efflux pump AcrAB of Salmonella typhimurium excretes only those beta-lactam antibiotics containing lipophilic side chains; Nikaido H et al.; We found that the previously reported SS-B drug-supersusceptible mutant of Salmonella typhimurium (S . Sukupolvi, M . Vaara, I . M . Helander, P . Viljanen, and P . H . Makela, J . Bacteriol . 159:704-712, 1984) had a mutation in the acrAB operon . Comparison of this mutant with its parent strain and with an AcrAB-overproducing strain showed that the activity of the AcrAB efflux pump often produced significant resistance to beta-lactam antibiotics in the complete absence of beta-lactamase . The effect of AcrAB activity on resistance was more pronounced with agents containing more lipophilic side chains, suggesting that such compounds were better substrates for this pump . This correlation is consistent with the hypothesis that only those molecules that become at least partially partitioned into the lipid bilayer of the cytoplasmic membrane are captured by the AcrAB pump . According to this mechanism, the pump successfully excretes even those beta-lactams that fail to traverse the cytoplasmic membrane, because these compounds are likely to become partitioned into the outer leaflet of the bilayer . Even the compounds with lipophilic side chains were shown to penetrate across the outer membrane relatively rapidly, if the pump was inactivated genetically or physiologically . The exclusion of such compounds, exemplified by nafcillin, from cells of the wild-type S . typhimurium was previously interpreted as the result of poor diffusion across the outer membrane (H . Nikaido, Biochim . Biophys . Acta 433:118-132, 1976), but it is now recognized as the consequence of efficient pumping out of entering antibiotics by the active efflux process.

J Bacteriol, 1998 Sep, 180(17), 4564 - 70
Promoter substitution and deletion analysis of upstream region required for rpoS translational regulation; Cunning C et al.; The RpoS sigma factor of enteric bacteria is required for the increased expression of a number of genes that are induced during nutrient limitation and growth into stationary phase and in response to high osmolarity . RpoS is also a virulence factor for several pathogenic species, including Salmonella typhimurium . The activity of RpoS is regulated at both the level of synthesis and protein turnover . Here we investigate the posttranscriptional control of RpoS synthesis by using rpoS-lac protein and operon fusions . Substitution of the native rpoS promoters with the tac or lac UV5 promoters allowed essentially normal regulation after growth into stationary phase in rich medium or after osmotic challenge . Regulation of these fusions required the function of hfq, encoding the RNA-binding protein host factor I (HF-I) . Short deletions from the 5' end of the rpoS transcript did not affect regulation very much; however, a larger deletion mutation that still retains 220 bp upstream of the rpoS ATG codon, including a proposed antisense element inhibitory for rpoS translation, was no longer regulated by HF-I . Several models for regulation of rpoS expression by HF-I are discussed.

FEBS Lett, 1998 Aug 7, 432(3), 228 - 30
Unfolding of bacteriophage P22 tailspike protein: enhanced thermal stability of an N-terminal fusion mutant; Carbonell X et al.; The tailspike protein (TSP) of bacteriophage P22 is a homotrimeric multifunctional protein responsible for cell attachment and hydrolysis of the Salmonella typhimurium host cell receptor . Despite the folding of TSP involves the formation of thermolabile intermediates, the mature protein is extremely resistant to heat and detergent denaturation . We have analyzed the thermal resistance and unfolding pathway of two mutant, functional TSPs carrying end-terminal peptide fusions . Whereas the C-terminal fusion has minor effects on the TSP stability, the presence of a 23-mer foreign peptide at the N terminus (protein ATSP) results in a significant enhancement of the thermal resistance by retarding the first transition step of the unfolding process . At 65 degrees C and in 2% SDS, the unfolding rate constant for the transition from the native to the unfolding intermediate is 9.3 x 10(-4) s(-1) for ATSP versus 1.7 x 10(-3) s(-1) for wild-type TSP . On the other hand, the electrophoretic mobility of ATSP intermediates is greatly affected, proving structural modifications induced by the fused peptide . These results suggest a critical participation of the N-terminal domain in the unfolding kinetic barriers generated during the TSP denaturation pathway.

Mol Microbiol, 1998 Jul, 29(2), 641 - 52
Limits to inducer exclusion: inhibition of the bacterial phosphotransferase system by glycerol kinase; Rohwer JM et al.; The uptake of methyl alpha-D-glucopyranoside by the phosphoenolpyruvate-dependent phosphotransferase system of Salmonella typhimurium could be inhibited by prior incubation of the cells with glycerol . Inhibition was only observed for glycerol preincubation times longer than 45 s and required the preinduction of both the glucose and the glycerol-catabolizing systems . Larger extents of inhibition by glycerol correlated with higher intracellular levels of glycerol kinase when the glp regulon had been induced to different extents . Preincubation with lactate did not inhibit methyl alpha-D-glucopyranoside uptake significantly, although both lactate and glycerol were oxidized by the cells . The cellular free-energy state of the cells (intracellular {ATP}/{ADP} ratio) was virtually identical for lactate and glycerol preincubation, suggesting that the inhibition of phosphotransferase-mediated uptake was not a metabolic effect . In vitro, phosphotransferase activity was inhibited to a maximal extent of 32% upon titrating cell-free extracts with high concentrations of commercial glycerol kinase . The results show that uptake systems that have hitherto been regarded merely as targets of the phosphotransferase system component IIA(Glc) also have the capacity themselves to retroinhibit the phosphotransferase system flux, presumably by sequestration of the available IIA(Glc), provided that these systems are induced to appropriate levels.

J Chemother, 1998 Aug, 10(4), 313 - 9
Successful treatment of murine listeriosis and salmonellosis with levofloxacin; Nichterlein T et al.; Levofloxacin (L-ofloxacin) is a fluoroquinolone derivative . It is the active substance contained in ofloxacin with a broad spectrum of antibacterial activity against both Gram-negative and Gram-positive bacteria . In this work we examined the activity of levofloxacin against the facultative intracellular bacteria Listeria monocytogenes and Salmonella typhimurium in vitro, in tissue culture and in animal models of infection . The minimum inhibitory concentrations (MICs) MIC90 for Salmonella enterica and L . monocytogenes were 0.078 mg/l and 4 mg/l, respectively . Levofloxacin was bactericidal against L . monocytogenes and S . typhimurium because 8 x MIC killed 90% of the initial inoculum of L . monocytogenes EGD and S . typhimurium LT2 within 4 hours and 3 hours, respectively . Levofloxacin was more effective than ampicillin against L . monocytogenes EGD infecting tissue culture cells . Also in tissue culture cells infected with S . typhimurium LT2, levofloxacin was slightly more effective than ciprofloxacin . In animal models of infection, levofloxacin was as potent as the reference substances . In conclusion, levofloxacin is a candidate for the treatment of infections caused by facultative intracellular Gram-positive and Gram-negative bacteria.

Biosci Biotechnol Biochem, 1998 Jul, 62(7), 1425 - 7
Suppression of the SOS-inducing activity of Trp-P-1 and aflatoxin B1 by meso-dihydroguaiaretic acid from Machilus thunbergii in the Salmonella typhimurium TA1535/pSK1002 umu test; Miyazawa M et al.; The methanol extract from Machilus thunbergii showed a suppressive effect on umu gene expression of the SOS response in Salmonella typhimurium TA1535/pSK1002 against the mutagen, 3-amino-1,4-dimethyl-5H-pyrido{4,3-b}indole (Trp-P-1), which requires liver metabolizing enzymes . The methanol extract from M . thunbergii was successively re-extracted with chloroform, butanol and water . A suppressive compound in the chloroform extract fraction was isolated by SiO2 column chromatography and identified as meso-dihydroguaiaretic acid by GC-MS, and 1H- and 13C-NMR spectroscopy . Meso-dihydroguaiaretic acid inhibited of the SOS-inducing activity of Trp-P-1 in the umu test . Gene expression was suppressed by 62% at less than 0.18 mumol/ml, the ID50 value being 0.08 mumol/ml . Compound 1 was also assayed with aflatoxin B1 (AfB1) and showed a suppressive effect.

Commun Dis Public Health, 1998 Mar, 1(1), 28 - 30
Protracted outbreak of Salmonella typhimurium definitive phage type 170 food poisoning related to tripe, 'pig bag', and chitterlings; Cornell J et al.; An outbreak of food poisoning around South Yorkshire due to Salmonella typhimurium definitive phage type 170 related to eating tripe, 'pig bag', and chitterlings was associated with a common supplier . Possible links with other suppliers were considered . Twenty-two cases occurred between 11 April 1995 and 7 May 1995 . The situation was complicated by the complex distribution network of suppliers and by possible cross contamination in the retail outlets . This complexity created difficulties in the collection, recording and subsequent analysis of the data . The investigation of the outbreak and the subsequent control measures are discussed . Some of the difficulties of monitoring foodborne intestinal disease are highlighted.

J Appl Microbiol, 1998 Jun, 84(6), 988 - 98
Assessment of bacterial viability status by flow cytometry and single cell sorting; Caron GN et al.; Rapid bacterial detection and viability measurements have been greatly enhanced by recent advances in the use of fluorescent stains in cytometry . It has previously been shown that four physiological states can be distinguished: reproductively viable, metabolically active, intact and permeabilized . Previous sorting experiments have shown that not all intact cells readily grow, but some intact cells can grow even when they fail to show metabolic activity, as determined by esterase turnover . To circumvent the limitations imposed by active dye extrusion or cell dormancy on viability measurements used to date (e.g., enzyme activity or cell polarization), a fast triple fluorochrome staining procedure has been developed that takes account of these problems . This allows further cellular characterization of intact cells by: active exclusion of ethidium bromide (EB) (metabolically active cells), uptake of EB but exclusion of bis-oxonol (BOX) (de-energized but with a polarized cell membrane) and uptake of both dyes (depolarized) . Permeabilized cells were identified by propidium iodide (PI) uptake . The method was validated using an electronically programmable single cell sorter (EPICS Elite) and aged Salmonella typhimurium cells . Reproductive viability was determined by sorting single cells to their staining pattern directly onto agar plates . Most polarized cells could be recovered as well as a significant fraction of the depolarized cells, demonstrating that depolarization is a sensitive measure of cell damage but a poor indicator of cell death.

Mutagenesis, 1998 Jul, 13(4), 385 - 9
Inhibition of metabolic activation of the promutagens, benzo{a}pyrene, 2-aminofluorene and 2-aminoanthracene by furanochromones in Salmonella typhimurium; Schimmer O et al.; Khellin, a naturally occurring furanochromone (Ammi visnaga fruits), inhibited the mutagenicity of the promutagens benzo{a}pyrene, 2-aminofluorene and 2-aminoanthracene in Salmonella typhimurium TA98 . The effect varied greatly and depended on the S9 fraction used . Cytosolic activation of 2-aminoanthracene was also inhibited . Khellin produced no effect or only weak activity against the direct acting mutagens 2-nitrofluorene, 4-nitro-o-phenylenediamine, 1-nitropyrene and ethylmethane sulfonate (in TA100) . Daunomycin mutagenicity was inhibited to a greater extent . Visnagin was more toxic, but showed similar effects . Khellol and its glucoside were inactive against all the mutagens tested . We conclude that khellin acts as an inhibitor or the microsomal cytochrome P450 sub-enzymes analogous to the related furanocoumarins and is also capable of inhibiting cytosolic enzymes . The extract from Ammi visnaga fruits showed a higher inhibition potency than khellin alone against 2-aminoanthracene, 1-nitropyrene and daunomycin . This might be due to additional inhibitors, e.g . coumarins, or to the synergistic effects of accompanying compounds.

Scand J Immunol, 1998 Aug, 48(2), 136 - 43
Priming of T-cell responses in mice by porins of Salmonella typhimurium; Gupta S; An imbalance in signals delivered to T cells via T-cell receptor and accessory molecules can lead to anergy, apoptosis, or both . In the present study we have demonstrated that Salmonella typhimurium infection in mice leads to a progressive loss of CD4+ T helper (Th) cell population, abnormal T-cell death by apoptosis and loss of accessory molecules (B7 and intracellular adhesion molecule-1) on macrophages . Quantification of interleukin-2 (IL-2), interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) secretion revealed a Th2-type of response in lymphocytes isolated from spleen . However, preimmunization of mice with porins resulted in an increased CD4+ Th cell population and accessory molecules on the surface of macrophages . Quantification of cytokines revealed a Th1-type of response . We conclude that preimmunization of mice with porins provides a microenvironment in which a well-balanced accessory molecule and cytokine network is established, which results in the prevention of cell death by apoptosis.

Mutat Res, 1998 Jul 31, 415(3), 201 - 11
Lack of mammalian mutagenicity of the potent bacterial mutagen tris(2,3-dibromopropyl) phosphate and its metabolite 2-bromoacrolein; van Beerendonk GJ et al.; The flame retardant tris(2,3-dibromopropyl)phosphate (Tris-BP) and its metabolite 2-bromoacrolein (2BA) are very potent bacterial mutagens in Salmonella typhimurium (S . typhimurium) TA 100 . In this study, we showed that 2BA and Tris-BP are also mutagenic in S . typhimurium TA 104, which detects mutations at AT base pairs, while TA 100 detects mutations at CG basepairs . We also studied the mutagenicity of 2BA in mammalian cells in vitro and in the rat in vivo . Firstly, 2BA was tested in the human lymphoblastoid cell line TK6 . The results showed that there was no increase in mutation frequency at the hprt locus, whereas there was a large decrease in cell survival . Secondly, a shuttle vector system was used to study the induction of mutations by 2BA:DNA adducts . The vector was modified by insertion of a single-stranded oligonucleotide containing on average one 2BA:DNA adduct . No increase in mutation frequency above background was detected after replication of this vector in SV40 transformed normal human fibroblasts . Because the liver is a major site for bioactivation of Tris-BP to 2BA in vivo, we tested the initiating capacity of Tris-BP in the rat liver in a modified Solt & Farber initiation and promotion system . Administration of Tris-BP resulted in a small increase in the number of preneoplastic gamma-glutamyl-transpeptidase positive (GGT+) foci in the liver compared to control animals (only significant in the lowest size class) . Modification of the experimental protocol by performing partial hepatectomy 24 h after the administration of Tris-BP, did not increase the number of GGT+ or glutathione S-transferase-P (GST-P+) positive foci above the control level . Taken together, these results indicate that, in spite of a high mutagenicity in S . typhimurium, 2BA and Tris-BP have low or negligible mutagenic effects in mammalian systems . The lack of mutagenic activity may explain why Tris-BP is not a carcinogen in the rat liver.

Ann Occup Hyg, 1998 May, 42(4), 259 - 66
Chemical degradation of wastes of antineoplastic agents amsacrine, azathioprine, asparaginase and thiotepa; Barek J et al.; As a part of a program devoted to the destruction of antineoplastic agents, three chemical methods readily available in the hospital environment, viz . oxidation with sodium hypochlorite (NaClO, 5%), hydrogen peroxide (H2O2, 30%), and Fenton reagent (FeCl2.2H2O; 0.3 g in 10 ml H2O2, 30%), were tested for the degradation of four anticancer drugs: Amsacrine, Azathioprine, Asparaginase and Thiotepa . The efficiency of the degradation was monitored by high-performance liquid chromatography . The mutagenicity of the degradation residues were tested by Ames test using tester strains Salmonella typhimurium TA 97a, TA 98, TA 100 and TA 102 with and without an exogenous metabolic activation system . Using sodium hypochlorite, 98.5% of Amsacrine, 99.0% of Azathioprine, 99.5% of Asparaginase and 98.7% of Thiotepa were destroyed after 1 hr . The hydrogen peroxide treatment destroyed 99% of Asparaginase and 98.7% of Thiotepa in 1 hr . However, this procedure was not efficient for the treatment of Amsacrine (28% after 16 hr) and of Azathioprine (53% degradation in 4 hr) . The action of Fenton reagent resulted in the destruction of 98% of Amsacrine, and 99.5% of Azathioprine, 98.5% of Asparaginase and 98.7% of Thiotepa in 1 hr . In all cases where a high degree of degradation was achieved, the residues obtained weee non mutagenic.

Mutat Res, 1998 Jul 8, 415(1-2), 119 - 30
The mutagenic activity of unpolymerized resin monomers in Salmonella typhimurium and V79 cells; Schweikl H et al.; Dimethacrylate derivatives are used as monomers to polymerize dental composite materials and for a great variety of other industrial resins . Occupational exposure is likely in various ways because of the many areas of methacrylate application . Here, the mutagenicity of the monomers, bisphenol A-diglycidyl dimethacrylate (Bis-GMA), urethane dimethacrylate (UDMA), triethylene glycol dimethacrylate (TEGDMA), Bisphenol A (BPA), glycidyl methacrylate (GMA), methyl methacrylate (MMA), and 2-hydroxyethyl methacrylate (HEMA) was studied in a bacterial (Ames test) and a mammalian gene mutation assay (V79/HPRT assay) . Mutagenicity was determined in different Salmonella typhimurium strains (TA97a, TA98, TA100, TA102) and in V79 cells in the presence and in the absence of a metabolically active microsomal fraction from rat liver (S9) . No mutagenic effects were observed with Bis-GMA and UDMA, methyl methacrylate, 2-hydroxyethyl methacrylate and bisphenol A . Glycidyl methacrylate (GMA) was mutagenic in a dose-dependent manner in three Salmonella tester strains . The number of mutants was increased by a factor of 2 to 3 with strains TA97a and TA102 in the absence of S9 . Moreover, the numbers of mutants induced in S . typhimurium TA100 were about 8-fold higher than in solvent controls . GMA also induced an increase of mutants in V79 cells in the absence of S9 . However, GMA was inactivated by microsomal enzymes . Triethylenglycol dimethacrylate (TEGDMA) was not mutagenic in any S . typhimurium . In contrast, the compound induced a dose-dependent rise in mutant frequencies in V79 cell cultures . It is concluded that TEGDMA acted through a clastogenic mechanism which is not detected by Ames tester strains.

Mutat Res, 1998 Jul 8, 415(1-2), 13 - 23
Mutagenic and cytotoxic effects of exhaust particulate matter of biodiesel compared to fossil diesel fuel; Bunger J et al.; The mutagenic and cytotoxic effects of diesel engine exhaust (DEE) from a modern passenger car using rapeseed oil methyl esters (RME, biodiesel) as fuel were directly compared to DEE of diesel fuel (DF) derived from petroleum . Combustion particulate matter was collected on glass fiber filters coated with polytetrafluoroethylene (PTFE) from an exhaust dilution tunnel using three different engine test cycles on a chassis dynamometer . Filters were extracted with dichloromethane in a soxhlet apparatus for 12 h . The mutagenicity of the extracts was tested in the Salmonella typhimurium/mammalian microsome plate-incorporation assay using strains TA97a, TA98, TA100, and TA102 . The toxicity to the established cell line L929 (mouse lung fibroblasts) was investigated in the neutral red assay . In the tester strains TA98 and TA100 a significant increase of mutations resulted for the particle extracts of both fuels, but for DF the revertants were significantly higher compared to RME . The highest levels of revertants were observed in tests including a cold start phase . This was probably due to incomplete combustion in the cold engine and a lower conversion rate of the cold catalytic converter . Testing with activated liver S9 fraction induced a slightly lower increase of revertants in most experiments . TA97a and TA102 showed no significant enhancement of spontaneous mutations . In the FTP-75 test cycle RME extracts showed slightly higher toxic effects to the L929 cells than DF, whereas in the other tests no significant differences were observable . These results indicate a higher mutagenic potency of DEE of DF compared to RME . This is probably due to the lower content of polycyclic aromatic compounds (PAC) in RME exhaust, although the emitted masses of RME were higher in most test procedures applied in this study.

Arch Toxicol, 1998 Jun, 72(7), 395 - 401
Induction of cytochrome P450 1A in hamster liver and lung by 6-nitrochrysene; Chen RM et al.; The present study has determined the effect of 6-nitrochrysene (6-NC) on hepatic and pulmonary cytochrome P450 (P450)-dependent monooxygenases using hamsters pretreated with the nitrated polycyclic aromatic hydrocarbon (nitro-PAH) at 5 mg/kg per day for 3 days . Pretreatment with 6-NC elevated serum gamma-glutamyltranspeptidase, lactate dehydrogenase, and bilirubin levels . Liver S9 fractions prepared from controls and hamsters pretreated with 6-NC markedly increased mutagenicity of the nitro-PAH in Salmonella typhimurium tester strains TA98, TA100, and TA102 . The pretreatment selectively increased 1-nitropyrene reductase activities of lung cytosol and liver and lung microsomes . Pretreatment with 6-NC resulted in increases of microsomal 7-ethoxyresorufin and methoxyresorufin O-dealkylases activities in liver and lung without affecting the monooxygenase activities in kidney . Immunoblot analysis of microsomal proteins using mouse monoclonal antibody 1-12-3 to rat P450 1A1 revealed that 6-NC induced P450 1A-immunorelated proteins in liver and lung . RNA blot analysis using mouse P450 1A1 cDNA showed that 6-NC increased liver and lung P450 1A mRNA . 6-NC had no effect on the kidney P450 protein and mRNA . The present study demonstrates that the hamster enzymes can support 6-NC metabolic activation and the nitro-PAH induces liver and lung P4501A via a pretranslational mechanism.

J Food Prot, 1998 Mar, 61(3), 350 - 3
Salmonella penetration through eggshell associated with freshness of laid eggs and refrigeration; Miyamoto T et al.; Effects of egg age after laying and refrigeration on penetration of the eggshell by Salmonella enteritidis (SE) and Salmonella typhimurium (ST) were examined . Eggs 0.25 to 3 h, 3.25 to 6 h, 1 day, and 7 days old held at two temperatures were immersed in SE or ST suspensions containing 10(3) or 10(6) CFU/ml at 25 degrees C for 10 min . After holding at 25 degrees C for 2 h, the inner eggshell and egg contents were examined for Salmonella cells . The recovery rates of Salmonella cells from both the inner eggshell and egg contents of the 0.25- to 3-h-old eggs were significantly higher than those of other groups, especially at the high-exposure dose . There was no significant difference noted between SE and ST in ability to penetrate through eggshell . Salmonella penetration was significantly decreased by cooling the eggs at 4 degrees C for 15 min prior to immersing them in SE or ST suspension . The data suggested that Salmonella cells readily penetrated through the shell of freshly laid eggs, but that this penetration was suppressed by cooling the eggs before they were exposed to Salmonella suspensions.

J Food Prot, 1998 Mar, 61(3), 272 - 5
Spraying chicken skin with selected chemicals to reduce attached Salmonella typhimurium; Xiong H et al.; Aqueous solutions of 5% and 10% trisodium phosphate (TSP), 0.1% and 0.5% cetylpyridinium chloride (CPC), 1% and 2% lactic acid (LA), and 0.1% and 0.5% grapefruit seed extract (DF-100) were evaluated in prechill spraying for reducing Salmonella typhimurium attached on chicken skins . Chicken skins were inoculated with S . typhimurium and then sprayed with the selected chemical solutions for 30 sec at 206 kPa and 20 degrees C . After chemical spraying, the skins were rinsed by spraying tap water for 30 sec . Each skin was stomached in buffered peptone water (BPW) for 1 min . The stomaching water was then diluted serially, inoculated onto both xylose lysine tergitol (XLT4) agar and Aerobic Plate Count (APC) Petrifilm, and incubated for 24 hr at 37 degrees C . The results showed that the numbers of Salmonella on the chicken skins after the chemical spraying were significantly lower than those without spray (P < 0.05) . The CPC reduced Salmonella by 1.5 to 1.9 log10 . TSP resulted in a 2.1 to 2.2 log10 reduction of Salmonella and DF-100 produced a 1.6 to 1.8 log10 reduction of Salmonella . The LA had a number of Salmonella with a 2.2 log10 reduction . The 0.5% CPC resulted a significantly greater reduction in Salmonella than 0.1% CPC . There were no significant differences in Salmonella reduction between different concentrations of the other three chemicals.

J Food Prot, 1998 Feb, 61(2), 152 - 7
Destruction of Escherichia coli O157:H7 and Salmonella typhimurium in Lebanon bologna by interaction of fermentation pH, heating temperature, and time; Ellajosyula KR et al.; Fermented meats have caused food-borne illness due to enterohemorrhagic Escherichia coli . Consumption of Lebanon bologna was epidemiologically associated wit a recent outbreak of salmonellosis . The present study was conducted to determine the effect of pH (after the fermentation step), final heating temperature, and time on destruction of E . coli O157:H7 and Salmonella typhimurium in Lebanon bologna . Raw Lebanon bologna mix was inoculate with either of the pathogens (ca.10(8) CFR/g and fermented for 12 h at 80 degrees F (26.7 degrees C) and then at 100 degrees F (37.8 degrees C) unit the pH reached wither 5.2 or 4.7 . The mix was then heated to 110, 115, or 120 degrees F (43.3, 46.1, or 48.9 degrees C) . The bologna was sampled at various times, decimally diluted, and plated on either McConkey sorbitol agar or XLD agar to enumerate E . coli O157:H7 and S . typhimurium, respectively . Fermentation alone reduced populations of both pathogens by < 2 log units and heating alone reduced populations of E . coli O157:H7 by < 3 log units . A combination of fermenting to either pH 5.2 or 4.7, followed by heating at 110 degrees F (43.3 degrees C) for 20h, 115 degrees F (46.1 degrees C) for 10 h, or 120 degrees F (48.9 degrees C) for 3 h reduced populations of both pathogens by > 7 log units . Overall S . typhimurium cells were either equally or significantly less resistant (P < 0.01) than cells of E . coli O157:H7 . Significantly interactions (P < 0.01) among the three factors for the destruction of E . coli O157:H7 were observed . A process-specific regression equation was developed to predict the destruction of E . coli O157:H7 in Lebanon bologna.

Immunology, 1998 May, 94(1), 5 - 13
Th1 and Th2 cell involvement in immune response to Salmonella typhimurium porins; Galdiero M et al.; In understanding the regulation of the specific immune response to Salmonella typhimurium, the role of a surface major component (porins) was studied . In this study we demonstrate that purified porins are able to induce a different response to that induced by the porins present on the S . typhimurium cell surface . Porin-treated or orally infected mice show anti-porin antibodies with bactericidal activity . The complete adoptive transfer of resistance to S . typhimurium is achieved only using splenic T cells from survivor mice after experimental infection . After stimulation with specific antigen in vitro CD4+ cells from porin-immunized mice released large amounts of interleukin-4 (IL-4), at a time when CD4+ cells from S . typhimurium-infected mice predominantly secreted interferon-gamma (IFN-gamma) . Limiting dilution analysis showed that infection resulted in a higher precursor frequency of IFN-gamma-producing CD4+ T cells and a lower precursor frequency of IL-4-producing CD4+ T cells, while immunization with porins resulted in a higher precursor frequency of IL-4-producing cells and a low frequency of IFN-gamma-producing cells . Analysis of polymerase chain reaction-amplified cDNA from the spleens of infected mice revealed that IFN-gamma, IL-2 and IL-12 p40 mRNA were found 5 days after in vitro challenge and increased after 15 days; IL-10 expression was barely present after both 5 and 15 days, while IL-4 mRNA expression was not detected . In immunized mice, the IL-4 mRNA expression increased after 15 days, IFN-gamma mRNA expression disappeared entirely after 15 days, while IL-2, IL-10 and IL-12 mRNA remained relatively unchanged.

Poult Sci, 1998 Aug, 77(8), 1217 - 27
Effect of dietary energy level and oil source on broiler performance and response to an inflammatory challenge; Korver DR et al.; Broiler chicks were fed one of five diets from 3 d of age: a low energy diet containing 7% cellulose (ME 2,714 kcal/kg), or high energy diets containing approximately 7% of either tallow, corn oil, safflower oil, or fish oil (each 3,302 kcal/kg) . Half of the chicks were injected intra-abdominally with Salmonella typhimurium lipopolysaccharide (LPS) on Day 11, sephadex on Day 13, and Freund's complete adjuvant on Day 15; samples were collected on Day 16 . The uninjected chicks served as controls . In a second experiment, 3-d-old chicks were fed one of eight isocaloric diets containing tallow as the sole supplemental fat source, or tallow plus either 2% corn oil, 1, 1.5, or 2% fish oil, or fish meal at an amount to provide 1, 1.5 or 2% supplemental oil . Half of the chicks were injected intra-abdominally with S . typhimurium LPS on Days 10, 12 and 14; the uninjected chicks served as controls . Samples were taken on Day 15 . In Experiment 1, the cellulose diet decreased performance to 10 d of age relative to the other diets, whereas immunogen injection decreased weight gain and feed efficiency and increased indices of inflammation among all dietary groups . Fish oil at approximately 7% of the diet did not improve weight gain . Fish oil diets improved weight gain and feed efficiency in Experiment 2 relative to the other diets, in spite of minimal effects on indices of inflammation . Injection of LPS decreased performance and increased inflammation across dietary treatment, although the second LPS injection was less potent in altering performance responses and inflammation compared to the first injection, indicating that repeated injections of LPS amy cause the chicks to become refractory to that stimulus . The fish meal diets resulted in poorer performance than similar levels of lipid provided as fish oil . Lower levels of dietary fish oil were more efficacious in improving broiler performance than higher levels, and fish oil provided from fish meal was not as efficacious as oil per se, possibly due to nonlipid components of the meal.

Mol Microbiol, 1998 Jul, 29(1), 311 - 20
Expression and transcriptional control of the Salmonella typhimurium Ipf fimbrial operon by phase variation; Norris TL et al.; The lpf operon mediates adhesion of Salmonella typhimurium to murine Peyer's patches . To investigate expression of this virulence factor, a transcriptional fusion between the lpf operon and the genes lacZYA of Escherichia coli was constructed and introduced into the S . typhimurium chromosome . The resulting strain yielded both Lac+ and Lac- colony phenotypes . Alternation between Lac+ (phase ON) and Lac- (phase OFF) phenotypes occurred by a heritable phase variation mechanism, as inoculation of broth cultures with bacteria picked from a Lac+ colony gave rise to a considerably higher proportion of Lac+ colonies than inoculation with bacteria picked from a Lac- colony . During growth in vitro, phase transition from ON to OFF and from OFF to ON occurred at rates 6.8 x 10(-3) and 2.4 x 10(-4) events per cell per generation respectively . In a murine intestinal organ culture model, selection for the ON expression state occurred when attached bacteria were recovered from Peyer's patches, suggesting that Lac phase variation correlated with expression of lpf mediated adherence . Selection for either the ON or the OFF expression state of the Ipf operon in vivo was studied in mice immunized with either GST or GST-LpfA fusion protein . A strong selection against phase ON cells occurred only in animals immunized with GST-LpfA . The ability of animals immunized with GST-LpfA to distinguish between phase ON and phase OFF bacteria provided evidence for the presence of LpfA fimbrial protein in phase ON cells and for its exposure to the immune system.

Acta Biochim Pol, 1998, 45(1), 163 - 72
Structural insights into the regulation of SOS mutagenesis; Gonzalez M et al.; The Escherichia coli Umu proteins are best characterized by their role in damage inducible mutagenesis . Recently, we discovered that the intracellular levels of the UmuD and UmuC proteins are kept to a minimum by the Lon serine protease . Studies with the Salmonella typhimurium UmuD protein (which is 73% homologous with its E . coli counterpart) revealed that it too is degraded by Lon, suggesting that both UmuD proteins share conserved structural motifs . In contrast, E . coli UmuD' is removed from the cell by the ClpXP serine protease, but only when it is in a heterodimer complex with UmuD . We have generated deletion mutants of UmuD' and have coexpressed the mutant proteins with UmuD1 (a non-cleavable UmuD protein) . By assaying the sensitivity of the mutant UmuD'-UmuD1 complex to ClpXP, we have been able to map regions of UmuD' that appear essential for efficient UmuD'-UmuD heterodimer formation . Previous experiments have suggested that the in vivo posttranslational processing of UmuD to UmuD' is inefficient . We have, however, discovered that limited cleavage occurs in an undamaged cell, but that these small amounts of UmuD' are rapidly degraded by ClpXP, thus giving rise to the appearance of inefficient cleavage . The ClpXP protease therefore plays dual roles in regulating SOS mutagenesis: it keeps the basal levels of UmuD' to a minimum in undamaged cells but it also acts in damaged cells to reduce the elevated levels of mutagenically active UmuD' protein, thereby returning the cell to a resting non-mutable state.

Nitric Oxide, 1997 Apr, 1(2), 158 - 66
Quantification of diazeniumdiolate mutagenicity in four different in vitro assays; Donovan PJ et al.; Diazeniumdiolates are under investigation as possible prodrugs of the multifaceted bioregulatory agent nitric oxide . This study was undertaken to assess further the mutagenic potential of two diazeniumdiolates, DEA/NO (Et2N{N(O)NO}Na) and SPER/NO ({H2N(CH2)3NH(CH2)4N{N(O)NO-}(CH2)3 NH3+}), which generate NO spontaneously with half-lives at 37 degrees C and pH 7.4 of 2 and 39 min, respectively . The genotoxic potential of these compounds was investigated with the Ames bacterial reverse mutation assay, two mammalian cell gene mutation assays (CHO/HGPRT and L5178Y TK+/-), and an assay for sister chromatid exchange (SCE) using Chinese hamster ovary (CHO) cells . Both diazeniumdiolates had previously been shown to be mutagenic in the Ames Salmonella plate assay . In the experiments reported here, Salmonella typhimurium strain TA1535 was exposed to the compounds in a liquid incubation assay for either 15 min or 48 h without an S-9 fraction . With the 15-min exposure, DEA/NO was mutagenic at concentrations of 0.625 mM (3.5 x control) and greater, while SPER/NO was mutagenic at 0.5 mM (2.7 x control) and above . In the CHO/HGPRT assay, DEA/NO was weakly mutagenic only at the highest concentration used, 20 mM, inducing a mutant frequency per survivor that was 2.5 x control, while SPER/NO was mutagenic at 0.5 mM with a mutant frequency of 2.5 x control . When the CHO cells were given 10 repetitive 20 mM DEA/NO exposures (3 min each), HGPRT mutant frequency was 4.1 x control . In the L5178Y mouse lymphoma cell TK+/- assay, DEA/NO doubled the mutation rate at 1.82 mM, while SPER/NO's mutation frequency was more than twice that of control at 0.63 mM . DEA/NO was positive in the SCE assay without metabolic activation, yielding significant SCE at 1.25, 2.5, and 5 mM that was 1.8, 2.2, and 2.6 times control, respectively . SPER/NO increased the SCE by 1.2, 1.4, and 1.3 times at 1.5, 2.0, and 2.5 mM . The results suggest that the two diazeniumdiolates, although mutagenic in the bacteria, are much weaker mutagens in mammalian cells.

Plant Mol Biol, 1998 Aug, 37(6), 955 - 66
A Brassica cDNA clone encoding a bifunctional hydroxymethylpyrimidine kinase/thiamin-phosphate pyrophosphorylase involved in thiamin biosynthesis; Kim YS et al.; We report the characterization of a Brassica napus cDNA clone (pBTHI) encoding a protein (BTHI) with two enzymatic activities in the thiamin biosynthetic pathway, thiamin-phosphate pyrophosphorylase (TMP-PPase) and 2-methyl-4-amino-5-hydroxymethylpyrimidine-monophosphate kinase (HMP-P kinase) . The cDNA clone was isolated by a novel functional complementation strategy employing an Escherichia coli mutant deficient in the TMP-PPase activity . A biochemical assay showed the clone to confer recovery of TMP-PPase activity in the E . coli mutant strain . The cDNA clone is 1746 bp long and contains an open reading frame encoding a peptide of 524 amino acids . The C-terminal part of BTH1 showed 53% and 59% sequence similarity to the N-terminal TMP-PPase region of the bifunctional yeast proteins Saccharomyces THI6 and Schizosaccharomyces pombe THI4, respectively . The N-terminal part of BTH1 showed 58% sequence similarity to HMP-P kinase of Salmonella typhimurium . The cDNA clone functionally complemented the S . typhimurium and E . coli thiD mutants deficient in the HMP-P kinase activity . These results show that the clone encodes a bifunctional protein with TMP-PPase at the C-terminus and HMP-P kinase at the N-terminus . This is in contrast to the yeast bifunctional proteins that encode TMP-PPase at the N-terminus and 4-methyl-5-(2-hydroxyethyl)thiazole kinase at the C-terminus . Expression of the BTH1 gene is negatively regulated by thiamin, as in the cases for the thiamin biosynthetic genes of microorganisms . This is the first report of a plant thiamin biosynthetic gene on which a specific biochemical activity is assigned . The Brassica BTH1 gene may correspond to the Arabidopsis TH-1 gene.

Braz J Med Biol Res, 1998 Apr, 31(4), 545 - 54
Immunization against the colonization factor antigen I of enterotoxigenic Escherichia coli by administration of a bivalent Salmonella typhimurium aroA strain; Guillobel HC et al.; An expression plasmid (pCFA-1) carrying the cfaB gene that codes for the enterotoxigenic Escherichia coli (ETEC) fimbrial adhesin colonization factor antigen I (CFA/I) subunit was constructed and used to transform a derivative of the attenuated Salmonella typhimurium aroA vaccine strain SL3261 carrying an F'lacIq . Treatment of the transformed strain with isopropyl-beta-D-thiogalactopyranoside (IPTG) resulted in elevated in vitro expression of the CFA/I subunit . Although flagellar function and lipopolysaccharide (LPS) synthesis were similar in both the parental and the recombinant strains, spleen colonization was reduced in the recombinant strain . All BALB/c mice parenterally inoculated with the recombinant strain developed significant anti-CFA/I and anti-LPS serum antibody titers (P < 0.05) . Moreover, 2 of 5 mice orally inoculated with the engineered Salmonella strain developed anti-CFA/I intestinal IgA (P > 0.05) while 4/5 of the same mice developed anti-LPS IgA (P < 0.05) . The results indicate that the vaccine strain elicited an antibody response against the bacterial host both after oral and intravenous immunization while the response against the CFA/I antigen was significant only after inoculation by the intravenous route.

Immunol Lett, 1998 Jun, 62(2), 81 - 6
DNA-mobility shift assay and the detection of anti-DNA IgG in systemic lupus erythematosus patients; Keyhani J et al.; Although the presence of antibodies against double-stranded (ds) DNA is highly specific of systemic lupus erythematosus (SLE), it is not detected in all SLE patients, perhaps due to a lack of sensitivity of the tests routinely used to assay anti-(ds) DNA . Looking for an alternative assay, this study explored the applicability of a DNA-mobility shift assay for the detection of anti-(ds) DNA; furthermore, the study compared the use of Salmonella typhimurium DNA with that of calf thymus DNA in the assay . After electrophoresis, samples containing S . typhimurium DNA and IgG from SLE sera showed marked alterations in DNA electrophoretic mobility when compared to DNA alone . In our sampling, SLE patients who tested negative for anti-(ds) DNA antibodies with routinely used assays such as Crithidia luciliae immunofluorescence test, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA), tested positive for anti-(ds) DNA with the DNA mobility shift assay using S . typhimurium DNA . Incubation with IgG from control sera in the same proportions as above did not affect S . typhimurium DNA electrophoretic mobility . When S . typhimurium DNA was replaced by calf thymus DNA, the effect on the DNA mobility was less pronounced and less reliable . These results indicated that a DNA-mobility shift assay would be a useful alternative for the unequivocal detection of abnormal titers of anti-(ds) DNA antibodies . Furthermore, data indicated a greater ability of the IgG from SLE patients to form complexes with S . typhimurium DNA than with calf thymus DNA, suggesting an alternative testing DNA which may lead to a more sensitive anti-(ds) DNA detection.

Microbiology, 1998 Jul, 144 ( Pt 7), 1835 - 43
Magnesium transport in Salmonella typhimurium: regulation of mgtA and mgtCB during invasion of epithelial and macrophage cells; Smith RL et al.; Salmonella typhimurium contains two inducible Mg2+ transport systems, MgtA and MgtB, the latter encoded by a two-gene operon, mgtCB . Mg2+ deprivation of S . typhimurium increases transcription of both mgtA and mgtCB over a thousandfold and a similar increase occurs upon S . typhimurium invasion of epithelial cells . These increases are mediated by the phoPQ two-component signal transduction system, an essential system for S . typhimurium virulence . It was therefore hypothesized that expression of MgtA and MgtCB is increased upon invasion of eukaryotic cells because of a lack of intravacuolar Mg2+ . However, when S . typhimurium was grown at pH 5.2, the capacity of the constitutive CorA transporter in mediating Mg2+ was greater than that at pH 7.4 . Furthermore, induction of mgtA and mgtCB transcription was greater in the presence of a wild-type corA allele than in its absence . This implies that intravacuolar S . typhimurium could obtain sufficient Mg2+ via the CorA system . The effect of acid pH on mgtA and mgtCB transcription was also measured . Compared to induction at pH 7.4, exposure to pH 5.2 almost completely abolished induction of mgtA at low Mg2+ concentrations but diminished induction of mgtCB only twofold . Adaptation of cells to acid pH by overnight growth resulted in normal levels of induction of mgtA and mgtCB at low Mg2+ concentrations . These results imply an additional level of regulation for mgtA that is not present for mgtCB . Conversely, repression of mgtA and mgtCB expression by increased extracellular Mg2+ was relatively insensitive to acid . Transcription of both loci was strongly induced upon invasion of the Hep-2 or CMT-93 epithelial-like or J774 macrophage-like cell lines . However, the presence or absence of functional alleles of either or both mgtA or mgtCB had no effect on invasion efficiency or short-term survival of S . typhimurium within the eukaryotic cells . It was concluded that the strong Mg(2+)-dependent induction of mgtA and mgtCB upon invasion of eukaryotic cells is not required because S . typhimurium lacks sufficient Mg2+ during eukaryotic cell invasion and initial intravacuolar growth.

Microbiology, 1998 Jul, 144 ( Pt 7), 1823 - 33
Regulation of the spvR gene of the Salmonella typhimurium virulence plasmid during exponential-phase growth in intracellular salts medium and at stationary phase in L broth; Wilson JA et al.; The authors previously showed that the SpvR-regulated spvABCD operon of the Salmonella typhimurium virulence plasmid is highly induced during exponential-phase growth by salmonellae intracellularly in mammalian cells and in a medium designed to mimic the intracellular environment of mammalian cells, intracellular salts medium (ISM), as well as at stationary phase in L broth (LB) . The most relevant signal(s) for spv gene expression in vivo is not known . To elucidate the means by which salmonellae regulate the spv genes in response to the environment during the disease process, expression of the spvR gene, encoding the positive regulatory protein SpvR, was examined under these same growth conditions by using RNAse-protection analysis . spvR was expressed at a low, basal level during exponential growth in LB but was induced during exponential growth in ISM and during stationary phase in LB, the same conditions that increased expression of the spvABCD operon . Basal expression of spvR during exponential growth in LB was independent of both SpvR and the alternative sigma factor RpoS, whereas maximal induction of spvR was dependent on both SpvR and RpoS . In an RpoS- background, spvR message was decreased in stationary phase, whereas spvR exhibited residual RpoS-independent induction during exponential growth in ISM . Deletion of spvA from the virulence plasmid of S . typhimurium increased expression of spvR during stationary phase in LB, but not during exponential growth in ISM . These results suggest that expression of spvR is controlled by different regulatory factors, depending on the growth conditions encountered by the salmonellae.

J Biol Chem, 1998 Aug 14, 273(33), 20721 - 7
Cloning and characterization of the Hakata antigen, a member of the ficolin/opsonin p35 lectin family; Sugimoto R et al.; The Hakata antigen is a novel, thermolabile beta2-macroglycoprotein that reacts with sera from patients suffering from systemic lupus erythematosus . In this study we present the structure and the function of the Hakata antigen . We have identified cDNA clones encoding the Hakata antigen and analyzed its function . The cDNA included a possible open reading frame of 897 nucleotides, encoding 299 amino acids . The Hakata antigen consisted of a collagen-like domain in the middle section and a fibrinogen-like domain in the COOH terminus, both of which are homologous to human ficolin-1 and opsonin P35, indicating that these three molecules form a distinct family . The molecular mass of the Hakata antigen expressed in transfected cells was 35 kDa under reduced conditions, and it formed ladder bands under nonreducing conditions compatible with the previous result that the Hakata antigen exists in serum as homopolymers . Purified Hakata antigen sustained lectin activity, showing affinity with GalNAc, GlcNAc, D-fucose as mono/oligosaccharide, and lipopolysaccharides from Salmonella typhimurium and Salmonella minnesota . These results suggest that the Hakata antigen, a new member of the ficolin/opsonin P35 family, plays a role in the serum exerting lectin activity under physiological conditions.

J Mol Evol, 1998 Aug, 47(2), 230 - 4
Identification of glyoxalase I sequences in Brassica oleracea and Sporobolus stapfianus: evidence for gene duplication events; Clugston SL et al.; Glyoxalase I (GlxI) is the first of two enzymes involved in the cellular detoxification of methylglyoxal . A recent search of the National Center for Biotechnology Information (NCBI) databases with the protein sequence of Salmonella typhimurium GlxI identified two new hypothetical proteins with unassigned function . These two sequences, from Brassica oleracea and Sporobolus stapfianus, have significant sequence similarity to known GlxI sequences, suggesting that these two open reading frames encode for GlxI in these plants . Interestingly, analysis of these two new sequences indicates that they code for a protein composed of two fused monomers, a situation previously found solely in the yeast GlxI enzymes.

Regul Toxicol Pharmacol, 1998 Jun, 27(3), 217 - 39
Evaluation of possible genotoxic mechanisms for acrylonitrile tumorigenicity; Whysner J et al.; Acrylonitrile (ACN) exposure is associated with tumors in rat brain, Zymbal gland, and mammary gland . Adducts affecting base pairing were formed in isolated DNA exposed in vitro to the ACN metabolite cyanoethylene oxide (CNEO) . DNA from liver, which is not a cancer target organ in ACN-exposed rats, contained low levels of 7-(2-oxoethyl)guanine, and adduct believed not to interfere with base pairing . No adducts have been detected in brain DNA from ACN-exposed rats, suggesting that brain tumors may have arisen by mechanisms other than ACN-DNA reactivity . Genotoxicity assays of ACN have indicated no particular carcinogenic mechanism . Positive reverse mutagenesis in Salmonella typhimurium HisG46 base substitution tester strains by ACN is attributable to CNEO . Other in vitro genotoxicity test assays of ACN have yielded mixed results, without consistent effect of metabolic activation . Some positive genotoxicity data for ACN appear to result from artifacts or from non-DNA-reactive mechanisms . In vivo micronucleus, chromosome aberration, and autoradiographic unscheduled DNA synthesis assays were negative for ACN . The comparative genotoxicity of vinyl chloride and ACN indicates that despite other similarities, they cause rodent tumors by different mechanisms . Also, they absence of ACN-DNA adduct formation in the rat brain suggests the operation of epigenetic mechanisms.

Biochemistry, 1998 Jul 28, 37(30), 10597 - 604
Cysteine 42 is important for maintaining an integral active site for O-acetylserine sulfhydrylase resulting in the stabilization of the alpha-aminoacrylate intermediate; Tai CH et al.; O-Acetylserine sulfhydrylase-A (OASS-A) is a pyridoxal 5'-phosphate (PLP) dependent enzyme from Salmonella typhimurium that catalyzes the beta-replacement of acetate in O-acetyl-L-serine (OAS) by sulfide to give L-cysteine . The reaction occurs via a ping-pong kinetic mechanism in which alpha-aminoacrylate in Schiff base with the active site PLP is an intermediate {Cook, P . F., Hara, S., Nalabolu, S . R., and Schnackerz, K . D . (1992) Biochemistry 31, 2298-2303} . The sequence around the Schiff base lysine (K41) has been determined {Rege, V . D., Kredich, N . M., Tai, C.-H., Karsten, W . E., Schnackerz, K . D., & Cook, P . F . (1996) Biochemistry 35, 13485-13493}, and the sole cysteine in the primary structure is immediately C-terminal to the lysine . In an effort to assess the role of C42, it has been changed to serine and alanine by site-directed mutagenesis . The mutant proteins are structurally nearly identical to the wild-type enzyme on the basis of UV-visible, fluorescence, far-UV and cofactor-induced CD, and 31P NMR studies, but subtle structural differences are noted . Kinetic properties of both mutant proteins differ significantly from those of the wild-type enzyme . The C42S mutant exhibits a > 50-fold increase in the OAS:acetate lyase activity and a 17-fold decrease in V for the cysteine synthesis compared to the wild-type enzyme, while decreases of > 200-fold in the OAS: acetate lyase activity and a 30-fold decrease in V for the cysteine synthesis are found for the C42A mutant enzyme . In both cases, however, the pH dependence of kinetic parameters for cysteine synthesis and OAS: acetate lyase activity yield, within error, identical pK values . In the three-dimensional structure of OASS-A, cysteine 42 is located behind the cofactor, pointing away from the active site, toward the interior of the protein . The dramatic change in the OAS:acetate lyase activity of OASS-A in the C42S and C42A mutant proteins likely results from a localized movement of the serine hydroxyl (compared to the cysteine thiol) toward additional hydrophilic, hydrogen-bonding groups in C42S, or away from hydrophilic groups for C42A, repositioning structure around and including K41 . Subtle movement of the epsilon-amino group of K41 may change the geometry for nucleophilic displacement of the amino acid from PLP, leading to changes in overall activity and stability of the alpha-aminoacrylate intermediate . Data indicate that single amino acid substitutions that yield only subtle changes in structure can produce large differences in reaction rates and overall mechanism.

J Membr Biol, 1998 Aug 1, 164(3), 263 - 74
Study of sugar binding to the sucrose-specific ScrY channel of enteric bacteria using current noise analysis; Andersen C et al.; ScrY, an outer membrane channel of enteric Gram-negative bacteria, which confers to the bacteria the rapid uptake of sucrose through the outer membrane was reconstituted into lipid bilayer membranes and the current noise was investigated in the open and in the carbohydrate-induced closed state of the channel . The open state of the channel exhibited up to about 200 Hz 1/f-noise with a rather small spectral density . Upon addition of carbohydrates to the aqueous phase the current through the ScrY channels decreased in a dose-dependent manner . Simultaneously, the spectral density of the current noise increased drastically, which indicated interaction of the carbohydrates with the binding site inside the channel and its reversible block . The frequency dependence of the spectral density was of the Lorentzian type but very often two Lorentzians were observed, from which the slow one may not be related to carbohydrate binding . Analysis of the power density spectra of the second Lorentzian using a previously proposed simple model of carbohydrate binding allowed the evaluation of the on- and the off-rate constants for the carbohydrate association with the binding site inside the ScrY channel and of a mutant (ScrYDelta3-72), in which 70 amino acids at the N-terminus are deleted . The binding of carbohydrates to ScrY was compared to those of the closely related maltoporin channels of Escherichia coli and Salmonella typhimurium by assuming that only the time constant and spectral density of the high frequency Lorentzian is related to carbohydrate transport.

Antimicrob Agents Chemother, 1998 Aug, 42(8), 1980 - 4
CTX-M-5, a novel cefotaxime-hydrolyzing beta-lactamase from an outbreak of Salmonella typhimurium in Latvia; Bradford PA et al.; At a children's hospital in Riga, Latvia, isolates identified as Salmonella typhimurium were found to be resistant to expanded-spectrum cephalosporins . Two of the resistant strains were analyzed for the mechanism of cephalosporin resistance . Isoelectric focusing revealed a common beta-lactamase with a pI of 8.8 . In addition, one of the strains produced a pI 7.6 beta-lactamase . A transconjugant producing only the pI 7.6 enzyme was susceptible to expanded-spectrum cephalosporins; therefore, this enzyme was most likely SHV-1 . Transformants producing only the pI 8.8 beta-lactamase were resistant to cefotaxime and aztreonam but were susceptible or intermediate to ceftazidime . A substrate profile determined spectrophotometrically with purified enzyme revealed potent activity against cefotaxime, with a relative kcat value of 95 (benzylpenicillin equal to 100) . The enzyme showed lower relative kcat values for ceftazidime (3.3) and aztreonam (9.3) . In addition, the enzyme was inhibited by clavulanate, sulbactam and tazobactam, with 50% inhibitory concentrations of 19, 100, and 3.4 nM, respectively . These results indicated the presence of an unusual extended-spectrum beta-lactamase . The gene expressing the pI 8.8 beta-lactamase was cloned . Nucleotide sequencing revealed a beta-lactamase gene that differs from the gene encoding CTX-M-2, which also originated from S . typhimurium, by 11 nucleotides, 4 of which result in amino acid substitutions: Ala27Thr, Val230Gly, Glu254Ala, and Ile278Val . These results indicated the presence of a novel extended-spectrum beta-lactamase, designated CTX-M-5, that specifically confers resistance to cefotaxime.

J Antimicrob Chemother, 1998 Jun, 41(6), 635 - 41
Role of mutation in the gyrA and parC genes of nalidixic-acid-resistant salmonella serotypes isolated from animals in the United Kingdom; Piddock LJ et al.; To answer the question as to whether isolates of salmonella from animals with decreased susceptibility to quinolones possess the same mechanism of resistance as isolates from humans, the polymerase chain reaction (PCR) was used to amplify a 347 base pair fragment equivalent to codons 39-159 covering the quinolone resistance determining region (QRDR) of the gyrA gene of Salmonella typhimurium NCTC 74, and 196 veterinary isolates of 26 serotypes of salmonella (109 from turkeys, 29 from chickens, the remainder from cattle, parrots, pigs, dogs, a pigeon, an exotic bird, a duck, a partridge, a sheep, a horse and environmental samples including litter, a nest and water) . All PCR products were electrophoresed for single-stranded conformational polymorphism (SSCP) . The DNA sequence of all eight novel SSCP patterns was determined . Nucleotide substitutions were identified in the gyrA gene of all except two isolates; all substitutions conferred a substitution at serine 83 or aspartate 87, as has previously been shown for isolates from humans . No mutations conferring an amino acid substitution in parC (the gene encoding the secondary target for quinolones) were revealed in the 48 isolates requiring 0.5-1 mg/L ciprofloxacin for inhibition, or the isolates for which no mutation in gyrA was revealed . The accumulation of enrofloxacin and ciprofloxacin was determined for 30 representative isolates (of which five were multiply drug resistant) . Five isolates accumulated one-quarter to one-half the concentration achieved by the corresponding wild-type serotype . For two of these five isolates no mutation in gyrA was revealed, and one lacked OmpF.

Mutat Res, 1998 May 25, 400(1-2), 259 - 69
Iron-chlorophyllin-mediated conversion of 3-hydroxyamino-1-methyl-5H-pyrido{4,3-b}indole (Trp-P-2(NHOH)) into its nitroso derivative; Arimoto-Kobayashi S et al.; Early work from our laboratory has shown that the mutagenicity of heterocyclic amines in Salmonella can be inhibited by hemin and chlorophyllins . We have speculated that the inhibition is a result of complex formation between heterocyclic amines and the pigments, and the speculation has been given a line of experimental evidence . We have now found that ferric-chlorophyllin (Fe-chlorophyllin) can modify the mutagenicity of 3-hydroxyamino-1-methyl-5H-pyrido{4, 3-b}indole (Trp-P-2(NHOH)), a metabolically activated form of 3-amino-1-methyl-5H-pyrido{4,3-b}indole (Trp-P-2) . The mutagenicity of Trp-P-2(NHOH) in Salmonella typhimurium TA 98 (without S9) was strongly inhibited by an addition of an equimolar Fe-chlorophyllin in the pre-incubation mixture . Fe-chlorophyllin also inhibited the mutagenicity of 2-hydroxyamino-6-methyldipyrido{1,2-a:3',2'-d} imidazole (Glu-P-1(NHOH)) . A rapid change in the UV spectrum of a mixture of Trp-P-2(NHOH) and Fe-chlorophyllin was observed . Analysis by high performance liquid chromatography showed that Trp-P-2(NHOH) was converted into 3-nitroso-1-methyl-5H-pyrido{4,3-b}indole (Trp-P-2(NO)), the mutagenic potency of which is a quarter of that of Trp-P-2(NHOH) . Furthermore, the mutagenicity of Trp-P-2(NO), in turn, was inhibited by Fe-chlorophyllin . We conclude that the suppression of the mutagenicity of Trp-P-2(NHOH) is ascribable to the oxidative function of Fe-chlorophyllin, coupled with its ability to form complex formation with the planar surface of the heterocyclic amine molecules .

Mutat Res, 1998 May 25, 400(1-2), 89 - 97
Detection of mutator subpopulations in Salmonella typhimurium LT2 by reversion of his alleles; LeClerc JE et al.; Defects in the methyl-directed mismatch repair lead to both the hypermutability phenotype and removal of a barrier to genetic exchange between species . Mutator bacteria carrying such defects occur frequently among bacterial pathogens, suggesting that subpopulations of mutators are contained within pathogen clones and give rise to the genetic variants that are acted upon by selective forces to allow survival or successful infection . We report here on the detection of the mutator subpopulation in Salmonella typhimurium and determination of its frequency in laboratory cultures . The analysis involved screening for mutators among revertants of S . typhimurium histidine auxotrophs selected for the His+ phenotype, since the frequency of mutators is expected to be increased in the selected mutant population they helped to spawn . The increases in spontaneous reversion of histidine mutations were first measured in isogenic strains carrying mismatch repair-defective mutH, mutL, mutS, or uvrD alleles, relative to their mismatch repair-proficient counterparts . Screening for the mutator phenotype in nearly 12,000 revertants of repair-proficient strains carrying his mutations highly stimulated for reversion in mutator backgrounds, the base substitution in hisG428 and frameshift in hisC3076, yielded five mutator strains (0.04%) . The his+ reversion mutations contained within the newly-arisen mutator strains were characteristic of the predominant nucleotide changes expected in such mutators, as assessed by comparison with the spectra for reversion events in wild-type and mismatch correction-defective backgrounds . The results show that subpopulations of mutators, residing in normal populations at a finite frequency, can be culled from the culture by strong selection for a required phenotype . We calculate that the frequency of mutators in the unselected population of S . typhimurium is 1-4x10-6, an incidence 10-fold lower than that expected based on studies of laboratory cultures of Escherichia coli .

Biosens Bioelectron, 1998 Mar 15, 13(5), 495 - 500
Acoustic standing-wave enhancement of a fiber-optic Salmonella biosensor; Zhou C et al.; An enhanced fluorescent fiber-optic biosensor system using ultrasonic concentration of particles and cells has been developed and applied in the detection of Salmonella typhimurium . A biosensor test chamber also serves as an ultrasonic standing-wave cell that allows microspheres or cells to be concentrated in parallel layers or in a column along the axis of the cell . A fiber probe along the axis delivers laser excitation to fluorescent-labeled antibodies of Salmonella and collects the fluorescent signal . The labeled-antibodies themselves do not respond to the ultrasound, but, when attached to Salmonella cells, the Salmonella-antibody complexes can be moved acoustically to the axis of the cell, increasing the fluorescent signal . In a second, more robust, type of immunoassay, the Salmonella-labeled-antibody complexes attach to unlabeled antibodies that have been immobilized on the surface of polystyrene microspheres . This entire structure can be manipulated acoustically and the increase in the fluorescent signal, which can be an order of magnitude, indicates the presence of Salmonella.

J Bacteriol, 1998 Aug, 180(15), 3845 - 52
Identification of a new site for ferrichrome transport by comparison of the FhuA proteins of Escherichia coli, Salmonella paratyphi B, Salmonella typhimurium, and Pantoea agglomerans; Killmann H et al.; The fhuA genes of Salmonella paratyphi B, Salmonella typhimurium, and Pantoea agglomerans were sequenced and compared with the known fhuA sequence of Escherichia coli . The highly similar FhuA proteins displayed the largest difference in the predicted gating loop, which in E . coli controls the permeability of the FhuA channel and serves as the principal binding site for the phages T1, T5, and phi80 . All the FhuA proteins contained the region in the gating loops required in E . coli for ferrichrome and albomycin transport . The three subdomains required for phage binding were contained in the gating loop of S . paratyphi B which is infected by the E . coli phages, whereas two of the subdomains were deleted in S . typhimurium and P . agglomerans which are resistant to the E . coli phages . Small deletions in a surface loop adjacent to the gating loop, residues 236 to 243 and 236 to 248, inactivated E . coli FhuA with regard to transport of ferrichrome and albomycin, but sensitivity to T1 and T5 was fully retained and sensitivity to phi80 and colicin M was reduced 10-fold . Full-size FhuA hybrid proteins of S . paratyphi B and S . typhimurium displayed S . paratyphi B FhuA activity when the hybrids contained two-thirds of either the N- or the C-terminal portions of S . paratyphi B and displayed S . typhimurium FhuA activity to phage ES18 when the hybrid contained two-thirds of the N-terminal region of the S . typhimurium FhuA . The central segment of the S . paratyphi B FhuA flanked on both sides by S . typhimurium FhuA regions conferred full sensitivity only to phage T5 . The data support the essential role of the gating loop for the transport of ferrichrome and albomycin, identified an additional loop for ferrichrome and albomycin uptake, and suggest that several segments and their proper conformation, determined by the entire FhuA protein, contribute to the multiple FhuA activities.

Vaccine, 1998 Jul, 16(11-12), 1193 - 202
Protective immune responses against protease-like antigens of the murine malaria parasite Plasmodium vinckei; Gor DO et al.; The Plasmodium falciparum proteins serine repeat antigen (SERA) and serine repeat protein homologue (SERPH) have similarity in sequence with cysteine proteases in a well-conserved protease domain . We identified three SERA homologues from the murine malaria parasite Plasmodium vinckei and evaluated immune responses to the protease domains of these proteins . Mice that developed protective immunity to P . vinckei after serial infection and cure demonstrated humoral and cell-mediated responses against the SERA homologue protease domains . Mice immunized with Salmonella typhimurium expressing the protease domain of one of these antigens demonstrated cellular responses against the antigen and increased survival against lethal challenge with P . vinckei . Our results suggest that the protease domains of SERA and SERPH are worthy of additional study as potential components of a malaria vaccine.

Mol Microbiol, 1998 Jun, 28(6), 1367 - 80
The Salmonella typhimurium InvH protein is an outer membrane lipoprotein required for the proper localization of InvG; Daefler S et al.; The secretion of pathogenicity factors by Salmonella typhimurium is mediated by a type III secretion system that includes an outer membrane protein of the secretin family . Related secretins are also required for f1 phage assembly and type II secretion . When the C-terminal 43 amino acids of the S . typhimurium secretin InvG are added to f1 pIV, the chimeric f1 pIV-'InvG43 protein becomes dependent on the co-expression of another gene, invH, for function in phage assembly . {3H}-palmitic acid labelling, globomycin sensitivity and density gradient flotation were used to demonstrate that InvH is an outer membrane lipoprotein that is processed by signal peptidase II . A complex between chimeric f1 pIV-'InvG43 and InvH was demonstrated in vivo . InvH was shown to be required for the proper localization of InvG in the outer membrane and for the secretion of the virulence factor SipC . These results suggest that InvH and InvG are part of the functional outer membrane translocation complex in type III secretion systems.

J Food Prot, 1998 Jul, 61(7), 796 - 801
Dosage titration of a characterized competitive exclusion culture to inhibit Salmonella colonization in broiler chickens during growout; Corrier DE et al.; Broiler chicks were spray treated on the day of hatch with titrated dosages (10(6), 10(7), or 10(8) anaerobic CFU) of a characterized competitive exclusion culture (CF3) and challenged orally on day 3 with 10(4) CFU of Salmonella typhimurium . On day 10, cecal contents from control and CF3-treated chicks were cultured for S . typhimurium to determine the minimal efficacious dosage of the CF3 culture . The experiment was repeated in three replicated trials . Resistance to Salmonella cecal colonization was dosage related and progressively enhanced at the 10(7)- and 10(8)-CFU dosages compared with the 10(6)-CFU dosage . The 10(7)-CFU dosage was selected as the minimal effective dosage and evaluated for efficacy during a 43-day broiler growout study . Six hundred broilers were spray treated on the day of hatch and compared with 600 controls . One-half of the control and CF3-treated birds were challenged orally on day 3 with 10(4) CFU of S . typhimurium and designated "seeders." The remaining unchallenged birds were designated "contacts." Compared with the controls, the recovery of Salmonella cells from the ceca of the CF3-treated broilers was significantly decreased (P < 0.01) in the challenged seeders on days 21 and 43 of growout . Salmonella contamination of floor pen litter was significantly reduced (P < 0.05) in pens of CF3-treated birds compared with controls . The transmission of Salmonella cells from seeder to contact birds in the same pens was decreased significantly (P < 0.01) . The results indicated that treatment of broiler chicks on the day of hatch with the 10(7)-CFU dosage of CF3 culture effectively increased resistance to S . typhimurium challenge during growout to market age.

J Food Prot, 1998 Jul, 61(7), 829 - 32
Use of antimicrobial spray applied with an inside-outside birdwasher to reduce bacterial contamination on prechilled chicken carcasses; Yang Z et al.; Antimicrobial sprays applied using a modified inside-outside birdwasher to reduce Salmonella typhimurium and total aerobic bacteria on prechilled chicken carcasses were evaluated in a poultry processing pilot plant . Four chemicals, including trisodium phosphate (TSP, 10%), lactic acid (LAC, 2%), cetylpyridinium chloride (CPC, 0.5%), and sodium bisulfate (SBS, 5%) were selected to be tested as antimicrobial agents . Each chicken carcass was inoculated by spraying the outside and inside of each carcass with S . typhimurium at 10(5) CFU per carcass . The inoculated carcasses then were passed through the birdwasher and sprayed with selected chemicals at 35 degrees C at a pressure of 413 kPa for 17 s . After a 60-s setting time on a shackle line, the carcasses were sprayed with tap water to rinse off chemical residue . All the chemical treatments reduced Salmonella on the chicken carcasses by approximately 2 log10 CFU per carcass . Total aerobes on the chicken carcasses, however, were reduced by 2.16, 1.66, 1.03, and 0.74 log10 CFU per carcass after spraying with 0.5% CPC, 5% SBS, 2% LAC, or 10% TSP, respectively . Spray treatments of both SBS and LAC caused slight discoloration in part of the chicken skin . The most effective antimicrobial spray treatment for reducing both Salmonella and total aerobes on prechilled chicken carcasses was 0.5% CPC.

J Mol Biol, 1998 Jul 24, 280(4), 571 - 82
pyrH-encoded UMP-kinase directly participates in pyrimidine-specific modulation of promoter activity in Escherichia coli; Kholti A et al.; The carAB operon of the enterics Escherichia coli K-12 and Salmonella typhimurium LT2, encoding the sole carbamoylphosphate synthetase (CPSase) of these organisms, is transcribed from two promoters in tandem, carP1 upstream and carP2 downstream, repressed respectively by pyrimidines and arginine . We present evidence that the pyrH gene product (the hexameric UMP-kinase) directly participates in the pyrimidine-specific control of carP1 activity . Indeed, we have isolated in E . coli a particular type of pyrH mutation (pyrH41) that retains a quasi-normal UMP-kinase activity, but yet is impaired in the pyrimidine-specific repression of the P1 promoter of the carAB operon of E . coli and of S . typhimurium . Moreover, the pyrimidine-dependent inhibition of in vivo Dam methylase modification of adenine -106 upstream of the carP1 promoter is altered in this pyrH mutant . The recessive pyrH41 allele bears a single C-G to A-T transversion that converts alanine 94 into glutamic acid (A94E) . Although overexpression of pyrH41 results in UMP-kinase levels far above that of a wild-type strain, pyrimidine-specific repression of the carAB operon is not restored under these conditions . Similarly, overexpression of the UMP-CMP-kinase gene of Dictyostelium discoideum in the pyrH41 mutant does not restore pyrimidine-mediated control of carP1 promoter activity, in spite of the elevated UMP-kinase activity measured in such transformants . These results indicate that besides its catalytic function in the de novo pyrimidine biosynthesis, E . coli UMP-kinase fulfils an additional, but previously unrecognized role in the regulation of the carAB operon . UMP-kinase might function as the real sensor of the internal pyrimidine nucleotide pool and act in concert with the integration host factor (IHF) and aminopeptidase A (PepA alias CarP and XerB) in the elaboration of the complex nucleoprotein structure required for pyrimidine-specific repression of carP1 promoter activity .

Science, 1998 Jul 24, 281(5376), 565 - 8
Delivery of epitopes by the Salmonella type III secretion system for vaccine development; Russmann H et al.; Avirulent strains of Salmonella typhimurium are being considered as antigen delivery vectors . During its intracellular stage in the host, S . typhimurium resides within a membrane-bound compartment and is not an efficient inducer of class I-restricted immune responses . Viral epitopes were successfully delivered to the host-cell cytosol by using the type III protein secretion system of S . typhimurium . This resulted in class I-restricted immune responses that protected vaccinated animals against lethal infection . This approach may allow the efficient use of S . typhimurium as an antigen delivery system to control infections by pathogens that require this type of immune response for protection.

Pharmazie, 1998 Jun, 53(6), 410 - 2
Postantibiotic effects of norfloxacin and netilmicin and their influence on the biological properties of Salmonella strains; Majtan V et al.; The postantibiotic effect (PAE) and the postantibiotic sub-MIC effect (PA SME) of norfloxacin and netilmicin on two clinical strains--Salmonella typhimurium and Salmonella enteritidis was investigated . After both PAE and PA SME of antibiotics were studied, we determined their effect on the induction of a prophage in the lysogenic S . typhimurium and on Congo red binding by both serovars, as an indicator of invasive ability in vitro . The PAE was induced by 2.MIC and 4.MIC of norfloxacin and netilmicin for 0.5 h . Norfloxacin induced a longer lasting PAE on both Salmonella serovars as compared to netilmicin . Supra-subinhibitory concentrations (PA SMEs) delayed regrowth of tested strains . The PA SMEs of norfloxacin as well as of netilmicin (except 2.MIC + 0.1.MIC concentration) did not allow regrowth of S . enteritidis . The prophage-inductive ability of norfloxacin was more expressive after PA SMEs than PAE . The PA SMEs of netilmicin caused loss of Congo red binding by S . typhimurium cells and decreased this binding by S . enteritidis cells.

Mutat Res, 1998 Jun 18, 402(1-2), 247 - 58
Antimutagenic and anticarcinogenic potentials of some Thai vegetables; Kusamran WR et al.; Fifteen kinds of commonly consumed Thai vegetables were sequentially extracted with hexane, chloroform and methanol, and then tested for antimutagenic activities against direct-acting (AF-2 and NaN3) and indirect-acting (AFB1 and B(a)P) mutagens using Ames' Salmonella mutagenicity test with Salmonella typhimurium TA100 as tester strain . It was found that only the methanol extract of neem leaves contain weak antimutagen inhibiting the mutagenicities of both direct-acting mutagens . Interestingly, all vegetables studied were found to contain chemical compounds, mainly nonpolar ones, capable of inhibiting the mutagenicity of AFB1, while only some vegetables contain chemical compounds capable of inhibiting the mutagenicity of B(a)P, which is also an indirect-acting mutagen . Studies on anticarcinogenic potentials demonstrated that Thai bitter gourd fruits, but not sweet basil leaves, at the concentration of 6.25% and 12.5% in the diet, partially inhibited DMBA-induced mammary gland carcinogenesis in female Sprague-Dawley rats when fed to the animals 2 weeks prior to DMBA . Results in the present study therefore demonstrated that most Thai vegetables contain antimutagens inhibiting the mutagenicity of some indirect-acting mutagen, particularly AFB1 . The mechanism of their antimutagenicity may probably be the inhibition of the activity of metabolic-activating enzymes in rat liver homogenates . Very interestingly, our results clearly reveal that Thai bitter gourd fruits, which possess Phase II enzymes inducing property, as well as the ability to reduce Phase I enzyme activities in rat liver, contain some anticarcinogens or chemopreventive agents . However, sweet basil leaves that possess both Phase I and Phase II enzyme-inducing properties may not contain any anticarcinogen, at least against DMBA-induced mammary gland carcinogenesis .

Mutat Res, 1998 Jun 18, 402(1-2), 231 - 6
Antigenotoxicity of galangin against N-methyl-N-nitrosourea; Sohn SJ et al.; Using the Ames bacterial mutagenicity test and an in vivo micronucleus test, we investigated the antigenotoxic effect of galangin against the genotoxicity of N-methyl-N-nitrosourea (MNU) . In the Ames assay, galangin showed an antimutagenic effect towards MNU-induced mutagenicity of Salmonella typhimurium TA 100 . In mice, galangin showed an anticlastogenic effect against MNU-induced micronuclei in polychromatic erythrocytes in the MNPCE in mouse bone marrow cells . On the other hand, galangin is neither mutagenic nor clastogenic in both assays . Results from our in vitro and in vivo studies indicate that galangin is capable of suppressing the mutagenicity and clastogenicity of MNU . Therefore, galangin may be a useful chemopreventive agent against potential long-term health effects from genotoxic environmental agents .

J Appl Microbiol, 1998 May, 84(5), 777 - 83
The specific and sensitive detection of bacterial pathogens within 4 h using bacteriophage amplification; Stewart GS et al.; This paper describes a novel approach, termed the 'phage amplification assay', for the rapid detection and identification of specific bacteria . The technique is based on the phage lytic cycle with plaque formation as the assay end-point . It is highly sensitive, quantitative and gives results typically within 4 h . The assay comprises four main stages: (1) phage infection of target bacterium; (2) destruction of exogenous phage; (3) amplification of phage within infected host and (4) plaque formation from infected host with the aid of helper bacteria . A key component of this assay is a potent virucidal agent derived from natural plant extracts, pomegranate rind extract (PRE) . In combination with ferrous sulphate PRE can bring about an 11 log-cycle reduction in phage titre within 3 min . This is achieved without any injury to the infected target bacteria . Subsequently, any resulting plaques are derived only from infected target organisms . Data are presented for a range of bacterial hosts including Pseudomonas aeruginosa, Salmonella typhimurium and Staphylococcus aureus . The detection limit for Ps . aeruginosa was 40 bacteria ml-1 in a time of 4 h and 600 bacteria m-1 for Salm . typhimurium . Application of the principles of this technology to other bacterial genera is discussed.

Infect Immun, 1998 Aug, 66(8), 3802 - 9
Magnesium and the role of MgtC in growth of Salmonella typhimurium; Moncrief MB et al.; Salmonella typhimurium has three distinct transport systems for Mg2+: CorA, MgtA, and MgtB . The mgtCB operon encodes two proteins, MgtC, a hydrophobic protein with a predicted molecular mass of 22.5 kDa, and MgtB, a 102-kDa P-type ATPase Mg2+ transport protein . The mgtCB locus has been identified as part of a new Salmonella pathogenicity island, SPI-3 . Transcription of mgtCB is regulated by extracellular Mg2+ via the two-component PhoPQ regulatory system important for virulence . To elucidate MgtC's role in a low-Mg2+ environment, we looked at growth and transport in strains lacking the CorA and MgtA Mg2+ transporters but expressing MgtB, MgtC, or both . mgtC mgtB+ and mgtC+ mgtB+ strains exhibited growth in N minimal medium without added Mg2+ with a 1- to 2-h lag phase . An mgtC+ mgtB strain was also able to grow in N minimal medium without added Mg2+ but only after a 24-h lag phase . In N minimal medium containing 10 mM Mg2+, all strains grew after a short lag phase; the mgtC+ mgtB strain grew to a higher optical density at 600 nm than an mgtC+ mgtB+ strain and was comparable to wild type . The lengthy lag phase before growth in an mgtC+ mgtB strain was not due to lack of expression of MgtC . Western blot analysis indicated that substantial MgtC protein is present by 2 h after suspension in N minimal medium . Surprisingly, in an mgtC+ mgtB+ strain, MgtC was undetectable during Mg2+ starvation, although large amounts of MgtB were observed . The lack of expression of MgtC is not dependent on functional MgtB, since a strain carrying a nonfunctional MgtB with a mutation (D379A) also did not make MgtC . Since, during invasion of eukaryotic cells, S . typhimurium appears to be exposed to a low-pH as well as a low-Mg2+ environment, the growth of an mgtC+ mgtB strain was tested at low pH with and without added Mg2+ . While significant quantities of MgtC could be detected after suspension at pH 5.2, the mgtC+ mgtB strain was unable to grow at pH 5.2 whether or not Mg2+ was present . Finally, using 63Ni2+ and 57Co2+ as alternative substrates for the unavailable 28Mg2+, cation uptake could not be detected in an mgtC+ mgtB strain after Mg2+ starvation . We conclude that MgtC is not a Mg2+ transporter and that it does not have a primary role in the survival of S . typhimurium at low pH.

Infect Immun, 1998 Aug, 66(8), 3758 - 66
Interactions of the invasive pathogens Salmonella typhimurium, Listeria monocytogenes, and Shigella flexneri with M cells and murine Peyer's patches; Jensen VB et al.; Invasive enteric bacteria must pass through the intestinal epithelium in order to establish infection . It is becoming clear that a common target for intestinal mucosa penetration is the specialized epithelial cell of Peyer's patches, the M cell . In order to gain a better understanding of how bacteria interact with M cells, we have compared the interactions of Salmonella typhimurium, Listeria monocytogenes, and Shigella flexneri with M cells by using a murine ligated-loop model . Our results indicate that S . typhimurium possesses a highly efficient mechanism for M cell entry that targets and destroys these cells, while L . monocytogenes and S . flexneri appear to be internalized into M cells in a less disruptive fashion . Early uptake of Listeria or Shigella into M cells appeared to lead to the death of some cells, as evidenced by the appearance of holes in the intestinal epithelium . At later time points, the follicle-associated epithelium of animals infected with these bacteria displayed extensive destruction . These data indicate that enteric pathogens use different strategies to interact with M cells and initiate infection of a host.

FEMS Microbiol Lett, 1998 Jun 15, 163(2), 143 - 8
In vivo effects of anti-inducers of the cysteine regulon in Salmonella typhimurium; Oppezzo OJ; Growth on readily utilizable sulfur sources reduces expression of the cysteine regulon in Salmonella typhimurium . Inhibition of serine transacetylase by cysteine and direct actions of the anti-inducers sulfide and thiosulfate are responsible for reduction of expression . In order to evaluate individual contributions of each mechanism, the inhibitory effects of Na2S and Na2S2O3 were studied in strains with or without the capacity to synthesize cysteine from these compounds, using a transcriptional fusion to the cysDNC operon . In a cysK cysM strain, although cysteine synthesis from sulfide and thiosulfate was blocked, Na2S and Na2S2O3 efficiently reduced expression of the cysDNC operon . The inhibitory effect observed in this mutant was equivalent to 70-100% of that found in a strain carrying the fusion in a wild-type context grown in the same conditions . The actions of sulfide and thiosulfate as anti-inducers seem therefore to be responsible for most of the reduction of expression caused by these agents in vivo.

FEMS Microbiol Lett, 1998 Jun 15, 163(2), 129 - 34
A Salmonella typhimurium mutant unable to utilize fatty acids and citrate is avirulent and immunogenic in mice; Utley M et al.; Salmonella typhimurium SR-11 is extremely virulent at a dose as low as 10(5) colony forming units (cfu) when administered perorally to BALB/c mice . Utilizing mini-transposon mutagenesis, a mutant of S . typhimurium SR-11 was isolated that was unable to utilize oleate and citrate as carbon sources . This mutant, designated S . typhimurium SR-11 Fad- (Fatty acid), was found to utilize sugars under cya/crp control as sole carbon sources, suggesting that the mutation is not in either of these genes . In addition, SR-11 Fad- utilized pyruvate and succinate, but was unable to utilize either acetate or isocitrate as sole carbon source . In contrast to SR-11, SR-11 Fad- was found to be avirulent, i.e . BALB/c mice were completely healthy after oral infection with 10(9) S . typhimurium SR-11 Fad- cells . Moreover, 21 days after SR-11 Fad- infection, BALB/c mice were found to be protected against an oral challenge with 10(9) cells of S . typhimurium SR-11.

J Mol Biol, 1998 Jul 31, 280(5), 847 - 58
Catalytic defects in mutants of class II histidyl-tRNA synthetase from Salmonella typhimurium previously linked to decreased control of histidine biosynthesis regulation; Francklyn C et al.; The expression of histidine biosynthetic genes in enteric bacteria is regulated by an attenuation mechanism in which the level of histidyl-tRNA serves as a key sensor of the intracellular histidine pool . Among the early observations that led to the formation of this model for Salmonella typhimurium were the identification of mutants in the gene (hisS) encoding histidyl-tRNA synthetase . We report here the detailed biochemical characterization of five of these S . typhimurium bradytrophic mutants isolated by selection for resistance to histidine analogs, including identification of the deduced amino acid substitutions and determination of the resulting effects on the kinetics of adenylation and aminoacylation . Using the crystal structure of the closely related Escherichia coli histidyl-tRNA synthetase (HisRS) as a guide, two mutants were mapped to a highly conserved proline residue in motif 2 (P117S, P117Q), and were correlated with a fivefold decrease in the kcat for the pyrophosphate exchange reaction, as well as a tenfold increase in the Km for tRNA in the aminoacylation reaction . Another mutant substitution (A302T) mapped to a residue adjacent to the histidine binding pocket, leading to a tenfold increase in Km for histidine in the pyrophosphate exchange reaction . The remaining two mutants (S167F, N254T) substitute residues in or directly adjacent to the hinge region, which joins the insertion domain between motif 2 and motif 3 to the catalytic core, and cause the Km for tRNA to increase four- to tenfold . The kinetic analysis of these mutants establishes a direct link between critical interactions within the active site of HisRS and regulation of histidine biosynthesis, and provides further evidence for the importance of local conformational changes during the catalytic cycle .

Arch Pediatr Adolesc Med, 1998 Jul, 152(7), 659 - 64
Epidemiology and molecular identification of Salmonella infections in children; Schutze GE et al.; OBJECTIVE: To explore the role of foods and the environment in the development of infections with Salmonella in infants and children . DESIGN: Case-controlled survey and the use of pulsed-field gel electrophoresis to establish DNA fingerprint patterns . SETTING: Ambulatory and hospitalized patients at a children's hospital . PATIENTS OR OTHER PARTICIPANTS: A consecutive sample of children younger than 4 years old who were infected with Salmonella and 3 age-matched controls per patient were to be surveyed . Of the 103 eligible cases of salmonellosis, 90 cases and 264 controls were included in the study . DATA ANALYSIS: Univariate analysis was done using the Mantel-Haenszel chi2 test or the Fisher exact test . The Bonferroni correction was used for multiple comparisons . DNA fingerprints were inspected for identical banding . RESULTS: Results demonstrated similar diets between cases and controls with the exception of more potato or macaroni salad or coleslaw consumption in the control group (P<.001) . DNA fingerprints of Salmonella newport and Salmonella typhimurium demonstrated that all cases were due to unique isolates except in 5 instances involving 12 patients . Seven of these patients could be connected geographically . CONCLUSIONS: Most of the cases of salmonellosis in children younger than 4 years are of a sporadic nature and the major source of infection remains unidentified . For patients infected with identical isolates of Salmonella, a common food source could not be incriminated with the methods used . Environmental contamination or other sources of Salmonella are suggested by these epidemiological data.

J Neuroimmunol, 1998 Jun 15, 86(2), 171 - 81
Effect of macrophage depletion by liposomes containing dichloromethylene-diphosphonate on endotoxin-induced uveitis; Pouvreau I et al.; Footpad injection of lipopolysaccharide (LPS) from Salmonella typhimurium in Lewis rats induces an acute anterior and posterior endotoxin-induced uveitis (EIU) . To investigate the role of macrophages in the pathogenesis of EIU, we eliminated macrophages by means of liposomes containing dichloromethylene-diphosphonate (Cl2MDP), a drug which depletes macrophages but not other immunocompetent cells . Intravenous injection of CL2MDP-liposomes clearly inhibited clinical and histological manifestations of uveitis in the anterior segment of the eye (iris/ciliary body) and reduced TNF level in aqueous humor . Specific immunostaining showed that CL2MDP-liposome injections decreased the number of ED2 + resident macrophages in the iris/ciliary body and the choroid . After LPS injection, CL2MDP-liposome treatment reduced the density of infiltrating ED1 + cells (mainly monocytes/macrophages) in the iris/ciliary body but not in the choroid; little or no effect was detected on the OX42 + cellular infiltration (mainly polymorphonuclear leukocytes) . The inflammatory cellular infiltration of the retina was not modified by the treatment . These findings suggest that macrophages play a key role in the pathogenesis of ocular inflammation.

Bioorg Khim, 1998 May, 24(5), 381 - 7
{Protein engineering of uridine phosphorylase from Escherichia coli K-12 . I . Cloning and expression of uridine phosphorylase genes from Klebsiella aerogenes and Salmonella typhimurium in E . coli}; Veiko VP et al.; Genes of uridine phosphorylases (udp) from Klebsiella aerogenes and Salmonella typhimurium were cloned and expressed . Highly effective producer strains of the corresponding proteins were constructed . Enzymic properties of the UPases obtained were studied and compared with those from the Escherichia coli enzyme . Mutant forms of UPase from E . coli (D5E, D5N, D5A) were prepared by site-directed mutagenesis techniques . It was shown that the Asp5 residue plays an insignificant role in the formation of the active form of the protein.

Mol Cell, 1998 May, 1(6), 795 - 805
Alkyl hydroperoxide reductase subunit C (AhpC) protects bacterial and human cells against reactive nitrogen intermediates; Chen L et al.; In Salmonella typhimurium, ahpC encodes subunit C of alkyl hydroperoxide reductase, an enzyme that reduces organic peroxides . Here, we asked if ahpC could protect cells from reactive nitrogen intermediates (RNI) . Salmonella disrupted in ahpC became hypersusceptible to RNI . ahpC from either Mycobacterium tuberculosis or S . typhimurium fully complemented the defect . Unlike protection against cumene hydroperoxide, protection afforded by ahpC against RNI was independent of the reducing flavoprotein, AhpF . Mycobacterial ahpC protected human cells from necrosis and apoptosis caused by RNI delivered exogenously or produced endogenously by transfected nitric oxide synthase . Resistance to RNI appears to be a physiologic function of ahpC . ahpC is the most widely distributed gene known that protects cells directly from RNI, and provides an enzymatic defense against an element of antitubercular immunity.

Eur J Biochem, 1998 Jun 1, 254(2), 341 - 6
Cobalamin (vitamin B12) biosynthesis--cloning, expression and crystallisation of the Bacillus megaterium S-adenosyl-L-methionine-dependent cobalt-precorrin-4 transmethylase CbiF; Raux E et al.; The Bacillus megaterium cbiF, encoding the cobalt-precorrin-4 S-adenosyl-L-methionine-dependent transmethylase of the anaerobic cobalamin biosynthetic pathway, has been cloned and overexpressed as a His-tagged recombinant protein in Escherichia coli . The protein was purified to homogeneity by a combination of metal chelate chromatography and high-resolution anion-exchange chromatography . The protein migrated with a subunit mass of 31 kDa by SDS/PAGE and with a molecular mass of 62 kDa by analytical gel filtration, suggesting that the native recombinant protein is a homodimer . The His-tagged protein was physiologically active as it was able to complement a Salmonella typhimurium cbiF mutant . However, the protein did not bind S-adenosyl-L-methionine with the same avidity as observed with other corrin biosynthetic transmethylases . A crystallisation screen of the purified protein led to the identification of two discrete crystal forms . One of these forms has been characterised and a full data set collected.

J Bacteriol, 1998 Jul, 180(14), 3517 - 21
The apeE gene of Salmonella typhimurium encodes an outer membrane esterase not present in Escherichia coli; Carinato ME et al.; Salmonella typhimurium apeR mutations lead to overproduction of an outer membrane-associated N-acetyl phenylalanine beta-naphthyl ester-cleaving esterase that is encoded by the apeE gene (P . Collin-Osdoby and C . G . Miller, Mol . Gen . Genet . 243:674-680, 1994) . This paper reports the cloning and nucleotide sequencing of the S . typhimurium apeE gene as well as some properties of the esterase that it encodes . The predicted product of apeE is a 69.9-kDa protein which is processed to a 67-kDa species by removal of a signal peptide . The predicted amino acid sequence of ApeE indicates that it is a member of the GDSL family of serine esterases/lipases . It is most similar to a lipase excreted by the entomopathogenic bacterium Photorhabdus luminescens . The Salmonella esterase catalyzes the hydrolysis of a variety of fatty acid naphthyl esters and of C6 to C16 fatty acid p-nitrophenyl esters but will not hydrolyze peptide bonds . A rapid diagnostic test reported to be useful in distinguishing Salmonella spp . from related organisms makes use of the ability of Salmonella to hydrolyze the chromogenic ester substrate methyl umbelliferyl caprylate . We report that the apeE gene product is the enzyme in Salmonella uniquely responsible for the hydrolysis of this substrate . Southern blot analysis indicates that Escherichia coli K-12 does not contain a close analog of apeE, and it appears that the apeE gene is contained in a region of DNA present in Salmonella but not in E . coli.

Arch Toxicol, 1998 May, 72(6), 342 - 6
Mutagenic properties of 1,2,3,4-tetrahydronaphthaline-1-hydroperoxide, a model compound for organic peroxides in Diesel exhaust; Stock S et al.; The genotoxicity of the organic peroxide 1,2,3,4-tetrahydronaphthaline-1-hydroperoxide (or tetraline-1-hydroperoxide, THP) was investigated in the Ames assay without a metabolic activating system using Salmonella typhimurium strains TA 98, TA 100, and TA 102 . THP served as a model compound for higher organic peroxides, which can arise from autoxidation of hydrocarbons, e.g . in Diesel exhaust . While THP induced no mutagenic response in S . typhimurium TA 98, it was directly mutagenic in strains TA 100 and TA 102 . These data, along with findings on mutagenic properties of other alkyl hydroperoxides, suggest that such compounds deserve further investigation regarding their genotoxic potential and occurrence in the environment.

Vet Immunol Immunopathol, 1998 Jun 30, 64(1), 83 - 95
Fluorescein isothiocyanate staining and characterization of avian heterophils; Rath NC et al.; Fluorescein isothiocyanate (FITC) was found to stain cytoplasmic granules of avian heterophilgranulocytes . In tissue sections, the fluorescent granulocytes were predominantly distributed adjacent to trabecular bones . The fluorescein stained granulocytes were abundant in synovial fluids of chickens with synovitis . A significant correlation was observed in the percent of fluorescein labeled granulocytes in blood smears and the percent of heterophils determined using an automated counting method, in unstained blood from normal and Escherichia coli-infected turkeys . The fluorescein-binding heterophils purified from chickens showed a time dependent increases in the oxidation of 2',7'-dichlorofluorescin diacetate (DCF-DA) and the reduction of nitroblue tetrazolium (NBT) which were indicative of changes in oxidative burst in response to phorbol 12-myristate 13-acetate (PMA), Salmonella typhimurium lipopolysaccharide (LPS), and zymosan A (ZA) . These heterophil-activating agents, also, caused significant degranulation at 16 h post-treatment, as indicated by the loss fluorescence . There were microscopically visible alterations in the cell shapes and a decrease in the density of granules due to treatment with LPS, PMA or ZA . In addition, these cells also showed phagocytic response which was evident at 30 min of incubation with fluorescent latex particles . Both chicken and turkey heterophils produced interleukin-6 in vitro at 24 h in response to LPS but not to PMA, FMLP or ZA . The chicken heterophils showed spontaneous production of matrix metalloproteinases (MMP) which was significantly enhanced by treatment with LPS, PMA, and ZA; however, LPS appeared to be most effective in inducing MMP production . These results demonstrate that the functions of heterophils can be differentially regulated by different activating agents and the fluorescein binding property of these cells may be useful for their histochemical identification.

Environ Mol Mutagen, 1998, 31(4), 383 - 9
Unicellular green alga Chlamydomonas reinhardtii as an activation system for 2-aminofluorene; Miadokova E et al.; Despite the promutagenic/procarcinogenic potential, polycyclic aromatic amines are widely spread in the environment . Biotransformation of the polycyclic aromatic amine 2-aminofluorene (2-AF) was proved in mammals and higher plants . The algal cell/microbe coincubation assay is an additional system that complemented those proved in mammals and higher plants, useful for detection and conversion of environmental promutagens, mainly in aquatic environments . The unicellular green algae may be a good activating system in coincubation assays in that the algal cells exist as a natural system . To increase the effectiveness of this metabolizing system, different modifications of the standard experimental procedure were conducted . Algae can accumulate and metabolize promutagenic pollutants, some of which may differ from those activated by the animal microsome metabolizing system (S9 mix) and by the plant cell/microbe coincubation assay . 2-AF was activated in the algal cell/ microbe coincubation assay in which wild-type Chlamydomonas reinhardtii cells were used as an activating system and the bacteria Salmonella typhimurium TA98, YG1024, and yeast Saccharomyces cerevisiae D7 as the genetic indicator organisms . It was converted to the mutagenic product(s) for the strain YG1024, but the strain TA98 did not exhibit any increase in the mutant yield of His+ revertants . Consequently, metabolites from 2-AF are substrates for O-acetyltransferase . A direct comparison of algal 2-AF activation with mammalian activation system (S9 mix) proved the higher activity of mammalian microsome system (S9 mix) . After the combination of both activation systems, a slight synergetic effect was found . Although the genetic endpoints induced by 2-AF using both modifications of the algal cell/S . cerevisiae coincubation assay and those obtained in intact yeast cells were similar at the equitoxic concentrations, 2-AF activation by the algal supernatant slightly increased the genetic endpoints studied.

Environ Mol Mutagen, 1998, 31(4), 327 - 32
Specificity of base substitution mutations induced by the dietary carcinogens 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine (PhlP) and 2-amino-3-methylimidazo{4,5-f}quinoline (IQ) in Salmonella; Koch WH et al.; The base pair substitution mutational profiles induced by the heterocyclic amine cooked food mutagens PhlP and IQ in Salmonella typhimurium strains TA100 and TA1535 were determined by colony hybridization analysis . Both PhlP and IQ induced predominantly GC-->TA transversions in strain TA100 (rfa,delta uvrB/pKM101) with a pronounced preference for the second codon position (CCC--> CAC; 72% of total) . PhlP also reverted strain TA1535 (rfa, delta uvrB) efficiently at concentrations similar to those required for strain TA100 . In contrast to the PhlP-induced mutational profile observed in strain TA100, in strain TA1535 PhlP induced exclusively GC-->AT transitions at the second codon position (CCC-->CTC; 96-99% of total) . Base substitution mutagenesis induced by heterocyclic amines related to PhlP is generally SOS-dependent, requiring the presence of plasmid pKM101 in Salmonella hisG46 strains . Thus, the SOS dependent reversion of S . typhimurium strain TA100 probably reflects error-prone lesion bypass at the major PhlP- guanosine adduct at the C-8 position . The GC-->AT transition mutations induced by PhlP in strain TA1535 appear to be SOS-independent, however, suggesting that these mutations may arise from the formation of PhlP-DNA adducts other than the replication-blocking C8-dG lesion.

Microb Drug Resist, 1998 Summer, 4(2), 113 - 8
Molecular epidemiology of antibiotic resistance genes in multiresistant epidemic Salmonella typhimurium DT 104; Ridley A et al.; The epidemiology of antibiotic resistance genes in epidemic multiresistant S . typhimurium DT 104 of human and animal origin was investigated . DNA prepared from 45 human and 21 animal strains isolated between 1984 and 1997, including eight isolated in other European countries, the USA, Trinidad, and South Africa and resistant to ampicillin, chloramphenicol, streptomycin, sulphonamides, spectinomycin, tetracyclines (R-type ACSSuSpT) were examined for the presence of integrons by PCR . Integron hot spots were observed in all strains conferring resistance to ACSSuSpT in two copies, determined by two discrete bands of approximately 1.0 and 1.2 kb . Direct nucleotide sequencing of the individual amplicons of selected strains indicated that the 1.0 kb gene product was ant (3")-Ia, responsible for resistance to streptomycin and spectinomycin; the 1.2 kb amplicon contained the gene blaPSE-1, encoding the beta-lactamase PSE-1 (CARB-2) . Both integrons were encoded on a single XbaI macrorestriction fragment of approximately 10 kb . All isolates of DT 104 of this resistance phenotype contained the same inserted gene cassettes, irrespective of source and country of origin, supporting the suggestion of the spread of an epidemic clone . Sequence analysis of the quinolone resistance determining region (QRDR) of gyrA of 15 multiresistant strains conferring additional resistance to nalidixic acid and ciprofloxacin (R-type ACSSuSpTNxCp) identified two discrete base substitutions at codon Asp-87 . Conversion of Asp-87 --> Asn was most commonly observed, in 7/10 human and 4/5 animal isolates, suggesting that this codon plays a major role in the development of ciprofloxacin resistance in multiresistant S . typhimurium DT 104.

Food Chem Toxicol, 1998 Apr, 36(4), 305 - 14
Identification of 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF) and 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF) with DNA breaking activity in soy sauce; Li X et al.; Components with DNA breaking activity in soy sauce were investigated . It was found that there were water soluble high molecular weight DNA breaking components in soy sauce . Two DNA breaking components in the ethyl acetate extract of soy sauce were identified as fragrant components, 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF) and 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF), in addition to the previously characterized DNA breaking fragrant component 4-hydroxy-5-methyl-3(2H)-furanone (HMF) (Hiramoto et al., 1996b) . Characterization of DNA breaking activity of HEMF was performed, and the mechanisms for the breaking were considered . HEMF cleaved the single strands of supercoiled pBR 322 DNA at pH 7.4 dose dependently and time dependently . DNA breaking was inhibited by superoxide dismutase, catalase, hydroxyl radical scavengers, spin trapping agents and metal chelators, and enhanced by Fe(III) ion . Electron spin resonance-spin trapping technique revealed the generation of hydroxyl radical . Hence, active oxygen species derived from interaction of HEMF with metal ions and oxygen participated in the cleavage . HEMF exhibited mutagenicity to Salmonella typhimurium TA100 without metabolic activation and induced micronucleated mouse peripheral reticulocytes.

Neuroimmunomodulation, 1997 Sep-Dec, 4(5-6), 298 - 304
Afferent signals to the CNS appear not to condition the modulation of interleukin-1 receptors in the hippocampus; Demissie S et al.; Conditioned alteration of natural killer (NK) cell activity was used as an indicator of the functional bidirectional communication between the immune and central nervous systems . Poly I:C and lipopolysaccharide (LPS) from Escherichia coli and Salmonella typhimurium were used as unconditioned stimuli and odor of camphor as the conditioned stimulus . An attempt was made to demonstrate the role of central interleukin (IL-1) receptors in this communication process . Brain IL-1 receptors were down-regulated by treatment with 50 microg/mouse of LPS from S . typhimurium, but not with the same dose of LPS from E . coli or poly I:C . A significant level of conditioned augmentation of NK cell activity was observed with poly I:C . Conditioned alteration in NK cell activity was also observed with LPS from E . coli, but at much lower level than poly I:C . NK cell activity was not conditioned with LPS from S . typhimurium at the same dose as E . coli LPS, but conditioned enhancement of NK cell activity was observed with a higher dose (100 microg) of S . typhimurium LPS . These results suggest that modulation of central IL-1 receptors do not seem to play a role in the conditioned augmentation of NK cell activity.

Chemosphere, 1998 Jul, 37(2), 209 - 18
Cytotoxicity and mutagenicity of a 2,4,6-trinitrotoluene (TNT) and hexogen contaminated soil in S . typhimurium and mammalian cells; Berthe-Corti L et al.; The toxicity and mutagenicity of aqueous and organic extracts of soil contaminated with TNT, TNT metabolites and hexogen was determined in mammalian cell lines and in prokaryotic cells . The prokaryotic toxicity was determined via the colony forming ability of Salmonella typhimurium (strains TA 98 and TA 100) . The same strains were used to test mutagenicity in the Ames test . The mammalian toxicity was analyzed in human fibroblasts by the inhibition of cell growth and cell viability (MTT assay) . The mammalian mutagenicity was tested with the HPRT test in V79 cells (hamster lung) . The aqueous soil extract did not reveal toxicity or mutagenicity in any of the tests performed . The DMSO/ethanol extract showed toxicity and mutagenicity in S . typhimurium . Thereby strain TA 98 was more sensitive than strain TA 100 . In human fibroblasts cell growth was strongly inhibited, whereas no reduction of cell viability was found in the MTT test . Mutagenicity of the DMSO/ethanol extract of the soil was demonstrated in V79 cells.

Biochemistry, 1998 Jun 30, 37(26), 9305 - 15
Insights into the mechanism of catalysis by the P-C bond-cleaving enzyme phosphonoacetaldehyde hydrolase derived from gene sequence analysis and mutagenesis; Baker AS et al.; Phosphonoacetaldehyde hydrolase (phosphonatase) catalyzes the hydrolysis of phosphonoacetaldehyde to acetaldehyde and inorganic phosphate . In this study, the genes encoding phosphonatase in Bacillus cereus and in Salmonella typhimurium were cloned for high-level expression in Escherichia coli . The kinetic properties of the purified, recombinant phosphonatases were determined . The Schiff base mechanism known to operate in the B . cereus enzyme was verified for the S . typhimurium enzyme by phosphonoacetaldehyde-sodium borohydride-induced inactivation and by site-directed mutagenesis of the catalytic lysine 53 . The protein sequence inferred from the B . cereus phosphonatase gene was determined, and this sequence was used along with that from the S . typhimurium phosphonatase gene sequence to search the primary sequence databases for possible structural homologues . We found that phosphonatase belongs to a novel family of hydrolases which appear to use a highly conserved active site aspartate residue in covalent catalysis . On the basis of this finding and the known stereochemical course of phosphonatase-catalyzed hydrolysis at phosphorus (retention), we propose a mechanism which involves Schiff base formation with lysine 53 followed by phosphoryl transfer to aspartate (at position 11 in the S . typhimurium enzyme and position 12 in the B . cereusphosphonatase) and last hydrolysis at the imine C(1) and acyl phosphate phosphorus.

Biosci Biotechnol Biochem, 1998 May, 62(5), 970 - 7
Antimutagenicity of flavones and flavonols to heterocyclic amines by specific and strong inhibition of the cytochrome P450 1A family; Kanazawa K et al.; We found the mechanism in flavonoids that can strongly suppress the mutagenicity of one of the food-derived and carcinogenic heterocyclic amines, 3-amino-1-methyl-5H-pyrido{4,3-b}indole (Trp-P-2) . The antimutagenicity was evaluated by IC50 value, the amount required for 50% inhibition of the mutagenicity of 0.1 nmol Trp-P-2, with Salmonella typhimurium TA98 strain in the presence of S9 mix . The flavones and flavonols were two orders stronger as antimutagens that such antimutagenic phytochemicals as chlorophylls and catechins . We had previously found flavonoids to be a desmutagen to neutralize Trp-P-2 before or during attack of DNA, because they had no effect on either the ultimate mutagenic form of Trp-P-2 (N-hydroxy-Trp-P-2) or the mutated cells . The desmutagenicity of the flavonoids did not depend on the hydroxy number or position that should be associated with antioxidative potency, and was also unaffected by the solubility of Trp-P-2 in the assay solution . The inhibitory effect of the flavonoids on the metabolic activation of Trp-P-2 to N-hydroxy-Trp-P-2 was almost in parallel with the antimutagenic IC50 value, when determined with a Saccharomyces cerevisiae AH22 cell simultaneously expressing both rat cytochrome P450 1A1 and yeast reductase . The Ki values of flavones and flavonols for the enzyme were less than 1 microM, while the K(m) value of Trp-P-2 was 25 microM . The antimutagenicity of the flavones and flavonols was thus concluded to be due to inhibition of the activation process of Trp-P-2 by P450 1A1 to the ultimate carcinogenic form . They were also able to act as antimutagens toward other indirect mutagens that were activated by P450 1A1.

Appl Environ Microbiol, 1998 Jul, 64(7), 2697 - 700
Effectiveness of SYTOX Green stain for bacterial viability assessment; Lebaron P et al.; The effectiveness of SYTOX Green nucleic acid stain for measuring bacterial viability was tested on starved populations of Escherichia coli and Salmonella typhimurium . This stain underestimates the fraction of dead cells within starved populations containing cells with damaged nucleic acids or membranes . Its application to natural samples should be considered with caution.

Avian Dis, 1998 Apr-Jun, 42(2), 257 - 64
Comparison of Salmonella typhimurium challenge models in chickens; Muir WI et al.; Experimental infection of chickens with controlled quantities of Salmonella typhimurium is often achieved by administration of a single oral inoculum of live bacteria to caged chickens . However, this method is a poor simulation of the natural process of S . typhimurium infection in the field, making the practical application of results obtained under such conditions tenuous . This experiment was designed to evaluate the use of horizontal transmission for the challenge/infection of chickens with S . typhimurium with the expectation that it may more closely resemble the natural situation and, therefore, the in-field physiological response of chickens . Further, the experiment allowed for comparison of both the kinetics and magnitude of the mucosal immune response following each mode of challenge by exposing the chickens to challenge by placing them on litter with S . typhimurium-infected seeder birds . Overall, birds challenged via seeded litter exhibited a slower rate of infection and a more gradual increase in serum antibody production compared with birds receiving a single oral inoculum.

J Toxicol Sci, 1998 May, 23(2), 113 - 9
Formation of 8-oxodeoxyguanosine in liver DNA and hepatic injury by peroxisome proliferator clofibrate and perfluorodecanoic acid in rats; Kim SC et al.; In this study, we examined whether the production of hydrogen peroxide by peroxisome proliferators causes oxidative DNA damage in the form of 8-oxodeoxyguanosine (8-oxodG) and hepatic injury, and whether it is related to their tumor-promoting or carcinogenic activities in female rats treated with the peroxisome proliferators clofibrate and perfluorodecanoic acid (PFDA) . Clofibrate has tumor-promoting and carcinogenic activities, whereas PFDA does not . We also tested whether peroxisome proliferators directly induce mutagenic events in Salmonella typhimurium strains TA 98 and TA 1537 . Rats were treated either by 5% clofibrate in diet or by an i.p . injection of corn oil containing 10 mg/kg body weight of PFDA every week for 2 or 8 weeks . 8-OxodG in liver DNA was analyzed by HPLC coupled with an electrochemical detector . Hepatic injury was evidenced by liver enlargement and by levels of serum enzymes, aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and hepatic gamma-glutamylpeptidase (gamma-GT) activity . Clofibrate and PFDA increased the activity of catalase about or less than 2-fold, whereas FAO activity was increased about 6 to 7-fold by clofibrate and about 3 to 4-fold by PFDA . Neither clofibrate nor PFDA induced mutation at any dose tested . Clofibrate significantly increased the formation of 8-oxodG, but PFDA only slightly increased . Serum AST and ALT levels, and hepatic gamma-GT activity were not significantly changed at both time points, whereas the ratio of liver/body weight was significantly increased by clofibrate and PFDA at 8 weeks . These data imply that the magnitude of the production of hydrogen peroxide-generated FAO is related to the induction of oxidative DNA damage by peroxisome proliferators, and their tumor-promoting or carcinogenic activities . However, the effect of hydrogen peroxide in hepatic injury is not clear.

J Bacteriol, 1998 Jul, 180(13), 3295 - 303
Transcriptional analysis of essential genes of the Escherichia coli fatty acid biosynthesis gene cluster by functional replacement with the analogous Salmonella typhimurium gene cluster; Zhang Y et al.; The genes encoding several key fatty acid biosynthetic enzymes (called the fab cluster) are clustered in the order plsX-fabH-fabD-fabG-acpP-fabF at min 24 of the Escherichia coli chromosome . A difficulty in analysis of the fab cluster by the polar allele duplication approach (Y . Zhang and J . E . Cronan, Jr., J . Bacteriol . 178:3614-3620, 1996) is that several of these genes are essential for the growth of E . coli . We overcame this complication by use of the fab gene cluster of Salmonella typhimurium, a close relative of E . coli, to provide functions necessary for growth . The S . typhimurium fab cluster was isolated by complementation of an E . coli fabD mutant and was found to encode proteins with > 94% homology to those of E . coli . However, the S . typhimurium sequences cannot recombine with the E . coli sequences required to direct polar allele duplication via homologous recombination . Using this approach, we found that although approximately 60% of the plsX transcripts initiate at promoters located far upstream and include the upstream rpmF ribosomal protein gene, a promoter located upstream of the plsX coding sequence (probably within the upstream gene, rpmF) is sufficient for normal growth . We have also found that the fabG gene is obligatorily cotranscribed with upstream genes . Insertion of a transcription terminator cassette (omega-Cm cassette) between the fabD and fabG genes of the E . coli chromosome abolished fabG transcription and blocked cell growth, thus providing the first indication that fabG is an essential gene . Insertion of the omega-Cm cassette between fabH and fabD caused greatly decreased transcription of the fabD and fabG genes and slower cellular growth, indicating that fabD has only a weak promoter(s).

J Bacteriol, 1998 Jul, 180(13), 3393 - 9
Identification of a specific chaperone for SptP, a substrate of the centisome 63 type III secretion system of Salmonella typhimurium; Fu Y et al.; Salmonella typhimurium uses of a type III protein secretion system encoded at centisome 63 of its chromosome to deliver effector molecule into the host cell . These proteins stimulate host cell responses such as reorganization of the actin cytoskeleton and activation of transcription factors . One of these effector proteins is SptP, a tyrosine phosphatase that causes disruption of the host cell actin cytoskeleton . A characteristic feature of many substrates of type III secretion systems is their association with specific cytoplasmic chaperones which appears to be required for secretion and/or translocation of these proteins into the host cell . We report here the identification of SicP, a 13-kDa acidic polypeptide that is encoded immediately upstream of sptP . A loss-of-function mutation in sicP resulted in drastically reduced levels of SptP but did not affect sptP expression, indicating that SicP exerts its effect posttranscriptionally . Pulse-chase experiments demonstrated that the loss of SicP leads to increased degradation of SptP . In addition, we show that SicP binds to SptP directly and that the binding site is located between residues 15 and 100 of the tyrosine phosphatase . Taken together, these results indicate that SicP acts as a specific chaperone for SptP.

Wei Sheng Wu Xue Bao, 1996 Aug, 36(4), 263 - 8
{Cloning and identification of fliCi gene of Salmonella typhimurium}; Cai X et al.; Purified Salmonella typhimurium 8705 chromosomal DNA was digested with EcoRI . DNA fragments above 400 bp were obtained by Sepharcyl S-400 chromatography and cloned into vector pGEM-3Zf(-), then transformed into host cell LC2a (hag- is recA-) . One out of 6013 transformants was found to be motile and this clone was named pGI4015 . Miniprep proved that pGI4015 contained an inserted fragment about 15.3 kb . Motility/inhibition tests as well as Southern blot hybridization showed that pGI4015 bear the H-1i gene of S . typhimurium . With the aid of BamHI and SalI site, sequences unrelated to flagellin in the cloned DNA fragment was removed, and a 3.8kb fragment containing fliC gene was subcloned.

Wei Sheng Wu Xue Bao, 1996 Oct, 36(5), 398 - 400
{Regulation of purine biosynthetic genes expression in Salmonella typhimurium . V . Nucleotide sequences evidence without purJ gene}; Huang Y et al.; Previous genetic analysis showed that AICAI transformylase, IMP cyclohydrolase and GAR synthetase are encoded by purJ, purH and purD respectively, and which constitute a operon, mapped on 90 min in genetic map of Salmonella typhimurium But recent study in E . coli indicated that the genes encoding for above three enzymes only have purH and purD, without purJ gene . Report here is the DNA sequences evidence for abence of purJ gene in Salmonella typhimurium.

Wei Sheng Wu Xue Bao, 1996 Jun, 36(3), 234 - 6
{Immunogenicity of recombinant S . typhimurium ex . pressing a hybrid antigen of Plasmodium falciparum}; Huang J et al.; We have expressed a 74-peptide hybrid Plasmodium falciparum antigen as fusion protein in attenuated Salmonella typhimurium SL3261 . Live organisms were orally immunized Rabbits with a dose of 2 x 10(9)cfu . Specific anti-serum were detected by ELISA after immunization . Obvious Delayed type hypersensitivity (DTH) could be induced by PfAg and GZ-C antigen . The recombinant vaccine had no evident side-effects to the hosts . Our studies indicate that attenuated Salmonella typhimurium SL3261 can express synthetic P . falciparum antigen with several epitopes and live organisms can activate special cell-mediated immunity and humoral immunity.

Mutat Res, 1998 Mar 16, 413(2), 129 - 42
In vitro antimutagenic and in vivo anticlastogenic effects of carotenoids and solvent extracts from fruits and vegetables rich in carotenoids; Rauscher R et al.; The water insoluble residues of some carotenoid-rich fruits and vegetables, such as apricots, oranges, brussels sprouts, carrots, yellow-red peppers, and tomatoes, were sequentially extracted with n-hexane, dichloromethane, acetone, and 2-propanol, and solvent extracted materials were tested for inhibition of mutagenicities induced by aflatoxin B1 (AFB1), benzo{a}pyrene (BaP), 2-amino-3-methylimidazo{4,5-f}quinoline (IQ), and cyclophosphamide (CP) in histidine-deficient strains of Salmonella typhimurium . Antimutagenic activities were found in many extracts, but especially in the n-hexane extracts . For example, in the case of oranges, 100 microg of this extract reduced the bacterial mutagenicity of AFB1, BaP, CP and IQ by 72, 67, 53, and 27%, respectively . Separation by semi-preparative HPLC of the n-hexane extracts of carrots, tomatoes, and oranges indicated that the antimutagenicity was mainly associated with the fractions of the hydrocarbon carotenoids (alpha-, beta-carotene, lycopene), the xanthophylls (beta-cryptoxanthin, lutein), and also the carotenolesters (oranges) . When 16 reference carotenoids were investigated as described above, the following results were obtained: In the case of BaP, antimutagenic activity, quantified by dose-response curves, was exhibited by 8'-apo-beta-carotenal, alpha- and beta-carotene, beta-cryptoxanthin, lutein, retinal, and retinol (ID50-values: 20-100 nmol ml-1 top agar, 50-70% maximum inhibition at 1 micromol ml-1 top agar), while the maximum inhibition by torularhodin did not exceed 40% . Astaxanthin, 10'- and 12'-apo-beta-carotenal, bixin, canthaxanthin, ethyl-8'-apo-beta-caro-ten-8'-oate, lycopene, and zeaxanthin were inactive or at best marginally active (<20% inhibition) . Closely similar results were obtained with AFB1 . The bacterial mutagenicity of CP was strongly reduced by alpha- and beta-carotene, canthaxanthin, and retinol (ID50-values: 67-112 nmol ml-1 top agar, 50-63% maximum inhibition at 1 micromol ml-1 top agar), moderately by beta-cryptoxanthin, and lutein (45% and 28%, respectively), and only marginally or, not at all, by all remaining carotenoids . In the case of IQ, the carotenoids exhibited the weakest antimutagenic potency (7-43%, ID50-values of retinal and retinol: 160 and 189 nmol ml-1 top agar, 60% and 55% inhibition, respectively) . The mutagenic activity of the proximal mutagen of IQ, N-OH-IQ, in S . typhimurium TA 98NR was not significantly reduced by any carotenoid tested . These observations as well as the inhibition of various cytochrome P-450 linked 7-alkoxyresorufin-O-dealkylase activities (EROD, MROD, PROD) by four selected carotenoids (retinol>beta-cryptoxanthin>beta-carotene>lutein, IC50-values: 19-109 microM), indicate that the inhibition of the metabolic activation of the different promutagens could cause antimutagenicity . Finally, it could be demonstrated that the number of BaP or CP induced micronuclei in polychromatic erythrocytes in bone-marrow of mice was reduced significantly by the carotenoids lycopene, canthaxanthin, lutein and beta-cryptoxanthin (25-46%) . These results clearly show that carotenoids possess biological activities in vitro and in vivo distinct from their function as precursors of vitamin A or antioxidants suggesting effects on activation of promutagens .

Arch Biochem Biophys, 1998 Jun 15, 354(2), 281 - 7
The PutA protein of Salmonella typhimurium catalyzes the two steps of proline degradation via a leaky channel; Surber MW et al.; Proline utilization in Salmonella typhimurium requires two proteins encoded by the put operon: PutP, the major proline permease, and PutA . PutA is a multifunctional, peripheral membrane protein which acts both as a transcriptional repressor for the put operon and enzyme catalyzing the two-step conversion of proline to glutamate . In the first enzymatic reaction catalyzed by PutA, proline oxidation to pyrroline-5-carboxylate (P5C) is coupled with the reduction of a tightly associated FAD . In the second reaction, P5C oxidation to glutamate is coupled with reduction of soluble NAD . Although PutA can use exogenous P5C, the concentration of exogenous P5C required for the P5C dehydrogenase reaction is much greater than the steady-state P5C concentration accumulated during proline degradation . Furthermore, exogenous P5C does not efficiently compete against endogenous P5C for the production of glutamate, and the endogenous P5C produced directly from proline is preferentially used by PutA for the production of glutamate . Kinetic assays indicate that in the presence of NAD the two enzymatic reactions of PutA function synchronously to increase the overall reaction rate over that of the two independent reactions, and the second reaction proceeds in the absence of a lag phase . These results indicate that PutA directly transfers the intermediate P5C between the two enzymatic functions via a "leaky channel" mechanism . Because both the reduction of FAD and the intermediate P5C stimulate membrane association of PutA, channeling of P5C may also contribute to the regulation of proline utilization .

Infect Immun, 1998 Jul, 66(7), 3378 - 83
Identification of three highly attenuated Salmonella typhimurium mutants that are more immunogenic and protective in mice than a prototypical aroA mutant; Valentine PJ et al.; A panel of Salmonella typhimurium 14028s mutants, which were previously shown to be highly attenuated in the BALB/c mouse model of infection, were analyzed for their potential as live Salmonella oral-vaccine candidates . A prototypical aroA mutant was chosen as a basis of comparison . From the panel of mutants initially chosen for this study, three mutants with comparable levels of attenuation elicited higher Salmonella-specific serum immunoglobulin G (IgG) and/or mucosal secretory-IgA antibody titers than the aroA vaccine strain . The three mutants, CL288, CL401, and CL554, also elicited a better protective immune response than the aroA control strain, after a single oral dose of 1 x 10(9) to 2 x 10(9) bacteria.

Infect Immun, 1998 Jul, 66(7), 3372 - 7
At least four percent of the Salmonella typhimurium genome is required for fatal infection of mice; Bowe F et al.; Salmonella typhimurium infection of mice is an established model system for studying typhoid fever in humans . Using this model, we identified S . typhimurium genes which are absolutely required to cause fatal murine infection by testing independently derived transposon insertion mutants for loss of virulence in vivo . Of the 330 mutants tested intraperitoneally and the 197 mutants tested intragastrically, 12 mutants with 50% lethal doses greater than 1, 000 times that of the parental strain were identified . These attenuated mutants were characterized by in vitro assays which correlate with known virulence functions . In addition, the corresponding transposon insertions were mapped within the S . typhimurium genome and the nucleotide sequence of the transposon-flanking DNA was obtained . Salmonella spp . and related bacteria were probed with flanking DNA for the presence of these genes . All 12 attenuated mutants had insertions in known genes, although the attenuating effects of only two of these were previously described . Furthermore, the proportion of attenuated mutants obtained in this study suggests that mutations in about 4% of the Salmonella genome lead to 1,000-fold or greater attenuation in the mouse typhoid model of infection . Most of these genes appear to be required during the early stages of a natural infection.

Infect Immun, 1998 Jul, 66(7), 3355 - 64
Susceptibility to Salmonella typhimurium infection and effectiveness of vaccination in mice deficient in the tumor necrosis factor alpha p55 receptor; Everest P et al.; Mice defective in the ability to produce the tumor necrosis factor alpha p55 receptor (TNFalphap55R) were orally challenged with a number of Salmonella typhimurium HWSH derivatives that differ in virulence . In comparison to TNFalphap55R+/+ mice, TNFalphap55R-/- mice succumbed earlier to challenge with wild-type S . typhimurium HWSH and S . typhimurium HWSH purE . In contrast, TNFalphap55R-/- mice were able to control an S . typhimurium HWSH aroA challenge, although greater numbers of Salmonella organisms were present in the tissues for a longer time period than was observed with TNFalphap55R+/+ mice . Vaccination of normal and TNFalphap55R knockout animals with S . typhimurium HWSH aroA showed that TNFalphap55R-/- mice, unlike TNFalphap55R+/+ mice, were not protected against a virulent S . typhimurium HWSH challenge . Splenocytes from TNFalphap55R-/- mice exhibited a reduced ability to proliferate in the presence of S . typhimurium antigen compared to TNFalphap55R+/+ mice . Thus, TNFalphap55R is essential for controlling Salmonella growth in tissues and for recall of immunity in murine salmonellosis.

Infect Immun, 1998 Jul, 66(7), 3208 - 17
The Salmonella typhimurium AhpC polypeptide is not essential for virulence in BALB/c mice but is recognized as an antigen during infection; Taylor PD et al.; The OxyR regulon is known to mediate protection against oxidizing agents in Salmonella typhimurium . We reported previously that ahp, one of the OxyR-regulated loci, is induced during macrophage interaction (K . P . Francis, P . D . Taylor, C . J . Inchley, and M . P . Gallagher, J . Bacteriol . 179:4046-4048, 1997) . We now report on the effects of disrupting ahp or oxyR on virulence in a BALB/c mouse model . Surprisingly, insertion of a Mudlux derivative within ahpC was found to result in attenuation, while irreversible inactivation of the locus through insertion of a cml cassette did not . An SL1344 derivative carrying an oxyR::kan disruption was also found to be as virulent as the parental strain . Moreover, both cell-mediated and humoral responses to AhpC were found to develop during the course of infection, probably through T-helper-cell (type I) activation . These results indicate that, although not essential for virulence, AhpC is expressed by S . typhimurium during infection of BALB/c mice and constitutes a target for the immune system.

Infect Immun, 1998 Jul, 66(7), 3080 - 7
Oral vaccination against tetanus: comparison of the immunogenicities of Salmonella strains expressing fragment C from the nirB and htrA promoters; Roberts M et al.; We have found the in vivo-regulated nirB promoter (PnirB) to be effective for directing expression of a number of antigens in salmonella in vivo . We wished to determine if other in vivo-regulated promoters have utility for antigen expression in salmonella and to compare the effectiveness of these promoters with that of PnirB . To this end, we have devised a scheme that allows the promoter element of the PnirB-fragment C plasmid pTETnir15 to be swapped with other promoters of interest . We demonstrate the usefulness of this system by replacing PnirB with PhtrA to create plasmid pTEThtrA1 . htrA is a stress response gene that is required for virulence of salmonella in mice and survival within macrophages . Expression of fragment C in Salmonella typhimurium BRD509 (aroA aroD) harboring pTEThtrA1 (strain BRD937) correlated with growth temperature in vitro . A comparison was made of the immune responses to fragment C elicited in mice immunized orally with BRD937 or BRD847 (BRD509/pTETnir15) or subcutaneously with purified fragment C plus alhydrogel . High levels of anti-fragment C antibodies that persisted for at least 12 weeks were present in all groups of mice . Vaccination with BRD937 was the most effective means of immunization: the serum immunoglobulin G (IgG), IgA, and IgM anti-fragment C titers were higher in the BRD937-immunized mice throughout the duration of the study than in mice in the other groups . The kinetics of the serum anti-fragment C responses were different in different groups . The response was most rapid in the BRD937 group, with the titers almost at peak levels at 2 weeks postimmunization . Only the mice immunized with BRD937 or BRD847 developed an intestinal IgA response to fragment C . Again, the response was superior in the BRD937 group . The peak of the intestinal response was delayed with respect to the serum response . Analysis of the IgG subtype response to fragment C revealed a dominant IgG2a response in the salmonella-immunized mice, indicating a type 1 helper T-cell response to fragment C, whereas the major subtype in the group parenterally immunized with fragment C plus alhydrogel was IgG1 . The IgG1/IgG2a ratio was much higher in sera of BRD937-immunized mice than in sera of BRD847-immunized mice . At 15 to 20 weeks after immunization, the mice immunized with BRD937 or BRD847 were solidly immune to tetanus toxin and salmonella . The immune responses to fragment C seen in mice immunized with BRD937 are the strongest we have observed and indicate that the htrA promoter may be very useful for expressing foreign antigens in salmonella vaccine strains.

J Appl Microbiol, 1998 Apr, 84(4), 577 - 84
Improved detection of bacteria by flow cytometry using a combination of antibody and viability markers; Clarke RG et al.; A proprietary fluorogenic marker for cell viability (Chemchrome) was investigated for the detection of bacteria using flow cytometry . This marker was used in combination with fluorescently labelled monoclonal antibodies (against Salmonella typhimurium) . Owing to the former's broad band emission spectrum, it was necessary to use the novel dye RED613 for the antibodies . This combined protocol, being sensitive only to the live Salm . typhimurium cells, reduced errors due to intrinsic fluorescence and non-specific binding . Detection of the order of 100 cells ml-1 was achieved in 30 min . This level was achieved even in the presence of large numbers of non-target or dead organisms.

J Appl Microbiol, 1998 Apr, 84(4), 478 - 84
Radiation inactivation of some food-borne pathogens in fish as influenced by fat levels; Kamat A et al.; The influence of low (0.39-1.1%), medium (4.25%) and high (7.1-32.5%) fat levels in fish on radiation inactivation of four food-borne pathogens was investigated . Cells of Listeria monocytogenes 036, Yersinia enterocolitica F5692, Bacillus cereus and Salmonella typhimurium at logarithmic phase were inoculated in 10% fish homogenates and subjected to gamma irradiation at ice temperature (0-1 degree C) with doses ranging from 0.05 to 0.8 kGy . The radiation survival curves of L . monocytogenes and B . cereus were characterized by shoulders, while a tailing effect was depicted by cells of Y . enterocolitica and B . cereus . The D10 values in kGy calculated on the exponential part of the curve ranged from 0.2 to 0.3, 0.15 to 0.25, 0.1 to 0.15 and 0.09 to 0.1 for L . monocytogenes 036, B . cereus, Salm . typhimurium and Y . enterocolitica F5692, respectively . This order (D10) of radiation resistance of each organism was not affected by the fat content of the fish . Inoculated pack studies carried out separately with each pathogen in fatty (Indian sardine, 7.1%) and lean (Golden anchovy, 0.39%) fish showed no difference in their survival after exposure to 1 kGy and 3 kGy doses, which corroborated the above observation . The practical significance of these results in the application of the technology is discussed.

Biochemistry, 1998 May 26, 37(21), 7686 - 95
Three-dimensional structure of adenosylcobinamide kinase/adenosylcobinamide phosphate guanylyltransferase from Salmonella typhimurium determined to 2.3 A resolution,; Thompson TB et al.; The X-ray structure of adenosylcobinamide kinase/adenosylcobinamide phosphate guanylyltransferase (CobU) from Salmonella typhimurium has been determined to 2.3 A resolution . This enzyme of subunit molecular weight 19 770 plays a central role in the assembly of the nucleotide loop for adenosylcobalamin where it catalyzes both the phosphorylation of the 1-amino-2-propanol side chain of the corrin ring and the subsequent attachment of GMP to form the product adenosylcobinamide-GDP . The kinase activity is believed to be associated with a P-loop motif, whereas the transferase activity proceeds at a different site on the enzyme via a guanylyl intermediate . The enzyme was crystallized in the space group C2221 with unit cell dimensions of a = 96.4 A, b = 114.4 A, and c = 106.7 A, with three subunits per asymmetric unit . The structure reveals that the enzyme is a molecular trimer and appears somewhat like a propeller with overall molecular dimensions of approximately 64 A x 77 A x 131 A . Each subunit consists of a single domain that is dominated by a seven-stranded mixed beta-sheet flanked on either side by a total of five alpha-helices and one helical turn . Six of the seven beta-strands run parallel . The C-terminal strand lies at the edge of the sheet and runs antiparallel to the others . Interestingly, CobU displays a remarkable structural and topological similarity to the central domain of the RecA protein, although the reason for this observation is unclear . The structure contains a P-loop motif located at the base of a prominent cleft formed by the association of two subunits and is most likely the kinase active site . Each subunit of CobU contains a cis peptide bond between Glu80 and Cys81 where Glu80 faces the P-loop and might serve to coordinate the magnesium ion of the triphosphate substrate . Interestingly, His46, which is the putative site for guanylylation, lies approximately 21 A from the P-loop and is solvent-exposed . This suggests that the enzyme undergoes a conformational change when the substrates bind to bring these two active sites into closer proximity.

J Mol Biol, 1998 May 8, 278(3), 507 - 14
The N terminus of the flagellar switch protein, FliM, is the binding domain for the chemotactic response regulator, CheY; Bren A et al.; A key event in signal transduction during chemotaxis of Salmonella typhimurium and related bacterial species is the interaction between the phosphorylated form of the response regulator CheY (CheY approximately P) and the switch of the flagellar motor, located at its base . The consequence of this interaction is a shift in the direction of flagellar rotation from the default, counterclockwise, to clockwise . The docking site of CheY approximately P at the switch is the protein FliM . The purpose of this study was to identify the CheY-binding domain of FliM . We cloned 17 fliM mutants, each defective in switching and having a point mutation at a different location, and then overexpressed and purified their products . The CheY-binding ability of each of the FliM mutant proteins was determined by chemical crosslinking . All the mutant proteins with an amino acid substitution at the N terminus, FliM6LI, FliM7SY and FliM10EG, bound CheY approximately P to a much lesser extent than did wild-type FliM . CheY approximately P-binding of the other mutant proteins was similar to wild-type FliM . To investigate whether the FliM domain that includes these three mutations is indeed the CheY-binding domain, we synthesized a peptide composed of the first 16 amino acid residues of FliM, including a highly conserved region of FliM (residues 6 to 15) . The peptide bound CheY and, to a larger extent, CheY approximately P . It also competed with full-length FliM on CheY approximately P . These results indicate that the CheY-binding domain of FliM is located at the N terminus, within residues 1 to 16, and suggest that FliM monomers can form a complete site for CheY binding .

J Biol Chem, 1998 May 15, 273(20), 12543 - 7
Role for the Salmonella flavohemoglobin in protection from nitric oxide; Crawford MJ et al.; Hemoglobin homologs are being identified in an expanding number of unicellular prokaryotic and eukaryotic organisms . Many of these hemoglobins are twodomain proteins that possess a flavin-containing reductase in their C terminus . Determination of a function for these flavohemoglobins has been elusive . A Salmonella typhimurium strain harboring a deletion in the flavohemoglobin gene shows no difference in growth under oxidative stress conditions but displays an increased sensitivity to acidified nitrite and S-nitrosothiols, both of which produce nitric oxide . The effect is seen aerobically or anaerobically, indicating that oxygen is not required for flavohemoglobin function . These results suggest a role for the bacterial flavohemoglobins that is independent of oxygen metabolism and provide evidence for a bacterial route of protection from nitric oxide that is distinct from oxidative stress responses.

Nat Biotechnol, 1996 Jun, 14(6), 765 - 9
An Escherichia coli hemolysin transport system-based vector for the export of polypeptides: export of Shiga-like toxin IIeB subunit by Salmonella typhimurium aroA; Tzschaschel BD et al.; The export of Escherichia coli hemolysin across the cytoplasmic and the outer membranes requires the COOH-terminal signal sequence of HlyA, the two specific translocator proteins HlyB and HlyD, and the outer membrane protein TolC . We have developed an export cloning system that is composed of two vectors: one in which the fusion of the desired gene with the 3'-end of hlyA is generated, and a second in which the sequences containing the fusion are combined with the accessory genes hlyB and hlyD, thereby reconstructing the natural organization of the hly locus . In the second vector the fusion and the accessory genes are flanked by Notl sites, allowing subcloning of the whole cluster into a variety of minitransposons to achieve the stable integration of the constructs into the chromosome of Gram-negative bacteria . Since some applications may require the production of transcriptional fusions, an alternative version of the system provides the efficient translation initiation region of T7 phage gene 10 upstream of the fusion protein coding sequence . The usefulness of the system was assessed by constructing a fusion between the gene encoding the B subunit of Shiga-like toxin lle and the 3'-end of hlyA . An attenuated Salmonella typhimurium vaccine strain harboring the resulting construct, either in multicopy or monocopy, efficiently expressed and exported the chimeric protein . We anticipate that this system will lead to a higher stability of the engineered function and permit a faithful monitoring of the export of the recombinant peptide under physiologic single-copy conditions.

Environ Res, 1998 Jul, 78(1), 43 - 9
Correlation between the amounts of polycyclic aromatic hydrocarbons and mutagenicity of airborne particulate samples from Taichung City, Taiwan; Kuo CY et al.; Taichung is the largest city in the central part of Taiwan, and its air pollution problems are similar to those in other large cities around the world . To evaluate the potential of the air pollution and identify major pollutant sources in this city, 181 airborne particulate samples were collected biweekly from seven locations around Taichung over an entire year . The mutagenicity of acetone extracts of the air samples was evaluated using the Salmonella/microsomal test with Salmonella typhimurium TA98 in the presence and absence of S9 mixtures . The air samples from September 1994 showed the highest direct and indirect mutagenicity among the 12 months, whereas those from October and June had the lowest direct and indirect mutagenicity, respectively . To elucidate the correlation between mutagenicity and polycyclic aromatic hydrocarbons (PAHs), high-performance liquid chromatography was used to determine the amount of each of 10 PAHs in the air samples . Among the 10 PAHs, the monthly average amount of B{g,h,i}P in the samples was the highest, followed by B{a}FA, B{a}P, and B{k}FA . Linear regression analysis showed a positive correlation between monthly average total amounts of PAHs and indirect mutagenicity . The monthly average amount of B{g,h,i}P was correlated more with indirect mutagenicity than with other PAHs . B{g,h,i}P is an indicator PAH emitted from both diesel and gasoline engine exhaust . Thus, we suggest that mobile air pollutant sources in Taichung City may be more significant than stationary ones . Moreover, B{g,h,i}P seems to act as a mutagenicity indicator compound in air samples from Taichung City.

Arch Microbiol, 1998 Jun, 169(6), 530 - 3
A temperature-sensitive DNA adenine methyltransferase mutant of Salmonella typhimurium; Brawer R et al.; A temperature-sensitive mutant of Salmonella typhimurium was isolated earlier after transposon mutagenesis with Tn10d Tet . The mutant D220 grows well at 28 degreesC but has a lower growth rate and forms filaments at 37 degreesC . Transposon-flanking fragments of mutant D220 DNA were cloned and sequenced . The transposon was inserted in the dam gene between positions 803 and 804 (assigned allele number: dam-231 : : Tn10d Tet) and resulted in a predicted ten-amino-acid-shorter Dam protein . The insertion created a stop codon that led to a truncated Dam protein with a temperature-sensitive phenotype . The insertion dam-231 : : Tn10d Tet resulted in a dam "leaky" phenotype since methylated and unmethylated adenines in GATC sequences were present . In addition, the dam-231 : : Tn10d Tet insertion rendered dam mutants temperature-sensitive for growth depending upon the genetic background of the S . typhimurium strain . The wild-type dam gene of S . typhimurium exhibited 82% identity with the Escherichia coli dam gene.

DNA Res, 1998 Feb 28, 5(1), 1 - 9
Complete nucleotide sequences of 93-kb and 3.3-kb plasmids of an enterohemorrhagic Escherichia coli O157:H7 derived from Sakai outbreak; Makino K et al.; Enterohemorrhagic Escherichia coli (EHEC) O157:H7, derived from an outbreak in Sakai city, Japan in 1996, possesses two kinds of plasmids: a 93-kb plasmid termed pO157, found in clinical EHEC isolates world-wide and a 3.3-kb plasmid termed pOSAK1, prevalent in EHEC strains isolated in Japan . Complete nucleotide sequences of both plasmids have been determined, and the putative functions of the encoded proteins and the cis-acting DNA sequences have been analyzed . pO157 shares strikingly similar genes and DNA sequences with F-factor and the transmissible drug-resistant plasmid R100 for DNA replication, copy number control, plasmid segregation, conjugative functions and stable maintenance in the host, although it is defective in DNA transfer by conjugation due to the truncation and deletion of the required genes and DNA sequences . In addition, it encodes several proteins implicated in EHEC pathogenicity such as an EHEC hemolysin (HlyA), a catalase-peroxidase (KatP), a serine protease (EspP) and type II secretion system . pOSAK1 possesses a ColE1-like replication system, and the DNA sequence is extremely similar to that of a drug-resistant plasmid, NTP16, derived from Salmonella typhimurium except that it lacks drug resistance transposons.

Nat Struct Biol, 1998 Jun, 5(6), 446 - 50
Chemotaxis receptor recognition by protein methyltransferase CheR; Djordjevic S et al.; Signal transduction processes commonly involve reversible covalent modifications of receptors . Bacterial chemotaxis receptors are reversibly methylated at specific glutamate residues within coiled-coil regions of their cytoplasmic domains . Methylation is catalyzed by an S-adenosylmethionine-dependent protein methyltransferase, CheR, that binds to a specific sequence at the C-termini of some chemotaxis receptors . From this tethering point, CheR methylates neighboring receptor molecules . We report the crystal structure, determined to 2.2 A resolution, of a complex of the Salmonella typhimurium methyltransferase CheR bound to the methylation reaction product, S-adenosylhomocysteine (AdoHcy), and the C-terminal pentapeptide of the aspartate receptor, Tar . The structure indicates the basis for the specificity of interaction between the chemoreceptors and CheR and identifies a specific receptor binding motif incorporated in the CheR methyltransferase domain.

Plant J, 1998 Apr, 14(2), 203 - 13
Cloning and functional expression of AtCOQ3, the Arabidopsis homologue of the yeast COQ3 gene, encoding a methyltransferase from plant mitochondria involved in ubiquinone biosynthesis; Avelange-Macherel MH et al.; A mutant of Saccharomyces cerevisiae deleted for the COQ3 gene was constructed . COQ3 encodes a 3,4-dihydroxy-5-hexaprenylbenzoate (DHHB) methyltransferase that catalyses the fourth step in the biosynthesis of ubiquinone from p-hydroxybenzoic acid . A full length cDNA encoding a homologue of DHHB-methyltransferase was cloned from an Arabidopsis thaliana cDNA library by functional complementation of a yeast coq3 deletion mutant . The Arabidopsis thaliana cDNA (AtCOQ3) was able to restore the respiration ability and ubiquinone synthesis of the mutant . The product of the 1372 bp cDNA contained 322 amino acids and had a molecular mass of 35,360 Da . The predicted amino acid sequence contained all consensus regions for S-adenosyl methionine methyltransferases and presented 26% identity with Saccharomyces cerevisiae DHHB-methyltransferase and 38% identity with the rat protein, as well as with a bacterial (Escherichia coli and Salmonella typhimurium) methyltransferase encoded by the UBIG gene . Southern analysis showed that the Arabidopsis thaliana enzyme was encoded by a single nuclear gene . The NH2-terminal part of the cDNA product contained features consistent with a putative mitochondrial transit sequence . The cDNA in Escherichia coli was overexpressed and antibodies were raised against the recombinant protein . Western blot analysis of Arabidopsis thaliana and pea protein extracts indicated that the AtCOQ3 gene product is localized within mitochondrial membranes . This result suggests that at least this step of ubiquinone synthesis takes place in mitochondria.

Scand J Immunol, 1998 May, 47(5), 444 - 52
Adjuvant is the major parameter influencing the isotype profiles generated during immunization with a protein antigen, the Schistosoma mansoni Sm28-GST; Comoy EE et al.; We have previously shown that immunization of mice with the vaccine candidate, the 28-kDa glutathione-S-transferase of Schistosoma mansoni (Sm28-GST), in alum or complete Freund's adjuvant, or with recombinant Salmonella typhimurium expressing Sm28-GST, induced type 2, mixed, or type 1 immune responses, respectively . In the present study we examined whether the genetic background, the dose and the route of antigen administration could modulate the profile of the immune response induced during these immunizations . Our results show that the nature of the adjuvant is the major factor that determines the profile of the response . Surprisingly, the genetic background did not influence the response, while the route of immunization, and to a lesser extent the dose of the antigen, weakly modulated the adjuvant-dependent orientation of the immune response.

Mutat Res, 1998 Feb 26, 398(1-2), 33 - 42
Roles of the mutagenesis proteins SamA'B and MucA'B in chemically induced frameshift mutagenesis in Salmonella typhimurium hisD3052; Gruz P et al.; The mutagenesis induced by ultraviolet light and many chemicals in Escherichia coli is largely dependent upon the proteins encoded by the umuDC operon and their analogs . In Salmonella typhimurium, there are two sets of umuDC-like operons: the umuDC(ST) operon in the chromosome and the samAB operon located on the 60-MDa cryptic plasmid . The former operon, but not the latter, confers UV mutability on S . typhimurium . Nevertheless, the samAB operon, when carried on high-copy-number plasmids, can efficiently promote UV mutagenesis . In order to characterize the function of samAB in greater detail, we have compared the abilities of MucA'B and a putative activated form of SamAB, i.e . SamA'B, to promote chemically induced frameshift mutagenesis in S . typhimurium hisD3052 . MucAB is an activated form of the products of mucAB, which is the most potent umuDC analog characterized so far . We have used four plasmids, each carrying samA', samB, mucA' or mucB with a lac promoter instead of their own promoters . The results indicated that under the conditions of elevated expression, SamA'B can promote chemically induced frameshift mutagenesis by furylfuramide, aflatoxin B1, 1-nitropyrene, and 1,8-dinitropyrene, with efficiencies comparable to, or even better than, MucA'B . Increase of the levels of expression enhanced the ability of SamA'B to promote the mutagenesis, while it decreased that of MucA'B . Surprisingly, the elevated expression of MucB alone significantly enhanced the frameshift mutagenesis induced by 1-nitropyrene and 1,8-dinitropyrene, whereas the elevated expression of SamB, MucA' and SamA' did not enhance it . These results suggest that the abilities of SamA'B and MucA'B to promote mutagenesis strongly depend on their levels of expression . The possible roles of these mutagenesis proteins in chemically induced frameshift mutagenesis are discussed.

Ecotoxicol Environ Saf, 1998 May-Jun, 40(1-2), 126 - 36
Correlation between on-line PAH detection in airborne particle samples and their bacterial genotoxicity; Wasserkort R et al.; The photoelectric aerosol sensor (PAS) is a technique suitable for on-line monitoring of particle-bound polycyclic aromatic hydrocarbons (PPAHs) . Although this is a very fast and inexpensive technique, it does not measure individual PAH species but gives a measure of the total amount of PPAHs . Because of the suitability of this sensor for air-pollution screening, it is desirable to know whether a correlation exists between the PPAHs detected with this method and the biological relevance of the respective particle samples . To test the DNA damaging potential of the organic fraction of collected particles, the umuC test with Salmonella typhimurium TA1535/pSK1002 was used . The primary source for particle sampling was a stationary diesel engine, but samples from a parking garage and two locations in the city of Zurich have also been included . The total mass of PPAHs as determined by the PAS was plotted against the induced genotoxicity . This resulted in a linear correlation (r2 = 0.82), indicating that the PAS detects biologically relevant PPAHs.

Proc Natl Acad Sci U S A, 1998 Jun 9, 95(12), 7046 - 50
Quorum sensing in Escherichia coli and Salmonella typhimurium; Surette MG et al.; Escherichia coli and Salmonella typhimurium strains grown in Luria-Bertani medium containing glucose secrete a small soluble heat labile organic molecule that is involved in intercellular communication . The factor is not produced when the strains are grown in Luria-Bertani medium in the absence of glucose . Maximal secretion of the substance occurs in midexponential phase, and the extracellular activity is degraded as the glucose is depleted from the medium or by the onset of stationary phase . Destruction of the signaling molecule in stationary phase indicates that, in contrast to other quorum-sensing systems, quorum sensing in E . coli and S . typhimurium is critical for regulating behavior in the prestationary phase of growth . Our results further suggest that the signaling factor produced by E . coli and S . typhimurium is used to communicate both the cell density and the metabolic potential of the environment . Several laboratory and clinical strains of E . coli and S . typhimurium were screened for production of the signaling molecule, and most strains make it under conditions similar to those shown here for E . coli AB1157 and S . typhimurium LT2 . However, we also show that E . coli strain DH5alpha does not make the soluble factor, indicating that this highly domesticated strain has lost the gene(s) or biosynthetic machinery necessary to produce the signaling substance . Implications for the involvement of quorum sensing in pathogenesis are discussed.

Proc Natl Acad Sci U S A, 1998 Jun 9, 95(12), 7030 - 4
Ammonia acquisition in enteric bacteria: physiological role of the ammonium/methylammonium transport B (AmtB) protein; Soupene E et al.; Homologues of the amtB gene of enteric bacteria exist in all three domains of life . Although their products are required for transport of the ammonium analogue methylammonium in washed cells, only in Saccharomyces cerevisiae have they been shown to be necessary for growth at low NH4+ concentrations . We now demonstrate that an amtB strain of Escherichia coli also grows slowly at low NH4+ concentrations in batch culture, but only at pH values below 7 . In addition, we find that the growth defect of an S . cerevisiae triple-mutant strain lacking the function of three homologues of the ammonium/methylammonium transport B (AmtB) protein {called methylammonium/ammonium permeases (MEP)} that was observed at pH 6.1 is relieved at pH 7.1 . These results provide direct evidence that AmtB participates in acquisition of NH4+/NH3 in bacteria as well as eucarya . Because NH3 is the species limiting at low pH for a given total concentration of NH4+ + NH3, results with both organisms indicate that AmtB/MEP proteins function in acquisition of the uncharged form . We confirmed that accumulation of {14C}methylammonium depends on its conversion to gamma-N-methylglutamine, an energy-requiring reaction catalyzed by glutamine synthetase, and found that at pH 7, constitutive expression of AmtB did not relieve the growth defects of a mutant strain of Salmonella typhimurium that appears to require a high internal concentration of NH4+/NH3 . Hence, contrary to previous views, we propose that AmtB/MEP proteins increase the rate of equilibration of the uncharged species, NH3, across the cytoplasmic membrane rather than actively transporting-that is, concentrating-the charged species, NH4+.

J Clin Invest, 1998 May 1, 101(9), 1860 - 9
Pathogen-induced chemokine secretion from model intestinal epithelium is inhibited by lipoxin A4 analogs; Gewirtz AT et al.; Enteric pathogens induce intestinal epithelium to secrete chemokines that direct movement of polymorphonuclear leukocytes . Mechanisms that might downregulate secretion of these proinflammatory chemokines and thus contain intestinal inflammation have not yet been elucidated . The antiinflammatory activities exhibited by the arachidonate metabolite lipoxin A4 (LXA4) suggests that this eicosanoid, which is biosynthesized in vivo at sites of inflammation, might play such a role . We investigated whether chemokine secretion could be regulated by stable analogs of LXA4 . Monolayers of T84 intestinal epithelial cells were infected with Salmonella typhimurium, which elicits secretion of distinct apical (pathogen-elicited epithelial chemoattractant) and basolateral (IL-8) chemokines . Stable analogs of LXA4 inhibited S . typhimurium-induced (but not phorbol ester-induced) secretion of both IL-8 and pathogen-elicited epithelial chemoattractant . LXA4 stable analogs did not alter bacterial adherence to nor internalization by epithelia, indicating that LXA4 stable analogs did not block all signals that Salmonella typhimurium activates in intestinal epithelia, but likely led to attenuation of signals that mediate chemokine secretion . Inhibition of S . typhimurium-induced IL-8 secretion by LXA4 analogs was concentration- (IC50 approximately 1 nM) and time-dependent (maximal inhibition approximately 1 h) . As a result of these effects, LXA4 stable analogs inhibited the ability of bacteria-infected epithelia to direct polymorphonuclear leukocyte movement . These data suggest that LXA4 and its stable analogs may be useful in downregulating active inflammation at mucosal surfaces.

Mol Microbiol, 1998 Apr, 28(2), 217 - 26
Microbial magnesium transport: unusual transporters searching for identity; Smith RL et al.; Mg2+ is unique among biological cations because of its charge density and solution chemistry . This is abundantly reflected in its transport systems, studied primarily in Salmonella typhimurium . The constitutively expressed CorA transport system is the primary Mg2+ influx pathway for the Bacteria and the Archaea . Its structure of a large N-terminal soluble periplasmic domain with three transmembrane segments at the C-terminus is unique among membrane carriers, and its protein sequence bears no resemblance to other known proteins . The MgtE transport system can also mediate Mg2+ uptake, but whether this is its primary function is not known . MgtE also lacks homology to other known proteins . In contrast, the MgtA and MgtB Mg2+ transport systems of enteric bacteria are P-type ATPases by sequence homology, mediating Mg2+ influx with, rather than against, the Mg2+ electrochemical gradient . They are closely related to mammalian Ca2+-ATPases . Expression of MgtA and MgtB is under the control of the PhoPQ two-component regulatory system, important in bacterial virulence . In S . typhimurium, MgtB is encoded by a two-gene operon mgtCB; the function of the MgtC protein is unknown, and it lacks close homologues . The ligand for the PhoQ membrane sensor kinase is Mg2+ and, at decreased extracellular Mg2+ concentrations, transcription of mgtA and mgtCB are enormously induced . All three genes are also induced upon S . typhimurium invasion of epithelial or macrophage cells . Mutation of these genes has no effect on invasion efficiency, but an insertion in mgtC renders S . typhimurium essentially avirulent in the mouse . The physiological roles of the known Mg2+ transport systems are not yet completely defined . Nonetheless, the singular sequence and apparent structure of the CorA and MgtE transport proteins, the complex regulation of MgtA, MgtB and MgtC and their involvement in pathogenesis suggests that further study will be rewarding.

J Bacteriol, 1998 Jun, 180(12), 3144 - 51
The ms2io6A37 modification of tRNA in Salmonella typhimurium regulates growth on citric acid cycle intermediates; Persson BC et al.; The modified nucleoside 2-methylthio-N-6-isopentenyl adenosine (ms2i6A) is present in position 37 (adjacent to and 3' of the anticodon) of tRNAs that read codons beginning with U except tRNA(i.v . Ser) in Escherichia coli . In Salmonella typhimurium, 2-methylthio-N-6-(cis-hydroxy)isopentenyl adenosine (ms2io6A; also referred to as 2-methylthio cis-ribozeatin) is found in tRNA, most likely in the species that have ms2i6A in E . coli . Mutants (miaE) of S . typhimurium in which ms2i6A hydroxylation is blocked are unable to grow aerobically on the dicarboxylic acids of the citric acid cycle . Such mutants have normal uptake of dicarboxylic acids and functional enzymes of the citric acid cycle and the aerobic respiratory chain . The ability of S . typhimurium to grow on succinate, fumarate, and malate is dependent on the state of modification in position 37 of those tRNAs normally having ms2io6A37 and is not due to a second cellular function of tRNA (ms2io6A37)hydroxylase, the miaE gene product . We suggest that S . typhimurium senses the hydroxylation status of the isopentenyl group of the tRNA and will grow on succinate, fumarate, or malate only if the isopentenyl group is hydroxylated.

J Toxicol Sci, 1998 May, 23 Suppl 1, 81 - 90
{Mutagenicity studies of magnesium sulfate--reverse mutation test with bacteria and chromosomal aberration test with mammalian cells in culture}; Oguma Y et al.; The mutagenicity potential of magnesium sulfate was re-assessed using the current procedure of the reverse mutation test with bacteria and chromosomal aberration test with mammalian cells (a Chinese hamster lung fibroblast cell line; CHL/IU) in culture . In the reverse mutation test with bacteria, Salmonella typhimurium TA100, TA98, TA1535 and TA1537 and Escherichia coli WP2 uvrA were use and the maximum dose level was set at 5000 micrograms/plate irrespective of the absence or presence of metabolic activation . Five dose levels (313-5000 micrograms/plate) were selected for all strains except for TA98 without metabolic activation and for TA1537 with metabolic activation, for which 6 dose levels (156-5000 micrograms/plate) were selected . Magnesium sulfate induced no increase in the number of colonies with reverse mutation in any of the strains irrespective of the absence of presence of metabolic activation in the dose-range-finding study or in the main study . In the chromosomal aberration test with mammalian cells, a Chinese hamster lung fibroblast cell line (CHL/IU) in culture was used and the maximum dose level was set at 5.0 mg/mL both in the direct and metabolic activation methods . Three dose levels (1.25-5.0 mg/mL) were selected . Magnesium sulfate induced no increase in the incidence of cells with chromosomal aberration or those with genome mutation (polyploidy) in any of the strains irrespective of the absence of presence of metabolic activation . Thus, it is concluded that magnesium sulfate does not have mutagenic potential under the presence experimental conditions.

J Epidemiol Community Health, 1998 Apr, 52(4), 272 - 6
Use of sequential case-control studies to investigate a community Salmonella outbreak in Wales; Llewellyn LJ et al.; STUDY OBJECTIVE: To establish the source of a community outbreak of Salmonella typhimurium definitive type 124 . DESIGN: Two stage case-control study . SETTING: Three districts in south east Wales . SUBJECTS: Cases of salmonella food poisoning and community controls . MAIN RESULTS: An initial case-control study identified an association between illness and eating ham (odds ratio 4.50, 95% confidence intervals 1.10, 21.8) and also found a possible association between illness and food bought from delicatessen stores (odds ratio 5.03, 95% confidence intervals 1.01, 32.3) . However, only after a second stage case-control study was a single common ham producer identified as the source (odds ratio 25.0, 95% confidence intervals 2.33, 1155) . CONCLUSION: Sequential case-control studies are an important and underused tool in the investigation of community outbreaks.

Anticancer Res, 1998 Mar-Apr, 18(2A), 829 - 31
Salmonella mutagenicity of musk ambrette depends on both microsomal and bacterial enzyme activity; Mersch-Sundermann V et al.; Former studies revealed musk ambrette as a mutagen in Salmonella typhimurium TA 100 in the presence (+S9) but not in the absence (-S9) of an exogenous metabolizing system . To clarify the role of bacterial nitroreductases (NR) in the toxification of musk ambrette to mutagenic metabolites the compound was examined with the Salmonella/mammalian microsome assay using the NR deficient strain S.typhimurium TA 100 NR in the presence and absence of S9 . Musk ambrette showed mutagenicity in Salmonella typhimurium TA 100 (+S9) but no mutagenicity in the NR deficient strain TA 100 NR (+S9) . Additionally, no mutagenicity was detected in both TA 100 (-S9) and TA 100 NR (-S9) . These results indicate the need for both mammalian microsomal enzymes and bacterial nitroreductases to cause the mutagenicity of musk ambrette.

J Bacteriol, 1998 Jun, 180(11), 2936 - 42
Translation of the flagellar gene fliO of Salmonella typhimurium from putative tandem starts; Schoenhals GJ et al.; The flagellar gene fliO of Salmonella typhimurium can be translated from an AUG codon that overlaps the termination codon of fliN (K . Ohnishi et al., J . Bacteriol . 179:6092-6099, 1997) . However, it had been concluded on the basis of complementation analysis that in Escherichia coli a second start codon 60 bp downstream was the authentic one (J . Malakooti et al., J . Bacteriol . 176:189-197, 1994) . This raised the possibility of tandem translational starts, such as occur for the chemotaxis gene cheA; this possibility was increased by the existence of a stem-loop sequence covering the second start, a feature also found with cheA . Protein translated from the first start codon was detected regardless of whether the second start codon was present; it was also detected when the stem-loop structure was disrupted or deleted . Translation from the second start codon, either as the natural one (GUG) or as AUG, was not detected when the first start and intervening sequence were intact . Nor was it detected when the first codon was attenuated (by conversion of AUGAUG to AUAAUA; in S . typhimurium there is a second, adjacent, AUG) or eliminated (by conversion to CGCCGC); disruption of the stem-loop structure still did not yield detectable translation from the second start . When the entire sequence up to the second start was deleted, translation from the second start was detected provided the natural codon GUG had been converted to AUG . A fliO null mutant could be fully complemented in swarm assays whenever the first start and intervening sequence were present, regardless of the state of the second start . Reasonably good complementation occurred when the first start and intervening sequence were absent provided the second start was intact, either as AUG or as GUG; thus translation from the GUG codon must have been occurring even though protein levels were too low to be detected . The translated intervening sequence is rather divergent between S . typhimurium and E . coli and corresponds to a substantial cytoplasmic domain prior to the sole transmembrane segment, which is highly conserved; the sequence following the second start begins immediately prior to that transmembrane segment . The significance of the data for FliO is discussed and compared to the equivalent data for CheA . Attention is also drawn to the fact that given an optimal ribosome binding site, AUA can serve as a fairly efficient start codon even though it seldom if ever appears to be used in nature.

J Bacteriol, 1998 Jun, 180(11), 2915 - 23
Characterization of the hca cluster encoding the dioxygenolytic pathway for initial catabolism of 3-phenylpropionic acid in Escherichia coli K-12; Diaz E et al.; We have identified, cloned, and sequenced the hca cluster encoding the dioxygenolytic pathway for initial catabolism of 3-phenylpropionic acid (PP) in Escherichia coli K-12 . This cluster maps at min 57.5 of the chromosome and is composed of five catabolic genes arranged as a putative operon (hcaA1A2CBD) and two additional genes transcribed in the opposite direction that encode a potential permease (hcaT) and a regulator (hcaR) . Sequence comparisons revealed that while hcaA1A2CD genes encode the four subunits of the 3-phenylpropionate dioxygenase, the hcaB gene codes for the corresponding cis-dihydrodiol dehydrogenase . This type of catabolic module is homologous to those encoding class IIB dioxygenases and becomes the first example of such a catabolic cluster in E . coli . The inducible expression of the hca genes requires the presence of the hcaR gene product, which acts as a transcriptional activator and shows significant sequence similarity to members of the LysR family of regulators . Interestingly, the HcaA1A2CD and HcaB enzymes are able to oxidize not only PP to 3-(2,3-dihydroxyphenyl)propionate (DHPP) but also cinnamic acid (CI) to its corresponding 2, 3-dihydroxy derivative . Further catabolism of DHPP requires the mhp-encoded meta fission pathway for the mineralization of 3-hydroxyphenylpropionate (3HPP) (A . Ferrandez, J . L . Garcia, and E . Diaz, J . Bacteriol . 179:2573-2581, 1997) . Expression in Salmonella typhimurium of the mhp genes alone or in combination with the hca cluster allowed the growth of the recombinant bacteria in 3-hydroxycinnamic acid (3HCI) and CI, respectively . Thus, the convergent mhp- and hca-encoded pathways are also functional in S . typhimurium, and they are responsible for the catabolism of different phenylpropanoid compounds (3HPP, 3HCI, PP, and CI) widely available in nature.

J Bacteriol, 1998 Jun, 180(11), 2862 - 5
Adaptive mutagenesis at ebgR is regulated by PhoPQ; Hall BG; Adaptive mutations are mutations that occur in nondividing or very slowly dividing microbial cells during prolonged nonlethal selection and that are specific to the challenge of the selection in the sense that the only mutations that can be detected are those that provide a growth advantage to the cell . The phoPQ genes encode a two-component positively acting regulatory system that controls expression of at least 25 to 30 genes in Escherichia coli and Salmonella typhimurium . PhoPQ responds to a variety of environmental stress signals including Mg2+ starvation and nutritional deprivation . Here I show that disruption of phoP or phoQ by Tn10dCam significantly reduces the adaptive mutation rate to ebgR, indicating that the adaptive mutagenesis machinery is regulated, directly or indirectly, by phoPQ . The finding that it is regulated implies that adaptive mutagenesis does not simply result from a failure of various error correction mechanisms during prolonged starvation.

J Bacteriol, 1998 May, 180(10), 2788 - 91
Functional similarity between archaeal and bacterial CorA magnesium transporters; Smith RL et al.; The constitutively expressed CorA Mg2+ transporter is the major Mg2+ influx system of Salmonella typhimurium and Escherichia coli . Genomic sequence data indicated the presence of a homolog in the archaeal organism Methanococcus jannaschii . The putative M . jannaschii CorA was expressed in an Mg2+-transport-deficient strain of S . typhimurium to determine its functional characteristics . The archaeal CorA homolog is a functional Mg2+ uptake system when expressed in S . typhimurium and has properties which are highly similar to those of the normal CorA transporter of S . typhimurium despite having a low level of sequence identity with the protein and being expressed in a lipid membrane of quite different composition than normal . This implies that the overall function of the proteins is the same and further suggests that their structures are very similar.

Mol Gen Genet, 1998 Apr, 258(1-2), 178 - 81
Directed formation of chromosomal deletions in Salmonella typhimurium: targeting of specific genes induced during infection; Julio SM et al.; In vivo expression technology (IVET) has resulted in the isolation of more than 100 Salmonella typhimurium genes that are induced during infection . Many of these in vivo induced (ivi) genes, as well as other virulence genes, are clustered in regions of the chromosome that are specific for Salmonella and are not present in Escherichia coli (e.g., pathogenicity islands) . It would be desirable to be able to delete such putative virulence regions of the chromosome, and if the deletion removes genes that play a role in pathogenesis subsequent efforts can then be focused on individual genes that reside within that region . We therefore have developed a strategy for constructing chromosomal deletions which are not limited in size, have defined endpoints with a selectable marker at the joint point, and are not dependent on prior knowledge of sequences contained within the deleted region . Such deletion strategies can be applied to almost any bacterium with homologous recombination and to plasmid-based mutational systems where homologous recombination is not desired or feasible.

Mol Gen Genet, 1998 Apr, 258(1-2), 174 - 7
Integration host factor positively regulates cycJIH transcription; Sirko A et al.; We report the identification of integration host factor (IHF) as an additional element involved in the regulation of the cysJIH promoter of Salmonella typhimurium and Escherichia coli . An IHF-binding site was located in the regulatory region of the cysJIH promoter by using two methods, protection against DNase I digestion and hydroxyl radical cleavage . The positive influence of IHF on in vitro run-off transcription from the cysJIH promoter is shown . The in vivo observations suggest that IHF is necessary for full expression of cysJIH in stationary phase but not during exponential growth.

Gene, 1998 Jun 8, 212(2), 189 - 96
The influence of the 5' codon context on translation termination in Bacillus subtilis and Escherichia coli is similar but different from Salmonella typhimurium; Mottagui-Tabar S et al.; The last two amino acids in the nascent peptide influence translation termination in E . coli (Mottagui-Tabar et al., 1994; Bjornsson et al., 1996) . We have compared the effects on termination in Escherichia coli, Bacillus subtilis and Salmonella typhimurium obtained by varying the -1 and -2 codons upstream of the weak UGAA stop signal . The peptide effect from the penultimate amino acid on translation termination in B . subtilis is similar to that seen in E . coli (with 66.5% RF-2 amino acid sequence similarity), whereas the influence in S . typhimurium (with 95.3% similarity to E . coli) is weaker . The effect of changing the -1 codon (P-site) is weaker in S . typhimurium as compared to those in E . coli and B . subtilis . RF-2s from E . coli and S . typhimurium have a threonine or alanine at position 246, respectively . This amino acid exchange in RF-2 can explain the difference in efficiency and toxicity during overexpression when E . coli and S . typhimurium are compared (Uno et al., 1996) . However, B . subtilis RF-2 also has an alanine at that position, yet the sensitivity to the nascent peptide is similar to that in E . coli . Thus, the amino acid difference at position 246 in the RF-2 sequences cannot explain why termination in E . coli and B . subtilis is similar in peptide sensitivity while being different from that in S . typhimurium . Sequence alignments of RF-2 from the three bacteria show other regions of the molecule that could be involved in the functional interactions with the C-terminal end of the nascent peptide.

Biochemistry, 1998 May 19, 37(20), 7062 - 9
Direct measurement of small ligand-induced conformational changes in the aspartate chemoreceptor using EPR; Ottemann KM et al.; Ligand-binding-induced conformational changes in the Salmonella typhimurium aspartate receptor were studied using spin-labeling electron paramagnetic resonance . Cysteine residues, introduced by site-directed mutagenesis at several positions in the aspartate receptor periplasmic domain, were used to attach covalently a thiol-specific spin label . The electron paramagnetic resonance spectra of these labeled proteins were obtained in the presence and absence of the ligand aspartate, and used to calculate the distance change between spin labels . The results support a model in which transmembrane signaling is executed by a combined movement of alpha helix 4 (which leads into transmembrane domain 2) relative to alpha helix 1 (connected to transmembrane domain 1), as well as a coming together of the two subunits . Ligand binding causes spin labels at position 39 and 179 (within one subunit) to move further from each other and spin labels at position 39 and 39' (between two subunits) to move closer to each other . Both of these changes are very small-less than 2.5 A . No similar changes were detected in any aspartate receptor samples solubilized in detergent, suggesting that the membrane is required for these conformational changes . This is the first case of physically measured ligand-induced changes in a full-length 1-2 transmembrane domain receptor, and the results suggest that very small ligand-induced movements can result in large effects on the activity of downstream proteins.

Food Chem Toxicol, 1998 Mar, 36(3), 183 - 90
Chemical and biological studies of a new cigarette that primarily heats tobacco . Part 2 . In vitro toxicology of mainstream smoke condensate; Bombick BR et al.; The genotoxic and cytotoxic potential of mainstream cigarette smoke condensate (CSC) from a new cigarette that primarily heats tobacco (TOB-HT) was compared with that of CSC from a Kentucky reference low "tar" cigarette (1R4F) representative of the current US cigarette market, and Kentucky Reference 1R5F, representative of ultra-low "tar" cigarettes on the US market . TOB-HT was evaluated at concentrations which induced concentration-dependent positive responses with 1R4F and 1R5F in an in vitro toxicology test battery which included sister chromatid exchange, chromosome aberration, and neutral red cytotoxicity assays in CHO cells, and the Ames bacterial mutagenicity assay . CSC from 1R4F and 1R5F was positive in the Ames assay with Salmonella typhimurium strains TA98, TA100, TA1538 and TA1537, and negative with TA1535, while CSC from TOB-HT was negative in all five strains . CSC from 1R4F and 1R5F cigarettes was positive in sister chromatid exchange (SCE), chromosome aberration (CA) and neutral red cytotoxicity assays, while CSC from the TOB-HT cigarette yielded negative results in all the above endpoints . These data indicate that in these assays the genotoxic and cytotoxic potential of CSC from the new cigarette that primarily heats tobacco is significantly less than CSC from Kentucky reference 1R4F and 1R5F cigarettes, which are representative of cigarettes currently sold in the US.

Vaccine, 1998 Jan, 16(1), 45 - 54
Immune responses in calves immunised orally or subcutaneously with a live Salmonella typhimurium aro vaccine; Villarreal-Ramos B et al.; Salmonella aro vaccines are able to confer solid protection against homologous virulent challenge in several animal species . Calves were protected against virulent S . typhimurium challenge following administration of a single oral dose of live BRD562 vaccine . Immune responses elicited by the S . typhimurium aro vaccine strain BRD562 were studied following administration to calves by either the oral or subcutaneous route . Serum antibodies to Salmonella polypeptides, following oral or subcutaneous vaccination, were detected by immunoblotting and the route of inoculation found to affect both the antibody isotype and the antigens detected . Oral, but not subcutaneous, immunisation induced bovine serum IgA antibodies against Salmonella antigens of 30 kDa and 65 kDa and bovine IgG2 antibodies against a 35 kDa antigen . Subcutaneous vaccination triggered responses against antigens of 52 kDa, 54 kDa and 57 kDa which were not detected by immune plasma of animals immunised orally . Antibody responses to LPS were poor in animals inoculated by either route . Subcutaneous vaccination elicited T-cell responses against Salmonella antigens as measured by in vitro peripheral blood cell thymidine incorporation . These studies show that the S . typhimurium vaccine strain BRD562 is capable of inducing both humoral and cellular immune responses . Further studies are necessary to identify the nature of the antigens responsible for protection . Oral or subcutaneous inoculation of BRD562(pTETnir15) failed to induce serum antibodies against the fragment C of tetanus toxin (TetC) but was effective in mice . Oral vaccination with this recombinant vaccine induced mucosal IgA against TetC . This is the first time that Salmonella recombinant vaccines have been shown to successfully elicit antibodies against a guest antigen in cattle after one single oral inoculation.

Biochemistry (Mosc), 1998 Feb, 63(2), 195 - 9
Isolation and initial characterization of the uridine phosphorylase from Salmonella typhimurium; Molchan OK et al.; The structural udp gene encoding uridine phosphorylase (UPase) was cloned from the Salmonella typhimurium chromosome and overexpressed in E . coli cells . The S . typhimurium UPase was purified to an apparently homogeneous state, and some physicochemical characteristics of the enzyme were studied . The molecular weight of one subunit of UPase is 27.5 kD, and the optimal pH for its activity is 7.2--7.4 . The native S . typhimurium UPase consists of six identical subunits, and its molecular weight is about 165 kD . According to these parameters, the S . typhimurium UPase is similar to the E . coli UPase . However, these enzymes differ substantially from one another by the substrate sensitivity and sensitivity to polarity of the medium . The S . typhimurium UPase has much higher phosphorylation activity toward thymidine, deoxyuridine, and 5;-bromide- or 5;-fluoride-containing analogs of nucleosides than that of E . coli UPase.

Scand J Gastroenterol, 1998 Apr, 33(4), 406 - 14
Role of reactive oxygen species in Salmonella typhimurium-induced enterocyte damage; Mehta A et al.; BACKGROUND: Reactive oxygen species (ROS) are potent mediators of inflammatory cell-mediated tissue destruction and may be of pathophysiologic importance in Salmonella typhimurium-induced tissue damage . METHODS: In this study the ligated rat ileal loops were injected with Salmonella live culture or toxin . The ROS generation was detected by measuring the mucosal myeloperoxidase (MPO) activity; the enterocyte xanthine oxidase (XO) activity, and the chemiluminescence response of gut macrophages . The enterocyte damage was estimated by measuring the extent of lipid peroxidation and cell viability . RESULTS: Treatment with Salmonella live culture or toxin resulted in an increase in the mucosal MPO activity, the enterocyte XO activity, and the chemiluminescence response of macrophages . Treated loop enterocytes had an increased extent of lipid peroxidation and decreased cell viability . Cell viability was also decreased when the enterocytes were co-cultured with macrophages isolated from the treated loops . Lipid peroxidation decreased, and cell viability increased in the presence of superoxide dismutase (SOD) or catalase . CONCLUSIONS: The S . typhimurium-mediated intestinal infection is accompanied by an increased generation of ROS, which may induce the lipid peroxidation of the enterocyte membrane, thereby leading to a loss of cell viability.

Lab Invest, 1998 May, 78(5), 523 - 34
Anti-inflammatory and antisecretory potential of histidine in Salmonella-challenged mouse small intestine; Peterson JW et al.; Challenge of mouse small intestinal loops with Salmonella typhimurium invoked the accumulation of luminal fluid, acute inflammation, and extensive structural damage to the small intestinal mucosa, as determined by histology and electron microscopy . Intraperitoneal and intestinal luminal injection of L-histidine, a known antioxidant, reduced the amount of fluid accumulating in the intestinal lumen and protected the intestinal tissue from S . typhimurium-induced damage . The reduction in S . typhimurium-induced fluid accumulation by L-histidine was specific for the L-enantiomer because D-histidine had no significant protective effect . Efficacy of L-histidine in protecting the infected intestinal tissue was attributed to the capacity of the imidazole ring to scavenge reactive oxygen species (ROS) generated by cells in the intestine during the acute inflammatory response . Glutathione levels were markedly reduced in S . typhimurium-challenged, inflamed intestinal tissues as a result of ROS generation . Importantly, after dosing the S . typhimurium-challenged mice with L-histidine, the glutathione content of the small intestinal tissue was not significantly different from mock-challenged controls . Further evidence favoring this mechanism included the capacity of L-histidine to scavenge ROS produced as a result of lipopolysaccharide (LPS) exposure of mononuclear cells (U937), as demonstrated with a redox-sensitive fluorescent dye (2'7'-dichlorodihydrofluorescein {DCF}) . Addition of L-histidine, and to a lesser extent D-histidine, to the culture media of U937 cells before LPS exposure, resulted in a significant dose-dependent reduction in LPS-induced intracellular DCF fluorescence, as measured quantitatively by flow cytometry . The potential therapeutic value of anti-inflammatory drugs containing an L-histidine-like structure could protect infected mucosal tissues irrespective of microbial etiology.

Mol Gen Genet, 1998 Apr, 257(6), 693 - 6
A gene, yaeQ, that suppresses reduced operon expression caused by mutations in the transcription elongation gene rfaH in Escherichia coli and Salmonella typhimurium; Wong KR et al.; RfaH is a specialised transcription elongation protein . We isolated from a Salmonella typhimurium rfaH suppressor strain a gene that complements in trans the attenuated transcription of the hlyCABD hemolysin operon caused by an rfaH mutation . The gene is homologous to the uncharacterised Escherichia coli gene yaeQ . YaeQ did not increase hlyCABD-encoded hemolysin activity in the RfaH+ wild type, nor did it increase the level of RfaH protein . YaeQ also complements a rfaH::Tn5 null mutation, indicating that it compensates for loss of RfaH function without interaction between the two proteins.

J Mol Biol, 1998 Apr 10, 277(4), 871 - 82
Effect of cellular level of FliK on flagellar hook and filament assembly in Salmonella typhimurium; Muramoto K et al.; Frameshift mutations in the fliK gene of Salmonella result in abnormal elongation of the hook and the failure to assemble filament (polyhook phenotype) . Second-site suppressor mutations restore filament assembly, but the cells often remain defective in hook-length control (polyhook-filament phenotype) . Where the suppressor mutations are intragenic, the second mutation restores the original frame, generating a region of frameshifted sequence, but restoring the natural C terminus . Some of these frameshifted sequences contain a UGA (opal) termination codon . These cells have few flagella and swarm poorly . We suspected that readthrough of UGA by tRNATrp might be the reason for the partial function . When the UGA codon was changed to the Trp codon UGG, flagellar assembly and function were restored to wild-type levels . Conversely, underexpression of the wild-type fliK gene, achieved by changing the sole Trp codon in the sequence (Trp271) to UGA, decreased both the number of flagella and the ability to swarm . These results validate the readthrough hypothesis and indicate that low levels of FliK sustain some degree of flagellation and motility . At low levels of FliK, most flagella had polyhooks . With increasing amounts, the morphology progressively changed to polyhook-filament, and eventually to wild-type hook-filament . When FliK was overproduced, the hook length was slightly shorter (46(+/-7) nm) than that of the wild-type strain (55(+/-9) nm) . FliK levels were measured by immunoblotting . Wild-type levels were about 40 to 80 molecules/cell . FliK synthesized by UGA readthrough could be detected when overproduced from plasmid fliK-W271opal, and the levels indicated a probability of readthrough of 0.002 to 0.01 . This value was used to estimate the cellular level of underexpressed FliK, which could partly restore function to a fliK mutant, at about 0.07 to 0.8 molecule/cell . These results suggest that FliK does not form a large structure in the cytoplasm and may function as a regulatory protein for protein export . A model for hook-length control is presented that involves feedback from the assembly point to the export apparatus .

Infect Immun, 1998 Jun, 66(6), 2791 - 7
Secretory phospholipase A2 is the principal bactericide for staphylococci and other gram-positive bacteria in human tears; Qu XD et al.; We examined human tears for molecules that killed gram-positive bacteria . The principal mediator of bactericidal activity against staphylococci proved to be a calcium-dependent enzyme, secretory phospholipase A2 . Whereas the concentration of secretory phospholipase A2 in the normal tear film exceeded 30 microg/ml, only 1.1 ng (<0.1 nM) of the enzyme per ml sufficed to kill Listeria monocytogenes and 15 to 80 ng/ml killed Staphylococcus aureus . Despite its efficacy against gram-positive bacteria, secretory phospholipase A2 lacked bactericidal activity against gram-negative organisms (Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa) when tested in the ionic environment of tears . Given the presence of secretory phospholipase A2 in tears, intestinal secretions, and leukocytes, this enzyme may play a substantial role in innate mucosal and systemic bactericidal defenses against gram-positive bacteria.

Infect Immun, 1998 Jun, 66(6), 2547 - 52
Mitogenic response of murine B lymphocytes to Salmonella typhimurium lipopolysaccharide requires protein kinase C-dependent late tyrosine phosphorylations; Mey A et al.; Unlike the cross-linking of membrane immunoglobulins, the activation of B cells by lipopolysaccharide (LPS) does not involve the phosphoinositol turnover and the initial activation of tyrosine kinases . However, LPS-induced B-cell proliferation was inhibited by the tyrosine kinase inhibitors genistein and herbimycin A even when added 48 h after the beginning of the culture . Tyrosyl-phosphorylated proteins were detected by Western blotting after 24 h of culture with LPS, reaching a maximum concentration after 72 h . Late tyrosine phosphorylations were also detected in B cells activated for 72 h with anti-immunoglobulin M antibody and were abrogated by the protein synthesis inhibitor cycloheximide, the tyrosine kinase inhibitors genistein and herbimycin A, and the protein kinase C inhibitor chelerythrine . The role of protein kinase C in late tyrosine kinase activation is independent of Ca2+ mobilization and was confirmed by detection of a comparable but restricted pattern of tyrosine-phosphorylated substrates in B cells treated with phorbol myristate acetate alone or in association with ionomycin . Tyrosine kinase activation was dependent on de novo protein synthesis . However, culture supernatants of LPS-activated B cells were devoid of mitogenic activity and induced a phosphorylation pattern more restricted than that achieved by LPS . Altogether these data indicate that proliferation signals induced by LPS or by the cross-linking of membrane immunoglobulins are controlled by late tyrosine phosphorylations occurring throughout the first 3 days of culture, controlled in part by protein kinase C activation, and dependent on the synthesis of an intermediate protein(s) either not secreted in the culture supernatant or present but biologically inactive in naive B cells.

Infect Immun, 1998 Jun, 66(6), 2471 - 85
Analysis of host cells associated with the Spv-mediated increased intracellular growth rate of Salmonella typhimurium in mice; Gulig PA et al.; The 90-kb virulence plasmid of Salmonella typhimurium encodes five spv genes which increase the growth rate of the bacteria within host cells within the first week of systemic infection of mice (P . A . Gulig and T . J . Doyle, Infect . Immun . 61:504-511, 1993) . The presently described study was aimed at identifying the host cells associated with Spv-mediated virulence by manipulating the mouse host and the salmonellae . To test the effects of T cells and B cells on the Spv phenotype, salmonellae were orally inoculated into nude and SCID BALB/c mice . Relative to normal BALB/c mice, nude and SCID BALB/c mice were unaffected for splenic infection with either the Spv+ or Spv- S . typhimurium strains at 5 days postinoculation . When mice were pretreated with cyclophosphamide to induce granulocytopenia, there was a variable increase in total salmonella infection, but the relative splenic CFU of Spv+ versus Spv- S . typhimurium was not changed after oral inoculation . In contrast, depletion of macrophages from mice by treatment with cyclophosphamide plus liposomes containing dichloromethylene diphosphate resulted in equivalent virulence of Spv+ and Spv- salmonellae . To examine if the spv genes affected the growth of salmonellae in nonphagocytic cells, an invA::aphT mutation was transduced into Spv+ and Spv- S . typhimurium strains . InvA- Spv+ salmonellae were not significantly affected for splenic infection after subcutaneous inoculation compared with the wild-type strain, and InvA- Spv- salmonellae were only slightly attenuated relative to InvA+ Spv- salmonellae . Invasion-defective salmonellae still exhibited the Spv phenotype . Therefore, infection of nonphagocytes is not involved with the Spv virulence function . Taken together, these data demonstrate that macrophages are essential for suppressing the infection by Spv- S . typhimurium, by serving as the primary host cell for Spv-mediated intracellular replication and possibly by inhibiting the replication of salmonellae within other macrophages.

Genetics, 1998 May, 149(1), 37 - 44
How optimized is the translational machinery in Escherichia coli, Salmonella typhimurium and Saccharomyces cerevisiae?
Xia X.
The optimization of the translational machinery in cells requires the mutual adaptation of codon usage and tRNA concentration, and the adaptation of tRNA concentration to amino acid usage . Two predictions were derived based on a simple deterministic model of translation which assumes that elongation of the peptide chain is rate-limiting . The highest translational efficiency is achieved when the codon recognized by the most abundant tRNA reaches the maximum frequency . For each codon family, the tRNA concentration is optimally adapted to codon usage when the concentration of different tRNA species matches the square-root of the frequency of their corresponding synonymous codons . When tRNA concentration and codon usage are well adapted to each other, the optimal content of all tRNA species carrying the same amino acid should match the square-root of the frequency of the amino acid . These predictions are examined against empirical data from Escherichia coli, Salmonella typhimurium, and Saccharomyces cerevisiae.

Genetics, 1998 May, 149(1), 17 - 36
Spectra of spontaneous frameshift mutations at the hisD3052 allele of Salmonella typhimurium in four DNA repair backgrounds; DeMarini DM et al.; To characterize the hisD3052 -1 frameshift allele of Salmonella typhimurium, we analyzed approximately 6000 spontaneous revertants (rev) for a 2-base deletion hotspot within the sequence (CG)4, and we sequenced approximately 500 nonhotspot rev . The reversion target is a minimum of 76 bases (nucleotides 843-918) that code for amino acids within a nonconserved region of the histidinol dehydrogenase protein . Only 0.4-3.9% were true rev . Of the following classes, 182 unique second-site mutations were identified: hotspot, complex frameshifts requiring DeltauvrB + pKM101 (TA98-specific) or not (concerted), 1-base insertions, duplications, and nonhotspot deletions . The percentages of hotspot mutations were 13.8% in TA1978 (wild type), 24.5% in UTH8413 (pKM101), 31.6% in TA1538 (DeltauvrB), and 41.0% in TA98 (DeltauvrB, pKM101) . The DeltauvrB allele decreased by three times the mutant frequency (MF, rev/10(8) survivors) of duplications and increased by about two times the MF of deletions . Separately, the DeltauvrB allele or pKM101 plasmid increased by two to three times the MF of hotspot mutations; combined, they increased this MF by five times . The percentage of 1-base insertions was not influenced by either DeltauvrB or pKM101 . Hotspot deletions and TA98-specific complex frameshifts are inducible by some mutagens; concerted complex frameshifts and 1-base insertions are not; and there is little evidence for mutagen-induced duplications and nonhotspot deletions . Except for the base substitutions in TA98-specific complex frameshifts, all spontaneous mutations of the hisD3052 allele are likely templated . The mechanisms may involve (1) the potential of direct and inverted repeats to undergo slippage and misalignment and to form quasi-palindromes and (2) the interaction of these sequences with DNA replication and repair proteins.

Mutat Res, 1998 Feb 13, 412(3), 299 - 305
Genotoxicity evaluation of trimethylbenzenes; Janik-Spiechowicz E et al.; The three trimethyl isomers of benzene (hemimellitene, 1,2,3-TMB; pseudocumene, 1,2,4-TMB and mesitylene, 1,3,5-TMB) were investigated for different genotoxicity endpoints: in vitro, in the Ames test with Salmonella typhimurium TA97a, TA98, TA100 and TA102 strains in the presence and absence of rat liver S9 metabolic activation; in vivo, in the micronucleus and sister chromatid exchange (SCE) tests with bone marrow cells of Imp:Balb/c mice . Only the isomer of benzene with the methyl-group at position 1, 2, 3 was found to have mutagenic effect on S . typhimurium cells . Increase in bacterial reversions was observed in four conventional strains used in this study, but most clearly in TA97a . The mutagenic responses of 1,2,3-TMB with the SalmonellaL tester strains were observed in the experiments performed in the absence of enzymatic activation . None of the compounds had an influence on the frequency of micronucleated polychromatic erythrocytes in bone marrow cells of mice . However, all the three compounds were observed to have a cytogenetic potential of increasing the SCE level in these cells . Significant responses in SCE induction, compared with the level of those changes in corresponding solvent-administered controls, were obtained at three test doses of 1,2,3-TMB (730, 1470, 2200 mg/kg) and 1,2,4-TMB (900, 1800, 2700 mg/kg) and at two doses of 1,3,5-TMB (1800, 2700 mg/kg) . These data provided a limited evidence for the genotoxic activity of 1,2,3-TMB and inadequate evidence for genotoxic activity of 1,2,4-TMB and 1,3,5-TMB.

Mutat Res, 1998 Feb 13, 412(3), 251 - 60
Chemicals mutagenic in Salmonella typhimurium strain TA1535 but not in TA100; Prival MJ et al.; The standard Salmonella mutagenicity test uses two strains of Salmonella typhimurium (TA1535 and TA100) containing the same base pair substitution mutation (hisG46) . These strains differ only in that strain TA100 contains the plasmid pKM101, whose mucAB gene products enhance SOS mutagenesis . This makes strain TA100, in general, the more sensitive of the two for mutagen detection, raising the question as to whether or not to include strain TA1535 in the core battery of strains in routine testing . Out of 659 chemicals judged as mutagens in the S . typhimurium assay when subjected to the National Toxicology Program's screening protocol, 36 (5%) were evaluated as positive in strain TA1535 but not in strain TA100 . Of these, 23 were judged as negative and 13 as equivocal in strain TA100, and 5 were positive or equivocal in at least one other strain (TA97 or TA98) . In general, the data on these chemicals indicate that the absolute increases in revertants per plate induced in strain TA1535 were too small to have been judged as positive if similar increases occurred in strain TA100, which has a much higher spontaneous background . For three chemicals (acetaldehyde oxime, 6-mercaptopurine, and 1,3-butadiene) the absolute increases in revertants in strain TA1535 greatly exceeded those in strain TA100 . Evaluation of the reproducibility of these findings and of the mechanisms and relevance of unique TA1535 positives should be useful when decisions are made as to whether this strain should be kept as a part of the core battery of strains in the S . typhimurium assay.

Int J Food Microbiol, 1998 Mar 3, 40(1-2), 117 - 21
Behavior of Salmonella typhimurium DT104 during the manufacture and storage of pepperoni; Ihnot AM et al.; Pepperoni batter (ca . 70% pork:30% beef) was prepared and subsequently inoculated with a six-strain cocktail (ca . 4.4 x 10(7) per gram batter) of Salmonella typhimurium DT104 . After fermentation at 36 degrees C and 92% relative humidity (RH) to < or = pH 4.8, counts of the pathogen decreased by about 1.3 log10 units . An additional 1.6 log10 unit decrease was observed following drying at 13 degrees C and 65% RH to a moisture protein ratio (M/Pr) of 1.6:1 . After storage of pepperoni sticks for 56 days under vacuum at 4 or 21 degrees C, counts of the pathogen were about 4.6 and 6.6 log10 units lower, respectively, compared with starting levels in the batter . These data establish that fermentation and drying result in about a 3.0 log10 reduction in numbers of S typhimurium DT104 in pepperoni sticks and that storage of pepperoni sticks under vacuum at ambient temperature is more severe on the pathogen than refrigerated storage.

J Med Invest, 1998 Feb, 44(3-4), 193 - 8
Activation of 1-nitropyrene by nitroreductase increases the DNA adduct level and mutagenicity; Arimochi H et al.; 1-Nitropyrene (1-NP) is a mutagenic nitro compound in the environment . We studied correlations between the mutagenicity of 1-NP for three strains of Salmonella typhimurium, the activity of bacterial nitroreductases and the amount of 1-NP-derived DNA adducts . Bacterial strains used in this study were S . typhimurium strains TA98, nitroreductase-less mutant TA98NR and YG1021 carrying a nitroreductase-producing plasmid . The mutagenicity of 1-NP was measured using the Ames assay, and the nitroreductase activities of these strains were assayed by quantification of 1-aminopyrene produced from 1-NP . The DNA adducts were measured by the 32P-postlabeling method . Among the three bacterial strains, strain YG1021 was the highest in mutagenicity of 1-NP, the nitroreductase activity and the DNA adduct level . However, S . typhimurium strain TA98NR had the lowest values of these three parameters . Nitroreductase activity, DNA adduct level and mutagenicity were strongly correlated with each other . These results indicate that bacterial nitroreductase plays an important role in forming the DNA adducts, and that the higher the adduct level the higher the level of mutagenicity.

Antimicrob Agents Chemother, 1998 May, 42(5), 1259 - 62
Sequence of the gene encoding a plasmid-mediated cefotaxime-hydrolyzing class A beta-lactamase (CTX-M-4): involvement of serine 237 in cephalosporin hydrolysis; Gazouli M et al.; The sequence of the gene encoding a novel cefotaxime-hydrolyzing beta-lactamase (CTX-M-4) was determined . It was located in a plasmid harbored by a Salmonella typhimurium strain . CTX-M-4 was similar to the plasmidic cefotaxime-hydrolyzing beta-lactamases CTX-M-2 and Toho-1 and related to the chromosomal beta-lactamase of Klebsiella oxytoca . A Ser-237-->Ala substitution, introduced by site-directed mutagenesis, caused minor alterations in the interaction of CTX-M-4 with beta-lactams, reducing slightly the relative hydrolytic activity against cefotaxime and the susceptibility to inhibition by clavulanate.

Environ Mol Mutagen, 1998, 31(3), 282 - 91
Mutagenic activation of aromatic amines by molluscs as a biomarker of marine pollution; Diaz-Mendez FM et al.; Mutagenic activation of arylamines by mollusc S9 fractions was evaluated as a biomarker for marine pollution . Two bivalve species were used as bioindicators, the common mussel (Mytilus edulis) and the striped venus (Chameleo gallina) . A strain of Salmonella typhimurium overproducing O-acetyltransferase was used as indicator of mutagenicity . Mussels from an area of the North Atlantic Spanish zone that was exposed to an accidental crude oil spill were compared to bivalves from a reference area . C . gallina samples were from low polluted and highly polluted areas of the South Atlantic Spanish littoral . The promutagen 2-aminoanthracene (2-AA) was activated to mutagenic derivative(s) by S9 fractions from both C . gallina and M . edulis . Animals from contaminated sites showed higher arylamine activation capabilities than reference animals . This was further correlated with the mutagenic activities of corresponding cyclopentone-dichloromethane animal extracts . 2-AA activation by mollusc S9 was potentiated by alpha-naphthoflavone (ANF), known to inhibit PAH-inducible CYP1A cytochromes from vertebrates, but inhibited by methimazole (MZ), a substrate of the flavin monooxygenase (FMO) system . 2-AA-activating enzymes were mainly cytosolic; this localization clearly suggests that such activity could be attributed to soluble enzymes, different from the CYP1A or FMO systems . In conclusion, mutagenic activation of arylamines by mollusc S9, using as indicator a strain of Salmonella typhimurium that overproduces O-acetyltransferase, could be a reliable biomarker for marine pollution.

Biochemistry, 1998 Apr 21, 37(16), 5394 - 406
Loop closure and intersubunit communication in tryptophan synthase; Schneider TR et al.; Crystal structures of wild-type tryptophan synthase alpha2beta2 complexes from Salmonella typhimurium were determined to investigate the mechanism of allosteric activation of the alpha-reaction by the aminoacrylate intermediate formed at the beta-active site . Using a flow cell, the aminoacrylate (A-A) intermediate of the beta-reaction () was generated in the crystal under steady state conditions in the presence of serine and the alpha-site inhibitor 5-fluoroindole propanol phosphate (F-IPP) . A model for the conformation of the Schiff base between the aminoacrylate and the beta-subunit cofactor pyridoxal phosphate (PLP) is presented . The structure is compared with structures of the enzyme determined in the absence (TRPS) and presence (TRPSF-IPP) of F-IPP . A detailed model for binding of F-IPP to the alpha-subunit is presented . In contrast to findings by Hyde et al . {(1988) J . Biol . Chem . 263,17857-17871} and Rhee et al . {(1997) Biochemistry 36, 7664-7680}, we find that the presence of an alpha-site alone ligand is sufficient for loop alphaL6 closure atop the alpha-active site . Part of this loop, alphaThr183, is important not only for positioning the catalytic alphaAsp60 but also for coordinating the concomitant ordering of loop alphaL2 upon F-IPP binding . On the basis of the three structures, a pathway for communication between the alpha- and beta-active sites has been established . The central element of this pathway is a newly defined rigid, but movable, domain that on one side interacts with the alpha-subunit via loop alphaL2 and on the other side with the beta-active site . These findings provide a structural basis for understanding the allosteric properties of tryptophan synthase.

Proc Natl Acad Sci U S A, 1998 Apr 14, 95(8), 4641 - 5
Differential patterns of acquired virulence genes distinguish Salmonella strains; Conner CP et al.; Analysis of several Salmonella typhimurium in vivo-induced genes located in regions of atypical base composition has uncovered acquired genetic elements that cumulatively engender pathogenicity . Many of these regions are associated with mobile elements, encode predicted adhesin and invasin-like functions, and are required for full virulence . Some of these regions distinguish broad host range from host-adapted Salmonella serovars and may contribute to inherent differences in host specificity, tissue tropism, and disease manifestation . Maintenance of this archipelago of acquired sequence by selection in specific hosts reveals a fossil record of the evolution of pathogenic species.

J Biol Chem, 1998 Apr 10, 273(15), 8946 - 50
thiBPQ encodes an ABC transporter required for transport of thiamine and thiamine pyrophosphate in Salmonella typhimurium; Webb E et al.; In Salmonella typhimurium, thiamine pyrophosphate (TPP) is a required cofactor for several enzymes in central metabolism . Herein we identify a new thi operon, thiBPQ (designated sfuABC in Escherichia coli), required for the transport of thiamine and TPP into the cell . Insertions in the operon result in strains that are phenotypically and biochemically defective in thiamine and TPP transport . Data presented herein show that this operon is transcriptionally repressed in the presence of exogenous thiamine, with TPP the likely regulatory molecule . This work represents the first identification of thiamine transport genes in bacteria and demonstrates the function of a proposed ABC transporter in E . coli.

Protein Expr Purif, 1998 Apr, 12(3), 371 - 80
Purification, characterization, and crystallization of an N-hydroxyarylamine O-acetyltransferase from Salmonella typhimurium; Sinclair JC et al.; The N-hydroxyarylamine O-acetyltransferase from Salmonella typhimurium has been expressed as a histidine-tagged fusion protein in Escherichia coli and purified to apparent homogeneity using single-step immobilized metal ion chromatography . Sufficient quantities of the purified protein have been obtained to allow its characterization by physical methods including dynamic light scattering and electrospray mass spectrometry . The substrate specificity and temperature sensitivity of the enzymatic activity have also been assessed . The enzyme has been crystallized from sodium, potassium tartrate and X-ray diffraction data have been obtained to allow the identification of an orthorhombic unit cell, point group P21212, with dimensions a = 137 A, b = 223 A, and c = 105 A . These crystals will provide a route to a crystallographic determination of the structure of the protein .

Cancer Res, 1998 May 1, 58(9), 1833 - 8
Establishment of a Salmonella tester strain highly sensitive to mutagenic heterocyclic amines; Suzuki A et al.; Heterocyclic amines (HCAs) that are present in cooked foods require metabolic activation to exert their genotoxicity . They undergo activation via N-hydroxylation by cytochrome P450 1A2 (CYP1A2), followed by O-esterification by O-acetyltransferase (OAT) . To develop a Salmonella tester strain that is highly sensitive to mutagenic HCAs, we introduced a coexpression plasmid (p1A2OR) carrying human CYP1A2 and NADPH-CYP reductase cDNAs and an expression plasmid (pOAT) carrying Salmonella OAT to Salmonella typhimurium TA1538 to yield a TA1538/ARO strain . The TA1538/ARO strain was proven to express the enzymes, as indicated by high activities of 7-ethoxyresorufin O-deethylase and isoniazid N-acetylase . The TA1538/ARO strain exhibited very high sensitivity to mutagenic HCAs 2-amino-3,4-dimethylimidazo{4,5-f}quinoline, 2-amino-3-methylimidazo{4,5-f}quinoline (IQ), and 2-amino-3,8-dimethylimidazo{4,5-f}quinoxaline and a somewhat higher sensitivity to 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine compared with the parent Ames tester strain TA1538 . The minimum concentrations of 2-amino-3,4-dimethylimidazo{4,5-f}quinoline, IQ, 2-amino-3,8-dimethylimidazo{4,5-f}quinoxaline, and 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine giving positive results were defined by evidence that the number of colonies increased in a dose-dependent manner and reached a number two times higher than that obtained by vehicle alone as a control in the TA1538/ARO strain at concentrations of 0.3, 3, 30, and 1000 pM, respectively . When the membrane and cytosol fractions prepared from TA1538/ARO were added to a mixture containing the parental TA1538, the sensitivity of TA1538 to IQ was much lower than that seen with TA1538/ARO . These results indicate that the intracellular expression of drug-metabolizing enzymes makes the established strain of Salmonella highly sensitive to mutagenic HCAs.

Mol Biol Evol, 1998 May, 15(5), 574 - 82
Evolutionary rates for tuf genes in endosymbionts of aphids; Brynnel EU et al.; The gene encoding elongation factor Tu (tuf) in aphid endosymbionts (genus Buchnera) evolves at rates of 1.3 x 10(-10) to 2.5 x 10(-10) nonsynonymous substitutions and 3.9 x 10(-9) to 8.0 x 10(-9) synonymous substitutions per position per year . These rates, which are at present among the most reliable substitution rates for protein-coding genes of bacteria, have been obtained by calibrating the nodes in the phylogenetic tree produced from the Buchnera EF-Tu sequences using divergence times for the corresponding ancestral aphid hosts . We also present data suggesting that the rates of nonsynonymous substitutions are significantly higher in the endosymbiont lineages than in the closely related free-living bacteria Escherichia coli and Salmonella typhimurium . Synonymous substitution rates for Buchnera approximate estimated mutation rates for E . coli and S . typhimurium, as expected if synonymous changes act as neutral mutations in Buchnera . We relate the observed differences in substitution frequencies to the absence of selective codon preferences in Buchnera and to the influence of Muller's ratchet on small asexual populations.

J Vet Diagn Invest, 1998 Apr, 10(2), 158 - 63
Use of muscle fluid as a source of antibodies for serologic detection of Salmonella infection in slaughter pig herds; Nielsen B et al.; Fluid drained from a muscle tissue sample was used as an alternative to serum for the detection of specific anti-Salmonella antibodies in an indirect LPS enzyme-linked immunosorbent assay (ELISA) . In the first study, serum and muscle fluid from 3 pigs experimentally infected with Salmonella typhimurium showed parallel dilution-response relationships when ELISA optical density (OD) values were plotted against sample dilution . ELISA results obtained with serum diluted 1:400 corresponded to those from muscle fluid diluted 1:30 . In a second study, using the predetermined dilutions of individually paired serum and muscle fluid samples from 103 pigs, a high degree of concordance between the serum ELISA and the muscle fluid ELISA was observed . Limits of agreement between the 2 methods were calculated as -8.9 to 12.3 OD%, which was considered acceptable . The muscle fluid ELISA had specificities of 0.91-1.0 and sensitivities of 0.80-0.89 at various cutoff values as compared with the serum ELISA . Muscle fluid is a useful postmortem alternative to serum when used with an ELISA to detect anti-Salmonella antibodies.

Infect Immun, 1998 May, 66(5), 1839 - 47
Helicobacter pylori glutamine synthetase lacks features associated with transcriptional and posttranslational regulation; Garner RM et al.; Helicobacter pylori urease, produced in abundance, is indispensable for the survival of H . pylori in animal hosts . Urea is hydrolyzed by the enzyme, resulting in the liberation of excess ammonia, some of which neutralizes gastric acid . The remaining ammonia is assimilated into protein by glutamine synthetase (EC 6.3.1.2), which catalyzes the reaction: NH3 + glutamate + ATP-->glutamine + ADP + Pi . We hypothesized that glutamine synthetase plays an unusually critical role in nitrogen assimilation by H . pylori . We developed a phenotypic screen to isolate genes that contribute to the synthesis of a catalytically active urease . Escherichia coli SE5000 transformed with plasmid pHP808 containing the entire H . pylori urease gene cluster was cotransformed with a pBluescript plasmid library of the H . pylori ATCC 43504 genome . A weakly urease-positive 9.4-kb clone, pUEF728, was subjected to nucleotide sequencing . Among other genes, the gene for glutamine synthetase was identified . The complete 1,443-bp glnA gene predicts a polypeptide of 481 amino acid residues with a molecular weight of 54,317; this was supported by maxicell analysis of cloned glnA expressed in E . coli . The top 10 homologs were all bacterial glutamine synthetases, including Salmonella typhimurium glnA . The ATP-binding motif GDNGSG (residues 272 to 277) of H . pylori GlnA exactly matched and aligned with the sequence in 8 of the 10 homologs . The adenylation site found in the top 10 homologs (consensus sequence, NLYDLP) is replaced in H . pylori by NLFKLT (residues 405 to 410) . Since the Tyr (Y) residue is the target of adenylation and since the H . pylori glutamine synthetase lacks that residue in four strains examined, we conclude that no adenylation occurs within this motif . Cloned H . pylori glnA complemented a glnA mutation in E . coli, and GlnA enzyme activity could be measured spectrophotometrically . In an attempt to produce a GlnA-deficient mutant of H . pylori, a kanamycin resistance cassette was cloned into the Tth111I site of H . pylori glnA . By using the standard technique of allelic exchange mutagenesis, no verifiable glutamine synthetase double-crossover mutant of strain UMAB41 could be isolated, suggesting that the mutation is lethal . We conclude that glutamine synthetase is critical for nitrogen assimilation in H . pylori and is active under all physiologic conditions.

J Bacteriol, 1998 May, 180(9), 2409 - 17
A low pH-inducible, PhoPQ-dependent acid tolerance response protects Salmonella typhimurium against inorganic acid stress; Bearson BL et al.; The acid tolerance response enables Salmonella typhimurium to survive exposures to potentially lethal acidic environments . The acid stress imposed in a typical assay for acid tolerance (log-phase cells in minimal glucose medium) was shown to comprise both inorganic (i.e., low pH) and organic acid components . A gene previously determined to affect acid tolerance, atbR, was identified as pgi, the gene encoding phosphoglucoisomerase . Mutations in pgi were shown to increase acid tolerance by preventing the synthesis of organic acids . Protocols designed to separate the stresses of inorganic from organic acids revealed that the regulators sigma38 (RpoS), Fur, and Ada have major effects on tolerance to organic acid stress but only minor effects on inorganic acid stress . In contrast, the two-component regulatory system PhoP (identified as acid shock protein ASP29) and PhoQ proved to be important for tolerance to inorganic {corrected} acid stress but had little effect against organic acid stress . PhoP mutants also failed to induce four ASPs, confirming a role for this regulator in acid tolerance . Acid shock induction of PhoP appears to occur at the transcriptional level and requires the PhoPQ system . Furthermore, induction by acid occurs even in the presence of high concentrations of magnesium, the ion known to be sensed by PhoQ . These results suggest that PhoQ can sense both Mg2+ and pH . Since phoP mutants are avirulent, the low pH activation of this system has important implications concerning the pathogenesis of S . typhimurium . The involvement of four regulators, two of which are implicated in virulence, underscores the complexity of the acid tolerance stress response and further suggests that features of acid tolerance and virulence are interwoven.

Infect Immun, 1998 May, 66(5), 2099 - 106
Identification of Salmonella typhimurium genes required for colonization of the chicken alimentary tract and for virulence in newly hatched chicks; Turner AK et al.; From a collection of 2,800 Tn5-TC1 transposon mutants of Salmonella typhimurium F98, 18 that showed reduced intestinal colonization of 3-week-old chicks were identified . The sites of transposon insertion were determined for most of the mutants and included insertions in the lipopolysaccharide biosynthesis genes rfaK, rfaY, rfbK, and rfbB and the genes dksA, clpB, hupA, and sipC . In addition, identification was made of an insertion into a novel gene that encodes a protein showing similarity to the IIC component of the mannose class of phosphoenolpyruvate-carbohydrate phosphotransferase systems, which we putatively called ptsC . Transduction of most of the transposon mutations to a fresh S . typhimurium F98 genetic background and construction of defined mutations in the rfbK, dksA, hupA, sipC, and ptsC genes of S . typhimurium F98 supported the role in colonization of all but the pts locus . The virulence of the rfbK, dksA, hupA, sipC, and ptsC defined mutants and clpB and rfaY transductants in 1-day-old chicks was tested . All but the ptsC and rfaY mutants were attenuated for virulence . A number of other phenotypes associated with some of the mutations are described.

Infect Immun, 1998 May, 66(5), 2007 - 17
Cell-contact-stimulated formation of filamentous appendages by Salmonella typhimurium does not depend on the type III secretion system encoded by Salmonella pathogenicity island 1; Reed KA et al.; The formation of filamentous appendages on Salmonella typhimurium has been implicated in the triggering of bacterial entry into host cells (C . C . Ginocchio, S . B . Olmsted, C . L . Wells, and J . E . Galan, Cell 76:717-724, 1994) . We have examined the roles of cell contact and Salmonella pathogenicity island 1 (SPI1) in appendage formation by comparing the surface morphologies of a panel of S . typhimurium strains adherent to tissue culture inserts, to cultured epithelial cell lines, and to murine intestine . Scanning electron microscopy revealed short filamentous appendages 30 to 50 nm in diameter and up to 300 nm in length on many wild-type S . typhimurium bacteria adhering to both cultured epithelial cells and to murine Peyer's patch follicle-associated epithelia . Wild-type S . typhimurium adhering to cell-free culture inserts lacked these filamentous appendages but sometimes exhibited very short appendages which might represent a rudimentary form of the cell contact-stimulated filamentous appendages . Invasion-deficient S . typhimurium strains carrying mutations in components of SPI1 (invA, invG, sspC, and prgH) exhibited filamentous appendages similar to those on wild-type S . typhimurium when adhering to epithelial cells, demonstrating that formation of these appendages is not itself sufficient to trigger bacterial invasion . When adhering to cell-free culture inserts, an S . typhimurium invG mutant differed from its parent strain in that it lacked even the shorter surface appendages, suggesting that SPI1 may be involved in appendage formation in the absence of epithelia . Our data on S . typhimurium strains in the presence of cells provide compelling evidence that SPI1 is not an absolute requirement for the formation of the described filamentous appendages . However, appendage formation is controlled by PhoP/PhoQ since a PhoP-constitutive mutant very rarely possessed such appendages when adhering to any of the cell types examined.

Infect Immun, 1998 May, 66(5), 1910 - 7
Genetic control of immune response to recombinant antigens carried by an attenuated Salmonella typhimurium vaccine strain: Nramp1 influences T-helper subset responses and protection against leishmanial challenge; Soo SS et al.; Attenuated strains of Salmonella typhimurium have been widely used as vehicles for delivery and expression of vaccine antigens in murine models of infectious disease . In mice, early bacterial replication following infection with S . typhimurium is controlled by the gene (Nramp1, formerly Ity/Lsh/Bcg) encoding the natural-resistance-associated macrophage protein (Nramp1) . Nramp1 regulates macrophage activation and has multiple pleiotropic effects, including regulation of tumor necrosis factor alpha, interleukin 1beta (IL-1beta), and major histocompatibility complex class II molecules, all of which influence antigen processing and presentation . Nramp1 also has a direct effect on antigen processing, possibly by regulating the activity of proteases in the late endosomal compartment . Hence, there are multiple ways (regulation of bacterial load or recombinant antigen dose, class II molecule expression, costimulatory or adjuvant activity, and antigen processing) that Nramp1 might influence responses to recombinant salmonella vaccines . To test the hypothesis that Nramp1 influences responses to vaccination, congenic mouse strains have been used to analyze immune responses to recombinant antigens (tetanus toxoid antigen and leishmanial gp63) carried by live attenuated S . typhimurium aroA aroD mutants . Results show that congenic mice carrying the wild-type (S . typhimurium resistance) Nramp1 allele mount a predominantly T-helper-1 (IL-2 and gamma interferon) response to vaccination and show enhanced resolution of lesions following challenge infection with Leishmania major . In contrast, mice carrying mutant (S . typhimurium susceptibility) Nramp1 mount a T-helper-2 (immunoglobulin E and IL-4) response and show exacerbated lesion growth upon challenge.

Science, 1998 Apr 24, 280(5363), 602 - 5
Supramolecular structure of the Salmonella typhimurium type III protein secretion system; Kubori T et al.; The type III secretion system of Salmonella typhimurium directs the translocation of proteins into host cells . Evolutionarily related to the flagellar assembly machinery, this system is also present in other pathogenic bacteria, but its organization is unknown . Electron microscopy revealed supramolecular structures spanning the inner and outer membranes of flagellated and nonflagellated strains; such structures were not detected in strains carrying null mutations in components of the type III apparatus . Isolated structures were found to contain at least three proteins of this secretion system . Thus, the type III apparatus of S . typhimurium, and presumably other bacteria, exists as a supramolecular structure in the bacterial envelope.

Proc Natl Acad Sci U S A, 1998 Mar 31, 95(7), 3949 - 53
Virulence of antibiotic-resistant Salmonella typhimurium; Bjorkman J et al.; We show that most Salmonella typhimurium mutants resistant to streptomycin, rifampicin, and nalidixic acid are avirulent in mice . Of seven resistant mutants examined, six were avirulent and one was similar to the wild type in competition experiments in mice . The avirulent-resistant mutants rapidly accumulated various types of compensatory mutations that restored virulence without concomitant loss of resistance . Such second-site compensatory mutations were more common then reversion to the sensitive wild type . We infer from these results that a reduction in the use of antibiotics might not result in the disappearance of the resistant bacteria already present in human and environmental reservoirs . Thus, second-site compensatory mutations could increase the fitness of resistant bacteria and allow them to persist and compete successfully with sensitive strains even in an antibiotic-free environment.

Proc Natl Acad Sci U S A, 1998 Mar 31, 95(7), 3495 - 500
In vitro disassembly and reassembly of an ABC transporter, the histidine permease; Liu PQ et al.; The membrane-bound complex of the Salmonella typhimurium periplasmic histidine permease, a member of the ABC transporters (or traffic ATPases) superfamily, is composed of two integral membrane proteins, HisQ and HisM, and two copies of an ATP-binding subunit, HisP . The complex hydrolyzes ATP upon induction of the activity by the liganded soluble receptor, the periplasmic histidine-binding protein, HisJ . Here we take advantage of the modular organization of this system to show that the nucleotide-binding component can be stripped off the integral membrane components, HisQ and HisM . The complex can be reconstituted by using the HisP-depleted membranes containing HisQ and HisM and pure soluble HisP . We show that HisP has high affinity for the HisP-depleted complex, HisQM, and that two HisP molecules are recruited independently of each other for each HisQM unit . The in vitro reassembled complex has entirely normal properties, responding to HisJ and ATPase inhibitors with the same characteristics as the original complex and in contrast to those of soluble HisP . These results show that HisP is absolutely required for ATP hydrolysis, that HisQM cannot hydrolyze ATP, that HisP depends on HisQM to relay the inducing signal from the soluble receptor, HisJ, and that HisQM regulates the ATPase activity of HisP . We also show that HisP changes conformation upon exposure to phospholipids.

J Mol Biol, 1998 Mar 6, 276(4), 759 - 73
Non-helical perturbations of the flagellar filament: Salmonella typhimurium SJW117 at 9.6 A resolution; Trachtenberg S et al.; Using a liquid-helium-cooled superconducting electron cryo-microscope, we obtained low-dose images of negatively stained preparations at 4 K and collected structural data to 1/9.6 -1 for flagellar filaments from the strain SJW117 of Salmonella typhimurium (serotype gt) . The subunits of this left-handed, straight filament are non-helically perturbed in a pairwise manner . The perturbation corresponds to an alternating conformation in every other row of subunits . These are the 5-start rows and, necessarily, the resulting structure has a seam . The perturbation is not confined to the outside but extends into the structure . We separated the non-symmetric and symmetric parts of the structural data and generated a three-dimensional reconstruction from the latter . The resulting density map is a structure similar in domain organization to the left-handed filament of S . typhimurium SJW1660 . Filtered images generated from the non-symmetric component show an ordered and polar structure . The nature of the perturbation was analyzed by model building using a sphere to represent the subunit at low resolution . A lateral shift of approximately 10 degrees mimics the perturbation .

J Appl Toxicol, 1998 Mar-Apr, 18(2), 129 - 42
Investigations on the in vitro and in vivo genotoxic potential of 5-vinyl-2-norbornene; Vergnes JS et al.; 5-Vinyl-2-norbornene (VNB: CAS no . 3048-64-4), an industrial chemical that produces hyaline droplet nephropathy in the male rat with associated alpha2u-globulin increases, was investigated in vitro and in vivo for its genotoxic potential . A Salmonella typhimurium reverse mutation assay (strains TA98, 100, 1535, 1537, 1538) was negative both without (dose range 0.003-0.3 mg per plate) and with (0.003-0.3 mg per plate) metabolic activation . A forward gene mutation test in Chinese Hamster Ovary (CHO) cells (HGPRT locus) did not show any significant concentration-related increases in mutation frequencies in the absence (0.01-0.1 mg ml{-1}) or presence (0.005-0.1 mg ml{-1}) of metabolic activation . In a sister chromatid exchange (SCE) test, VNB did not produce statistically significant or dose-related increases in the incidence of SCEs in the absence (0.02-0.06 mg ml{-1}) or presence (0.005-0.03 mg ml{-1}) of metabolic activation . A bone marrow chromosome aberration test was conducted in groups of 10 male and 10 female Sprague-Dawley rats exposed for 6 hr/day for 5 consecutive days to mean (+/- SD) VNB vapor concentrations of 0 (air control), 48.1 +/- 1.29, 146 +/- 9.2, or 336 +/- 8.5 ppm . Marrow was collected 6 and 24 hr after the final exposure . No statistically significant or dose-related increases in chromosomal aberrations occurred in the VNB-exposed animals . 5-Vinyl-2-norbornene did not show any potential for genotoxic activity with this in vitro-in vivo battery of tests.

J Immunol, 1998 Feb 1, 160(3), 1285 - 9
Protective effect on Leishmania major infection of migration inhibitory factor, TNF-alpha, and IFN-gamma administered orally via attenuated Salmonella typhimurium; Xu D et al.; The genes encoding murine macrophage migration inhibitory factor (MIF), IL-2, IFN-gamma or TNF-alpha were cloned individually into an expression plasmid under the control of the inducible promoter nirB and transfected into the aroA- aroD- deletion mutant strain of Salmonella typhimurium (BRD509) . These S . typhimurium derivatives (henceforward called constructs and termed GIDMIF, GIDIL2, GIDIFN and GIDTNF) expressed their respective cytokines in vitro under anaerobic conditions and stably colonized BALB/c mice up to 14 days after oral administration . The highly susceptible BALB/c mice that had received the constructs orally and that had been subsequently infected via the footpad with Leishmania major, developed significantly reduced disease compared with control mice administered the untransfected Salmonella strain (BRD509) . Importantly, a combination of GIDMIF, GIDIFN, and GIDTNF administered orally after L . major infection was able to significantly limit lesion development and reduced parasite loads by up to three orders of magnitude . Spleen and lymph node cells of mice administered this combination expressed markedly higher levels of inducible nitric oxide synthase (iNOS) compared with those from mice receiving an equivalent dose of the control strain of Salmonella (BRD509) . These data therefore demonstrate the feasibility of therapeutic treatment in an infectious disease model using cytokines delivered by attenuated Salmonella . The protective effect observed correlates with the induction of inducible nitric oxide synthase in vivo.

Mol Microbiol, 1998 Mar, 27(6), 1171 - 82
PmrA-PmrB-regulated genes necessary for 4-aminoarabinose lipid A modification and polymyxin resistance; Gunn JS et al.; Antimicrobial peptides are distributed throughout the animal kingdom and are a key component of innate immunity . Salmonella typhimurium regulates mechanisms of resistance to cationic antimicrobial peptides through the two-component systems PhoP-PhoQ and PmrA-PmrB . Polymyxin resistance is encoded by the PmrA-PmrB regulon, whose products modify the lipopolysaccharide (LPS) core and lipid A regions with ethanolamine and add aminoarabinose to the 4' phosphate of lipid A . Two PmrA-PmrB-regulated S . typhimurium loci (pmrE and pmrF) have been identified that are necessary for resistance to polymyxin and for the addition of aminoarabinose to lipid A . One locus, pmrE, contains a single gene previously identified as pagA (or ugd) that is predicted to encode a UDP-glucose dehydrogenase . The second locus, pmrF, is the second gene of a putative operon predicted to encode seven proteins, some with similarity to glycosyltransferases and other complex carbohydrate biosynthetic enzymes . Genes immediately flanking this putative operon are also regulated by PmrA-PmrB and/or have been associated with S . typhimurium polymyxin resistance . This work represents the first identification of non-regulatory genes necessary for modification of lipid A and subsequent antimicrobial peptide resistance, and provides support for the hypothesis that lipid A aminoarabinose modification promotes resistance to cationic antimicrobial peptides.

Mol Microbiol, 1998 Mar, 27(6), 1129 - 39
Flagellar filament elongation can be impaired by mutations in the hook protein FlgE of Salmonella typhimurium: a possible role of the hook as a passage for the anti-sigma factor FlgM; Saito T et al.; Among motile revertants isolated from flagellar hook-deficient (flgE) mutants of Salmonella typhimurium, one produced only short flagellar filaments in L broth, despite the fact that flagellin itself has the ability to polymerize into long filaments in vitro . This pseudorevertant has an intragenic suppressor, resulting in a two-amino-acid substitution (Asp-Gln-->Ala-Arg) in the C-terminal region of the hook protein, FlgE . The flagellation of the pseudorevertant was greatly affected by the concentration of NaCl in the culture media: we observed no filaments in the absence of NaCl, short filaments in 1% NaCl and full-length filaments in 2% NaCl . Electron microscopy of osmotically shocked cells showed that the number of hook-basal bodies on cells was constant under various NaCl conditions . Furthermore, we found that the mutant hook was straight rather than curved . We monitored the cellular flagellin level of this pseudorevertant under various NaCl concentrations by immunoblotting . It was revealed that little flagellin was present under NaCl-free conditions in contrast with the ordinary amounts of flagellin present in 2% NaCl . As the expression of flagellin is regulated by competitive interaction of a sigma factor, FliA, and a corresponding anti-sigma factor, FlgM, we also observed the effect of NaCl on the secretion of FlgM . FlgM was secreted into the media in more than 1% NaCl but accumulated inside the cells in the absence of NaCl, indicating that the failure of secretion of FlgM in the absence of salt was the cause of the impaired elongation of filaments.

Mol Microbiol, 1998 Mar, 27(6), 1099 - 105
Non-genetic population heterogeneity studied by in situ polymerase chain reaction; Tolker-Nielsen T et al.; Expression of a lac operon in Salmonella typhimurium single cells was monitored using lac mRNA targeting in situ reverse transcription-polymerase chain reaction (RT-PCR) . It is demonstrated that suboptimal induction of the lac operon in a culture of S . typhimuriuml/F'lac+ cells generates a subpopulation in which transcription of the lac operon occurs and another subpopulation in which transcription of the lac operon is repressed, whereas suboptimal induction of the lac operon in a culture of S . typhimuriuml/F'lacY cells generates a population with uniform levels of lac mRNA . The outcome of the single-cell lac mRNA detection assay was compared with the outcome of a single-cell beta-galactosidase assay . In cultures grown under different suboptimal lac induction conditions, the fraction of cells in which transcription of the lac operon occurred was concurrent with the fraction of cells showing beta-galactosidase activity . Besides supporting the hypothesis that the lactose permease has a role in generating non-genetic heterogeneity in suboptimally induced cultures of Lac+ cells, these results demonstrate the usefulness of in situ RT-PCR for the study of non-genetic population heterogeneities.

Cancer Lett, 1997 Nov 25, 120(1), 117 - 23
Potent suppressing activity of the non-polyphenolic fraction of green tea (Camellia sinensis) against genotoxin-induced umu C gene expression in Salmonella typhimurium (TA 1535/pSK 1002)--association with pheophytins a and b; Okai Y et al.; Antigenotoxic and antimutagenic activities of green tea extract and tea-derived polyphenols have been studied using in vitro and in vivo experiments . However, antigenotoxic substances in the non-polyphenolic fraction of green tea have been poorly elucidated . In the present study, the effect of the non-polyphenolic fraction of green tea on genotoxin-induced umu C gene expression was analyzed using a tester bacteria, and potent antigenotoxic substances in the non-polyphenolic fraction were identified . The non-polyphenolic fraction of green tea showed strong suppressive activities against umu C gene expression in Salmonella typhimurium (TA 1535/pSK 1002) induced by 3-amino-1,4-dimethyl-5H-pyrido{4,3-b}indol (Trp-P-1) or mitomycin C (MMC) in the presence or absence of S9 metabolizing enzyme mixture . The non-polyphenolic fraction of green tea exhibited major two-color bands in a silica gel TLC and they were identified as chlorophyll-related compounds, pheophytins a and b, judged by their specific colors, Rf values in silica gel TLC and absorption spectra . These pigments showed significant suppressive activities against umu C gene expression in tester bacteria induced by Trp-P- and MMC in a dose-dependent manner . These results suggest that the non-polyphenolic fraction of green tea contains pheophytins a and b as potent antigenotoxic substances.

Mutagenesis, 1998 Mar, 13(2), 181 - 5
Microsomal metabolism and activation of the environmental carcinogen 2-amino-3-methyl-9H-pyrido{23-b}indole; Frandsen H et al.; 2-Amino-3-methyl-9H-pyrido{2,3-b}indole (MeA alpha C) is a mutagenic and carcinogenic heterocyclic amine formed as a pyrolysis product during cooking of food and combustion of tobacco . Hepatic microsomes from PCB-induced rats metabolized MeA alpha C to four products, of which three were non-mutagenic and one was mutagenic without S9 activation . The three non-mutagenic products, which accounted for 83% of the metabolism of MeA alpha C, were characterized by mass spectrometry and NMR spectroscopy as 6-hydroxy-MeA alpha C, 7-hydroxy-MeA alpha C and 3-hydroxy-methyl-A alpha C . The mutagenic metabolite, accounting for 17% of the metabolism of MeA alpha C, was characterized as N2-hydroxy-MeA alpha C by comparison with the HPLC retention time and UV spectrum of N2-hydroxy-MeA alpha C obtained by chemical synthesis . N2-Hydroxy-MeA alpha C was very reactive and a part of it bound covalently to microsomal proteins during incubation and a part was degraded to other products during incubation or chromatography . N2-Hydroxy-MeA alpha C was mutagenic in Salmonella typhimurium TA98 without metabolic activation, resulting in 5070 revertants/microgram, which was > 20 times the specific mutagenic activity of the parent compound.

Mutagenesis, 1998 Mar, 13(2), 157 - 66
Comparative investigation on the mutagenicities of organophosphate, phthalimide, pyrethroid and carbamate insecticides by the Ames and lactam tests; Hour TC et al.; The Salmonella lactam test is a newly developed method for detecting genotoxins . This technique is based on the ability of DNA damaging agents to reverse expression of the beta-lactamase gene, an important gene that enables microbes to resist beta-lactam antibiotics . A construct p-SELECT Control DNA plasmid containing a beta-lactamase gene site was constructed in many mutant forms, including point and frameshift mutants . These mutant constructs were introduced into Salmonella tester strains whose mutagenicity is based on their ability to reverse expression of the beta-lactamase gene . Fourteen pesticides were evaluated for genotoxicity using our newly developed Salmonella typhimurium strains JK947 and JK3, which are useful for detecting base substitution mutations . Six pesticides, namely allethrin, captan, folpet, monocrotophos, acephate and carbofuran, proved highly mutagenic in strain JK947, while the first four pesticides were more weakly mutagenic in strain JK3 . In comparison, results from the Ames test show strain JK947 to be more sensitive to these pesticides than strains TA100 and TA1535 . Strains TA98 and JK1 proved insensitive to allethrin, captan, folpet, acephate, carbofuran and monocrotophos . Among the many advantages of the lactam test are: large numbers of cells can treated and the test is operationally simple and inexpensive; revertant colonies form faster in the lactam test (16 h) than in the Ames test (48 h); the lactam test can detect mutagens present in biological specimens contaminated by histidine and biotin, samples that may give false positive results in the Ames test.

Chem Biol Interact, 1998 Feb 20, 109(1-3), 255 - 65
Inhibitory effect of hemin on mutagenicity of the electrophilic sulfuric acid ester of 6-hydroxymethylbenzo{a}pyrene; Hong ST et al.; In the present study, we examined the effects of hemin on the mutagenicity of 6-sulfooxymethylbenzo{a}pyrene (SMBP) in Salmonella typhimurium TA98 and Chinese hamster lung fibroblast (V79) cells . The compound was tested for the possible chemoprotective activity against mutagenesis induced by SMBP and its precursor, 6-hydroxymethylbenzo{a}pyrene (HMBP), activated by hepatic cytosol and PAPS in S . typhimurium TA98 . Hemin not only inhibited the mutagenic activity of SMBP in V79 cells but repressed the cytotoxicity induced by this reactive ester as demonstrated by increased cell growth . The intracellular accumulation of radioactivity in V79 cells exposed to {3H}SMBP was reduced by approximately 50% when hemin (10 microM) was added to the medium . Likewise, the formation of SMBP-DNA adducts in these cells was significantly attenuated by treatment with hemin . The covalent complex formation of hemin with SMBP was confirmed by solvent extraction and reverse-phase HPLC.

Chem Biol Interact, 1998 Feb 20, 109(1-3), 249 - 53
Expression of human estrogen sulfotransferase in Salmonella typhimurium: differences between hHST and hEST in the enantioselective activation of 1-hydroxyethylpyrene to a mutagen; Hagen M et al.; Various human sulfotransferases (hP-PST, hM-PST, hHST) and rat sulfotransferases (rPST-IV, rHSTa) have already been expressed in Ames' Salmonella strains (in particular in TA1538) . Now a further strain, TA1538-hEST, which expresses the human estrogen sulfotransferase (hEST), has been constructed . This strain activated the primary benzylic alcohol 1-hydroxymethylpyrene (1-HMP) and the secondary benzylic alcohol 1-hydroxyethylpyrene (1-HEP) to mutagens . Human sulfotransferases hEST and hHST both activated 1-HEP, but they differed substantially in their enantioselectivity for this compound.

Chem Biol Interact, 1998 Feb 20, 109(1-3), 195 - 219
Sulfotransferase-mediated activation of mutagens studied using heterologous expression systems; Glatt H et al.; Sulfation is a common final step in the biotransformation of xenobiotics and is traditionally associated with inactivation . However, the sulfate group is electron-withdrawing and may be cleaved off heterolytically in some molecules leading to electrophilic cations which may form adducts with DNA and other important cellular structures . Since endogenous sulfotransferases do not appear to be expressed in indicator cells of standard mutagenicity tests, rat and human sulfotransferases have been stably expressed in his- Salmonella typhimurium strain TA1538 and Chinese hamster V79 cells . Using these recombinant indicator cells, sulfotransferase-dependent genotoxic activities were detected with N-hydroxy-2-acetylaminofluorene, 2-acetylaminofluorene (in the presence of co-expressed rat cytochrome P450 1A2), hycanthone, 1'-hydroxysafrole, alpha-hydroxytamoxifen and various benzylic alcohols derived from polycyclic aromatic hydrocarbons . In several cases, it was critical that the reactive sulfuric acid conjugates were formed directly within the indicator cells, owing to the inefficient penetration of cell membranes . In other cases, spontaneous benzylic substitution reactions with medium components, such as halogenide ions or amino acids, led to secondary, membrane-penetrating reactive species . Different sulfotransferases, including related forms from rat and human, substantially differed in their substrate specificity towards the investigated promutagens . It is known that some sulfotransferases are expressed with high tissue and cell type specificities . This site-dependent expression together with the limitations in the distribution of reactive sulfuric acid conjugates may explain organotropic effects of compounds activated by this metabolic pathway.

FEMS Immunol Med Microbiol, 1998 Mar, 20(3), 191 - 9
Macrophages recognize and adhere to an OmpD-like protein of Salmonella typhimurium; Negm RS et al.; Murine peritoneal macrophages bind to Salmonella typhimurium in vitro in the absence of exogenous opsonins . We have identified an outer membrane protein of S . typhimurium that mediates this adhesion . Biotin-labeled macrophages were used to probe electroblotted envelope proteins of S . typhimurium that had been previously resolved by polyacrylamide electrophoresis under denaturing and reducing conditions . Macrophages bound to an outer membrane protein with an apparent molecular mass of 44 kDa . The protein was purified to homogeneity and free of detectable lipopolysaccharide . Limited microsequencing of this protein resulted in a 15-amino acid query sequence of A-E-V-Y-N-K-D-G-N-K-L-D-L-Y-G, which shares complete identity with a 15-mer of both the OmpD of S . typhimurium SH 7454 and the OmpC polypeptide of Escherichia coli K-12 . Picomolar concentrations of this purified protein significantly inhibited the subsequent adherence of 35S-labeled S . typhimurium to macrophages in monolayers . We propose that this 44-kDa protein is involved in the recognition of S . typhimurium by macrophage during the initial stages of infection.

FEMS Immunol Med Microbiol, 1998 Mar, 20(3), 175 - 80
Salmonella infection-induced non-responsiveness of murine splenic T-lymphocytes to interleukin-2 (IL-2) involves inhibition of IL-2 receptor gamma chain expression; Matsui K et al.; In a previous study we demonstrated that Salmonella typhimurium-induced immunosuppression involved T-cell non-responsiveness to interleukin-2 (IL-2) . In this study we observed that Salmonella-induced T-cell non-responsiveness to IL-2 was not reversed completely by treatment with N(G)-monomethyl-L-arginine, which is known to inhibit nitric oxide (NO) secretion by macrophages in culture . Furthermore, when purified splenic T-lymphocytes from Salmonella-infected mice were activated with an anti-CD3 antibody, the responsiveness of these T-cells to IL-2 was suppressed significantly . Results of flow cytometric analysis using an anti-IL-2 receptor gamma chain (IL-2Rgamma) antibody showed that IL-2Rgamma expression in mitogen-activated T-cells was down-regulated by Salmonella infection . These results suggest that Salmonella infection-induced T-cell non-responsiveness to IL-2 involves a defective function of T-cells themselves and appears to be regulated by inhibition of IL-2Rgamma expression in T-cells.

Chem Res Toxicol, 1998 Apr, 11(4), 375 - 80
Chemical synthesis of a novel aromatic amine mutagen isolated from water of the Nishitakase River in Kyoto and a possible route of its formation; Shiozawa T et al.; Among five mutagenic compounds isolated from water samples, taken at sites below the sewage plants of the Nishitakase River in Kyoto, Japan, the structure of compound I has been determined to be 2-{2-(acetylamino)-4-{bis(2-methoxyethyl)amino}-5-methoxyphenyl}-5-am ino-7-bromo-4-chloro-2H-benzotriazole (PBTA-1) . Since this novel aromatic amine mutagen has characteristic substituents in its molecule, it is postulated that the azo dye, 2-{(2-bromo-4, 6-dinitrophenyl)azo}-4-methoxy-5-{bis(2-methoxyethyl)amino}acetoanili de (AZO DYE-1), used as an industrial material, is converted to the corresponding 2-phenylbenzotriazole derivative with a reducing reagent and subsequently to PBTA-1 by chlorination . In fact, AZO DYE-1 changed to the dechlorinated derivative of PBTA-1 (deClPBTA-1) on treatment with sodium hydrosulfite, and this reacted with sodium hypochlorite to produce PBTA-1 . Moreover, the presence of deClPBTA-1 was confirmed in a river water sample, along with PBTA-1 . PBTA-1 showed potent mutagenic activities in Salmonella typhimurium TA98 and YG1024, inducing 88 000 and 3 000 000 revertants, respectively, per microg, with S9 mix . deClPBTA-1 was also mutagenic, but less potent . From these observations, it is suggested that PBTA-1 is produced from AZO DYE-1 through deClPBTA-1, during industrial processes at dyeing factories and the treatment of wastewater at sewage plants.

Biochemistry, 1998 Mar 24, 37(12), 4181 - 8
Conversion of a cosubstrate to an inhibitor: phosphorylation mutants of nicotinic acid phosphoribosyltransferase; Rajavel M et al.; Nicotinic acid phosphoribosyltransferase (NAPRTase; EC 2.4.2.11) forms nicotinic acid mononucleotide (NAMN) and PPi from 5-phosphoribosyl 1-pyrophosphate (PRPP) and nicotinic acid (NA) . The Vmax NAMN synthesis activity of the Salmonella typhimurium enzyme is stimulated about 10-fold by ATP, which, when present, is hydrolyzed to ADP and Pi in 1:1 stoichiometry with NAMN formed . The overall NAPRTase reaction involves phosphorylation of a low-affinity form of the enzyme by ATP, followed by generation of a high-affinity form of the enzyme, which then binds substrates and produces NAMN . Hydrolysis of E-P then regenerates the low-affinity form of the enzyme with subsequent release of products . Our earlier studies {Gross, J., Rajavel, M., Segura, E., and Grubmeyer, C . (1996) Biochemistry 35, 3917-3924} have shown that His-219 becomes phosphorylated in the N1 (pi) position by ATP . Here, we have mutated His-219 to glutamate and asparagine and determined the properties of the purified mutant enzymes . The mutant NAPRTases fail to carry out ATPase, autophosphorylation, or ADP/ATP exchanges seen with wild-type (WT) enzyme . The mutants do catalyze the slow formation of NAMN in the absence of ATP with rates and KM values similar to those of WT . In striking contrast to WT, NAMN formation by the mutant enzymes is competitively inhibited by ATP . Thus, the NAMN synthesis reaction may occur at a site overlapping that for ATP . Previous studies suggest that the yeast NAPRTase does not catalyze NAMN synthesis in the absence of ATP . We have cloned, overexpressed, and purified the yeast enzyme and report its kinetic properties, which are similar to those of the bacterial enzyme.

Am J Vet Res, 1998 Apr, 59(4), 416 - 20
Prevention of fatal salmonellosis in neonatal calves, using orally administered chicken egg yolk Salmonella-specific antibodies; Yokoyama H et al.; OBJECTIVE: To protect neonatal calves against fatal salmonellosis within the first 2 weeks after birth, using chicken egg yolk antibodies specific against Salmonella typhimurium or S dublin . ANIMALS: 38 neonatal Holstein calves from Salmonella-free farms . PROCEDURE: After removal of the lipid components with hydroxypropylmethylcellulose phthalate, egg yolk antibodies were spray dried . At 4 days of age, calves were challenge exposed by oral inoculation with 10(11) virulent S typhimurium (experiment 1) or S dublin (experiment 2) . Starting from the challenge-exposure day, egg yolk antibody preparations were administered orally 3 times a day for 7 to 10 days . RESULTS: In passive immunization trials, the orally administered antibodies conferred dose-dependent protection against infection with each of the homologous strains of Salmonella . Within 7 to 10 days after challenge exposure, all control calves died, whereas low-titer antibody-treated calves had 60 to 100% mortality . Only fever and diarrhea, but no deaths (P < 0.01), were observed in calves given the highest titer of antibody . CONCLUSIONS AND CLINICAL RELEVANCE: Compared with that in control calves, survival was significantly higher among calves given antibodies with titers of 500 (P < 0.05) and 1,000 (P < 0.01) homotypic for S typhimurium and with titer of 5,000 (P < 0.01) for S dublin . Egg yolk antibodies specific for whole cell S typhimurium or S dublin are protective against fatal salmonellosis when given in sufficiently high concentration, and may be clinically useful during a salmonellosis outbreak.

Mol Gen Genet, 1998 Mar, 257(5), 529 - 33
Relief of transcriptional polarity by a mutation that creates a promoter in the hisG gene of Salmonella typhimurium LT2; Bianco R et al.; Six mutant strains were independently isolated in Salmonella typhimurium as non-polar revertants of a polar Tn10 insertion in hisG . DNA sequence analysis showed that all six mutants result from the same nucleotide alteration: a G/C to A/T transition 4 bp from the end of the hisG coding sequence . We present data suggesting that the mutation (hisG10225) acts by creating a new transcriptional initiation signal . Furthermore, the hisG10225 mutation renders the hisG gene product cold sensitive.

Vet Parasitol, 1998 Jan 31, 74(2-4), 179 - 89
Depressed immunity to a Salmonella typhimurium vaccine in mice experimentally parasitized by Taenia crassiceps; Rubio M et al.; To assess the immunological status of mice parasitized with Taenia crassiceps metacestodes, 6-month old female BALB/c mice experimentally parasitized with T . crassiceps and immunized with Salmonella typhimurium antigens were infected with S . typhimurium virulent bacilli (1.6 x LD50) . Both T . crassiceps-parasitized and immunized and parasitized mice showed a very high susceptibility to infection (**P < 0.01) with higher bacteremia than control and immunized-control animals and produced a reduced IgG response to S . typhimurium, antigens (* P < 0.05) . This indicates that T . crassiceps is able to preclude development of immunity to S . typhimurium, because appropriate antibody production to a heterologous antigenic stimulus did not take place, and the bacteremia results suggest the parasitosis altered the mononuclear phagocyte system . It has been demonstrated that Taenia solium metacestodes produce a small RNA molecule in culture which suppresses humoral and cellular responses against homologous antigens in mice . We propose that T . crassiceps may be actively synthesizing such a factor, apart from other simultaneously acting immunomodulatory mechanisms, to induce an immunosuppressed state favorable to its development in the host.

Roum Arch Microbiol Immunol, 1997 Jan-Jun, 56(1-2), 17 - 26
Experimental studies on bacterial product CANTASTIM derived from Pseudomonas aeruginosa . II . Protective effect in Salmonella typhimurium infection; Salageanu A et al.; Stimulation of the host defense system in a nonspecific way may provide effective treatment of recurrent infections . CANTASTIM is a bacterial product that has been successfully used in cancer immunotherapy as well as in chronic infections treatment . The nonspecific protective effect of CANTASTIM was investigated in two models of experimental infection with Salmonella typhimurium in mice . Prophylactic administration of CANTASTIM (three days before challenge) enhanced peritoneal macrophages bactericidal activity and significantly increased survival of treated mice . When CANTASTIM was administered 72 h after bacterial challenge, in a sublethal infection model with Salmonella typhimurium, by activating macrophages, NK and T cells, it increased the survival rate . The cell populations and molecular mechanisms involved in the prophylactic and therapeutic protective effect CANTASTIM seem to be partially different.

Am J Epidemiol, 1998 Apr 15, 147(8), 774 - 82
Epidemiology of Salmonella typhimurium O:4-12 infection in Norway: evidence of transmission from an avian wildlife reservoir; Kapperud G et al.; In 1987, a nationwide outbreak of Salmonella typhimurium O:4-12 infection traced to contaminated chocolate bars occurred in Norway . In the 5 years after the outbreak, elevated numbers of sporadic cases caused by the epidemic strain of Salmonella were detected, followed by a decline in subsequent years . To characterize the epidemiology of this infection, the authors analyzed information concerning all sporadic cases reported in Norway from 1966 to 1996 . Of the 153 patients infected by the outbreak strain, 43% were less than 5 years of age, and only three persons had acquired the infection abroad . In contrast, 46% of the cases attributable to other S . typhimurium O:4-12 variants and 90% of the total number of Salmonella infections were related to foreign travel . A distinct seasonality was observed: 76% of the cases appeared between January and April . At the same time of year, the epidemic strain was regularly encountered as the etiologic agent of fatal salmonellosis among wild passerine birds, suggesting an epidemiologic link between the avian and human cases . The strain was rarely isolated from other sources . From 1990 to 1992, the authors conducted a prospective case-control study of sporadic indigenous infections to identify risk factors and obtain guidance for preventive efforts . Forty-one case-patients, each matched by age, sex, and geographic area with two population controls, were enrolled . In conditional logistic regression analysis, the following environmental factors were independently related to an increased risk of infection: drinking untreated water, having direct contact with wild birds or their droppings, and eating snow, sand, or soil . Cases were also more likely than controls to report having antecedent or concurrent medical disorders . Forty-six percent of the study patients were hospitalized for their salmonellosis.

J Immunol, 1998 Jan 1, 160(1), 455 - 66
Apical secretion of a pathogen-elicited epithelial chemoattractant activity in response to surface colonization of intestinal epithelia by Salmonella typhimurium; McCormick BA et al.; Modeling Salmonella-epithelial cell interaction in vitro has led to the realization that epithelial cells are crucial in orchestrating neutrophil (PMN) responses, in part by stimulating basolateral release of epithelial chemokines, including IL-8 . However, such basolaterally released chemokines, while likely important in orchestration of PMN movement across the subepithelial matrix, are unlikely to be responsible for the final step of transepithelial migration of PMN and entry into the apical compartment . We now show that S . typhimurium attachment to T84 cell apical epithelial membranes induces polarized apical secretion of a pathogen-elicited epithelial chemoattractant (PEEC) bioactivity . Experiments employing semipurified PEEC indicate that it is released in a polarized apical fashion and is sufficient to explain the observed final step of transepithelial migration of PMN induced by Salmonella-apical membrane interaction . By preliminary physical characterization and profiles of PMN activation, PEEC appears to be a novel PMN chemotactic bioactivity . This 1- to 3-kDa nominal molecular mass chemokine-like bioactivity directly stimulates PMN via a pertussis toxin-sensitive receptor and elicits a Ca2+ signal . While these latter features are shared by most other chemokines, analysis of PEEC-elicited PMN activation reveals that, unlike these other agonists, PEEC, even at saturating concentrations, elicits chemotactic activity in the absence of stimulation of superoxide production and/or release of primary and/or secondary granules . These data suggest that the apically released PEEC activity appears to represent a novel epithelial-derived chemoattractant that directs PMN movement across epithelial monolayers.

J Immunol, 1998 Jan 1, 160(1), 449 - 54
Regulation of chemokine gene expression in human peripheral blood neutrophils phagocytosing microbial pathogens; Hachicha M et al.; Production of chemokines (chemotactic cytokines) by neutrophils is likely to be important in the regulation of inflammation and the control of infection . In this study we show that exposure of human neutrophils to various microbial pathogens leads to the production of both macrophage inflammatory protein 1alpha (MIP-1alpha) and IL-8 . The bacterial microbes, Salmonella typhimurium and Pseudomonas aeruginosa, and Staphylococcus aureus all strongly induced both IL-8 and MIP-1alpha secretion, whereas Streptococcus pneumoniae, Staphylococcus epidermidis, and the opportunistic yeast Candida albicans were less potent . Saccharomyces cerevisiae and zymosan both induced IL-8 secretion but failed to stimulate that of MIP-1alpha . Coincubation of neutrophils with the proinflammatory cytokine TNF-alpha and the micro-organisms also led to differential expression of MIP-1alpha and IL-8 . Significant enhancement of the induction of both MIP-1alpha and IL-8 by S . typhimurium, P . aeruginosa, and S . pneumoniae as well as by C . albicans was observed . In contrast, while IL-8 production in response to S . cerevisiae and zymosan was enhanced in the presence of TNF-alpha, no MIP-1alpha was produced . These combined results indicate that while neutrophils exposed to some micro-organisms alone or in the presence of inflammatory cytokines such as TNF-alpha will produce both MIP-1alpha and IL-8, resulting in generation of signals for the recruitment of mononuclear leukocytes and neutrophils, respectively, certain types of microorganisms can skew this response toward synthesis of IL-8.

EMBO J, 1998 Apr 15, 17(8), 2359 - 67
The supercoiling sensitivity of a bacterial tRNA promoter parallels its responsiveness to stringent control; Figueroa-Bossi N et al.; In Salmonella typhimurium, expression of the hisR locus, a tRNA operon, decreases upon inhibiting DNA gyrase . Here, the hisR promoter dependence on negative DNA supercoiling was examined in vivo and in vitro . Mutant analysis showed the sequence determinants of this dependence to lie in the region between the -10 box and the transcription start site . As with most promoters subject to stringent control, this portion of the hisR promoter is C-G-rich . Replacing a C/G bp with T/A at position -7 partially relieves the supercoiling response while changing the sequence between -5 and + 1 (-CCCCCG-) for -GTTAA- abolishes the response in vitro and in vivo . The relief of the supercoiling dependence closely correlates with increased promoter susceptibility to melting in vivo and a lesser requirement for initiating nucleotides in the formation of stable initiation complexes in vitro . Studies in isoleucine-starved cells showed that such sequence changes mitigate and abolish the hisR promoter response to stringent control, respectively . The data presented suggest that the hisR promoter's sensitivity to stringent regulation arises from the same physical property that confers supercoiling sensitivity, i.e . resistance to melting . We propose that the stringent control mechanism acts by hampering the ability of RNA polymerase to melt the DNA helix.

Vet Rec, 1998 Mar 7, 142(10), 235 - 9
Investigations into field cases of porcine colitis with particular reference to infection with Serpulina pilosicoli; Thomson JR et al.; Investigations into the possible causes of colitis and typhlocolitis were carried out on 85 pig units in the United Kingdom between 1992 and 1996 . Serpulina pilosicoli was identified most commonly, occurring as the suggested primary agent on 21 (25 per cent) of the units but forming part of mixed infections on another 23 (27 per cent) of the units, the main co-infections being Yersinia pseudotuberculosis (eight units), proliferative enteropathy (six units), Salmonella species (four units) or Serpulina hyodysenteriae (two units) . 'Atypical' Serpulina species, S hyodysenteriae, Salmonella typhimurium, Y pseudotuberculosis and Lawsonia intracellularis (proliferative enteropathy) were the suggested primary agents on seven, six, four, four and three units, respectively . Various combinations of mixed infections involving the latter organisms and other possibly incidental agents were recorded on another 10 units . Investigations on a further six units failed to detect any recognised pathogens . On units where S pilosicoli was the suggested primary agent, pigs ranging between 20 to 40 kg (eight to 16 weeks of age), but occasionally up to 50 kg, had diarrhoea and grew poorly over a period of two to three weeks . The prevalence was estimated to be between 5 and 15 per cent in affected batches, with a mortality of approximately 1 per cent . The clinical signs usually developed seven to 14 days after the moving and mixing of pigs . At postmortem examination, affected pigs had liquid contents in their colon, which contained accumulations of mucus in some chronic cases . Gross and histological lesions of colitis were prominent in the mid-spiral region of the colon . In mixed infections with Y pseudotuberculosis, Salmonella typhimurium or S hyodysenteriae, lesions were more extensive and affected the caecum as well as the colon . In the colon, lesions of proliferative enteropathy were usually confined to the proximal half of the ascending spiral but mixed infection with S pilosicoli caused more extensive colitis . Mixed infections were reported to prolong the time taken for pigs to recover naturally and to have a more detrimental effect on growth rates than S pilosicoli infection alone . Despite the successful treatment of batches of pigs with tiamulin or lincomycin, S pilosicoli infection persisted as a chronic problem on many units, with diarrhoea and colitis in successive batches of pigs unless prophylactic medication was used.

Mutat Res, 1998 Feb 2, 397(2), 293 - 301
Synthesis, metabolism and structure-mutagenicity relationships of novel 4-nitro-(imidazoles and pyrazoles) in Salmonella typhimurium; Hrelia P et al.; A new series of 4-nitro-(imidazoles and pyrazoles) were synthesized as novel antimycotics and tested for their activation to mutagenic forms using Salmonella typhimurium TA98 and TA100, in the presence and in the absence of metabolic activation . TA100NR, TA100/1,8-DNP6, YG1026 and YG1029 strains were employed to identify a specific metabolic reaction which governs the mutagenic potency . Derivatives in the pyrazole group were generally found to be non mutagenic and active imidazoles were weak-direct-acting mutagens . For most of the compounds the mutagenic responses in TA98 were absent or 12- to 22-fold lower compared to TA100 . The presence of a methyl or a benzylic group on the imidazole ring and substituents on the N1 and N3 positions were determinant for mutagenicity . Metabolism by bacterial enzyme systems was important to the expression of genotoxicity . Active compounds showed no mutagenicity toward the strain defective in classical nitroreductase and increased mutagenicity, from 2- to 7-fold depending on the test compound, toward the corresponding overproducing bacteria . On the other hand, compounds displayed reduced mutagenicity to the O-acetyltransferase strain without having increased activity in the corresponding overproducing bacteria, YG1029.

Mutat Res, 1998 Feb 2, 397(2), 263 - 9
Mutagenicity of 6-sulfooxymethylbenzo{a}pyrene in Salmonella typhimurium and Chinese hamster V79 cells; Cho YS et al.; 6-Sulfooxymethylbenzo{a}pyrene (SMBP) is an ultimate and reactive form of 6-hydroxymethybenzo{a}pyrene (HMBP), which is converted into SMBP by the mediation of sulfotransferase . SMBP and HMBP with metabolic activation were mutagenic to S . typhimurium TA98 and TA100 . The number of mutation per plate in strain TA98 was proportional to the concentrations of SMBP ranging from 0.2 to 1.0 nmol/plate, whereas that in strain TA100 was decreased at concentrations above 0.6 nmol/plate . The mutation frequencies by HMBP was also increased in a dose dependent manner in both strains . Furthermore, SMBP and HMBP were highly mutagenic and cytotoxic to Chinese hamster lung fibroblast (V79) cells . A dose-dependent increase in mutation frequencies at both hypoxanthine:guanine phosphoribosyltransferase (HGPRT) and sodium/potassium-ATPase (Na/K-ATPase) loci were found in V79 cells treated with SMBP and HMBP . The cytotoxicity of SMBP was increased with the increasing concentrations up to 2.5 microM, where the survival frequency and growth rate were decreased to almost 40% and 30% of the control value, respectively . The survival frequencies of V79 cells by HMBP were also decreased in a dose dependent manner up to 180 microM as similar to those of SMBP but the effects were less remarkable . SMBP was progressively accumulated in V79 cells, reaching plateau in just 30 min . A dose dependent increase in complex formation with DNA or proteins was observed by treatment with SMBP . The mutagenicity and cytotoxicity of SMBP and HMBP may be derived from their binding capacity to DNA in V79 cells and S . typhimurium.

J Immunol, 1997 Dec 1, 159(11), 5550 - 9
Involvement of mitogen-activated protein kinase pathways in the nuclear responses and cytokine production induced by Salmonella typhimurium in cultured intestinal epithelial cells; Hobbie S et al.; Central to the pathogenesis of Salmonella typhimurium is its ability to engage the host cell in a two-way biochemical interaction . As a consequence of this interaction, a dedicated protein secretion system, termed type III, is activated in these bacteria and directs the translocation of signaling proteins into the host cell . Secretion of these proteins stimulates host cell signal transduction pathways that lead to a variety of cellular responses . An important feature of S . typhimurium pathogenesis is the induction of a profound inflammatory response in the intestinal epithelium . In this report, we show that S . typhimurium induces host cell signal transduction pathways that lead to the activation of the transcription factors NF-kappaB and AP-1, resulting in the production of proinflammatory cytokines such as IL-8 . We also show that S . typhimurium infection of cultured intestinal epithelial cells results in the activation of the mitogen-activated protein (MAP) kinases ERK, JNK, and p38 . Induction of these signaling pathways and the synthesis of IL-8 was strictly dependent on the function of the invasion-associated type III protein secretion system encoded by S . typhimurium . Pretreatment of cells with the highly specific p38 MAP kinase inhibitor SB 203580 prevented S . typhimurium-induced IL-8 production . These results indicate that the inflammatory response induced by S . typhimurium may be due to the specific stimulation of MAP kinase signaling pathways leading to nuclear responses.

Mol Cell Biochem, 1998 Jan, 178(1-2), 345 - 52
Intestinal mucosal lipid peroxidation and absorptive function in Salmonella typhimurium mediated intestinal infection; Mehta A et al.; S . typhimurium infection is associated with neutrophil infiltration within the intestinal mucosa . Neutrophil activation provides a major source of reactive oxygen species (ROS) . The mucosal pathology of S . typhimurium infection may be in part due to the excessive production of these reactive species . This study was carried out to investigate if ROS play a role in mediating the changes in the structural components and functional properties of brush border membrane (BBM) in rats during S . typhimurium infection . This was done by determining the changes in the BBM extent of lipid peroxidation and absorptive function . A significant increase in the extent of lipid peroxidation of BBM during S . typhimurium infection was observed as judged by malondialdehyde (MDA) and conjugated diene formation and depletion of alpha-tocopherol and protein associated thiol groups . A significant decrease in the BBMV (brush border membrane vesicle) transport of amino acids was also observed . However there was no change in the transport of D-glucose . The decrease in amino acid transport further led to a significant decrease in the enterocyte level of protein synthesis . Exposure of BBMV to a free radical donor, cumene hydroperoxide, also led to an increase in the extent of lipid peroxidation and a decrease in the amino acid transport . Possibly ROS might play a significant role in mediating the mucosal damage during S . typhimurium infection.

Appl Environ Microbiol, 1998 Apr, 64(4), 1323 - 7
A competitive microflora increases the resistance of Salmonella typhimurium to inimical processes: evidence for a suicide response; Aldsworth TG et al.; The presence of a viable competitive microflora at cell densities of 10(8) CFU ml-1 protects an underlying population of 10(5) CFU of Salmonella typhimurium ml-1 against freeze injury . The mechanism of enhanced resistance was initially postulated to be via an RpoS-mediated adaptive response . By using an spvRA:: luxCDABE reporter we have shown that although the onset of RpoS-mediated gene expression was brought forward by the addition of a competitive microflora, the time taken for induction was measured in hours . Since the protective effect of a competitive microflora is essentially instantaneous, the stationary-phase adaptive response is excluded as the physiological mechanism . The only instantaneous effect of the competitive microflora was a reduction in the percent saturation of oxygen from 100% to less than 10% . For both mild heat treatment (55 degrees C) and freeze injury this change in oxygen tension affords Salmonella a substantive (2 orders of magnitude) enhancement in survival . By reducing the levels of dissolved oxygen through active respiration, a competitive microflora reduces oxidative damage to exponential-phase cells irrespective of the inimical treatment . These results have led us to propose a suicide hypothesis for the destruction of rapidly growing cells by inimical processes . In essence, the suicide hypothesis proposes that a mild inimical process leads to the growth arrest of exponential-phase cells and to the decoupling of anabolic and catabolic metabolism . The result of this is a free radical burst which is lethal to unadapted cells.

Environ Mol Mutagen, 1998, 31(2), 163 - 8
Formation of direct-acting mutagens from mixtures of N-nitrosomorpholine and carboxylates by UVA irradiation; Arimoto-Kobayashi S et al.; Previously, we found that a directly mutagenic compound is produced from N-nitrosopiperidine (NPIP) in phosphate buffer on exposure to near-ultraviolet light (UVA) and we identified its structure as alpha-hydroxy-N-nitrosopiperidine phosphate ester . In the present study, we show that a similar photoactivation of an N-nitrosamine can take place with carboxylates in place of phosphate . When a neutral solution of a mixture of N-nitrosomorpholine (NMOR) and sodium acetate was irradiated with UVA, the solution became directly mutagenic towards Salmonella typhimurium TA1535 . O6-Alkylguanine-DNA alkyltransferase-deficient strains of S . typhimurium showed remarkably higher mutagenesis responses to this mutagen than the proficient strains . Citrate, succinate, and several other biological carboxylates were also effective in producing the mutagens . Since a treatment of the "NMOR plus acetate" photoproduct with carboxylic ester hydrolase resulted in a loss of the mutagenicity, the active principle is suggested to be an acetate-esterified derivative of NMOR . The role of the esters as intermediates in the photomutagenesis of nitrosamines is discussed.

FEMS Immunol Med Microbiol, 1998 Feb, 20(2), 111 - 9
A Salmonella typhimurium strain genetically engineered to secrete effectively a bioactive human interleukin (hIL)-6 via the Escherichia coli hemolysin secretion apparatus; Hahn HP et al.; Human interleukin-6 (hIL-6) cDNA was genetically fused with the Escherichia coli hemolysin secretorial signal (hlyA{S}) sequence in a plasmid vector . Recombinant E . coli XL-1 Blue and attenuated Salmonella typhimurium secreted a 30 kDa hIL-6-HlyA(S) fusion protein, with an additional form of higher apparent molecular mass produced by S . typhimurium . In S . typhimurium cultures hIL-6-HlyA(S) concentrations entered a plateau at 500 to 600 ng ml(-1) culture supernatant . In contrast to E . coli XL-1 Blue, in S . typhimurium culture supernatants hIL-6-HlyA(S) was accumulated faster reaching three-fold higher maximal concentrations . The cell proliferating activity of hIL-6-HlyA(S) fusion protein(s) was equivalent to that of mature recombinant hIL-6 . Furthermore . hIL-6-secreting S . typhimurium were less invasive than the attenuated control strain . Therefore, the bulky hemolysin secretorial peptide at the C-terminus of the fusion protein does not markably affect hIL-6 activity, suggesting that the hemolysin secretion apparatus provides an excellent system to study immunomodulatory effects of in situ synthesized IL-6 in Salmonella vaccine strains.

Immunopharmacol Immunotoxicol, 1998 Feb, 20(1), 147 - 58
Evaluation of nitric oxide production by Leishmania infantum-infected dog macrophages; Panaro MA et al.; Protozoa of the genus Leishmania (L.) infect reticuloendothelial cells of several mammalian species, including dogs, in which they often give rise to a chronic, not self-healing visceral disease . Since the parasitocidal mechanism of macrophages towards Leishmania in dog has not yet been well investigated, in this work we have evaluated in Leishmania infantum-infected macrophage cultures from 10 healthy dogs, killing capacity and nitric oxide (NO) production, in terms of nitrite (NO2) levels . Parallel experiments were performed on macrophages stimulated with both Concanavalin A (ConA)-activated PBMC supernatants and Salmonella typhimurium lipopolysaccharide (LPS), and in the same conditions, but in the presence of the NO synthase inhibitor L-N monomethylarginine (L-NMMA) . In L . infantum-infected macrophages, nitric oxide production was observed at a concentration significantly higher after stimulation with both Con A-activated PBMC supernatants and LPS than that observed in uninfected cells cultured in medium alone, or infected cells unstimulated or stimulated by PBMC supernatants or LPS alone, respectively . Moreover, NO production was abolished in the presence of the NO synthase inhibitor L-NMMA . Finally, killing of Leishmania by macrophages was significantly reduced in the presence of L-NMMA.

Protein Sci, 1998 Mar, 7(3), 600 - 4
Cadmium-induced crystallization of proteins: II . Crystallization of the Salmonella typhimurium histidine-binding protein in complex with L-histidine, L-arginine, or L-lysine; Trakhanov S et al.; To further investigate favorable effects of divalent cations on the formation of protein crystals, three complexes of Salmonella typhimurium histidine-binding protein were crystallized with varying concentrations of cadmium salts . For each of the three histidine-binding protein complexes, cadmium cations were found to promote or improve crystallization . The optimal cadmium concentration is ligand specific and falls within a narrow concentration range . In each case, crystals grown in the presence of cadmium diffract to better than 2.0 angstroms resolution and belong to the orthorhombic space group P2(1)2(1)2(1) . From our results and from the analysis of cadmium sites in well-refined protein structures, we propose that cadmium addition provides a generally useful technique to modify crystal morphology and to improve diffraction quality.

Dig Dis Sci, 1998 Mar, 43(3), 646 - 51
Impairment of intestinal mucosal antioxidant defense system during Salmonella typhimurium infection; Mehta A et al.; The mucosal pathology of Salmonella typhimurium infection may in part be due to the excessive production of reactive oxygen species (ROS) . The influence of S . typhimurium infection on the intestinal mucosal antioxidant defense system was investigated . We injected ligated rat ileal loops with Salmonella live culture or toxin . After 18 hr of infection, the animals were killed and enterocytes isolated from the ileal loops . The enterocyte-reduced glutathione (GSH) content and activities of the enzymes superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase, glutathione-S-transferase (GST), glutathione reductase (GR), and glucose-6-phosphate dehydrogenase (G6PDH) were spectrophotometrically estimated . The vitamin E and A contents were determined by high-performance liquid chromatography (HPLC) . In both the Salmonella live culture and toxin-treated groups, the enterocyte GSH and vitamin E contents and activities of the enzymes SOD, GSH-Px, catalase, GR, and G6PDH were significantly decreased as compared to the control group . However there was a significant increase in the enterocyte activity of GST . There was no change in the vitamin A content of the enterocytes . These findings might indicate a decreased endogenous intestinal protection against ROS in S . typhimurium-mediated infection, which could contribute to the pathogenesis of the disease.

FEMS Immunol Med Microbiol, 1997 Dec, 19(4), 289 - 96
Construction and characterization of type 1 non-fimbriate and non-adhesive mutants of Salmonella typhimurium; Hancox LS et al.; Mutations in the fimH gene of Salmonella typhimurium result in a non-fimbriate, non-adhesive phenotype . This phenotype was shown to be due to the lack of both fimH and fimF expression since disruption of the fimH gene by insertion of a DNA cassette into this determinant results in mutants that are complemented by plasmids carrying both fimH and fimF . Deletion mutations within the S . typhimurium fimH gene carried on a recombinant plasmid can be used to complement the mutant, and these transformants are non-adhesive but fully fimbriate, consistent with the role of FimH as being necessary for fimbrial adhesin expression . Adherence to erythrocytes, HeLa, and Hep-2 cells is associated with expression of the FimH polypeptide, and fimbriate strains that cannot synthesize FimH are non-adhesive . Discrete differences in the amino acid sequences of the adhesive type 1 and the non-hemagglutinating type 2 FimH polypeptides were detected, and are most likely responsible for the differences in hemagglutinating activity.

FEMS Immunol Med Microbiol, 1997 Dec, 19(4), 261 - 5
Influence of blood transfusion on bactericidal activity of human leukocytes and sera against Yersinia enterocolitica and Salmonella typhimurium; Markova N et al.; Patients undergoing joint surgery and blood transfusion were studied . Serum and leukocyte bactericidal tests in vitro against Salmonella typhimurium and Yersinia enterocolitica were carried out preoperatively as well as on the 1st, 3rd and 7th days after the operation . The serum complement (C3 and C4) concentrations were determined at the same intervals . It was found that after blood transfusion the bactericidic activity of sera and the serum C3 complement concentrations were increased . In contrast the killing ability of leukocytes was suppressed.

J Bacteriol, 1998 Apr, 180(7), 1862 - 8
Chemotactic adaptation is altered by changes in the carboxy-terminal sequence conserved among the major methyl-accepting chemoreceptors; Okumura H et al.; In Escherichia coli and Salmonella typhimurium, methylation and demethylation of receptors are responsible for chemotactic adaptation and are catalyzed by the methyltransferase CheR and the methylesterase CheB, respectively . Among the chemoreceptors of these species, Tsr, Tar, and Tcp have a well-conserved carboxy-terminal motif (NWET/SF) that is absent in Trg and Tap . When they are expressed as sole chemoreceptors, Tsr, Tar, and Tcp support good adaptation, but Trg and Tap are poorly methylated and supported only weak adaptation . It was recently discovered that CheR binds to the NWETF sequence of Tsr in vitro . To examine the physiological significance of this binding, we characterized mutant receptors in which this pentapeptide sequence was altered . C-terminally-mutated Tar and Tcp expressed in a receptorless E . coli strain mediated responses to aspartate and citrate, respectively, but their adaptation abilities were severely impaired . Their expression levels and attractant-sensing abilities were similar to those of the wild-type receptors, but the methylation levels of the mutant receptors increased only slightly upon addition of attractants . When CheR was overproduced, both the adaptation and methylation profiles of the mutant Tar receptor became comparable to those of wild-type Tar . Furthermore, overproduction of CheR also enhanced adaptive methylation of wild-type Trg, which lacks the NWETF sequence, in the absence of any other chemoreceptor . These results suggest that the pentapeptide sequence facilitates effective adaptation and methylation by recruiting CheR.

J Bacteriol, 1998 Apr, 180(7), 1793 - 802
Identification of a novel Salmonella invasion locus homologous to Shigella ipgDE; Hong KH et al.; Genes essential for Salmonella typhimurium invasion have been localized to Salmonella pathogenicity island 1 (SPI1) on the chromosome . However, it is clear that other genes are required for the invasion process . Mutations that abolish the SPI1 invasion type III secretion system do not significantly reduce invasion into Chinese hamster ovary tissue culture cells . Two invasion defective mutants were isolated by screening 2,500 Tn10dTc insertion mutants of S . typhimurium in the tissue culture invasion assay . One of the invasion mutants, SVM167, has an insertion between centisomes 24.5 and 25.5 in an operon homologous to the ipgDEF operon of the Shigella flexneri and Shigella sonnei virulence plasmid . A second mutant, SVM168, has an insertion in an IS3-type element with homology to the Salmonella enteritidis IS1351 element and Yersinia enterocolitica IS1400 element from a high-pathogenicity island . Further characterization of SVM167 showed that culture supernatants from this mutant lack a previously uncharacterized protein that is also missing from culture supernatants of a SPI1 mutant, suggesting it can be secreted by the SPI1 type III secretion system . In addition, transcription of this operon, sigDE (Salmonella invasion gene), is dependent on the presence of sirA, an activator of hilA expression . HilA activates transcription of several of the SPI1 genes but does not appear to have a major role in activation of transcription from the sigDE promoter.

Indian J Exp Biol, 1998 Jan, 36(1), 86 - 90
Antibacterial activity of the antiinflammatory agent diclofenac sodium; Annadurai S et al.; Antimicrobial property of ten antiinflammatory drugs was tested with eleven sensitive bacteria belonging to both Gram positive and Gram negative types . Since most of the bacteria were moderate to highly sensitive to diclofenac (Dc), this compound was tested in vitro against 397 bacteria, most of which were inhibited by Dc at 50-100 micrograms/ml level . When tested in vivo, Dc at 1.5 and 3.0 micrograms/g body weight of a Swiss strain of white mice, could significantly protect the animals challenged with 50 MLD of Salmonella typhimurium NCTC 74 . According to chi 2 test the in vivo data were highly significant (P < 0.001).

Microbiology, 1998 Mar, 144 ( Pt 3), 697 - 704
Expression of the cold-shock gene cspB in Salmonella typhimurium occurs below a threshold temperature; Craig JE et al.; Previous studies have shown that several bacterial species exhibit a multigenic response following temperature downshift (cold shock) . Evidence for such a response in Salmonella typhimurium is reported, based on the isolation of a range of low-induction-temperature gene fusions containing Mudlux insertions . The fusions exhibited different levels of basal light at 30 degrees C, and were induced at different rates and to different degrees over several hours following a reduction in temperature to 10 degrees C . Of the Mudlux gene fusions isolated, one was found which produced essentially no light when grown at 30 degrees C but exhibited rapid and high-level induction when the temperature was reduced to 10 degrees C . The target of this gene fusion (which was named cspB) was shown to lie adjacent to the umuDC operon and to encode a homologue of the major cold-shock protein of Escherichia coli, CspA . Luminescence studies revealed that substantial light production occurred from the cspB::Mudlux fusion at or below 22 degrees C but not at higher temperatures, even following a temperature drop from 30 degrees C . Moreover, cspB mRNA levels were found to mimic this pattern of luminescence, suggesting that cspB expression occurs below a defined temperature threshold . The cspB mRNA was also found to be very stable at 10 degrees C but to become highly unstable when the temperature was raised towards the threshold temperature, even in the presence of rifampicin . Existing cellular RNases therefore appear to mediate the decay of cspB mRNA at high temperatures, but are incapable of this at low temperatures.

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