Salmonella typhimurium

Indian Pediatr, 1995 Aug, 32(8), 881 - 5
An outbreak of multidrug resistant . Salmonella typhimurium in a nursery; Kumar A et al.; A nursery epidemic caused by multidrug resistant Salmonella typhimurium is reported . In total, 21 infants developed symptomatic illness; of these, 17 had septicemia (7 blood culture positive) and 4 had diarrhea alone . Asymptomatic carrier state was identified in 13 infants . Male sex and birth asphyxia increased the risk for symptomatic illness . Fever, lethargy, and diarrhea were the most common clinical features . Amongst the septicemic infants there was no difference in clinical profile whether the blood culture was positive or negative for S . typhimurium . In the symptomatic group, S . typhimurium was isolated from feces in 19 cases and from blood in 7 cases . In both symptomatic and asymptomatic infants, all isolates of S . typhimurium, whether obtained from feces and/or from blood, were resistant to ampicillin, chloramphenicol, and trimethoprim, and a significant number (almost one-fifth) of them also showed resistance to third generation cephalosporins . More than 90% of isolates were sensitive to aminoglycosides and ciprofloxacin . On a combination of third generation cephalosporin (cefotaxime or ceftriaxone) and amikacin, 17 (81%) infants recovered, 2 succumbed to their illness, and 2 failed to improve and required ciprofloxacin . The origin of epidemic was traced to a carrier staff nurse working in nursery.

Mol Microbiol, 1995 Aug, 17(3), 523 - 31
Salmonella typhimurium responses to a bactericidal protein from human neutrophils; Qi SY et al.; Bactericidal/permeability-increasing protein {BPI} is a cationic antimicrobial protein from neutrophils that specifically binds to the surfaces of Gram-negative bacteria via the lipid A component of lipopolysaccharide . To obtain information about the responses of Salmonella typhimurium to cell-surface damage by BPI, two-dimensional gel electrophoresis and N-terminal microsequencing were used to identify proteins that were induced or repressed following BPI treatment . The majority of the affected proteins are involved in central metabolic processes . Upon addition of BPI, the beta-subunit of the F1 portion of Escherichia coli ATP synthase was repressed threefold whereas six proteins were induced up to 11-fold . Three of the latter were identified as lipoamide dehydrogenase, enoyl-acyl carrier protein reductase, and the heat-shock protein HtpG . Additionally, a novel protein, BipA, was identified that is induced over sevenfold by BPI; sequence analysis suggests that it belongs to the GTPase superfamily and interacts with ribosomes . A conserved direct-repeat motif is present in the regulatory regions of several BPI-inducible genes, including the bipA gene . Only one of the BPI-responsive proteins was induced when cells were treated with polymyxin B, which also binds to lipid A . We therefore conclude that BPI and polymyxin B affect different global regulatory networks in S . typhimurium even though they bind with high affinity to the same cell-surface component.

Cent Eur J Public Health, 1995 Aug, 3(3), 161 - 2
Salmonella phage types distribution in the Czech Republic in 1991-1994; Karpiskova R et al.; The Salmonella enteritidis and Salmonella typhimurium phage types in the Czech Republic during the monitored period 1991-1994 are described . The total number of 2318 strains were examined . From 652 Salmonella typhimurium strains 24 various phage types were identified . PT 104 was predominating in both human and non-human strains . 1666 of Salmonella enteritidis strains were identified and were representative of 14 different phage types . PT 8 was the most common type in humans (91.7%) and in animals (79.1%).

Am J Vet Res, 1995 Aug, 56(8), 1012 - 8
Tumor necrosis factor-alpha production in swine after oral or respiratory challenge exposure with live Salmonella typhimurium or Salmonella choleraesuis; Stabel TJ et al.; A series of experiments was conducted to document tumor necrosis factor-alpha (TNF) activity in serum of swine after inoculation with Salmonella spp endotoxin and after oral or respiratory tract challenge exposure with live Salmonella spp . For experiment 1, a potentially lethal dose of S typhimurium endotoxin (25 micrograms/kg of body weight) was administered IV, and serum TNF activity was measured . High TNF (approx 700 IU/ml) activity at 1 to 2 hours after administration of the inoculum was associated with death, whereas lower TNF (approx 30 IU/ml) activity was associated with a general prolonged state of shock . For experiment 2, pigs were administered a nonlethal dose (5 micrograms/kg, IV) of either S typhimurium or S choleraesuis endotoxin . Difference in the ability to induce porcine serum TNF activity was not observed between strains . During experiment 3, pigs were inoculated with 10(4) colony-forming units of S typhimurium chi 4232 either orally by gelatin capsule (GC) or by intranasal (IN) instillation . A late serum TNF response (17 IU/ml) was measured at 6 weeks after IN inoculation . A serum TNF response was not detected in GC-inoculated pigs . All tissues and feces were test-negative for S typhimurium prior to the 6-week TNF response . Serum TNF activity may be related to clearance of S typhimurium after respiratory tract exposure, but it is not important to or indicative of clearance of orally presented S typhimurium in swine . During experiment 4, pigs were inoculated with 10(6) colony-forming units of S typhimurium chi 4232 similarly as for experiment 3.(ABSTRACT TRUNCATED AT 250 WORDS)

J Bacteriol, 1995 Aug, 177(16), 4628 - 37
Fur regulon of Salmonella typhimurium: identification of new iron-regulated genes; Tsolis RM et al.; In order to identify genes belonging to the Fur regulon of Salmonella typhimurium, a bank of 10,000 independent S . typhimurium MudJ insertion mutants was screened for lacZ fusions regulated by the iron response regulator Fur . In parallel, a plasmid gene bank of S . typhimurium consisting of 10,000 independent clones was screened for Fur-regulated promoters or iron binding proteins by the Fur titration assay (FURTA) . Fur-regulated MudJ insertions and Fur-regulated promoters were mapped . In addition, iron-regulated promoter activities of transcriptional fusions from MudJ insertions and FURTA-positive clones were quantified . The nucleotide sequences of 11 FURTA-positive plasmids and of short fragments of DNA flanking three MudJ insertions were determined . By these methods we identified 14 Fur-regulated genes of S . typhimurium . For 11 of these genes, Fur-regulated homologs have been described in Escherichia coli or Yersinia enterocolitica, including fhuA,fhuB,fepA,fes,fepD,p43,entB,fur ,foxA,hemP, and fhuE . In addition, we identified three genes with homologs in other bacteria which have not previously been shown to be Fur regulated.

J Bacteriol, 1995 Aug, 177(15), 4524 - 7
Involvement of cysB and cysE genes in the sensitivity of Salmonella typhimurium to mecillinam; Oppezzo OJ et al.; cysB and cysE strains were obtained as spontaneous mecillinam-resistant mutants of Salmonella typhimurium . The resistance to mecillinam was caused by the cys mutations which also conferred tolerance to lethal cell shape mutations . Most, but not all, cysB and cysE mutations from other origins displayed the same behavior . Resistance was abolished by O- and N-acetylserine in cysE mutants; by thiosulfate, sulfite, and sulfide in cysB mutants; and by cysteine in both types of mutants . It is concluded that an event involved in mecillinam action requires the inducer and the activator protein of the cysteine regulon.

J Bacteriol, 1995 Aug, 177(15), 4508 - 13
Fine-structure deletion analysis of the transcriptional silencer of the proU operon of Salmonella typhimurium; Fletcher SA et al.; Transcriptional control of the osmotically regulated proU operon of Salmonella typhimurium is mediated in part by a transcriptional silencer downstream from the promoter (D.G . Overdier and L.N . Csonka, Proc . Natl . Acad . Sci . USA 89:3140-3144, 1992) . We carried out a fine-structure deletion analysis to determine the structure and the position of the silencer, which demonstrated that this regulatory element is located between nucleotide positions +73 to +274 downstream from the transcription start site . The silencer appears to be made up of a number of components which have cumulative negative regulatory effects . Deletions or insertions of short nucleotide sequences (< 40 bp) between the proU promoter and the silencer do not disrupt repression exerted by the silencer, but long insertions (> or = 0.8 kbp) result in a high level of expression from the proU promoter, similar to that imparted by deletion of the entire silencer . The general DNA-binding protein H-NS is required for the full range of repression of the proU operon in media of low osmolality . Although in the presence of the silencer hns mutations increased basal expression from the proU promoter three- to sixfold, in the absence of the silencer they did not result in a substantial increase in basal expression from the proU promoter . Furthermore, deletion of the silencer in hns+ background was up to 10-fold more effective in increasing basal expression from the proU promoter than the hns mutations . These results indicate that osmotic control of the proU operon is dependent of some factor besides H-NS . We propose that the transcriptional regulation of this operon is effected in media of low osmolality by a protein which makes the promoter inaccessible to RNA polymerase by forming a complex containing the proU promoter and silencer.

J Bacteriol, 1995 Aug, 177(15), 4297 - 302
Salmonella typhimurium pgtB mutants conferring constitutive expression of phosphoglycerate transporter pgtP independent of pgtC; Niu S et al.; PgtC is one of the three components of the atypical "two-component" pgt regulatory system . To investigate whether functional PgtC required for the induction of pgtP expression could be bypassed in the signal transduction process, we sought, and succeeded in isolating, intergenic suppressors arising in the low-copy mini-F plasmid, pSJ11, bearing the entire pgt system except for a 168-bp deletion near the end of the pgtC gene . By transport assays, these suppressors were found to confer constitutive pgtP expression . Intriguingly, all five mutations reside near the 5' end of the pgtB gene, at codons 19 and 21 . One mutation alters Arg-19 to Gln, two alter Ala-21 to Thr, one alters Ala-21 to Val, and one alters Ala-21 to Ile . Appropriate strains in which the pgtP promoter was fused to lacZ and which bore the pgtB mutations with and without mutations in pgtC and pgtA genes were constructed, and the epistatic relationships of the wild-type pgtC allele, a mutant pgtA allele, and an essentially total deletion of pgtC to the constitutive pgtB mutations were determined . In the mutant strains bearing the Ala-21 --> Ile and Ala-21 --> Val substitutions, the level of constitutive pgtP-lacZ reporter expression was not affected by the presence of the wild-type pgtC allele, nor was it affected by the total absence of PgtC in the case of the Ala-21 --> Val alteration examined; however, in the mutant strains bearing the Ala-21 --> Thr and the Arg-19 --> Gln substitutions, the extent of constitutive pgtP-lacZ reporter expression was markedly enhanced by the presence of wild-type pgtC allele and, in the case of the Arg-19 -->Gln change examined, by the total absence of PgtC as well . These results indicate that PgtC contains no domain necessary for the kinase activity; that PgtB can be activated in the absence of PgtC mutational alterations of the protein itself; and that PgtB and PgtC interact in the signaling process, with PgtC functioning to activate and modulate the kinase activity of Pgtb . In all strains, the replacement of the wild type pgtA allele with a mutant pgtA allele completely abolished expression of the pgtP-lacZ reporter, indicating that functional pgtA is essential for the constitutivity . His-457 of PgtB, a potential site of autophosphorylation, is also required for the constitutivity because its change to Val drastically reduced pgtP-lacZ reporter expression . The structural basis for the activation of the altered PgtB is discussed in terms of putative structure of PgtB in the membrane.

J Infect Dis, 1995 Aug, 172(2), 490 - 6
Deferoxamine B but not deferoxamine G1 inhibits cytokine production in murine bone marrow macrophages; Autenrieth IB et al.; The iron chelator deferoxamine (DFO) B enhances virulence of Yersinia enterocolitica and modulates cellular immune responses . Since cytokines mediate effector mechanisms in resolution of yersiniae from infected tissues, the impact of DFO B and DFO G1 on cytokine production by murine bone marrow macrophages (BMM) was investigated . BMM were stimulated with lipopolysaccharide (LPS) of Salmonella typhimurium or infected with Y . enterocolitica . DFO B inhibited interleukin (IL)-6, IL-12, and tumor necrosis factor (TNF)-alpha mRNA production 4-fold (shown by semiquantitative reverse transcription polymerase chain reaction) . TNF-alpha and IL-6 protein production was reduced 50% by DFO B . In contrast, DFO G1 had no effect on cytokine production . Moreover, cytokine production by Yersinia-infected BMM was decreased by plasmid-encoded Yersinia proteins . Thus, plasmid-cured strains induced higher cytokine responses in BMM than did the wild type strain . These results suggest that DFO B acts in a bimodal fashion in yersiniosis: iron supply to the pathogen and immunosuppression of the host.

Infect Immun, 1995 Aug, 63(8), 3196 - 8
Exogenous tumor necrosis factor alpha and interleukin-1 alpha increase resistance to Salmonella typhimurium: efficacy is influenced by the Ity and Lps loci; Morrissey PJ et al.; Interleukin-1 alpha (IL-1 alpha) or tumor necrosis factor alpha (TNF-alpha) administered prior to infection with Salmonella typhimurium increases survival in mice that are Ityr, not in susceptible Lpsd or Itys mice . Combined IL-1 alpha and TNF-alpha pretreatment results in greater survival than that seen with either cytokine alone in Ityr mice . Treatment after infection with TNF-alpha and/or IL-1 alpha increases the mean time to death but not the survival fraction of Lpsd mice and was ineffective in either Ityr or Itys mice.

Infect Immun, 1995 Aug, 63(8), 2859 - 66
A novel synthetic lipid A analog with low endotoxicity, DT-5461, prevents lethal endotoxemia; Sato K et al.; Bacterial endotoxin (lipopolysaccharide {LPS}) causes severe damage to the host organism as a result of excessive release of inflammatory cytokines, including interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha), from mononuclear phagocytes during gram-negative bacterial infection . We evaluated the ability of a novel synthetic lipid A analog with low endotoxicity, DT-5461, to antagonize LPS-induced IL-1 and TNF-alpha production in cells of monocyte/macrophage lineage and examined the protective effect of DT-5461 against lethal endotoxic shock in mice . The IL-1- or TNF-alpha-inducing activity of DT-5461 is 100,000 to 10,000 times less active than that of Escherichia coli LPS (EcLPS) or synthetic lipid A . DT-5461 significantly inhibited EcLPS-induced IL-1 and TNF-alpha release when murine peritoneal macrophages were incubated with DT-5461 2 h prior to EcLPS stimulation at the same concentration (1 microgram/ml) . The antagonistic effect of DT-5461 on the production of IL-1 and TNF-alpha induced by EcLPS occurred in a concentration-dependent manner . DT-5461 also inhibited IL-1 and TNF-alpha induction when murine peritoneal macrophages were stimulated by LPS from Salmonella typhimurium or synthetic lipid A, as well as by EcLPS, but not by muramyl dipeptides . This indicated that DT-5461 specifically antagonized the action of LPS . DT-5461 also antagonized EcLPS-mediated activation of human peripheral blood monocytes . DT-5461 blocked the binding of fluorescein isothiocyanate-labelled LPS to murine peritoneal macrophages as well as it did the binding of EcLPS and synthetic lipid A, i.e., in a concentration-dependent fashion . Injection of DT-5461 2 h before EcLPS challenge prevented the production of serum IL-1 and TNF-alpha in D-galactosamine-treated mice . Furthermore, this treatment modality protected mice against LPS-induced lethal toxicity . This study suggests that DT-5461 possesses a potent LPS antagonistic effect and may be useful in a protective strategy against lethal endotoxemia caused by gram-negative bacterial infection.

Mutat Res, 1995 Aug, 335(1), 21 - 6
Modifying role of trace elements on the mutagenicity of benzo{a}pyrene; Olson B et al.; Benzo{a}pyrene (BaP) is a polycyclic aromatic hydrocarbon that is found in tobacco smoke and various environmental contaminants and has been shown to be carcinogenic and mutagenic in animal and cell culture studies, respectively . Research studies suggest that various nutritional factors such as the antioxidant vitamins and selenium are very promising as potential anticarcinogenic agents . Moreover, some evidence exists showing that both iron and germanium, at specific dosage levels, may possess antimutagenic potential . This study examined the influence of ferrous sulfate and germanium oxide, independently, upon the mutagenic potential of BaP in the Ames test . Four test strains of Salmonella typhimurium were exposed to BaP (15 micrograms/plate) in the presence of different dosage levels of iron (0-1000 micrograms/plate) and germanium (0-600 micrograms/plate) . In the case of iron, it was observed that, depending upon the strain tested, iron reduced BaP's mutagenicity . In strain TA98, this was a significant effect at 100 micrograms/plate and higher . In strains TA97a and TA100, iron concentrations had to reach 250 micrograms/plate or higher to produce significant effects . Iron was much less effective in reducing BaP mutagenicity in strain TA102 . In general, germanium was not as effective in reducing the mutagenic potential of BaP . Only in the case of the highest concentrations tested (400 and 600 micrograms/plate) was any effect noted, and this in only three of the four strains evaluated.

J Appl Bacteriol, 1995 Aug, 79(2), 128 - 34
The effect of transient temperatures on the growth of Salmonella typhimurium LT2 . II: Excursions outside the growth region; Mitchell GA et al.; The effect of fluctuating temperatures on microbial growth is important in the passage of foods from production to consumption . Suspensions of Salmonella typhimurium have been subjected to sinusoidally time-varying temperatures of periods from 60 to 240 min between 4 degrees and 22 degrees C, that is within and below the growth temperature range . The suspensions were prepared with two concentrations of sodium chloride and adjusted to two different values of pH . The change in the numbers of viable bacteria was measured with time and the experimental growth curves and average generation times compared with predictions based on isothermal growth data . Generally, the experimental average generation times exceeded the predictions by not more than 10% . In enumerating viable bacteria in the suspensions containing 3.5% (w/v) sodium chloride it was necessary to use sodium chloride in the diluent and recovery medium in order to recover the bacteria quantitatively.

Genetika, 1995 Aug, 31(8), 1073 - 8
{Isolation and characteristics of Salmonella typhimurium mutants with a disrupted process of generating nonculturable forms}; Romanova IuM et al.; A laboratory model of the induction of nonculturable forms in Salmonella typhimurium has been developed . Mutants of S . typhimurium were obtained using insertion mutagenesis via the TnPhoA transposon . These mutants were impaired in the cell transition from the vegetative to the nonculturable state assayed in this model . Mutants have various phenotypes and are located in different regions of the chromosome, as shown by the data obtained using pulsed-field electrophoresis of genomic DNA.

Microbiology, 1995 Aug, 141 ( Pt 8), 1937 - 45
I-CeuI recognition sites in the rrn operons of the Bacillus subtilis 168 chromosome: inherent landmarks for genome analysis; Toda T et al.; The Bacillus subtilis 168 circular chromosome yielded ten fragments on I-CeuI endonuclease digestion . I-CeuI recognizes a 26 bp sequence that is located within the gene encoding the 23S subunit of the rRNA in Chlamydomonas eugametos, Escherichia coli and Salmonella typhimurium . The precise locations of the I-CeuI sites of the B . subtilis chromosome were determined on a NotI-SfiI physical map by (i) double digestion analyses with I-CeuI and SfiI, (ii) comparison of mutant strains lacking a specific rrn operon, (iii) using an I-CeuI linking clone and (iv) analysis of nucleotide sequence data of some rrn operons . In conclusion, all the I-CeuI sites were located within the B . subtilis rrn operons and the I-CeuI sites were conserved in all the B . subtilis 168 derivatives tested . Thus, variations in size of the I-CeuI fragments must be due to genome alterations . A B . subtilis 168 strain was investigated with I-CeuI . We demonstrated that the aberrant structure was the outcome of the inversion of an approximately 1700 kb DNA segment.

J Bacteriol, 1995 Aug, 177(15), 4364 - 71
Transcriptional autoregulation of the Salmonella typhimurium phoPQ operon; Soncini FC et al.; The Salmonella typhimurium PhoP-PhoQ two-component regulatory system controls the expression of several genes, some of which are necessary for virulence . During a screening for PhoP-regulated genes, we identified the phoPQ operon as a PhoP-activated locus . beta-Galactosidase activity originating from phoPQ-lac transcriptional fusions required the presence of both the transcriptional regulator PhoP and its cognate sensor-kinase PhoQ . At low concentrations, PhoQ stimulated expression of phoPQ-lac transcriptional fusions . However, larger amounts of PhoQ protein without a concomitant increase in PhoP failed to activate phoPQ-lac fusions . Two different transcripts are produced from the phoPQ operon during exponential growth . These transcripts define two promoters: phoPp1, which requires both PhoP and PhoQ for activity and which is environmentally regulated, and phoPp2, which remains active in the absence of PhoP and PhoQ but which is slightly stimulated by these proteins . The pattern of transcriptional autoregulation was also observed at the protein level with anti-PhoP antibodies . In sum, autoregulation of the phoPQ operon provides several levels of control for the PhoP-PhoQ regulon . First, environmental signals would stimulate PhoQ to phosphorylate the PhoP protein that is produced at basal levels from the PhoP-PhoQ-independent promoter . Then, phospho-PhoP would activate transcription of phoPp1, resulting in larger amounts of PhoP and PhoQ and increased expression of PhoP-activated genes . A return to basal levels could be mediated by a posttranscriptional mechanism by which translation of the mRNA produced from phoPp1 is inhibited.

Infect Immun, 1995 Aug, 63(8), 2818 - 25
Clearance of Pseudomonas aeruginosa from the murine gastrointestinal tract is effectively mediated by O-antigen-specific circulating antibodies; Pier GB et al.; The colonization of mucosal surfaces by Pseudomonas aeruginosa can lead to local or disseminated disease . Secretory immunoglobulin A (IgA) has been assumed to be responsible for preventing mucosal colonization by interfering with the binding of bacterial ligands to epithelial surface receptors . However, the efficacy of this mechanism of immunity derives little actual support from in vivo experiments . In an investigation of the role of local and systemic immunization strategies in reducing colonization of the gastrointestinal tract of mice by P . aeruginosa, the bacterial antigens that were potential targets for immune effectors promoting mucosal clearance were identified . Levels of gastrointestinal colonization were reduced when immunity to homologous O antigens, but not that to pili or flagella, was elicited . Oral vaccination with attenuated Salmonella typhimurium expressing P . aeruginosa serogroup O11 antigen elicited mucosal and serum IgA antibodies and serum IgG antibodies specific for the recombinant antigen . Oral challenge of immunized mice with P . aeruginosa serogroup O11 demonstrated protection against gastrointestinal colonization . Intraperitoneal immunization with a serogroup O11 high-molecular-weight O-polysaccharide antigen elicited only serum IgG and IgM antibodies yet was as effective as oral vaccination in protecting mice against gastrointestinal colonization . This finding was confirmed by the demonstration that intraperitoneal immunization with purified lipopolysaccharide was also protective against mucosal surface colonization . These results call into question the need for local immune effectors, particularly secretory IgA, directed at bacterial ligands for epithelial surface components, in protecting a mucosal surface from bacterial challenge.

Curr Opin Immunol, 1995 Aug, 7(4), 474 - 8
Entry of microbes into the host: using M cells to break the mucosal barrier; Jones B et al.; Enteric microbial pathogens interact with the gut epithelium to establish infection . Recently, it has become clear that many microorganisms that colonize or traverse the intestinal mucosa do so via the specialized M cells . Recent work has shown that Shigella flexneri and Salmonella typhimurium specifically target M cells to initiate infection of the host.

Biol Reprod, 1995 Aug, 53(2), 462 - 71
Oral immunization with attenuated Salmonella expressing human sperm antigen induces antibodies in serum and the reproductive tract; Srinivasan J et al.; Induction of immune responses in the reproductive tract will be crucial for a functional gamete antigen-based antifertility vaccine . Here we describe the construction and development of an avirulent Salmonella as an oral vaccine delivery vector to elicit sperm-specific immune responses in reproductive tract secretions . A cDNA sequence encoding the human sperm antigen SP10 was cloned on an asd+vector and expressed to a high level in an avirulent delta cya, delta crp, and delta asd vaccine strain of Salmonella typhimurium . Oral immunization of female BALB/c mice with this recombinant Salmonella elicited high-titer anti-SP10 IgG antibodies in serum and IgA antibodies in vaginal secretions . Anti-SP10 antibody titers could be increased by secondary and tertiary oral administrations of the recombinant Salmonella . Induction of sperm-specific antibodies in the reproductive tract following oral administration of a recombinant Salmonella could lead to the development of a simple, safe, efficient, and easy-to-use antifertility vaccine.

AIDS Res Hum Retroviruses, 1995 Aug, 11(8), 909 - 20
Highly attenuated HIV type 2 recombinant poxviruses, but not HIV-2 recombinant Salmonella vaccines, induce long-lasting protection in rhesus macaques; Franchini G et al.; Immunization schemes employing priming with vector-based vaccine candidates followed by subunit booster administrations have been explored and shown to have merit in the human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus systems . In this study, we have assessed the priming capacity of highly attenuated poxvirus vector (NYVAC and ALVAC)-based HIV-2 recombinants, as well as Salmonella typhimurium HIV-2 recombinants in rhesus macaques . ALVAC- and NYVAC-based vaccine candidates expressing the HIV-2 gag, pol, and env genes or NYVAC-based recombinants expressing either gp160 or gp120 were used to immunize rhesus macaques in combination protocols with alum-adjuvanted HIV-2 rgp160 . Following intravenous challenge exposure with 100 infectious doses of the HIV-2SBL6669 parental virus genotype mixture, seven of eight animals were protected from infection . The seven protected animals were rechallenged 6 months postprimary challenge, without additional booster inoculations, with the same dose of the HIV-2SBL6669 parental virus . Five of the seven animals remained protected against HIV-2 infection at 6 months following the second challenge . In contrast, oral immunization with recombinant Salmonella expressing the HIV-2 gag and the gp120 portion of the envelope either alone or in combination with alum-adjuvanted rgp160 failed to confer protection . These results suggest that the NYVAC- and ALVAC-based recombinants may confer long-lasting protection and that these two highly attenuated poxvirus vaccine vectors may represent promising candidates for developing an acquired immunodeficiency syndrome vaccine.

Biochemistry, 1995 Jul 25, 34(29), 9466 - 76
Monovalent metal ions play an essential role in catalysis and intersubunit communication in the tryptophan synthase bienzyme complex; Woehl EU et al.; This investigation shows that the alpha 2 beta 2 tryptophan synthase bienzyme complex from Salmonella typhimurium is subject to monovalent metal ion activation . The effects of the monovalent metal ions Na+ and K+ were investigated using rapid scanning stopped-flow (RSSF), single-wavelength stopped-flow (SWSF), and steady-state techniques . RSSF measurements of individual steps in the reaction of L-serine and indole to give L-trytophan (the beta-reaction) as well as the reaction of 3-indole-D-glycerol 3'-phosphate (IGP) with L-serine (the alpha beta-reaction) demonstrate that monovalent metal ions such as Na+ and K+ change the distribution of intermediates in both the transient and steady states . Therefore the metal ion effect alters relative ground-state energies and the relative positions of ground- and transition-state energies . The RSSF spectra and SWSF time courses show that the turnover of indole is significantly reduced in the absence of either Na+ or K+ . The alpha-aminoacrylate Schiff base species, E(A-A), is in a less active state in the absence of monovalent metal ions . Na+ decreases the steady-state rate of IGP cleavage (the alpha-reaction) to about 30% of the value obtained in the absence of metal ions . Steady-state investigations show that in the absence of monovalent metal ions the alpha- and alpha beta-reactions have the same activity . Na+ binding gives a 30-fold stimulation of the alpha-reaction when the beta-site is in the E(A-A) form.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1995 Jul 25, 34(29), 9459 - 65
Monovalent cations affect dynamic and functional properties of the tryptophan synthase alpha 2 beta 2 complex; Peracchi A et al.; Monovalent cations affect both conformational and catalytic properties of the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium . Their influence on the dynamic properties of the enzyme was probed by monitoring the phosphorescence decay of the unique Trp-177 beta, a residue located near the beta-active site, at the interface between alpha- and beta-subunits . In the presence of either Li+, Na+, Cs+, or NH4+, the phosphorescence decay is biphasic and the average lifetime increases indicating a decrease in the flexibility of the N-terminal domain of the beta-subunit . Since amplitudes but not lifetimes are affected, cations appear to shift the equilibrium between preexisting enzyme conformations . The effect on the reaction between indole and L-serine was studied by steady state kinetic methods at room temperature . We found that cations: (i) bind to the L-serine--enzyme derivatives with an apparent dissociation constant, measured as the concentration of cation corresponding to one-half of the maximal activity, that is in the millimolar range and decreases with ion size; (ii) increase kcat with the order of efficacy Cs+ > K+ > Li+ > Na+; (iii) decrease KM for indole, Na+ being the most effective and causing a 30-fold decrease; and (iv) cause an increase of the kcat/KM ratio by 20-40-fold . The influence on the equilibrium distribution between the external aldimine and the alpha-aminoacrylate, intermediates in the reaction of L-serine with the beta-subunits of the enzyme, was found to be cation-specific.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1995 Jul 25, 34(29), 9403 - 12
An expanded two-state model accounts for homotropic cooperativity in biosynthetic threonine deaminase from Escherichia coli; Eisenstein E et al.; The linkage between substrate and regulatory effector binding to separate sites on allosteric enzymes results in shifts in their sigmoidal kinetics to regulate metabolism . Control of branched chain amino acid biosynthesis in Escherichia coli occurs in part through shifts in the sigmoidal dependence of alpha-ketobutyrate production promoted by isoleucine and valine binding to biosynthetic threonine deaminase . The structural similarity of threonine, valine, and isoleucine have given rise to suggestions that there may be competition among different ligands for the same sites on this tetrameric enzyme, resulting in a complex pattern of regulation . In an effort to provide a coherent interpretation of the cooperative association of ligands to the active sites and to the effector sites of threonine deaminase, binding studies using single amino acid variants were undertaken . A previously-isolated, feedback-resistant mutant identified in Salmonella typhimurium, ilvA219, has been cloned and sequenced . The phenotype is attributable to a single amino acid substitution in the regulatory domain of the enzyme in which leucine at position 447 is substituted with phenylalanine . The mutant exhibits hyperbolic saturation curves in both ligand binding and steady-state kinetics . These results, in addition to calorimetric and spectroscopic measurements of isoleucine and valine binding, indicate that the low affinity (T) state is destabilized in the mutant and that it exists predominantly in the high affinity (R) conformation in the absence of ligands, providing an explanation for its resistance to isoleucine . Chemical and spectroscopic analyses of another mutant, in which alanine has replaced an essential lysine at position 62 that forms a Schiff base with pyridoxal phosphate, indicate that the cofactor is complexed to exogenous threonine and is therefore unable to bind additional amino acids at the active sites . Isoleucine and valine binding to this inactive, active site-saturated enzyme revealed that it too was stabilized in the R state, yielding binding constants in excellent agreement with the leucine to phenylalanine mutant . The lysine to alanine mutant was further utilized to demonstrate that both threonine and 2-aminobutyrate bind with stronger affinity to the regulatory sites than to the active sites . A direct consequence of these results is that substrates and analogs have a synergistic effect on the allosteric transition since, in effect, they act as both homotropic and heterotropic effectors.(ABSTRACT TRUNCATED AT 250 WORDS)

Science, 1995 Jul 21, 269(5222), 400 - 3
Simultaneous identification of bacterial virulence genes by negative selection; Hensel M et al.; An insertional mutagenesis system that uses transposons carrying unique DNA sequence tags was developed for the isolation of bacterial virulence genes . The tags from a mixed population of bacterial mutants representing the inoculum and bacteria recovered from infected hosts were detected by amplification, radiolabeling, and hybridization analysis . When applied to a murine model of typhoid fever caused by Salmonella typhimurium, mutants with attenuated virulence were revealed by use of tags that were present in the inoculum but not in bacteria recovered from infected mice . This approach resulted in the identification of new virulence genes, some of which are related to, but functionally distinct from, the inv/spa family of S . typhimurium.

J Mol Biol, 1995 Jul 21, 250(4), 392 - 406
carP, involved in pyrimidine regulation of the Escherichia coli carbamoylphosphate synthetase operon encodes a sequence-specific DNA-binding protein identical to XerB and PepA, also required for resolution of ColEI multimers; Charlier D et al.; The carP gene involved in pyrimidine-specific regulation of the upstream P1 promoter of the Escherichia coli carAB operon has been cloned in vivo on a mini-Mu replicon, sequenced and shown to be identical to the xerB (pepA) gene encoding aminopeptidase A, a protein also involved in the Xer-mediated site-specific recombination at ColEI cer . The trans-dominant allele carP6 was cloned as well and shown to bear a single G-->A transition that converts the TGG codon (Trp473) into a TAG amber stop codon . The truncated mutant protein, missing the 31 C-terminal amino acid residues, was shown to be partially active; in the multicopy state the carP6 allele can restore pyrimidine repressibility of the carAB promoter P1 . The trans-dominant character of the single copy carP6 allele was found to be suppressed in the presence of multiple copies of the wild-type gene . The carP (pepA) control region was sequenced and transcription shown to be initiated at three promoters, the most upstream one of which was shown to be subject to negative autoregulation . The aminopeptidase activity of CarP (PepA) was found to be dispensable for its role in pyrimidine-mediated repression of carAB transcription . CarP (PepA) was shown to be a sequence-specific DNA-binding protein that does not require, at least not in vitro, any pyrimidine cofactor to bind to the DNA . Mobility-shift and DNase I footprinting experiments have revealed a specific binding of purified CarP (PepA) to two sites in each one of the control regions of the E . coli and Salmonella typhimurium carAB operons and to a single site in the carP (pepA) control region . We propose that integration host factor and CarP/PepA-induced structural modifications in the carAB control region cause conformational changes required to assemble a pyrimidine-specific nucleo-protein regulatory complex.

J Mol Biol, 1995 Jul 21, 250(4), 383 - 91
Pyrimidine regulation of the Escherichia coli and Salmonella typhimurium carAB operons: CarP and integration host factor (IHF) modulate the methylation status of a GATC site present in the control region; Charlier D et al.; By measuring the protection against Dam methylase modification of a GATC sequence located 106 bp upstream of the startpoint of promoter P1 in the control region of the carAB operon (encoding carbamoylphosphate synthetase) we have obtained evidence for a direct correlation between the degree of in vivo occupancy of a specific regulatory target site and the repressibility of the P1 promoter by pyrimidine residues . A high uridine nucleotide pool as well as binding of the carP (alias xerB/pepA) gene product and of the integration host factor (IHF) to the carAB control region are prerequisites to observe this in vivo protection . Purified CarP binds in vitro to the carAB control region and protects against DNase I two approximately 25 bp long stretches, one of which is located just downstream of the GATC sequence . Mutations in this site strongly impair the pyrimidine regulation of the P1 promoter and the interference with Dam methylase modification . These processes are also strongly impaired in the absence of integration host factor and in mutants affected in the IHF site located some 200 bp upstream of this Dam methylase modification site . IHF therefore exerts at least part of its antagonistic effects on P1, i.e . increased expression in minimal medium but increased repression in the presence of pyrimidine residues, indirectly by influencing the formation or the stability of a particular protein-DNA complex . Furthermore, we demonstrate that the distance separating the IHF and Dam methylase target sites is crucial for the in vivo protection and for pyrimidine-mediated regulation of the promoter expression . Mutations altering this distance result in severe reductions of the degree of in vivo protection and, concomitantly, of the repressibility by pyrimidine residues of promoter P1 activity in a way indicative of the formation of a complex nucleoprotein structure . Since neither IHF nor CarP require pyrimidine residues to bind to the carAB control region, at least not in vitro, it is tempting to suggest that IHF and CarP-induced bending and looping provide changes in DNA topology that are required for assembling a specific pyrimidine-dependent nucleoprotein complex that modulates P1 activity.

Cancer Lett, 1995 Jul 20, 94(1), 33 - 40
Chemoprotective properties of chlorophyllin against vinyl carbamate, p-nitrophenyl vinyl ether and their electrophilic epoxides; Park KK et al.; Chlorophyllin (CHL), a water-soluble sodium and copper derivative of chlorophyll, has been shown to be a strong antimutagen in several test systems, but its mechanism of antimutagenic action is largely unknown . In the present study, we have found the protective properties of CHL against vinyl carbamate, p-nitrophenyl vinyl ether and their electrophilic epoxides . CHL exhibited dose-related inhibition of his+ reversion in Salmonella typhimurium TA 1535 induced by these mutagens . Formation of DNA adducts from vinyl carbamate epoxide (VCO) and 2'-(4-nitrophenoxy)oxirane (NPO) was also markedly attenuated in the presence of CHL . Oral administration of CHL prior to the topical application of each of the above carcinogens resulted in significant reduction in both incidence and multiplicity of skin tumors in mice . The effective protection by CHL against VCO and NPO suggest that its formation of inactive complexes with these carcinogens is mediated by mechanisms other than pi-pi interactions.

FEMS Microbiol Lett, 1995 Jul 15, 130(1), 25 - 30
Production and identification of recombinant proteins of Salmonella typhimurium and their use in detection of antibodies in experimentally challenged animals; Kwang J et al.; Antibodies to experimental Salmonella typhimurium challenge in cattle and sheep were assessed by 15 recombinant flagellum proteins . The 15 DNA fragments selected for gene expression were derived from external flagellin, hook, hook-associated protein, and basal body gene domains . Our efforts were focused on characterizing the humoral immune response of Salmonella infected and vaccinated animals and identifying immunodominant antigenic determinants . This communication reports that the 159-261 amino acids of external flagellum (FliCi-1), 285-331 amino acids of hook protein (FlgE-2), and 309-391 amino acids and 440-537 amino acids of hook-associated protein (FlgK-1 and -2) appeared to be the most immunoreactive proteins and were recognized by all of the experimental animal sera tested in this study.

J Mol Biol, 1995 Jul 7, 250(2), 276 - 90
Crystal structure of the catalytic domain of the chemotaxis receptor methylesterase, CheB; West AH et al.; Signaling activity of bacterial chemotaxis transmembrane receptors is modulated by reversible covalent modification of specific receptor glutamate residues . The level of receptor methylation results from the activities of a specific S-adenosylmethionine-dependent methyltransferase, CheR, and the CheB methylesterase, which catalyzes hydrolysis of receptor glutamine or methylglutamate side-chains to glutamic acid . The CheB methylesterase belongs to a large family of response regulator proteins in which N-terminal regulatory domains control the activities of C-terminal effector domains . The crystal structure of the catalytic domain of the Salmonella typhimurium CheB methylesterase has been determined at 1.75 A resolution . The domain has a modified, doubly wound alpha/beta fold in which one of the helices is replaced by an anti-parallel beta-hairpin . Previous biochemical and mutagenesis data, suggest that the methylester hydrolysis catalyzed by CheB proceeds through a mechanism involving a serine nucleophile . The methylesterase active site is tentatively identified as a cleft at the C-terminal edge of the beta-sheet containing residues Ser164, His190 and Asp286 . The three-dimensional fold, and the arrangement of residues within the catalytic triad distinguishes the CheB methylesterase from any previously described serine protease or serine hydrolase.

J Mol Biol, 1995 Jul 7, 250(2), 123 - 7
Differential codon usage for conserved amino acids: evidence that the serine codons TCN were primordial; Diaz-Lazcoz Y et al.; The availability of specialized sequence databanks for Escherichia coli, Saccharomyces cerevisiae and Bacillus subtilis made it possible to build a set of 105 protein-coding genes that are homologous in these three species . An analysis of the triplets at both the nucleotide and amino acid level revealed that the codon bias of some amino acids are significantly higher at conserved rather than at non-conserved positions . Comparisons of homologous genes in E . coli and Salmonella typhimurium, and in S . cerevisiae and Drosophila melanogaster, led to the same conclusion . A special case was made for serine in E . coli, whose major codon is AGC for non-conserved and TCC for conserved residues . We interpret this observation as evidence that the primordial codons for serine were TCN, while codons AGY appeared later . This conclusion is substantiated by an analysis of the codon usage of catalytic serine residues in ancient, ubiquitous and essential proteins (ATP synthases and topoisomerases) . It is shown that in these proteins the proportion of the catalytic serine residues coded by TCN is significantly higher than the one expected from the overall codon usage of serine residues.

J Biol Chem, 1995 Jul 7, 270(27), 16097 - 106
Structure/function analysis of the periplasmic histidine-binding protein . Mutations decreasing ligand binding alter the properties of the conformational change and of the closed form; Wolf A et al.; The periplasmic histidine-binding protein, HisJ, is a receptor for the histidine permease of Salmonella typhimurium . Receptors of this type are composed of two lobes that are far apart in the unliganded structure (open conformation) and drawn close together in the liganded structure (closed conformation) . The binding of the ligand, in a cleft between the lobes, stabilizes the closed conformation . Such receptors have several functions in transport: interaction with the membrane-bound complex, transmission of a transmembrane signal to hydrolyze ATP, and receiving a signal to open the lobes and release the ligand . In this study the mechanism of action of HisJ was further investigated using mutant proteins defective in ligand binding activity and closed form-specific monoclonal antibodies (Wolf, A., Shaw, E . W., Nikaido, K., and Ames G . F.-L . (1994) J . Biol . Chem . 269, 23051-23058) . Y14H is defective in stabilization of the closed form, does not assume the closed empty form, and assumes an altered closed liganded form . T121A and G119R are similar to Y14H, but assume a normal closed liganded form . S72P binds the ligand to the open form, but does not assume a recognizable closed form . S92F is defective in the ability to undergo conformational change and to stabilize the closed form . All other mutant proteins appear to fall within one of these four categories . The biochemical characterization of these mutant proteins agrees with the structural analysis of the protein . We suggest that mutant proteins that do not assume the normal closed form, in addition to their defect in ligand binding, fail to interact with the membrane-bound complex and/or to transmit transmembrane signals.

Gene, 1995 Jul 4, 160(1), 123 - 8
A single tyrosine differentiates active and inactive Trypanosoma cruzi trans-sialidases; Cremona ML et al.; Several genes encode members of the Trypanosoma cruzi (Tc) trans-sialidase (TS) family . These proteins contain an enzymatic domain on the N terminus, the only one required for TS activity, and an antigenic domain (SAPA (shed acute phase antigen) amino acid (aa) repeats) on the C terminus . Only some members of this glycoprotein family are enzymatically active . The complete sequence of two clones encoding the enzymatic domain of active and inactive protein from each of two Tc strains has now been obtained . Comparison of these sequences showed a limited divergence among them: 20 out of the 642 deduced aa in the enzymatic domain were found to differ . From these 20 aa, only one was found to be essential for enzymatic activity . A Tyr342 residue is deduced in both active proteins while a His342 is present in both inactive ones . This naturally occurring Tyr342-->His substitution completely abolished the TS activity . In addition to Tyr342, a second deduced aa, Pro231, was found to be necessary for full enzymatic TS activity; a Pro231-->Ala change rendered the TS protein partially active . Fourteen aa residues, including Tyr342, out of the 16 aa in the active site of a sialidase from Salmonella typhimurium are present at the same or very similar positions in the Tc TS.

Proc Natl Acad Sci U S A, 1995 Jul 3, 92(14), 6399 - 403
Genetic and redox determinants of nitric oxide cytotoxicity in a Salmonella typhimurium model; De Groote MA et al.; Paradoxically, nitric oxide (NO) has been found to exhibit cytotoxic, antiproliferative, or cytoprotective activity under different conditions . We have utilized Salmonella mutants deficient in antioxidant defenses or peptide transport to gain insights into NO actions . Comparison of three NO donor compounds reveals distinct and independent cellular responses associated with specific redox forms of NO . The peroxynitrite (OONO-) generator 3-morpholinosydnonimine hydrochloride mediates oxygen-dependent Salmonella killing, whereas S-nitrosoglutathione (GSNO) causes oxygen-independent cytostasis, and the NO . donor diethylenetriamine-nitric oxide adduct has no antibacterial activity . GSNO has the greatest activity for stationary cells, a characteristic relevant to latent or intracellular pathogens . Moreover, the cytostatic activity of GSNO may best correlate with antiproliferative or antimicrobial effects of NO, which are unassociated with overt cell injury . dpp mutants defective in active dipeptide transport are resistant to GSNO, implicating heterolytic NO+ transfer rather than homolytic NO . release in the mechanism of cytostasis . This transport system may provide a specific pathway for GSNO-mediated signaling in biological systems . The redox state and associated carrier molecules are critical determinants of NO activity.

Rev Latinoam Microbiol, 1995 Jul-Sep, 37(3), 227 - 36
Kdp-like system in Salmonella typhimurium LT-2; Garcia-Cuellar C et al.; Salmonella typhimurium LT-2, as Escherichia coli K12, was able to grow in a potassium concentration-dependent manner, down to a very low concentration (< 5 microM) . Its metabolic swelling also was {K+}-dependent . When the cells were subjected to hyperosmotic shock, this ion was uptaken rapidly, probably due to a K(+)-high affinity transport-system, similar to the E . coli Kdp system . The shrinkage in presence of 0.6 M NaCl, however, was more noticeable in S . typhimurium, which expressed a smaller level of intracellular K+ than E . coli . The genetic locus responsible for the ability of S . typhimurium to grow in low {K+}, was mapped in nitrosoguanidine mutants and localized around min 18, close to the gal operon . This asseveration was confirmed by experiments of reversion, conjugation, and transduction . The mutants required considerably more {K+} to grow and to swell than the parental strain; in addition, below 1 mM {K+}, they showed less internal {K+}.

Rev Inst Med Trop Sao Paulo, 1995 Jul-Aug, 37(4), 297 - 302
Lysotypes and plasmidial profile of Salmonella serovar typhimurium isolated from children with enteric processes in the cities of Rio de Janeiro, RJ, and Salvador, BA - Brazil; Asensi MD et al.; The lysotypes, plasmidial profiles, and profiles of resistance to antimicrobial agents were determined in 111 Salmonella Typhimurium strains isolated from feces and blood of children treated in Rio de Janeiro and in Salvador . Six distinct lysotypes (19, 41, 97, 105, 120 and 193) were recognized, with a predominance of lysotype 193 (59.7%) in Rio de Janeiro and of phage type 105 (38.4) in Salvador . Approximately 86.7% of the lysotype 193 strains presented multiple resistance to more than six antimicrobial agents, whereas 93% of lysotype 105 strains were fully susceptible . More than 90% of the strains presented plasmids distributed into 36 different profiles in Rio de Janeiro and into 10 profiles in Salvador . A 40 MDa plasmid was the most frequent (47%) in the strains from Rio de Janeiro, whereas a 61 MDa plasmid predominated (14.5%) in Salvador . Combined analysis of plasmid profile and classification into lysotypes (especially those belonging to types 105 and 103, proved to be more discriminatory than the other methods applied).

Avian Dis, 1995 Jul-Sep, 39(3), 627 - 30
Egg dipping in hydrogen peroxide solution to eliminate Salmonella typhimurium from eggshell membranes; Padron M; The effectiveness of egg dipping in a 6% hydrogen peroxide solution to eliminate Salmonella typhimurium from eggshell membranes was evaluated . The first step was to assess the water uptake from broiler hatching eggs dipped in tap water once or twice using a positive-pressure differential method in order to determine the best method to use in the disinfection trials . Double dipping increased water uptake by 86% more than single dipping . Dipping S . typhimurium-contaminated eggs twice in a 6% hydrogen peroxide solution reduced the average number of organisms in eggshell membranes by 95% and the number of S . typhimurium-positive eggs by 55% compared with the infected untreated group . Dipping the eggs in a 6% hydrogen peroxide solution did not adversely affect hatchability.

Mikrobiol Z, 1995 Jul-Aug, 57(4), 66 - 72
{The evaluation of the mutagenic activity of gaseous particles in the atmospheric air by using a microbiological test}; Dugan AM et al.; Total mutagen activity of a gas component in chemical pollutions of the atmospheric air in a number of industrially developed towns of Ukraine has been studied and assessed in a microbiological test on Salmonella typhimurium . The towns were chosen proceeding from the specificity of industry: metallurgical industry (Mariupol, Zaporozhye, Donetsk, Krivoi Rog, Makeevka); chemical industry (Cherkassy, Chernigov; Kremenchug, Severodonetsk, Lisichansk, Gorlovka, Rovno, Sumy) and conditionally control ones (Simferopol, Sevastopol, Nikolaev, Poltava, Zhitomir) . The air samples, 100 m3, have been taken in each town weekly during a month by special absorbers of Polysorb-2 type . The extraction of chemical matters from absorbers was carried out by traditional methods . Chemical matters were dissolved in dimethyl sulphoxide and tested for its ability to induce the gene mutations . The studies have shown that the atmospheric air samples from the group of "metallurgical" towns prove the mutagen activity, classified as the "middle" one (the number of revertant colonies in the experiment exceeded the control 10.3 to 22.2 times) . The mutagenicity of "chemical" towns was on the level of "middle" and "weak", that of conditionally control ones was on the level of "weak" only.

Infect Immun, 1995 Jul, 63(7), 2770 - 2
Salmonella typhimurium displays normal invasion of mice with defective epidermal growth factor receptors; McNeil A et al.; The role of the epidermal growth factor (EGF) receptor in cell invasion by Salmonella typhimurium was examined in vitro and in vivo by using waved-2 mice which express an EGF receptor with reduced kinase activity . S . typhimurium invaded fibroblasts from waved-2 mice as efficiently as fibroblasts from wild-type control animals . In vivo, S . typhimurium both invaded the gastrointestinal tract and penetrated through to the spleen of waved-2 mice . Our studies suggest that the EGF receptor has only a limited role, if any, in cell invasion by S . typhimurium.

Infect Immun, 1995 Jul, 63(7), 2743 - 54
Characterization of intestinal invasion by Salmonella typhimurium and Salmonella dublin and effect of a mutation in the invH gene; Watson PR et al.; The relative levels of invasiveness of two bovine isolates each of Salmonella typhimurium and Salmonella dublin and of invH mutants of S . typhimurium were determined in MDCK and Int 407 cultured-cell assays and in bovine ileal loops . S . dublin was found to be significantly less invasive in cultured cells than S . typhimurium, but this difference was not observed in bovine intestines . The invH mutants exhibited a significant reduction in invasion in both cultured cells and bovine intestines . The invasive phenotypes of the strains were confirmed by fluorescent microscopy and scanning and transmission electron microscopy . The wild-type strains were observed in the laminae propriae of the intestinal villi, while in contrast the invH mutants were generally associated with the enterocyte layer . The degree of damage in the bovine ileum was related to the magnitude of the invasion . There was no difference in the amount of S . typhimurium or S . dublin recovered from the bovine ileum either with or without Peyer's patches 3 h after inoculation of the loop.

Infect Immun, 1995 Jul, 63(7), 2564 - 9
Influence of preimmunization with tetanus toxoid on immune responses to tetanus toxin fragment C-guest antigen fusions in a Salmonella vaccine carrier; Chabalgoity JA et al.; We have previously described a new system for the delivery of recombinant antigens in live Salmonella vaccines as genetic fusions to the C terminus of fragment C of tetanus toxin (TetC) driven by the anaerobically inducible nirB promoter . It has been reported that preimmunization with tetanus toxoid (TT) can suppress the antibody response to peptides chemically coupled to TT (epitope-specific suppression) in both animals and humans, which could interfere with efficacy of the Salmonella-TetC delivery system . We report that preimmunization of BALB/c mice with TT in alum did not suppress the response to either of two protective antigens of Schistosoma mansoni, the full-length S . mansoni P28 glutathione S-transferase (P28) and a construct consisting of eight tandem copies of the protective peptide comprising amino acids 115 to 131 of P28 . The guest antigens were expressed in the aroA Salmonella typhimurium SL3261 vaccine strain as fusions to TetC . Preimmunization with TT 10 weeks before administration of the recombinant salmonellae did not alter the antibody response to the full-length P28, whereas the response to the peptide comprising amino acids 115 to 131 was increased by preimmunization with TT, with the increase seen mainly in the immunoglobulin G1 isotype . The antitetanus response was increased by preimmunization with TT in all groups receiving salmonellae expressing TetC . The results could be important when one is considering the use of the Salmonella-TetC delivery system in populations preimmunized with TT.

Trop Doct, 1995 Jul, 25(3), 104 - 6
Multidrug resistant non-typhoidal Salmonella infections; Udgaonkar US et al.; Over a period of 2 years, 28 patients admitted to Government General Hospital (GGH), Sangli (which is attached to Government Medical College (GMC), Miraj) yielded multi-drug resistant non-typhoidal salmonellae from their clinical material . The pediatric age group predominated in the study, accounting for 93% of cases . Salmonella typhimurium was the main isolate (86%), the other being Salmonella newport (14%) . Gastroenteritis was the commonest presentation . Septicaemia was seen with 100% mortality, in infants below 1 month of age . Two cases of meningitis were also seen.

J Invertebr Pathol, 1995 Jul, 66(1), 68 - 71
Safety testing of Bacillus thuringiensis preparations, including thuringiensin, using the Salmonella assay; Carlberg G et al.; The aim of this study was to test genotoxicity aspects of the safety of two strains of Bacillus thuringiensis . The strains were of serotype H-1, producing thuringiensin, toxic to flies, and serotype H-14, producing endotoxin, toxic to mosquitoes, but not thuringiensin . Four preparations were tested: Tenfold concentrated cell-free culture media of serotypes H-1 and H-14, prepurified thuringiensin, and purified endotoxin . No increases in revertant colony numbers of the tester strains of Salmonella typhimurium TA98 or TA100 were observed at the dose levels used with or without metabolic activation . Infrequent slight increases in revertant colony numbers in strains TA98 and TA1538 at a very high test dose of 50 mg/plate, both in the presence and absence of thuringiensin, were probably caused by histidine present in the growth medium of B . thuringiensis . Furthermore, the effects were at most slightly dose related and not reproducible, and therefore the preparations can be considered nonmutagenic.

Appl Environ Microbiol, 1995 Jul, 61(7), 2614 - 9
A rapid, direct method for enumerating respiring enterohemorrhagic Escherichia coli O157:H7 in water; Pyle BH et al.; Simple, rapid methods for the detection and enumeration of specific bacteria in water and wastewater are needed . We have combined incubation using cyanoditolyl tetrazolium chloride (CTC) to detect respiratory activity with a modified fluorescent-antibody (FA) technique, for the enumeration of specific viable bacteria . Bacteria in suspensions were captured by filtration on nonfluorescent polycarbonate membranes that were then incubated on absorbent pads saturated with CTC medium . A specific antibody conjugated with fluorescein isothiocyanate was reacted with the cells on the membrane filter . The membrane filters were mounted for examination by epifluorescence microscopy with optical filters designed to permit concurrent visualization of fluorescent red-orange CTC-formazan crystals in respiring cells which were also stained with the specific FA . Experiments with Escherichia coli O157:H7 indicated that both respiratory activity and specific FA staining could be detected in logarithmic- or stationary-phase cultures, as well as in cells suspended in M9 medium or reverse-osmosis water . Following incubation without added nutrients in M9 medium or unsterile reverse-osmosis water, the E . coli O157:H7 populations increased, although lower proportions of the organisms reduced CTC . Numbers of CTC-positive, FA-positive cells compared with R2A agar plate counts gave a strong linear regression (R = 0.997) . Differences in injury did not appear to affect CTC reduction . The procedure, which can be completed within 3 to 4 h, has also been performed successfully with Salmonella typhimurium and Klebsiella pneumoniae.

Appl Environ Microbiol, 1995 Jul, 61(7), 2521 - 6
Flow cytometric assessment of Escherichia coli and Salmonella typhimurium starvation-survival in seawater using rhodamine 123, propidium iodide, and oxonol; Lopez-Amoros R et al.; The use of flow cytometry in microbiology allows rapid characterization of cells from a nonhomogeneous population . A method based on flow cytometry to assess the effects of lethal agents and the bacterial survival in starved cultures through the use of membrane potential-sensitive dyes and a nucleic acid marker is presented . The use of propidium iodide, rhodamine, and oxonol has facilitated the differentiation of cells of Escherichia coli and Salmonella typhimurium of various states of vitality following various treatments (heat, sonication, electroporation, and incubation with gramicidin) and during starvation in artificial seawater . The fluorescence intensity is directly correlated with viable cell counts for rhodamine 123 labelling, whereas oxonol and propidium iodide labelling is inversely correlated with viable counts . The distribution of rhodamine and oxonol uptake during starvation-survival clearly indicates that single-species starved bacteria are heterogeneous populations, and flow cytometry can be a fundamental tool for quantifying this heterogeneity.

J Leukoc Biol, 1995 Jul, 58(1), 32 - 9
Dietary modulation of Kupffer cell and splenocyte function during a Salmonella typhimurium challenge in mice; Eicher SD et al.; Oils from cold-water fish are rich in (n-3) polyunsaturated fatty acids, in particular eicosapentaenoic acid (20:5) and docosahexaenoic acid (22:6) . Although those fatty acids are beneficial in the prevention of cardiac disease and have anti-inflammatory properties, they can also decrease survival rates of mice during challenges with food-borne pathogens . This study was designed to determine dietary fat effects on Kupffer cells and splenocytes during a Salmonella typhimurium challenge . Mice were fed a low corn oil diet (3%, control), high corn oil diet (20%, HCO), or a menhaden fish oil diet (17% + 3% corn oil, FO) for 28 days and then orally given 3.1 x 10(8) colony-forming units of S . typhimurium . Kupffer cells and splenocytes were separated immediately prior to and on days 6, 10, and 14 postchallenge . Fish oil decreased Kupffer cell phagocytosis and oxidative burst early in the infection and adhesion molecule (CD18) expression at the end of the infection . In splenocytes, fish oil affected Ia expression prior to and late in the infection and depressed CD18 expression late in the infection . These data suggest that the diet affected Kupffer cells most early in the infection but affected splenocytes primarily later in the infection . Therefore, because the greatest death rate during an S . typhimurium infection occurs early, the reduced function of the Kupffer cells is probably a major factor.

J Toxicol Environ Health, 1995 Jul, 45(3), 337 - 47
Mouse bone marrow micronucleus assay: relationships with in vitro mutagenicity and rodent carcinogenicity; Benigni R; In this article, the relationship was studied between the in vivo mutagenicity assay of mouse bone marrow micronucleus (MIC), and four in vitro assays: Salmonella typhimurium, chromosomal aberrations in Chinese hamster ovary (CHO) cells, sister chromatid exchanges (SCE) in CHO cells, and mutation in mouse lymphoma L5178Y cells . A comparison with the rodent carcinogenicity data was also undertaken . The MIC data on 49 chemicals were generated by Shelby et al . (1993) . The MIC assay system employed three daily exposures by intraperitoneal injection; bone marrow samples were obtained 24 h following the final exposure . A preliminary analysis indicated that the 49 chemicals selected by Shelby et al . (1993) are a representative subset of the National Toxicology Program database . This study showed that MIC has a number of particular characteristics that are not shared by other biological systems . MIC is basically different from rodent carcinogenicity, despite being an in vivo system . At the same time, it responds to the chemicals in a different way from that of the in vitro genotoxicity assays . These in vitro assays mainly differ from each other in their different sensitivities to the genotoxins: MIC gives just a few positives (limited sensitivity), but, at the same time, some of these positives are detected only by the most sensitive assays, like the mouse lymphoma or SCE assays . In terms of risk assessment, MIC does not complement Salmonella for predicting chemical carcinogenicity, and would be better used to verify if the in vitro positive chemicals are able to exert their genotoxic potential in vivo.

J Bacteriol, 1995 Jul, 177(14), 3985 - 91
Role of methylation in aerotaxis in Bacillus subtilis; Wong LS et al.; Taxis to oxygen (aerotaxis) in Bacillus subtilis was characterized in a capillary assay and in a temporal assay in which the concentration of oxygen in a flow chamber was changed abruptly . A strong aerophilic response was present, but there was no aerophobic response to high concentrations of oxygen . Adaptation to a step increase in oxygen concentration was impaired when B . subtilis cells were depleted of methionine to prevent methylation of the methyl-accepting chemotaxis proteins . There was a transient increase in methanol release when wild-type B . subtilis, but not a cheR mutant that was deficient in methyltransferase activity, was stimulated by a step increase or a step decrease in oxygen concentration . The methanol released was quantitatively correlated with demethylation of methyl-accepting chemotaxis proteins . This indicated that methylation is involved in aerotaxis in B . subtilis in contrast to aerotaxis in Escherichia coli and Salmonella typhimurium, which is methylation independent.

J Bacteriol, 1995 Jul, 177(14), 3965 - 71
Homologs of the Shigella IpaB and IpaC invasins are required for Salmonella typhimurium entry into cultured epithelial cells; Kaniga K et al.; Entry into host cells is an essential feature in the pathogenicity of Salmonella spp . The inv locus of Salmonella typhimurium encodes several proteins which are components of a type III protein secretion system required for these organisms to gain access to host cells . We report here the identification of several proteins whose secretion into the culture supernatant of S . typhimurium is dependent on the function of the inv-encoded translocation apparatus . Nucleotide sequence analysis of the genes encoding two of these secreted proteins, SipB and SipC, indicated that they are homologous to the Shigella sp . invasins IpaB and IpaC, respectively . An additional gene was identified, sicA, which encodes a protein homologous to IpgC, a Shigella protein that serves as a molecular chaperone for the invasins IpaB and IpaC . Nonpolar mutations in sicA, sipB, and sipC rendered S . typhimurium unable to enter cultured epithelial cells, indicating that these genes are required for bacterial internalization.

Mutat Res, 1995 Jul, 329(2), 205 - 12
Emodin inhibits the mutagenicity and DNA adducts induced by 1-nitropyrene; Su HY et al.; Polygonum cuspidatum S . (PC) is frequently used as a laxative and an anticancer drug in Chinese medicine . The inhibitory effect of this herb and its component, emodin, on the direct-acting mutagenicity of 1-nitropyrene (1-NP) was examined using the Ames/microsomal test with Salmonella typhimurium TA98 and the genotoxicity of 1-NP was evaluated using the SOS chromotest with E . coli PQ37 . Emodin and water extracts of PC markedly decreased the mutagenicity of 1-NP in a dose-dependent manner in both assay systems . Furthermore, emodin and the extracts of PC significantly inhibited the formation of 1-NP DNA adducts in S . typhimurium TA98 in the 32P-postlabeling study . The results suggest that PC extracts and emodin act as blocking and/or suppressing agents to reduce the direct-acting mutagenicity of 1-NP.

Eur J Pharmacol, 1995 Jul 1, 293(2), 173 - 81
Activation of benzylic alcohols to mutagens by rat and human sulfotransferases expressed in Escherichia coli; Glatt H et al.; Human hydroxysteroid sulfotransferase, human phenol-sulfating form of phenol sulfotransferase, rat hydroxysteroid sulfotransferase a and rat phenol sulfotransferase IV were expressed in Escherichia coli . Cytosol preparations of transformed bacteria were used as activating systems in mutagenicity tests with Salmonella typhimurium TA98 . All test compounds, 1-hydroxymethylpyrene, 2-hydroxymethylpyrene, 1-(1-pyrenyl)ethanol, 9-hydroxymethylanthracene, 7-hydroxymethyl-12-methylbenz{a}anthracene and 4H-cyclopenta{def}chrysen-4-ol, were activated by both hydroxysteroid sulfotransferases investigated . However, 1-(1-pyrenyl)ethanol was 67-fold more efficiently activated by the human enzyme, whereas 7-hydroxymethyl-12-methylbenz{a}anthracene was 27-fold more efficiently activated by the rat enzyme . The phenol sulfotransferases showed relatively low activities with the benzylic alcohols investigated . The only exception was 4H-cyclopenta{def}chrysen-4-ol, which was activated efficiently by rat phenol sulfotransferase IV . We had previously tested the ability of rat and human hepatic cytosol preparations to activate the same compounds . The results of a statistical analysis suggest that the activities of human hydroxysteroid sulfotransferase, rat hydroxysteroid sulfotransferase a and phenol sulfotransferase IV can account for a substantial portion of the activation of benzylic alcohols in human, female rat and male rat liver, respectively.

Clin Microbiol Rev, 1995 Jul, 8(3), 336 - 56
Recovery of uncommon bacteria from blood: association with neoplastic disease; Beebe JL et al.; Table 6 is a summary of the organisms discussed with a listing of the environmental source, the endogenous source, the predisposing factors including neoplasms, and the postulated mechanisms by which the organism can gain access to the circulation . The evidence considered indicates that the entrance of one of these microorganisms into the bloodstream of a human being depends on the presence of multiplicity of predisposing factors . In the majority of cases of bacteremia due to one of these unusual organisms, two or more predisposing factors are present . Certain predisposing factors, such as cancer chemotherapy or intravenous catheterization, often provide a barrier break, while others, such as liver disease, may render the host immune system less capable of clearing organisms from the circulation . For organisms such as Campy-lobacter, Listeria, and Salmonella spp., attributes that allow the invasion of a healthy host are present and seem to be enhanced by the simultaneous presence of a predisposing condition, such as liver disease, in the host . Although somewhat fragmentary, a number of individual case reports describe bacteremia due to one of these organisms occurring weeks to years after surgery and after other therapeutic measures had effected a supposed cure of a cancer . It may be speculated that cancer patients, even after a cure, are still susceptible to bloodstream invasion by one of the aforementioned organisms by virtue of the presence of one or more predisposing metabolic, physiologic, or immunologic factors, even though these factors may be cryptic . The predominance of hematologic malignancies among cases of bacteremia due to these unusual organisms is also apparent . Although, as pointed out by Keusch (169), the reduction in the performance of immune function in hematologic malignancies compared with solid tumors is likely to be responsible, other associations of certain organisms with specific neoplasms warrant further examination . The frequency of bloodstream infections of Salmonella typhimurium and Capno-cytophaga canimorsus in Hodgkin's disease patients seems likely due to a particular mechanism which infection by these species is favored . The specific nature of these mechanisms remains to be determined . The recovery of any unusual bacterium from blood should warrant a careful consideration of the possibility of underlying disease, especially cancer . Microbiologists should advise clinicians of the unusual nature of the identified organism and provide the counsel that certain neoplastic processes, often accompanied by neutropenia, render the human host susceptible to invasion by almost any bacterium . The recovery of such organisms as C . septicum or S . bovis should prompt the clinician to aggressively seek to identify an occult neoplasm if one has not yet been diagnosed.

Microbiology, 1995 Jul, 141 ( Pt 7), 1715 - 22
Functional analysis of the flagellar genes in the fliD operon of Salmonella typhimurium; Yokoseki T et al.; The fliD genes of Salmonella typhimurium and Escherichia coli encode the filament-cap protein of the flagellar apparatus, which facilitates the polymerization of endogenous flagellin at the tips of the growing filaments . Previous sequence analysis of this operon in both organisms has revealed that the fliD gene constitutes an operon together with two additional genes, fliS and fliT . Based on the gene-disruption experiment in E . coli, both the fliS and fliT genes have been postulated to be necessary for flagellation . In the present study, we constructed S . typhimurium mutants in which either fliS or fliT on the chromosome was specifically disrupted . Both mutants were found to produce functional flagella, indicating that these genes are dispensable for motility development in S . typhimurium . However, flagellar filaments produced by the fliS mutant were much shorter than those produced by the wild-type strain . This indicates that the fliS mutation affects the elongation step of filament assembly . The excretion efficiency of flagellin was examined in the fliD-mutant background, where the exported flagellin molecules cannot assemble onto the hooks, resulting in their excretion into the culture media . We found that the amount of flagellin excreted was much reduced by the fliS mutation . Based on these results, we conclude that FliS facilitates the export of flagellin through the flagellum-specific export pathway.

Microbiology, 1995 Jul, 141 ( Pt 7), 1655 - 61
A homologue to the Escherichia coli alkyl hydroperoxide reductase AhpC is induced by osmotic upshock in Staphylococcus aureus; Armstrong-Buisseret L et al.; Four major proteins are induced in Staphylococcus aureus in response to hyperosmotic shock caused by the presence of two different osmolytes, sucrose and NaCl . The gene encoding one of these proteins was isolated using a novel PCR procedure . The derived protein sequence shows extensive similarity to a subunit of alkyl hydroperoxide reductase (AhpC) from both Escherichia coli and Salmonella typhimurium . Exposure of S . aureus to varying concentrations of H202 did not result in the detectable induction of AhpC.

Turk J Pediatr, 1995 Jul-Sep, 37(3), 229 - 33
Prognostic factors in Salmonella typhimurium septicemia . A 10-year retrospective study; Secmeer G et al.; In this study, 74 S.typhimurium septicemia cases were evaluated retrospectively from their records, and the age and sex distribution, presence of underlying disease, signs and symptoms, complete blood count, liver function tests and case fatality rate were documented and prognostic factors determined . It has been shown that S.typhimurium is the most common strain causing Salmonella septicemia, which is more fatal in the newborn period and in the presence of an associated disease, while hemoglobin and leukocyte counts do not play an important role in the prognosis . In Salmonella septicemia, congenital heart disease was the second-most common associated disease, which may be attributed to probable underlying immunodeficiency.

Antimicrob Agents Chemother, 1995 Jul, 39(7), 1621 - 3
Salmonella typhimurium gyrA mutations associated with fluoroquinolone resistance; Reyna F et al.; Spontaneous quinolone-resistant mutants obtained from Salmonella typhimurium Su694 were screened for mutations by direct DNA sequencing of an amplified PCR gyrA fragment . Substitutions Ser-83-->Phe (Ser83Phe), Ser83Tyr, Asp87Tyr, and Asp87Asn and double mutation Ala67Pro-Gly81Ser, which resulted in decreased sensitivities to ciprofloxacin, enoxacin, pefloxacin, norfloxacin, ofloxacin, and nalidixic acid, were found . The levels of resistance to quinolones for each mutant were determined.

Mutagenesis, 1995 Jul, 10(4), 357 - 64
Biotransformation of genotoxic agents in marine sponges . Mechanisms and modulation; De Flora S et al.; Marine sponges do not appear to suffer from neoplastic diseases, in spite of possible high exposures resulting from their nature as sessile bottom filter feeders which pump large volumes of sea water . The assessment of several parameters related to the biotransformation of mutagens/carcinogens showed that the metabolic machinery of sponge medulla cells is mainly oriented towards detoxification, with some differences depending on species (Geodia cydonium or Tethya aurantium) . Glutathione (GSH) levels were unexpectedly high in these cells, especially in Geodia, in which the concentration of this tripeptide was more than twice that measured in liver preparations from untreated rats, at least when related to the protein content . The oxidoreductive enzyme activities involved in the glutathione cycle were balanced in such a way as to favour a high GSH: oxidized glutathione (GSSG) ratio . GSH S-transferase activity was conversely rather low, compared to that of rat liver, and the dehydrogenases involved in the hexose monophosphate shunt were high in Tethya but low in Geodia . The metabolism of mutagens was investigated by using the Salmonella typhimurium his- strains TA100, TA98 and YG1024 . Sponge S12 fractions failed to activate aflatoxin B1, benzo{a}pyrene and the two heterocyclic amines 3-amino-1-methyl-5H-pyrido{4,3-b}indole and 2-amino-3,4-dimethyl-imidazo{4,5-f}quinoline . Although far less efficiently than untreated rat liver S12 fractions, Geodia and especially Tethya preparations weakly activated the three aromatic amines 2-acetyl-aminofluorene, 2-aminofluorene and 2-aminoanthracene . On the other hand, sponge S12 fractions were remarkably efficient in decreasing the mutagenic potency of the direct-acting mutagens 4-nitroquinoline 1-oxide and sodium dichromate.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutagenesis, 1995 Jul, 10(4), 343 - 51
Genotoxicity of 17 gyrase- and four mammalian topoisomerase II-poisons in prokaryotic and eukaryotic test systems; Albertini S et al.; The genotoxic potency of certain classes of topoisomerase II poisons is correlated with their affinity to the topoisomerase protein rather than with the presence of 'classical' structural alerts for DNA reactivity: bacterial topoisomerase II poisons (specifically named gyrase inhibitors) are highly genotoxic in prokaryotic systems; mammalian topoisomerase II poisons are potent mutagens/clastogens in eukaryotic systems . Studies with bacterial, lower eukaryotic and mammalian genotoxicity tests were performed to draw structure-activity conclusions and address risk-benefit considerations for the class of quinolone gyrase inhibitors . All 17 gyrase inhibitors investigated in this study showed genotoxic activity in Salmonella typhimurium strain TA102 and the SOS test . The genotoxic and the toxic activities increased in a highly parallel fashion from the parent compounds, nalidixic acid and oxolinic acid, to the new generation fluoroquinolones . Generally, the most potent fluoroquinolones also show clear-cut positive effects in eukaryotic test systems, although at concentrations 100-1000-fold higher than those effective in bacteria and also 100-1000-fold higher than the minimal genotoxic concentrations of antitumour topoisomerase II inhibitors (ellipticine, teniposide, mAMSA) used as reference compounds . However, subtle structural modifications of the quinolones can strongly diminish the preferential genotoxicity in the prokaryotic test systems.

Mutagenesis, 1995 Jul, 10(4), 333 - 41
Use of the SOS chromotest, the Ames-fluctuation test and the newt micronucleus test to study the genotoxicity of four trihalomethanes; Le Curieux F et al.; Three short-term assays (the SOS chromotest, the Ames-fluctuation test and the newt micronucleus test) were carried out to evaluate the genotoxicity of four trihalomethanes (chloroform, bromodichloromethane, chlorodibromomethane and bromoform) . With the SOS chromotest, all the chemicals studied except chloroform were found to induce primary DNA damage in Escherichia coli PQ37 . In the Ames-fluctuation test, only bromoform showed mutagenic activity on Salmonella typhimurium strain TA100 . The newt micronucleus assay detected a clastogenic effect on the peripheral blood erythrocytes of Pleurodeles waltl larvae for bromodichloromethane and bromoform . It appeared that the presence of bromine substituent(s) generally led to significant genotoxic activity . Moreover, the use of the metabolic system significantly increased the genotoxicity of the brominated trihalomethanes in the SOS chromotest . Unlike previous investigations in which the SOS chromotest was always the least interesting assay, this study exhibited the good efficiency of this in vitro test on E.coli for the detection of trihalomethanes with bromine substituents.

Mutagenesis, 1995 Jul, 10(4), 321 - 3
Lack of uniformity in the mutational spectra of chlorohydroxyfuranones in Salmonella typhimurium strain TA100; Hyttinen JM et al.; The mutational specificity of three chlorohydroxyfuranones found in chlorinated drinking water, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), 3-chloro-4(chloromethyl)-5-hydroxy-2(5H)-furanone (CMCF) and 3,4-dichloro-5-hydroxy-2(5H)-furanone (mucochloric acid, MCA), was examined in Salmonella typhimurium strain TA100 . DNA colony-hybridization of TA100 revertants showed that MX and CMCF both induced predominantly G:C-->T:A transversions (87 and 75% of total, respectively) with a 3:1 preference for the second position of the hisG46 (CCC) target codon . By contrast, MCA produced primarily G:C-->A:T transitions (66% of the total) with a 4:1 preference for the second position of the CCC codon . The mutational specificity of MCA is consistent with the idea that chloroacetaldehyde, a degradation product of MCA, is responsible for the observed mutations . The chemical mechanism by which either MX or CMCF induces G:C-->T:A transversions remains unknown.

Mol Microbiol, 1995 Jul, 17(1), 49 - 56
Co-ordinate, temperature-sensitive regulation of the three Yersinia enterocolitica flagellin genes; Kapatral V et al.; Yersinia enterocolitica cells, when cultured at 30 degrees C or below, are flagellated and motile . Cells cultured at 37 degrees C or above lack flagella and are non-motile . To identify flagellin genes that are a target of this temperature-dependent regulation, a library of Y . enterocolitica genomic inserts in a phage lambda vector was probed with the Salmonella typhimurium fliC (flagellin) gene . A DNA fragment subcloned from a recombinant phage which hybridizes with the probe complements a non-motile S . typhimurium fliC-fljB- (flagellin-minus) mutant . DNA sequence analysis shows that Y . enterocolitica contains three tandem flagellin genes, designated fleA, fleB and fleC . All three genes are co-ordinately transcribed at low, but not high, temperature from fliA-dependent (sigma F) promoters . Flagellin transcription arrests rapidly after upshift to 37 degrees C (host temperature) . In contrast, flagellin transcription resumes only after several generations when cells cultured at 37 degrees C are downshifted to 28 degrees C.

Mol Microbiol, 1995 Jul, 17(1), 169 - 81
PhoP/PhoQ transcriptional repression of Salmonella typhimurium invasion genes: evidence for a role in protein secretion; Pegues DA et al.; Previously, the PhoP-repressed locus prgH was identified as important for signalling epithelial cells to endocytose Salmonella typhimurium . Characterization of prgH revealed that it is an operon of four genes encoding polypeptides of 392 (prgH), 80 (prgI), 101 (prgJ) and 252 amino acid residues (prgK) . Synthesis of the 2.6 kb prgHIJK transcript was repressed in bacteria that activate PhoP/PhoQ, indicating that PhoP/PhoQ regulates prgHIJK by transcriptional repression . The prgI, prgJ and prgK predicted gene products were similar to Shigella flexneri and Yersinia enterocolitica proteins required for secretion of Ipa and Yop virulence factors . Analysis of the culture supernatants from wild-type S . typhimurium demonstrated that at least 25 polypeptides larger than 14 kDa could be detected . In contrast, prgH1::TnphoA, phoP-constitutive and hil-deletion mutants had significant defects in their supernatant protein profiles . The invasion and supernatant protein profile defects of the prgH1::TnphoA mutant were both complemented by a 5.1 kb plasmid that included prgHIJK . These results suggest that PhoP/PhoQ regulates extracellular transport of proteins by transcriptional repression of secretion determinants and that secreted proteins may be involved in signalling epithelial cells to endocytose bacteria.

Mol Microbiol, 1995 Jul, 17(1), 155 - 67
The stationary-phase sigma factor sigma S (RpoS) is required for a sustained acid tolerance response in virulent Salmonella typhimurium; Lee IS et al.; The acid tolerance response (ATR) of log-phase Salmonella typhimurium is induced by acid exposures below pH 4.5 and will protect cells against more extreme acid . Two systems are evident: a transiently induced system dependent on the iron regulator Fur that provides a moderate degree of acid tolerance and a more effective sustained ATR that requires the alternate sigma factor sigma S encoded by rpoS . Differences between the acid responses of virulent S . typhimurium and the attenuated laboratory strain LT2 were attributed to disparate levels of RpoS caused by different translational starts . The sustained ATR includes seven newly identified acid shock proteins (ASPs) that are dependent upon sigma S for their synthesis . It is predicted that one or more of these ASPs is essential for the sustained system . The sustained ATR also provided cross-protection to a variety of other environmental stresses (heat, H2O2 and osmolarity); however, adaptation to the other stresses did not provide significant acid tolerance . Therefore, in addition to starvation, acid shock serves as an important signal for inducing general stress resistance . Consistent with this model, sigma S proved to be induced by acid shock . Our results also revealed a connection between the transient and sustained ATR systems . Mutations in the regulator atbR are known to cause the overproduction of ten proteins, of which one or more can suppress the acid tolerance defect of an rpoS mutant . One member of the AtbR regulon, designated atrB, was found to be co-regulated by sigma S and AtbR . Both regulators had a negative effect on atrB expression . The results suggest AtrB serves as a link between the sustained and transient ATR systems . When sigma S concentrations are low, a compensatory increase in AtrB is required to engage the transiently induced, RpoS-independent system of acid tolerance . Results also suggest different acid-sensitive targets occur in log-phase versus stationary-phase cells.

Mol Gen Genet, 1995 Jun 25, 247(6), 680 - 92
RNA polymerase (rpoB) mutants selected for increased resistance to gyrase inhibitors in Salmonella typhimurium; Blanc-Potard AB et al.; Some rifampicin-resistance (RifR) mutations make bacteria slightly resistant to the gyrase inhibitors novobiocin (Nov) and nalidixic acid (Nal) . This suggested that it might be possible to isolate rpoB mutants using either drug for positive selection . In an initial test, we confirmed the presence of Rif-resistant isolates among clones selected for Nov resistance . These mutants are also more resistant to Nal . In a subsequent experiment, we found that mutants selected for low-level resistance to Nal include isolates harboring mutations genetically linked to the rpoB locus; of two such mutants studied, one is temperature-sensitive for growth . These two mutants, which are only marginally affected in their response to Nov, are normally sensitive to Rif and thus might be representative of a new class of rpoB alleles . The Rif-resistant and Rif-sensitive rpoB alleles that increase resistance to gyrase inhibitors have one property in common: they all suppress, to varying degrees, the defect in his operon regulation (transcriptional deattenuation) caused by a gyrase defect or inhibition by novobiocin . To further analyse the transcription-supercoiling relationships in these mutants, we examined the ability of RNA polymerase to recruit gyrase activity during transcription . This was done by two independent approaches: (i) observing transcription-induced accumulation of hyper-negatively supercoiled plasmid DNA in a topA mutant background and (ii) measuring transcription-induced plasmid DNA cleavage in the presence of oxolinic acid . Results indicate that the rpoB alleles described in this study diminish the recruitment of gyrase activity by the transcription process . This property correlates with a decrease in the rate of transcription initiation.(ABSTRACT TRUNCATED AT 250 WORDS)

J Mol Biol, 1995 Jun 16, 249(4), 700 - 13
Constitutive forms of the enhancer-binding protein NtrC: evidence that essential oligomerization determinants lie in the central activation domain; Flashner Y et al.; Nitrogen regulatory protein C (NtrC) is a bacterial enhancer-binding protein that activates transcription by the sigma 54-holoenzyme . To activate transcription, NtrC must hydrolyze ATP, a reaction that depends upon its being phosphorylated and forming an appropriate oligomer . In this paper we characterize "constitutive" mutant forms of the NtrC protein from Salmonella typhimurium; unlike wild-type NtrC, these forms are able to hydrolyze ATP and activate transcription in vitro without being phosphorylated . The amino acids altered in NtrCconstitutive proteins are located in both the N-terminal regulatory domain and the central domain, which is directly responsible for transcriptional activation . The residues that are altered are not conserved among activators of the sigma 54-holoenzyme, and are not identical even among NtrC proteins from members of different subgroups of the proteobacteria (purple bacteria) . NtrCconstitutive proteins are phosphorylated normally; phosphorylation increases their ability to hydrolyze ATP and activate transcription . Moreover, the oligomerization of these proteins that occurs when they bind to an enhancer also increases the ATPase activity of both unmodified and phosphorylated forms . Removal of the N-terminal regulatory domain from two NtrCconstitutive proteins with amino acid substitutions in the central domain (NtrCS160F and NtrCV2881) leaves them active, indicating that essential oligomerization determinants lie outside the regulatory domain . This conclusion is confirmed by the observation that the ATPase activity of delta N-NtrCS160F is greatly stimulated when it binds to an enhancer, and by the ability of this protein to activate transcription synergistically with a form of NtrC incapable of DNA-binding . Together with previous results indicating that oligomerization determinants do not lie in the C-terminal DNA-binding domain of NtrC; these results provide evidence that they lie in the central domain.

J Mol Biol, 1995 Jun 9, 249(3), 529 - 34
Cold denaturation of CheY; DeKoster GT et al.; The thermal stability of the bacterial chemotaxis protein CheY from Salmonella typhimurium has been examined by thermal denaturation at pH 7.0 in the presence of guanidine-HCl and urea . For both denaturants, thermal denaturation monitored by circular dichroism spectropolarimetry consists of transitions both above and below 25 degrees C, which is strong evidence for a heat capacity change that is > or = 1500 cal/(mol K) upon unfolding . While many data for chemical and thermal denaturation are consistent with data for CheY from Escherichia coli, the observation of cold denaturation for S . typhimurium CheY is inconsistent with the small heat capacity change, 600 to 850 cal/(mol K), reported for denaturation of the E . coli protein.

J Natl Cancer Inst, 1995 Jun 7, 87(11), 836 - 41
Mutagens from heated Chinese and U.S . cooking oils; Shields PG et al.; BACKGROUND: The lung cancer incidence in Chinese women is among the highest in the world, but tobacco smoking accounts for only a minority of the cancers . Epidemiologic investigations of lung cancer among Chinese women have implicated exposure to indoor air pollution from wok cooking, where the volatile emissions from unrefined cooking oils are mutagenic . PURPOSE: This study was conducted to identify and quantify the potentially mutagenic substances emitted from a variety of cooking oils heated to the temperatures typically used in wok cooking . METHODS: Several cooking oils and fatty acids were heated in a wok to boiling, at temperatures (for the cooking oils) that ranged from 240 degrees C to 280 degrees C (typical cooking temperatures in Shanghai, China) . The oils tested were unrefined Chinese rapeseed, refined U.S . rapeseed (known as canola), Chinese soybean, and Chinese peanut in addition to linolenic, linoleic, and erucic fatty acids . Condensates of the emissions were collected and tested in the Salmonella mutation assay (using Salmonella typhimurium tester strains TA98 and TA104) . Volatile decomposition products also were subjected to gas chromatography and mass spectroscopy . Aldehydes were detected using high-performance liquid chromatography and UV spectroscopy . RESULTS: 1,3-Butadiene, benzene, acrolein, formaldehyde, and other related compounds were qualitatively and quantitatively detected, with emissions tending to be highest for unrefined Chinese rapeseed oil and lowest for peanut oil . The emission of 1,3-butadiene and benzene was approximately 22-fold and 12-fold higher, respectively, from heated unrefined Chinese rapeseed oil than from heated peanut oil . Lowering the cooking temperatures or adding an antioxidant, such as butylated hydroxyanisole, before cooking decreased the amount of these volatile emissions . Among the individual fatty acids tested, heated linolenic acid produced the greatest quantities of 1,3-butadiene, benzene, and acrolein . Separately, the mutagenicity of individual volatile emission condensates was correlated with linolenic acid content (r = .83; P = .0004) . Condensates from heated linolenic acid, but not linoleic or erucic acid, were highly mutagenic . CONCLUSIONS: These studies, combined with experimental and epidemiologic findings, suggest that high-temperature wok cooking with unrefined Chinese rapeseed oil may increase lung cancer risk . This study indicates methods that may reduce that risk . IMPLICATIONS: The common use of wok cooking in China might be an important but controllable risk factor in the etiology of lung cancer . In the United States, where cooking oils are usually refined for purity, additional studies should be conducted to further quantify the potential risks of such methods of cooking.

Med Oncol, 1995 Jun, 12(2), 103 - 8
Immunotherapy of a plasmacytoma with attenuated salmonella; Eisenstein TK et al.; An attenuated strain of Salmonella typhimurium, SL3235, developed as a prototypic typhoid vaccine, is shown to retard growth of a murine plasmacytoma, TEPC-183, and to prolong survival of tumor-bearing mice . Live salmonella, but not acetone-killed organisms, had antitumor activity . The immunotherapeutic effect was demonstrable when the tumor was injected intralesionally or intraperitoneally . Increased survival, longer mean time to death, and retardation of tumor growth were found when the salmonella were given intralesionally as late as the sixth day post-tumor injection . Timing of salmonella inoculation, as well as the salmonella dose, had an effect on treatment efficacy . Injection of salmonella intraperitoneally exerted a strong antitumor effect when given as late as the third day post-tumor inoculation . The highest dose (2 x 10(6)) of salmonella was less effective than doses 10- or 100-fold lower . TEPC-183 plasmacytoma is rapidly growing and highly immunosuppressive, so the ability of the salmonella to exert therapeutic activity against it is a measure of the potency of the vaccine . These observations are of interest, as they show that a genetically engineered, avirulent strain of Salmonella has immunotherapeutic properties similar to those of BCG and other biological response modifiers, and might have clinical potential as an antitumor agent.

Food Chem Toxicol, 1995 Jun, 33(6), 491 - 500
Evaluation of the genotoxicity potential and chronic inhalation toxicity of 1,1-dichloro-1-fluoroethane (HCFC-141b); Millischer RJ et al.; A battery of in vitro and in vivo tests were conducted on HCFC-141b as a vapour . Bacterial gene mutation assays with Escherichia coli and Salmonella typhimurium were negative in all tester strains . In vitro chromosomal aberration assays were positive on CHO cells but negative on human lymphocytes . Moreover, HCFC-141b was negative in vivo in a mouse micronucleus inhalation assay . On the basis of these data and previously reported genotoxicity testing, HCFC-141b is considered non-genotoxic . Groups of 80 male and 80 female Sprague-Dawley rats were exposed, by inhalation (6 hr/day, 5 days/wk) to vapours of HCFC-141b for 104 wk at target concentrations of 0 (control), 1500, 5000 and 20,000 ppm (increased from 15,000 ppm after 17 wk of exposure) . No exposure-related effects of toxicological significance were noted with respect to survival, clinical signs, ophthalmoscopy, haematology, clinical chemistry, urinalysis or organ weight analysis . Reduced food intake and body weight gain were noted in both sexes of the 15,000 ppm group during the first 16 wk; thereafter, body weight gains in all groups were similar although the intergroup differences in body weight remained evident . Reduced food intake persisted in both sexes through wk 52 and in females during the second year of exposure . Treatment-related effects on macroscopic pathology were confined to increased incidences of testicular masses and altered appearance . Microscopic pathology examinations confirmed the testes as the target organ with findings of increased incidences of benign interstitial cell tumours and hyperplasia at 5000 and 20,000 ppm . The no-observable-adverse-effect level (NOAEL) was 1500 ppm . The testicular changes at high exposure levels were considered to be due to a change of the senile hormonal imbalance in geriatric rats and of little significance for the assessment of human health effects.

Invest Ophthalmol Vis Sci, 1995 Jun, 36(7), 1344 - 51
Systemic immunization with Hsp60 alters the development of chlamydial ocular disease; Rank RG et al.; PURPOSE: To determine whether immunization with recombinant Hsp60 would exacerbate ocular pathology on challenge with viable chlamydial elementary bodies . METHODS: Guinea pigs were immunized either subcutaneously with recombinant Hsp60 or both subcutaneously with recombinant Hsp60 and ocularly with attenuated Salmonella typhimurium expressing the guinea pig inclusion conjunctivitis (GPIC) Hsp60 antigen . All animals were challenged in the conjunctiva with the agent of GPIC, and the degree of gross ocular pathology was determined . Immunoglobulin G (IgG) and immunoglobulin A (IgA) antibody titers to Hsp60 were measured in ocular secretions as a measure of the degree of immunization . RESULTS: In primary and challenge GPIC infection, the degree of gross ocular pathology was lower in the immunized group . The presence of high IgA and IgG antibody titers to Hsp60 in tears suggested that the response may have been modified by the presence of blocking antibodies that either may have removed the antigen quickly or prevented interaction with sensitized T cells . In contrast to subcutaneous immunization, the combined immunization regimen, consisting of subcutaneous recombinant Hsp60 followed by ocular inoculation of the attenuated Salmonella, resulted in no difference in gross pathology after reinfection of guinea pigs with GPIC . CONCLUSIONS: These data indicated that the immunization with Hsp60 did not produce exacerbated disease on challenge with viable organisms; however, the data suggested that the route of administration, form of antigen, or both may be critical in the disease process.

Mutat Res, 1995 Jun, 329(1), 1 - 9
Specific disruption of samAB genes in a 60-megadalton cryptic plasmid of Salmonella typhimurium; Nohmi T et al.; The expression of umuDC operon is required for UV and most chemical mutagenesis in Escherichia coli . The closely related species Salmonella typhimurium has two sets of umuDC-like operons, i.e., umuDCST in the chromosome and samAB in a 60-megadalton cryptic plasmid . In this study, we specifically disrupted the samAB genes to investigate their exact roles in UV mutagenesis in S . typhimurium . The specific gene disruption was carried out by the preligation method . Deletion of samAB did not lower the UV mutability of S . typhimurium TA2659 but rather increased the UV mutability about twofold . The samAB-umuDCST double deletion mutant as well as the umuDCST deletion mutant was UV nonmutable . These results suggest that the samAB genes do not considerably contribute to the UV mutability of S . typhimurium and raise the question of why such quiet umuDC-like genes are present in the cryptic plasmid of S . typhimurium.

J Bacteriol, 1995 Jun, 177(12), 3485 - 95
Regulated underexpression of the FliM protein of Escherichia coli and evidence for a location in the flagellar motor distinct from the MotA/MotB torque generators; Tang H et al.; The FliM protein of Escherichia coli is essential for the assembly and function of flagella . Here, we report the effects of controlled low-level expression of FliM in a fliM null strain . Disruption of the fliM gene abolishes flagellation . Underexpression of FliM causes cells to produce comparatively few flagella, and most flagella built are defective, producing subnormal average torque and fluctuating rapidly in speed . The results imply that in a normal flagellar motor, multiple molecules of FliM are present and can function independently to some degree . The speed fluctuations indicate that stable operation requires most, possibly all, of the normal complement of FliM . Thus, the FliM subunits are not as fully independent as the motility proteins MotA and MotB characterized in earlier work, suggesting that FliM occupies a location in the motor distinct from the MotA/MotB torque generators . Several mutations in fliM previously reported to cause flagellar paralysis in Salmonella typhimurium (H . Sockett, S . Yamaguchi, M . Kihara, V.M . Irikura, and R . M . Macnab, J . Bacteriol . 174:793-806, 1992) were made and characterized in E . coli . These mutations did not cause flagellar paralysis in E . coli; their phenotypes were more complex and suggest that FliM is not directly involved in torque generation.

J Bacteriol, 1995 Jun, 177(11), 3355 - 7
I-CeuI reveals conservation of the genome of independent strains of Salmonella typhimurium; Liu SL et al.; The enzyme I-CeuI, encoded by a class I mobile intron inserted in the gene for 23S rRNA in Chlamydomonas eugamatos, cleaves a specific 19-bp sequence in this gene . This sequence is present only in the seven genes for rRNA in Salmonella typhimurium and Escherichia coli . Partial digestion with I-CeuI of DNA from 17 wild-type strains of S . typhimurium indicates that the chromosome of these strains is strongly conserved, for the digestion products closely resemble those of strain LT2 . The lengths and order of chromosomal segments are conserved in 15 of the strains; 2 show some rearrangements . XbaI digestion indicated heterogeneity without revealing the genomic structure . Because of conservation of I-CeuI sites in genes for rRNA and conservation of the number and locations of these genes, I-CeuI provides an excellent tool for the rapid examination of the chromosomes of related species of bacteria; differences in the fingerprints indicate the occurrence of chromosomal rearrangements such as insertions or inversions.

J Bacteriol, 1995 Jun, 177(11), 3326 - 31
Cloning and characterization of the Escherichia coli hemN gene encoding the oxygen-independent coproporphyrinogen III oxidase; Troup B et al.; Coproporphyrinogen III oxidase, an enzyme involved in heme biosynthesis, catalyzes the oxidative decarboxylation of coproporphyrinogen III to form protoporphyrinogen IX . Genetic and biochemical studies suggested the presence of two different coproporphyrinogen III oxidases, one for aerobic (HemF) and one for anaerobic (HemN) conditions . Here we report the cloning of the hemN gene encoding the oxygen-independent coproporphyrinogen III oxidase from Escherichia coli by complementation of a Salmonella typhimurium hemF hemN double mutant . An open reading frame of 1,371 bp encoding a protein of 457 amino acids with a calculated molecular mass of 52.8 kDa was identified . Sequence comparisons revealed 92% amino acid sequence identity to the recently cloned S . typhimurium hemN gene and 35% identity to the Rhodobacter sphaeroides gene . The hemN gene was mapped to 87.3 min of the E . coli chromosome and found identical to open reading frame o459 previously discovered during the genome sequencing project . Complementation of S . typhimurium hemF hemN double mutants with the E . coli hemN gene was detected under aerobic and anaerobic conditions, indicating an aerobic function for HemN . The previously cloned E . coli hemF gene encoding the oxygen-dependent enzyme complemented exclusively under aerobic conditions . Primer extension experiments revealed a strong transcription initiation site 102 bp upstream of the translational start site . DNA sequences with homology to a sigma 70-dependent promoter were detected . Expression of the hemN gene in response to changing environmental conditions was evaluated by using lacZ reporter gene fusions . Under anaerobic conditions, hemN expression was threefold greater than under aerobic growth conditions . Removal of iron from the growth medium resulted in an approximately fourfold decrease of aerobic hemN expression . Subsequent addition of iron restored normal expression.

J Bacteriol, 1995 Jun, 177(11), 3259 - 68
Functional equivalence of Escherichia coli sigma E and Pseudomonas aeruginosa AlgU: E . coli rpoE restores mucoidy and reduces sensitivity to reactive oxygen intermediates in algU mutants of P . aeruginosa; Yu H et al.; Mucoid colony morphology is the result of the overproduction of the exopolysaccharide alginate and is considered to be a major pathogenic determinant expressed by Pseudomonas aeruginosa during chronic respiratory infections in cystic fibrosis . Conversion to mucoidy can be caused by mutations in the second or third gene of the stress-responsive system algU mucA mucB . AlgU is 66% identical to the alternative sigma factor RpoE (sigma E) from Escherichia coli and Salmonella typhimurium and directs transcription of several critical alginate biosynthetic and regulatory genes . AlgU is also required for the full resistance of P . aeruginosa to reactive oxygen intermediates and heat killing . In this work, we report that E . coli sigma E can complement phenotypic defects of algU inactivation in P . aeruginosa: (i) the rpoE gene from E . coli complemented an algU null mutant of P . aeruginosa to mucoidy; (ii) the presence of the E . coli rpoE gene in P . aeruginosa induced alginate production in the standard genetic nonmucoid strain PAO1; (iii) the plasmid-borne E . coli rpoE gene induced transcription of algD, a critical algU-dependent alginate biosynthetic gene; and (iv) when present in algU::Tcr mutants, E . coli rpoE partially restored resistance to paraquat, a redox cycling compound that increases intracellular levels of superoxide radicals . A new gene, mclA, encoding a polypeptide with an apparent molecular mass of 27.7 kDa was identified immediately downstream of rpoE in E . coli . The predicted product of this gene is 28% identical (72% similar) to MucA, a negative regulator of AlgU activity in P . aeruginosa . The results reported in this study demonstrate that RpoE and AlgU are functionally interchangeable in P . aeruginosa and suggest that elements showing sequence similarity to those known to regulate AlgU activity in P . aeruginosa are also present in other bacteria.

J Bacteriol, 1995 Jun, 177(11), 3080 - 6
The superinfection exclusion gene (sieA) of bacteriophage P22: identification and overexpression of the gene and localization of the gene product; Hofer B et al.; Previous work has shown that the sieA gene of Salmonella bacteriophage P22 is located between the genes mnt and 16 . We cloned DNA fragments of the region into multicopy vectors and tested the transformants for mediating superinfection exclusion . Subcloning, phenotypical tests, and DNA sequencing resulted in the identification of the sieA gene . There are two possible initiation codons within one open reading frame of 492 or 480 bp . The deduced amino acid sequence leads to a hypothetical polypeptide with a calculated molecular mass of 18.8 or 18.3 kDa, respectively . According to three hydrophobic regions, all of which are long enough to span the membrane, the product of sieA should be a protein of the inner membrane of a P22-lysogenic cell of Salmonella typhimurium . The SieA protein was moderately overproduced from an expression vector in cultures of Escherichia coli and could be recovered from the membrane fraction.

Infect Immun, 1995 Jun, 63(6), 2302 - 9
Transepithelial signaling to neutrophils by salmonellae: a novel virulence mechanism for gastroenteritis; McCormick BA et al.; Salmonella serotypes which elicit human enteritis cannot be distinguished from those that do not on the basis of their in vitro interactions with eukaryotic cells . We have recently reported that an enteritis-producing strain of Salmonella typhimurium signals intact intestinal epithelium to recruit subepithelial neutrophils to migrate across the epithelial (B . A . McCormick, S . P . Colgan, C . D . Archer, S . I . Miller, and J . L . Madara, J . Cell Biol . 123:895-907, 1993) . We now utilize a cell culture model of human intestinal epithelium (with T84 cells) to examine whether such transepithelial signaling to neutrophils by salmonellae is predictive of potential to elicit gastroenteritis . Various Salmonella serotypes, including S . typhimurium, S . enteritidis, S . pullorum, S . arizonae, S . typhi, and S . paratyphi, as well as invasion-defective mutants of S . typhimurium, were studied . Strains or serotypes which elicit diffuse enteritis in humans (defined histologically as transepithelial migration of neutrophils) exhibited transepithelial signaling to neutorphils across epithelial cell monolayers, while those which do not elicit diffuse enteritis in humans did not display transepithelial signaling . In contrast, the ability to enter the apical surface of T84 cells did not differentiate strains or serotypes which induce diffuse enteritis from those which do not . These results strongly suggest that the ability of salmonellae to elicit transepithelial signaling to neutrophils is a key virulence mechanism underlying Salmonella-elicited enteritis.

Jpn J Antibiot, 1995 Jun, 48(6), 868 - 77
{Cytotoxicity and mutagenicity studies of T-3761}; Nakamura S et al.; We investigated cytotoxicity and mutagenicity of T-3761 . The mutagenicity was evaluated using reverse mutation test with bacteria, chromosome aberration test with cultured cells and micronucleus test with mice . The following results were obtained . 1 . Cytotoxicity test: The cell growth was examined using Chinese hamster (V79) cells . The 50% inhibition doses of T-3761 for cell growth (ID50) were 490 micrograms/ml (cultured for 24 hours) and 220 micrograms/ml (cultured for 48 hours) . The inhibitory effect of T-3761 was 2-4 times lower than those of ciprofloxacin or norfloxacin and approximately equal to cephalothin . 2 . Reverse mutation test with bacteria: The preincubation method with Salmonella typhimurium TA100, TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA, and the induced mutation frequency (IMF) test with Salmonella typhimurium TA100, TA98 were performed . The number of revertant colonies were not increased in any strains treated with T-3761 in the presence or absence of S9 mix . 3 . Chromosome aberration test: V79 cells were treated with 50-200 micrograms/ml of T-3761 for 24 or 48 hours, and were treated with 400-3,200 micrograms/ml of T-3761 for 6 hours with S9 mix . The number of cells showing chromosomal aberrations were not increased in any conditions tested for T-3761 . 4 . Micronucleus test: The male ICR mice were given a single (500-5,000 mg/kg) or five consecutive (150-1,500 mg/kg) oral administration of T-3761 . The number of polychromatic erythrocytes with micronuclei were not increased at any dosage groups of T-3761 . From these results, it is concluded that T-3761 has low cytotoxicity, and has no mutagenicity.

Vet Microbiol, 1995 Jun, 45(1), 19 - 26
In vitro susceptibility to antimicrobial drugs of 62 Salmonella strains isolated from horses in The Netherlands; van Duijkeren E et al.; The in vitro activity of 17 antimicrobial drugs against strains of Salmonella typhimurium (n = 52), Salmonella thompson (n = 2), Salmonella heidelberg (n = 3), Salmonella hadar (n = 2), Salmonella enteritidis (n = 1), Salmonella infantis (n = 1) and Salmonella derby (n = 1) was tested using the agar dilution method . The strains were isolated from horses admitted to the Large Animal Clinics of Utrecht University . The majority of strains were susceptible to gentamicin, amikacin, kanamycin, enrofloxacin, ciprofloxacin, flumequine, colistine, furazolidone and ceftiofur . However, all strains of Salmonella typhimurium phage type 200 (n = 14), were multiresistant i.e . were resistant to ampicillin amoxycillin, amoxycillin in combination with clavulanic acid, chloramphenicol, nitrofurantoin, trimethoprim, aditoprim and baquiloprim . Two of these strains were also resistant to gentamicin . Based on the susceptibility data found in the present study in combination with pharmacokinetic data available in the literature a rationale for antimicrobial therapy in equine salmonellosis is given . As first choice, gentamicin at a dosage of 3 mg/kg combined with ampicillin at a dosage of 20 mg/kg given with a 8-12 hour dosing interval by intravenous route is advised . As an alternative, the intravenous administration of trimethoprim/sulfonamide combinations given twice daily at a combined dose of 30 mg/kg is suggested.

J Small Anim Pract, 1995 Jun, 36(6), 279 - 81
Chronic carriage of multidrug resistant Salmonella typhimurium in a cat; Wall PG et al.; Gastroenteritis caused by a multidrug-resistant Salmonella typhimurium was diagnosed in a 12-week-old kitten . Although the kitten recovered from the acute episode after seven days, it continued to shed salmonella in its faeces for a further 12 weeks . Carriage was finally cleared by administering a 14-day course of treatment with parenteral enrofloxacin . The public health implications of an infection and subsequent carriage in a cat of a salmonella strain known to cause illness in humans is discussed.

Poult Sci, 1995 Jun, 74(6), 916 - 24
Control of Salmonella typhimurium colonization in broiler chicks with a continuous-flow characterized mixed culture of cecal bacteria; Corrier DE et al.; A continuous-flow culture system was used to isolate and maintain a mixed culture of cecal bacteria from adult broilers composed of 29 bacterial strains representing 10 genera . Broiler chicks were treated with the mixed culture in the drinking water on the day of hatch and challenged orally with 10(4) Salmonella typhimurium 2 d after treatment . The experiment was repeated in four separate trials using newly hatched chicks . The concentration of propionic acid and total volatile fatty acid (VFA) in the cecal contents was determined 2 d after treatment and at 10 d of age . Compared with controls, the number of treated chicks that were cecal culture-positive for Salmonella decreased (P < .01) in each of the trials . Additionally, the number of Salmonella in the cecal contents of the treated chicks at 10 d of age was decreased (P < .005) compared with controls in each trial . The decreased number of Salmonella in the cecal contents of the 10-d-old treated chicks was correlated with elevated concentrations of propionic acid (P < .05) and total VFA (P < .1) in the cecal contents of the treated chicks 2 d after treatment . The results indicated that VFA-producing bacteria present in the mixed culture became rapidly established in the ceca of the treated chicks and effectively increased resistance to S . typhimurium challenge.

J Appl Bacteriol, 1995 Jun, 78(6), 601 - 8
Growth of food-borne pathogenic bacteria in oil-in-water emulsions: I--Methods for investigating the form of growth; Parker ML et al.; Methods are presented for investigating the site and form of growth of bacteria in model oil-in-water emulsions and in dairy cream . Following growth of the bacteria, the continuous aqueous phase is gelled using agarose and the oil phase removed using a mixture of chloroform and methanol . Using this method, the authors have found that Listeria monocytogenes, Salmonella typhimurium and Yersinia enterocolitica grow in the form of colonies in concentrated oil-in-water emulsions . Colonies of L . monocytogenes and Y . enterocolitica also form in artificially-inoculated fresh and tinned dairy cream . If information about the precise site of growth is not required, the authors have discovered that intact colonies can be liberated from the model emulsions by dissolving away the oil phase with chloroform:methanol.

Eur J Biochem, 1995 Jun 1, 230(2), 517 - 24
Conformation of MgATP bound to 5-phospho-alpha-D-ribose 1-diphosphate synthetase by two-dimensional transferred nuclear Overhauser effect spectroscopy; Jarori GK et al.; The conformation of MgATP bound at the active site of Salmonella typhimurium 5-phospho-alpha-D-ribose 1-diphosphate synthetase (PRibPP synthetase) has been investigated by two-dimensional transferred-NOE spectroscopy (TRNOESY) . Inter-proton NOEs of the ligand were measured in the presence of the protein at several mixing times in the range of 40-300 ms at 500 MHz and 10 degrees C . Measurements were made at low ligand concentrations (approximately 1 mM) in order to avoid weak non-specific ligand-protein interactions and to ensure that the NOE arises from the ligand bound at the active site . The inter-proton distances were determined from the experimentally observed NOE buildup curves by comparing them with theoretical simulations obtained by using the complete relaxation matrix . These distances were used as constraints in molecular modeling and energy minimization calculations to deduce the structure of the bound ligand . PRibPP synthetase is known to appreciably aggregate so that it exists in multiple oligomeric forms in solution . The structure was determined under the assumption that the ligand assumes the same conformation on each subunit of every oligomer regardless of its size . On the basis of the rotational correlation time deduced for the enzyme-nucleotide complexes, it is estimated that the average oligomer of PRibPP synthetase, in the sample used for the TRNOESY measurements, consists of about 30 subunits, whereas the smallest active form of the protein is a pentamer . The conformation of enzyme-bound MgATP is described by a glycosidic torsion angle chi = 50 +/- 5 degrees and phase angle of pseudorotation P = 114.9 degrees corresponding to a 1T degree sugar pucker . It is noteworthy that the value of the glycosidic torsion angle obtained in this pyrophosphoryl transfer enzyme complex agrees well with those obtained previously for MgATP complexes of creatine kinase, pyruvate kinase (active and ancillary sites), and arginine kinase . The sugar pucker, on the other hand, differs from one enzyme complex to another.

Microbiol Rev, 1995 Jun, 59(2), 241 - 303
Genetic map of Salmonella typhimurium, edition VIII; Sanderson KE et al.; We present edition VIII of the genetic map of Salmonella typhimurium LT2 . We list a total of 1,159 genes, 1,080 of which have been located on the circular chromosome and 29 of which are on pSLT, the 90-kb plasmid usually found in LT2 lines . The remaining 50 genes are not yet mapped . The coordinate system used in this edition is neither minutes of transfer time in conjugation crosses nor units representing "phage lengths" of DNA of the transducing phage P22, as used in earlier editions, but centisomes and kilobases based on physical analysis of the lengths of DNA segments between genes . Some of these lengths have been determined by digestion of DNA by rare-cutting endonucleases and separation of fragments by pulsed-field gel electrophoresis . Other lengths have been determined by analysis of DNA sequences in GenBank . We have constructed StySeq1, which incorporates all Salmonella DNA sequence data known to us . StySeq1 comprises over 548 kb of nonredundant chromosomal genomic sequences, representing 11.4% of the chromosome, which is estimated to be just over 4,800 kb in length . Most of these sequences were assigned locations on the chromosome, in some cases by analogy with mapped Escherichia coli sequences.

Mutat Res, 1995 Jun, 347(1), 17 - 9
Mutagenic activity of dopamine after nitrosation; Su C et al.; Dopamine hydrochloride is reported to be a new mutagen precursor in this study . After treatment with nitrite under acidic conditions, dopamine hydrochloride showed direct-acting mutagenicity on Salmonella typhimurium TA100, TA98 and Escherichia coli WP2uvra . The addition of S9 mix did not affect the mutagenicity of nitrosated dopamine significantly in these three strains . Meanwhile, a comparison of the mutagenicity of nitrosated dopamine with nitrosated tyramine was carried out.

Mutat Res, 1995 Jun, 347(1), 1 - 7
dinP, a new gene in Escherichia coli, whose product shows similarities to UmuC and its homologues; Ohmori H et al.; A new gene, designated dinP, was found during E . coli genomic sequencing around the 5.5 min region . Its coding region is preceded by a sequence similar to the consensus binding sequence for LexA, the so-called SOS box sequence . The amino acid sequence of DinP (351 amino acid residues) has a strong similarity to the C . elegans hypothetical protein F22B7.6 and weaker similarities to the UmuC homologues in E . coli and Salmonella typhimurium and also to REV1 of Saccharomyces cerevisiae . Another SOS operon (dinJ1 and dinJ2 genes) found in this region is also described.

J Chemother, 1995 Jun, 7(3), 201 - 6
In vitro and in vivo antimicrobial action of fluphenazine; Dastidar SG et al.; The antipsychotic drug fluphenazine was obtained in a dry powder form and was screened with respect to 482 strains of bacteria, which included 170 Gram-positive and 326 Gram-negative strains . Nutrient agar plates containing increasing concentrations of fluphenazine (0-200 micrograms/ml) were used for the determination of the minimum inhibitory concentration (MIC) which was demonstrated by inoculating a loopful of an overnight peptone water culture of the organism on nutrient agar plates and determining the MIC against a control . Fluphenazine was detected to possess pronounced action against both Gram-positive and Gram-negative bacteria at 20-100 micrograms/ml . In the in vivo studies it was seen that when fluphenazine was used at a concentration of 1.5 micrograms/g and 3 micrograms/g mouse body weight both the levels offered significant protection to Swiss strain of white mice when challenged with 50 minimum lethal dose (MLD) of a virulent strain of Salmonella typhimurium 74 . The in vivo data with fluphenazine were highly significant (p < 0.001) according to the chi-square test.

Mutat Res, 1995 Jun, 329(1), 37 - 47
The effect of antioxidants on bleomycin treatment in in vitro and in vivo genotoxicity assays; Anderson D et al.; Antioxidants are thought to be important in protecting against damage from active oxygen species . The effects of the antioxidant nutrients vitamins C and E have been investigated after bleomycin treatment in the Salmonella typhimurium bacterial mutation assay, in the human peripheral lymphocyte chromosome aberration assay, and in the mouse micronucleus assay in peripheral blood and bone marrow cells . There were no protective effects from vitamins C and E in the bacterial mutation assay, but vitamin C and not vitamin E abolished chromosome damaging responses in human peripheral lymphocytes, and both vitamins reduced responses in micronuclei from peripheral blood cells in mice . This would suggest that in human cells in vitro and mouse cells in vivo these vitamins could have a protective role.

Mutat Res, 1995 Jun, 329(1), 19 - 27
Microbial mutagenic effects of the DNA minor groove binder pibenzimol (Hoechst 33258) and a series of mustard analogues; Ferguson LR et al.; A series of aniline mustards and half-mustards targeted to DNA by linkage (through a polymethylene chain) to the bisbenzimidazole chromophore of pibenzimol (Hoechst 33258) have been evaluated for their mutagenic properties, as estimated in three strains of Salmonella typhimurium, and for their mitotic crossing-over and petite mutagenesis activities in Saccharomyces cerevisiae strain D5 . Agarose gel electrophoresis studies showed that only the derivative with the longest linker chain cross-linked DNA, with the remaining compounds being monoalkylators . The parent (non-alkylator) minor groove binding ligand (Hoechst 33258) was inactive in the bacterial strains TA98 or TA100 but weakly mutagenic in TA102, and caused neither mitotic crossing-over nor 'petite' mutagenesis in yeast . Aniline half-mustard itself (monoalkylator) was an effective base-pair substitution mutagen (events in S . typhimurium strain TA100) with some frameshift mutagenesis activity in TA98, but showed only weak effects in the yeast assays, whereas aniline mustard (cross-linker) was inactive in these bacterial systems but caused substantial amounts of mitotic crossing-over in yeast . The composite molecules studied here showed effects more characteristic of the minor groove binding chromophore than of alkylating moieties . All showed weak mutagenic activity in TA102 and none in TA98 . The only compound to show significant mitotic crossing-over ability was the long-chain derivative which cross-linked DNA . For most of the compounds, the mutagenicity data provided no supportive evidence for DNA alkylation . Since other evidence suggests this does occur readily, it is likely to have a different target to that seen with untargeted aniline mustards . The significant antitumor activity and low mutagenic potential shown by these compounds make them worthy of further study.

Regul Toxicol Pharmacol, 1995 Jun, 21(3), 375 - 81
Safety evaluation of pullulanase enzyme preparation derived from Bacillus licheniformis containing the pullulanase gene from Bacillus deramificans; Modderman JP et al.; Pullulanase enzyme is an amylopectin debranching enzyme used in starch hydrolysis . This article describes studies conducted to investigate the safety of a pullulanase enzyme preparation produced by a strain of Bacillus licheniformis that has been transformed by introduction of genetic material from another Bacillus species, B . deramificans . A 4-week dietary toxicity study in rats was conducted in which test animals received pullulanase in the feed at concentrations of 0.2, 1.0, and 5.0% . No adverse treatment-related effects were observed . Lack of genetic toxicity potential was demonstrated by the results of a bacterial mutation assay in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538, in an in vitro histidine forward mutation study in mouse lymphoma cells, and in in vivo mouse bone marrow chromosome aberration and micronucleus assays . The enzyme preparation also has been shown to be a nonirritant in eye and primary dermal irritation tests in rabbits and is nontoxic by inhalation exposure . Finally, the genetically altered B . licheniformis has been demonstrated to be nonpathogenic upon single intraperitoneal injection to rats of both live and killed cells at doses up to 10(11) cells/kg . The results of these studies demonstrate that the enzyme preparation may be considered safe when employed in starch processing.

Gene, 1995 May 26, 158(1), 141 - 2
An arabinose-inducible expression vector, pAR3, compatible with ColE1-derived plasmids; Perez-Perez J et al.; Arabinose-inducible genetic elements from the Salmonella typhimurium arabinose operon were inserted into pACYC184 . The resultant plasmid, pAR3, is compatible with ColE1-derived plasmids and allows efficient expression of recombinant (re) genes upon induction with arabinose . These features make it convenient for use in combination with standard gene expression vectors for the independently controlled production of two or more re-polypeptides in Escherichia coli.

J Mol Biol, 1995 May 26, 249(1), 88 - 110
Structure of bacterial flagellar filaments at 11 A resolution: packing of the alpha-helices; Morgan DG et al.; Recent advances in the analysis of electron micrographs of frozen, hydrated bacterial filaments have allowed us to average data from more than 150 images and to reconstruct the bacterial flagellar filament of Salmonella typhimurium at a resolution of approximately 11 A . In addition to the outermost features seen in earlier lower resolution maps of the filament, we find a pair of concentric tubes which surround a approximately A diameter channel at the center of the structure . The walls of these tubes are composed of rod-like features which we have interpreted as columns of individual alpha-helices stacked end-to-end . Each column runs approximately parallel to the helix axis . The wall of the innermost tube, at a radius of approximately 20 A, is formed from 11 such columns . The wall of the second tube is formed from 22 columns which occur alternately at radii of approximately 43 and approximately 47 A . The two concentric tubes are held apart by spacers . These are short, rod-like features, which run approximately parallel to the helix axis . We have interpreted these as additional alpha-helices . By symmetry, each flagellin monomer contributes an alpha-helix to the inner tube, two alpha-helices to the outer tube and a fourth alpha-helix to the spacer . We have tentatively assigned one type of alpha-helix in the outer tube to the approximately 30 C-terminal residues of flagellin while the remaining three alpha-helices are assigned to the approximately 70 N-terminal residues . This interpretation of the reconstruction is consistent with available biochemical, biophysical and amino acid sequence information . We also present details of improved methodology to extract and evaluate the original data and also to assess the statistical significance of features in the three-dimensional map.

J Mol Biol, 1995 May 26, 249(1), 69 - 87
The structure of the R-type straight flagellar filament of Salmonella at 9 A resolution by electron cryomicroscopy; Mimori Y et al.; The supercoiled forms of the flagellar filaments are thought to be constructed from a mixture of two distinct subunit conformations arranged in a regular manner . We analyzed the structure of one of the two straight flagellar filaments, each of which is built up with all its subunits in one of the two conformations . The filament we studied was isolated from the strain SJW1655 of Salmonella typhimurium and had a right-handed helical symmetry . With recent advancements in electron cryomicroscopy, such as a liquid helium temperature stage for frozen hydrated specimens and a stable field emission source, and also by averaging high resolution data with a proper correction of the contrast transfer function, the density distribution map of this straight flagellar filament was generated in far more detail than before by including data up to 9 A resolution . The structure shows a densely packed core region from about 15 to 55 A in radius, where a pair of concentric tubular features of high density is present without well-defined subunit boundaries, and an outer part from 55 to 115 A, where the subunits are mostly well separated from each other . The outer tube in the core region, from 35 to 55 A in radius, contains many rod-like features with near-axial orientation and closest lateral distances of around 10 A, which are most likely to represent the alpha-helical bundles that were predicted in our previous report . In the inner tube, from 15 to 30 A in radius, the rod-like features are less clear . Between the inner and outer tubes are the short spoke-like densities, which are radially tilted and are connecting the two tubes . The outer part, from 55 to 115 A, contains an axially elongated column density and a slewed projection with a narrow neck region . When compared with the other straight filament having left-handed helical symmetry, this outer part does not show any significant changes in orientation, suggesting that the switch in the subunit conformation and packing involved in the polymorphic transitions is quite subtle and only occurs within the core region . Reassignment of each structural domain to the amino acid sequence is suggested, based on the volume of each domain, which was determined rather precisely by a proper correction of the contrast transfer function for both amplitudes and phases.

Biochemistry, 1995 May 16, 34(19), 6552 - 61
Allosteric linkages between beta-site covalent transformations and alpha-site activation and deactivation in the tryptophan synthase bienzyme complex; Leja CA et al.; This work examines two aspects of the catalytic mechanism and allosteric regulation of the tryptophan synthase bienzyme complex from Salmonella typhimurium: (a) the chemical mechanism by which indole and other nucleophiles react with the enzyme-bound alpha-aminoacrylate Schiff base intermediate, E(A-A), to form quinonoidal intermediates, E(Q), and (b) the effects of covalent transformations at the beta-site on the catalytic activity of the alpha-site . Transient kinetic studies in combination with alpha-secondary deuterium isotope effects are undertaken to determine the mechanism of nucleophile addition to E(A-A) . These studies establish that nucleophilic attack is best described by a two-step reaction sequence consisting of a binding step that is followed by Michael addition to the conjugated double bond of E(A-A) . Analysis of isotope effects suggests that the transition state for indole addition gives an E(A-A) beta-carbon that resembles an sp3 center, while the stronger nucleophiles, indoline and beta-mercaptoethanol, have transition states that appear to more closely resemble an sp2 beta-carbon . The effects of beta-site covalent transformations on alpha-site catalysis were studied using quasi-stable beta-site intermediates and the alpha-site substrate analogue 3-{6-nitroindole}-D-glycerol 3'-phosphate (6-nitro-IGP) . It was found that the cleavage of 6-nitro-IGP is strongly activated by the formation of E(A-A) and various E(Q) species at the beta-site but not by external aldimine species . Therefore, we conclude that the conversion of the L-Ser external aldimine to E(A-A) is the beta-site process which activates the alpha-site, while conversion of E(Q) to the L-Trp external aldimine triggers deactivation of the alpha-site . These findings are discussed within the context of allosteric regulation of substrate channeling in tryptophan synthase catalysis.

FEMS Microbiol Lett, 1995 May 15, 128(3), 247 - 53
Characterization of the superoxide dismutase gene and its upstream region from Methanobacterium thermoautotrophicum Marburg; Meile L et al.; A gene (sod) encoding superoxide dismutase (SOD) was isolated from the strictly anaerobic archaeon Methanobacterium thermoautotrophicum Marburg . Its identify was confirmed by functional complementation of an Escherichia coli mutant strain lacking SOD activity and by DNA sequence analysis of a cloned fragment . Upstream of sod, separated by a 5-bp intergenic region, lies the open reading frame orfk which potentially codes for a protein of 209 amino acid residues . The amino acid sequence for this presumptive product had a similarity coefficient of 55.5% to a subunit of the alkyl hydroperoxide reductase (encoded by the ahpC gene) from Salmonella typhimurium.

Eur J Biochem, 1995 May 15, 230(1), 170 - 82
Control of glucose metabolism by the enzymes of the glucose phosphotransferase system in Salmonella typhimurium; van der Vlag J et al.; The quantitative role of the phosphoenolpyruvate:glucose phosphotransferase system (glucose phosphotransferase system) in glucose uptake and metabolism, and phosphotransferase-system-mediated regulation of glycerol uptake, was studied in vivo in Salmonella typhimurium . Expression plasmids were constructed which contained the genes encoding enzyme I (ptsI), HP (ptsH), IIAGlc (crr), and IICBGlc (ptsG) of the glucose phosphotransferase system behind inducible promoters . These plasmids allowed the controlled expression of each of the glucose phosphotransferase system proteins from about 30% to about 300% of its wild-type level . When enzyme I, HPr or IIAGlc were modulated between 30% and 300% of their wild-type value, hardly any effects on the growth rate on glucose, the glucose oxidation rate, the rate of methyl alpha-D-glucopyranoside (a glucose analog) uptake or the phosphotransferase-system-mediated inhibition of glycerol uptake by methyl alpha-D-glucopyranoside were observed . Employing the method of metabolic control analysis, it was shown that the enzyme flux control coefficients of these phosphotransferase system components on the different measured processes were close to zero . The enzyme flux control coefficient of IICBGlc on growth on glucose or glucose oxidation was also close to zero . In contrast, the enzyme flux control coefficient of IICBGlc on the flux through the glucose phosphotransferase system (transport and phosphorylation) was 0.72 . The experimentally determined enzyme flux control coefficients allowed us to calculate the flux control coefficients of the phosphoenolpyruvate/pyruvate and methyl alpha-D-glucopyranoside/methyl alpha-D-glucopyranoside 6-phosphate couples and the process control coefficients of the phosphotransfer reactions of the glucose phosphotransferase system . We discuss the implications of these values and the possible control points in the glucose phosphotransferase system.

Mol Gen Genet, 1995 May 10, 247(3), 275 - 81
Transcriptional analysis of the flgK and fliD operons of Salmonella typhimurium which encode flagellar hook-associated proteins; Kutsukake K et al.; In Salmonella typhimurium, three hook-associated proteins, HAP1, HAP2 and HAP3, are known to be essential for formation of flagellar filament . HAP1 and HAP2 are encoded by the flgK and flgL genes, respectively, which together constitute an operon, called the flgK operon . HAP3 is encoded by the fliD gene which forms part of the fliD operon together with the fliS and fliT genes . In the flagellar regulon, the operons are divided into three classes, 1, 2 and 3, based on their positions within a transcriptional hierarchy . Transcriptional analysis suggested that the flgK and fliD operons should belong to class 3, whose expression is dependent on the flagellum-specific sigma factor FliA . However, biochemical data indicated that these HAP proteins are detectable even in the hook-basal body structures produced by the fliA mutant . This work was carried out to resolve this discrepancy . More careful examination of transcription revealed that the fliA mutation reduces but does not eliminate the expression of these operons, whereas a mutation in the flhD operon, which encodes activator proteins for the class 2 operons, eliminates their expression . This suggests that the flgK and fliD operons may be transcribed from both class 2 and class 3 promoters . Primer extension analysis indicated that the promoter region of fliD contains both class 2 and class 3 promoters, while that of flgK contains only a class 3 promoter . Transposon insertion into the flgB operon, which belongs to class 2 and lies upstream of the flgK operon, was found to decrease the expression of the flgK operon to the basal level.(ABSTRACT TRUNCATED AT 250 WORDS)

Photochem Photobiol, 1995 May, 61(5), 471 - 8
On the induction of protective responses in Salmonella typhimurium strain TA1535/pSK1002 by UVA (365 nm); Rahman S et al.; Exposure to UVA (365 nm) led to growth delay, loss of viability and inhibition of 3H-thymidine incorporation into the cells of Salmonella typhimurium strain TA1535 containing multiple copies of a plasmid pSK1002 carrying a umuC'-'lacZ fusion gene . Ultraviolet-A induced umu gene expression, as monitored by the estimation of beta-galactosidase, in a linear fluence-dependent manner . The induction of umu gene expression increased with the increase of postirradiation incubation period of the cells in the LB-ampicillin (LBA) medium at 37 degrees C and leveled off from 2 h onward . The induction of gene expression depended on concomitant protein synthesis and represented the induction of the SOS response in the particular S . typhimurium cells used . The exposure to low fluences (sublethal) of UVA also led to the induction of an adaptive response in the same bacterial cells, which made them resistant to subsequent challenge by a much higher fluence of the same radiation . The adaptive response, as monitored by the assays of viability and beta-galactosidase units, increased with the period of exposure to sublethal fluences of UVA, attained a maximum at the UVA exposure of 4.5 kJ/m2 (15 min) and thereafter gradually decreased with further increase of UVA exposure period . Modulation studies involving D2O, LBA growth medium, different scavengers of free radicals and quenchers of activated oxygen species indicated the involvement of both hydroxyl free radicals and singlet oxygen in the UVA-induced umu gene expression.

J Appl Bacteriol, 1995 May, 78(5), 495 - 500
The effect of step changes in sucrose concentration on the growth of Salmonella typhimurium LT2; Brocklehurst TF et al.; Water activity is a method of preservation that can affect microbial growth in foods and that may fluctuate during their processing, distribution and storage . Sucrose has been used to change the water activity of microbiological culture media . Suspensions of Salmonella typhimurium LT2 in the exponential phase of growth have been subjected to step changes in sucrose concentration at 20 degrees C . The changes in the numbers of viable bacteria were measured with time and the experimental growth curves compared with predictions based on growth data obtained at constant sucrose concentrations . Steps down in sucrose concentration showed some apparent loss of viability after the step followed by growth at a rate close to the expected value . Steps up in sucrose concentration resulted in a greater apparent loss of viability after the step and either growth or the inducement of lag, depending on the final concentration of sucrose . A series of small steps up in sucrose concentration to 45% (w/v) was able to sustain growth where it was not possible by inoculation directly into this concentration . Improved recovery of bacteria subject to osmotic stress was possible with a medium containing sodium chloride.

J Med Microbiol, 1995 May, 42(5), 348 - 52
Cytokine stimulation during Salmonella typhimurium sepsis in Itys mice; Jotwani R et al.; Cytokine production was measured in mice during Salmonella typhimurium sepsis and intoxication . In mice given live S . typhimurium (10 cfu/mouse), by intra-peritoneal injection, serum levels of tumour necrosis factor (TNF)-alpha and interleukin-6 increased steadily from day 1 until day 4 . Interferon-gamma levels showed a transient peak on day 3 . Interleukin-1-alpha levels were very low . There were high bacterial counts in the livers at day 3 and deaths occurred from day 4 onwards . Intraperitoneal injection of lipopolysaccharide or heat-killed bacteria also induced all of the cytokines, but their time of appearance and levels varied greatly . Cytokine induction by heat-killed bacteria was more marked . Endotoxaemia decreased with time during intoxication and increased during sepsis . Bioactive TNF, as measured by a cytotoxicity assay, was found only in mice given heat-killed bacteria.

J Bacteriol, 1995 May, 177(10), 2813 - 20
Sequence analysis of the phs operon in Salmonella typhimurium and the contribution of thiosulfate reduction to anaerobic energy metabolism; Heinzinger NK et al.; The phs chromosomal locus of Salmonella typhimurium is essential for the dissimilatory anaerobic reduction of thiosulfate to hydrogen sulfide . Sequence analysis of the phs region revealed a functional operon with three open reading frames, designated phsA, phsB, and phsC, which encode peptides of 82.7, 21.3, and 28.5 kDa, respectively . The predicted products of phsA and phsB exhibited significant homology with the catalytic and electron transfer subunits of several other anaerobic molybdoprotein oxidoreductases, including Escherichia coli dimethyl sulfoxide reductase, nitrate reductase, and formate dehydrogenase . Simultaneous comparison of PhsA to seven homologous molybdoproteins revealed numerous similarities among all eight throughout the entire frame, hence, significant amino acid conservation among molybdoprotein oxidoreductases . Comparison of PhsB to six other homologous sequences revealed four highly conserved iron-sulfur clusters . The predicted phsC product was highly hydrophobic and similar in size to the hydrophobic subunits of the molybdoprotein oxidoreductases containing subunits homologous to phsA and phsB . Thus, phsABC appears to encode thiosulfate reductase . Single-copy phs-lac translational fusions required both anaerobiosis and thiosulfate for full expression, whereas multicopy phs-lac translational fusions responded to either thiosulfate or anaerobiosis, suggesting that oxygen and thiosulfate control of phs involves negative regulation . A possible role for thiosulfate reduction in anaerobic respiration was examined . Thiosulfate did not significantly augment the final densities of anaerobic cultures grown on any of the 18 carbon sources tested . on the other hand, washed stationary-phase cells depleted of ATP were shown to synthesize small amounts of ATP on the addition of the formate and thiosulfate, suggesting that the thiosulfate reduction plays a unique role in anaerobic energy conservation by S typhimurium.

J Bacteriol, 1995 May, 177(10), 2737 - 43
Characterization of the umu-complementing operon from R391; Kulaeva OI et al.; In addition to conferring resistances to antibiotics and heavy metals, certain R factors carry genes involved in mutagenic DNA repair . These plasmid-encoded genes are structurally and functionally related to the chromosomally encoded umuDC genes of Escherichia coli and Salmonella typhimurium . Three such plasmid operons, mucAB, impCAB, and samAB, have been characterized at the molecular level . Recently, we have identified three additional umu-complementing operons from IncJ plasmid R391 and IncL/M plasmids R446b and R471a . We report here the molecular characterization of the R391 umu-complementing operon . The nucleotide sequence of the minimal R plasmid umu-complementing (rum) region revealed an operon of two genes, rumA(R391) and rumB(R391), with an upstream regulatory signal strongly resembling LexA-binding sites . Phylogenetic analysis revealed that the RumAB(R391) proteins are approximately equally diverged in sequence from the chromosomal UmuDC proteins and the other plasmid-encoded Umu-like proteins and represent a new subfamily . Genetic characterization of the rumAB(R391) operon revealed that in recA+ and recA1730 backgrounds, the rumAB(R391) operon was phenotypically indistinguishable from mucAB . In contrast, however, the rumAB(R391) operon gave levels of mutagenesis that were intermediate between those given by mucAB and umuDC in a recA430 strain . The latter phenotype was shown to correlate with the reduced posttranslational processing of the RumA(R391) protein to its mutagenically active form, RumA'(R391) . Thus, the rumAB(R391) operon appears to possess characteristics that are reminiscent of both chromosome and plasmid-encoded umu-like operons.

J Bacteriol, 1995 May, 177(10), 2654 - 62
Magnesium transport in Salmonella typhimurium: mgtA encodes a P-type ATPase and is regulated by Mg2+ in a manner similar to that of the mgtB P-type ATPase; Tao T et al.; Salmonella typhimurium has three distinct Mg2+ transport systems: CorA, MgtA, and MgtB, each encoded by its respective gene . corA and mgtB have been previously sequenced and characterized . This report details the sequence and properties of mgtA . Like mgtB, mgtA encodes a P-type ATPase . The mgtA gene encodes a slightly smaller protein than does mgtB, with a predicted molecular mass of about 95 kDa, running at 91 kDa on protein gels, which compares with values of 101 and 102 kDa, respectively, for the MgtB protein . The deduced amino acid sequence of MgtA is only 50% identical to that of MgtB, with a further 25% conservative amino acid substitutions, surprisingly low for such otherwise functionally similar proteins from the same organism . Codon usage for each gene is normal for S . typhimurium, however, indicating that neither gene is the result of a recent acquisition from another organism . A single open reading frame at mgtA encodes MgtA, in contrast to mgtB, which is shown to be an operon encoding (5' to 3') the 22.5-kDa MgtC and the MgtB proteins . Genetic constructs were used to show that deletion of MgtC does not alter the expression or transport properties of MgtB, making the role of the companion MgtC protein unclear . (The S . typhimurium homolog of treR, which encodes a putative repressor for trehalose uptake, is encoded by a gene adjacent to mgtA, and its sequence is also reported . Finally, exteremely strong Mg(2+) regulation of the mgtA and mgtB promoters but not of the corA or treR promoters was demonstrated by cloning the appropriate DNA sequences with luxAB and measuring enhancement of light production as a function of extracellular Mg(2+) concentration . Lowering the extracellular Mg(2+) concentration from 10 mM to 1 or 10 microM elicited a transcriptional response of several thousandfold from both the mgtA and mgtB promoters.

Mutat Res, 1995 May, 328(2), 183 - 91
Structure-activity relationships of anthraquinones as inhibitors of 7-ethoxycoumarin O-deethylase and mutagenicity of 2-amino-3-methylimidazo{4,5-f}quinoline; Hao NJ et al.; The antimutagenicity of 17 natural and synthetic anthraquinones was determined using Salmonella typhimurium TA98 against 2-amino-3-methylimidazo{4,5-f}quinoline (IQ) in the presence of Aroclor 1254-induced rat hepatic S9 . In general, the relationship between the chemical structures of anthraquinones and their antimutagenicity was found to contain one or more of the following features: (i) C9 carbonyl group, (ii) hydroxyl group at C1 and C4, (iii) C2 ethyl group, and (iv) C3 methyl group . The inhibitory effect of anthraquinones on 7-ethoxycoumarin O-deethylase (ECD) of Aroclor, 1254-induced hepatic microsomes was also examined . In addition, we studied the effect of anthraquinones on the metabolism of IQ by Aroclor 1254-induced microsomes using high-performance liquid chromatography . The antimutagenicity correlated with the inhibition of cytochrome P-450IA2-linked ECD activity in hepatic microsomes, and with the inhibition of N-hydroxy-IQ formation of IQ metabolism by hepatic microsomes . Moreover, we also examined the antimutagenicity of anthraquinones against synthetic N-hydroxy-IQ . Quinizarin and anthraflavic acid were shown to have more effect on the direct mutagenicity of N-hydroxy-IQ than that of the anthraquinones tested . This might explain why both anthraquinones showed higher antimutagenicity; although they inhibited ECD less . These results suggest that there exist at least two mechanisms of action in modifying roles of anthraquinones on the mutagenicity of IQ: (i) mediation through interaction with microsomal activating enzymes to inhibit the major active metabolite of N-hydroxy-IQ formation and (ii) direct interaction with the proximate metabolite of IQ, N-hydroxy-IQ, to block its attack on DNA.

J Bacteriol, 1995 May, 177(9), 2343 - 53
Hydroxyl radical footprints and half-site arrangements of binding sites for the CysB transcriptional activator of Salmonella typhimurium; Hryniewicz MM et al.; CysB is a transcriptional activator for the cysteine regulon and negatively autoregulates its own gene, cysB . Transcription activation also requires an inducer, N-acetyl-L-serine . CysB is known to bind to activation sites just upstream of the -35 regions of the positively regulated cysJIH, cysK, and cysP promoters and to a repressor site centered at about +1 in the cysB promoter . Additional accessory sites have been found in positively regulated promoters . The hydroxyl radical footprinting experiments reported here indicate that the activation sites CBS-J1, CBS-K1, and CBS-P1 in the cysJIH, cysK, and cysP promoters are composed of two convergently oriented 19-bp half-sites separated by 1 or 2 bp . N-Acetyl-L-serine stimulates binding to these sites as well as to the accessory sites CBS-J2 and CBS-P2, both of which share a similar topology with activation sites . A second topology is found in the accessory site CBS-K2 and the repressor site CBS-B, which contain divergently oriented 19-bp half-sites separated by one or two helical turns . N-Acetyl-L-serine inhibits binding to these two sites . A third topology is present in the cysK and cysP promoters, where an additional half-site is oriented toward the activation site and separated from it by one helical turn . Here, CysB binds to all three half-sites, bending the DNA, and N-acetyl-L-serine decreases the extent of bending . The marked dissimilarities of these half-site arrangements and of their responses to N-acetyl-L-serine suggest that CysB, a homotetramer, binds to them with different combinations of subunits.

J Bacteriol, 1995 May, 177(9), 2335 - 42
Transcriptional control of the nuo operon which encodes the energy-conserving NADH dehydrogenase of Salmonella typhimurium; Archer CD et al.; The 14 nuo genes encode the subunits of the type I (energy-conserving) NADH dehydrogenase, a key component of the respiratory chain . Salmonella typhimurium, like Escherichia coli, has two enzymes that can oxidize NADH and transfer electrons to ubiquinone, but only the type I enzyme translocates protons across the membrane to generate a proton motive force . Cells with the type I enzyme are energetically more efficient; the role of the type II enzyme (encoded by ndh) is not established, but it may function like a relief valve to allow more rapid NADH recycling . Here, we have investigated transcription of the nuo gene cluster, primarily in S . typhimurium . Studies with polar insertion mutants demonstrate that these genes are arranged as a single, large operon that is expressed from a complex promoter region upstream of nuoA . The DNA sequence of the promoter region was determined, and primer extension analysis of nuo transcripts was used to map four major RNA 5' ends to this region . A set of lac operon fusions to various DNA segments from the nuo promoter region was also constructed . Analysis of these fusions confirmed the presence of at least two nuo promoters . Mutations in the global regulatory genes arcA, oxrA (fnr), crp, cya, and katF were tested for effects on expression of the nuo operon . However, none of the mutations tested had a large effect on expression of type I NADH dehydrogenase.

Infect Immun, 1995 May, 63(5), 2047 - 53
Listeria monocytogenes p60 supports host cell invasion by and in vivo survival of attenuated Salmonella typhimurium; Hess J et al.; The extracellular protein p60 is a major virulence factor of the intracellular bacterium Listeria monocytogenes . Its roles in pathogen survival in vivo and host cell invasion in vitro were studied . To this end, Salmonella typhimurium SL7207 was used as carrier for secreted p60-HlyA fusion protein by Escherichia coli HlyB and HlyD transport proteins . C57BL/6 mice infected intravenously with this strain suffered from increased bacterial numbers in livers and spleens compared with the p60-nonexpressing control strain, but only transiently . In vitro experiments showed that p60 promotes invasion of recombinant S . typhimurium SL7207 p60 into hepatocytes and resting macrophages independent from complement . Moreover, the uptake of wild-type L . monocytogenes EGD and L . monocytogenes BUG 8, an internalin-deficient strain, into hepatocytes was partially blocked by anti-p60 antibodies . The impaired invasion of dissociated bacterial chains of L . monocytogenes RIII, a p60 expression mutant, into hepatocytes and macrophages was partially restored by addition of p60- or p60-HlyA-enriched bacterial supernatants . These data suggest that the L . monocytogenes surface-associated proteins, p60 and internalin, act in concert to achieve optimal uptake into nonprofessional phagocytes and macrophages . Together, these experiments reveal a substantial impact of p60 on cell invasion and virulence and thus emphasize the importance of the intracellular habitat for survival of L . monocytogenes in the host.

Infect Immun, 1995 May, 63(5), 2004 - 11
Oral immunization with recombinant Salmonella typhimurium expressing surface protein antigen A of Streptococcus sobrinus: dose response and induction of protective humoral responses in rats; Redman TK et al.; An attenuated, recombinant Salmonella typhimurium mutant, chi 4072(pYA2905), expressing the surface protein antigen A (SpaA) of Streptococcus sobrinus was investigated for its effectiveness in inducing protective immune responses against S . sobrinus-induced dental caries in an experimental caries model . Fischer rats were orally immunized with either 10(8) or 10(9) CFU of S . typhimurium chi 4072(pYA2905) . Persistence of salmonellae in Peyer's patches and spleens and the induction of immune responses were determined . Maximum numbers of salmonellae were recovered from Peyer's patches of rats within the first week of immunization, with higher numbers recovered from rats given 10(9) CFU than from those given 10(8) CFU . Serum anti-Salmonella and anti-SpaA responses increased more rapidly in rats given 10(9) CFU than in rats given 10(8) CFU . The salivary antibody response to SpaA increased with time, but the response varied in the two groups . In a separate study, rats were orally immunized with the recombinant Salmonella mutant and then challenged with cariogenic S . sobrinus 6715 . The levels of serum and salivary antibody and caries activity were assessed at the termination of the experiment . Higher levels of salivary immunoglobulin A antibody to SpaA and Salmonella carrier were detected in rats given 10(9) CFU than in those given 10(8) CFU, and these responses were higher than those in nonimmunized controls . Mandibular molars from immunized rats had lower numbers of recoverable streptococci and less extensive carious lesions than those from nonimmunized, control rats . These data indicate that oral immunization with an attenuated recombinant S . typhimurium expressing SpaA of S . sobrinus induces the production of antigen-specific mucosal antibody and confers protection against dental caries.

Infect Immun, 1995 May, 63(5), 1739 - 44
Role of Salmonella typhimurium Mn-superoxide dismutase (SodA) in protection against early killing by J774 macrophages; Tsolis RM et al.; The Salmonella typhimurium gene for Mn-cofactored superoxide dismutase (sodA) was cloned by complementation of an Escherichia coli sodA sodB mutant for growth on minimal medium . Sequence analysis revealed an open reading frame of 618 bp encoding a polypeptide with 97% identity to E . coli SodA . A S . typhimurium sodA mutant was created by allelic exchange and tested for the ability to survive in the murine macrophage-like cell line J774 . Growth of bacteria under iron-limiting conditions, inactivation of the Fur repressor, or expression of sodA from a plasmid resulted in increased resistance to early killing by J774 cells, which was abolished in the sodA mutant . These results suggest that resistance to the early oxygen-dependent microbicidal mechanisms of phagocytes involves the SodA gene product . The S . typhimurium sodA mutant was not significantly attenuated in mice, however, which suggests that resistance to early oxygen-dependent microbicidal mechanisms in vivo may play only a minor role in Salmonella pathogenesis.

Mutagenesis, 1995 May, 10(3), 179 - 83
Effects of Maillard reaction products on mutagen formation in boiled pork juice; Lee H et al.; The three IQ (2-amino-3-methylimidazo{4,5-f}quinoline) compounds IQ, MeIQx (2-amino-3,4-dimethyl{4,5-f}quinoxaline) and MeIQ (2-amino-3,4-dimethylimidazo{4,5-f}quinoline) have been found in boiled pork juice . To determine which Maillard reaction products are important in the formation of IQ-type mutagens in boiled pork juice, six Maillard reaction products were separately added to the pork juice before reflux boiling and then the mutagenicity of each sample was examined with Salmonella typhimurium TA98 in the presence of S9 mix . The addition of four Maillard reaction products enhanced the mutagenicity of pork juice 1.2-2.9-fold after reflux boiling . The highest level of enhancement was observed with tetrahydrothiophene, followed by 2,3-dimethylpyrazine, 3-methylpyridine and 2-methylpyridine . However, the addition of 2-acetylpyrrole and imidazole greatly inhibited the mutagenicity of pork juice . To confirm which IQ-type mutagens were changed, four major mutagenic fractions were monitored after HPLC separation by their mutagenicity with Salmonella typhimurium TA98 . By comparing the retention time of authentic IQ compounds from boiled pork juice with added tetrahydrothiophene or 2,3-dimethylpyrazine, we show that four major HPLC fractions have significantly increased mutagenicity compared with the same fractions in boiled pork juice alone . In contrast, the mutagenicity of these fractions was considerably reduced with the addition of imidazole or 2-acetylpyrrole to the pork juice before refluxing . The residual amounts of tetrahydrothiophene and 2,3-dimethylpyrazine added to the boiled pork juice after heating were measured by gas chromatography and found to be inversely correlated with the mutagenicity of the pork juice.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutagenesis, 1995 May, 10(3), 171 - 7
Analysis of metabolism and genotoxicity of 5-nitro-3-thiophenecarboxanilides in bacterial, mammalian and human cells; Hrelia P et al.; 5-nitro-3-thiophenecarboxanilide (NTCA3) was clearly mutagenic in Salmonella typhimurium strains TA98, YG1021 (the strain with elevated nitroreductase) and YG1024 (the strain with elevated O-acetyltransferase) and only slightly mutagenic at the gpt locus in AS52 cells . Clastogenic activity in human lymphocytes was dependent on the length of exposure: detectable chromosome aberrations were observed following a 24 h treatment period, but not after 3 h exposure . S9 increased genotoxicity in both mammalian cells and human lymphocytes . Metabolites formed by incubation of NTCA3 with the different cell systems were examined . A time-course study in cell whole extracts showed that bacterial and mammalian cells can acetylate NTCA3 forming 5-acetylamino-3-thiophene-carboxanilide . The formation of this metabolite in human lymphocyte extracts was not confirmed . These data support the conclusions that: (i) both bacterial and mammalian activation pathways play a role in mutations by NTCA3; (ii) the N-acetylated derivative is generated by acyl-transferase after reduction and is the end product of the metabolism in both bacterial and mammalian cells; and (iii) different levels of reductase and acetyltransferase activity may contribute to the differential sensitivity of the different cellular species to the genotoxicity of NTCA3 . The fact that NTCA3 serves as substrate for enzymatic activities of importance also in human metabolism needs consideration in assessing the potential risk posed by NTCAs.

Food Addit Contam, 1995 May-Jun, 12(3), 331 - 6
Procedures for destruction of patulin in laboratory wastes; Fremy JM et al.; Patulin is immunosuppressive and there is limited evidence of its carcinogenicity in experimental animals . The International Agency for Research on Cancer (IARC) initiated a programme for the development of degradation techniques for the commonly investigated mycotoxins . As a part of this programme, the following techniques were tested for the degradation of patulin: treatment with ammonia, treatment with ascorbic acid, and treatment with potassium permanganate in acidic or in alkaline conditions . Patulin analysis was performed by using HPLC with UV detection . Mutagenic activity of degradation residues was tested by in Salmonella typhimurium strains TA 97a, TA 98, TA 100, and TA 102 . Complete disappearance of patulin was not achieved after 92 h of treatment with ascorbic acid . All the other methods tested led to complete removal of the molecule . However, the technique using potassium permanganate in acidic conditions produced residues which were mutagenic without activation to Salmonella typhimurium strains TA 100 and TA 102, which was attributed later to Mn2+ . The two other techniques gave satisfactory results and were selected for further validation studies.

Clin Diagn Lab Immunol, 1995 May, 2(3), 307 - 13
Detection of bacterial pyrogens on the basis of their effects on gamma interferon-mediated formation of neopterin or nitrite in cultured monocyte cell lines; Werner-Felmayer G et al.; In a number of mammalian cell types, pteridine biosynthesis from guanosine 5'-triphosphate and formation of nitric oxide from L-arginine are induced by gamma interferon (IFN-gamma) and bacterial lipopolysaccharide (LPS) . We assessed the possibility of using such metabolic alterations for the in vitro detection of pyrogens . Products from gram-negative and gram-positive bacteria and related synthetic compounds were tested for their potential to induce either of these pathways . Stimulation of pteridine biosynthesis was monitored as the formation of neopterin in the human myelomonocytic cell line THP-1 . The formation of nitric oxide was determined as nitrite in murine J774A.1 macrophage cultures . The substances tested included toxic and detoxified parts of LPS and lipid A from Escherichia coli, Salmonella typhimurium, Salmonella minnesota, and Klebsiella pneumoniae as well as lipoteichoic acid and toxic shock syndrome toxin 1 from Staphylococcus aureus . Furthermore, two cell wall compounds from Mycobacterium tuberculosis, trehalose 6,6'-dimycolate and N-acetylmuramyl-L-alanyl-D-isoglutamine, which are active components of Freund's adjuvant, were used . When applied as a single stimulus, only the whole LPS molecule potently stimulated neopterin or nitrite formation . Lipid A and products from gram-positive bacteria were weakly active . For neopterin formation, lipid A required the presence of fetal calf serum . Besides detoxified LPS and independently from the presence of serum, all bacterial compounds tested strongly increased the effects mediated by IFN-gamma . Our results show that bacterial pyrogens can be detected by monitoring the formation of neopterin or nitrite . This may provide a basis for the development of an in vitro assay for the detection of pyrogenic contamination with the aim of replacing the currently used animal test.

Appl Environ Microbiol, 1995 May, 61(5), 1853 - 8
Influence of the RpoS (KatF) sigma factor on maintenance of viability and culturability of Escherichia coli and Salmonella typhimurium in seawater; Munro PM et al.; The sigma factor RpoS is essential for stationary-phase-specific, multiple-stress resistance . We compared the viabilities (direct viable counts) and culturabilities (colony counts) in seawater of Escherichia coli and Salmonella typhimurium strains and those in which rpoS was deleted or which were deficient in guanosine 3',5'-bispyrophosphate (ppGpp) synthesis (relA spoT) . RpoS, possibly via ppGpp regulation, positively influenced the culturability of these bacteria in oligotrophic seawater . This influence closely depended, however, upon the growth state of the cells and the conditions under which they were grown prior to their transfer to seawater . The protective effect of RpoS was observed only in stationary-phase cells grown at low osmolarity . A previous exposure of cells to high osmolarity (0.5 M NaCl) also had a strong influence on the effect of RpoS on cell culturability in seawater . Both E . coli and S . typhimurium RpoS mutants lost the ability to acquire a high resistance to seawater, as observed in both logarithmic-phase and stationary-phase RpoS+ cells grown at high osmolarity . A previous growth of S . typhimurium cells under anoxic conditions also modulated the incidence of RpoS on their culturability . When grown anaerobically at high osmolarity, logarithmic-phase S . typhimurium RpoS+ cells partly lost their resistance to seawater through preadaptation to high osmolarity . When grown anaerobically at high osmolarity until stationary phase, both RpoS+ and RpoS- cells retained very high levels of both viability and culturability and then did not enter the viable but nonculturable state for over 8 days in seawater because of an RpoS-independent, unknown mechanism.

Immunology, 1995 May, 85(1), 1 - 7
Protection against Leishmania major infection in genetically susceptible BALB/c mice by gp63 delivered orally in attenuated Salmonella typhimurium (AroA- AroD-); Xu D et al.; The gene encoding the Leishmania major (L . major) promastigote surface glycoprotein, gp63, was introduced into the Salmonella typhimurium (S . typhimurium) aroA- aroD- live oral vaccine strain BRD509 and expressed under the control of a constitutive tac promoter in plasmid pKK233-2 . This construct (GID101) expressed gp63 in vitro and was used to immunize highly susceptible BALB/c mice by the oral route . The plasmid was relatively stably inherited by bacteria growing or persisting in the mesenteric lymph nodes of immunized mice . Mice immunized with GID101 developed significant resistance against a challenge infection with L . major compared to controls immunized with BRD509 alone . Spleen and lymph node cells from immunized mice developed a strong in vitro proliferative T-cell response to killed or live L . major . The activated T cells secreted interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) which was abrogated by treatment with anti-CD4 but not with anti-CD8 antibody . The cells did not produce detectable levels of interleukin-4 (IL-4) . The immunized mice also produced significant amounts of leishmanial specific IgG2a antibody but did not develop delayed-type hypersensitivity (DTH) to live parasites . No IgG1 antibody was detected . These data therefore demonstrate that gp63 gene delivered orally by a vaccine strain of S . typhimurium can preferentially induce the development of Th-1 subset of CD4+ T cells and protective immunity in the highly susceptible BALB/c mice.

J Clin Microbiol, 1995 May, 33(5), 1292 - 5
Detection of Salmonella typhimurium from rectal swabs of experimentally infected beagles by short cultivation and PCR-hybridization; Stone GG et al.; A rapid and sensitive cultivation and PCR-hybridization procedure for the detection and identification of Salmonella typhimurium was evaluated over a 42-day period with eight experimentally infected beagles . Rectal swabs were taken at several times postinfection, inoculated into selenite-cystine broth, and plated onto Hektoen-Enteric Enteric agar immediately after incubation for 4 and 24 h . PCRs and hybridizations were also conducted with each sample, and the results were compared with those of standard culture techniques to evaluate the efficiency of the PCR-hybridization procedure . The PCR-hybridization procedure was more sensitive than standard culture techniques at each enrichment incubation (P < 0.05) . In addition, the PCR-hybridization procedure was significantly better than culture up through 3 days postinfection (P < 0.05) . A nonspecific amplified product, relatively close in size to the 457-bp specifically amplified product, did not hybridize to an internal oligonucleotide probe or to a random-primed labeled probe . Subsequent sequence information revealed that the product had very little similarity to the 457-bp product but had significant similarity to an Escherichia coli aldehyde dehydrogenase gene . This study indicated that a cultivation and PCR-hybridization procedure is significantly better than culture for the identification of S . typhimurium . Additionally, the results confirm the importance of determining specificities of PCR products beyond the gel electrophoresis level by hybridization with a specific probe.

Biophys J, 1995 May, 68(5), 2181 - 9
Spatio-temporal patterns generated by Salmonella typhimurium; Woodward DE et al.; We present experimental results on the bacterium Salmonella typhimurium which show that cells of chemotactic strains aggregate in response to gradients of amino acids, attractants that they themselves excrete . Depending on the conditions under which cells are cultured, they form periodic arrays of continuous or perforated rings, which arise sequentially within a spreading bacterial lawn . Based on these experiments, we develop a biologically realistic cell-chemotaxis model to describe the self-organization of bacteria . Numerical and analytical investigations of the model mechanism show how the two types of observed geometric patterns can be generated by the interaction of the cells with chemoattractant they produce.

J Dairy Res, 1995 May, 62(2), 339 - 48
Inhibition of proliferative responses of mouse spleen lymphocytes and rabbit Peyer's patch cells by bovine milk caseins and their digests; Otani H et al.; The modulating effect of bovine milk casein components and their digests on the proliferative responses of mouse spleen lymphocytes and rabbit Peyer's patch cells induced or not induced by mitogens has been studied with a colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide . All the casein components and their digests tested had little mitogenic effect on the proliferative responses of mouse spleen lymphocytes and rabbit Peyer's patch cells . Intact kappa-casein significantly inhibited the proliferative responses of mouse spleen lymphocytes and Peyer's patch cells induced by mitogens such as lipopolysaccharide from Salmonella typhimurium, concanavalin A, phytohaemagglutinin and pokeweed mitogen . In contrast, intact alpha s1-casein and beta-casein had little effect . kappa-Casein had an inhibitory effect after digestion by pancreatin or trypsin, but not after pepsin or chymotrypsin digestion . Both pancreatin and trypsin digests of alpha s1-casein and beta-casein significantly inhibited the proliferative responses of mouse spleen lymphocytes and rabbit Peyer's patch cells induced by mitogens, whereas pepsin and chymotrypsin digests of both caseins were without effect . Moreover, the trypsin digest of each casein component had an inhibitory effect on mouse spleen lymphocyte proliferation in the absence of mitogen . Since trypsin is a major proteinase in pancreatin, the substrate specificity of trypsin seems to be important for the formation of the inhibitory peptides from casein components . These observations suggest that intact kappa-casein and some peptides formed from milk casein components by the action of trypsin may suppress the immune responsiveness of neonates.

Mol Microbiol, 1995 May, 16(3), 587 - 95
Resistance to mecillinam produced by the co-operative action of mutations affecting lipopolysaccharide, spoT, and cya or crp genes of Salmonella typhimurium; Anton DN; Lipopolysaccharide (LPS), spoT, and cya or crp mutations individually do not affect the minimum inhibitory concentration of mecillinam on Salmonella typhimurium . However, when mutations of two of these types were combined in the same strain, high-level resistance appeared, and increased even further when all three types of mutations were present . Most mutations affecting LPS (rfa, rfb, rfc) showed this behaviour, although to different degrees . The highest resistance to mecillinam was caused by galE and rfc mutations whereas almost no effect was noticed with rfaB or rfaK mutations . This phenomenon appears to be specific for mecillinam since none of several other antibiotics elicited it . Reduction of guanosine tetraphosphate (ppGpp) levels by introduction of a relA mutation did not significantly affect the MIC of mecillinam on strains carrying different combinations of spoT, galE, and cya or crp mutations . All the strains produced spherical cells in medium with a low concentration (0.05 microgram ml-1) of the antibiotic . These results suggest that the antibacterial action of mecillinam on S . typhimurium is somehow dependent on the interaction of LPS, cyclic AMP/cyclic AMP receptor protein (cAMP/CRP), and SpoT . The reported resistance to mecillinam of cya and crp mutants of Escherichia coli K-12 is probably due to the natural LPS defectiveness of this strain.

Mol Microbiol, 1995 May, 16(3), 465 - 76
The phoP locus influences processing and presentation of Salmonella typhimurium antigens by activated macrophages; Wick MJ et al.; The destruction and processing of bacteria by activated macrophages facilitates the presentation of antigens to T cells and thereby promotes the induction of specific immunity . The PhoP-PhoQ regulatory system that controls the synthesis of many Salmonella proteins required for virulence and survival within macrophages is one mechanism that this particular intracellular pathogen has evolved to resist destruction . To address whether the phoP locus also influences antigen processing during the interaction of Salmonella typhimurium with macrophages, we tested the effect of phoP mutations on the processing and presentation of model antigens expressed by the bacteria . Activated macrophages processed phoP- bacteria with greater efficiency than wild-type bacteria, as measured by the response of antigen-specific T-hybridoma cells; Salmonella constitutively expressing PhoP were processed even less efficiently than wild-type Salmonella . After heat-inactivation, however, both wild-type and phoP- bacteria were efficiently processed . The altered processing and presentation efficiency was not due to differences in the level of antigen expressed by the bacteria or differences in the level of bacterial uptake by the macrophages . In addition, phoP-regulated gene expression was shown to influence processing of antigen phagocytosed independently of the bacteria . Thus, phoP-regulated gene products decrease the processing and presentation of S . typhimurium antigens, demonstrating a role for this virulence locus in the inhibition of the induction of specific immunity.

Mol Microbiol, 1995 May, 16(3), 397 - 404
The role of anti-sigma factors in gene regulation; Brown KL et al.; Despite the isolation of an anti-sigma factor over 20 years ago, it is only recently that the concept of an anti-sigma factor emerged as a general mechanism of transcriptional regulation in prokaryotic systems . Anti-sigma factors bind to sigma factors and inhibit their transcriptional activity . Studies on the mechanism of action of anti-sigma factors has shed new light on the regulation of gene expression in bacteria, as the anti-sigma factors add another layer to transcriptional control via negative regulation . Their cellular roles are as diverse as FIgM of Salmonella typhimurium, which can be exported to sense the structural state of the flagellar organelle, to SpoIIAB of Bacillus subtilis participating in the switch from one cell type to another during the process of sporulation . Additionally, the bacteriophage T4 uses an anti-sigma factor to sabotage the Escherichia coli E.sigma 70 RNA polymerase in order to direct exclusive transcription of its own genes . Cross-linking, co-immunoprecipitations, and co-purification indicate that the anti-sigma factors directly interact with their corresponding sigma factor to negatively regulate transcription . In B . subtilis, anti anti-sigma factors regulate anti-sigma factors by preventing an anti-sigma factor from interacting with its cognate sigma factors, thereby allowing transcription to occur.

Eur J Clin Microbiol Infect Dis, 1995 May, 14(5), 448 - 51
Extra-intestinal infections with multiply drug-resistant Salmonella typhimurium in hospitalized patients in Jordan; Shehabi AA; During the 12-year period from 1978 to 1989, Salmonella typhimurium was the most frequently isolated serotype (592/1,500; 39.5%) among all clinical Salmonella isolates at Jordan University Hospital . Extra-intestinal infections due to Salmonella typhimurium accounted for 68 (11.5%) isolates . A high percentage of Salmonella typhimurium strains (52-90%) were resistant to commonly used drugs in Jordan . Most of the antibiotic-resistant strains of Salmonella typhimurium (10/12) examined which were from extra-intestinal sources contained a large plasmid (55 MDa) in addition to two to four small plasmids . These strains were also able to transfer most or part of their drug resistance in vitro . It is concluded that the invasive potential of Salmonella typhimurium isolates is probably associated with the presence of a large virulence plasmid and multiple antibiotic resistance.

Mutagenesis, 1995 May, 10(3), 235 - 41
Collaborative study of interlaboratory variability in Salmonella typhimurium TA102 and TA2638 and Escherichia coli WP2/pKM101 and WP2 uvrA/pKM101 . Association of Microbial Mutation Testing Laboratory C.O; Watanabe K et al.; A collaborative study of interlaboratory variability in bacterial mutagenicity induced by mitomycin C (MMC) and bleomycin (BLM) was performed using the four strains Salmonella typhimurium TA102 and TA2638 and Escherichia coli WP2/pKM101 and WP2 uvrA/pKM101 . Thirteen laboratories participated in this study . The four strains and two chemicals were sent from a central source to each laboratory . Each strain was cultured in nutrient broth containing antibiotics and laboratories were requested to ensure that the spontaneous counts for marker check fell within a specified range in the preparation of the original stock culture . Concerning the response to the chemicals, most interlaboratory variability was within a 4-fold range for tests with TA102, TA2638 and WP2/pKM101 . From the results of the statistical analysis using the linear regression test, positive results in TA102, TA2638 and WP2/pKM101 and negative results in WP2 uvrA/pKM101 were obtained by MMC testing in all of the laboratories . On the other hand, with BLM testing, considerable interlaboratory variability was observed between the strains, largely because of the variation in results of the repeat experiments . Overall mean spontaneous revertant counts in all laboratories were within acceptable ranges for all four bacterial strains . In conclusion, it is judged that among Japanese laboratories, there is excellent agreement in the tested response of these bacterial strains to a strong mutagen such as MMC, as well as uniformity in the spontaneous reversion rates . However, for chemicals such as BLM that induce a weak response, it may be necessary to repeat experiments several times to obtain clear results . In this study, TA102 was shown to be a useful strain for routine mutagenicity testing . This necessitated control over culture maintenance by the addition of tetracycline and strict selection in the preparation of the original stock cultures.

J Biol Chem, 1995 Apr 28, 270(17), 9978 - 81
Sugar recognition by a glucose/galactose receptor . Evaluation of binding energetics from molecular dynamics simulations; Aqvist J et al.; A new theoretical method for free energy calculations is used to compute the absolute binding constants for beta-D-glucose and methyl-beta-D-galactoside to the periplasmic glucose/galactose receptor from Salmonella typhimurium . The computer simulation results agree well with available experimental data and make it possible to assess the sources of both the high affinity as well as the specificity for glucose . It was found that the major contribution to the binding energy comes from electrostatic interactions and particularly hydrogen bonds of the charge-dipole type . We also predict the structure of the complex with methyl-galactoside as this has not yet been experimentally determined.

J Biol Chem, 1995 Apr 28, 270(17), 9819 - 27
Proline dehydrogenase activity of the transcriptional repressor PutA is required for induction of the put operon by proline; Muro-Pastor AM et al.; The proline utilization (put) operon from Salmonella typhimurium consists of the putP gene, encoding a proline transporter, and the putA gene, encoding an enzyme with both proline dehydrogenase and 1-pyrroline-5-carboxylate dehydrogenase activities . In addition to these two enzymatic activities, the PutA protein is a transcriptional repressor that regulates the expression of putP and putA in response to the availability of proline . We report the isolation of super-repressor mutants of PutA that decrease expression from the putA promoter in the presence or absence of proline . None of the mutants exhibited increased affinity for the DNA in the put regulatory region in vitro . Although DNA binding by wild-type PutA was prevented by the addition of proline and an artificial electron acceptor, DNA binding by the two strongest super-repressors was not prevented under identical conditions . The proline dehydrogenase activity of the purified mutant proteins showed altered kinetic properties (increased Km(Pro), reduced Vmax, or a completely null phenotype) . The observation that these mutations simultaneously affect induction by proline and proline dehydrogenase activity suggests that a single proline-binding site is involved in both proline dehydrogenase activity and induction of the expression of the put operon . Furthermore, the results indicate that the proline dehydrogenase activity of PutA is essential for induction of the put operon by proline.

Biochem Biophys Res Commun, 1995 Apr 26, 209(3), 996 - 1002
Bioactivation of 5-hydroxymethyl-2-furaldehyde to an electrophilic and mutagenic allylic sulfuric acid ester; Lee YC et al.; 5-Hydroxymethyl-2-furaldehyde (HMF), a ubiquitous food contaminant, has been proposed to be metabolically activated through sulfonation of its allylic hydroxyl functional group . In support of this idea, we have found the strong direct mutagenicity of chemically synthesized sulfuric acid ester, 5-sulfooxymethylfurfural (SMF), in Salmonella typhimurium TA104 . The intrinsic mutagenicity of this reactive ester was significantly inhibited by glutathione and glutathione S-transferase activity in dialyzed rat liver cytosol . The metabolic formation of SMF was elucidated by enhanced mutagenicity of HMF in the presence of rat hepatic cytosol enriched with the sulfo-group donor, 3'-phosphoadenosine-5'-phosphosulfate (PAPS) . The PAPS- and cytosol-dependent mutagenicity of HMF was markedly lessened by sulfotransferase inhibitors such as 2,6-dichloro-4-nitrophenol and dehydroepiandrosterone . These results suggest that HMF can be metabolically activated to an allylic sulfuric acid ester which may play a role as an ultimate electrophilic metabolite in toxification of the parent compound in vivo.

Biochem J, 1995 Apr 15, 307 ( Pt 2), 377 - 81
Mammalian antioxidant protein complements alkylhydroperoxide reductase (ahpC) mutation in Escherichia coli; Tsuji K et al.; The MER5 {now called the Aop1 (antioxidant protein 1) gene} was cloned as a transiently expressed gene of murine erythroleukaemia (MEL) cell differentiation and its antisense expression inhibited differentiation of MEL cells . We found that the Aop1 gene shows significant nucleotide sequence similarity to the gene coding for the C22 subunit of Salmonella typhimurium alkylhydroperoxide reductase, which is also found in other bacteria, suggesting it functions as an antioxidant protein . Expression of the Aop1 gene product in E . coli deficient in the C22-subunit gene rescued resistance of the bacteria to alkylhydroperoxide . The human and mouse Aop1 genes are highly conserved, and they mapped to the regions syntenic between mouse and human chromosomes . Sequence comparisons with recently cloned mammalian Aop1 homologues suggest that these genes consist of a family that is responsible for regulation of cellular proliferation, differentiation and antioxidant functions.

Gene, 1995 Apr 14, 156(1), 53 - 7
Cloning and characterization of a gene cluster, phsBCDEF, necessary for the production of hydrogen sulfide from thiosulfate by Salmonella typhimurium; Alami N et al.; We cloned, by complementation of an H2S- mutant, a cluster of Salmonella typhimurium genes, phsBCDEF, that appears to be essential for the anaerobic production of hydrogen sulfide from thiosulfate . Tn5 mutagenesis and ExoIII deletion analysis showed that approx . the entire region of a 3.3-kb subclone was necessary for H2S production . Subsequent sequencing revealed the presence of five potential translationally coupled open reading frames (ORFs) . Their putative protein products were confirmed by synthesis from a phage T7 expression system . Comparison of the encoded sequences with previously determined sequences suggests that these genes constitute part of a thiosulfate-reducing operon coding for a membrane-associated electron transport chain which contains proteins potentially capable of ligating iron-sulfur clusters and heme . Immediately upstream from these genes, a region encoding the C-terminal portion of an ORF (OrfA) was identified that showed a high degree of similarity to some other anaerobic terminal reductases, polysulfide reductase (PsrA) of Wolinella succinogenes and dimethylsulfoxide reductase (DmsA), formate dehydrogenase (formate-hydrogene-lyase linked) (FdhF) and nitrate reductase (NarG) of Escherichia coli.

Biochim Biophys Acta, 1995 Apr 13, 1243(3), 549 - 51
Identification of SPT14/CWH6 as the yeast homologue of hPIG-A, a gene involved in the biosynthesis of GPI anchors; Vossen JH et al.; Cwh6 is a temperature-sensitive cell wall mutant of Saccharomyces cerevisiae . CWH6 was found to be identical to SPT14, a gene that is highly homologous to both human PIG-A and to RFAK from Salmonella typhimurium . PIG-A and RFAK are involved in transferring N-acetylglucosamine to, respectively, a GPI anchor precursor and to lipopolysaccharides . Because cell walls of cwh6 are greatly reduced in mannose, and because some cell wall proteins are known to be incorporated into the cell wall through a GPI-anchor dependent mechanism, we propose that Spt14p/Cwh6p is involved in transferring N-acetylglucosamine to a precursor of GPI anchors . We further propose that the majority of cell wall proteins are incorporated into the cell wall through a GPI anchor.

Toxicology, 1995 Apr 12, 98(1-3), 215 - 23
Immunological effects of 2-methoxyethanol administered dermally or orally to Fischer 344 rats; Williams WC et al.; Exposure of rats to 2-methoxyethanol (ME) by gavage for 10 consecutive days results in immunotoxicity . To determine whether dermal exposure to ME also induces immunotoxicity, undiluted ME was applied to Fisher 344 male rats at dose levels of 150, 300, 600, 900 or 1200 mg/kg/day on shaved occluded test sites for 4 consecutive days . Decreased thymus weights were produced by all doses of ME, while reductions in spleen weight were observed at doses of 900 mg/kg/day ME or greater . The alterations in these lymphoid organ weights were produced in the absence of loss in body weight . The lymphoproliferative (LP) responses to phytohemagglutinin (PHA) and pokeweed mitogen (PWM) were enhanced at 1200 mg/kg/day ME compared with water controls . Separate groups of rats, employed for the antibody plaque-forming cell (PFC) response to either trinitrophenyl-lipopolysaccharide (TNP-LPS) or sheep red blood cells (SRBC), were exposed dermally to 150, 300 or 600 mg/kg/day ME for 4 consecutive days . A reduction in the PFC response to TNP was observed at 600 mg/kg/day ME, whereas decreases in the PFC response to SRBC were observed at dosages of 300 and 600 mg/kg/day ME . To compare the immunotoxic effects of dermally applied ME to those effects caused by ME administered orally, rats were dosed by gavage with 25, 50, 100 or 200 mg/kg/day ME in distilled water for 4 consecutive days . Reductions in thymus weights were observed at oral dosages ranging from 50-200 mg/kg/day, while spleen weights were reduced in rats dosed at 200 mg/kg/day ME . LP responses to PHA, PWM and Salmonella typhimurium were increased at the 200 mg/kg/day ME dose level . PFC responses to TNP-LPS and SRBC were suppressed at the 50, 100 and 200 mg/kg/day ME dosages . These results indicate that, like oral exposure, dermal exposure to ME compromises the ability of the immune system to mount an effective humoral immune response.

Mol Gen Genet, 1995 Apr 10, 247(1), 7 - 16
Involvement of umuDCST genes in nitropyrene-induced -CG frameshift mutagenesis at the repetitive CG sequence in the hisD3052 allele of Salmonella typhimurium; Nohmi T et al.; Expression of the umuDC operon is required for UV and most chemical mutagenesis in Escherichia coli . The closely related species Salmonella typhimurium has two sets of umuDC-like operons, umuDCST on the chromosome and samAB on a 60-MDa cryptic plasmid . The roles of the umuDC-like operons in chemically induced frameshift mutagenesis of the hisD3052 allele of S . typhimurium were investigated . Introduction of a pBR322-derived plasmid carrying umuDCST increased the rate of reversion of hisD3052, following treatment with 1-nitropyrene (1-NP) or 1,8-dinitropyrene (1,8-DNP) tenfold and fivefold, respectively, whereas it did not substantially increase the rate of reversion induced by other frameshift mutagens, i.e . 2-nitrofluorene (2-NF) and 2-amino-3-methyldipyrido{1,2-a:3',2'-d}imidazole (Glu-P-1) . Introduction of a pBR322-derived plasmid carrying samAB did not increase the incidence of reversion of hisD3052 observed with any of the mutagens examined . Deletion of umuDCST substantially lowered the reversion rate induced by 1-NP or 1,8-DNP, but it did not affect reversion induced by 2-NF, Glu-P-1 or N-hydroxyacetylaminofluorene (N-OH-AAF) . Deletion of samAB had little impact on reversion incidence induced by any of the five frameshift mutagens . DNA amplification using the polymerase chain reaction technique followed by restriction enzyme analysis using BssHII, suggested that the mutations induced by the five frameshift mutagens were all CG deletions at the CGCGCGCG sequence in hisD3052 . These results suggest that umuDCST, but not samAB, is involved in the -2 frameshift mutagenesis induced by 1-NP and 1,8-DNP at the repetitive CG sequence, whereas neither operon participates in induction of the same type of mutations by 2-NF, Glu-P-1 or N-OH-AAF.

J Biol Chem, 1995 Apr 7, 270(14), 8285 - 9
Transcriptional activation of the Escherichia coli adaptive response gene aidB is mediated by binding of methylated Ada protein . Evidence for a new consensus sequence for Ada-binding sites; Landini P et al.; The Escherichia coli aidB gene is part of the adaptive response to DNA methylation damage . Genes belonging to the adaptive response are positively regulated by the ada gene; the Ada protein acts as a transcriptional activator when methylated in one of its cysteine residues at position 69 . Through DNaseI protection assays, we show that methylated Ada (meAda) is able to bind a DNA sequence between 40 and 60 base pairs upstream of the aidB transcriptional startpoint . Binding of meAda is necessary to activate transcription of the adaptive response genes; accordingly, in vitro transcription of aidB is dependent on the presence of meAda . Unmethylated Ada protein shows no protection against DNaseI digestion in the aidB promoter region nor does it promote aidB in vitro transcription . The aidB Ada-binding site shows only weak homology to the proposed consensus sequences for Ada-binding sites in E . coli (AAANNAA and AAAGCGCA) but shares a higher degree of similarity with the Ada-binding regions from other bacterial species, such as Salmonella typhimurium and Bacillus subtilis . Based on the comparison of five different Ada-dependent promoter regions, we suggest that a possible recognition sequence for meAda might be AATnnnnnnG-CAA . Higher concentrations of Ada are required for the binding of aidB than for the ada promoter, suggesting lower affinity of the protein for the aidB Ada-binding site . Common features in the Ada-binding regions of ada and aidB are a high A/T content, the presence of an inverted repeat structure, and their position relative to the transcriptional start site . We propose that these elements, in addition to the proposed recognition sequence, are important for binding of the Ada protein.

Rev Latinoam Microbiol, 1995 Apr-Jun, 37(2), 153 - 60
Induction of humoral immune response to Salmonella typhimurium in mouse Peyer's patches; Camacho Villarreal NG et al.; The mechanisms of immune response generation and regulation at the intestinal level are not well known, mainly due to the lack of suitable and reproducible methods to measure local immune responses . The Cunningham direct and indirect hemolytic plaque assay for the quantification of antibody producing cells against Salmonella in Peyer's patches of mice orally infected with Salmonella typhimurium was used . After infection IgM and IgG producing cells were determined on days 6, 9, 12 and 19 . Specific antibody producing cells appeared after bacterial invasion of Peyer's patches, nine days after infection . At this time, there were more antibody producing cells in the distal part of the intestine, which correlated with a higher infection of these Peyer's patches as detected by bacterial culture . After day nine, the number of plaque forming cells was similar in both parts of the intestine . The peak of response was on day 12 and diminished on day 19 . The number of IgM and IgG producing cells was similar in all days analyzed . Histological analysis of Peyer's patches of infected mice showed inflammation with disorganization and tumefaction.

J Bacteriol, 1995 Apr, 177(7), 1903 - 5
Salmonella typhimurium LT7 and LT2 strains carrying the imp operon on colIa; Koch WH et al.; The imp operon is carried on a transmissible plasmid, ColIa, in original isolates of Salmonella typhimurium LT7 . LT2 strain recipients of F' factors from LT7 strains harboring ColIa can acquire ColIa and imp under nonselective conditions . Thus, S . typhimurium LT2 strains that have received plasmids by conjugal transfer from LT7 strains might be inadvertently harboring ColI factors.

J Bacteriol, 1995 Apr, 177(7), 1879 - 82
The umpA gene of Escherichia coli encodes phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (lgt) and regulates thymidylate synthase levels through translational coupling; Gan K et al.; Using a combination of biochemical, physical, and genetic techniques, we have shown that the umpA gene of Escherichia coli is allelic with the lgt (phosphatidylglycerol:prolipoprotein diacylglyceryl transferase) of Salmonella typhimurium . These genes are essential for the viability of the respective organism and exhibit 92.8% sequence identity at the amino acid level . In E . coli, lgt and thyA (thymidylate synthase) form an operon . Thymidylate synthase levels are regulated by transcription from the lgt promoter and by translational coupling.

J Bacteriol, 1995 Apr, 177(7), 1834 - 9
Characterization of a second MetR-binding site in the metE metR regulatory region of Salmonella typhimurium; Wu WF et al.; Transcription of the metE gene in Salmonella typhimurium and Escherichia coli is positively regulated by the MetR protein, with homocysteine serving as a coactivator . It was shown previously that MetR binds to and protects from DNase I digestion a 24-bp sequence in the metE metR regulatory region from nucleotides -48 to -71 relative to the metE transcription initiation site (designated as site 1) . In this study, we show that purified MetR protein also binds to and protects a second 24-bp sequence adjacent to the original site, from nucleotides -24 to -47 relative to the metE transcription initiation site (designated as site 2) . Single and multiple base changes were introduced into sites 1 and 2 in a metE-lacZ fusion . Base pair changes in site 1 or site 2 away from the MetR consensus binding sequence resulted in decreased metE-lacZ expression, suggesting that both sites are necessary for expression . DNase I footprint analysis showed that MetR bound at the high-affinity site 1 enhances MetR binding at the low-affinity site 2 . A 2-bp change in site 2 toward the MetR consensus binding sequence resulted in high metE-lacZ expression; the increased expression was MetR dependent but homocysteine independent.

J Bacteriol, 1995 Apr, 177(7), 1683 - 91
Tar-dependent and -independent pattern formation by Salmonella typhimurium; Blat Y et al.; When Salmonella typhimurium cells were allowed to swarm on either a minimal or complex semisolid medium, patterns of cell aggregates were formed (depending on the thickness of the medium) . No patterns were observed with nonchemotactic mutants . The patterns in a minimal medium were not formed by a mutant in the aspartate receptor for chemotaxis (Tar) or by wild-type cells in the presence of alpha-methyl-D,L-aspartate (an aspartate analog), thus resembling the patterns observed earlier in Escherichia coli (E . O . Budrene and H . C . Berg, Nature {London} 349:630-633, 1991) and S . typhimurium (E . O . Budrene and H . C . Berg, Abstracts of Conference II on Bacterial Locomotion and Signal Transduction, 1993) . Distinctively, the patterns in a complex medium had a different morphology and, more importantly, were Tar independent . Furthermore, mutations in any one of the genes encoding the methyl-accepting chemotaxis receptors (tsr, tar, trg, or tcp) did not prevent the pattern formation . Addition of saturating concentrations of the ligands of these receptors to wild-type cells did not prevent the pattern formation as well . A tar tsr tcp triple mutant also formed the patterns . Similar results (no negative effect on pattern formation) were obtained with a ptsI mutant (defective in chemotaxis mediated by the phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system {PTS}) and with addition of mannitol (a PTS ligand) to wild-type cells . It therefore appears that at least two different pathways are involved in the patterns formed by S . typhimurium: Tar dependent and Tar independent . Like the Tar-dependent patterns observed by Budrene and Berg, the Tar-independent patterns could be triggered by H(2)O(2), suggesting that both pathways of pattern formation may be triggered by oxidative stress.

Infect Immun, 1995 Apr, 63(4), 1611 - 4
Oral vaccination with an attenuated Salmonella typhimurium strain expressing Borrelia burgdorferi OspA prevents murine Lyme borreliosis; Dunne M et al.; Borrelia burgdorferi is the causative agent of Lyme disease . In the mouse model, protection is correlated with the development of antibodies to a major outer surface protein, OspA . In this study, we expressed OspA in an attenuated strain of Salmonella typhimurium and tested the efficacy of the transformed strain in protecting against disease . We show that mice inoculated by gavage developed high titers of anti-OspA antibodies and were protected against an intradermal challenge with the spirochete.

Infect Immun, 1995 Apr, 63(4), 1462 - 7
Association with MDCK epithelial cells by Salmonella typhimurium is reduced during utilization of carbohydrates; Schiemann DA; Association of Salmonella typhimurium with MDCK epithelial cells in monolayers, represented primarily by intracellular bacteria after 30 min of contact, with centrifugation followed by vigorous washing, was measured during aerobic and anaerobic growth of the bacteria in brain heart infusion broth . Cell association was greatest during a short period in the late log phase of growth under aerobic conditions . At this time, the pH of the growth medium was changing from acid to alkaline and glucose (0.2% initially) was exhausted . Addition of excess glucose (0.5%) to brain heart infusion broth, which was not exhausted before the bacteria entered the stationary phase of growth, in which cell association dropped sharply, resulted in repression of cell association by the bacteria . The repressive effect of glucose on cell association could not be reversed by exogenous cyclic AMP in the bacterial growth medium . Under anaerobic conditions, the effect of glucose on cell association by the bacteria was not as great and the glucose was not exhausted before the bacteria entered the stationary phase . When S . typhimurium was grown in a rich but carbohydrate-free medium, cell association by the bacteria increased earlier in the growth cycle under both aerobic and anaerobic conditions . The addition of glucose and certain other utilizable carbohydrates to this medium caused a repression of cell association by S . typhimurium that was greater under aerobic growth conditions . These results show that cell association by S . typhimurium, which is accompanied by rapid internalization (cell invasion), is the same under aerobic and anaerobic conditions if the bacteria are grown to the log phase in a carbohydrate-free medium . This suggests that prior reports of greater cell invasion by S . typhimurium during anaerobic growth may have arisen from the use of media containing carbohydrates which were found to be more repressive during aerobic growth of the bacteria.

Mutat Res, 1995 Apr, 334(2), 145 - 56
Development of high sensitive umu test system: rapid detection of genotoxicity of promutagenic aromatic amines by Salmonella typhimurium strain NM2009 possessing high O-acetyltransferase activity; Oda Y et al.; A highly sensitive umu test system for the detection of carcinogenic/mutagenic aromatic amines has been developed utilizing a new tester strain, Salmonella typhimurium NM2009, possessing an elevated O-acetyltransferase (O-AT) level . NM2009 was constructed by subcloning the bacterial O-AT gene into a plasmid vector pACYC184 and introducing the plasmid into the original strain S . typhimurium TA1535/pSK1002 harboring an umuC'-'lacZ fusion gene . The system is based on the ability of DNA-damaging agents (genotoxins) to induce umuC gene expression and monitored by measuring the cellular beta-galactosidase activity evoked by the fusion gene . Twenty-two aromatic amine compounds including arylamines, aminoazo dyes, and heterocyclic aromatic amines were tested for inducibility of DNA damage after metabolic activation by rat liver S9 in strain NM2009 and the sensitivity was compared with those of the parent strain TA1535/pSK1002 and the O-AT-defective strain NM2000 . NM2009 had about 400 times higher O-AT activity than the parent strain . It was found that NM2009 was much more sensitive to aromatic amines than other strains to induce umuC gene expression after metabolic activation; the chemicals which were extremely sensitive in strain NM2009 include 2-aminoanthracene, 2-aminofluorene, 2-acetylaminofluorene, benzidine, 6-aminochrysene, 2,4-diaminotoluene, 2,6-diaminotoluene, 1-naphthylamine, o-tolidine, 3-MeO-AAB, o-aminoazotoluene, Glu-P-1, Trp-P-1, MeA alpha C, A alpha C, MeIQ, MeIQx, and IQ . In contrast, Trp-P-2 and PhIP showed almost similar sensitivities in three tester strains used in this study . These results suggest that strain NM2009 with high O-acetyltransferase activity is very useful to detect the genotoxic activities of potential mutagenic aromatic amine compounds, which require metabolic activation via the cytochrome P-450/acetyltransferase system.

Biophys J, 1995 Apr, 68(4 Suppl), 168S - 171S; discussion 171S-172S
Conserved machinery of the bacterial flagellar motor; Stahlberg A et al.; Novel periplasmic and cytoplasmic structural modules of the bases of bacterial flagella have been observed in situ and isolated using new biochemical protocols . Flagellar rotation may depend upon interactions of these modules with the intramembrane particle rings, a ubiquitous feature of flagellar bases necessary for torque generation . The outer membrane-associated basal disk of the Wolinella succinogenes polar flagellum has architecture well suited for interaction with the ring particles . However, antibody against the main W . succinogenes basal disk protein did not cross-react with flagella-enriched fractions from Salmonella typhimurium and Bacillus firmus; nor have such structures been observed in these species thus far . Antibodies against two S . typhimurium proteins, FliG and FliM, known to be involved in motor function and part of the cytoplasmic module in this species cross-reacted with flagella-enriched fractions from both W . succinogenes and B . firmus . In addition, flagellar cytoplasmic structure could be isolated from B . firmus . The basal disk may anchor the flagellar motor to the cell wall in some polar bacteria, but this does not seem to be a unique strategy . In contrast, the data indicate that the cytoplasmic module is conserved.

FEMS Microbiol Lett, 1995 Apr 1, 127(3), 235 - 42
Cloning, sequencing and analysis of a gene encoding Escherichia coli proline dehydrogenase; Xia M et al.; Using a genomic subtraction technique, we cloned a DNA sequence that is present in wild-type Escherichia coli strain CSH4 but is missing in a presumptive proline dehydrogenase deletion mutant RM2 . Experimental evidence indicated that the cloned fragment codes for proline dehydrogenase (EC 1.5.99.8) since RM2 cells transformed with a plasmid containing this sequence was able to survive on minimal medium supplemented with proline as the sole nitrogen and carbon sources . The cloned DNA fragment has an open reading frame of 3942 bp and encodes a protein of 1313 amino acids with a calculated Mr of 143,808 . The deduced amino acid sequence of the E . coli proline dehydrogenase has an 84.9% homology to the previously reported Salmonella typhimurium putA gene but it is 111 amino acids longer at the C-terminal than the latter.

Mutat Res, 1995 Apr, 346(4), 215 - 20
Increased mutability by oxidative stress in OxyR-deficient Escherichia coli and Salmonella typhimurium cells: clonal occurrence of the mutants during growth on nonselective media; Blanco M et al.; Escherichia coli and Salmonella typhimurium strains deficient in the OxyR-regulated adaptive response to oxidative stress were used to study the mode in which spontaneous SOS-dependent mutations are generated in a distressed bacterial population . When assayed on supplemented selective medium, the E . coli strain IC3821 (trpE65), carrying the delta oxyR30 mutation and containing the plasmid pRW144 (mucA/B), showed a frequency of spontaneous Trp+ revertants similar to that of the oxyR+ control . Instead, the IC3821 strain exhibited an enhancement in the clonal occurrence of spontaneous revertants arising at random during growth on a nonselective medium . A similar enhancement was observed for the S . typhimurium strain TA4125 (hisG428 delta oxyR2) . The mutator effect observed in oxyR- cells would be induced by an increased background of reactive oxygen species; it provides a model for studying the mutability of a cell population constantly exposed to mutation-inducing agents . In the IC3821 strain, revertants were induced by t-butyl hydroperoxide with higher efficiency than in oxyR+ . We suggest that strain IC3821 could be useful for the detection of SOS-dependent mutagenesis induced by chemical oxidants.

Appl Environ Microbiol, 1995 Apr, 61(4), 1637 - 40
Frequency of generalized transducing phages in natural isolates of the Salmonella typhimurium complex; Schicklmaier P et al.; From 85 natural isolates of the Salmonella typhimurium complex, including the Salmonella reference collection A (P . Beltran, S . A . Plock, N . H . Smith, T . S . Whittam, D . C . Old, and R . K . Selander, J . Gen . Microbiol . 137:601-606, 1991), 65 strains (76.5%) released 71 different temperate phages . Forty-three (93.5%) of 46 tested phages were able to transduce the chromosomal markers his+ and trp+ and the cloning vector pBR325.

Appl Environ Microbiol, 1995 Apr, 61(4), 1220 - 5
Protein aggregation kinetics in an Escherichia coli strain overexpressing a Salmonella typhimurium CheY mutant gene; Klein J et al.; The tendency of recombinant protein in bacteria to partition into soluble and insoluble forms is attributed, in general, to a kinetic competition between protein folding and aggregation . However, little experimental work has actually been performed in vivo on the kinetics and mechanisms of protein folding and aggregation . Results are presented here from radiolabeling experiments which monitored the kinetics of recombinant protein aggregation in actively growing cultures . The strain used was an Escherichia coli strain overexpressing a Salmonella typhimurium CheY mutant gene . The rate of CheY aggregation was found to be time dependent in that the tendency of CheY to aggregate was greater for newly translated molecules, i.e., those translated within the previous several minutes, than for molecules translated less recently . CheY protein molecules that were translated less recently continued to aggregate for several hours but at a lower rate . The movement of soluble CheY to the insoluble form was enhanced at elevated growth temperatures and inhibited by the presence of chloramphenicol . The latter observation suggests that ongoing translation facilitates the movement of soluble CheY to the insoluble form . The implications of these results for the mechanism of protein aggregation in vivo, i.e., inclusion body formation, are discussed.

J Appl Bacteriol, 1995 Apr, 78(4), 402 - 8
Salmonella typhimurium DT 193: differentiation of an epidemic phage type by antibiogram, plasmid profile, plasmid fingerprint and salmonella plasmid virulence (spv) gene probe; Hampton MD et al.; Of over 2000 isolates of Salmonella typhimurium DT 193 from humans examined in the 2 year period 1991-92, 93% were antibiotic-resistant with the most common R-types being ASSuT (38%) and T (29%) . Fourteen plasmid profiles were identified in DT 193 R-type ASSuT with the majority of isolates being characterized by a single plasmid of 80 MDa (pDEP 34) which in addition to coding for ASSuT, also hybridized with a spv gene probe prepared from the 50 MDa Salm . dublin serovar-specific plasmid . On the basis of restriction fragment length polymorphisms, two variant lines of pDEP 34-like plasmids were identified and a third line which had lost the genes coding for resistance to ampicillin, streptomycin and sulphonamides, was recognized . Although 18 plasmid profile types were identified in DT 193 R-type T, all isolates carried a high mol . wt plasmid which coded for tetracycline resistance only . Further discrimination was achieved on the basis of hybridization of tetracycline resistance plasmids with the spv gene probe and restriction enzyme fingerprinting . These results demonstrate that Salm . typhimurium DT 193 can be rapidly subdivided by antibiogram and that further subdivision can be achieved on the basis of plasmid profile, plasmid fingerprint and hybridization with a spv gene probe.

Arch Environ Contam Toxicol, 1995 Apr, 28(3), 391 - 5
Genotoxic risk assessment of drinking water consumed in the city of Tehran, Iran; Sabouni F et al.; Two hundred liters of drinking water of the city of Tehran were collected as tap water in a glass container and passed through a XAD-2 resin column at a flow rate of 60-100 ml/minute . The adsorbed materials were eluted with acetone, dried and dissolved in 2 ml dimethylsulfoxide (DMSO) . Doses up to 10 microliters did not elevate sister chromatid exchanges (SCE) in CHO fibroblasts, while 20 microliters caused a significant increase in SCE . However, this dose did not show any chromosomal aberration (CA) in V79 fibroblasts . Doses up to 50 microliters of the extract did not increase his+ spontaneous revertant colonies in Salmonella typhimurium tester strain TA98, TA100 and TA102 in the absence of S9 metabolizing mixture, neither induced DNA alkaline labile sites in V79 fibroblasts.

J Bacteriol, 1995 Apr, 177(8), 2087 - 97
Identification and sequence analysis of lpfABCDE, a putative fimbrial operon of Salmonella typhimurium; Baumler AJ et al.; A chromosomal region present in Salmonella typhimurium but absent from related species was identified by hybridization . A DNA probe originating from 78 min on the S . typhimurium chromosome hybridized with DNA from Salmonella enteritidis, Salmonella heidelberg, and Salmonella dublin but not with DNA from Salmonella typhi, Salmonella arizonae, Escherichia coli, and Shigella serotypes . Cloning and sequence analysis revealed that the corresponding region of the S . typhimurium chromosome encodes a fimbrial operon . Long fimbriae inserted at the poles of the bacterium were observed by electron microscopy when this fimbrial operon was introduced into a nonpiliated E . coli strain . The genes encoding these fimbriae were therefore termed lpfABCDE, for long polar fimbriae . Genetically, the lpf operon was found to be most closely related to the fim operon of S . typhimurium, both in gene order and in conservation of the deduced amino acid sequences.

Toxicol Appl Pharmacol, 1995 Apr, 131(2), 332 - 41
Nitrofurantoin-stimulated reactive oxygen species production and genotoxicity in digestive gland microsomes and cytosol of the common mussel (Mytilus edulis L.); Garcia Martinez P et al.; The ability of nitrofurantoin (NF) to produce reactive oxygen species (ROS) was investigated in subcellular fractions of digestive gland of the mussel Mytilus edulis in terms of oxygen consumption and the formation of superoxide anion radical (O2-) (measured as SOD-sensitive cytochrome c reduction or SOD-sensitive sensitive .OH production), H2O2 (effect of catalase), and hydroxyl radical (.OH) (iron/EDTA-mediated oxidation of KMBA to ethylene) . Additionally, the genotoxic effects of NF were examined using the Salmonella typhimurium umu mutagenicity assay . Microsomal NAD(P)H-dependent oxygen consumption was stimulated by NF, leading to the formation of H2O2 . Stimulation of microsomal O2- production by NF was evident for NADH but not NADPH, confirming redox cycling at least with the former coenzyme . No stimulation of O2- production was obvious for cytosolic fraction with either coenzyme . NF stimulated microsomal NAD(P)H-dependent .OH production; the rates of .OH production were greater for NADH than NADPH; and the .OH was indicated to be formed, at least in part, by an iron-catalyzed Haber-Weiss reaction . A role was indicated for a free radical driven Fenton reaction in the NF-stimulated microsomal production of .OH from NADPH . The production of mutagenic species from NF was observed for cytosol but not for microsomes, and the former effects were greater for NADH than NADPH . Overall, the NAD(P)H-dependent microsomal generation of ROS, and the lack of correlation of ROS production with mutagenicity, are considered indicative of the potential of digestive gland to metabolize NF by both one-electron and two-electron reductive pathways . From this and other studies, enhanced ROS production by NF and other redox cycling xenobiotics is indicated to be a widespread phenomenon in aquatic organisms and a potential mechanism of pollutant-mediated toxicity.

Mutat Res, 1995 Apr, 342(3-4), 179 - 90
A comparison of the mutagenicity of mainstream cigarette smoke condensates from a representative sample of the U.S . cigarette market with a Kentucky reference cigarette (K1R4F); Steele RH et al.; The Salmonella mutagenicity assay has been used to investigate the mutagenicity of cigarette smoke and cigarette smoke condensate . The Kentucky reference (K1R4F) cigarette is designed to be representative of full-flavor, low-tar cigarettes sold in the U.S . and to serve as a reference standard for comparative studies on the chemistry and biological activities of cigarette smoke and condensate . The objective of this study was to determine if the mutagenicity of mainstream smoke condensate from the K1R4F, as measured by the Salmonella mutagenicity assay, is representative of the mutagenic activity of U.S . cigarettes . Mainstream smoke condensates prepared in dimethyl sulfoxide from the K1R4F and 73 brand styles (representing greater than 70% of the total U.S . cigarette market) were assayed using Salmonella typhimurium TA98 and TA100 (+S9) at concentrations of 0, 25, 50, 75, 100, 125 and 250 micrograms/plate . Revertants/mg condensate were determined by calculating the slopes of the dose-response curves using linear and nonlinear regression models . Revertants/cigarette were determined by multiplying the revertants/mg condensate by the mg condensate/cigarette . No significant differences (p > 0.05) were observed between the mean mutagenicity of U.S . market and K1R4F mainstream smoke condensates in terms of revertants/mg condensate or revertants/cigarette . Increased variability in mutagenicity was observed among the U.S . brands versus that of the K1R4F . This is not surprising since variability among the U.S . brands would be expected to have both measurement error and brand style variability while the K1R4F variability contains only the measurement error portion . These results demonstrate that the K1R4F is a representative model for the U.S . cigarette market in comparative Salmonella mutagenicity studies using mainstream smoke condensates.

Mutat Res, 1995 Apr, 342(3-4), 141 - 6
Comparative study on Salmonella mutagenicity and on cytogenetic and antineoplastic effects induced by cyclophosphamide and 3-aminobenzamide in cells of three transplantable tumours in vivo; Eliopoulos P et al.; Synergistically enhanced sister chromatid exchange (SCE) frequency by cyclophosphamide (CP) was observed when L1210 lymphoid tumor cells were exposed in vivo to a non-toxic concentration of 3-aminobenzamide (3-AB) . Additive effects in SCE induction in vivo were observed when either Ehrlich ascites tumor (EAT) cells or P388 lymphocytic leukemia cells treated with CP were exposed to 3-AB in vivo . 3-AB enhanced the survival time of L1210 tumor bearing BDF1 mice treated with CP . However, the combined CP plus 3-AB treatment did not increase the survival of either EAT BALB/c- or P388 BDF1-tumor bearing mice compared with the effect on survival by CP alone . Therefore the in vivo differential antitumor effect, by CP in conjunction with 3-AB, appears to correlate well with the in vivo differential effect on cytogenetic damage caused by the combined CP plus 3-AB treatment . In the Salmonella typhimurium/mammalian microsome test CP appears to have a dose dependent ability to induce base-pair substitutions in strains TA 100 and TA 1535 and frameshift mutations in strains TA 98 and TA 1537 . Both types of mutation were synergistically increased in the presence of 3-AB.

J Cell Biol, 1995 Apr, 129(1), 81 - 97
Targeting of Salmonella typhimurium to vesicles containing lysosomal membrane glycoproteins bypasses compartments with mannose 6-phosphate receptors; Garcia-del Portillo F et al.; Salmonella typhimurium is an intracellular bacterial pathogen that remains enclosed in vacuoles (SCV) upon entry into the host cell . In this study we have examined the intracellular trafficking route of S . typhimurium within epithelial cells . Indirect immunofluorescence analysis showed that bacteria initiated fusion with lysosomal membrane glycoprotein (lgp)-containing compartments approximately 15 min after bacterial internalization . This process was completed approximately 75 min later and did not require microtubules . Cation-independent (CI)- or cation-dependent (CD)-mannose 6-phosphate receptors (M6PRs) were not observed at detectable levels in SCV . Lysosomal enzymes showed a different distribution in SCV: lysosomal-acid phosphatase (LAP) was incorporated into these vacuoles with the same kinetics as lgps, while cathepsin D was present in a low proportion (approximately 30%) of SCV . Uptake experiments with fluid endocytic tracers such as fluorescein-dextran sulphate (F-DX) or horseradish-peroxidase (HRP) showed that after 2 h of uptake, F-DX was present in approximately 75% of lgp-containing vesicles in uninfected cells, while only approximately 15% of SCV contained small amounts of the tracer during the same uptake period . SCV also showed only partial fusion with HRP-preloaded secondary lysosomes, with approximately 30% of SCV having detectable amounts of HRP at 6 h after infection . These results indicate that SCV show limited accessibility to fluid endocytic tracers and mature lysosomes, and are therefore functionally separated from the endocytic route . Moreover, the unusual intracellular trafficking route of S . typhimurium inside epithelial cells has allowed us to establish the existence of two different lgp-containing vesicles in Salmonella-infected cells: one population is separated from the endocytic route, fusogenic with incoming SCV and may arise from a secretory pathway, while the second involves the classical secondary or mature lysosomes.

Avian Dis, 1995 Apr-Jun, 39(2), 309 - 16
Synergism of lactate and succinate as metabolites utilized by Veillonella to inhibit the growth of Salmonella typhimurium and Salmonella enteritidis in vitro; Hinton A Jr et al.; The inhibition of salmonellae growth by a Veillonella bacterium isolated from the cecal contents of adult chickens was examined . The Veillonella isolate was grown on an agar medium supplemented with 175 mumol of lactate or succinate/ml . Either 0, 100, 125, 150, or 175 mumol of succinate/ml was added to the lactate medium; either 0, 100, 125, 150, or 175 mumol of lactate/ml was added to the succinate medium; and the pH of all media was adjusted to 6.0 . Agar overlays of Veillonella cultures grown on the media were inoculated with Salmonella typhimurium or S . enteritidis . The largest zones of inhibition of salmonellae growth were produced by Veillonella cultures grown on medium supplemented with 175 mumol/ml of both lactate and succinate . The widths of the zones of inhibition decreased as the concentration of lactate was reduced in the succinate medium and as the concentration of succinate was reduced in the lactate medium . Analyses of lactate broth and succinate broth inoculated with Veillonella indicated that inhibition of salmonellae growth on the agar media was related to the production of volatile fatty acids by Veillonella, the presence of residual succinate in the media, and the final pH of the media.

Avian Dis, 1995 Apr-Jun, 39(2), 230 - 8
Cell-mediated and humoral immune responses in chickens infected with Salmonella typhimurium; Lessard M et al.; The cellular immune responses of leghorn and New Hampshire chickens following oral challenge at 4 weeks of age with Salmonella typhimurium were measured using assays for lymphocyte proliferative responses to mitogens and cytotoxic activity of natural killer (NK) cells . In addition, the humoral immune response to Newcastle disease virus (NDV) vaccine was assessed in S . typhimurium-infected chickens . At 8 and 20 days after infection with S . typhimurium, lymphocyte proliferative responses to various mitogens were significantly higher in S . typhimurium-infected chickens than in uninfected birds of the same breeds . Cytotoxic activity of NK cells was also significantly increased in infected birds on these days . Thirteen days after S . typhimurium infection, infected NDV-vaccinated chickens had higher antibody titers in response to NDV vaccination than uninfected vaccinated birds . NDV titers did not differ among groups at any other time of testing . These results show that both cellular and humoral immune functions are activated in the first 3 weeks following infection of 4-week-old chickens with S . typhimurium.

Pediatr Res, 1995 Apr, 37(4 Pt 1), 469 - 75
Failure of premature rabbits to increase lung antioxidant enzyme activities after hyperoxic exposure: antioxidant enzyme gene expression and pharmacologic intervention with endotoxin and dexamethasone; Sosenko IR et al.; Premature rabbits, unlike full-term rabbits, are unable to mount a protective increase in pulmonary antioxidant enzyme (AOE) activities in response to 48 h of hyperoxic exposure and demonstrate increased pulmonary O2 toxicity compared with full-term rabbits . To examine AOE gene expression of CuZn superoxide dismutase (SOD), Mn SOD, catalase, and glutathione peroxidase in preterm versus term rabbits in response to hyperoxia, 29.5 d preterm rabbits (delivered by hysterotomy) and term rabbits (spontaneously vaginally delivered) were exposed to 48 h of > 90% O2 or room air . Preterm rabbits had a significant increase in CuZn SOD mRNA without corresponding AOE activity increases, suggesting translational/posttranslational inhibition . In full-term rabbits, the magnitude of lung AOE mRNA changes was associated with concordant magnitude changes in activities of CuZn SOD, Mn SOD, and catalase, suggesting pretranslational regulation of AOE gene expression; glutathione peroxidase, however, appears to be regulated translationally/posttranslationally . To investigate potential pharmacologic means of overcoming the susceptibility of the preterm rabbit to O2 toxicity, 29.5 d preterm rabbits received 20-40 micrograms/kg of Salmonella typhimurium endotoxin or diluent S.C . (after birth and at 24 h); in separate experiments, pregnant rabbits received intramuscular injections of dexamethasone (0.01-0.05 mg/kg) or saline on gestational d 27.5 and 28.5 and underwent hysterotomy at 29.5 d.(ABSTRACT TRUNCATED AT 250 WORDS)

Chem Res Toxicol, 1995 Apr-May, 8(3), 465 - 72
Enhancement of bacterial mutagenicity of bifunctional alkylating agents by expression of mammalian glutathione S-transferase; Thier R et al.; Recently, we inserted the plasmid vector pKK233-2 containing rat GSH S-transferase (GST) 5-5 cDNA into Salmonella typhimurium TA1535 and found that these bacteria {GST 5-5(+)} expressed the protein and produced mutations when ethylene or methylene dihalides were added {Thier, R., Taylor, J . B., Pemble, S . E., Ketterer, B., Persmark, M., Humphreys, W . G., and Guengerich, F . P . (1993) Proc . Natl . Acad . Sci . U.S.A . 90, 8576-8580} . After exposure to the known GST 5-5 substrate 1,2-epoxy-3-(4'-nitrophenoxy)propane, the GST 5-5(+) strain showed fewer mutants than the bacteria transfected with the cDNA clone in a reverse orientation {GST 5-5(-)}, suggesting a protective role of GST 5-5 . However, mutations were considerably enhanced in the GST 5-5(+) strain {as compared to GST 5-5(-)} when 1,2,3,4-diepoxybutane (butadiene diepoxide) or 1,2-epoxy-4-bromobutane was added . The GST 5-5(+) and GST 5-5(-) bacterial stains showed similar responses to 1,2-epoxypropane, 3,4-epoxy-1-butene, and 1,4-dibromobutane . The results suggest that some bifunctional activated butanes are transformed to mutagenic products through GSH conjugation . We also found that the GST 5-5(+) strain showed enhanced mutagenicity with 1,4-dibromo-2,3-epoxybutane, 1,2-epoxy-3-bromopropane (epibromohydrin), and (+/-)-1,4-dibromo-2,3-dihydroxybutane.(ABSTRACT TRUNCATED AT 250 WORDS)

Chem Res Toxicol, 1995 Apr-May, 8(3), 328 - 32
Aflatoxin M1 8,9-epoxide: preparation and mutagenic activity; Bujons J et al.; Treatment of aflatoxin M1 (AFM1) with dimethyldioxirane in an anhydrous mixture of CH2-Cl2 and acetone afforded the corresponding aflatoxin M1 8,9-epoxide (AFM1-E) in practically quantitative yield . This highly reactive intermediate was identified by 1H NMR and characterized by its neat conversion into the corresponding trans-methoxyhydrin derivative 1 . The analysis of the 1H NMR spectrum of the above epoxide revealed that one stereoisomer, which should be that with the exo configuration, was present as major component . The mutagenicities of AFM1-E, the parent mycotoxin (AFM1), aflatoxin B1 (AFB1), and its epoxide (AFB1-E) were assessed by using a sensitive improved Ames test with the Salmonella typhimurium strain TA-100 . AFM1 and AFB1 had specific mutagenic activities (SMA) of 13 and 121 revertants/ng, respectively, with S9 metabolic activation . AFM1-E was mutagenic with and without metabolic activation showing SMA of 13 and 12 revertants/ng, respectively . AFB1-E had a SMA of 42 and 29 revertants/ng, with and without S9 metabolic enzymes, respectively . These results suggest that the epoxidation of AFM1 can constitute a major route accounting for the cytotoxic effects elicited by this mycotoxin and that AFM1-E is not as active as AFB1-E in reacting with the constituents of the mutagenicity assay.

Proteins, 1995 Apr, 21(4), 345 - 50
Purification, crystallization, and preliminary X-ray diffraction analyses of the bacterial chemotaxis receptor modifying enzymes; West AH et al.; Bacterial chemotaxis receptor modifying enzymes from Salmonella typhimurium have been crystallized using microseeding techniques . The crystals of the S-adenosyl-L-methionine-dependent methyltransferase, CheR, belong to the monoclinic space group P21 with cell constants a = 55.1 A, b = 48.1 A, c = 63.1 A, beta = 112.3 degrees . The crystals of the catalytic domain of the methylesterase, CheB, belong to the trigonal space group P3(2)21 or P3(1)21 with unit cell dimensions of a = b = 63.4 A, c = 86.8 A . Both crystals contain one molecule per asymmetric unit and have calculated Matthews' volumes of 2.4 A3/Da.

Indian J Exp Biol, 1995 Apr, 33(4), 309 - 10
Prolonged stability of R plasmids KR61 and KR61-A in Salmonella host; Khatoon H et al.; The R plasmids, KR61 and KR61-A, that were originally isolated from a clinical strain of Aerobacter aerogenes in 1971 and determined resistance to kanamycin (Km), neomycin (Nm), streptomycin (Sm), tetracycline (Tc); and ampicillin (Ap) respectively were found stable in Salmonella typhimurium LT2 even after 22 years of cultivation on antibiotic free media . KR61, carrying resistance to KmNmSmTc, not only maintained all its resistances but also maintained its conjugal transferability (RTF) as indicated by its subsequent transfer to Escherichia and Salmonella hosts . KR61-A that carried resistance to Ap and lacked an RTF could be mobilized by KR61 from S . typhimurium LT2, constructed to bear KR61-A and KR61, to E . coli recipients . S . typhimurium LT2 carrying KR61-A + KR61 (ApKmNmSmTc), showed the characteristic conjugal transfer of resistances in following three patterns: (i) Ap, (ii) KmNmSmTc and (iii) ApKmNmSmTc . The findings reported here are based on conjugal isolation of plasmids . Physical isolation of KR61 and KR61-A was never made.

Int J Food Microbiol, 1995 Apr, 25(2), 119 - 29
Effects of high levels of selenite and Escherichia coli on enrichment of Salmonella and detection of Salmonella by polymyxin-cloth enzyme immunoassay; Chen H et al.; The effect of various concentrations of selenite on the recovery of Salmonella typhimurium and Escherichia coli was examined in a rich medium containing 0.5% cholate . For heat-injured S . typhimurium, longer preincubation periods were required for recovery in higher concentrations of selenite . When 0.7% selenite was added to 6-h preincubated cultures of heat-injured cells, S . typhimurium was fully recovered while E . coli, whether heat-injured or uninjured, did not recover when present at numbers at less than 2 x 10(7) colony forming units (CFU) . The polymyxin-cloth enzyme immunoassay detected S . typhimurium grown from a small number (as low as 1 CFU) of cells in the presence of a large excess of E . coli.

J Interferon Cytokine Res, 1995 Apr, 15(4), 297 - 300
Action of quail and chicken interferons on a quail cell line, QT35; Fulton RW et al.; Production of interferon (IFN) in quail cells (QT35) and the activity of quail IFN and heterologous avian IFN (chicken) on QT35 cells were examined . Quail cells produced IFN after induction by bluetongue virus serotype 10; chicken and quail IFN conferred antiviral resistance on the quail cells . Both chicken and quail IFN induced increased levels of 2',5'-oligoadenylate synthetase (2',5'-OAS) in QT35 cells and reduced levels of intracellular Salmonella typhimurium postchallenge . These results indicate that the quail cells produce IFN and respond to homologous and heterologous avian IFN as evidenced by enhanced (1) resistance to viral infection, (2) production of 2',5'-OAS, and (3) resistance to invasion by bacteria . QT35 quail cell monolayer cultures offer an alternative to primary chicken embryo fibroblasts cultures used for avian IFN studies.

J Bacteriol, 1995 Apr, 177(8), 2021 - 32
Sequence, regulation, and functions of fis in Salmonella typhimurium; Osuna R et al.; The fis operon from Salmonella typhimurium has been cloned and sequenced, and the properties of Fis-deficient and Fis-constitutive strains were examined . The overall fis operon organization in S . typhimurium is the same as that in Escherichia coli, with the deduced Fis amino acid sequences being identical between both species . While the open reading frames upstream of fis have diverged slightly, the promoter regions between the two species are also identical between -49 and +94 . Fis protein and mRNA levels fluctuated dramatically during the course of growth in batch cultures, peaking at approximately 40,000 dimers per cell in early exponential phase, and were undetectable after growth in stationary phase . fis autoregulation was less effective in S . typhimurium than that in E . coli, which can be correlated with the absence or reduced affinity of several Fis-binding sites in the S . typhimurium fis promoter region . Phenotypes of fis mutants include loss of Hin-mediated DNA inversion, cell filamentation, reduced growth rates in rich medium, and increased lag times when the mutants are subcultured after prolonged growth in stationary phase . On the other hand, cells constitutively expressing Fis exhibited normal logarithmic growth but showed a sharp reduction in survival during stationary phase . During the course of these studies, the sigma 28-dependent promoter within the hin-invertible segment that is responsible for fljB (H2) flagellin synthesis was precisely located.

J Bacteriol, 1995 Apr, 177(8), 1967 - 75
The methylthio group (ms2) of N6-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms2io6A) present next to the anticodon contributes to the decoding efficiency of the tRNA; Esberg B et al.; A Salmonella typhimurium LT2 mutant which harbors a mutation (miaB2508::Tn10dCm) that results in a reduction in the activities of the amber suppressors supF30 (tRNA(CUATyr)), supD10 (tRNA(CUASer)), and supJ60 (tRNA(CUALeu)) was isolated . The mutant was deficient in the methylthio group (ms2) of N6-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms2io6A), a modified nucleoside that is normally present next to the anticodon (position 37) in tRNAs that read codons that start with uridine . Consequently, the mutant had i6A37 instead of ms2io6A37 in its tRNA . Only small amounts of io6A37 was found . We suggest that the synthesis of ms2io6A occurs in the following order: A-37-->i6A37-->ms2i6A37-->ms2io6A37 . The mutation miaB2508::Tn10dCm was 60% linked to the nag gene (min 15) and 40% linked to the fur gene and is located counterclockwise from both of these genes . The growth rates of the mutant in four growth media did not significantly deviate from those of a wild-type strain . The polypeptide chain elongation rate was also unaffected in the mutant . However, the miaB2508::Tn10dCm mutation rendered the cell more resistant or sensitive, compared with a wild-type cell, to several amino acid analogs, suggesting that this mutation influences the regulation of several amino acid biosynthetic operons . The efficiencies of the aforementioned amber suppressors were decreased to as low as 16%, depending on the suppressor and the codon context monitored, demonstrating that the ms2 group of ms2io6A contributes to the decoding efficiency of tRNA . However, the major impact of the ms2io6 modification in the decoding process comes from the io6 group alone or from the combination of the ms2 and io6 groups, not from the ms2 group alone.

Proc Natl Acad Sci U S A, 1995 Mar 28, 92(7), 2904 - 8
Macrophage-inducible expression of a model antigen in Salmonella typhimurium enhances immunogenicity; Hohmann EL et al.; Attenuated Salmonella are useful oral vaccine vectors capable of carrying multiple heterologous antigen genes, but optimal expression of foreign antigens has not yet been achieved . We hypothesized that Salmonella phoP-activated genes, which are transcriptionally activated within antigen-processing macrophages, could prove useful for delivery of heterologous antigens to the immune system . We have created a suicide vector that allows the stable chromosomal insertion of heterologous antigen genes within the phoP-activated gene C (pagC) of Salmonella and permits the expression of heterologous antigens as fusion proteins between the first 84 amino acids of PagC and the chosen antigen . The Escherichia coli phoA gene encoding alkaline phosphatase was cloned into this vector; the resultant plasmid was used to construct Salmonella typhimurium strains that express PagC-alkaline phosphatase fusion proteins from a single chromosomal gene copy . Such strains were administered orally and i.p . as vaccines to BALB/c mice and compared with control strains expressing alkaline phosphatase constitutively . After 3 weeks, mouse sera were analyzed for IgG responses to S . typhimurium lipopolysaccharide and alkaline phosphatase . Remarkably, though all mice had comparable antibody responses to lipopolysaccharide, only mice immunized with strains bearing phoP-activated fusion genes had antibody responses to the heterologous antigen . We conclude that expression of a heterologous antigen from an S . typhimurium in vivo-induced promoter that is activated within macrophages markedly enhances the immunogenicity of a model antigen expressed from a single chromosomal gene copy.

Proc Natl Acad Sci U S A, 1995 Mar 14, 92(6), 1970 - 4
Motility protein interactions in the bacterial flagellar motor; Garza AG et al.; Five proteins (MotA, MotB, FliG, FliM, and FliN) have been implicated in energizing flagellar rotation in Escherichia coli and Salmonella typhimurium . One model for flagellar function envisions that MotA and MotB comprise the stator of a rotary motor and that FliG, FliM, and FliN are part of the rotor . MotA probably functions as a transmembrane proton channel, and MotB has been proposed to anchor MotA to the peptidoglycan of the cell wall . To study interactions between the Mot proteins themselves and between them and other components of the flagellar motor, we attempted to isolate extragenic suppressors of 13 dominant or partially dominant motB missense mutations . Four of these yielded suppressors, which exhibited widely varying efficiencies of suppression . The pattern of suppression was partially alleles-specific, but no suppressor seriously impaired motility in a motB+ strain . Of 20 suppressors from the original selection, 15 were characterized by DNA sequencing . Fourteen of these cause single amino acid changes in MotA . Thirteen alter residues in, or directly adjacent to, the putative periplasmic loops of MotA, and the remaining one alters a residue in the middle of the fourth predicted transmembrane helix of MotA . We conclude that the MotA and MotB proteins form a complex and that their interaction directly involves or is strongly influenced by the periplasmic loops of MotA . The 15th suppressor from the original selection and 2 motB suppressors identified during a subsequent search cause single amino acid substitutions in FliG . This finding suggests that the postulated Mot-protein complex may be in close proximity to FliG at the stator-rotor interface of the flagellar motor.

Lik Sprava, 1995 Mar-Apr, (3-4), 92 - 4
{The participation of the renin-angiotensin-aldosterone system in the pathogenesis of acute kidney failure in endotoxemia}; Krishtal' NV et al.; It was found in acute experiments on white rats that injection of Salmonella typhimurium lipopolysaccharide activates renin-angiotensin-aldosterone system (RAAS) and causes oligohydruria form of acute renal failure (ARF) . Enalapril, angiotensin-converting enzyme, being injected simultaneously with endotoxin, increases diuresis, glomerular filtration rate, electrolytes excretion and reabsorption by proximal tubules, decreases proteinuria and blood nitrogen-down to the normal . Thus, RAAS takes part in endotoxemia ARF induction and enalapril has protective effect.

Mutat Res, 1995 Mar, 342(1-2), 9 - 16
Mutagenicity of benzo{a}pyrene and dibenzopyrenes in the Salmonella typhimurium TM677 and the MCL-5 human cell forward mutation assays; Busby WF Jr et al.; The mutagenicity of benzo{a}pyrene (B{a}P), dibenzo{ae}pyrene (DB{ae}P), dibenzo{ah}pyrene (DB{ah}P), dibenzo{ai}pyrene (DB{ai}P), and dibenzo{al}pyrene (DB{al}P) was measured in quantitative forward mutation assays with bacteria (Salmonella typhimurium TM677) and a metabolically competent cell line derived from human B-lymphoblastoid cells (MCL-5) that contained activity for five cytochrome P450s and microsomal epoxide hydrolase found in human liver . DB{al}P and B{a}P, both potent animal carcinogens, were the most mutagenic substances in both assays . DB{al}P was nearly 50-fold more potent than B{a}P in human cells, but only 60% more mutagenic in Salmonella . The carcinogenic isomer DB{ah}P, though nonmutagenic in bacteria, was active in human cells . The following mutagenic potency series, expressed as the minimum detectable mutagen concentration (MDMC) in nmol/ml, was obtained with Salmonella in the presence of rat liver postmitochondrial supernatant (PMS): DB{al}P (3.7), B{a}P (5.8), DB{ae}P (6.9), DB{ai}P (14.9), DB{ah}P (> 100) . None of the compounds were mutagenic in the absence of PMS . In human MCL-5 cells the potency series was: DB{al}P (3.1 x 10(-4)), B{a}P (1.5 x 10(-2)), DB{ae}P (2.5 x 10(-2)), DB{ah}P (0.5), DB{ai}P (3.2) . The human cell assay thus exhibited over a 10,000-fold range between the most mutagenic and least mutagenic compound, whereas in the bacterial assay there was only a corresponding four-fold difference if the nonmutagenic DB{ah}P was excluded . The results were discussed in terms of their concordance with animal carcinogenicity studies.

J Clin Invest, 1995 Mar, 95(3), 1047 - 53
DNA repair is more important than catalase for Salmonella virulence in mice; Buchmeier NA et al.; Pathogenic microorganisms possess antioxidant defense mechanisms for protection from reactive oxygen metabolites such as hydrogen peroxide (H2O2), which are generated during the respiratory burst of phagocytic cells . These defense mechanisms include enzymes such as catalase, which detoxify reactive oxygen species, and DNA repair systems which repair damage resulting from oxidative stress . To determine the relative importance of these two potentially protective defense mechanisms against oxidative stress encountered by Salmonella during infection of the host, a Salmonella typhimurium double mutant unable to produce either the HPI or HPII catalase was constructed, and compared with an isogenic recA mutant deficient in DNA repair . The recA mutant was hypersusceptible to H2O2 at low cell densities in vitro, while the catalase mutant was more susceptible to high H2O2 concentrations at high cell densities . The catalase mutant was found to be resistant to macrophages and retained full murine virulence, in contrast to the recA mutant which previously was shown to be macrophage-sensitive and attenuated in mice . These observations suggest that Salmonella is subjected to low concentrations of H2O2 while at relatively low cell density during infection, conditions requiring an intact DNA repair system but not functional catalase activity.

J Bacteriol, 1995 Mar, 177(6), 1638 - 40
Distribution of the CorA Mg2+ transport system in gram-negative bacteria; Smith RL et al.; The CorA Mg2+ transport system is the dominant constitutive uptake mechanism in Salmonella typhimurium and Escherichia coli . Southern blot hybridization and PCR techniques were used to screen a panel of 18 additional gram-negative bacterial species for corA homologs . Virtually all strains tested positive for the presence of corA . Thus, corA appears to be ubiquitous within gram-negative bacteria and is likely their major Mg2+ influx system.

J Bacteriol, 1995 Mar, 177(6), 1511 - 9
Construction and characterization of mutants of Salmonella typhimurium deficient in DNA repair of O6-methylguanine; Yamada M et al.; Escherichia coli has two O6-methylguanine DNA methyltransferases that repair alkylation damage in DNA and are encoded by the ada and ogt genes . The ada gene of E . coli also regulates the adaptive response to alkylation damage . The closely related species Salmonella typhimurium possesses methyltransferase activities but does not exhibit an adaptive response conferring detectable resistance to mutagenic methylating agents . We have previously cloned the ada-like gene of S . typhimurium (adaST) and constructed an adaST-deletion derivative of S . typhimurium TA1535 . Unexpectedly, the sensitivity of the resulting strain to the mutagenic action of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was similar to that of the parent strain . In this study, we have cloned and sequenced the ogt-like gene of S . typhimurium (ogtST) and characterized ogtST-deletion derivatives of TA1535 . The ogtST mutant was more sensitive than the parent strain to the mutagenicity of MNNG and other simple alkylating agents with longer alkyl groups (ethyl, propyl, and butyl) . The adaST-ogtST double mutant had a level of hypersensitivity to these agents similar to that of the ogtST single mutant . The ogtST and the adaST-ogtST mutants also displayed a two to three times higher spontaneous mutation frequency than the parent strain and the adaST mutant . These results indicate that the OgtST protein, but not the AdaST protein, plays a major role in protecting S . typhimurium from the mutagenic action of endogenous as well as exogenous alkylating agents.

J Bacteriol, 1995 Mar, 177(6), 1461 - 9
The end of the cob operon: evidence that the last gene (cobT) catalyzes synthesis of the lower ligand of vitamin B12, dimethylbenzimidazole; Chen P et al.; The cob operon of Salmonella typhimurium includes 20 genes devoted to the synthesis of adenosyl-cobalamin (coenzyme B12) . Mutants with lesions in the promoter-distal end of the operon synthesize vitamin B12 only if provided with 5,6-dimethylbenzimidazole (DMB), the lower ligand of vitamin B12 . In the hope of identifying a gene(s) involved in synthesis of DMB, the DNA base sequence of the end of the operon has been determined; this completes the sequence of the cob operon . The cobT gene is the last gene in the operon . Four CobII (DMB-) mutations mapping to different deletion intervals of the CobII region were sequenced; all affect the cobT open reading frame . Both the CobT protein of S . typhimurium and its Pseudomonas homolog have been shown in vitro to catalyze the transfer of ribose phosphate from nicotinate mononucleotide to DMB . This reaction does not contribute to DMB synthesis but rather is the first step in joining DMB to the corrin ring compound cobinamide . Thus, the phenotype of Salmonella cobT mutants conflicts with the reported activity of the affected enzyme, while Pseudomonas mutants have the expected phenotype . J . R . Trzebiatowski, G . A . O'Toole, and J . C . Escalante Semerena have suggested (J . Bacteriol . 176:3568-3575, 1994) that S . typhimurium possesses a second phosphoribosyltransferase activity (CobB) that requires a high concentration of DMB for its activity . We support that suggestion and, in addition, provide evidence that the CobT protein catalyzes both the synthesis of DMB and transfer of ribose phosphate . Some cobT mutants appear defective only in DMB synthesis, since they grow on low levels of DMB and retain their CobII phenotype in the presence of a cobB mutation . Other mutants including those with deletions, appear defective in transferase, since they require a high level of DMB (to activate CobB) and, in combination with a cobB mutation, they eliminate the ability to join DMB and cobinamide . Immediately downstream of the cob operon is a gene (called ORF in this study) of unknown function whose mutants have no detected phenotype . Just counterclockwise of ORF is an asparagine tRNA gene (probably asnU) . Farther counterclockwise, a serine tRNA gene (serU or supD) is weakly cotransducible with the cobT gene.

Mutat Res, 1995 Mar, 327(1-2), 75 - 86
Mutagenicity and mutation spectra of 2-acetylaminofluorene at frameshift and base-substitution alleles in four DNA repair backgrounds of Salmonella; Shelton ML et al.; We used colony probe hybridization procedures to determine the mutations in approximately 600 revertants of the -1 frameshift allele hisD3052 and approximately 200 revertants of the base-substitution allele hisG46 of Salmonella typhimurium induced by 2-acetylaminofluorene (2-AAF) in the presence of Aroclor-induced rat liver S9 . 2-AAF was primarily a frameshift mutagen, exhibiting 5 times more frameshift than base-substitution activity . The only frameshift mutation 2-AAF induced at the hisD3052 allele was a hotspot (-2) deletion within the sequence CGCGCGCG . The addition of the pKM101 plasmid had a small effect on the mutagenic potency of 2-AAF at this allele in a uvr+ background and no effect on the mutation spectra in either a uvr+ or uvr- background . The small amount of base-substitution activity exhibited by 2-AAF at the hisG46 allele required the presence of both the pKM101 plasmid and the uvrB mutation . The base substitutions were G.C-->T.A transversions (86%) and G.C-->A.T transitions (14%), and 85% of the substitutions were at the second position of the CCC target of the hisG46 allele; the remainder were at the first position . We propose that the hotspot frameshift may be initiated by N-acetyl-2-aminofluorene adducts located at the C(8) position of any of the guanines except the first one in the CGCGCGCG hotspot sequence . The mutation might then result from correct incorporation of cytosine opposite the adducted guanine, followed by a 2-base slippage according to our recently proposed correct-incorporation/slippage model . The hotspot mutation may also result from a 2-AAF-induced B- to Z-DNA transition at the repeating GpC site as well as by the action of enzymes involved in DNA metabolism, such as DNA resolvases or topoisomerases, on DNA structures that have been distorted by 2-AAF adducts . The small amount of 2-AAF-induced base-substitution activity may be due to mispairing of adenine opposite the minor aminofluorene adduct at the C(8) position of guanine.

J Bacteriol, 1995 Mar, 177(5), 1383 - 7
MudSacI, a transposon with strong selectable and counterselectable markers: use for rapid mapping of chromosomal mutations in Salmonella typhimurium; Lawes M et al.; The transposable bacteriophage Mu and its mini-Mu derivatives are useful tools for the genetic analysis of many bacteria . A variety of antibiotic-resistant Mu derivatives have been constructed, allowing direct selection for cells which contain the transposon . However, in many cases a counterselection against the transposon would greatly facilitate further genetic analysis . In this paper we report the construction of MudSacI, a mini-Mu derived transposon containing the sacB (secretory levansucrase) gene of Bacillus subtilis, which confers sucrose sensitivity upon gram-negative bacteria . We describe the use of this transposon as a tool for rapid genetic mapping of chromosomal genes in Salmonella typhimurium . Simple modifications of this approach should facilitate rapid mapping in many other bacteria as well.

J Bacteriol, 1995 Mar, 177(5), 1357 - 66
Ethanolamine utilization in Salmonella typhimurium: nucleotide sequence, protein expression, and mutational analysis of the cchA cchB eutE eutJ eutG eutH gene cluster; Stojiljkovic I et al.; A fragment of the Salmonella typhimurium ethanolamine utilization operon was cloned and characterized . The 6.3-kb nucleotide sequence encoded six complete open reading frames, termed cchA, cchB, eutE, eutJ, eutG, and eutH . In addition, the nucleotide sequences of two incomplete open reading frames, termed eutX and eutI, were also determined . Comparison of the deduced amino acid sequences and entries in the GenBank database indicated that eutI encodes a phosphate acetyltransferase-like enzyme . The deduced amino acid sequences of the EutE and EutG proteins revealed a significant degree of homology with the Escherichia coli alcohol dehydrogenase AdhE sequence . Mutations in eutE or eutG completely abolished the ability of mutants to utilize ethanolamine as a carbon source and reduced the ability to utilize ethanolamine as a nitrogen source . The product of eutE is most probably an acetaldehyde dehydrogenase catalyzing the conversion of acetaldehyde into acetyl coenzyme A . The product of the eutG gene, an uncommon iron-containing alcohol dehydrogenase, may protect the cell from unconverted acetaldehyde by converting it into an alcohol . The deduced amino acid sequence of cchA resembles that of carboxysome shell proteins from Thiobacillus neapolitanus and Synechococcus sp . as well as that of the PduA product from S . typhimurium . CchA and CchB proteins may be involved in the formation of an intracellular microcompartment responsible for the metabolism of ethanolamine . The hydrophobic protein encoded by the eutH gene possesses some characteristics of bacterial permeases and might therefore be involved in the transport of ethanolamine . Ethanolamine-utilization mutants were slightly attenuated in a mouse model of S . typhimurium infection, indicating that ethanolamine may be an important source of nitrogen and carbon for S . typhimurium in vivo.

J Bacteriol, 1995 Mar, 177(5), 1285 - 91
Mutational analysis of the redox-sensitive transcriptional regulator OxyR: regions important for DNA binding and multimerization; Kullik I et al.; OxyR is a LysR-type transcriptional regulator which negatively regulates its own expression and positively regulates the expression of proteins important for the defense against hydrogen peroxide in Escherichia coli and Salmonella typhimurium . Using random mutagenesis, we isolated six nonrepressing OxyR mutants that were impaired in DNA binding . Five of the mutations causing the DNA binding defect mapped near the N-terminal helix-turn-helix motif conserved among the LysR family members, confirming that this region is a DNA binding domain in OxyR . The sixth nonrepressing mutant (with E-225 changed to K {E225K}) was found to be predominantly dimeric, in contrast to the tetrameric wild-type protein, suggesting that a C-terminal region defined by the E225K mutation is involved in multimerization.

J Bacteriol, 1995 Mar, 177(5), 1233 - 8
Cloning and characterization of MgtE, a putative new class of Mg2+ transporter from Bacillus firmus OF4; Smith RL et al.; The MM281 strain of Salmonella typhimurium which possesses mutations in each its three known Mg2+ transport systems and requires 100 mM Mg2+ for growth was used to screen a genomic library from the gram-positive alkaliphilic bacterium Bacillus firmus OF4 for clones that could restore the ability to grow without Mg2+ supplementation . Of the clones obtained, five also conferred sensitivity to Co2+, similar to the phenotype of mutants with mutations in the S . typhimurium corA Mg2+ transport locus . All five contained identical inserts by restriction analysis . Using 63Ni2+ as a surrogate for the unavailable 28Mg2+, the plasmid insert was shown to restore cation uptake with properties similar but not identical to those of the S . typhimurium CorA Mg2+ transporter . Sequence analysis of one clone identified a single open reading frame with multiple possible initiation sites . Deletion and mutation analysis identified a minimum open reading frame of 939 bp encoding a polypeptide with a predicted molecular mass of 34 kDa . Disruption of the open reading frame eliminated cation influx activity and restored resistance to Co2+ . This putative transporter, designated MgtE, has no sequence similarity to any known protein including CorA and appears to represent a new class of Mg2+ transport system.

Infect Immun, 1995 Mar, 63(3), 762 - 9
Biological and genetic characterization of TnphoA mutants of Salmonella typhimurium TML in the context of gastroenteritis; Lodge J et al.; TnphoA transposon insertion mutants of phoN-negative derivatives of Salmonella typhimurium TML (of human gastroenteritic origin) were selected by growing mutagenized recipient bacteria under a variety of growth conditions . Ninety-seven individual mutants, which expressed alkaline phosphatase, were collected and tested for their ability to invade HEp-2 cells . Seven smooth mutants had a reduced ability to invade HEp-2 cells, and three smooth mutants were consistently more invasive than their corresponding parental strains . One rough mutant was of similar invasiveness and two were of reduced invasiveness when compared with that of parental strains . The seven smooth hypoinvasive mutants, the three smooth hyperinvasive mutants, and the three rough mutant strains were tested for their abilities to invade ileal enterocytes by the rabbit ileal invasion assay described previously (3) . All smooth mutants exhibited parental levels of invasiveness . The rough mutants were hypoinvasive in the rabbit ileal invasion assay . The HEp-2 system is therefore not a good predictor of behavior in gut tissue in this model . DNA sequences flanking the transposon were determined for five mutants which were hypoinvasive in the HEp-2 cell assay . The mutations were found to be insertions in two previously identified invasion genes, invG and invH, and in a gene not normally associated with invasion, pagC . These observations lead one to be cautious in the interpretation of the biological significance of data obtained from invasion of tissue culture monolayers when extrapolated to gut tissue.

Infect Immun, 1995 Mar, 63(3), 1134 - 7
Cholera toxin and Salmonella typhimurium induce different cytokine profiles in the gastrointestinal tract; Klimpel GR et al.; Salmonella infection of the gastrointestinal tract (GT) results in fluid secretion and inflammation . In contrast, cholera toxin (CT) induces fluid secretion but no inflammation . Using a murine ligated intestinal loop model, we investigated cytokine production (interleukin-1 {IL-1}, IL-2, IL-4, IL-6, IL-10, gamma interferon, and tumor necrosis factor alpha) in the GT following exposure to these agents . Salmonella typhimurium induced a Th1-like cytokine profile in loops obtained from either nonimmune mice or Salmonella-immunized mice . CT induced only IL-6 and IL-10 production in ligated loops from nonimmune mice but induced a Th2-like cytokine profile in ligated loops obtained from CT-immunized mice . These results show that CT and S . typhimurium induce very different cytokine profiles in the GT.

Z Ernahrungswiss, 1995 Mar, 34(1), 22 - 6
{Improved microbiological assay of heterocyclic aromatic amines in cooked food}; Wild D; Heating of protein, especially muscle meat and meat extracts, can result in the formation of heterocyclic aromatic amines (HA) which are carcinogenic in animals . They are therefore unwelcome in human food . Here, an improved method for the microbiological assay of HA is reported; it makes use of the high mutagenic potency of HA in the Ames test and of the new Salmonella typhimurium strain YG1024 instead of strain TA98 . The high sensitivity of the new strain is a consequence of its high acetyltransferase activity which results in a more efficient formation of genotoxic HA metabolites . The mutagenic activity of three selected HA in YG1024 is 10-20 times higher than that in TA98 . This method can be used for the analysis of the HA content of food . A study of meat patties revealed significant differences in the mutagenic activity in the center and crust and of home-made and commercial patties; in both, the mutagenic activity was localized in the crust.

Biomaterials, 1995 Mar, 16(4), 337 - 40
Cytotoxicity and mutagenicity of Kevlar: an in vitro evaluation; Wening JV et al.; Toxicity and mutagenicity of Kevlar 49 (PPPT; poly-para-phenylene-terephthalamide) was tested in six strains of Salmonella typhimurium (Ames test; TA97, TA98, TA100, TA102, TA1535, TA1537) with and without an external metabolic activation system (S9), as well as in a mammalian cell mutagenesis assay using V79 Chinese hamster cells . For the Ames test, liquid preincubation, which is considered particularly sensitive, was used . The cells were incubated for 24 h at a temperature of 37 degrees C either directly with Kevlar49 or with ethanol- or chloroform-extracted Kevlar49 . The experiments were performed at least twice . The Ames test with six different Salmonella typhimurium strains featuring either base pair substitution or frameshift mutations revealed no cytotoxic or mutagenic activity of Kevlar49 . In the mammalian cell mutagenesis assay, using 8-azaguanine (AG) as a selective agent, Kevlar49 was also devoid of cytotoxic or mutagenic activity . Both tests have to be regarded as an initial exploratory screening due to the chosen testing conditions and should be supplemented by tests at different temperatures.

Chem Res Toxicol, 1995 Mar, 8(2), 269 - 77
Identification of two N2-deoxyguanosinyl DNA adducts upon nitroreduction of the environmental mutagen 1-nitropyrene; Herreno-Saenz D et al.; 1-Nitropyrene, the most abundant nitro-polycyclic aromatic hydrocarbon in the environment, is a known mammalian and bacterial mutagen and a tumorigen in animals . Early studies on DNA adduct characterization for 1-nitropyrene identified N-(deoxyguanosin-8-yl)-1-aminopyrene as the major product from the modification of calf thymus DNA with N-hydroxy-1-aminopyrene, the activated metabolite from nitroreduction of 1-nitropyrene . In this paper, we report the identification of two N2-deoxyguanosinyl adducts, in addition to N-(deoxyguanosin-8-yl)-1-aminopyrene, formed from the reaction of N-hydroxy-1-aminopyrene, prepared in situ, with calf thymus DNA . These DNA adducts were identified as 6-(deoxyguanosin-N2-yl)-1-aminopyrene and 8-(deoxyguanosin-N2-yl)-1-aminopyrene . The two N2-deoxyguanosinyl adducts were also identified in an ascorbic acid-catalyzed activation of 1-nitrosopyrene and in the mammary gland of female Sprague-Dawley rats administered 1-nitropyrene . The DNA adducts were also formed when 1-nitropyrene was metabolized by xanthine oxidase in the presence of calf thymus DNA, and when 1-nitropyrene was activated by rat liver microsomes and cytosols, as well as from DNA isolated from Salmonella typhimurium suspension cultures incubated with 1-nitropyrene.

Res Vet Sci, 1995 Mar, 58(2), 152 - 7
In vitro studies with lymphocytes from sheep orally inoculated with an aromatic-dependent mutant of Salmonella typhimurium; Brennan FR et al.; It was previously shown that a live aroA-strain of Salmonella typhimurium of ovine origin was a safe and effective vaccine against salmonellosis in sheep . The protective effect was observed in the apparent absence of a detectable, systemic T cell response . In the present study, populations of B and T cells from the peripheral blood of sheep vaccinated with S25/1aroA were separated and their responsiveness in vitro to Salmonella was examined . The purified T cells proliferated very weakly in response to Salmonella in the absence of interferon-gamma and interleukin 2/4 production . However, whole peripheral blood mononuclear cells and purified B cells proliferated strongly in response to Salmonella, and Salmonella-specific IgM antibodies could be detected in cell supernatants . Furthermore, Salmonella-specific IgM-producing cells were detected at low frequency by enzyme linked immunospot techniques . These observations extend the earlier findings that oral vaccination with S25/1aroA primes predominantly antigen-specific B cells in the absence of strong Salmonella-specific T cell responses.

Transgenic Res, 1995 Mar, 4(2), 87 - 104
Expression of bacterial cysteine biosynthesis genes in transgenic mice and sheep: toward a new in vivo amino acid biosynthesis pathway and improved wool growth; Bawden CS et al.; It is possible to improve wool growth through increasing the supply of cysteine available for protein synthesis and cell division in the wool follicle . As mammals can only synthesise cysteine indirectly from methionine via trans-sulphuration, expression of transgenes encoding microbial cysteine biosynthesis enzymes could provide a more efficient pathway to cysteine synthesis in the sheep . If expressed in the rumen epithelium, the abundant sulphide, produced by ruminal microorganisms and normally excreted, could be captured for conversion to cysteine . This paper describes the characterisation of expression of the cysteine biosynthesis genes of Salmonella typhimurium, cysE, cysM and cysK, and linked cysEM, cysME and cysKE genes as transgenes in mice and sheep . The linked transgenes were constructed with each gene driven by a separate promoter, either with the Rous sarcoma virus long terminal repeat (RSVLTR) promoter or the mouse phosphoglycerate kinase-1 (mPgk-1) promoter, and with human growth hormone (hGH) polyadenylation sequences . Transgenesis of mice with the RSVLTR-cysE gene afforded tissue-specific, heritable expression of the gene . Despite high levels of expression in a number of tissues, extremely low levels of expression occurred in the stomach and small intestine . Results of a concurrent sheep transgenesis experiment using the RSVLTR-cysEM and -cysME linked transgenes revealed that the RSVLTR promoter was inadequate for expression in the rumen . Moreover, instability of transgenes containing the RSVLTR sequence was observed . Expression of mPgk-cysME and -cysKE linked transgenes in most tissues of the mice examined, including the stomach and small intestine, suggested this promoter to be a better candidate for expression of these transgenes in the analogous tissues of sheep . However, a subsequent sheep transgenesis experiment indicated that use of the mPgk-1 promoter, active ubiquitously and early in development, may be inappropriate for expression of the cysteine biosynthesis transgenes . In summary, these results indicate that enzymically active bacterial cysteine biosynthesis gene products can be coexpressed in mammalian cells in vivo but that expression of the genes should be spatio-temporally restricted to the adult sheep rumen epithelium.

Mutat Res, 1995 Mar, 346(3), 135 - 44
Mutagenicity of particulates from the laboratory combustion of plastics; Lee H et al.; Carcinogenic polycyclic aromatic hydrocarbons (PAHs) and nitropolycyclic aromatic hydrocarbons (nitro-PAHs) have been identified in airborne particulate organic matter extracts . The pollutant sources were generally contributed by motor vehicles and industrial activity . Massive quantities of urban solid wastes, containing plastic materials such as PVC, PET, PS, and PE, burnt in the open air in local garbage dumps are frequently found in developing countries . In this study, the smog particulates from the combustion of these synthetic polymers were produced in a laboratory combustion chamber . The mutagenicity of acetone extracts from the smog particulates was evaluated with Salmonella typhimurium TA98 and TA100 in the presence and absence of S9 mix . Four samples in TA98 exhibited higher mutagenicity than those in TA100 . The greatest mutagenicity was observed from the extracts of particulates from combustion of PVC followed by that of PS, PET, and PE . To determine the major mutagenic compounds in these samples, mutagens were partially purified through TLC and their mutagenicity was monitored with TA98 . 1-NP and DNPs in the above samples were also determined by HPLC . The amounts of 1-NP and DNPs generally corresponded with their mutagenicity . Higher levels of 1-NP and DNPs were generated from the combustion of PVC, PET, and PS . The combustion of synthetic polymer wastes might be responsible for the presence of high levels of 1-NP and DNPs in Taiwan urban air.

Carcinogenesis, 1995 Mar, 16(3), 643 - 8
Metabolic activation of heterocyclic aromatic amines catalyzed by human arylamine N-acetyltransferase isozymes (NAT1 and NAT2) expressed in Salmonella typhimurium; Wild D et al.; Heterocyclic aromatic amines formed during the cooking of meat and meat-derived products can be activated to reactive metabolites which bind to DNA, induce mutations and cause tumors in animals . A principal route of metabolic activation is N-oxidation to hydroxylamines, and their subsequent activation by acetyltransferase-catalyzed O-acetylation . We have used mutagenicity assays to study O-acetylation of heterocyclic arylhydroxylamines by the two isozymes of human N-acetyltransferase, NAT1 and NAT2, expressed in Salmonella typhimurium . N-Acetylation was also examined, using an HPLC method . In addition, Salmonella strains with endogenous acetyltransferase and lacking this activating activity were used . Hydroxylamines of nine heterocyclic aromatic amines, IQ, isoIQ, MeIQ, MeIQx, NI, PhIP, Glu-P-1, Glu-P-2, and Trp-P-2 were generated in situ by rat liver S9 mix . The strains expressing human NAT1 and lacking acetyltransferase activity showing little or no ability to activate these substrates . The strains expressing human NAT2 and Salmonella acetyltransferase supported to different extents the activation of all the compounds except PhIP and Trp-P-2 . N-Acetylation of IQ, MeIQx and PhIP was slow or not detectable . In conclusion, human NAT2 but not NAT1 can O-acetylate heterocyclic hydroxylamines . NAT2 probably plays a key role in the genotoxic effects of the above heterocyclic amines except for PhIP and Trp-P-2, which have NAT2-independent mutagenic activity.

J Commun Dis, 1995 Mar, 27(1), 10 - 4
Nosocomial outbreak of Salmonella typhimurium infection in a nursery intensive care unit (NICU) and paediatric ward; Mahajan R et al.; A nosocomial outbreak of multidrug resistant Salmonella typhimurium in a Nursery Intensive Care Unit (NICU) and Paediatric ward is reported . Eight (16.6%) out of a total of 48 babies taken ill during this outbreak expired . Clinical manifestations included diarrhoea and fever . The organism was isolated from stool samples/rectal swabs of all the 48 cases and blood of 6 cases . An investigation was undertaken to trace the source of infection . The organism was isolated in pure cultures from three suction machines of NICU . The epidemic was immediately controlled with identification of the source but the question of how the suction machines were infected by this organism remained unsolved.

Int J Food Microbiol, 1995 Mar, 25(1), 1 - 9
Lactic acid decontamination of fresh pork carcasses: a pilot plant study; van Netten P et al.; Lactic acid decontamination (LAD) was carried out in an abattoir on pork carcasses, artificially contaminated with Salmonella typhimurium in faeces suspensions . The surface contamination with S . typhimurium ranged from 1-2 log10 cfu/cm2 . Before cold and hot LAD was undertaken, the inoculum was allowed to adhere to the meat surface for 20 min . Cold LAD consisted of treatment for 60 s with 2% (pH 2.3) or 5% (pH 1.9) lactic acid (LA); for hot LAD the exposure times were 30, 60, 90 and 120 s . The spray nozzle temperatures were 11 degrees C and 55 degrees C, and that of the treated meat surface 16-18 degrees C and 36-38 degrees C, respectively . Treatment with cold 2% and 5% LA for 60 s eliminated S . typhimurium from pork carcasses inoculated with ca . 1 log10 cfu/cm2, but not from those inoculated at ca . 2 log10 cfu/cm2 . However, this could be achieved by hot 2% and 5% LA sprayed for 60-120 s . Also exposures of at least 30 s using these hot LA solutions eliminated S . typhimurium consistently from carcasses inoculated with ca . 1 log10 cfu/cm2 . Rinsing-off contributed only marginally to contamination reduction . Application of 2% or 5% LA for 120 s led to an unacceptable deterioration of the organoleptic qualities of the meat . Addition of nicotinic and ascorbic acid as colour stabilizers to the spraying solutions reduced these changes to just acceptable levels when 2% LA was used.

J Ind Microbiol, 1995 Mar-Apr, 14(3-4), 252 - 8
Interaction of lead nitrate and cadmium chloride with Escherichia coli K-12 and Salmonella typhimurium global regulatory mutants; LaRossa RA et al.; To investigate the interactions of heavy metals with cells, a minimal medium for the growth of enteric bacteria using glycerol-2-phosphate as the sole phosphorus source was developed that avoided precipitation of Pb2+ with inorganic phosphate . Using this medium, spontaneous mutants of Escherichia coli resistant to addition of Pb(NO3)2 were isolated . Thirty-five independent mutants all conferred a low level of resistance . Disk diffusion assays on solid medium were used to survey the response of E . coli and Salmonella typhimurium mutants altered in global regulatory networks to Pb(NO3)2 and CdCl2 . Strains bearing mutations in oxyR and rpoH were the most hypersensitive to these compounds . Based upon the response of strains completely devoid of isozymes needed to inactivate reactive oxygen species, this hypersensitivity to lead and cadmium is attributable to alteration in superoxide dismutase rather than catalase levels . Similar analysis of chaperone-defective mutants suggests that these metals damage proteins in vivo.

Poult Sci, 1995 Mar, 74(3), 586 - 90
Effect of lyophilization in sucrose plus dextran and rehydration in thioglycollate broth on performance of competitive exclusion cultures in broiler chicks; Hollister AG et al.; Cecal bacteria grown under continuous-flow anaerobic conditions were lyophilized in skim milk (SM), Reagent 20 (R-20; sucrose and bovine serum albumin fraction V), or sucrose plus dextran (SDx), rehydrated in drinking water or thioglycollate-beef broth (TGB), and compared with fresh broth culture for control of Salmonella typhimurium enteric colonization in broiler chicks . All groups were challenged on Day 3 with 10(4) cfu of S . typhimurium per chick . Mean Salmonella colony-forming units in cecal contents of groups provided lyophilized cultures rehydrated in TGB were not different from those in groups provided fresh broth culture and were reduced (P < .05) compared with controls at 10 d of age . Likewise, groups treated with the cultured lyophilized in R-20 and SDx and rehydrated in TGB had fewer Salmonella than similar groups treated with culture rehydrated in water . The results of this study indicated that the culture was as effective when lyophilized in SDx as it was when lyophilized in SM or R-20; culture lyophilized in SDx, SM, or R-20 and rehydrated in TGB was more effective than when it was rehydrated in water for the reduction of Salmonella colonization in broiler chicks.

Cancer Res, 1995 Feb 15, 55(4), 799 - 802
Bioactivation of aromatic amines by recombinant human cytochrome P4501A2 expressed in Ames tester strain bacteria: a substitute for activation by mammalian tissue preparations; Josephy PD et al.; The most widely used bioassay in genetic toxicology is the Ames test, which combines a bacterial mutagenicity assay (reversion of Salmonella typhimurium histidine-auxotrophic tester strains) with an exogenous bioactivation system (hepatic postmitochondrial supernatant or "S9") . The enzymatic activities of S9 prepared from the tissues of experimental animals are difficult to control . We show that the requirement for S9 can be obviated by the engineered expression of enzymes of bioactivation within the bacterial cell . With this strategy, reactive metabolites are produced inside the bacterial cell, proximate to the genetic target . Species boundaries can be crossed, and chimeric or mutant enzymes can be studied . We have constructed an Ames tester strain, expressing both aromatic amine N-acetyltransferase and human cytochrome P4501A2, which detects aromatic amine mutagenicity in the absence of S9.

FEMS Microbiol Lett, 1995 Feb 15, 126(2), 171 - 6
The live oral typhoid vaccine Ty21a is a rpoS mutant and is susceptible to various environmental stresses; Robbe-Saule V et al.; The rpoS (katF) gene, which encodes a RNA polymerase sigma factor (sigma s), regulates the virulence of Salmonella typhimurium in mice . In the present study, we show that rpoS mutants can be frequently found among laboratory strains of Salmonella . In addition, a rpoS mutation was identified in the S . typhi live oral vaccine Ty21a . Introduction of a wild-type rpoS gene in Ty21a allowed the bacteria to survive better under starvation conditions and increased their resistance to other stresses . These results contribute to a better understanding of the genetic background of the live typhoid oral vaccine Ty21a and suggest that the rpoS mutation may contribute to the safety of this strain in humans.

Proc Natl Acad Sci U S A, 1995 Feb 14, 92(4), 1018 - 22
Rearrangements in the genome of the bacterium Salmonella typhi; Liu SL et al.; We have determined the genomic map of the bacterium Salmonella typhi Ty2, the causal organism of typhoid fever, by using pulsed-field gel electrophoresis . Digestion of the Ty2 genome with endonucleases Xba I, Bln I, and Ceu I yielded 33, 26, and 7 fragments, respectively, that were placed in order on a circular chromosome of 4780 kb . Transposon Tn10 was inserted in specific genes of Salmonella typhimurium and transduced into S . typhi, and thus, the positions of 37 S . typhi genes were located through the Xba I and Bln I sites of the Tn10 . Gene order on chromosomes of Escherichia coli K-12 and S . typhimurium LT2 is remarkably conserved; however, the gene order in S . typhi Ty2 is different, suggesting it has undergone major genomic rearrangements during its evolution . These rearrangements include inversions and transpositions in the 7 DNA fragments between the seven rrn operons for rRNA (postulated to be due to homologous recombination in these rrn genes), another inversion that covers the replication terminus region (resembling inversions found in other enteric bacteria), and at least three insertions, one as large as 118 kb . Partial digestion of genomic DNA with the intron-encoded endonuclease I-Ceu I, which cuts only in rrn genes, shows chromosomal rearrangements, apparently due to homologous recombination in the rrn genes, that were detected in all wild-type strains of S . typhi tested . These rearrangements may have been selected to compensate for the insertions that otherwise would have altered the locations of genes with respect to the origin and terminus of replication . These observations are relevant to our view of the evolution of the bacterial genome and may be significant in the virulence of S . typhi.

Gene, 1995 Feb 3, 153(1), 63 - 5
A versatile vector for controlled expression of genes in Escherichia coli and Salmonella typhimurium; Velterop JS et al.; We have constructed two expression vectors based on the pJF118HE vector developed for Escherichia coli by Furste et al . {Gene 48 (1986) 119-131} . The tac promoter (ptac) was exchanged for the trc promoter (ptrc) and an NdeI site was created at the appropriate distance from the ribosome-binding site . The NdeI site permits cloning of a gene at its translation start point without altering the amino-acid sequence of the synthesized protein, while ptrc and the lacIQ gene confer inducible and controlable expression . We have tested these plasmids in E . coli and Salmonella typhimurium.

Mutat Res, 1995 Feb, 346(2), 99 - 105
Mutagenic activity of 6-aminoquinoxalines in Salmonella typhimurium; Terao Y et al.; Mutagenicity of 6-aminoquinoxaline derivatives was tested with Salmonella typhimurium strains TA98 and TA100 in the presence and absence of S9 mix from the viewpoint that the 6-aminoquinoxaline skeleton is a common unit of mutagenic imidazoquinoxalines . We tested nine compounds: 5-methyl-6-methylaminoquinoxaline (1), 3,5-dimethyl-6-methylaminoquinoxaline (2), 2,5-dimethyl-6-methylaminoquinoxaline (3), 6-methylamino-2,3,5-trimethylquinoxaline (4), 2,3-diethyl-5-methyl-6-methylaminoquinoxaline (5), 5-methyl-6-methylamino 3-phenylquinoxaline (6), 6-amino-2,3,5-trimethylquinoxaline (7), 6-dimethylamino-2,3,5- trimethylaminoquinoxaline (8), 6-amino-2,3-dimethylquinoxaline (9) . These compounds showed the mutagenic activity for both TA98 and TA100 in the presence of S9 mix, where they were more sensitive for TA100 strain . Methyl groups at the 2, 3 and/or 5 positions increased the potency of mutagenicity (1 < 2 < 3 << 4, 9 < 7) . However, ethyl groups at the 2 and 3 positions lowered the mutagenicity of the methyl substitute but elevated it of the parental compound (1 < 5 < 4) . A methyl group at the N6 position decreased the mutagenicity (7 > 4 > 8).

J Bacteriol, 1995 Feb, 177(4), 981 - 6
The ars operon of Escherichia coli confers arsenical and antimonial resistance; Carlin A et al.; The chromosomally encoded arsenical resistance (ars) operon subcloned into a multicopy plasmid was found to confer a moderate level of resistance to arsenite and antimonite in Escherichia coli . When the operon was deleted from the chromosome, the cells exhibited hypersensitivity to arsenite, antimonite, and arsenate . Expression of the ars genes was inducible by arsenite . By Southern hybridization, the operon was found in all strains of E . coli examined but not in Salmonella typhimurium, Pseudomonas aeruginosa, or Bacillus subtilis.

J Bacteriol, 1995 Feb, 177(4), 921 - 5
Purification and initial characterization of the ATP:corrinoid adenosyltransferase encoded by the cobA gene of Salmonella typhimurium; Suh S et al.; The cobA gene of Salmonella typhimurium and its product were overexpressed to approximately 20% of the total cell protein . CobA was purified to 98% homogeneity; N-terminal sequence analysis (21 residues) of homogeneous protein confirmed the predicted amino acid sequence . ATP:corrinoid adenosyltransferase activity was demonstrated in vitro to be associated with CobA . This activity was optimal at pH 8 and 37 degrees C . A quantitative preference was determined for Mn(II) cations and ATP . The apparent Km of CobA for ATP was 2.8 microM, and that for cob(I)alamin was 5.2 microM . Vmax was measured at 0.43 nmol/min . Cobinamide served as the substrate for CobA to yield adenosylcobinamide . Activity was stable at 4 degrees C for several weeks but was lost rapidly at room temperature (50% overnight) . Dithiothreitol was required to maintain the enzymatic activity of CobA.

J Bacteriol, 1995 Feb, 177(4), 1090 - 3
Flagellar filament structure and cell motility of Salmonella typhimurium mutants lacking part of the outer domain of flagellin; Yoshioka K et al.; We have isolated spontaneous mutants of Salmonella typhimurium which can swim in the presence of antifilament antibodies . The molecular masses of flagellins isolated from these mutants were smaller than that (52 kDa) of wild-type flagellin . Two mutants which produced the smallest flagellins (42 and 41 kDa) were selected, and the domain structures of the flagellins were analyzed by trypsin digestion and then subjected to amino acid sequencing . The two flagellins have deletions at Ala-204 to Lys-292 and Thr-183 to Lys-279, respectively . These deleted parts belong to the outer domain (D3) of flagellin, which is believed to be at the surface of the filament . These mutant filaments aggregated side by side in the presence of salt, resulting in disordered motility.

Infect Immun, 1995 Feb, 63(2), 729 - 32
Functional conservation among members of the Salmonella typhimurium InvA family of proteins; Ginocchio CC et al.; InvA, which is essential for Salmonella spp . to enter cultured epithelial cells, is a member of a family of proteins involved in either flagellar biosynthesis or the secretion of virulence determinants by a number of plant and mammalian pathogens . The predicted overall secondary structures of these proteins show significant similarities and indicate a modular construction with a hydrophobic amino-terminal half, consisting of six to eight potential transmembrane domains, and a hydrophilic carboxy terminus which is predicted to reside in the cytoplasm . These proteins can be aligned over the entire length of their polypeptide sequences, with the highest degree of homology found in the amino terminus and clusters of conserved residues in the carboxy terminus . We examined the functional conservation among members of this protein family by assessing the ability of MxiA of Shigella flexneri and LcrD of Yersinia pseudotuberculosis to restore invasiveness to an invA mutant of Salmonella typhimurium . We found that MxiA was able to complement the entry defect of the invA mutant strain of S . typhimurium . In contrast, LcrD failed to complement the same strain . However, a plasmid carrying a gene encoding a chimeric protein consisting of the amino terminus of LcrD and the carboxy terminus of InvA complemented the defect of the Salmonella invA mutant . These results indicate that the secretory systems in which these proteins participate are functionally similar and that the Salmonella and Shigella systems are very closely related . These data also suggest that determinants of specificity may be located at the carboxy termini of these proteins.

Infect Immun, 1995 Feb, 63(2), 563 - 8
Immunization with live recombinant Salmonella typhimurium aroA producing F1 antigen protects against plague; Oyston PC et al.; An attenuated Salmonella typhimurium strain which expressed the F1 capsular antigen of Yersinia pestis was constructed by transformation of S . typhimurium SL3261 with plasmid pFGAL2a, a derivative of pUC18 which contained the caf1 gene without the leader sequence . The recombinant was used to vaccinate mice intragastrically and intravenously . The immunity induced was able to protect mice against challenge with a virulent strain of plague . Protection correlated with the induction of high titers of immunoglobulin G in serum samples and a specific T-cell response.

Cent Eur J Public Health, 1995 Feb, 3(1), 21 - 4
Salmonellae in gulls and other free-living birds in the Czech Republic; Hubalek Z et al.; Cloacal swabs, collected from 756 wild synanthropic and exoanthropic birds of 57 species in the Czech Republic, yielded 32 strains of Salmonella typhimurium {phage types (PT) 141, 104 and 41}, six isolates of S . enteritidis (PT 8, 4 and 6e), and one each of S . panama and S . anatum . Except for one S . enteritidis isolate from a grey-lag goose (Anser anser) and one S . typhimurium isolate from a coot (Fulica atra), all of the other strains were derived from black-headed gulls (Larus ridibundus), of which 24.7% were found to be infected . The black-headed gull might play a role in the dispersal of pathogenic salmonellae.

Mol Microbiol, 1995 Feb, 15(4), 749 - 59
A 40 kb chromosomal fragment encoding Salmonella typhimurium invasion genes is absent from the corresponding region of the Escherichia coli K-12 chromosome; Mills DM et al.; Many Salmonella typhimurium genes are required for bacterial entry into host cells . P22 transduction analysis has localized several invasion loci near minute 59 on the S . typhimurium chromosome . To further characterize the 59-60 min chromosomal region, we determined the physical and genetic map of 85 kb of S . typhimurium DNA between srl and cysC . It was previously shown that some of the invasion genes from this region are not present in Escherichia coli K-12 . We examined whether other S . typhimurium genes on the 85 kb of DNA were similarly absent from E . coli . We found that a contiguous 40 kb fragment of the S . typhimurium chromosome which encodes invasion genes is absent from the corresponding region of the E . coli K-12 chromosome and may represent a 'pathogenicity island' . We speculate that acquisition of the 40 kb region must have significantly advanced the evolution of Salmonella as a pathogen.

Mol Microbiol, 1995 Feb, 15(3), 507 - 17
Overexpression of the Shigella flexneri genes coding for DNA topoisomerase IV compensates for loss of DNA topoisomerase I: effect on virulence gene expression; McNairn E et al.; Introducing the Escherichia coli topA20::Tn10 allele to Shigella flexneri results in osmotic sensitivity, a reduced growth rate, an increase in reporter plasmid supercoiling (all common to the E . coli mutants), an inability to grow on MacConkey agar and a loss of virulence gene expression . E . coli mutants harbouring this topA allele often compensate for the loss of DNA topoisomerase I by amplifying the genes coding for topoisomerase IV . Unlike the E . coli topA mutants, derivatives of S . flexneri harbouring this topA allele did not appear to acquire any compensatory mutations . We investigated the possibility that this was due in part to an inability of the S . flexneri topoisomerase IV genes to compensate for loss of DNA topoisomerase I when overexpressed . The S . flexneri genes encoding the alpha- and beta subunits of topoisomerase IV were detected and cloned in separate multicopy plasmids . These plasmids complemented well-characterized Salmonella typhimurium temperature-sensitive topoisomerase IV mutations, showing that the S . flexneri and S . typhimurium proteins are capable of combining to form active complexes . When the S . flexneri topoisomerase IV genes were cloned in the same multicopy plasmid and introduced into a S . flexneri topA mutant, the plasmid restored osmotic tolerance, improved the growth rate, allowed growth on MacConkey indicator plates and resulted in a relaxation of reporter plasmid supercoiling . The same plasmid also partially restored S . flexneri virulence gene transcription . These data show that overexpression of the S . flexneri topoisomerase IV genes can compensate for the loss of topoisomerase I in terms of general viability of the cell, DNA supercoiling, and (partially) virulence gene expression . The fact that S . flexneri does not exploit topoisomerase IV gene amplification as E . coli does points to a significant difference in the abilities of these species to adapt to the loss of topoisomerase I.

Mol Microbiol, 1995 Feb, 15(3), 483 - 94
Genetic suppression and phenotypic masking of a Myxococcus xanthus frzF- defect; Kashefi K et al.; An insertion of transposon Tn5-lac, omega 4519, generates a lacZ fusion with a Myxococcus xanthus promoter expressed during both vegetative growth and development . Sequence analysis of the junction of omega 4519 with M . xanthus DNA shows that the insertion is in frzF, a homologue of cheR from Salmonella typhimurium . When frzF- (or frzCD-) cells are starved for nutrients at modest densities, they aggregate to form a radial pattern and produce fewer than 1% of the wild-type complement of spores . At higher densities, frzF::omega 4519 cells form 'frizzy' aggregates and produce 80-90% of the wild-type complement of spores . In contrast, when cells with both a frzF- (or frzCD-) and an sglA1 mutation are allowed to develop at either low or high cell densities, they produce frizzy aggregates containing a near wild-type complement of heat-resistant spores . In addition to suppressing the density dependence of fruiting-body morphogenesis, the sglA1 mutation also suppresses the sporulation defect caused by two different frzF- mutations and a frzCD- mutation . In contrast, a mutation in a different S motility gene, sglG1, does not suppress the frz- mutations . Thus, the suppression of frz- mutations by sgl- mutations is allele-specific, and depends on the sgl allele, but not the frz allele . Because the phenotypes of frz- mutations have been determined in a (suppressing) sglA1 genetic background, the frz genes may play more central roles in development than initially recognized.

FEMS Immunol Med Microbiol, 1995 Feb, 10(3-4), 227 - 34
Immunosuppression induced by Salmonella infection is correlated with augmentation of interleukin-2 receptor alpha chain expression in murine splenic lymphocytes; Matsui K et al.; In a previous study, we observed that the suppression of T-cell proliferation induced by Salmonella cell-free extract was associated with augmentation of IL-2 receptor (IL-2R) alpha chain expression . In this study, we also observed this kind of augmentation of IL-2R alpha in Salmonella-infected mice . Phytohaemagglutinin (PHA)-stimulated proliferation of murine spleen cells was significantly suppressed when the mice were infected with Salmonella typhimurium . However, expression of the alpha chain but not the beta chain of IL-2R in lymphocytes was augmented by the infection . Analysis of the IL-2R-positive cell-population showed that the augmentation of IL-2R alpha was not specific to certain cell subpopulations . Furthermore, the inhibition of PHA-stimulated murine spleen cell proliferation and the augmentation of IL-2R alpha expression induced by the infection in lymphocytes was completely reversed by treatment with anti-interferon-gamma monoclonal antibody (anti-IFN-gamma Ab) . These results suggest that the suppression of T-cell proliferation induced by Salmonella infection was associated with augmentation of IL-2R alpha expression in an IFN-gamma production-dependent manner in the same way as the suppression of T-cell proliferation induced by Salmonella cell-free extract.

Mol Biochem Parasitol, 1995 Feb, 69(2), 139 - 48
Partial protection against malaria by immunization with Leishmania enriettii expressing the Plasmodium yoelii circumsporozoite protein; Wang HH et al.; Since infection with Leishmania species induces CD4+ and CD8+ anti-leishmania T cells, we assessed protection against malaria by immunization with Leishmania enriettii transfected with the gene encoding the Plasmodium yoelii circumsporozoite protein (PyCSP) . The recombinant plasmid appeared to be a circular episome in the host cells . Reverse transcription PCR showed that the PyCSP was trans-spliced by the addition of the 39-bp spliced leader of L . enriettii at its 5' end . The transfectant expressed a protein in a pattern similar to that found in the sporozoite itself . Immunofluorescence and immunoelectron microscopy indicated that PyCSP was abundantly expressed on the surface of the parasite . Mice immunized with the transfectant produced antibodies to sporozoites, had a delay in onset of parasitemia after challenge, and 4 of 22 (18%) were completely protected . The protected mice had cytotoxic T lymphocytes against the PyCSP . Immunization with recombinant vaccinia, Salmonella typhimurium, and pseudorabies virus expressing the PyCSP induces excellent immune responses, but has not been shown to protect against challenge . Thus, the modest protection found in these initial studies represents a step forward . After further work Leishmania may prove to be an important live vector vaccine system for induction of protective immune responses.

Protein Expr Purif, 1995 Feb, 6(1), 10 - 4
Functional purification of a bacterial ATP-binding cassette transporter protein (MalK) from the cytoplasmic fraction of an overproducing strain; Schneider E et al.; The malK gene of Salmonella typhimurium encoding the ATP-hydrolyzing subunit of the ATP-Binding Cassette (ABC) transporter for maltose was subcloned into the pRSET5d expression vector . Subsequently, the resulting plasmid (pES67) was introduced into Escherichia coli strain BL21(DE3)/pLysS . When strain BL21-(DE3)/pLysS/pES67 was grown at 30 degrees C in a tryptone-phosphate medium (J.T . Moore, A . Uppal, F . Maley, and G . F . Maley, Protein Expression Purif . 4, 160-163, 1993), the addition of isopropyl beta-thiogalactoside resulted in the synthesis of large amounts of MalK protein . After cell disrupture about 60% of MalK was recovered with the soluble (cytoplasmic) fraction . The protein was purified by ion exchange chromatography and dye ligand affinity chromatography . The purified MalK protein displayed enzymatic properties similar to those of a preparation purified and renatured from inclusion bodies (S . Morbach, S . Tebbe, and E . Schneider, J . Biol . Chem . 268, 18617-18621, 1993) . Thus, our results disprove the view that the biochemical properties of a protein renatured from inclusion bodies might be artefactual . In addition, we provide further evidence that the modification of growth conditions and the use of a T7 expression system can be a useful approach to overcome at least in part the formation of inclusion bodies.

Vet Immunol Immunopathol, 1995 Feb, 44(3-4), 369 - 76
Proliferative responses of splenocytes from wild and domestic northern bobwhites (Colinus virginianus) to T- and B-cell mitogens; Dabbert CB et al.; Baseline information on the functional responses expected for assays used to assess immunocompetence in the Northern bobwhite (Colinus virginianus) are largely unavailable . Our primary objective was to develop an in vitro lymphoproliferative response assay for assessing cell-mediated immunocompetence in the Northern bobwhite . Culture conditions were optimised for domestic Northern bobwhites and field tested on splenocytes from wild-caught quail . Results indicated that increasing cell concentration and media volume in culture, as well as decreasing concentrations of serum in media, improved splenocyte responses to Con A stimulation . Optimum culture conditions were attained with 1 million cells per well cultured in 200 microliters of AIM-V serum-free media for 72 h . Five micrograms concanavalin A (Con A) or 2.5 micrograms Salmonella typhimurium mitogen (STM) per well provided maximum stimulation as measured by 3H-thymidine incorporation . Stimulation indices of splenocyte cultures of wild-caught Northern bobwhites to 5 micrograms Con A were approximately four-fold greater than levels observed for domestic quail (P = 0.0055) . Alternatively, stimulation indices of splenocyte cultures obtained from wild-caught and domestic Northern bobwhites to 2.5 micrograms STM per well were not different (P = 0.3938).

Biol Pharm Bull, 1995 Feb, 18(2), 363 - 7
Mutagenicity of condensed pyridazines with different substituents; Morita T et al.; A total of 24 compounds were prepared by introducing an N-oxide, a hydrazino group, a methoxy group or a chloro group into 3 kinds of condensed pyridazines: pyrido{3,4-d}pyridazines, pyrido{2,3-d}pyridazines and phthalazines . The mutagenicity of these 24 compounds was assessed by the Ames method using two tester strains (Salmonella typhimurium TA98 and TA100) . No mutagenic activity was detected with any of the 3 condensed pyridazines without substituents or any of the 5 condensed pyridazines with a methoxy group . The compounds with N-oxide in the pyridazine ring showed no or only very weak mutagenicity . However, when an oxide was introduced into the nitrogen of the pyridine ring, the mutagenicity against strain TA98 was higher than that of any other test compound . All compounds with a hydrazino group were mutagenic against strains TA98 and TA100, irrespective of the presence or absence of S9 mix-induced metabolic activation . 1-Hydrazinophthalazine (hydralazine) which has been clinically used as an antihypertensive agent was weakly mutagenic . The introduction of a chloro group increased the bactericidal effects of the condensed pyridazines, thus hampering the assessment of mutagenicity . A majority of the compounds which were found to be mutagenic in this study required no metabolic activation with S9 mix.

Genetika, 1995 Feb, 31(2), 268 - 72
{Interaction between mutagenic activity and chemical structure in a series of biphenyl derivatives}; Liubimova IK et al.; A comparative study of the mutagenic activity of 21 derivatives of biphenyl was performed in the strain TA1538 of Salmonella typhimurium . The position effect of carboxyl, amide, aldehyde, and ether groups was examined . The para position of substituents, among which at least one is a nitro group, causes mutagenic activity in most of the molecules studied . Derivatives of biphenyl that have no substituents in the para position were inactive . In addition, 4-nitrobiphenyl (4-NBP), 4-nitro-2'-carboxy biphenyl (4-N-2'-C-BP), 4,4'-dinitro-2'-carboxy biphenyl (4-4'-DN-2'-C-BP), and 4,4'-dinitro-2,2'-carboxy biphenyl (4-4'-DN-2,2'-DC-BP) induced no frameshift mutations in TA1538 . The most active was 2,4,4'-TN-6-C-6'-Ad-BP, giving up to 800 revertants per nmol; 2,4,6,2'-TN-4'-6'-DC-BP and 2,4,2',4'-TN-2'-C-BP, which induced 250 and 350 revertants per nmol, respectively, were highly active frameshift mutagens.

Arzneimittelforschung, 1995 Feb, 45(2), 198 - 9
Efficacy of new organic ammonium salts on Pseudomonas aeruginosa Salmonella typhimurium; Majtan V et al.; The inhibitory activities of four new homologous series of organic ammonium salts (OAS) were tested on bacterial strains isolated from patients . Two types of compounds are used: "hard" (group A) and three groups (B, C, D) of biodegradable "soft" OAS with metabolically labile CO or NH groups in their molecules . The strain Pseudomonas aeruginosa was isolated from the sputum of a patient with carcinoma . The strain Salmonella typhimurium was isolated from a patient with clinical diagnosis of diarrhea . In all homologous series, the antibacterial activity was increasing continuously with the length of alkyl chain up to dodecyl or tetradecyl, then the "cut off" effect was observed . The most active compounds from both "hard" and "soft" types had superior activity to commercial disinfectants . The strain of Pseudomonas aeruginosa was more sensitive to these compounds than that of Salmonella typhimurium.

Arch Environ Contam Toxicol, 1995 Feb, 28(2), 248 - 58
Mutagenicity and antimutagenicity testing of six chemicals associated with the pungent properties of specific spices as revealed by the Ames Salmonella/microsomal assay; Azizan A et al.; Three compounds, capsaicin, thymol and borneol, were initially screened for mutagenic activity using Salmonella typhimurium strains TA97, TA98 and TA100, with and without S9 metabolic activation, and 20 min standard preincubation time . Three other compounds, allyl isothiocyanate, eugenol and cinnamaldehyde, were screened for mutagenic activity as above, but with a prolonged, nonstandard preincubation time of up to 120 minutes . All six test compounds used in the assays are associated with the pungent properties of some specific spices in which the test compounds can be found to exist naturally . The first objective of this study was to observe if mutagenic activity can be correlated to the pungent properties of these six test compounds . However, due to toxicity and the observation that only capsaicin was mutagenic, using strain TA100 in the presence of S9 metabolic activation, it was not possible to deduce any relationship between mutagenicity and the test chemicals' pungent properties . Naturally occurring capsaicin, found in the spice Capsicum annum, was detected and quantified using thin layer and gas chromatographic techniques . The final objective was to detect the presence of antimutagenic factor(s) in C . annum that would suppress the mutagenicity of capsaicin . When the mutagenic capsaicin and 2-aminoanthracene were assayed in the presence of C . annum acetone extract, using strain TA100 with S9 metabolic activation, the mutagenic response of both the mutagens were reduced by approximately 50% . Assaying capsaicin and 2-aminoanthracene in the presence of chlorophyll, the mutagenic response of the two mutagens was reduced by less than 40% . From this observation it was inferred that chlorophyll can successfully suppress the mutagenicity activities of capsaicin and 2-aminoanthracene, together with other antimutagenic factors that were present in the acetone extract of C . annum.

Adv Dent Res, 1995 Feb, 9(1), 31 - 6
Interactions between Salmonella typhimurium, enteropathogenic Escherichia coli (EPEC), and host epithelial cells; Finlay BB; The interactions that occur between pathogenic micro-organisms and their host cells are complex and intimate . We have used two enteric pathogens, Salmonella typhimurium and enteropathogenic Escherichia coli (EPEC), to examine the interactions that occur between these organisms and epithelial cells . Although these are enteric pathogens, the knowledge and techniques developed from these systems may be applied to the study of dental pathogens . Both S . typhimurium and EPEC disrupt epithelial monolayer integrity, although by different mechanisms . Both pathogens cause loss of microvilli and re-arrangement of the underlying host cytoskeleton . Despite these similarities, both organisms send different signals into the host cell . EPEC signal transduction involves generation of intracellular calcium and inositol phosphate fluxes, and activation of host tyrosine kinases that results in tyrosine phosphorylation of a 90-kDa host protein . Bacterial mutants have been identified that are deficient in signaling to the host . We propose a sequence of events that occur when EPEC interacts with epithelial cells . Once inside a host cell, S . typhimurium remains within a vacuole . To define some of the parameters of the intracellular environment, we constructed genetic fusions of known genes with lacZ, and used these fusions as reporter probes of the intracellular vacuolar environment . We have also begun to examine the bacterial and host cell factors necessary for S . typhimurium to multiply within epithelial cells . We found that this organism triggers the formation of novel tubular lysosomes, and these structures are linked with intracellular replication.

Eur J Immunol, 1995 Feb, 25(2), 405 - 10
Intracellular processing and presentation of T cell epitopes, expressed by recombinant Escherichia coli and Salmonella typhimurium, to human T cells; Verjans GM et al.; Vaccines based on recombinant attenuated bacteria represent a potentially safe and effective immunization strategy . A carrier system was developed to analyze in vitro whether foreign T cell epitopes, inserted in the outer membrane protein PhoE of Escherichia coli and expressed by recombinant bacteria, are efficiently processed and presented via human leukocyte antigen (HLA) class I and II molecules by bacterial infected human macrophages . A well-defined HLA-B27-restricted cytotoxic T cell (CTL) epitope and an HLA-DR53 restricted T helper (Th) epitope of the fusion protein of measles virus were genetically inserted in a surface-exposed region of PhoE, and the chimeric proteins were expressed in E . coli and Salmonella typhimurium . Macrophages infected with both recombinant bacteria presented the Th epitope to the specific CD4+ T cell clone, but failed to present the CTL epitope to the specific CD8+ T cell clone . Presentation of the Th epitope by the infected macrophages was inhibited by cytochalasin D, indicating that phagocytic processing of intact bacteria within infected macrophages was essential for antigen presentation via HLA class II . Presentation of the Th epitope to the CD4+ T cell clone by infected macrophages was blocked by brefeldin A and cycloheximide, indicating the requirement of nascent HLA class II molecules for presentation . The efficiency of macrophages to process and present the inserted Th epitope was similar for both the recombinant E . coli and S . typhimurium strains.

Mutat Res, 1995 Feb, 341(4), 303 - 18
Modifying actions of solvent extracts from fruit and vegetable residues on 2-amino-3-methylimidazo{4,5-f}quinoline (IQ) and 2-amino-3,4-dimethylimidazo{4,5-f}quinoxaline (MeIQx) induced mutagenesis in Salmonella typhimurium TA 98; Edenharder R et al.; The edible parts of 13 fruits--apples, apricots, bananas, blackberries, sweet cherries, red currants, white grapes, oranges, peaches, pears, plums, raspberries, and strawberries--and of 12 vegetables--asparagus, green beans, broccoli, brussels sprouts, red and white cabbage, carrots, cauliflower, onions, green peppers, spinach, and tomatoes--were squeezed in order to separate juices and residues . The residues were washed, lyophilized, and extracted sequentially with n-hexane, dichloromethane, acetone, and 2-propanol . Solvent extracted materials were tested in Salmonella typhymurium TA 98 for antimutagenicity against IQ and MeIQx . We found antimutagenic activities in 96% of the n-hexane extracts, 64% of the dichloromethane extracts, 44% of the acetone extracts, and 36% of the 2-propanol extracts . Since no or only minor differences were seen between the mutagens IQ and MeIQx investigations were continued with IQ only . Additional antimutagenic activities were detected in a total of 29.6% of extracts tested when an enzyme preparation with glycosidase-activities (fecalase) was included in the assay . These activities were found in originally inactive or less active dichloromethane, acetone, and 2-propanol extracts, and are therefore strongly suggestive for the liberation of antimutagenic aglycones from inactive glycosides . The existence of possibly a multitude of antimutagenic factors in fruits and vegetables was further substantiated by: (1) solvent partitioning of the n-hexane extracts of cauliflower, peaches, and spinach; (2) separation of the n-hexane and dichloromethane extracts of cauliflower, peaches, and spinach into acidid, neutral, and basic compounds; (3) chromatographic analysis of the n-hexane and dichloromethane extracts of spinach . Taken together, antimutagenic activities were present in 32 of 36 subfractions, corresponding to 88.9% . In the green vegetables beans, broccoli, and spinach the known antimutagen chlorophyll was proven to contribute considerably to antimutagenic potency . Other important contributions may be caused by various fibers: (I) antimutagenicity of fruit and vegetable solvent extracts was extensively heat stable; (II) heating surprisingly caused an increase of antimutagenic potencies or generated new antimutagenic activities in several solvent fractions, especially of broccoli, white and red cabbage . Indeed, mutagenicity induced by IQ was strongly reduced by lignin, weakly by alginic acid and pectin A, while cellulose, gum arabic, gum guar, and xylan were ineffective . With respect to the mechanisms of antimutagenicity binding of IQ by various fibers and inhibition of cytochrome P-450-dependent monooxygenases might be of major importance since no solvent fraction of any fruit or vegetable was able to reduce mutagenic activity induced by N-OH-IQ in S . typhimurium TA 98NR.

Mutat Res, 1995 Feb, 341(4), 289 - 302
Study of the genotoxic activity of six halogenated acetonitriles, using the SOS chromotest, the Ames-fluctuation test and the newt micronucleus test; Le Curieux F et al.; Three short-term assays (the SOS chromotest, the Ames-fluctuation test and the newt micronucleus test) were carried out to evaluate the genotoxicity of six halogenated acetonitriles identified in chlorinated waters (monochloro-, dichloro-, trichloro-, monobromo-, dibromo- and bromochloroacetonitrile) . With the SOS chromotest, three of the chemicals studied (dichloro-, dibromo- and bromochloroacetonitrile) were found to induce primary DNA damage in Escherichia coli PQ37 . In the Ames-fluctuation test, all the compounds except dibromoacetonitrile showed mutagenic activity on Salmonella typhimurium strain TA100 . The newt micronucleus assay detected a clastogenic effect on the peripheral blood erythrocytes of Pleurodeles waltl larvae for all the six haloacetonitriles studied . Moreover, two structure-activity relationships were noted: (1) the genotoxic activity of haloacetonitriles containing bromine substituents appeared higher than the corresponding chlorinated acetonitriles and (2) the clastogenic activity of the chlorinated acetonitriles increased with the number of chlorine substituents.

Mutat Res, 1995 Feb, 341(4), 281 - 7
Mutagenicity of ethylene glycol ethers and of their metabolites in Salmonella typhimurium his-; Hoflack JC et al.; Ethylene glycol ethers, their aldehyde and their acid metabolites were evaluated for their mutagenicity with the Ames test . The Salmonella typhimurium his- tester strains TA 97a, TA 98, TA 100 and TA 102 were used with and without rat S9 mix . Ethylene glycol monomethyl ether, ethylene glycol monoethyl ether, ethylene glycol n-butyl ether and their corresponding aldehyde and acid derivatives were tested up to 10(-4) mol/plate (around 10 mg/plate) or up to cytotoxic concentrations . All tested substances gave negative results with TA 98, TA 100 and TA 102 either with or without S9 mix . In contrast, ethylene glycol n-butyl ether (EGBE) and the aldehyde metabolite of ethylene glycol monomethyl ether, methoxyacetaldehyde (MALD), displayed mutagenic potency in strain TA 97a with and without S9 mix at high concentrations . A significant number of revertants was obtained from 19 mumol/plate EGBE (2.2 mg/plate) and from 34 mumol/plate MALD (2.5 mg/plate) . At these concentrations the level of revertants reached up to 7-fold and 3-fold the control values for EGBE and MALD respectively.

Mutat Res, 1995 Feb, 326(2), 219 - 25
Screening of tea clones for inhibition of PhIP mutagenicity; Apostolides Z et al.; Standard black and green tea extracts have been known to inhibit mutagenicity caused by PhIP, in the Salmonella typhimurium TA98 assay containing S9 fraction from the liver of rats induced with alpha-naphthoflavone and phenobarbital . Breeding and selection programs for high yielding tea clones have successfully increased yields in many tea producing areas . Six clonal teas and three seedling teas were obtained from a tea producing area in Southern Africa . Standard black and green teas were used as controls . Dose-dependent inhibition of the bacterial mutagenicity elicited by two concentrations of PhIP was found in the extracts of all the teas tested . This indicates that the clonal teas have not lost their anti-mutagenic properties . Small differences were found amongst the clonal teas in their ability to inhibit mutagenicity . This indicates that it may be possible to enhance this trait in future breeding and selection programs.

Infect Immun, 1995 Feb, 63(2), 437 - 41
Acetylation (O-factor 5) affects the structural and immunological properties of Salmonella typhimurium lipopolysaccharide O antigen; Slauch JM et al.; The lipopolysaccharide (LPS) of gram-negative bacteria serves as a barrier between the cell and its environment . The LPS O antigen is the immunodominant portion of the molecule and thus has a significant effect on the interaction between a bacterial pathogen and the host organism . Antibodies directed against O antigen are vital to the immune response to infection . In this study, we have characterized the interaction between a series of monoclonal immunoglobulin A antibodies and the LPS of Salmonella typhimurium . Using one of these antibodies, we have previously shown that monoclonal immunoglobulin A is sufficient to protect against S . typhimurium infection, both in vivo and in vitro . Here, we show that recognition of LPS by the monoclonal antibodies is affected by acetylation of the O antigen on the abequose moiety, the determinant of the O5 epitope . Although recognition of LPS by several of the monoclonal antibodies is completely dependent on acetylation, the antibodies recognize clearly separable epitopes . This suggests that acetylation of O antigen affects the three-dimensional structure of the molecule and thus creates and destroys a series of conformational antigenic determinants . We have shown that a change in the acetylation state of LPS has no effect on virulence . However, acetylation has important consequences for the mucosal immune response and thus could potentially have profound implications for the ability of an immune host to respond to a subsequent infection.

Mutat Res, 1995 Feb, 334(1), 103 - 13
Predicting chemical carcinogenesis in rodents: the state of the art in light of a comparative exercise; Benigni R; Within a recent comparative exercise, different approaches to the prediction of rodent carcinogenicity were challenged on a common set of chemicals bioassayed by the U.S . National Toxicology Program . The approaches were of very different natures . Some prediction systems looked for relationships between carcinogenicity and other, more quickly detectable biological events (activity-activity relationships, AAR) . Some approaches tended to find structure-activity relationships (SAR) . To give an objective evaluation of the results of the exercise, we have analyzed the rodent results and the predictions with the multivariate data analysis methods . The calculated performances varied according to the adopted carcinogenicity classification of the chemicals . When the four rodent results were summarized into a final + or - call, the Tennant approach (AAR method) showed the best performance (about 75% accuracy), whereas the best SAR systems had 60-65% accuracy . A common limitation of almost all the systems was the lack of specificity (too many false positives) . Based on these results, better concordance was obtained when the input information was the very costly (and closer to the final endpoint) biological data, rather than the inexpensive (and farther from the endpoint) knowledge of the chemical structure . However, when the rodent results were summarized into a carcinogenicity classification that maintained, to some extent, the gradation intrinsic to the original experimental data, the performance of the AAR systems declined, and the SAR approaches showed a better performance . The difficulty in evaluating the various approaches was further complicated because of a fundamental difference in the approaches themselves: some approaches were 'pure' prediction methods (i.e . their predictions were rigorously based on information not inclusive of carcinogenicity); other approaches (e.g . Tennant, Weisburger) used 'mixed' information, inclusive of known carcinogenicity results from experiments performed before the NTP bioassays . As far as the SAR systems are concerned, their sets of predictions showed a fundamental similarity . This happened in spite of the extremely different procedures adopted to treat the chemical formula (initial information): very simple calculations (Benigni), intuition of the experts (Weisburger and Lijinsky), sophisticated computer programs (TOPKAT and CASE) . The results of the Bakale Ke method, based on the experimental measurement of the chemical electrophilicity, and of the Salmonella typhimurium mutagenicity assay were similar to the patterns of predictions of the SAR methods.

Proc Natl Acad Sci U S A, 1995 Jan 31, 92(3), 669 - 73
Antibiotic-based selection for bacterial genes that are specifically induced during infection of a host; Mahan MJ et al.; We have recently described a genetic system, termed in vivo expression technology (IVET), that uses an animal as a selective medium to identify genes that pathogenic bacteria specifically express when infecting host tissues . Here, the potential utility of the IVET approach has been expanded with the development of a transcriptional-fusion vector, pIVET8, which uses antibiotics resistance as the basis for selection in host tissues . pIVET8 contains promoterless chloramphenicol acetyltransferase (cat) and lacZY genes . A pool of Salmonella typhimurium clones carrying random cat-lac transcriptional fusions, produced with pIVET8, was used to infect BALB/c mice that were subsequently treated with intraperitoneal injections of chloramphenicol . Strains that survived the selection by expressing the cat gene in the animal were then screened for those that had low-level lacZY expression on laboratory medium . These strains carry operon fusions to genes that are specifically induced in vivo (ivi genes) . One of the ivi genes identified (fadB) encodes an enzyme involved in fatty acid oxidation, suggesting that this enzyme might contribute to the metabolism of bactericidal or proinflammatory host fatty acids . The pIVET8-based selection system was also used to identify S . typhimurium genes that are induced in cultured macrophages . The nature of ivi gene products will provide a more complete understanding of the metabolic, physiological, and genetic factors that contribute to the virulence of microbial pathogens.

Virology, 1995 Jan 10, 206(1), 479 - 84
Dual translational start motif evolutionarily conserved in the holin gene of Bacillus subtilis phage phi 29; Tedin K et al.; Holins represent phage encoded lysis functions required for transit of the phage murein hydrolases to the periplasm . The Lambda S, phage 21 S, and P22 13 holin genes contain a dual translational start motif, beginning with Met1-Lys2-X-Met3 . In all cases both start codons at the 5' end of the respective holin gene are utilized . The resulting polypeptides have opposing functions, with the longer product acting as an inhibitor of the shorter one . The 131-codon gene 14 of Bacillus subtilis phage phi 29 encodes the holin function, whereas the downstream gene 15 codes for a lysozyme . phi 29 Gene 14 begins with Met1-Lys2-Met3 . Here, we present in vitro and in vivo evidence for the expression of two protein 14 species consisting of 129 and 131 amino acids, respectively . These data suggest that the lysis control mechanism based on two holin species, which has been shown to be operational in the temperature Escherichia coli phages Lambda and 21, and in the Salmonella typhimurium phage P22, is evolutionarily conserved in the lytic B . subtilis phage phi 29.

Biochemistry, 1995 Jan 10, 34(1), 50 - 64
Structure of a duplex oligodeoxynucleotide containing propanodeoxyguanosine opposite a two-base deletion in the (CpG)3 frame shift hotspot of Salmonella typhimurium hisD3052 determined by 1H NMR and restrained molecular dynamics; Weisenseel JP et al.; Structural refinement from solution 1H NMR data was performed on the 5'-d{ATCGC(PdG)-CGGCATG}-3'.5'-d{CATGCCGCGAT}-3' duplex, in which the adducted oligodeoxynucleotide containing the exocyclic lesion 1,N2-propano-2'-deoxyguanosine (PdG) was annealed with the complementary strand which contained a CpG deletion . The resulting duplex required PdG and one adjacent cytosine to be unpaired . A total of 352 distances were utilized to restrain molecular dynamics calculations, of which 264 were NOE-derived . These distances were calculated using complete relaxation matrix methods from hybrid matrices, which were comprised of the experimentally determined distances and additional distances derived from either A-form or B-form DNA . A simulated annealing protocol combined with the distance restraints was able to refine a single structure with an average rms deviation of < 1.35 A . The accuracy of the refined structure was assessed using full relaxation matrix calculations, which gave good agreement with measured NOE intensities . PdG was found to be stacked into the helix below base pair C3.G18, whereas C5 was found to be unpaired and extruded toward the major groove and parallel to base pair G6.C17 . This created a localized bend in the DNA helix of approximately 20-35 degrees at the junction between PdG and C5 . The bending corroborated previous assays performed on this modified sequence {Moe, J . G., Reddy, G . R., Marnett, L . J., & Stone, M . P . (1994) Chem . Res . Toxicol . 7, 319-328}.

Acta Pol Pharm, 1995 Jan-Feb, 52(1), 31 - 3
Genetic effects of binazine and hydralazine in vitro and in vivo; Chlopkiewicz B et al.; The mutagenic and genotoxic activities of binazine and hydralazine were studied . In the Ames test, both with and without S-9 fraction, hydralazine was mutagenic in strains Salmonella typhimurium TA100 and TA1537, whereas binazine was not mutagenic in these strains . Both drugs were negative in mice micronucleus test.

Int Ophthalmol, 1995-96, 19(5), 303 - 6
Distribution of intercellular adhesion molecule-1 on leukocytes and corneal endothelium after endotoxin stimulation in rats; Yamaguchi K et al.; After stimulation with Salmonella typhimurium endotoxin, the intercellular adhesion molecule-1 (ICAM-1) was studied on the corneal endothelium and associated leukocytes in rats using immunoscanning electron microscopy . Two hundred micrograms of the endotoxin was injected in Lewis rats . The corneae were excised at 0-h and 16-h-postinjection time (n = 5, respectively) . The corneae were prepared in hypothermic University of Wisconsin (UW) solution for immunoscanning electron microscopy . Histotopographical examination visualized ICAM-1 antigen on cytoplasmic processes of the corneal endothelium, arranged along microfolds, especially at the peaks . In the leukocytes, ICAM-1 was located primarily in morphologically non-specialized domains of the cell body surface, and only rarely scattered on the surface of microvillar projections . We concluded that the endotoxin stimulation can increase ICAM-1 in both corneal endothelium and associated leukocytes . Increased ICAM-1 may be an important factor for the leukocytes to form clustering and adhering to the corneal endothelium.

Yi Chuan Xue Bao, 1995, 22(2), 152 - 60
{Regulation of purine biosynthetic genes expression in Salmonella typhimurium . III . Isolation of O+ and Oc fragments of pur JHD}; Li K et al.; It has been known that 10 enzymatic reactions are involved in de novo biosynthesis of IMP in Salmonella typhimurium . 10 genes encoding the enzymes have been localized on the Salmonella typhimurium chromosome . Seven structure genes including pur JHD operon are negatively regulated by purR . In this study the purJHD operon containing O+ and Oc were cloned in vivo respectively . Two preliminary clones, p2-9 (O+) and pC-12 (Oc) were identified to be the hybrid plasmids carrying intact pur JHD operon by genetic complementary test and reconstruction enzyme analysis . The fragments of O+ and Oc were subcloned on to pUC19 and DNA sequence were determined directly by DNA sequencer . The DNA sequence of O+ fragment revealed that a 16 base consensus sequence (named PUR box) GCGCAAACGTTTTCGT also existed in control region of pur JHD operon in Salmonella typhimurium . The DNA sequence of Oc fragment indicated that only one base change occurred at the 14th position of PUR box(C-->T) . Our result strongly support the idea that PUR box is binding region of purR protein.

Folia Microbiol (Praha), 1995, 40(2), 153 - 8
Subinhibitory concentrations of organic ammonium salts: the effect on biological properties of Salmonella typhimurium; Majtanova L et al.; The effect of subinhibitory concentrations (subMICs) of new organic ammonium salts of four homologous series of alkylammonium bromides (32 compounds) was determined with respect to the induction of lysogenic strain prophage, influence of permeability reactions in a rabbit skin test and cytotoxic changes of monolayers of Vero cells . The culture filtrates were prepared by 1-d cultivation of Salmonella typhimurium in a synthetic culture medium under conditions of intensive aeration at 37 degrees C after addition of subinhibitory concentrations of organic ammonium salts . The results showed that substances of the homologous series of 2-(10-undecenoyloxy)ethyl-alkyldimethylammonium bromides were characterized by a prophage-inducing effect in lysogenic strain cells . The induction of prophage raised with rising concentrations of subMICs of the substances, and its titer in the culture filtrates was mostly 4.10(6) PFU/mL . Substances C3, C9 and C12 of the same homologous series had the strongest effect on the permeability reaction in rabbit skin in 1/2 MICs . One-half MICs of four substances (B14, C3, C12, C14) and 1/4 MICs of substance A16 influenced cytotoxic changes on Vero cells, the other substances were ineffective.

Princess Takamatsu Symp, 1995, 23, 39 - 49
Identification of new mutagenic heterocyclic amines and quantification of known heterocyclic amines; Wakabayashi K et al.; 2-amino-1-methyl-6-(4-hydroxyphenyl)imidazo{4,5-b}pyridine (4'-OH-PhIP) was mutagenic, inducing 180 revertants of Salmonella typhimurium TA98 per 100 micrograms with S9 mix and was formed by heating a mixture of creatine, tyrosine and glucose . It was detected in broiled beef at a level of 21.0 ng per g of broiled beef, which is comparable to the level of 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine (PhIP) . Two new mutagens were isolated from bacteriological-grade beef extract using a new Salmonella tester strain, YG1024, which has a much higher O-acetyltransferase level than TA98 . These mutagens were identified as 2-amino-4-hydroxymethyl-3,8-dimethylimidazo{4,5-g}quinoxaline (4-CH2OH-8-MeIQx) and 2-amino-1,7,9-trimethylimidazo{4,5-g}quinoxaline(7,9-DiMeIgQx++ +) . The amounts of these mutagenic heterocyclic amines (HCAs) in beef extract were 6.0 ng and 53 ng per g of beef extract, respectively . 4-CH2OH-8-MeIQx induced 326,000 revertants of YG1024 and 99,000 revertants of TA98 per microgram with S9 mix, while 7,9-DiMeIgQx induced 13,800 and 670 revertants of YG1024 and TA98, respectively, per microgram in the presence of S9 mix . The levels of nine previously reported HCAs in cooked meats and fish and in beef extract were determined quantitatively . The level of PhIP was highest (0.56 approximately 69.2 ng/g), followed by that of 2-amino-3,8-dimethylimidazo{4,5-f}quinoxaline (MeIQx) (0.64 approximately 6.44 ng/g), and those of other HCAs were 0.03 approximately 2.50 ng/g . Mainstream smoke condensates of five Japanese brands of cigarettes contained four HCAs, 3-amino-1,4-dimethyl-5H-pyrido{4,3-b}indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido{4,3-b}indole (Trp-P-2), 2-amino-9H-pyrido{2,3-b}indole (A alpha C) and 2-amino-3-methyl-9H-pyrido{2,3-b}indole (MeA alpha C), at levels of 0.02 approximately 13.5 ng per cigarette and sidestream smoke condensates of two brands of cigarettes contained these HCAs at levels of 0.14 approximately 2.72 ng per cigarette . PhIP was not detected in any sample of mainstream or sidestream smoke condensate.

Nucleic Acids Symp Ser, 1995, (34), 231 - 2
Mechanism of mutagenesis induced by cytosine analogs bearing N(4)-substitutions; Negishi K et al.; The mechanism of mutagenesis induced by dihydropyrimido {4,5-c}{1,2}oxazin-7-one deoxyriboside, P-nucleoside, was studied . This analog is highly mutagenic toward Escherichia coli and Salmonella typhimurium . In E . coli, it induces GC-to-AT and AT-to-GC transitions specifically . No transversions are inducible . P-nucleoside was highly mutagenic to a wild-type E . coli, but little mutagenic in a strain lacking thymidine kinase . This indicates that P-nucleoside may be phosphorylated by thymidine kinase after its uptake into bacteria . The mutagenesis induced by P-nucleoside was efficiently inhibited by the addition of thymidine . This inhibition further confirmed the involvement of thymidine kinase in the first step of the metabolism of P-nucleoside in the bacterial cells . These findings indicate that P-nucleoside is a mutagen of a nucleoside-analog type, causing mutations by the erroneous incorporation and replication . The experiments to prove its ambiguous nature in DNA synthesis is now under way.

Folia Microbiol (Praha), 1995, 40(5), 457 - 61
Mutagenicity of cytostatic drugs in a bacterial system . I . Ames test; Marhan J; A set of nine cytostatic drugs were tested in the standard plate incorporation method of Ames reversion test using four Salmonella typhimurium his strains . Six of them were classical and commonly used cytostatics, three (cloturin, butocin and oracin) are at different stages of development . The results showed 6-mercaptopurine, cloturin, adriamycin, mitoxantron, cyclophosphamide and lomustine to be mutagenic; butocin, oracin and tris(2-chloroethyl)amine were negative in this test.

Crit Rev Microbiol, 1995, 21(4), 215 - 37
Low pH adaptation and the acid tolerance response of Salmonella typhimurium; Foster JW; Salmonella typhimurium periodically confronts acid environments during its life . These situations arise in chemically compromised ponds, soil, degradative cellular organelles, host digestive systems, and may even result from byproducts of their own metabolism . The levels of acid that are encountered range from mild to extreme . As a neutralophile, S . typhimurium prefers to grown in pH environments above pH 5.5 . They can survive down to pH 4 for extended periods of time . However, the limits of endurance can be stretched if the organisms are first adapted to a moderate acid pH before exposing them to acidity below pH 4.0 . This adaptation, called the acid-tolerance response (ATR), includes several log phase and stationary phase systems . Some of these systems are dependent on an alternate sigma factor for RNA polymerase called sigma s, whereas other systems are sigma s-independent . A key to the ATR is the synthesis of a series of acid shock inducible proteins (ASPs), 51 for log phase ATR and 15 for stationary phase ATR . Some of these ASPs require sigma s for their synthesis; others require the participation of the ferric uptake regulator protein Fur . Effective acid tolerance involves RecA-independent DNA repair systems, iron, and facets of fatty acid metabolism . Aspects of medium composition and carbon metabolism are also known to influence the nature of acid tolerance in this organism . In addition to aiding survival in the natural non-host environment, aspects of acid tolerance are also tied to virulence, as evidenced by the involvement of the mouse virulence locus mviA and the fact that acid-sensitive strains of S . typhimurium exhibit reduced virulence . This review summarizes these aspects of acid adaptation and includes a discussion of acid-regulated gene expression.

Nutr Cancer, 1995, 24(3), 249 - 56
Lack of aberrant crypt promotion and of mutagenicity in extracts of cooked casein, a colon cancer-promoting food; Corpet DE et al.; Dietary casein cooked at 180 degrees C promotes the growth of aberrant crypt foci and colon cancer in rats initiated with azoxymethane . We speculated that promotion was due to a product that could be extracted by a solvent, such as 5-hydroxymethyl-2-furaldehyde (HMF), with tumor- promoting activity or the carcinogenic heterocyclic aromatic amines (HAA) . This hypothesis was tested by extracting cooked casein with solvents and water . The extracts were then 1) assayed by high-performance liquid chromatography for HMF and HAA, 2) measured for mutagenicity on a frame-shift-sensitive strain of Salmonella typhimurium, and 3) fed for 100 days to azoxymethane-initiated rats to test the promoting effect on aberrant crypt foci . Data show that 1) no HMF or HAA was detected in cooked casein, 2) no mutagenicity was detected on strain TA98, with or without metabolic activation, and 3) promotion was not associated with the extracts but with the cooked casein residue . Therefore the promotion by cooked casein would not appear to be associated with a product that can be extracted by solvents.

Pharmacol Ther, 1995, 68(1), 87 - 104
Chemotaxis in Bacillus subtilis: how bacteria monitor environmental signals; Garrity LF et al.; Virtually all organisms have means of monitoring their environment and making use of information gained to aid their survival . Many organisms, from bacteria to animals, move from place to place and can alter their movements . Chemotaxis is a signal transduction system found in motile bacteria that allows them to sense changes in the concentrations of various extracellular compounds and change their swimming behavior in a way that moves them toward more favorable environments . Chemotaxis is the most ancient sensory-motor process in nature . For years, studies of enteric bacteria, such as Escherichia coli and Salmonella typhimurium, have served as the paradigm for understanding this process on a molecular level . Recent studies on the gram-positive bacterium, Bacillus subtilis, and other bacteria, suggest that a slightly more complex system may be ancestral to that of the more extensively studied enterics . Aspects of chemotaxis that are unique to B . subtilis include a more complex adaptation system, with protein-protein methyl group transfer, chemotaxis proteins having no counterparts in E . coli, and a very extensive repertoire of repellents that are sensed at very low concentrations by receptors.

Nutr Cancer, 1995, 24(2), 143 - 50
Dietary caffeine reduces the genotoxicity of MeIQx in the host-mediated assay in mice; Alldrick AJ et al.; The influence of dietary caffeine on the genotoxicity of the cooked food mutagen 2-amino-3,8-dimethylimidazo{4,5-f}-quinoxaline (MeIQx) was evaluated using the host-mediated assay in mice . For four weeks, BALB/c mice were fed a purified diet with or without caffeine (0.01% wt/wt in the diet) . In the host-mediated assay, Salmonella typhimurium TA98 was given intravenously immediately before an oral dose of MeIQx (1.5 mg/kg body wt) . After one hour, the mice were killed, the Salmonellae were recovered from the liver, and the number of mutants (his+ revertants) were determined . Consumption of caffeine led to a 47% reduction in the number of mutants induced by MeIQx (p < 0.001) . Subsequent in vitro experiments using S . typhimurium TA98 revealed that the capacity of hepatic S-9 fractions from the caffeine-fed mice to covert MeIQx to an active mutagen was reduced by approximately 35% . This effect was not attributable to caffeine in the S-9 preparation . These data suggest that consumption of caffeine modifies MeIQx mutagenicity by altering the spectrum of enzymes involved in its activation.

Annu Rev Microbiol, 1995, 49, 489 - 522
How bacteria sense and swim; Blair DF; Cells of Escherichia coli or Salmonella typhimurium can sense chemicals in their environment and respond by moving toward some and away from others . The ability to sense and swim requires the products of approximately 50 genes, about 10 for detecting and processing sensory cues and the rest for assembly and operation of the flagella . The function of each component in the chemosensory signaling pathway is well understood . Signaling is known to involve phosphorylation of a set of cytoplasmic proteins, but questions remain concerning the protein conformational changes and interactions that take place . Functions have been assigned to almost all of the approximately 40 flagellar proteins, and the sequence of events in flagellar assembly has been largely determined . Flagellar assembly depends on a specialized apparatus for exporting certain flagellar components to their appropriate locations . The structure and mechanism of this apparatus remain a mystery, as does the mechanism by which the flagellar motor generates torque.

Annu Rev Microbiol, 1995, 49, 145 - 74
How Salmonella survive against the odds; Foster JW et al.; The enteric pathogen Salmonella typhimurium faces daunting odds during its voyages in the natural environment and through an infected host . It must manage stresses ranging from feast to famine, acid to base, and high to low osmolarity, among others, as well as counter various types of oxidative stress and a variety of antimicrobial peptides . The defenses used to survive these encounters can be specific or can provide cross protection to a variety of hostile conditions . Once inside a host, Salmonella spp . escape the extracellular environment and thus humoral immunity by invading professional and nonprofessional phagocytes in which a new set of challenges await . Some of these stresses are similar to those encountered in the natural environment (e.g . acid, starvation) but the bacterial response is complicated by the simultaneous occurrence of multiple stresses . S . typhimurium appears to sense various in vivo cues and responds by seducing the host signal-transduction pathways that are required to phagocytize the bacterial cell . The pathogen then calls upon components of its stress-response arsenal to survive the intracellular environment . These survival strategies enable the organism to persist in nature, where conditions are usually suboptimal, and equip the bacterium with pathogenic properties that, if successful, will provide it with a very rich and stress-free growth environment, a dead host.

Arch Med Res, 1995 Winter, 26(4), 355 - 60
Salmonella typhimurium infection in mice: lymphoproliferative responses to fractionated protein antigens; Melendro EI et al.; The proliferative response of spleen cells of mice immunized with S . typhimurium by the oral route was analyzed using antigen fractions from a protein extract of the bacteria . Mice that survived the challenge with a virulent strain of Salmonella and normal mice were also studied . Mice were immunized with three doses of live S . typhimurium on consecutive days (3C) or once a week for 3 weeks (3W) . Fractions 12-100 kDa from the protein extract were separated by SDS-polyacrylamide gel electrophoresis, electroblotted to nitrocellulose membranes and processed to obtain particulate antigens for use in lymphocyte cultures . Mice immunized weekly showed a higher survival rate and responded to more antigenic fractions . We identified three fractions of 68-76, 50-52, and 42-45 kDa that were immunodominant for spleen cells from S . typhimurium immunized mice and from survivors to the challenge with the virulent strain.

J Biol Regul Homeost Agents, 1995 Jan-Mar, 9(1), 15 - 20
Orally administered interferon-gamma but not tumor necrosis factor-alpha suppress infection with Salmonella typhimurium in a mouse model; Degre M et al.; Intragastrically inoculated Salmonella typhimurium produces a systemic infection in mice with high mortality . We have examined the effect of interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha on the development of the disease . IFN-gamma reduced penetration of bacteria into the gut epithelial cells, reduced the development of bacteremia, reduced mortality and prolonged the length of survival of mice both after peroral and after intraperitoneal administration . On the other hand TNF-alpha had a similar effect only when given intraperitoneally but not by peroral route . These findings indicate that the mechanisms by which these two cytokines influence the development of S . typhimurium infection are different . This is the first observation that perorally administered cytokines may have local and systemic effects on bacterial infection.

Prog Clin Biol Res, 1995, 392, 317 - 26
Structure and functions of endotoxin-binding peptides derived from CAP18; Hirata M et al.; CAP18 (cationic antimicrobial protein, 18kDa) is a 142 amino acid protein originally isolated from rabbit granulocytes using agglutination of LPS-coated erythrocytes as an assay . CAP-18 is composed of an N-terminal domain of unknown function (CAP181-105) and a C-terminal LPS-binding domain (CAP18106-142) . Synthetic CAP18106-142 and CAP18106-137, a 32-amino acid peptide resulting from the truncation of 5 amino acids from the C-terminus of CAP18106-142, inhibited LPS-induced tissue factor generation, nitric oxide production and TNF release by macrophages . Mice treated with CAP18106-142 or CAP18106-137 were significantly protected from LPS lethality . Although CAP18106-142 and CAP18106-137 were highly active, other fragments of CAP18106-142, including CAP18110-142 with a truncated N-terminus, did not exhibit LPS-binding and LPS-neutralizing activities . Both peptides had broad anti-microbial activity against both Gram-negative bacteria such as Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae, Pseudomonas aeruginosa (IC50; 40-100 nM) and Gram-positive bacteria such as Staphylococcus aureus(Methicillin sensitive and resistant strains) and Streptococcus pneumoniae (IC50; 100-200nM) . We cloned a CAP18 family protein from human granulocytes . The cloned cDNA encoded 140 amino acid residues . Human CAP18 (CAP181-140) was highly homologous to that of rabbit . A 32- amino-acid C-terminal fragment (CAP18104-135) was shown to bind LPS, inhibit LPS-induced tissue factor generation by murine macrophages, and protect mice from LPS lethality . This peptide exhibited antimicrobial activity against both Gram-negative and Gram-positive bacteria . We hypothesize that CAP18 and the derived peptides bind to LPS and alter the capacity of LPS to initiate disseminated intravascular coagulation . In this regard, CAP may act as host defense protein against infectious diseases, and have therapeutic potential for sepsis and endotoxin shock.

Prog Clin Biol Res, 1995, 392, 3 - 14
Molecular genetics of polymyxin resistance in Salmonella typhimurium; Roland KL et al.; Resistance to Polymyxin and CAP are conferred by a point mutation, pmrA505, in position 81 that results in an Arg to His substitution in pmrA, a regulator protein . The protein, pmrB is expressed in the cell membrane . This is consistent with the hypothesis that it is a sensor/kinase protein . A newly discovered gene, pmrD, confers resistance to polymyxin B equal to that of pmrA505 when expressed on a multicopy plasmid, but such transformed strains are not resistant to CAP . Evidence is presented that pmrA505 can be expressed, but only suboptimally, in the absence of pmrD . However, there is an absolute requirement for pmrA+ in order for pmrD to express resistance to polymyxin B . It is therefore suggested that pmrD is regulated by the two component regulatory pair pmrAB.

Genetika, 1995 Jan, 31(1), 128 - 32
{The effect of various structural features in molecules of derivatives of pyrene and its heterocyclic analogs on its mutagenic activity}; Liubimova IK et al.; The mutagenic potency of five acetyl and acetylamine derivatives of nitropyrene and 15 heterocyclic analogues of pyrene was studied in the strain Salmonella typhimurium TA1538 . A significant impact of the electron-withdrawing acetyl group in the para-position on the mutagenic activity of derivatives of nitropyrene was shown . The para position of nitro and amino groups as well as of the amino and carboxyl group was also responsible for the mutagenic activity . A proposal for the existence of several cellular mechanisms of activation of molecules of heterocyclic analogues of pyrene is presented.

Immunology, 1995 Jan, 84(1), 8 - 15
Expression of TGF-beta in attenuated Salmonella typhimurium: oral administration leads to the reduction of inflammation, IL-2 and IFN-gamma, but enhancement of IL-10, in carrageenin-induced oedema in mice; Ianaro A et al.; Mice injected with carrageenin in the footpad developed local inflammation which peaked at 48 hr . This was significantly reduced in mice inoculated orally with an attenuated Salmonella construct expressing transforming growth factor-beta (TGF-beta) . Administration of the Salmonella construct alone had no effect on inflammation . High levels of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) were secreted by draining lymph node cells from mice injected with carrageenin following stimulation in vitro . Prior inoculation with Salmonella enhanced the production of IL-2 and IFN-gamma from the draining lymph node cells . Administration of the Salmonella-TGF-beta construct significantly inhibited the production of these cytokines . In contrast, IL-10 only was secreted from draining lymph node cells of animals inoculated with the Salmonella-TGF-beta construct . Thus, oral administration of TGF-beta can significantly inhibit local inflammation and alter the cytokine secretion pattern of cells from lymph nodes draining the site of inflammation.

Comp Immunol Microbiol Infect Dis, 1995 Jan, 18(1), 27 - 39
Systemic and mucosal intestinal antibody response of sheep immunized with aromatic-dependent live or killed Salmonella typhimurium; Mukkur TK et al.; Following the development of a suitable formulation capable of inhibiting intestinal proteolytic activity, the total anti-lipopolysaccharide (LPS) and anti-flagellin (Fla) antibody response and isotype in the sera and intestinal washings of sheep, immunized with live aromatic-dependent (aro-) Salmonella typhimurium strain CS332 by the intramuscular (live i.m.) or oral (live oral) route or acetone-killed virulent S . typhimurium by the intramuscular route (killed i.m.), were determined at various intervals post-immunization . The serum or intestinal anti-lipopolysaccharide (LPS) or anti-flagellin (Fla) antibody titres of immunized sheep, regardless of the route of immunization, were significantly greater (P < 0.01) than those of non-immune control sheep . Although significant differences between the serum anti-LPS or anti-Fla antibody titres of sheep in various immunization regimes were observed, they were not consistent for different periods post-immunization . The predominant isotype contributing to serum anti-LPS antibody activity was IgM whereas the serum antiflagellar antibody activity was confined to IgM, IgG1 and IgG2 . In either case, the contribution of the IgA antibody isotype was minimal . Antibody activity in the intestinal washings of immunized sheep, regardless of the route of immunization was significantly greater (P < 0.01) than that in non-immune control sheep . However, the titres in sheep immunized with the live S . typhimurium vaccines were significantly greater than those immunized with the killed vaccine . The major anti-LPS or anti-flagellin antibody isotype in the intestinal washings of sheep in the live i.m . or live oral groups was IgM at day 7 post-immunization followed by IgG1 and IgG2 at days 14 and 21 post-immunization, with only a minimal contribution by the IgA antibody isotype . On the other hand, the major antibody isotype in the intestinal washings of sheep immunized with the killed S . typhimurium was IgG1.

Environ Mol Mutagen, 1995, 25(1), 77 - 82
Frameshift mutations in Salmonella induced by the extracts of medicinal herbs Lannea edulis (Sond.) Engl . and Monotes glaber Sprague; Sohni YR et al.; Lannea edulis and Monotes glaber have been prescribed for various affectations in the traditional medical practice of Zimbabwe and other parts of Africa . Mutagenicity testing using Salmonella typhimurium strains TA97a, TA98, and TA100, indicated that the aqueous extracts of these plants induced frameshift mutations in Salmonella . The extract of L . edulis displayed marginal mutagenicity in strain TA97a while that of M . glaber showed a significant dose-dependent mutagenicity in both strains TA97a and TA98 . There was no mutagenic effect observed in strain TA100 . Two other plant extracts, those of Lannea discolor and Dolichos kilimandscharicus, were nonmutagenic in all three strains.

Environ Mol Mutagen, 1995, 25(1), 58 - 66
Metabolic reduction of novel 3,4-dichloro-5-nitrofurans in Salmonella typhimurium; Hatcher JF et al.; To gain insight on biochemical mechanisms of mutagenesis and carcinogenesis by the experimental carcinogens, 5-nitrofurans, a new series of 3,4-dichloro-5-nitrofurans, comprised of 3,4-dichloro-5-nitro-2-acetylfuran (I), 3,4-dichloro-5-nitro-2-bromoacetylfuran (II), methyl 3,4-dichloro-5-nitro-2-furoate (III), were synthesized and tested for their activation to mutagenic forms in the standard plate assay using Salmonella typhimurium TA98, TA100, and TA100NR, a derivative of TA100 deficient in nitroreductase activity . The mutagenic responses in TA98 were 2- to 6-fold lower compared to TA100 . Furthermore, I and II were less active in TA100NR, while compound III was about four times more mutagenic in TA100NR compared to the parent strain TA100 . Incubation of III with NADPH and bacterial lysates showed that the extent of reduction was greater in TA100 compared to TA100NR . High-pressure liquid chromatography analysis of the ethyl acetate extract obtained from incubation of III with lysates of TA100 revealed the formation of four metabolites with retention times of about 4.0, 5.7, 10.0, and 14.3 minutes . The spectroscopic and chromatographic properties of the components with retention times of 10.0 and 14.3 minutes were identical to two derivatives obtained by chemical reduction of III, and thus represent nitroreduction products . These derivatives have been identified as cis- and trans-oxime isomers of methyl 3,4-dichloro-2-furoate, based on spectroscopic analyses . These oximes were not mutagenic for TA100 . Furthermore, III was more mutagenic under anaerobic conditions, suggesting that secondary superoxide or nitroanion free radicals generated from nitroreduction are not responsible for the mutagenicity of III . In addition, the higher mutagenic response in TA100NR, and the lack of mutagenic activities of the amino and the oxime analogs of III suggest that the mutagenic activation of III might be due to the nitroso intermediate or involve mechanisms other than nitroreduction.

Environ Mol Mutagen, 1995, 25(1), 50 - 7
Metabolic activation of carcinogenic aromatic amines by fish exposed to environmental pollutants; Rodriguez-Ariza A et al.; Activation of arylamines to mutagenic metabolites by hepatic S9 fractions has been evaluated as a biomaker of fish exposure to pollutants, using gilthead seabream (Sparus aurata), a valuable fish species from the Spanish South Atlantic littoral, as model organism . To obtain maximal sensitivity to the mutagenic action of aromatic amines, a strain of Salmonella typhimurium overproducing O-acetyltransferase was used . Fish were treated with Aroclor 1254, pesticides (malathion and dieldrin), or copper(II), and compared to Aroclor 1254-treated rats . The promutagen activation capabilities of the S9 fractions were further characterized by studying the effect of two monooxygenase inhibitors, alpha-naphthoflavone, a well known inhibitor of aromatic hydrocarbon-inducible forms of cytochrome P450, and methimazole, a substrate for the flavin monooxygenase (FMO) system . This study shows that 2-aminoanthracene (2-AA) and 2-acetylaminofluorene (AAF) activation by gilthead liver is enhanced by treatment of fish with different xenobiotics . The catalyst responsible for this enhanced activation appears to be different for each promutagen and, at least for 2-AA, dependent on the type of xenobiotic . The data presented indicate further that treatment of gilthead with some compounds, such as malathion and dieldrin, enhances the activation of aromatic amines in liver, without inducing ethoxyresorufin-O-deethylase activity . The use of acetyltransferase-overproducing bacteria appears to be a useful tool in the study of arylamine activation by fish liver, where biotransformation capability is lower than in mammals.

FEMS Microbiol Lett, 1995 Jan 1, 125(1), 89 - 93
Kinetics of cobalamin repression of the cob operon in Salmonella typhimurium; Andersson D; The cob operon in Salmonella typhimurium encodes 25 proteins involved in the biosynthesis of cobalamin . Expression of the cob operon is negatively feedback regulated by cobalamin via a translational control mechanism . The concentration of cobalamin required to repress cob expression to half-maximal was determined in vivo and in vitro to 0.4 microM and 0.6 microM, respectively . These results suggest that cob expression in wild-type cells is partially repressed by de novo synthesized cobalamin.

Toxicol Lett, 1995 Jan, 75(1-3), 119 - 25
Comparative activation of aflatoxin B1 by mammalian pulmonary tissues; Ball RW et al.; Occupational exposures to respirable dusts contaminated with the mycotoxin aflatoxin B1 (AFB1) have been associated with an increased incidence of upper airway tumors . To investigate this possible etiology we compared the abilities of tracheal and lung S9 from rabbit, hamster and rat to activate AFB1 to mutagens in Salmonella typhimurium TA 98 . The activation of AFB1 was compared to that of benzo{a}pyrene (B{a}P), a known respiratory carcinogen . These species differ in airway morphology with respect to numbers of metabolically-active non-ciliated tracheal epithelial cells . Tracheas from hamster and rabbit and lung from rabbit were active in converting AFB1 to bacterial mutagens . Trachea from hamster was more efficient in activating AFB1 to mutagens than lung, while rabbit lung was over 4 times more active in converting AFB1 to mutagens than that from trachea . In all cases, AFB1 was more mutagenic than B{a}P . The relative capabilities of trachea to activate AFB1 were in agreement with the ability of cultured tracheas from these species to form to AFB1-DNA adducts . These results demonstrate that AFB1 is activated more efficiently than B{a}P in the lung, and that the metabolic capabilities of airway epithelium to activate AFB1 are not predictable by airway morphology.

Am J Med, 1995 Jan, 98(1), 13 - 21
Post-Salmonella reactive arthritis: late clinical sequelae in a point source cohort; Thomson GT et al.; PURPOSE: To define the natural history of post-Salmonella-infection reactive arthritis (ReA) in a point source cohort concurrently exposed to the same microorganism, and to determine any relationship between anti-Salmonella humoral immune response to the organism and clinical outcome at 5 years . PATIENTS AND METHODS: A cohort of 423 Ontario Provincial Police officers with a clinical diagnosis of Salmonella food poisoning were defined in 1984 . Five years following the food poisoning, a mail and telephone survey was carried out to determine all those who developed ReA within 3 months of the onset of dysentery . Medical and physiotherapy charts from an earlier study on the same cohort were incorporated . All patients with a history compatible with reactive arthritis were interviewed and examined . Serum was taken to determine the presence of isotypic antibodies to the lipopolysaccharide of the causative Salmonella typhimurium . RESULTS: Twenty-seven of the 423 individuals with dysentery were identified as developing acute ReA . In one third of them, the arthritis resolved within 4 months of onset . Two thirds continued to have subjective complaints, mostly of minor significance . However, symptoms were severe enough to force a change in work for 4 patients . Another 4 patients had objective damage to joints radiographically . Objective changes to joints were documented on physical examination in 37% of ReA patients 5 years following onset of disease . IgA antilipopolysaccharide antibodies correlated with the severity and duration of disease . Tests of cellular immune function did not correlate with clinical variables . CONCLUSIONS: Chronic symptoms persist 5 years after the onset of ReA in the majority of patients . Joint damage by physical examination and radiographic assessment correlate with functional disability . Some early clinical features of disease, including prolonged diarrhea during the acute illness, may predict a worse outcome . IgA antilipopolysaccharides may serve as a disease marker for late post-Salmonella-infection ReA.

J Bacteriol, 1995 Jan, 177(2), 390 - 400
Isolation and characterization of adenylate kinase (adk) mutations in Salmonella typhimurium which block the ability of glycine betaine to function as an osmoprotectant; Gutierrez JA et al.; Mutants of Salmonella typhimurium that were not protected by glycine betaine (GB) but could still use proline as an osmoprotectant in media of high osmolality were isolated . The mutations responsible for this phenotype proved to be alleles of the adenylate kinase (adk) gene, as shown by genetic mapping, sequencing of the cloned mutant alleles, complementation with the Escherichia coli adk gene, and assay of Adk enzyme activity in crude extracts . One of the mutations was in the untranslated leader of the adk mRNA, a second was in the putative Shine-Dalgarno sequence, and a third was in the coding region of the gene . The loss of osmoprotection by GB was shown to be due to the fact that the accumulation of this solute actually resulted in a severe inhibition of growth in the adk mutants . The addition of GB in the presence of 0.5 M NaCl resulted in a rapid decline in the ATP pool and a dramatic increase in the AMP pool in the mutants . Proline, which is not toxic to the adk mutants, did not have any significant effects on the cellular levels of ATP and AMP . The mutants exhibited two different phenotypes with respect to the utilization of other osmoprotectants: they were also inhibited by propiothiobetaine, L-carnitine, and gamma-butyrobetaine, but they were stimulated normally in media of high osmolality by proline, choline-O-sulfate, and stachydrine.

Infect Immun, 1995 Jan, 63(1), 99 - 103
Analysis of immunization route-related variation in the immune response to heat-killed Salmonella typhimurium in mice; Thatte J et al.; In examinations of the factors regulating the quality and quantity of the immune response to Salmonella typhimurium, we have shown previously that live and heat-killed preparations of S . typhimurium can induce gamma interferon-dominant and interleukin-4-dominant immune responses, respectively, upon intraperitoneal (i.p.) immunization of BALB/c mice . Using this system to investigate the role of the route of immunization in the immune response, we show in the present study that i.p . immunization with heat-killed S . typhimurium generates a quantitatively better immune response than does intradermal (i.d.) immunization . The quantitative differences observed between the i.p . and i.d . routes are apparent in the amount of S . typhimurium-specific antibodies produced, the extent of responses in T-cell proliferation assays, and the quantities of lymphokines generated . However, the ratios of immunoglobulin (Ig) isotypes {IgG1/IgG2a} are comparable and the relative dominance of interleukin-4 over gamma interferon is seen in both i.p.- and i.d.-immunized mice, suggesting that the predominant T-cell effector pathways triggered are not qualitatively dependent on the route of immunization . An examination of the antigenic profile recognised by the B-cell and T-cell responses in i.p.- versus i.d.-immunized mice shows that while the Western immunoblot patterns recognized by serum antibodies from the two groups of mice were not significantly different, T cells from i.p.-immunized mice recognized a broader spectrum of antigens in an immunoblot assay than did those from i.d.-immunized mice . These data suggest that there may be a significant difference in the antigen-processing ability of peritoneal and dermal antigen-presenting cells for complex antigenic formulations such as bacterial vaccines.

Infect Immun, 1995 Jan, 63(1), 356 - 9
Rapid disruption of epithelial barrier function by Salmonella typhimurium is associated with structural modification of intercellular junctions; Jepson MA et al.; Short-term infection of MDCK II monolayers with Salmonella typhimurium SL1344 caused a progressive decrease in transepithelial electrical resistance concomitant with decreased cation permselectivity and increased paracellular inulin flux . Cytochemical staining of F-actin, E-cadherin, and ZO-1 revealed the concentration of each junctional protein in invaded cells as a result of contraction at their apical poles and resultant distortion of adjacent uninvaded cells.

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