Salmonella typhimurium

Poult Sci, 1993 Nov, 72(11), 2064 - 8
Location of Salmonella typhimurium during incubation and hatching of inoculated eggs; Cason JA et al.; Location of Salmonella during hatching of broiler chicks was studied in three experiments . Unincubated, fertile hatching eggs heated to 42 C were inoculated by immersion for 15 min in a 16 C physiological saline solution containing approximately 1 x 10(5) cfu/mL of a nalidixic acid-resistant strain of Salmonella typhimurium . All eggs were sanitized externally by wiping with a paper towel wet with 70% ethanol . When incubating eggs were transferred to a hatcher, each was placed in a closed paper bag to minimize cross-contamination . Unhatched eggs were sampled by cutting away the shell over the air cell, sanitizing the inner air cell membrane with 70% ethanol, and removing the contents through the membrane . Shells and membranes were crushed and mixed in 10 mL buffered peptone (BP) . Yolks were dissected out, dipped in 70% ethanol, and mixed with 10 mL BP . Embryos or chicks and all surrounding fluid were rinsed in 100 mL BP . A total of 172 eggs was sampled . Shells and membranes were 100% Salmonella-positive 30 min after inoculation, but only 38% were positive after 17 to 21 days of incubation . No chick rinses were positive before pipping, but 15% were positive after pipping . Yolk samples were 2% positive before pipping versus 8% after pipping . A majority of chicks hatching from positive shells and membranes were Salmonella-negative.

Int Immunol, 1993 Nov, 5(11), 1431 - 6
Immunization with live versus killed Salmonella typhimurium leads to the generation of an IFN-gamma-dominant versus an IL-4-dominant immune response; Thatte J et al.; The mechanisms responsible for differential commitment of effector T cells to the production of either the IL-4/5/10 group or to the IL-2/IFN-gamma group of lymphokines during an immune response have not yet been clearly elucidated . We have used Salmonella typhimurium as a model murine bacterial parasite in BALB/c mice for live-cell versus killed-cell immunization and looked at the immune response in terms of delayed type hypersensitivity (DTH), IgG subclass distribution in the serum antibody response, and antigen-specific T cell proliferation and lymphokine secretion . The results indicate that the two forms of immunogen induce qualitatively different immune responses . Intraperitoneal immunization with live bacteria induces an IFN-gamma-dominant immune response associated with a strong DTH reaction and relatively higher levels of specific antibodies belonging to the IFN-gamma-dependent IgG2a isotype rather than the IL-4-dependent IgG1 isotype . Immunization with heat-killed bacteria gives rise to an IL-4-dominated response that shows excellent proliferative capacities in vitro, with lower levels of DTH responses and comparatively high levels of specific antibodies of the IgG1 isotype . IL-2 production in the responses generated by the two modes of immunization, however, is not preferentially associated with IFN-gamma production, unlike the reported profiles of long-lived murine T cell clones in vitro.

Fundam Appl Toxicol, 1993 Nov, 21(4), 535 - 45
Comparative effects of immunotoxic chemicals on in vitro proliferative responses of human and rodent lymphocytes; Lang DS et al.; In order to determine the comparability of human and rodent in vitro systems, the direct effects of various therapeutic or environmental chemicals on proliferative responses of lymphocytes of mouse, rat, and human origins were examined and analyzed by a detailed statistical approach . Four compounds of diverse structure and mechanism of action which are known to impair lymphocyte transformation, such as hydroquinone, T-2 toxin, lead nitrate, as well as the widely used immunosuppressive drug cyclosporin A, were chosen as model test substances . T cells were stimulated by phytohaemagglutinin as well as monoclonal antibodies directed at the T cell receptor/CD3 complex, while B cells were activated by the T-independent mitogens, including Staphylococcus aureus cells, Escherichia coli lipopolysaccharide, and Salmonella typhimurium mitogen with specificity for human, mouse, and rat lymphocytes, respectively . In almost all cases the chemicals altered lymphoproliferative responses in a concentration-related manner in all three species . In general, overall similarities in the relative sensitivity of lymphoblastogenesis were obtained when the human dose-response curves were compared to the rodent response curves . Frequent, statistically significant species-dependent discrepancies of the overall response curves between mice and rats were observed . Large, statistically significant differences were observed for inorganic lead, revealing obvious divergences of the effect patterns in all cases, across all species . In this case, rodent species, especially the rat, were very sensitive to immunomodulation by lead, whereas human cells were relatively resistant . It is suggested that direct interspecies comparisons of immunological effects due to chemical treatment in vitro can provide a greater understanding of the relationship between animal and human data, which will improve the confidence of extrapolation from findings in laboratory animals to human health risk.

Carcinogenesis, 1993 Nov, 14(11), 2303 - 7
Increased mutagenicity of 1,2-dibromo-3-chloropropane and tris(2,3-dibromopropyl)phosphate in Salmonella TA100 expressing human glutathione S-transferases; Simula TP et al.; We have expressed human glutathione S-transferases GSTA1-1 and GSTP1-1 in Salmonella typhimurium TA100 in order to assess the ability of these enzymes to modulate the mutagenicity of 1,2-dibromo-3-chloropropane (DBCP) and tris(2,3-dibromopropyl)phosphate (Tris-BP) . Both compounds were mutagenic when activated by Aroclor-induced rat liver microsomes . However, when Aroclor-induced rat liver microsomes were used together with the GST-expressing strains the mutagenicity of both DBCP and Tris-BP was markedly potentiated . Neither of the GST-expressing strains potentiated the mutagenicity in the absence of microsomes, indicating that cytochrome P450-mediated metabolism was a prerequisite for GST-mediated potentiation . With DBCP both isozymes had comparable effects on mutagenic frequency, although the highest dose of DBCP was toxic in strains expressing GSTP1-1 . In the case of Tris-BP, GSTP1-1 was much more active in potentiating the mutagenicity . These results indicate that human GSTs can play an important role in the activation of compounds such as DBCP and Tris-BP to mutagenic metabolites.

Carcinogenesis, 1993 Nov, 14(11), 2233 - 7
Bacterial and mammalian cell mutagenicity of four optically active bay-region 10,11-diol-8,9-epoxides of the nitrogen heterocycle dibenz{a,h}acridine; Chang RL et al.; The mutagenic activities of the enantiomers of the diastereomeric pair of bay-region 10,11-diol-8,9-epoxides of dibenz{a,h}acridine (DB{a,h}ACR) were evaluated in histidine-dependent strains of Salmonella typhimurium and in cultured Chinese hamster V79 cells . In strains TA98 and TA100 of S.typhimurium, the (-)-{8S,9R,10R,11S} diol-epoxide was the most mutagenic compound, inducing 1200 and 6900 His+ revertants/nmol respectively . The mutagenic activity of each of the remaining three isomers was essentially independent of the bacterial strain used and had 14-72% of the activity of the {S,R,R,S} isomer . However, in Chinese hamster V79 cells, the (+)-{8R,9S,10S,11R} diol-epoxide was the most mutagenic compound (68 8-azaguanine resistant variants/nmol/10(5) cells), inducing from 2 to 11 times as many mutations as the other three isomers . These results are analogous to previous studies with the bay-region diol-epoxides of other polycyclic hydrocarbons in that the isomer with {R,S,S,R} absolute configuration has had variable activity in the bacterial assays, but has generally been the most active in the mammalian cells . Furthermore, this isomer has almost always been highly tumorigenic in the mouse.

Proc Natl Acad Sci U S A, 1993 Nov 1, 90(21), 9983 - 7
Characterization of composite aminodeoxyisochorismate synthase and aminodeoxyisochorismate lyase activities of anthranilate synthase; Morollo AA et al.; Anthranilate synthase {chorismate pyruvatelyase (amino-accepting), E.C.4.1.3.27} catalyzes the formation of anthranilate (o-aminobenzoate) and pyruvic acid from chorismate and glutamine . A mutant form of the enzyme from Salmonella typhimurium accumulates a compound that we had isolated and identified as trans-6-amino-5-{(1-carboxyethenyl)-oxy}-1,3- cyclohexadiene-1-carboxylic acid, commonly called aminodeoxyisochorismate (ADIC) . Here we report that ADIC is formed by a reversible, Mg(2+)-dependent ADIC synthase activity of anthranilate synthase that can be functionally uncoupled from a Mg(2+)-dependent ADIC lyase activity of the enzyme by single amino acid substitutions in the TrpE subunit of the anthranilate synthase complex of S . typhimurium . Both of the component activities of the enzyme are sensitive to feedback inhibition by L-tryptophan . Purified ADIC is quantitatively converted to anthranilate and pyruvic acid by the ADIC lyase activity of wild-type anthranilate synthase . ADIC also serves as a substrate for the formation of chorismate by the enzyme in the absence of glutamine and (NH4)2SO4 . The rate of ADIC formation by the mutant enzyme and the steady-state parameters for ADIC utilization by the wild-type enzyme are consistent with a role for ADIC as an enzyme-bound intermediate that does not accumulate during the course of the anthranilate synthase reaction . The altered catalytic specificity of mutant anthranilate synthase enzymes suggests a potential role for ADIC in secondary metabolism.

Proc Natl Acad Sci U S A, 1993 Nov 1, 90(21), 9852 - 6
Crystal structure of a bacterial sialidase (from Salmonella typhimurium LT2) shows the same fold as an influenza virus neuraminidase; Crennell SJ et al.; Sialidases (EC 3.2.1.18 or neuraminidases) remove sialic acid from sialoglycoconjugates, are widely distributed in nature, and have been implicated in the pathogenesis of many diseases . The three-dimensional structure of influenza virus sialidase is known, and we now report the three-dimensional structure of a bacterial sialidase, from Salmonella typhimurium LT2, at 2.0-A resolution and the structure of its complex with the inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid at 2.2-A resolution . The viral enzyme is a tetramer; the bacterial enzyme, a monomer . Although the monomers are of similar size (approximately 380 residues), the sequence similarity is low (approximately 15%) . The viral enzyme contains at least eight disulfide bridges, conserved in all strains, and binds Ca2+, which enhances activity; the bacterial enzyme contains one disulfide and does not bind Ca2+ . Comparison of the two structures shows a remarkable similarity both in the general fold and in the spatial arrangement of the catalytic residues . However, an rms fit of 3.1 A between 264 C alpha atoms of the S . typhimurium enzyme and those from an influenza A virus reflects some major differences in the fold . In common with the viral enzyme, the bacterial enzyme active site consists of an arginine triad, a hydrophobic pocket, and a key tyrosine and glutamic acid, but differences in the interactions with the O4 and glycerol groups of the inhibitor reflect differing kinetics and substrate preferences of the two enzymes . The repeating "Asp-box" motifs observed among the nonviral sialidase sequences occur at topologically equivalent positions on the outside of the structure . Implications of the structure for the catalytic mechanism, evolution, and secretion of the enzyme are discussed.

Proc Natl Acad Sci U S A, 1993 Nov 1, 90(21), 10390 - 4
Salmonella typhimurium induces membrane ruffling by a growth factor-receptor-independent mechanism; Jones BD et al.; Invasive Salmonella typhimurium induces dramatic actin rearrangements on the membrane surface of mammalian cells as part of its entry mechanism . These changes, which are best characterized as membranous ruffles, closely resemble the membrane changes that occur when a growth factor binds to its receptor . Recently, inhibition of the function of the small GTPases rac and rho in quiescent serum-starved fibroblasts was demonstrated to abolish growth factor-mediated ruffling and stress-fiber formation, respectively . In addition, actin changes induced by the oncogene ras were also shown to be regulated by rac and rho . Because Salmonella-induced actin rearrangements resemble those caused by growth factors, we investigated whether ras, rho, or rac regulates the membrane ruffling elicited by S . typhimurium . Surprisingly, inhibition of the functions of these GTPases had no effect on the ability of invasive S . typhimurium to induce membrane ruffles on a variety of tissue culture cells including Madin-Darby canine kidney cells, Swiss 3T3 fibroblasts, and Hep-2 cells . These results led us to examine the interactions of S . typhimurium with Henle-407 intestinal cells, which lack epidermal growth factor receptor on their membrane surface . We found no difference in the ability of invasive S . typhimurium to induce membrane ruffling and to enter Henle-407 cells with or without the epidermal growth factor receptor on the membrane surface . We, therefore, conclude that invasive S . typhimurium induces membrane ruffling and its own internalization by a rac-independent, growth factor-receptor-independent signaling pathway.

J Cell Biol, 1993 Nov, 123(4), 895 - 907
Salmonella typhimurium attachment to human intestinal epithelial monolayers: transcellular signalling to subepithelial neutrophils; McCormick BA et al.; In human intestinal disease induced by Salmonella typhimurium, transepithelial migration of neutrophils (PMN) rapidly follows attachment of the bacteria to the epithelial apical membrane . In this report, we model those interactions in vitro, using polarized monolayers of the human intestinal epithelial cell, T84, isolated human PMN, and S . typhimurium . We show that Salmonella attachment to T84 cell apical membranes did not alter monolayer integrity as assessed by transepithelial resistance and measurements of ion transport . However, when human neutrophils were subsequently placed on the basolateral surface of monolayers apically colonized by Salmonella, physiologically directed transepithelial PMN migration ensued . In contrast, attachment of a non-pathogenic Escherichia coli strain to the apical membrane of epithelial cells at comparable densities failed to stimulate a directed PMN transepithelial migration . Use of the n-formyl-peptide receptor antagonist N-t-BOC-1-methionyl-1-leucyl-1- phenylalanine (tBOC-MLP) indicated that the Salmonella-induced PMN transepithelial migration response was not attributable to the classical pathway by which bacteria induce directed migration of PMN . Moreover, the PMN transmigration response required Salmonella adhesion to the epithelial apical membrane and subsequent reciprocal protein synthesis in both bacteria and epithelial cells . Among the events stimulated by this interaction was the epithelial synthesis and polarized release of the potent PMN chemotactic peptide interleukin-8 (IL-8) . However, IL-8 neutralization, transfer, and induction experiments indicated that this cytokine was not responsible for the elicited PMN transmigration . These data indicate that a novel transcellular pathway exists in which subepithelial PMN respond to lumenal pathogens across a functionally intact epithelium . Based on the known unique characteristics of the intestinal mucosa, we speculate that IL-8 may act in concert with an as yet unidentified transcellular chemotactic factor(s) (TCF) which directs PMN migration across the intestinal epithelium.

J Bacteriol, 1993 Nov, 175(22), 7290 - 300
dcp gene of Escherichia coli: cloning, sequencing, transcript mapping, and characterization of the gene product; Henrich B et al.; Dipeptidyl carboxypeptidase is a C-terminal exopeptidase of Escherichia coli . We have isolated the respective gene, dcp, from a low-copy-number plasmid library by its ability to complement a dcp mutation preventing the utilization of the unique substrate N-benzoyl-L-glycyl-L-histidyl-L-leucine . Sequence analysis of a 2.9-kb DNA fragment revealed an open reading frame of 2,043 nucleotides which was assigned to the dcp gene by N-terminal amino acid sequencing and electrophoretic molecular mass determination of the purified dcp product . Transcript mapping by primer extension and S1 protection experiments verified the physiological significance of potential initiation and termination signals for dcp transcription and allowed the identification of a single species of monocistronic dcp mRNA . The codon usage pattern and the effects of elevated gene copy number indicated a relatively low level of dcp expression . The predicted amino acid sequence of dipeptidyl carboxypeptidase, containing a potential zinc-binding site, is highly homologous (78.8%) to the corresponding enzyme from Salmonella typhimurium . It also displays significant homology to the products of the S . typhimurium opdA and the E . coli prlC genes and to some metalloproteases from rats and Saccharomyces cerevisiae . No potential export signals could be inferred from the amino acid sequence . Dipeptidyl carboxypeptidase was enriched 80-fold from crude extracts of E . coli and used to investigate some of its biochemical and biophysical properties.

J Bacteriol, 1993 Nov, 175(22), 7200 - 8
Two global regulatory systems (Crp and Arc) control the cobalamin/propanediol regulon of Salmonella typhimurium; Ailion M et al.; The genes for cobalamin (vitamin B12) biosynthesis (cob) are coregulated with genes for degradation of propanediol (pdu) . Both the cob and pdu operons are induced by propanediol by means of a positive regulatory protein, PocR . This coregulation of a synthetic and a degradative pathway reflects the fact that vitamin B12 is a required cofactor for the first enzyme in propanediol breakdown . The cob/pdu regulon is induced by propanediol under two sets of growth conditions, i.e., during aerobic respiration of a poor carbon source and during anaerobic growth . We provide evidence that, under aerobic conditions, the Crp/cyclic AMP system is needed for all induction of the pocR, cob, and pdu genes . Anaerobically, the Crp/cyclic AMP and ArcA/ArcB systems act additively to support induction of the same three transcription units . The fact that these global control systems affect expression of the gene for the positive regulatory protein (pocR) as well as the pdu and cob operons is consistent with our previous suggestion that these two global controls may act directly only on the pocR gene; their control over the cob and pdu operons may be an indirect consequence of their effect on the level of PocR activator protein . The reported experiments were made possible by the observation that pyruvate supports aerobic growth of all of the mutants tested (cya, crp, arcA, and arcB); pyruvate also supports anaerobic growth of these mutants if the alternative electron acceptor, fumarate, is provided . By using pyruvate as a carbon source, it was possible to grow all of these mutant strains under identical conditions and compare their expression of the cob/pdu regulon . The role of Crp in control of vitamin B12 synthesis suggests that the major role of vitamin B12 in Salmonella spp . is in catabolism of carbon sources; the coregulation of the cob and pdu operons suggests that propanediol is the major vitamin B12-dependent carbon source.

J Bacteriol, 1993 Nov, 175(21), 7086 - 91
Bacteriophage P22 transduction of integrated plasmids: single-step cloning of Salmonella typhimurium gene fusions; Mahan MJ et al.; Transcriptional fusions to Salmonella typhimurium chromosomal genes were constructed by integration of a suicide fusion vector into the chromosome by homologous recombination with random cloned chromosomal fragments . We describe here a transductional method using the generalized transducing phage of S . typhimurium, P22, to clone these fusions directly from the bacterial chromosome, in a single step, without the use of restriction enzymes . In this transduction, the phage packages the chromosomal fragment containing the integrated plasmid . Once introduced into the recipient, the plasmid circularizes by homologous recombination between the duplicated region determined by the cloned fragment . Although RecA mediates the majority of these events, the plasmid can circularize in a recA recipient . However, in this case, the event occurs at a much lower frequency and only when the transduction is done at a high multiplicity of infection . In addition to integrated fusion constructs, we also show that autonomously replicating low-copy-number plasmids can be transduced . In this case, transduction is dependent on homologous recombination between the plasmid and the donor chromosome via cloned sequences, in which the transducing particle effectively traps the integrated plasmid.

EMBO J, 1993 Nov, 12(11), 4053 - 62
Molecular genetic analysis of a locus required for resistance to antimicrobial peptides in Salmonella typhimurium; Parra-Lopez C et al.; The innate immunity of vertebrates and invertebrates to microbial infection is mediated in part by small cationic peptides with antimicrobial activity . Successful pathogens have evolved mechanisms to withstand the antibiotic activity of these molecules . We have isolated a set of genes from Salmonella typhimurium which are required for virulence and resistance to the antimicrobial peptides melittin and protamine . Sequence analysis of a 5.7 kb segment from the wild-type plasmid conferring resistance to protamine contained five open reading frames: sapA, sapB, sapC, sapD and sapF, organized in an operon structure and transcribed as a 5.3 kb mRNA . SapD and SapF exhibited similarity with the 'ATP binding cassette' family of transporters including the bacterial Opp and SpoOK, involved in the uptake of oligopeptides; the yeast STE6, necessary for the export of a peptide pheromone; and the mammalian mdr, which mediates resistance to chemotherapeutic agents in cancer cells . SapA showed identity with other periplasmic solute binding proteins involved in peptide transport . The SapABCDF system constitutes a novel transporter for enteric bacteria and the first one harboring a periplasmic component with a role in virulence.

Rev Med Chil, 1993 Nov, 121(11), 1245 - 51
{Mutagen extraction from bile of patients with inflammatory biliary pathology: Ames test using blue rayon}; Araya JC et al.; Gallbladder carcinoma is frequent in Chile . The aim of this study was to report the mutagenicity of whole human bile, using the Ames/Salmonella microsome assay with Salmonella typhimurium TA98 . The bile of 19 patients, aged 23 to 64 years old, subjected to cholecystectomy was examined, and mutagen activity was found in 13 (72%) . Mutagens were extracted using blue rayon and three dilutions for the eluted material from blue rayon were used (50, 100 and 200 ul) . The best result was obtained using 200 ul . In some cases, the amount of revertive colonies was very high (over 5 times the control value) . We propose that the bile from these patients possibly contains mutagenic substances with frame shift mutagenic activity and that these substances may be related to gallbladder carcinoma . Our results have addressed the importance of bile studies to elucidate the pathogenesis of gallbladder carcinoma.

Mutagenesis, 1993 Nov, 8(6), 577 - 81
Formaldehyde is a bacterial mutagen in a range of Salmonella and Escherichia indicator strains; O'Donovan MR et al.; Formaldehyde was examined for bacterial mutagenicity using Escherichia coli WP2(pKM101) and WP2uvrA(pKM101), and Salmonella typhimurium TA1535, TA1537, TA1538, TA98, TA100 and TA102, in the absence of any exogenous source of metabolic activation . Using pre-incubation exposure, clear mutagenicity was seen for TA98, TA100 and TA102, and both E . coli strains . In standard plate-incorporation assays, consistent mutagenicity was seen only for TA100 and WP2uvrA(pKM101) . No evidence of mutagenicity was seen for TA1535, TA1537 or TA1538 using either method of exposure . These data confirm the enhanced ability of the pre-incubation method to detect the mutagenicity of formaldehyde both quantitatively, as expressed by numbers of revertant colonies, and qualitatively, in terms of the range of indicator strains reverted . The relatively greater sensitivity of the pre-incubation assay is probably due to better containment of a volatile agent and/or lack of interaction with agar during the initial period of exposure . The findings are consistent with the suggestion that formaldehyde induces lesions in bacteria which are, at least to some extent, excision-repairable, and indicate that the presence of the R-factor plasmid may be required for the expression of its mutagenicity in excision repair-deficient Salmonella.

Mutagenesis, 1993 Nov, 8(6), 527 - 32
Genetic differences between the standard Ames tester strains TA100 and TA98; Jurado J et al.; The standard Ames tester strains of Salmonella typhimurium are separated by many steps in their pedigree, some involving mutagen treatments, and contain independently isolated uvrB-bio-gal deletions and rfa mutations . In this work the araD531 mutation was introduced into the Ames tester strains TA100 and TA98 . The responsiveness of the resulting strains (BA15 and BA14) to a number of chemical mutagens was then assessed by monitoring the induction of forward mutations to L-arabinose resistance (Ara test) . Here we have shown that these two strains of the Ames test differ greatly in their responses to mutagens, in ways that are not associated with the mutagenic specificities of the original his mutations . In general, the genetic background of strain TA100 appears to be more sensitive to the killing effects of chemicals than that of TA98 . The greatest differences were found with nifurtimox (NFX) and its analogue, compound 1K . The Ara test responded to the mutagenic effects of these two nitrofurans when carried out in the genetic background of strain TA98 but not in that of TA100 . A higher sensitivity to the lethal effects of NFX and 1K together with the greater nitro-reduction capability of strain TA100 as compared with TA98 might explain the differences . In conclusion, our results indicate that the standard Ames S . typhimurium tester strains are not isogenic and that genetic differences at loci other than his might be significant for mutagenicity testing.(ABSTRACT TRUNCATED AT 250 WORDS)

Chem Res Toxicol, 1993 Nov-Dec, 6(6), 895 - 9
The influence of delivery rate on the chemistry and biological effects of nitric oxide; Tamir S et al.; Nitric oxide can be introduced slowly and steadily into aqueous solutions, including cell culture media, over extended periods of time via semipermeable Silastic (a registered trademark of the Dow Corning Corp.) polymer membranes . The rates of introduction are predictable and reproducible and can approach rates of nitric oxide production by stimulated cells, such as macrophages, that express inducible nitric oxide synthases . DNA damage in Chinese hamster ovary cells by membrane-delivered nitric oxide is comparable to that observed in the DNA of stimulated macrophages . Toxicity and mutagenicity of nitric oxide toward Salmonella typhimurium, toxicity of nitric oxide toward Chinese hamster ovary cells, and nitrosation of dimethylmorpholine are all more efficient when nitric oxide is delivered by membrane than when an equivalent amount of gaseous nitric oxide is added by syringe.

Chem Res Toxicol, 1993 Nov-Dec, 6(6), 825 - 36
1H NMR of an oligodeoxynucleotide containing a propanodeoxyguanosine adduct positioned in a (CG)3 frameshift hotspot of Salmonella typhimurium hisD3052: Hoogsteen base-pairing at pH 5.8; Singh US et al.; The exocyclic DNA adduct 1,N2-propano-2'-deoxyguanosine (PdG) was inserted into the oligodeoxynucleotide 5'-CGC(PdG)CGGCATG-3' and annealed to the complementary oligodeoxynucleotide 5'-CATGCCGCGCG-3' . This sequence is derived from a spontaneous revertant of the hisD3052 gene in a frameshift-sensitive tester strain of Salmonella typhimurium and is a hotspot for two-base pair deletions . The solution structure of the modified duplex was examined by 1H NMR spectroscopy . The exocyclic lesions resulted in loss of Watson-Crick base-pairing capability . Modification resulted in an approximately 24 degrees C decrease in Tm of the duplex . NMR experiments revealed pH-dependent conformational equilibria, which involved the modified base pair and its 3'-neighbor base pair . At pH 5.8, the lesion resulted in a localized perturbation of the B-form helix . PdG was rotated about the glycosyl bond from the anti to the syn conformation, thus placing the propano moiety into the major groove . This resulted in the observation of a strong NOE between the imidazole proton of PdG and the anomeric proton of the attached deoxyribose . Additional NOEs were observed between the methylene protons of the propano moiety and H5 and H6 of the 5'-neighbor cytosine . An imino proton resonance from the cytosine complementary to PdG and protonated at N3, characteristic of a Hoogsteen base pair, was observed at 15 ppm, but was broadened due to exchange with water . The amino protons of the complementary cytosine were shifted downfield from the other cytosine amino protons, characteristic of a Hoogsteen-like conformation at the site of modification . A second equilibrium involved the 3'-neighbor base pair, which alternated between Watson-Crick and Hoogsteen pairing, also via rotation of the guanosine glycosyl bond from the anti to the syn conformer . The conformational exchange of the 3'-neighbor base pair was sufficiently slow on the NMR time scale to allow simultaneous observation of resonances from the Watson-Crick and the Hoogsteen conformers.

Mikrobiologiia, 1993 Nov-Dec, 62(6), 1093 - 100
{Antimutagenic effect of culture fluid obtained as a result of propionic acid fermentation}; Vorob'eva LI et al.; It was shown for first time that propionic acid bacteria excrete in the medium substances with antimutagenic activity against mutagenicity of 4-nitroquinoline-N-oxide in Salmonella typhimurium TA100 . The substances act as antimutagens . Antimutagenic action of filtrate of culture liquid increased with time preincubation with mutagen and dose increase . The most activity was found in the culture of logarithmic and early stationary phase (24-48 h) . The substances with antimutagenic activity are formed in the medium with glucose, lactose and lactate . Antimutagenic factor (factors) is dialysable, thermostable and is not presented with peptide.

Mol Microbiol, 1993 Nov, 10(3), 685 - 96
Chromosomal domains of supercoiling in Salmonella typhimurium; Pavitt GD et al.; The chromosomes of enteric bacteria are divided into about 50 independently supercoiled domains . It is not known whether the net level of DNA supercoiling is similar in each domain, or whether the domains are differentially supercoiled . We have addressed this question genetically, using a supercoiling-sensitive promoter to probe the relative levels of supercoiling at defined points around the Salmonella typhimurium chromosome . We conclude that, within the limits of resolution of this approach, the level of supercoiling does not differ significantly between chromosomal domains, and that each domain responds in a similar fashion to factors that perturb supercoiling . These findings have implications for the organization of the bacterial genome.

Mol Microbiol, 1993 Nov, 10(3), 655 - 64
The XbaI-BlnI-CeuI genomic cleavage map of Salmonella enteritidis shows an inversion relative to Salmonella typhimurium LT2; Liu SL et al.; We have established the genomic cleavage map of Salmonella enteritidis strain SSU7998 using pulsed-field gel electrophoresis . The chromosome of 4600 kb was analysed by XbaI (16 fragments), I-CeuI (7 fragments) and BlnI (12 fragments); the genome also contains a plasmid of 60 kb . Cleavage sites of I-Ceul, in the large subunit ribosomal RNA gene, are conserved from Salmonella typhimurium and Escherichia coli K-12, and the XbaI and BlnI sites in glt-tRNA are also conserved, but other sites are less conserved . Transposon Tn10, located at 60 different positions in the chromosome of S . typhimurium, was transduced by bacteriophage P22 into S . enteritidis and the insertion mapped using the XbaI and BlnI sites on Tn10 . Gene order in S . enteritidis is identical to S . typhimurium LT2 and similar to E . coli K-12 except for an inversion of 815 kb, which covers the terminus region including T1 and T2 . Endpoints are in the NDZs, or non-divisible zones, in which inversion endpoints were not detected in experiments in E . coli K-12 and S . typhimurium LT2 . This inversion resembles the inversion between S . typhimurium and E . coli, but is longer at both ends.

Drug Metab Dispos, 1993 Nov-Dec, 21(6), 1048 - 56
Characterization of cytochrome P-450 2B6 in human liver microsomes; Mimura M et al.; A cytochrome P-450 (P-450) enzyme of the CYP2B subfamily was partially purified from human liver microsomes and characterized with respect to immunochemical properties, N-terminal amino acid sequence, and catalytic activities toward typical P-450 substrates . P-450 enzymes were monitored in chromatographic fractions by immunoblotting analysis using antibodies raised against a monkey P-450 2B, as well as several purified human P-450 enzymes . The final P-450 2B preparation thus obtained was contaminated with P-450 3A4, but an N-terminal amino acid sequence matching the sequence predicted from the CYP2B6 cDNA was obtained . The apparent M(r) of this protein was 48 kDa, and the migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was the same as that of the P-450 2B6 protein expressed in a human lymphoblast cell line . Immunoblotting analysis of 50 human liver samples revealed that the protein band considered to be P-450 2B6 was detected in only 12 samples, with four of these having relatively high levels . Several activities toward typical P-450 substrates were determined in a reconstituted monooxygenase system containing partially purified P-450 2B6 and compared with those obtained using a highly purified preparation of P-450 3A4 enzyme; we found that most of the activities were similar in these preparations, except that the partially purified P-450 2B6 showed high rates of activation of the mutagens 6-aminochrysene and 3-methoxy-4-aminoazobenzene to genotoxic metabolites in Salmonella typhimurium NM2009 strain.(ABSTRACT TRUNCATED AT 250 WORDS)

Acta Trop, 1993 Nov, 55(3), 171 - 80
In vivo macrophage function in experimental infection with Trypanosoma cruzi subpopulations; Celentano AM et al.; The macrophage function was investigated in mice infected with Trypanosoma cruzi . Two subpopulations of the parasite were utilized, RA and K98 . Strain RA is efficiently internalized by macrophages and is lethal for mice, and clone K98 is poorly phagocytosed by macrophages and is not lethal . Treatment with silica enhanced parasitemia and mortality in mice infected with both parasite subpopulations . Parasitemia kinetics, however, were affected only in mice infected with RA, which suggests that macrophage effector mechanisms may play a more relevant role in this experimental group than in mice infected with K98 . Resistance to Salmonella typhimurium infection and bactericidal activity of macrophages depended upon the T . cruzi subpopulation utilized and the infection period . Infection with K98 induced only a trend towards enhanced resistance to bacterial challenge during both the acute and chronic phases, whereas a significantly enhanced bactericidal activity of spleen and liver phagocytes was observed . Mice acutely infected with RA showed significantly enhanced susceptibility to S . typhimurium infection and lower bactericidal activity . Mice surviving infection with this aggressive strain, however, showed significantly enhanced resistance and bactericidal activities . Mice acutely infected with the RA strain displayed a dissociation between macrophage capacities to control S . typhimurium and T . cruzi . A similar phenomenon was also observed in other parasitoses (schistosomiasis, African trypanosomiasis) . This fact may be due to differences in the lethal mechanisms through which macrophages control these parasites and S . typhimurium.

Mutat Res, 1993 Nov, 319(3), 223 - 36
Comparison of three short-term assays: results on seven chemicals . Potential contribution to the control of water genotoxicity; Le Curieux F et al.; Three short-term assays (the SOS chromotest, the Ames fluctuation test and the newt micronucleus test) were used to evaluate the genotoxicity of seven chemicals (4-nitroquinoline 1-oxide, potassium dichromate, formaldehyde, sodium hypochlorite, benzo{a}pyrene, cyclophosphamide and 2-naphthylamine) . In the SOS chromotest, all seven compounds except sodium hypochlorite and cyclophosphamide were found to induce primary DNA damage in E . coli . With the Ames fluctuation test, all seven chemicals except sodium hypochlorite showed mutagenic activity on Salmonella typhimurium TA100, TA102 or TA98 . The newt micronucleus assay detected a clastogenic effect of all seven compounds except formaldehyde on the peripheral blood erythrocytes of Pleurodeles waltl larvae . For each compound, the sensitivity of the tests was compared; (1) the SOS chromotest is the least sensitive assay in every case, (2) the Ames fluctuation test is the most sensitive assay for studied chemicals with direct genotoxic effect and (3) the newt micronucleus assay is the most sensitive test for tested compounds with indirect genotoxic activity . The potential contribution of these three tests in the monitoring of water genotoxicity is discussed.

Mutat Res, 1993 Nov, 303(3), 135 - 42
Mutagenicity and antimutagenicity of extracts of three spices and a medicinal plant in Thailand; Higashimoto M et al.; Three kinds of spices (caraway, coriander and black pepper seeds) and a medicinal plant called 'tong tak' in Thai (Baliospermum axillar, a species of the spurge family) were fractionated into hot water, methanol and hexane extracts . These extracts were not mutagenic for Salmonella typhimurium strains TA98 and TA100 by the Ames assay . However, when the extracts were treated with nitrite, samples of the water and methanol extracts were mutagenic for strain TA100 without metabolic activation . The mutagenicity of the nitrite-treated methanol and hot water extracts of black pepper was highest (8380 and 22,200 His+ per 0.1 g of spice powder, respectively), and that of the nitrite-treated hot water extracts of caraway and tong tak was moderate . The hot water extracts were examined for their antimutagenic activity against mutagenicity induced by various carcinogens by the Ames assay, using the preincubation technique . The tested samples (equivalent to 1-2 mg of spice powder) reduced the mutagenicity induced by 2.7 nmole (397 ng) of N-methyl-N'-nitro-N-nitrosoguanidine by more than 84%, and that induced by dimethylnitrosamine (1.48 mg) or ICR-170 (10 ng) by 30-60% . However, they did not inhibit the mutagenic activity of 1-nitropyrene, 3-nitrofluoranthene, AF-2, methyl methanesulfonate, N-ethyl-N'-nitro-N-nitrosoguanidine, 2-aminoanthracene, 2-acetylaminofluorene, benzo{a}pyrene or IQ.

Mutat Res, 1993 Nov, 303(3), 127 - 33
Dimethyl sulfoxide (DMSO) is mutagenic for bacterial mutagenicity tester strains; Hakura A et al.; We used the Ames method with the modification of pre-incubation to evaluate the potential mutagenicity of DMSO . We performed the assays using nine different Ames Salmonella strains and Escherichia coli strains WP2 and WP2uvrA . DMSO was found to be mutagenic for Salmonella typhimurium TA1537 and TA2637 (the latter strain being isogenic to TA1537 but carrying plasmid pKM101) and for E . coli WP2uvrA . The mutagenic activity of DMSO observed at a concentration of 33% was about 10 times higher than the background level (65 revertants induced) for TA1537 after 20 min of incubation, where some lethal toxicity was observed . The mutagenicity of DMSO was observed in the presence and absence of rat liver S9 mix.

J Bacteriol, 1993 Nov, 175(21), 7006 - 15
Transcription from two promoters and autoregulation contribute to the control of expression of the Salmonella typhimurium flagellar regulatory gene flgM; Gillen KL et al.; The flgM gene product has been shown to be a negative regulator of flagellin transcription in Salmonella typhimurium (K . L . Gillen and K . T . Hughes, J . Bacteriol . 173:2301-2310, 6453-6459, 1991; K . Ohnishi, K . Kutsukake, H . Suzuki, and T . Iino, Mol . Microbiol . 6:3149-3157, 1992) . Mud-lac fusions to the flgM gene were isolated and used to characterize the regulation of flgM gene expression . Transcription of the flgM gene was decreased more than 30-fold in strains with the flagellar master regulatory genes, flhC and flhD, deleted . A class 2 flagellar defect caused a slight increase of flgM gene transcription unless a wild-type copy of the flgM gene was present, in which case transcription was decreased threefold . A deletion in the gene for the alternative sigma factor sigma 28 (FliA) caused a fourfold decrease in flgM expression . Insertional inactivation of a gene upstream of the flgM gene (flgA) in a fliA mutant strain caused transcription of the flgM gene to be decreased to a basal level . Northern (RNA) blot analysis confirmed the presence of two transcripts through the flgM gene, one which initiates upstream of the flgM gene and a second which initiates upstream of the flgA gene.

Gene, 1993 Oct 29, 133(1), 103 - 8
Gene sequence, overproduction, purification and determination of the wild-type level of the Escherichia coli flagellar switch protein FliG; Roman SJ et al.; The flagellar motor switch in Escherichia coli and Salmonella typhimurium controls swimming behavior by regulating the direction of flagellar rotation . The switch is a complex apparatus composed of at least three proteins--FliG, FliM and FliN . During chemotactic behavior, the switch responds to signals transduced by the chemotaxis sensory signaling system . CheY, the chemotaxis response regulator, is thought to act directly on the switch to induce tumbles in the swimming pattern, but physical interaction of CheY and switch proteins has not been shown . We have undertaken this work to develop the molecular tools to investigate CheY binding to switch proteins, as well as to understand more about the structure and function of the switch . We present here the sequences of the fliG gene and its protein product, the engineering and amplification of fliG by the polymerase chain reaction (PCR) and its subcloning, and the overproduction, purification and determination of the wild-type (wt) level of the FliG protein . The sequence data revealed a 91.8% amino acid (aa) identity between E . coli and S . typhimurium FliG . Engineering and amplifying fliG by PCR allowed convenient cloning into an efficient expression vector . FliG was successfully overproduced and purified to > 98% purity . Polyclonal antibodies (Ab) were generated against purified FliG and used in quantitative Western blots to determine that the wt expression level of fliG results in about 3700 FliG copies per cell . Purified FliG and anti-FliG Ab will be useful for direct biochemical analyses of CheY-switch protein interaction.

J Biol Chem, 1993 Oct 25, 268(30), 22469 - 79
Membrane topology of a P-type ATPase . The MgtB magnesium transport protein of Salmonella typhimurium; Smith DL et al.; P-type ATPases are a family of cation transport enzymes present in all species from bacteria to mammals whose members mediate membrane flux of all common biologically relevant cations . More than 50 members of this family of transporters have been sequenced; extensive structural data are available, and several members have been analyzed by site-directed mutagenesis . Nonetheless, there is no current consensus regarding their membrane topology . In this work, the Salmonella typhimurium Mg2+ transporting P-type ATPase encoded by the MgtB locus has been used as a model for P-type ATPases . Unlike other prokaryotic P-type ATPases, the MgtB protein is similar in length, amino acid sequence, and hydropathy profile to known eukaryotic P-type ATPases . The membrane topology of MgtB was analyzed by several epitope insertions in MgtB and from the activity of 35 protein fusions between MgtB and the reporter enzymes BlaM (beta-lactamase) and LacZ (beta-galactosidase) . The epitope insertions within MgtB all retained function as assessed by cation uptake assays and were regulated normally by the level of Mg2+ within the growth medium . The epitope insertion and fusion protein data are completely incompatible with the numerous previously proposed models for P-type ATPases predicting 7, 8, 9, or 12 transmembrane segments . Rather, they indicate that MgtB contains 10 transmembrane segments with both amino and carboxyl termini residing within the cytosol . By extension, we suggest that all eukaryotic P-type ATPases contain 10 transmembrane segments with both termini within the cytosol.

J Biol Chem, 1993 Oct 25, 268(30), 22269 - 72
A novel intersubunit repair mechanism in the tryptophan synthase alpha 2 beta 2 complex . Critical role of the beta subunit lysine 167 in intersubunit communication; Yang XJ et al.; This study explores intersubunit communication in the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium . We find that an engineered mutation in the contact region between the alpha and beta subunits remarkably alters the catalytic and spectroscopic properties of the beta subunit in the alpha 2 beta 2 complex . Ligands that bind to the alpha subunit largely repair the deleterious effects of the beta subunit mutation in the alpha 2 beta 2 complex . The conserved residue chosen for mutation, beta subunit lysine 167, appears to form an ion pair with alpha subunit aspartate 56 in the crystal structure of the wild type alpha 2 beta 2 complex . Although replacement of beta subunit lysine 167 by threonine does not prevent formation of the alpha 2 beta 2 complex, this mutation reduces the rate of synthesis of L-tryptophan from L-serine and indole (beta reaction) 25-fold . Ligands that bind to the alpha subunit (indole-3-glycerol phosphate, indole-3-propanol phosphate, alpha-glycerol 3-phosphate, or potassium phosphate) largely restore the activity of the mutant alpha 2 beta 2 complex in the beta reaction . We conclude that beta subunit lysine 167 plays an important role in intersubunit communication in the alpha 2 beta 2 complex . The striking allosteric effects of alpha subunit ligands on the mutant beta subunit in the alpha 2 beta 2 complex may result from ligand-induced conformational changes in the alpha subunit that are transmitted to the beta subunit and repair the mutational defect in the beta subunit.

J Mol Biol, 1993 Oct 20, 233(4), 739 - 52
The 1.7 A refined X-ray structure of the periplasmic glucose/galactose receptor from Salmonella typhimurium; Zou JY et al.; The X-ray structure of the periplasmic glucose/galactose receptor (binding protein) of Salmonella typhimurium (GBP-S) has been refined at 1.7 A resolution with an R-factor of 19.0% . The model contains all 309 residues of the amino acid sequence, 153 water molecules, a calcium ion and beta-D-galactose . The protein consists of two very similar structural domains, each of which is composed a core of parallel beta-sheet flanked on both sides by alpha-helices . Three short stretches of amino acid chain (from symmetrically related portions of the structure) link the domains, and presumably act as a hinge to allow their relative movement in functionally important conformational changes . Galactose is bound between the domains, interacting with a number of side-chains from the loops lining the binding cleft . A combination of hydrogen bonding, hydrophobic and steric effects give rise to tight binding (dissociation constant 0.2 microM) and high specificity . Of nine hydrogen bonding groups, three are aspartate, three asparagine, one histidine (unprotonated), one arginine and one water, contributing 13 hydrogen bonds in total . Additional residues pack against (primarily) non-polar faces of the sugar molecule . The precise arrangement of the hydrogen bonding and hydrophobic residues results in an enclosed binding site with a shape that is a composite of those of the allowed sugar molecules . It is presumed that ligands bind to a more open form of the receptor that then closes by rotation in the hinge . Comparison with the GBP-S structure solved earlier in complex with glucose shows no significant changes, even for the aspartate residue most directly involved with the different sugars . Comparison with the galactose/glucose receptor of Escherichia coli indicates that these two proteins are very similar in overall structure, with the main difference being a 2 to 3 degrees rotation in the hinge . This difference appears to be the result of different crystal packing for the two proteins; it is likely that both conformations are normally found in solution.

FEBS Lett, 1993 Oct 18, 332(3), 260 - 2
The effect of sugars on the morphology of the bacterial flagellum; Seville M et al.; Using dark-field microscopy, we have found that certain sugars cause the normal-to-curly helical transition of bacterial flagella . Titration of flagella isolated from Salmonella typhimurium with 16 different carbohydrates showed that: (i) only certain sugars cause the transition . There is no obvious relationship between the simple physico-chemical properties of the sugar and whether the sugar causes the transition or not; (ii) the efficacies of sugars that do cause the transition differ markedly . For these sugars there is a relationship between efficacy and molecular size . These results suggest that the specific, though weak, binding of sugars to sites on flagella cause the morphological transition.

Cell Immunol, 1993 Oct 15, 151(2), 336 - 44
Tumor necrosis factor (TNF alpha) regulates intestinal mucus production during salmonellosis; Arnold JW et al.; Mucus production by goblet cells in the gastrointestinal tract following Salmonella typhimurium infection using a ligated ileal loop model in mice was investigated . Assessment of the morphology of the loop tissue after Salmonella challenge revealed generalized tissue inflammation, characterized by edema and an inflammatory cell infiltrate . Villi were shortened and blunted, and crypts contained an increased number of cells with mitotic figures . Production of TNF alpha in the loops followed Salmonella challenge and occurred at the same time as the pathological sequelae . A nearly 50% decrease in the number of goblet cells in infected tissue compared to tissue from noninfected controls was observed at these same times . The sulfation of mucins produced by the goblet cells in infected tissues was increased in the villi but was unchanged in the crypts compared to uninfected tissues . Treatment of mice with antibody to TNF alpha before Salmonella challenge abrogated tissue pathology and returned goblet cell numbers and mucin profiles to those observed in noninfected controls . Our results indicate that TNF alpha may mediate changes in goblet cell expression and mucin sulfation in response to Salmonella challenge.

Eur J Biochem, 1993 Oct 15, 217(2), 771 - 9
Comparison of X-ray powder-diffraction data of various bacterial lipopolysaccharide structures with theoretical model conformations; Kastowsky M et al.; X-ray powder-diffraction experiments have been performed on dry samples of lipid A and various rough-mutant lipopolysaccharides (LPS) of Salmonella minnesota, Salmonella typhimurium and Escherichia coli . The diffraction patterns obtained indicated exclusively lamellar, bilayered arrangements in all samples . The periodicities were found to be in the range 4.5 nm for lipid A to 8.8 nm for Ra-LPS . Upon treatment with water-saturated air, swelling of the lamellar structures was achieved, as indicated by shifts of reflections . The increase in bilayer dimensions normally was about 0.3 nm . X-ray intensities were used for the determination of the inner bilayer structure, i.e . for calculation of the one-dimensional electron-density distribution across the bilayer . For lipid A and several Re-LPS, Rd2-LPS, Rd1-LPS and Rc-LPS samples, a striking coincidence of the electron-density distributions in the lipid-A domain was found, suggesting that in all these structures the lipid-A portion is similarly arranged . For Rb1 and Ra-LPS the lipid-A domain could not be resolved due to the limited number of observed reflections . For other Re-mutant lipopolysaccharide samples, quite different X-ray patterns were obtained . Some samples yielded diffraction patterns indicating a very high state of order in the lipid-A domain, whereas, in others, a significantly reduced order in the lipid-A domain was inferred . Comparison of the X-ray data with features of a calculated three-dimensional molecular model of lipopolysaccharide revealed reasonable agreement in molecular dimensions and bilayer structure.

Biochemistry, 1993 Oct 12, 32(40), 10658 - 65
Inhibition of viral capsid assembly by 1,1'-bi(4-anilinonaphthalene-5-sulfonic acid); Teschke CM et al.; The precursor shells of dsDNA bacteriophages are assembled by the polymerization of competent states of coat and scaffolding subunits . The fluorescent dye 1,1'-bi(4-anilinonaphthalene-5-sulfonic acid) (bisANS) binds to both the coat and scaffolding proteins from the Salmonella typhimurium bacteriophage P22 . It displays little affinity for the polymerized forms of the proteins . The subunits with bound bisANS are incapable of assembling into procapsids . The binding constants of bisANS for both coat and scaffolding protein monomers have been measured and are 7 and 6 microM, respectively . Binding of bisANS to coat protein has little effect on the conformation as determined by circular dichroism and susceptibility to proteolysis . Binding of bisANS to scaffolding protein induces a change in the secondary structure consistent with a loss of alpha-helix, and an altered susceptibility to proteolysis . We suggest that the bisANS is probably binding at sites responsible for intersubunit interactions and thereby inhibiting capsid assembly.

Biochim Biophys Acta, 1993 Oct 6, 1202(2), 297 - 304
CheZ mutants with enhanced ability to dephosphorylate CheY, the response regulator in bacterial chemotaxis; Huang C et al.; CheZ is a component of the chemotaxis signal-transduction pathway in Escherichia coli and Salmonella typhimurium . It is responsible for accelerating dephosphorylation of CheY and thereby antagonizing the tumble-promoting activity of CheY . In the absence of functional CheZ, cells are non-chemotactic and tumble constantly . We characterized the effects of two mutations in CheZ, R54C and V166G, that are unusual in that they cause cells to have a smooth swimming bias . These mutations were isolated as second-site suppressors of mutations in the switch complex responsible for regulating the direction of flagellar rotation (Yamaguchi, S., Aizawa, S.-I., Kihara, M . Isomura, M., Jones, C.J . and Macnab, R.M . (1986) J . Bacteriol . 168, 1172-1179) . When produced at low levels in a delta cheZ host strain, CheZ R54C and CheZ V166G supported chemotaxis . However, when moderately overproduced they markedly inhibited chemotactic ability . In vitro studies revealed that these mutations enhanced the ability of CheZ to accelerate dephosphorylation of CheY . These results are discussed in relation to the possible roles and interactions of CheZ in the chemotaxis system.

Biochemistry, 1993 Oct 5, 32(39), 10404 - 13
Characterization of the functional role of a flexible loop in the alpha-subunit of tryptophan synthase from Salmonella typhimurium by rapid-scanning, stopped-flow spectroscopy and site-directed mutagenesis; Brzovic PS et al.; The function of a flexible loop (loop 6) in the alpha-subunit from the tryptophan synthase alpha 2 beta 2 bienzyme complex has been investigated utilizing rapid-scanning (RSSF) and single-wavelength (SWSF) stopped-flow spectroscopies . Loop 6 is an extended sequence of residues which connects beta-strand 6 with alpha-helix 6 in the beta/alpha-barrel fold of the alpha-subunit . Substitution of Leu for Arg179 near the base of loop 6 does not significantly affect either the association of the alpha- and beta-subunits to form the bienzyme complex or the kinetics of the reaction of indole with L-serine (L-Ser) to form L-tryptophan (L-Trp), the process catalyzed by the wild-type beta-subunit {Kawasaki, H., Bauerle, R., Zon, G., Ahmed, S., & Miles, E . W . (1987) J . Biol . Chem . 262, 10678-10683} . However, the alpha-subunit-specific ligand glycerol phosphate (GP), which is an inhibitor of the wild-type beta-reaction, is a much less effective inhibitor of the alpha R179L-catalyzed beta-reaction . Equilibrium titration studies show that the affinity of GP for the alpha-site when either L-Ser or glycine is bound at the beta-site has been reduced by nearly 100- and 200-fold, respectively . SWSF analysis of the reaction of IGP and L-Ser to form L-Trp catalyzed by the bienzyme complex revealed a 15-fold reduction in the binding affinity of the alpha-site substrate 3-indole-D-glycerol 3'-phosphate (IGP) in the reaction catalyzed by the alpha R179L mutant as compared to the wild-type enzyme . These studies show that loop 6 is important both for ligand binding to the alpha-site and for the ligand-induced conformational transition of the alpha-subunit from an "open" to a "closed" structure . Modeling studies, based on extensive structural homology of the alpha-subunit with the glycolytic enzyme triosephosphate isomerase (TIM), predict that closure of loop 6 induced by ligand binding at the alpha-active site would effectively sequester the bound substrate from the solvent and trap indole, produced from the cleavage of IGP, within the confines of the bienzyme complex . This conformational transition would promote the diffusion of indole to the beta-active site via the interconnecting tunnel and would help ensure the close coordination of alpha- and beta-subunit catalytic activities.

Toxicology, 1993 Oct 5, 82(1-3), 3 - 20
Heterologous expression of drug-metabolizing enzymes in cellular and whole animal models; Simula AP et al.; In this report we describe the heterologous expression of glutathione S-transferase (GST) and cytochrome P450 reductase (Red) in E . coli and Salmonella typhimurium . The same expression vectors could be applied to both systems and high levels of catalytically active GST and Red were obtained . Interestingly the level of expression was invariably higher in S . typhimurium . The level of the alpha class GST being up to 20% of the total bacterial protein . A further advantage of the salmonella system is that strains were used which can be applied to mutagenicity tests . This system was validated by demonstrating increasing mutation frequency of halogenated hydrocarbons in strains expressing the GST and increased cytotoxicity of mitomycin C in cells expressing P450 reductase.

J Nutr, 1993 Oct, 123(10), 1714 - 23
Dietary energy source and density modulate the expression of immunologic stress in chicks; Benson BN et al.; To determine how dietary energy level and source influence feed intake, growth and energy partitioning drug immunologic stress, growing chicks were fed diets based on cornstarch and casein with varying energy densities and injected every other day for 6 d with either saline (control), Salmonella typhimurium lipopolysaccharide or heat-killed Staphylococcus aureus . Salmonella typhimurium lipopolysaccharide decreased growth and feed consumption at low energy densities . When the dietary energy density was increased above 13.4 kJ/g using cornstarch, but not corn oil, the growth depressing effect of immunogens was eliminated . Immunologically stressed chicks had a greater proportion of gain in visceral organs and less in the carcass, regardless of the nutrient density of the diet . Immunologic stress decreased intake of metabolizable energy of chicks fed a diet with low nutrient density and increased it for those fed a diet with high nutrient density . Chicks injected with S . typhimurium lipopolysaccharide lost more energy as heat than controls when differences in metabolizable energy intakes were accounted for and modified their preference between two diets differing in metabolizable energy density and fat content as a result of the challenge . Control chicks selected between the 11.7 and 14.2 kJ/g diets to obtain an energy density of 13.2 kJ/g compared with 12.5 kJ/g in the S . typhimurium lipopolysaccharide-challenged chicks . The S . typhimurium lipopolysaccharide-challenged chicks consumed similar amounts of the low energy diet but decreased intake of the high energy diet.

J Bacteriol, 1993 Oct, 175(19), 6368 - 71
Cloning of the phs genetic locus from Salmonella typhimurium and a role for a phs product in its own induction; Fong CL et al.; The Salmonella typhimurium phs chromosomal locus essential for the reduction of thiosulfate to hydrogen sulfide was cloned, and some features of its regulation were examined . The phs locus conferred H2S production on Escherichia coli, suggesting that it contains the structural gene for thiosulfate reductase . H2S production by the E . coli host was, as in S . typhimurium, suppressed by nitrate or glucose in the growth medium . The presence of plasmid-borne phs genes in a S . typhimurium chl+ host containing a chromosomal phs::lacZ operon fusion was found to significantly increase the relative induction efficiency of beta-galactosidase by thiosulfate . These results are consistent with a model for phs regulation in which the true inducer is not thiosulfate per se and in which the action of a phs-encoded molybdoprotein, possibly the reductase itself, converts thiosulfate into a compound that resembles the true inducer more closely than does thiosulfate.

J Bacteriol, 1993 Oct, 175(19), 6328 - 36
cobU-dependent assimilation of nonadenosylated cobinamide in cobA mutants of Salmonella typhimurium; O'Toole GA et al.; The cobA locus of Salmonella typhimurium is involved in the assimilation of nonadenosylated cobinamide, (CN)2CBI, into cobalamin (CBL) under aerobic and anaerobic growth conditions . Aerobically, cobA mutants are unable to assimilate (CN)2CBI into CBL . However, under anaerobic conditions, cobA mutants assimilate (CN)2CBI into CBL as efficiently as cobA+ strains . On the basis of this observation, we postulated the existence of a cobA-independent pathway for the assimilation of (CN)2CBI into CBL that is functional under anaerobic growth conditions (J . C . Escalante-Semerena, S.-J . Suh, and J . R . Roth, J . Bacteriol . 172:273-280, 1990) . In this paper, we report the isolation and initial genetic characterization of derivatives of cobA mutants that are unable to assimilate (CN)2CBI into CBL during anaerobic growth . As demonstrated by complementation analysis, marker rescue, and DNA sequencing data, these mutations are alleles of cobU, a gene involved in the assembly of the nucleotide loop of CBL . We have shown that the block in CBL synthesis in these cobU cobA double mutant strains can be corrected by exogenous adenosyl-CBI . Our data indicate that this new class of cobU mutations blocks CBL biosynthesis but does not destroy the putative kinase-guanylyltransferase activities of the CobU protein . We propose that this new class of cobU mutations may affect an as yet unidentified ATP:corrinoid adenosyltransferase activity of the CobU protein . Alternatively, such mutations may alter the ability of CobU to use nonadenosylated CBI as a substrate.

Infect Immun, 1993 Oct, 61(10), 4489 - 92
Role of acid tolerance response genes in Salmonella typhimurium virulence; Garcia-del Portillo F et al.; The atp and fur genes are involved in the acid tolerance response of Salmonella typhimurium . An atp::Tn10 mutant was avirulent in the mouse typhoid model when assayed by oral and intraperitoneal routes . However, a fur mutant was completely virulent by the intraperitoneal route . No relevant differences in intracellular survival or invasion rates were observed for the two mutants in macrophages and epithelial cells . These data indicate that separate acid tolerance response genes may have different roles in S . typhimurium virulence.

Epidemiol Infect, 1993 Oct, 111(2), 325 - 35
Bacteriophage as models for virus removal from Pacific oysters (Crassostrea gigas) during re-laying; Humphrey TJ et al.; A study was undertaken to examine the feasibility of using naturally-occurring bacteriophages to assess the impact of re-laying on levels of viral contamination in Crassostrea gigas, the Pacific oyster . Two phages were chosen . One, male-specific (F+), was enumerated using Salmonella typhimurium . The other, a somatic phage, was detected using an, as yet, uncharacterized Escherichia coli . Investigations, using a variety of re-laying sites, demonstrated that numbers of F+ phage in oyster tissue declined more rapidly than those of somatic phage . For example, in oysters placed in commercially-used sea water ponds, F+ phage reached undetectable levels within 2-3 weeks, whereas somatic phage could still be detected 5 weeks after re-laying . The studies suggest that F+ phage may not be a suitable indicator for virus removal and that somatic phage may be better suited to this role.

Epidemiol Infect, 1993 Oct, 111(2), 189 - 97
A comparison of multiple drug resistance in salmonellas from humans and food animals in England and Wales, 1981 and 1990; Threlfall EJ et al.; For Salmonella typhimurium from humans in England and Wales, the incidence of multiple resistance more than doubled over the 8-year period 1981-8 and, over the next 2 years, increased by a further 7% . From 1981 to 1988 both resistance and multiple resistance also increased significantly in S . virchow and although multiple resistance did not increase over the next 2 years, the overall incidence of resistance has continued to rise . In 1990 the majority of S . typhimurium from cattle were multiply-resistant and the occurrence of such resistance has quadrupled since 1981 . Multiple resistance has also increased in S . typhimurium from pigs and, to a lesser extent, from poultry . In contrast, multiple resistance has remained uncommon in the poultry-associated serotype S . enteritidis . For S . virchow, multiple resistance was common in a phage type frequently associated with poultry meat imported from France . The continuing use of a range of different antimicrobials in calf husbandry has been an important factor in promoting the emergence of multiply-resistant strains of S . typhimurium in cattle . In contrast, multiple resistance has remained rare in those serotypes associated with poultry, where the use of such antimicrobials has been less intensive . It is hoped that recent recommendations discouraging, in veterinary medicine, the prophylactic use of antibiotics with cross resistance to those used in human medicine will result in a reduction in the occurrence of multiresistant strains in food animals and subsequently in humans.

EMBO J, 1993 Oct, 12(10), 3779 - 87
Cognate gene clusters govern invasion of host epithelial cells by Salmonella typhimurium and Shigella flexneri; Groisman EA et al.; The enteric pathogens Salmonella typhimurium and Shigella flexneri differ in most virulence attributes including infectivity, pathology and host range . We have identified a new assemblage of genes responsible for invasion properties of Salmonella which is remarkably similar in order, arrangement and sequence to the gene cluster controlling the presentation of surface antigens (spa) on the virulence plasmid of Shigella . In Salmonella, this chromosomally encoded complex consists of over 12 genes, mutations in which abolish bacterial entry into epithelial cells . Although these genera use distinct invasion antigens, a non-invasive spa mutant of Salmonella could be rescued by the corresponding Shigella homolog . While spa promotes equivalent functions in Shigella and Salmonella, this constellation of genes has been acquired independently by each genus and displays motifs used by diverse antigen export systems including those required for flagellar assembly and protein secretion.

Genetika, 1993 Oct, 29(10), 1640 - 5
{Effect of structural features of nitro-derivatives of fluorenone and biphenyl on frameshift mutagenesis in tester strains of Salmonella typhimurium}; Abilev SK et al.; Comparative mutagenic activity of 7 derivatives of biphenyl and fluorenone, 4,4'-dinitrobiphenyl-2,2'-dicarboxylic acid; 4,4',6,6'-tetranitrobiphenyl-2,2'-dicarboxylamide; 2-nitrofluorenone-5-carboxylic acid; 2,7-dinitrofluorenone-5-carboxylic acid; 2,7-dinitrofluorenone-5-carboxylamide; 2-nitrofluorenone-5,7-dicarboxylic acid; 2,4-dinitrofluorenone-5,7-dicarboxylic acid was studied . The highest activity was demonstrated for 2,7-DNF-5-KA and 2,7-DNF-5,7-DK which induced frameshift mutations in the tester strains Salmonella typhimurium TA1537, TA97, TA1538, TA98 . High mutagenicity of these compounds is correlated with the position of nitro-groups and the effects of carboxylic and carboxyamide groups.

Eur J Cell Biol, 1993 Oct, 62(1), 152 - 62
Epithelioid and fibroblastic cell lines derived from the ileum of an adult histocompatible miniature boar (d/d haplotype) and immortalized by SV40 plasmid; Kaeffer B et al.; Intestinal explants were maintained for weeks in a growth medium containing collagenase for progressive digestion to derive finite cell lines from the ileum (64 lines) or from the colon (8 lines) of a boar . Two ileal cell lines retaining either a fibroblastic or an epithelioid morphology have been used to derive heteroploid cell lines (IPI-1 and IPI-2) immortalized by transfection with an SV40 plasmid (pSV3-neo) . The IPI-1 cells were found of fibroblastic lineage . The IPI-2 cell line gave rise to morphologically heterogeneous colonies ranging from typical epithelial cells to colonies of more-elongated cells . A crisis occurred during subcultivation of IPI-2 leading to the isolation of the IPI-2I cell line with a 24 h doubling time and a 21% plating efficiency . Epithelial nature of IPI-2I cells was supported by ultrastructural analysis of the cell monolayers . Differentiated cells were found to express microvilli at the apical cellular membrane and desmosomes connecting adjacent cells . Stable epithelioid phenotypes were obtained only from the IPI-2I cell line by multiple subcloning . These cells were found to express characteristics of both epithelial and mesenchymal cells by positive immunostaining with monoclonal antibodies reacting either with keratin 18 filament of simple epithelia or with vimentin filament typical in vivo of mesoderm . The lack of villin expression and the absence of transepithelial resistance have to be related to a poor differentiated state of this cell line . All these immortalized cell lines were permissive to the replication of microorganisms pathogenic for pig (Salmonella chloleraesuis, Salmonella typhimurium and tissue culture-adapted strains of transmissible gastroenteritis virus) . The collection of finite and continuous cell lines will help to develop in vitro methods for long-term propagation of freshly isolated epithelium or three-dimensional organ culture in pig . In addition, the IPI-2I cell line provides a new model to study the conversion from a transformed to a nontransformed phenotype as incorporation of 2% dimethyl sulfoxide in the growth medium to repress large tumor antigen expression led to the progressive disappearance of cytokeratin 18 positive cells with, over a week, the death of the surviving vimentin-positive cells.

Can J Vet Res, 1993 Oct, 57(4), 281 - 7
Virulence of Salmonella enteritidis phagetypes 4, 8 and 13 and other Salmonella spp . for day-old chicks, hens and mice; Poppe C et al.; Virulence of three Canadian poultry strains of Salmonella enteritidis, namely phagetypes (PT) 4, 8 and 13, and one Salmonella heidelberg strain was assessed in orally and intraperitoneally inoculated one-day old chickens and compared to the virulence of a human S . enteritidis PT 4 strain from the United Kingdom (UK) . The two PT 4 strains were also compared in orally inoculated adult laying hens . In addition, orally inoculated Balb/c mice were used to evaluate virulence of the above strains and two strains of Salmonella typhimurium containing different plasmids . In orally inoculated one-day old chickens, the UK S . enteritidis PT 4 strain was more virulent than the Canadian PT 4 strain . The UK PT 4 strain was also more virulent and invasive in adult laying hens than the Canadian PT 4 strain . The S . enteritidis PT 8 strain and one S . typhimurium strain isolated from a chicken hatchery were the most virulent for orally inoculated Balb/c mice . This strain of S . typhimurium contained the 60 megadalton plasmid associated with virulence for Balb/c mice which was not present in the S . typhimurium strain isolated from a pig with septicemic disease.

Immunology, 1993 Oct, 80(2), 306 - 12
Influence of the antigen delivery system on immunoglobulin isotype selection and cytokine production in response to influenza A nucleoprotein; Brett SJ et al.; The influence of different antigen delivery systems on antibody isotype and lymphokine profile has been investigated using influenza nucleoprotein as a model antigen system . Mice exposed to live or inactivated influenza virus produced antibody against whole virus or recombinant nucleoprotein (rNP), which was predominantly of the IgG2a isotype . Spleen or lymph node cells from these mice rapidly produced large amounts of interferon-gamma (IFN-gamma), but no detectable interleukin-5 (IL-5) when stimulated in vitro with specific antigen . In contrast, after primary immunization with rNP or p206-229 in different adjuvants (CFA, quil A or alhydrogel), specific antibody was predominantly of the IgG1 isotype and relatively lower amounts of IFN-gamma but no IL-5 were detected following in vitro antigenic stimulation . Secondary immunization, however, resulted in detection of IgG2a antibodies and increased levels of IFN-gamma . IL-5 was only detected after secondary immunization with peptide in adjuvant . Mice infected with aro A- Salmonella typhimurium expressing NP produced antibody of both IgG1 and IgG2a isotypes and large amounts of IFN-gamma and no IL-5, following in vitro antigenic stimulation, and therefore parallelled the pattern seen with whole virus more closely than that seen following primary immunization with protein or peptide in conventional adjuvants . The results suggest that the antigen delivery vehicle influences both quantitative and qualitative differences in the type of immune response elicited, which may be important in determining the potency of protective immunity induced.

Antimicrob Agents Chemother, 1993 Oct, 37(10), 2251 - 3
Susceptibilities of oxyR regulon mutants of Escherichia coli and Salmonella typhimurium to isoniazid; Rosner JL; Escherichia coli and Salmonella typhimurium are normally resistant to > 500 micrograms of the antituberculosis drug isonicotinic acid hydrazide (isoniazid; INH) per ml . Susceptibility to INH (< 50 micrograms/ml) has now been found for mutants that are deficient in OxyR, the oxidative stress response regulator . Two OxyR-regulated enzymes, alkyl hydroperoxide reductase and hydroperoxidase I, were identified as playing important roles in INH resistance . OxyR regulon mutants should be useful for identifying other determinants of INH resistance in both E . coli and Mycobacterium tuberculosis and for finding new INH-like drugs.

Protein Sci, 1993 Oct, 2(10), 1643 - 7
Dominant role of local dipoles in stabilizing uncompensated charges on a sulfate sequestered in a periplasmic active transport protein; He JJ et al.; Electrostatic interactions are among the key factors determining the structure and function of proteins . Here we report experimental results that illuminate the functional importance of local dipoles to these interactions . The refined 1.7-A X-ray structure of the liganded form of the sulfate-binding protein, a primary sulfate active transport receptor of Salmonella typhimurium, shows that the sulfate dianion is completely buried and bound by hydrogen bonds (mostly main-chain peptide NH groups) and van der Waals forces . The sulfate is also closely linked, via one of these peptide units, to a His residue . It is also adjacent to the N-termini of three alpha-helices, of which the two shortest have their C-termini "capped" by Arg residues . Site-directed mutagenesis of the recombinant Escherichia coli sulfate receptor had no effect on sulfate-binding activity when an Asn residue was substituted for the positively charged His and the two Arg (changed singly and together) residues . These results, combined with other observations, further solidify the idea that stabilization of uncompensated charges in a protein is a highly localized process that involves a collection of local dipoles, including those of peptide units confined to the first turns of helices . The contribution of helix macrodipoles appears insignificant.

Appl Environ Microbiol, 1993 Oct, 59(10), 3509 - 12
Comparison of methods for specific depletion of ATP in Salmonella typhimurium; Johnson MS et al.; Three methods of ATP depletion in Salmonella typhimurium were compared . ATP concentrations were lowest after arsenate treatment . Arsenate or alpha-methylglucoside-plus-azide treatment nonspecifically lowered all nucleotide triphosphate levels . Histidine starvation in a hisF mutant was relatively specific for ATP depletion and therefore has potential in distinguishing ATP-dependent processes from processes dependent on other nucleotides.

Food Chem Toxicol, 1993 Oct, 31(10), 707 - 15
Bioassay of quinoline, 5-fluoroquinoline, carbazole, 9-methylcarbazole and 9-ethylcarbazole in newborn mice; Weyand EH et al.; Quinoline and carbazole are among the more prevalent aza-arenes present as components of environmental pollutants . Both of these aza-arenes are hepatocarcinogenic to mice when administered in the diet . The hepatocarcinogenic potential of quinoline is consistent with its mutagenic activity in Salmonella typhimurium TA100 and potential to induce unscheduled DNA synthesis (UDS) in rat hepatocytes . Structure-activity studies with fluorinated quinolines indicate that the presence of a fluorine atom at the 5-position of quinoline may inhibit detoxification and result in enhanced genotoxic potency . Quinoline and 5-fluoroquinoline were assayed in newborn CD-1 mice at a total dose of 1.75 mumol to establish their relative tumorigenic activity . Liver tumours developed in 60 and 90% of the male newborn mice treated with quinoline and 5-fluoroquinoline, respectively . The majority of liver tumours observed among the quinoline-treated mice were classified as adenomas . In contrast, liver carcinomas developed in most of the male mice treated with 5-fluoroquinoline . Unlike the well established genotoxic potential of quinoline, there is limited evidence for carbazole having either genotoxic or carcinogenic activity . Whereas carbazole is not mutagenic towards several strains of S . typhimurium, both 9-methylcarbazole and 9-ethylcarbazole are active as mutagens in S . typhimurium TA100 . Carbazole, 9-methylcarbazole and 9-ethylcarbazole were assayed in primary rat hepatocytes to assess their relative potential to induce UDS in rat hepatocytes; only 9-ethylcarbazole did so . These carbazole derivatives were also assayed in newborn CD-1 mice at a total dose of 1.75 mumol . Neither carbazole nor either of these 9-alkylcarbazoles produced a significant tumorigenic response in this bioassay system.

Carcinogenesis, 1993 Oct, 14(10), 2039 - 43
DNA adduct formation in Salmonella typhimurium, cultured liver cells and in Fischer 344 rats treated with o-tolyl phosphates and their metabolites; Mentzschel A et al.; 2-Phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide is an electrophilic and a neurotoxic metabolite of o-tolyl phosphates . In a previous paper we reported that 2-phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide is mutagenic in Salmonella typhimurium TA100 and forms DNA adducts in incubations with nucleotides, nucleosides and isolated DNA . In the present study we compare DNA adduct formation using 32P-post-labelling assays in 2-phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide-treated bacteria (S.typhimurium TA100) and hepatoma cells with DNA adducts formed in liver, kidney, lung and heart of tri-o-tolyl phosphate-exposed Fischer 344 male rats . In both bacteria and hepatoma cells two DNA adducts could be detected after treatment with 2-phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide . The minor adduct co-chromatographed with synthetic N3-(o-hydroxy-benzyl)deoxyuridine 3' monophosphate after postlabelling . The major DNA adduct was a cytidine adduct, most likely N3-(o-hydroxybenzyl)deoxycytidine 3' monophosphate . Male Fischer 344 rats were treated orally for 10 days with tri-o-tolyl phosphate (50 mg/kg/day) and DNA was isolated from liver, kidney, lung, heart, brain and testes 1, 4, 7 and 28 days after giving the last dose . Analysis by 32P-postlabelling revealed that two adducts were present in the DNA isolated from liver, kidney, lung and heart on the first day after giving the last dose; DNA adducts were not detected in the brain and testes . The adduct pattern after in vivo treatment with tri-o-tolyl phosphate was identical with that found in bacteria and hepatoma cells treated with 2-phenoxy-4H-1,3,2-benzo-dioxaphosphorin 2-oxide, the major adduct being N3-(o-hydroxybenzyl)deoxycytidine 3' monophosphate and the minor N3-(o-hydroxybenzyl)deoxyuridine 3' monophosphate . Both DNA adducts persisted in the lungs for the entire observation period, whereas in the kidney only the cytidine adduct could be detected 28 days after the last dose of tri-o-tolyl phosphate . In liver and heart the adducts were detectable only on the first day after completion of the treatment . The results indicate that in addition to the well established neurotoxicity, some o-tolyl phosphates may have a carcinogenic potential.

J Environ Pathol Toxicol Oncol, 1993 Oct-Dec, 12(4), 185 - 91
The involvement of reactive oxygen species in the direct-acting mutagenicity of 5-nitro-3-thiophenecarboxanilides in Salmonella typhimurium; Hrelia P et al.; The primary basis of 5-nitro-3-thiophenecarboxanilides (NTCAs) direct-acting mutagenicity in Salmonella typhimurium appears to be the reduction of the nitro function to the corresponding hydroxylamine via diamagnetic and free radicals intermediates . In Ames test conditions, mutagenicity of NTCAs may be partly attributed to the action of reactive oxygen species and other radicals generated during the bioreductive process . The nitro anion radical seems to be particularly susceptible to react with oxygen and generate superoxide, as shown by the inhibitory effects exerted by superoxide dismutase on genotoxicity by most NTCAs in the study . On the other hand, hydroxyl radical-trapping agents such as mannitol, the enzyme catalase and other scavengers as 3-tert-butyl-4-hydroxyanisole (BHA), and vitamins C, A, and E showed weak inhibitory potency, and rather increased the mutagenic activity of some NTCAs . Our results contribute to the mechanistic understanding of genotoxic activity of NTCAs.

Environ Health Perspect, 1993 Oct, 101 Suppl 3, 53 - 6
Mutagenicity and human chromosomal effect of stevioside, a sweetener from Stevia rebaudiana Bertoni; Suttajit M et al.; Leaves of Stevia rebaudiana Bertoni have been popularly used as a sweetener in foods and beverages for diabetics and obese people due to their potent sweetener stevioside . In this report, stevioside and steviol were tested for mutagenicity in Salmonella typhimurium strains TA98 and TA100 and for chromosomal effects on cultured human lymphocytes . Stevioside was not mutagenic at concentrations up to 25 mg/plate, but showed direct mutagenicity to only TA98 at 50 mg/plate . However, steviol did not exhibit mutagenicity in either TA98 or TA100, with or without metabolic activation . No significant chromosomal effect of stevioside and steviol was observed in cultured blood lymphocytes from healthy donors (n = 5) . This study indicates that stevioside and steviol are neither mutagenic nor clastogenic in vitro at the limited doses; however, in vivo genotoxic tests and long-term effects of stevioside and steviol are yet to be investigated.

Environ Health Perspect, 1993 Oct, 101 Suppl 3, 33 - 6
Evaluation of mutagenicity testing with Salmonella typhimurium TA102 in three different laboratories; Muller W et al.; Thirty compounds of various chemical classes were investigated for mutagenicity in a collaborative study (three laboratories) using Salmonella typhimurium TA102 . With five compounds, hydrazine sulfate, phenylhydrazine, hydralazine, glutardialdehyde, and glyoxal, mutagenicity was detected by all laboratories . Formaldehyde was assessed as weakly mutagenic in only one of three laboratories . The remaining 24 agents were uniformly described as non-genotoxic in TA102 . In spite of the overall good qualitative agreement in the mutagenicity results between the three laboratories, some quantitative discrepancies occurred in the dose response of the mutagenic compounds . Varying inter- and intralaboratory differences in the spontaneous rate of revertants were obtained . The usefulness of the tester strain TA102 in routine mutagenicity testing is discussed.

Environ Health Perspect, 1993 Oct, 101 Suppl 3, 27 - 31
Metabolism of 2-aminofluorene by human polymorphonuclear leukocytes: more evidence for the association between inflammation and cancer; Isola VJ et al.; Recent investigations have demonstrated the ability of leukocytes to metabolize promutagens or procarcinogens into their genotoxic forms . As a possible explanation for the association between inflammation and cancer, we and others have hypothesized that local accumulations of leukocytes could take up nearby promutagens, metabolize them, and release genotoxic agents that may cause damage in the surrounding tissue . Using a modified, two-step preincubation protocol with Salmonella, we have tested this hypothesis . We have shown that total human peripheral blood leukocytes, cultured in the presence of 2-aminofluorene for 18 hr, can metabolize 2-aminofluorene into agents mutagenic to Salmonella typhimurium strain TA98 . Furthermore, experiments in which polymorphonuclear leukocytes were separated from mononuclear leukocytes demonstrated that the PMNs metabolized 2-aminofluorene to a much greater extent than the MNs.

Environ Health Perspect, 1993 Oct, 101 Suppl 3, 21 - 6
Mutagenicity testing of 9-N-substituted adenines and their N-oxidation products; Gorrod JW et al.; Adenine together with certain 9-N-substituted derivatives such as 9-methyl, 9-benzyl, 9-benzhydryl, and 9-trityl were tested against Salmonella typhimurium strains TA97, TA98, and TA100 in the absence and presence of rat hepatic S9 prepared from Aroclor 1254 pretreated rats . All compounds were positive toward TA98 in the presence of the metabolic activating system, whereas they all lacked mutagenic activity in the absence of S9, and toward TA97 and TA100 with or without S9 when tested at 100 ng/plate . A similar pattern was observed for the corresponding 1-N-oxides . 6-Hydroxylaminopurine was not mutagenic toward TA100 at 100 ng/plate, whereas it was toxic toward TA97 and TA98 at this level . When tested at 1 ng/plate, hydroxylaminopurine was still toxic to TA98 but produced twice the spontaneous reversion rate to TA97 without metabolic activation . Surprisingly, 9-methyl-6-hydroxylaminopurine was only active toward TA98 in the presence of S9, whereas 9-benzyl-6-hydroxylaminopurine was highly active toward TA97 and TA100 in the absence of S9 and even more active in the presence of S9 . This compound was inactive toward TA98 in the absence of S9 . The results generally support the concept that nuclear N-oxidation of aminoazaheterocycles is a detoxication process, whereas N-hydroxylation of the exo amino group is a toxication reaction.

Avian Dis, 1993 Oct-Dec, 37(4), 1051 - 6
Effectiveness of dietary propionic acid in controlling Salmonella typhimurium colonization in broiler chicks; Hume ME et al.; Newly hatched broiler chicks were provided a corn/soybean meal-based ration treated with propionic acid at 30 mumol/g of feed ration . At 3 days of age, the chicks were challenged orally with 10(4) Salmonella typhimurium . Crop contents from 4-day-old chicks that were provided dietary propionic acid contained significantly higher concentrations of propionic acid (4.0 to 6.8 mumol/g crop contents) than crops from challenged control chicks provided untreated feed (0.9 to 1.5 mumol/g crop contents) . Provision of dietary propionic acid on feed as a dry powder in five trials or a liquid application in three trials had no significant effect on crop or cecal pH . Significant decreases in Salmonella in the crop and ceca were detected in one trial, but the decreases were likely the result of the presence of anti-salmonellae bacteria rather than the dietary propionic acid . Results indicate that propionic acid in the feed was ineffective in reducing Salmonella infection in the crop and ceca.

Rev Latinoam Microbiol, 1993 Oct-Dec, 35(4), 345 - 9
Evaluation of the in vitro susceptibility and emergence of mutants resistant to ciprofloxacin among multidrug resistant clinical isolates; Lopes CA et al.; Two hundred and seventy-seven multidrug resistant clinical isolates {K . pneumoniae, (N = 87); E coli, (N = 30); Salmonella typhimurium (N = 100); P . aeruginosa, (N = 30); S . aureus, (N = 30)} from hospitalized patients specimens, were tested in vitro for sensitivity to Ciprofloxacin . Application of the disk diffusion test and determination of the minimal inhibitory concentration by the microdilution method indicated that, almost all isolates were sensitive to the drug . Overall, S . aureus and P . aeruginosa were the less sensitive organisms . Ciprofloxacin-resistant mutants occurred at frequencies of > or = 10(-5)/CFU.

Mol Microbiol, 1993 Oct, 10(1), 75 - 86
The influence of DNA topology on the environmental regulation of a pH-regulated locus in Salmonella typhimurium; Karem K et al.; Salmonella typhimurium is exposed to major shifts in H+ concentration both in its natural and pathogenic environments . The organism undergoes extensive changes in gene expression in response to these pH fluctuations . A current question of regulatory biology is how a change in external pH selectively modulates transcription . We have analysed the expression of one such pH-regulated locus, aniG, and found it is controlled by several additional environmental conditions including osmolarity and oxygen . For factors such as osmolarity and anaerobiosis, an environmentally triggered change in DNA supercoiling has been suggested as a means for controlling gene expression . Thus, environmentally induced changes in DNA topology were explored as a possible common means for establishing the multiple controls on aniG . The involvement of DNA supercoiling in the genetic response of S . typhimurium to external pH has not previously been defined . This report establishes that alkaline environments lower the linking number of reporter plasmids when compared to acidic environments . A consistent pattern was then established whereby conditions or mutations leading to either increased or decreased negative supercoiling were associated with altered expression of aniG . A similar relationship was observed for another environmentally regulated locus, proU . The DNA topology effects on aniG expression were dependent on the presence of EarA, the negative regulator of aniG . These data can be explained by a model in which repressor-operator interactions are very sensitive to changes in operator conformation . These environmentally induced topological influences on operator DNA structure contribute to the magnitude of pH control exerted upon aniG.

Mol Microbiol, 1993 Oct, 10(1), 113 - 22
Integration host factor binds to a unique class of complex repetitive extragenic DNA sequences in Escherichia coli; Oppenheim AB et al.; Interspersed repeated DNA sequences are characteristic features of both prokaryotic and eukaryotic genomes . REP sequences are defined as conserved repetitive extragenic palindromic sequences and are found in Escherichia coli, Salmonella typhimurium and other closely related enteric bacteria . These REP sequences may participate in the folding of the bacterial chromosome . In this work we describe a unique class of 28 conserved complex REP clusters, about 100bp long, in which two inverted REPs are separated by a singular integration host factor (IHF) recognition sequence . We term these sequences RIP (for repetitive IHF-binding palindromic) elements and demonstrate that IHF binds to them specifically . It is estimated that there are about 70 RIP elements in E . coli . Our analysis shows that the RIP elements are evenly distributed around the bacterial chromosome . The possible function of the RIP element is discussed.

Mol Microbiol, 1993 Oct, 10(2), 273 - 82
Expression of the gene encoding the major bacterial nucleotide protein H-NS is subject to transcriptional auto-repression; Falconi M et al.; Expression of a promoterless cat gene fused to a DNA fragment of approximately 400 bp, beginning at -313 of Escherichia coli hns, was significantly repressed in E . coli and Salmonella typhimurium strains with wild-type hns but not in mutants carrying hns alleles . CAT expression from fusions containing a shorter (110 bp) segment of hns was essentially unaffected in the same genetic backgrounds . The stage of growth was found to influence the extent of repression which was maximum (approximately 75%) in mid-log cultures and negligible in cells entering the stationary phase . The level of repression in early-log phase was lower than in mid-log phase cultures, probably because of the presence of high levels of Fis protein, which counteracts the H-NS inhibition by stimulating hns transcription . The effects observed in vivo were mirrored by similar results obtained in vitro upon addition of purified H-NS and Fis protein to transcriptional systems programmed with the same hns-cat fusions . Electrophoretic gel shift assays, DNase I footprinting and cyclic permutation gel analyses revealed that H-NS binds preferentially to the upstream region of its own gene recognizing two rather extended segments of DNA on both sides of a bend centred around -150 . When these sites are filled by H-NS, an additional site between approximately -20 and -65, which partly overlaps the promoter, is also occupied . Binding of H-NS to this site is probably the ultimate cause of transcriptional auto-repression.

Am J Vet Res, 1993 Oct, 54(10), 1648 - 52
Use of random fragments of chromosomal DNA to highlight restriction site heterogeneity for fingerprinting isolates of Salmonella typhimurium from hospitalized animals; Hansen LM et al.; Random fragments of DNA were obtained from a cosmid library of Salmonella agona genomic DNA . From this library, 2 fragments were chosen and pooled to probe isolates of S typhimurium obtained during an episode of salmonellosis in a veterinary medical teaching hospital . Chromosomal DNA from the Salmonella isolates was digested with restriction endonucleases, and was probed with the random fragments of chromosomal DNA . This procedure resulted in a fingerprint pattern for each isolate . We found that the method permitted discrimination between isolates involved in the disease episode and S typhimurium obtained prior to the episode . We conclude that random fragments of chromosomal DNA are useful for fingerprinting isolates of S typhimurium . Analysis of plasmid DNA obtained from the isolates was not as useful . Some isolates found to be identical by restriction site analysis, had plasmids of different molecular weight . These results indicate that plasmid analysis may not be as useful a fingerprinting tool as previously reported.

Mutat Res, 1993 Oct, 303(2), 63 - 70
Identification of heterogenous antimutagenic activities in the extract of edible brown seaweeds, Laminaria japonica (Makonbu) and Undaria pinnatifida (Wakame) by the umu gene expression system in Salmonella typhimurium (TA1535/pSK1002); Okai Y et al.; A significant antimutagenic activity was found in the hot water-soluble extract from a common edible brown alga, Laminaria japonica (Makonbu in Japanese) which showed suppressive effects on umu gene expression of the SOS response against DNA damage in Salmonella typhimurium (TA1535/pSK1002) . The extract showed a drastic antimutagenic activity against 2-acetylaminofluorene (2-AAF)- or 3-amino-1,4-dimethyl-5H-pyrido{4,3-b}indole (Trp-P-1)-induced mutagenesis which requires liver-metabolizing enzymes, whereas the same extract exhibited weak but significant inhibitory effects on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)- or furylfuramide (AF-2)-induced mutagenesis in the absence of liver-metabolizing enzymes . Among these antimutagenic activities, the minor activity was found in the polysaccharide fraction of the extract which showed roughly equal antimutagenic activities against all the mutagens tested . The major activity was detected in the nonpolysaccharide fraction which exhibited a relatively strong antimutagenic activity against 2-AAF- or Trp-P-1-induced mutagenesis but a weak activity against MNNG- or AF-2-induced mutagenesis . The nonpolysaccharide fraction was further separated into high- or low-molecular-weight fractions and the latter fraction showed a much stronger activity than the former fraction . In addition, similar antimutagenic activities were detected in polysaccharide and nonpolysaccharide fractions from the extract of the other edible brown alga, Undaria pinnatifida (Wakame in Japanese) . These experimental results indicate that the hot water-soluble extract of Laminaria japonica or Undaria pinnatifida contains heterogenous antimutagenic activities against typical genotoxic substances . The significance of this finding is discussed from the viewpoint of the protection against genotoxic substances by traditional edible seaweeds in Japan.

Mutat Res, 1993 Oct, 303(2), 55 - 61
Mutagenic activity of urban air samples and its modulation by chili extracts; Espinosa-Aguirre JJ et al.; Different samples of ambient particulate organic matter were collected during the summer and winter of 1990 in Mexico City . After dichloromethane extraction, the samples were tested for mutagenicity with derivatives of Salmonella typhimurium possessing high activity of 'classical' nitroreductase (YG1021) or O-acetyltransferase (YG1024), and compared to the mutagenicity of the normal strain YG1020, and to that of a nitroreductase-deficient mutant TA98NR . The two enzyme-overproducing strains were more sensitive to the mutagenic effect of the extracts than the parent and deficient strains . The sensitivity order, i.e., YG1024 > YG1021 > YG1020 > TA98NR, emphasizes the usefulness of the new Salmonella strains in analyzing the mutagenicity of complex mixtures and suggests that some of the direct mutagenic compounds in the urban air samples are nitro-aromatics . Investigations were also conducted to analyze the effect of chili extract on the mutagenicity of an urban air sample . The extract itself showed moderate mutagenic activity and an additive effect was noted when both the chili and air extracts were present . On the other hand, the maximum volume of chili tested produced a decrease in the number of revertants without affecting the background lawn of bacterial growth . The same response was also observed when 1-nitropyrene, 1,6-dinitropyrene or 1,8-dinitropyrene was used as the genotoxic compound, although potentiation instead of addition occurred at low vegetable volumes . At the concentrations found in the chili extract, chlorophyllin and beta-carotene showed an antimutagenic effect against the nitro-aromatic compounds.

Mutat Res, 1993 Oct, 292(2), 155 - 63
A new Salmonella tester strain expressing a hamster acetyltransferase shows high sensitivity for arylamines; Ando M et al.; A hamster acetyltransferase, AT-I, has high activities for N-acetylation of arylamines, O-acetylation of N-hydroxyarylamines and N,O-acetyltransfer of N-hydroxyarylacetamides . In the present study, the cDNA was expressed in Salmonella typhimurium TA1538 . The new SAT138 strain expressing high levels of AT-I showed remarkably high sensitivity (> 10,000 fold) for a carcinogenic intermediate, N-hydroxy-2-acetylaminofluorene, in an Ames mutagenesis test as compared to the parental TA1538 strain . SAT138 had 650-1,600-fold higher sensitivities for mutagenesis induced by 2-acetylaminofluorene and benzidine in the presence of S9 mix . Higher sensitivities (32-560-fold) were also observed with N-hydroxy-2-aminofluorene, N-hydroxy-4-aminobiphenyl, N-hydroxy-4-acetylaminobiphenyl, N-hydroxy-4-propionylaminobiphenyl and N-hydroxy-phenacetin in the absence of S9 mix . These high sensitivities to arylamines and the related chemicals are accounted for by the efficient expression of AT-I in the cytosol of this bacterium . The unique characteristics of SAT138 having high N-hydroxyarylacetamide N,O-transacetylating activity, which is defective in Salmonella acetyltransferase, provide broadened and high sensitivities for the detection of mutagenic N-substituted chemicals in the Salmonella mutagenesis test.

J Biol Chem, 1993 Sep 25, 268(27), 19991 - 7
Purification and characterization of the periplasmic domain of the aspartate chemoreceptor; Milligan DL et al.; In order to facilitate biochemical studies of cell-surface receptors, a plasmid allowing the expression of the periplasmic domain of the aspartate receptor from Salmonella typhimurium as a soluble periplasmic protein has been constructed . This 18-kDa protein is exported to the periplasm, where it may be extracted by mild osmotic lysis . This isolated domain behaves as a normal, soluble protein and has been purified to homogeneity by standard techniques . The purified periplasmic domain binds aspartate with a kD similar to that of the full-length receptor, and the binding occurs with negative cooperativity, i.e . the binding of one molecule of aspartate induces a conformational change that interferes with the binding of the second aspartate . Unlike the full-length receptor, the periplasmic domain undergoes a protein concentration- and aspartate-dependent monomer-dimer equilibrium . At low protein concentrations and in the absence of aspartate, the protein is monomeric . At higher protein concentrations or in the presence of saturating aspartate, the protein is dimeric . Two charge variants of the protein have been identified on native polyacrylamide gels . The more acidic form is blocked to Edman degradation, indicating that modification of the amino terminus of this protein can occur after cleavage of the signal peptide in the periplasm.

J Biol Chem, 1993 Sep 25, 268(27), 20299 - 304
Active site lysines in orotate phosphoribosyltransferase; Grubmeyer C et al.; Orotate phosphoribosyltransferase (OPRTase; EC 2.4.2.10) catalyzes the formation of the nucleotide orotidine-5'-monophosphate from orotate and 5-phosphoribosyl-1-pyrophosphate (PRPP) . The bacterial enzyme, unlike its mammalian homolog, is monofunctional and is a dimer of M(r) 23,000 subunits . The availability of large amounts of highly purified crystalline Salmonella typhimurium OPRTase have enabled us to being structure/function studies on the enzyme . Like other phosphoribosyltransferases, OPRTase binds the highly charged MgPRPP complex, as well as anionic orotate, suggesting an active site containing basic residues . The S . typhimurium sequence (Scapin, G., Sacchettini, J . C., Dessen, A., Bhatia, M., and Grubmeyer, C . (1993) J . Mol . Biol . 230, 1304-1308) contains 12 lysine and 13 arginine residues, of which Lys-26, Arg-99, Lys-100, Lys-103, and Arg-156 are conserved as identities among the sequences of OPRTases from other organisms, with Lys-19 and Arg-161 replaced by the alternative basic residue in one or more sequences . The lysine modifier 2,4,6-trinitrobenzene sulfonate inactivated S . typhimurium OPRTase in a pseudo first-order process, and OMP and PRPP provided good protection against inactivation . Spectral quantitation of trinitrophenyl (TNP) group incorporation showed that inactivation was correlated with incorporation of one TNP group per subunit . Surprisingly, tryptic proteolysis followed by high performance liquid chromatography and amino acid sequence analysis revealed that four peptides, containing three distinct lysines, had been modified . Peptides modified at Lys-26, Lys-100 and Lys-103, as well as a doubly modified peptide containing TNP groups at Lys-100 and Lys-103, were identified . Inactivation kinetics showed that the 3 lysine residues were modified at equal rates . Protection studies demonstrated that Lys-26, and to a lesser extent Lys-100 and Lys-103, were protected against modification by OMP, whereas PRPP protected Lys-26, Lys-100 and Lys-103 . Pyrophosphate protected Lys-100 and Lys-103 . The results suggest active site locations for the sequence-conserved and TNP-modified lysine residues, with Lys-26 interacting with the ribose-5-phosphate moiety of OMP and PRPP, and Lys-100 and Lys-103 interacting with the pyrophosphate moiety of PRPP.

Proc Natl Acad Sci U S A, 1993 Sep 15, 90(18), 8576 - 80
Expression of mammalian glutathione S-transferase 5-5 in Salmonella typhimurium TA1535 leads to base-pair mutations upon exposure to dihalomethanes; Thier R et al.; Dihalomethanes can produce liver tumors in mice but not in rats, and concern exists about the risk of these compounds to humans . Glutathione (GSH) conjugation of dihalomethanes has been considered to be a critical event in the bioactivation process, and risk assessment is based upon this premise; however, there is little experimental support for this view or information about the basis of genotoxicity . A plasmid vector containing rat GSH S-transferase 5-5 was transfected into the Salmonella typhimurium tester strain TA1535, which then produced active enzyme . The transfected bacteria produced base-pair revertants in the presence of ethylene dihalides or dihalomethanes, in the order CH2Br2 > CH2BrCl > CH2Cl2 . However, revertants were not seen when cells were exposed to GSH, CH2Br2, and an amount of purified GSH S-transferase 5-5 (20-fold excess in amount of that expressed within the cells) . HCHO, which is an end product of the reaction of GSH with dihalomethanes, also did not produce mutations . S-(1-Acetoxymethyl)GSH was prepared as an analog of the putative S-(1-halomethyl)GSH reactive intermediates . This analog did not produce revertants, consistent with the view that activation of dihalomethanes must occur within the bacteria to cause genetic damage, presenting a model to be considered in studies with mammalian cells . S-(1-Acetoxymethyl)GSH reacted with 2'-deoxyguanosine to yield a major adduct, identified as S-{1-(N2-deoxyguanosinyl)methyl}GSH . Demonstration of the activation of dihalomethanes by this mammalian GSH S-transferase theta class enzyme should be of use in evaluating the risk of these chemicals, particularly in light of reports of the polymorphic expression of a similar activity in humans.

FEMS Microbiol Lett, 1993 Sep 15, 112(3), 251 - 4
Correlation between umuC induction and Salmonella mutagenicity assay for quinolone antimicrobial agents; Power EG et al.; Quinolone antimicrobial agents induce the SOS response in bacteria, including the umuDC genes necessary for error-prone repair . Consequently these drugs may be mutagenic in bacteria with a functional SOS response . Differential killing tests with Escherichia coli WP2 (trp) and its repair-deficient derivative CM871 (trp lexA recA uvrA) indicated that a functional DNA repair system was protective against the action of quinolones, implying that quinolones are causing some form of DNA damage (not necessarily directly) and are therefore genotoxic . Dose-dependent reversion from His- to His+ with quinolones was observed in the Ames test with Salmonella typhimurium TA102 (uvr+) but in no other Salmonella tester strains (all uvr-), suggesting that a functional excision repair system is essential for quinolone-induced bacterial mutagenesis . A significant correlation between SOS inducing potential (SOSIP) and mutagenic potential in the Ames test (r = 0.89) indicated that quinolone-induced mutagenic effects in bacteria are almost entirely due to SOS-processed DNA damage.

J Biol Chem, 1993 Sep 5, 268(25), 18633 - 6
Cloning and characterization of a 23-kDa stress-induced mouse peritoneal macrophage protein; Ishii T et al.; Exposure of mouse peritoneal macrophages to oxidative and sulfhydryl-reactive agents in vitro enhances synthesis of a few cellular proteins that may be important in a self-defense system . A cDNA encoding a novel stress-inducible protein, designated MSP23 (macrophage 23-kDa stress protein), was cloned from a cDNA library of the macrophages by differential screening . A 1.0-kilobase mRNA transcript hybridized with the MSP23 cDNA gradually increased in macrophages upon culture in vitro . Treatment with diethylmaleate or glucose/glucose oxidase, which generates H2O2, markedly enhanced the induction of the transcript after several hours . Cadmium chloride and sodium arsenite also induced the transcript . An antiserum raised against recombinant MSP23 reacted with the 23-kDa stress-inducible protein of the macrophages . The amounts of 23-kDa protein in the cells rapidly increased during culture with diethylmaleate . The mRNA was detected in various tissues, and it was especially high in content in the liver . A search of databases revealed that six proteins of various species from bacteria to the mouse have a sequence homology to MSP23 . One of the proteins is the C22 component of alkyl hydroperoxide reductase, which is induced by hydrogen peroxide in Salmonella typhimurium.

J Biol Chem, 1993 Sep 5, 268(25), 18617 - 21
The ATP-binding cassette (ABC) transporter for maltose/maltodextrins of Salmonella typhimurium . Characterization of the ATPase activity associated with the purified MalK subunit; Morbach S et al.; The ATPase activity associated with the purified MalK subunit of the maltose transport complex of Salmonella typhimurium, a bacterial ATP-Binding Cassette (ABC) transporter (Walter, C., Honer zu Bentrup, K., and Schneider, E . (1992) J . Biol . Chem . 267, 8863-8869), was characterized in detail . The analysis of the kinetics of ATP hydrolysis yielded a Km value of 70 +/- 4 microM and a Vmax of 1.3 +/- 0.3 mumol/min/mg of protein . Both GTP and CTP also served as substrates . While MalK exhibited nearly the same affinity for GTP as for ATP, the Michaelis constant for CTP as a substrate was much higher . ATP hydrolysis was strongly dependent on the presence of Mg2+ ions . Mn2+ at low concentrations, but neither Ca2+ nor Zn2+ partially substituted for Mg2+ . The ATPase activity was optimal at slightly alkaline pH and was stimulated in the presence of both glycerol (7.5%) and dimethyl sulfoxide (Me2SO) (5%) . ADP and the non-cleavable substrate analog ATP gamma S (adenosine 5'-O-(3-thiotriphosphate)) were identified as competitive inhibitors . The MalK-ATPase was resistant to specific inhibitors of F-, P-, and V-type ATPases, such as dicyclohexylcarbodiimide, azide, vanadate, or bafilomycin A1 . In contrast, micromolar concentrations of the sulfhydryl reagent N-ethylmaleimide strongly inhibited the enzymatic activity . This inhibition was blocked in the presence of ATP . These results suggest that the intrinsic ATPase activity of purified MalK can be clearly distinguished from other ATP-hydrolyzing enzymes, e.g . ion-translocating ATPases.

Mol Gen Genet, 1993 Sep, 240(3), 360 - 4
The pleiotropic effects of his overexpression in Salmonella typhimurium do not involve AICAR-induced mutagenesis; Flores A et al.; Inhibition of cell division associated with overexpression of hisH and hisF in Salmonella typhimurium is strongly reminiscent of a cellular response to DNA damage . On these grounds, we investigated the involvement of a metabolite which appeared to represent a possible candidate for an endogenous mutagen: the base analog 5-amino-4-carboxamide imidazole riboside 5'-phosphate (AICAR), a by-product of HisH and HisF activity . However, we showed that AICAR is not an endogenous mutagen in S . typhimurium . Other types of DNA damage induced by his overexpression seem also unlikely, since similar mutation rates are found in hisO+ and hisOc strains . We also show that AICAR production is not involved in the pleiotropic effects of his overexpression, since these are still observed in strains devoid of AICAR . Thus inhibition of cell division resulting from HisH and HisF overexpression must operate through a mechanism unrelated to the role of these proteins in histidine biosynthesis.

Mol Gen Genet, 1993 Sep, 240(3), 355 - 9
AICAR is not an endogenous mutagen in Escherichia coli; Fox M et al.; A number of observations in the Escherichia coli and Salmonella typhimurium literature could be explained by the hypothesis that a particular purine ribonucleotide precursor can be converted to the corresponding deoxyribonucleotide triphosphate, thereby becoming a base-analogue mutagen . The metabolite in question, AICAR (5-amino-4-carboxamide imidazole riboside 5'-phosphate), is also a by-product of histidine biosynthesis, and its (ribo)triphosphate derivative, ZTP, has been detected in E . coli . We constructed E . coli tester strains that had either a normal AICAR pool (pur+ his+ strains cultivated without purines or histidine) or no AICAR pool (purF hisG mutant strains, lacking the first enzyme of each pathway and cultivated in the presence of adenine and histidine) . Using a set of lacZ mutations, each of which can revert to Lac+ only by a specific substitution mutation, we found that no base substitution event occurs at a higher frequency in the presence of an AICAR pool . We conclude that the normal AICAR pool in E . coli is not a significant source of spontaneous base substitution mutagenesis.

Mol Gen Genet, 1993 Sep, 240(3), 348 - 54
Excess histidine enzymes cause AICAR-independent filamentation in Escherichia coli; Frandsen N et al.; High-level expression of the hisHAFI genes in Escherichia coli, cloned under the control of an IPTG-inducible promoter, caused filamentation, as previously reported in Salmonella typhimurium . We speculated that this filamentation might be produced by an action of the HisH and HisF enzymes on their product AICAR (amino-imidazole carboxamide riboside 5'-phosphate), a histidine by-product and normal purine precursor, possibly by favouring the formation of ZTP, the triphosphate derivative of AICAR . However, filamentation occurred even in the absence of carbon flow through the histidine and purine pathways, as observed in a hisG purF strain lacking the first enzyme in each pathway . Filamentation thus does not require either the normal substrate or products of the overproduced histidine enzymes and must reflect another activity.

J Clin Microbiol, 1993 Sep, 31(9), 2497 - 8
Wound isolate of Salmonella typhimurium that became chlorate resistant after exposure to Dakin's solution: concomitant loss of hydrogen sulfide production, gas production, and nitrate reduction; Lannigan R et al.; A strain of Salmonella typhimurium isolated from a decubitus ulcer that was being treated topically with half-strength Dakin's solution became H2S negative, nitrate negative, and unable to produce gas from glucose . Experimental data suggested that these effects were associated with the development of chlorate resistance . Thirty-five other strains of Salmonella spp . that were made chlorate resistant also became negative for these three tests.

FEMS Microbiol Lett, 1993 Sep 1, 112(2), 217 - 21
Molecular subtyping within a single Salmonella typhimurium phage type, DT204c, with a PCR-generated probe for IS200; Baquar N et al.; We report primers and conditions for the generation by PCR of a probe for the DNA insertion element IS200 . This probe was shown to be suitable for genotypic subtyping within a single phage type of Salmonella typhimurium . A collection of isolates of DT204c, a phage type implicated in the spread of multiple drug resistance in bovine animals and to man, was analyzed . Three IS200 profiles, corresponding to related chromosomal genotypes were characterized in DT204c . Molecular discrimination within a single phage type of S . typhimurium has general significance for genotypic typing, and for the definition of epidemiological clonality in Salmonella.

FEMS Microbiol Lett, 1993 Sep 1, 112(2), 179 - 83
The bifunctional NadR regulator of Salmonella typhimurium: location of regions involved with DNA binding, nucleotide transport and intramolecular communication; Foster JW et al.; NadR is the repressor protein that controls the expression of genes for NAD synthesis . It is also believed to be involved in nucleotide transport . Point mutations conferring different phenotypes were localized to six different regions within the nadR gene . That mutations affecting repression and transport all mapped within nadR confirms the bifunctional model for NadR action . The clustering of these mutations and 2 fusions revealed that those affecting repression lie in the amino terminal while those affecting transport occur in the carboxy-terminal . Mutations resulting in superrepression occurred within a central region of NadR that probably senses NAD concentrations . This region is predicted to direct the transition between NadR transport and repressor conformations.

Carcinogenesis, 1993 Sep, 14(9), 1875 - 80
In vitro DNA damage and mutations induced by a macrocyclic tetraamide chromium(V) complex: implications for the role of Cr(V) peptide complexes in chromium-induced cancers; Dillon CT et al.; Electron paramagnetic resonance and electronic absorption spectroscopies have shown that unlike the bidentate Cr(V) complex {Cr(ehba)2O}- (ehba = 2-hydroxy-2-ethylbutanoato(2-)), I, the macrocyclic tetradentate complex, {Cr (mampa-dcb)(O)}- (mampa-dcb = 5,6-(4,5-dichlorobenzo)-3,8,11,13-tetraoxo-2,2,9,9-tetrameth yl-12,12-diethyl-1, 4,7,10-tetraazacyclotridecane), II, is substitutionally inert . Low levels of DNA strand cleavage were observed after treatment with II under physiological conditions (50 mM sodium phosphate, pH 7.4, 37 degrees C) at concentrations as high as 2 mM for periods as long as 2 days . II also induces a lower number of revertants in mutation assays with Salmonella typhimurium TA100 than I when identical Cr concentrations are applied . The slopes of the linear portion of the dose-response curves are parallel, however, indicating that the mutagenicity of II is comparable to I . II is stable toward ligand exchange, reduction and disproportionation in the mutagenicity test medium and also in the presence of bacteria and the common cell reductant, glutathione . This indicates that ligand exchange with DNA and/or reduction to Cr(IV) are not responsible for the mutagenicity of II (unlike I) . It is believed that II reversibly but weakly intercalates with DNA placing the Cr(V) center in close proximity for hydrogen atom abstraction or oxo-transfer reactions to ensure . This tetraamide complex is a good structural and biomimetic model for non-sulfur-containing Cr(V) peptide species that may form in vivo from reactions of Cr(VI) with peptides . Hence, it is likely to be relevant to understanding one possible mechanism by which Cr(VI) causes cancer.

J Bacteriol, 1993 Sep, 175(18), 5862 - 6
Regulation of the Salmonella typhimurium metF gene by the MetR protein; Cowan JM et al.; The metF gene in Escherichia coli and Salmonella typhimurium is under negative transcriptional control by the MetJ repressor . Expression of an S . typhimurium metF-lacZ gene fusion is repressed up to 10-fold by methionine addition to the growth medium in E . coli hosts encoding wild-type MetJ repressor; this repression is not seen in metJ mutants . metR mutations which eliminate the MetR activator protein result in two- to threefold-more-severe repression by the MetJ repressor . In a metJ metR double mutant, however, the level of metF-lacZ expression is the same as in a metJ mutant, suggesting that MetR antagonizes MetJ-mediated methionine repression of the metF promoter . A DNA footprint analysis showed that MetR binds to a DNA fragment carrying the metF promoter and protects two separate regions from DNase I digestion: a 46-bp region from position -50 to -95 upstream of the transcription initiation site and a 24-bp region from about position +62 to +85 downstream of the transcription initiation site and within the metF structural gene . Nucleotide changes in each of the MetR-binding sites away from the consensus sequence disrupt MetR-mediated regulation of the metF-lacZ fusion.

J Bacteriol, 1993 Sep, 175(17), 5539 - 47
New method for gene disruption in Salmonella typhimurium: construction and characterization of an ada-deletion derivative of Salmonella typhimurium TA1535; Yamada M et al.; A new method for gene disruption in Salmonella typhimurium was developed . The key steps of this method are to produce restriction fragments with compatible ends, preligate to produce concatemers, and then transform by electrotransformation . We developed and used this method to construct a mutant of S . typhimurium TA1535 in which the resident ada-like (adaST) gene was replaced with a kanamycin resistance gene to produce an adaST-deletion mutant derivative . The S . typhimurium adaST-deletion strain did not exhibit a higher level of mutability upon treatment with N-methyl-N'-nitro-N-nitrosoguanidine than did its wild-type parent strain . However, it did exhibit a higher sensitivity with respect to killing by N-methyl-N'-nitro-N-nitrosoguanidine . The ability of AdaST to function as a transcriptional activator is discussed.

Infect Immun, 1993 Sep, 61(9), 3981 - 4
Adoptive transfer of immunity to oral challenge with virulent salmonellae in innately susceptible BALB/c mice requires both immune serum and T cells; Mastroeni P et al.; The mechanisms of immunity to salmonellae conferred by immunization with live vaccines were studied by adoptive transfer using the mouse-virulent strain Salmonella typhimurium C5 and innately susceptible BALB/c (ltys) mice . This organism cannot establish a sublethal infection in naive BALB/c mice . Animals immunized 2 to 3 months earlier with the S . typhimurium SL3261 aroA live vaccine were used as donors of serum, spleen cells, and mesenteric lymph node cells for naive recipients which were challenged orally with the virulent C5 strain . Simultaneous transfer of both immune serum and immune cells was necessary for protection . Simultaneously depleting the donors of CD4+ and CD8+ T cells by administration of antisera in vivo prior to cell harvesting showed that T cells were necessary for protection . The results demonstrate that both antibody and T cells are required for recall of immunity to oral challenge with virulent salmonellae in innately susceptible mice and suggest that the ability to elicit opsonizing antibody in addition to cell-mediated immunity is important for optimal protection induced by salmonella vaccines.

Mol Cell Biol, 1993 Sep, 13(9), 5245 - 54
Inducible transcriptional activation of the human immunodeficiency virus long terminal repeat by protein kinase inhibitors; Brown FL et al.; The protein kinase inhibitor 2-aminopurine (2-AP) greatly stimulated expression in human promonocytes-macrophages of plasmid constructs carrying various reporter genes (chloramphenicol acetyltransferase, lacZ, firefly luciferase {luc}, and Salmonella typhimurium histidinol dehydrogenase {his}) driven by the human immunodeficiency virus type 1 (HIV-1) long terminal repeat . Adenine, adenosine, and caffeine were also effective inducers, but other purine or pyrimidine derivatives were ineffective . Experiments with mutant derivatives of the HIV-1 long terminal repeat revealed no specific eukaryotic promoter elements necessary for 2-AP induction but indicated the need for some minimum combination of such elements . Induction of HIV-1-directed gene expression appeared not to require action of the transcription factor NF-kappa B . The mechanism of induction was investigated by using the luc and his genes linked to the HIV-1 long terminal repeat . 2-AP induced marked, steady rises in mRNA accumulation from both transfected and chromosomally integrated HIV-1 constructs but no increases from an endogenous gene encoding gamma-actin or glucose 6-phosphate dehydrogenase . Thus, induction is selective and not an artifact induced by transfecting DNA into cells . In run-on transcription experiments, the rates of transcription initiation of both transfected and integrated copies of the his gene increased about sixfold in cells treated with 2-AP . Thus, while increased initiation accounted for a portion of 2-AP induction, it could not cause the far greater increase in steady-state mRNA levels . 2-AP induction did not change mRNA decay rates and differed from the phorbol ester (phorbol myristate acetate)-induced activation of the protein kinase C-NF-kappa B pathway in its time course and in its requirement for new protein synthesis . Gel retardation assays showed that unlike phorbol myristate acetate induction, 2-AP induction is enhancer independent . Whereas many previous studies have implicated the activation of various protein kinases in gene induction, we here describe a mechanism of gene activation that appears to involve protein kinase inhibition as a component of the induction response.

Biomed Chromatogr, 1993 Sep-Oct, 7(5), 262 - 6
Chromatographic determination method for 1-nitropyrene and its metabolites in biological samples with fluorescence detection after on-line reduction; Hayakawa K et al.; A high performance liquid chromatographic (HPLC) method with fluorescence detection after on-line reduction for the determination of 1-nitropyrene (NP), 1-nitrosopyrene (NSP), 1-aminopyrene (AP) and N-acetylaminopyrene (AAP) has been developed . The reduction efficiency of NP and NSP on a zinc column was found to be higher than that of an electrochemical reducer . Using a HPLC system equipped with a zinc column (4.0 mm i.d . x 10 mm) and an imidazole/HClO4 (pH 6.8): acetonitrile mobile phase, detection limits (S/N = 3) of 20-30 fmol for NP, NSP and AP and 350 fmol for AAP were obtained . NP, NSP and AP were determined in the incubation mixture of NP and Salmonella typhimurium, YG1021, by this method . Time course studies showed that a large ratio of NP was metabolized in the pre-incubation step of the Ames test.

J Antimicrob Chemother, 1993 Sep, 32(3), 367 - 77
High-level fluoroquinolone resistance in a Salmonella typhimurium isolate due to alterations in both gyrA and gyrB genes; Heisig P; A clinical isolate of Salmonella typhimurium serovar copenhagen (80190) with high-level fluoroquinolone resistance (MIC ciprofloxacin 32 mg/L) was examined for the occurrence of alterations of gyrA and gyrB by a dominance test with the introduction of plasmids carrying either the gyrA or the gyrB gene of Escherichia coli K-12 . Either plasmid resulted in enhanced susceptibilities of each of the resulting heterodiploid strains . Introduction of a plasmid carrying both gyrA and gyrB genes into S . typhimurium 80190 restored the wildtype sensitivity . These observations provide evidence that alterations of both gyrA and gyrB are responsible for the high-level fluoroquinolone resistance of this isolate of S . typhimurium.

Vaccine, 1993 Sep, 11(12), 1221 - 8
Expression and immunogenicity of the V3 loop from the envelope of human immunodeficiency virus type 1 in an attenuated aroA strain of Salmonella typhimurium upon genetic coupling to two Escherichia coli carrier proteins; Charbit A et al.; A peptide comprising residues glu293 to ser334 from the principal neutralization determinant (V3 loop) of the envelope of human immunodeficiency virus type 1 (HIV1 LAVBRU isolate) has been inserted within internal permissive sites of either LamB or MalE, two envelope proteins from Escherichia coli K12 . The MalE hybrid protein (MalE133-V3 loop) was stably expressed in the periplasm of Escherichia coli K12, and the V3 loop peptide was detectable on the surface of the native protein by an anti-gp160 monoclonal antibody (mAb 110-A) . The disulfide bridge between the two cysteines of the loop was formed . In contrast, genetic coupling to the outer membrane protein LamB did not allow the expression of a stable hybrid protein, and major proteolytic cleavage products of the LamB153-V3 loop were detected by mAb 110-A . The two plasmid-encoded hybrid genes were transferred to an aroA mutant of Salmonella typhimurium . Constitutive expression of the MalE133-V3 loop had no detectable effect on cell growth and on the survival in vivo of the recipient strain . The LamB153-V3 loop was not stably expressed in Salmonella, either in vitro or in vivo . Live recombinant salmonellas expressing MalE-V3 and LamB-V3 loop hybrids were used to immunize mice . The MalE-V3 loop hybrid induced anti-HIV1 envelope antibodies detectable by Western blot and ELISA, while the anti-HIV1 envelope antibodies induced by the LamB-V3 loop hybrid were only detectable by Western blot . In addition, purified MalE-V3 loop hybrid protein was able to stimulate in vitro and induce in vivo a V3 loop-specific T-cell proliferative response.

J Nat Prod, 1993 Sep, 56(9), 1532 - 8
Selligueain A, a novel highly sweet proanthocyanidin from the rhizomes of Selliguea feei; Baek NI et al.; Selligueain A, a novel sweet trimeric proanthocyanidin with a doubly linked A unit, has been isolated from the rhizomes of Selliguea feei collected in Indonesia . The structure of this substance was established as epiafzelechin-(4 beta-->8, 2 beta-->O-->7)-epiafzelechin-(4 beta-->8)-afzelechin {1}, on the basis of a combination of spectral and chemical methods . The compound was not acutely toxic for mice and not mutagenic in a forward mutation assay utilizing Salmonella typhimurium strain TM677 . Selligueain A {1} was rated by a taste panel as exhibiting about 35 times the sweetness intensity of a 2% w/v aqueous sucrose solution, and at a concentration of 0.5% w/v in H2O was perceived as pleasant-tasting rather than astringent.

Genomics, 1993 Sep, 17(3), 667 - 75
Physical delineation of the minimal chromosomal segment encompassing the murine host resistance locus Bcg; Malo D et al.; The host resistance locus Bcg determines resistance of mice to infection with intracellular pathogens such as certain species of Mycobacteria, Salmonella typhimurium, and Leishmania donovani . Bcg maps on the proximal portion of mouse chromosome 1, very tightly linked to villin (Vil), with the gene order and intergene distances Tp-1-(1 cM)-D1Mcg105-(0.1 cM)-lambda Mm1C165/Vil/Bcg-(0.2 cM)-lambda Mm1C136-(0.3 cM)-Des-(0.1 cM)-Inha . In an effort to clone genomic sequences overlapping Bcg, we have used pulsed-field gel electrophoresis (PFGE) and fluorescence in situ hybridization to construct a physical map of the 3.9-Mb segment of proximal mouse chromosome 1, near Bcg . In situ hybridization to metaphase mouse chromosomes indicates that the mapped region is within band C5 . Physical mapping of the Tp-1-Vil and lambda Mm1C136-Inha intervals was carried out by PFGE analysis, whereas the Vil-Des interval was estimated by in situ hybridization to interphase nuclei . Results of these combined analyses indicate the following locus order and maximal interlocus distances: Tp-1-(1000 kb)-D1Mcg105-(160 kb)-lambda Mm1C165-(180 kb)-Vil-(800 kb)-lambda Mm1C136-(290 kb)-Des-(130 kb)-Inha . Detailed restriction mapping of this region identifies numerous putative CpG islands, suggesting that several transcription units are present in the vicinity of Bcg.

Chin Med Sci J, 1993 Sep, 8(3), 151 - 6
Protective effect of Salmonella typhimurium Re-LPS antiserum; Yu X et al.; There is increasing evidence that antiserum to LPS can reduce the morbidity and mortality of Gram-negative bacterial infections . We report that antiserum to S . typhimurium SL 1102 (Re mutant strain) has excellent cross-protective activity . Antisera to these bacteria and to their Re-LPS were prepared in rabbits immunized with heat-killed bacterial cells and with Re-LPS preparations . Re-LPS antibody titers were tested by immune hemagglutination (IHA) and by ELISA . These antisera were found to be capable of protecting ICR mice against lethal challenge with S-type S . typhimurium 50014 (100 LD50), E . coli 0111:B4 (32 LD50), Pseudomonas aeruginosa (8 LD50) and Klebsiella pneumonia (16 LD50) . We used gastric mucin (5%) as a virulence enhancing agent for the bacterial challenges . The IHA titer of antibody to the homologous strain proved to be much higher than that of other strains . Protection by the sera was 75-100%, 25% and 0% when injected 24, 48 or 72 h before the challenge, respectively . The survival rate was more than 50% when the antiserum was injected 5-7 h after challenge with a ten-fold or higher lethal dose . No protection was observed against such high challenge when the serum was injected later . According to these results, Re-LPS antiserum provides better protection than S-type specific antisera.

Tohoku J Exp Med, 1993 Sep, 171(1), 89 - 95
Mutagenicity of activated carbon adsorbate of drinking water in the Ames assay; Shibuya N et al.; Mutagenicity of activated carbon adsorbate from drinking water collected in Niigata City was assayed by the Ames assay . Adsorbate was extracted from activated carbon with benzene, and then with ethanol . Although the benzene extract was not mutagenic, the ethanol one showed the mutagenic activity for Salmonella typhimurium strains TA98 and TA100 with and without S9 mix . The ethanol extract was much more mutagenic on TA100 than TA98 both with and without S9 mix . The mutagenic activity per liter of water was found to be the strongest in winter and the weakest in summer.

Mutat Res, 1993 Sep, 303(1), 29 - 34
The protective role of gallic acid esters in bacterial cytotoxicity and SOS responses induced by hydrogen peroxide; Nakayama T et al.; The effects of gallic acid and its esters on H2O2-induced cytotoxicity, mutagenicity and SOS response were investigated in bacterial assay systems, i.e., the Ames test with Salmonella typhimurium TA104 and the SOS chromotest with E . coli PQ37 . In the Ames test, gallic acid esters showed protective effects against H2O2-induced cytotoxicity and no effects on the number of revertant colonies . In the SOS chromotest, gallic acid esters lowered the SOS induction factor raised by H2O2 . Throughout the study, the effects of gallic acid itself were weak or negligible, and lauryl gallate was most effective among the three gallic acid esters . This structure-activity relationship indicates the similarity of the protective effects of gallic acid esters on the H2O2-induced damages to both bacterial and mammalian cells.

Mutat Res, 1993 Sep, 319(1), 1 - 9
Isolation of substances from glossy privet (Ligustrum lucidum Ait.) inhibiting the mutagenicity of benzo{a}pyrene in bacteria; Niikawa M et al.; Methanol and hot-water extracts of glossy privet (Ligustrum lucidum Ait.) inhibited the mutagenic activity of benzo{a}pyrene in Salmonella typhimurium TA98 with S9 mix . The methanol extract was fractionated with ether and n-hexane . As the active components, oleanolic and ursolic acids were isolated, which were soluble in ether and insoluble in n-hexane . The hot-water extract was fractionated to water, 60% and 100% methanol fractions . Nuezhenide was isolated from the 60% methanol fraction as the active component.

Schweiz Rundsch Med Prax, 1993 Aug 31, 82(35), 946 - 8
{Diarrhea, erythema nodosum, arthralgia}; Brutsche M et al.; Following antibiotic treatment of febrile tonsillitis, a 20-year old man developed watery diarrhea during military service . He was admitted to the infirmary by the medical officer . During the last year the patient had traveled to Spain . The history of recent food intake was not remarkable . The clinical investigation revealed a slightly tender liver . A markedly elevated ESR, a consecutively developing erythema nodosum on both lower legs and arthralgias opened a broad spectrum of differential diagnoses that are discussed here . Stool cultures grew salmonella typhimurium . The clinical picture, the treatment and possible complications of this salmonellosis are discussed.

FEMS Microbiol Lett, 1993 Aug 15, 112(1), 113 - 8
Inhibition of mitogen-induced proliferation of spleen lymphocytes is correlated with the induction of cell-mediated immunity in Salmonella infection in mice; Matsui K et al.; The proliferation of murine spleen cells stimulated by a T-cell mitogen such as phytohemagglutinin (PHA) or concanavalin A (ConA) was significantly suppressed when the mice were immunized with either the viable cells or the sonicate of Salmonella typhimurium but not of Escherichia coli . The suppression of T-cell proliferation caused by the sonicate of S . typhimurium was completely restored by addition of phorbol 12-myristate-13-acetate (PMA), an activator of protein kinase C (PKC) . Western blots using anti-phosphotyrosine antibodies showed that the mitogen-induced tyrosine phosphorylation of 120-, 106-, 94-, 76-, 68- and 57-kDa proteins in murine splenic T-cells was inhibited in the mice immunized with the viable cells but not the sonicate of S . typhimurium . These results suggest that the inhibition caused by the sonicate involves suppression of PKC activity, whilst that produced by viable cells involves down-regulation of tyrosine phosphorylation, and both inhibitions correlate with the induction of cell-mediated immunity in mice, as evidenced by the induction of delayed-type hypersensitivity reactions.

Nature, 1993 Aug 12, 364(6438), 639 - 42
Ruffles induced by Salmonella and other stimuli direct macropinocytosis of bacteria; Francis CL et al.; Ruffles are specialized plasma membrane ultrastructures of mammalian cells though to be integral to growth, development and locomotion . Induced by growth factors, mitogens or oncogene expression, ruffles are sites of filamentous actin rearrangement and are temporally associated with enhanced pinocytosis . But the function of ruffles, their mechanism of induction and their role in pinocytosis are not understood . We have observed formation of structures resembling ruffles associated with the site of entry of invasive Salmonella typhimurium . Here we report that ruffles elicited by invasive Salmonella directly mediate internalization of non-invasive bacteria in a macropinocytotic fashion, a phenomenon we term 'passive entry' . Furthermore, ruffles induced in the absence of Salmonella also facilitate passive entry . We present evidence that ruffles, common to many signalling events, comprise the macropinocytotic machinery mediating pinocytosis and are subverted by Salmonella so as to enter mammalian cells.

J Biol Chem, 1993 Aug 5, 268(22), 16551 - 6
Characterization of a temperature-sensitive mutant of Salmonella typhimurium defective in apolipoprotein N-acyltransferase; Gupta SD et al.; On screening 440 temperature-sensitive (ts) mutants of Salmonella typhimurium, a mutant strain SE5312 which accumulated apolipoprotein (ALP) at 42 degrees C was identified . In vitro assay of apolipoprotein N-acyltransferase activity indicated that the mutant cell envelope contained reduced activity as compared to the wild-type strain . Transduction with a Mud-P22 mapping set placed the ts mutation to 14-17 min region of the S . typhimurium chromosome . P22 transduction using transposon insertions in this region revealed a linkage of the ts mutation to cobD (6%), nag (8%), and corC68 (99%) . The ts phenotype was complemented by a 2.3-kilobase EcoRI subclone derived from lambda-phage 170 of Kohara's bank of Escherichia coli . Restriction enzyme analysis of the cloned DNA revealed that this 2.3-kilobase EcoRI fragment included the copper transport (cutE) gene in E . coli . The mutant strain SE5312 was copper-sensitive at 30 degrees C, and the complementing clone conferred copper resistance and restored the ALP N-acyltransferase activity in the mutant cell . Wild-type strain of S . typhimurium harboring this clone exhibited elevated levels of ALP N-acyltransferase activity . These results suggest that the cloned gene encodes the ALP N-acyltransferase . Upon shift to the non-permissive temperature, the viability of the mutant cells decreased, and the mutant cells assumed anomalous morphology . Temperature-resistant revertants could be readily isolated, and a subset of tr revertants contained no detectable lipoprotein . A lpp::Tn10 derivative of the mutant SE5312 was also temperature-resistant . These observations suggest that ALP N-acyltransferase is essential for the growth and viability of S . typhimurium, and this requirement is decreased in the absence of major outer membrane lipoprotein.

J Biol Chem, 1993 Aug 5, 268(22), 16544 - 50
Isolation and characterization of a temperature-sensitive mutant of Salmonella typhimurium defective in prolipoprotein modification; Gan K et al.; A temperature-sensitive (ts) mutant of Salmonella typhimurium that accumulated unmodified murein prolipoprotein at 42 degrees C but not at 30 degrees C was identified . In vivo and in vitro studies of the biosynthesis of Braun's lipoprotein revealed that this mutant (SE5221) was defective in the glyceryl modification of prolipoprotein . The ts mutation was mapped to 60.6 min of the S . typhimurium chromosome and was linked to argA and cysH . A clone with a 1.4-kilobase S . typhimurium DNA insert that complemented the ts mutation and restored the prolipoprotein modification activity both in vivo and in vitro was isolated . DNA sequencing of the complementing region revealed an open reading frame encoding a protein with 291 amino acids lacking NH2-terminal signal sequence . This open reading frame is immediately 5' to the thyA gene and is allelic to umpA of Escherichia coli . Wild-type strains harboring the cloned gene exhibited elevated levels of prolipoprotein modification activity . At the non-permissive temperature, the mutation affected both growth and viability, and the mutant cells exhibited anomalous cell morphology . The ts phenotype was suppressed by the introduction of a lpp::Tn10 mutation . These results suggest that the cloned gene encodes prolipoprotein glyceryl transferase (lgt), and in the wild-type background, this prolipoprotein modification enzyme is essential for the growth and viability of S . typhimurium.

J Mol Biol, 1993 Aug 5, 232(3), 756 - 65
Deficiency of 1-methylguanosine in tRNA from Salmonella typhimurium induces frameshifting by quadruplet translocation; Hagervall TG et al.; The trmD gene encodes the tRNA(m1G37)methyltransferase, which methylates guanosine (G) to 1-methylguanosine (m1G) at position 37 of tRNAs that read CUN (leucine), CCN (proline), and CGG (arginine) codons . A mutant, trmD3, has previously been isolated, which at high temperature lacks m1G in tRNA, and this deficiency was correlated with a +1 frameshifting activity . In this study, the mechanism of this trmD3-induced frameshift involving mutant tRNA(Pro) and tRNA(Leu) species has been investigated . Potential frameshifting sites for proline tRNAs, CCC-N, were efficiently suppressed in the mutant strain . Hybrid beta-galactosidases encoded by plasmid constructs containing the sites CCC-U and CCC-A were subjected to amino-terminal sequencing . The protein sequences demonstrated that a quadruplet translocation had occurred and that a proline was inserted at these sites, suggesting that a tRNA(Pro) deficient in m1G is the frameshifting agent . Therefore, a mechanism involving a quadruplet codon-anticodon interaction is favoured for trmD3-dependent +1 frameshifting . Of the four potential sites for tRNA(Leu) (CCU-N), two, CCU-U and CCU-C, were significantly suppressed in the trmD3 mutant . Thus, species of tRNA(Leu) may also act as +1 frameshift suppressors . No -1 frameshifting activity was found with the trmD3 mutant.

Mol Microbiol, 1993 Aug, 9(3), 601 - 11
Chemotaxis plays a role in the social behaviour of Myxococcus xanthus; Shi W et al.; Myxococcus xanthus is a Gram-negative bacterium that glides on a solid surface and displays a wide range of social behaviour including microbial development . The frz genes are homologues to the chemotaxis genes of Escherichia coli and Salmonella typhimurium and have been shown to be involved in microbial development . However, chemotaxis has never been clearly demonstrated in Myxococcus . In this study, we showed that M . xanthus exhibited tactic movements to many chemicals when they were subjected to steep and stable chemical gradients . M . xanthus was observed to spread into areas with abundant nutrients like yeast extract or Casitone and avoid areas with no nutrients or repellents (short-chain alcohols or DMSO) . Responses to attractants and repellents were additive . Movement towards attractants or away from repellents required the frz genes and was correlated with methylation or demethylation of FrzCD, a methyl-accepting taxis protein . Furthermore, the frz genes were found to be required for both fruiting body formation during starvation and swarming in nutrient-rich medium . In wild-type strains, cells near the colony edge were observed to swarm towards the surrounding growth medium and to contain highly methylated FrzCD; cells near the colony centre contained mainly demethylated FrzCD and showed directed movement towards the colony edge . FrzCD was also found to be methylated during the aggregation stage of fruiting body formation on agar but largely demethylated in cells shaken in liquid starvation media . An frzE mutant failed to exhibit directed cell movements and no longer showed modification of FrzCD under these conditions . These observations suggest that M . xanthus does show chemotactic movements, that these movements require the frz genes, and that chemotaxis plays a very important role in the social behaviour of this organism.

Int J Immunopharmacol, 1993 Aug, 15(6), 657 - 64
Non-specific resistance induced by a low-toxic lipid A analogue, DT-5461, in murine salmonellosis; Onozuka K et al.; The ability of DT-5461, a chemically synthetic low-toxic lipid A analogue, to activate anti-Salmonella activity in C3H/HeN mice was examined . Previous intraperitoneal (i.p.) injection of DT-5461 (100 micrograms or more/mouse) significantly hindered the bacterial growth in the peritoneal cavities of the mice after the i.p . infection with Salmonella typhimurium LT2 strain . The effect was the maximum when DT-5461 was given 6 h before the challenge . The injection of DT-5461 6 h in advance could also confer protection against the infection . Bactericidal activity enhancement was also seen in mice that had been injected with a small amount of recombinant murine IFN-gamma (10(3) U per mouse) and non-effective dose (10 micrograms) of DT-5461 together 6 h before the challenge . Bactericidal effect enhancement was seen in mice that had been injected with IFN-gamma at 6 h and DT-5461 at 3 h before the challenge, while it could be hardly seen in mice injected with them in a reversed order . The i.p . injection of DT-5461 recruits the exudate cells into the peritoneal cavities, and the phagocytic and bactericidal abilities of the macrophages in the exudate cells are apparently elevated . The mechanisms of non-specific resistance enhancement induced by DT-5461 were discussed.

FEMS Microbiol Lett, 1993 Aug 1, 111(2-3), 331 - 5
Analysis of a cloned Francisella tularensis outer membrane protein gene and expression in attenuated Salmonella typhimurium; Leslie DL et al.; We have determined the nucleotide sequence of fopA from Francisella tularensis . Using the polymerase chain reaction fopA was detected in high and low virulence biotypes of F . tularensis . fopA was stably maintained in pBluescript in attenuated Salmonella typhimurium where FopA was expressed and located in the outer membrane . This recombinant will be suitable for studies on the role of FopA in immunity against tularaemia.

FEMS Microbiol Lett, 1993 Aug 1, 111(2-3), 225 - 31
Chromosomal DNA from both flagellate and non-flagellate Bordetella species contains sequences homologous to the Salmonella H1 flagellin gene; Leigh AF et al.; The genus Bordetella contains four species: two are non-motile, the human pathogens B . pertussis and B . parapertussis; and two are motile, the broad host-range mammalian pathogen B . bronchiseptica, and the avian pathogen B . avium . The motility of the latter two species is due to peritrichous flagella . Here we show that strains of all four species contain DNA sequences homologous to flagellin genes . Two types of gene probe were hybridised to Bordetella chromosomal DNa in Southern blots: the structural gene for H1 flagellin of Salmonella typhimurium and an oligonucleotide derived from the conserved N-terminal amino acid sequences of various flagellin proteins . ClaI-digested DNa from all four Bordetella species hybridised with both probes in Southern blots, although each species gave a characteristic pattern of hybridisation . This indicates that the non-motile B . pertussis and B . parapertussis species contain non-expressed flagellin genes.

FEMS Immunol Med Microbiol, 1993 Aug, 7(2), 111 - 8
Infection of differentiated U937 cells by Salmonella typhimurium: absence of correlation between oxidative burst and antimicrobial defence; Chateau MT et al.; The human histiocytic lymphoma cell line U937 can be induced to differentiate along the monocyte/macrophage pathway by either phorbol myristate acetate (PMA) or by the combination of retinoic acid (RA) and 1,25-dihydroxyvitamin D3 (VD) . U937 cells treated with either PMA or RA/VD were able to phagocytose Salmonella typhimurium in the presence of non-immune human serum . However, only cells differentiated by RA/VD were capable of developing an oxidative metabolic burst in response to infection . Since the oxidative burst is considered to be a potent antimicrobial mechanism, we investigated its effect on S . typhimurium . The oxidative burst failed to affect either the viability or the multiplication of S . typhimurium suggesting that it plays only a minor role in the host defence against S . typhimurium.

Can J Surg, 1993 Aug, 36(4), 311 - 4
Osteomyelitis of the spine due to Salmonella: case report, review of clinical aspects, pathogenesis and treatment; D'Souza CR et al.; Osteomyelitis of the spine caused by Salmonella is rare . The authors describe a case in which the patient had fusion of the L1 and L2 vertebrae, which were affected by osteomyelitis . The infecting organism was Salmonella typhimurium . The authors describe the relationship of the vascular anatomy to the development of osteomyelitis of the spine . They discuss the clinical features, diagnosis, pathogenesis and treatment of the disease . Although the association of osteomyelitis due to Salmonella and sickle cell disease has long been known, this patient did not have sickle cell disease or any other condition that would compromise the immune system.

Carcinogenesis, 1993 Aug, 14(8), 1697 - 9
The in vitro metabolic activation of the 11-trifluoromethyl analogue of the potent carcinogen 15,16-dihydro-11-methyl-cyclopenta{a}-phenanthren-17-one to mutagens; Boyd GW et al.; A strongly electronegative, bay-region analogue of the potent carcinogen 15,16-dihydro-11-methylcyclopenta{a}phenanthren-17-one, namely 15,16-dihydro-11-trifluoromethylcyclopenta{a}phenanthren-17-one, is mutagenic to Salmonella typhimurium TA100 . Also it is metabolized at the 1,2- and 3,4-positions in the A-ring as well as C-15 in the D-ring to give 3,4-dihydroxy-3,4,15,16-tetrahydro-11-trifluoromethyl- cyclopenta{a}phenanthren-17-one as the only mutagenic metabolite . In these respects its behaviour is closely similar to that of the 11-methyl compound, suggesting that the electronic nature of the bay-region substituent is rather less critical than its spatial configuration in influencing metabolism to genotoxic intermediates . It remains to be seen, however, whether the trifluoromethyl compound is also a carcinogen.

J Bacteriol, 1993 Aug, 175(16), 4990 - 9
An oxygen-dependent coproporphyrinogen oxidase encoded by the hemF gene of Salmonella typhimurium; Xu K et al.; The 8th step in the 10-step heme biosynthetic pathway of Salmonella typhimurium is the oxidation of coproporphyrinogen III to protoporphyrinogen IX . On the basis of genetic studies, we have suggested that this reaction may be catalyzed by either of two different enzymes, an oxygen-dependent one encoded by hemF or an oxygen-independent enzyme encoded by hemN . Here, we report the cloning of the S . typhimurium hemF gene and its DNA sequence . The predicted amino acid sequence of the HemF protein is 44% identical to that of the coproporphyrinogen oxidase encoded by the yeast HEM13 gene . The wild-type S . typhimurium strain LT-2 produces an oxygen-dependent coproporphyrinogen oxidase activity detectable in crude extracts, which is not found in hemF mutants and is overproduced in strains carrying the hemF gene on a multicopy plasmid . the hemF gene is the second gene in an operon with an upstream gene with an unknown function, whose amino acid sequence suggests a relation to amidases involved in cell wall synthesis or remodeling . The upstream gene and hemF are cotranscribed from a promoter which was mapped by primer extension . A weaker, hemF-specific promoter is inferred from the behavior of an omega-Cm insertion mutation in the upstream gene . Although this insertion decreases expression of beta-galactosidase about 7.5-fold when placed upstream of a hemF-lacZ operon fusion, it still allows sufficient HemF expression from an otherwise wild-type construct to confer a Hem+ phenotype . The hemF operon is transcribed clockwise with respect to the genetic map.

Food Chem Toxicol, 1993 Aug, 31(8), 561 - 7
Contribution of phenolic and quinonoid structures in the mutagenicity of the edible mushroom Agaricus bisporus; Papaparaskeva-Petrides C et al.; The objectives of this work were to establish the contribution of agaritine in the mutagenicity of ethanolic extracts from Agaricus bisporus and to examine the possible involvement of phenolic and quinonoid compounds in the mutagenic response to mushrooms . The mutagenic profile of agaritine in the Ames test, in the absence of an activation system, was different from that of the mushroom ethanolic extracts . Incorporation of rat hepatic cytosolic fractions as the activation system increased the mutagenicity of the mushroom ethanolic extracts in Salmonella typhimurium strain TA 104 but did not influence the mutagenicity of agaritine . It was concluded that agaritine is not the principal mutagenic component in the mushroom . The cytosol-induced mutagenicity of the mushroom extracts required NADPH, and was inhibited by dicoumarol and menadione . Moreover, the mutagenic response in the presence of cytosolic fractions was inhibited by superoxide dismutase, catalase, glutathione and dimethyl sulfoxide, thus implicating reactive oxygen species . Finally, tyrosinase, the enzyme converting mushroom phenols to quinones, increased the mutagenicity of the mushroom extracts . Collectively, the above results indicate that phenolic and quinonoid compounds, presumably through the generation of reactive oxygen species, may play a significant role in the mutagenicity of mushroom extracts.

Microb Pathog, 1993 Aug, 15(2), 93 - 101
Use of incompatible plasmids to control expression of antigen by Salmonella typhimurium and analysis of immunogenicity in mice; Ervin SE et al.; Salmonella spp . have been investigated as live vaccine vectors because they are heat stable and can elicit humoral, cellular, and secretory immune responses . However, the expression of some foreign antigens is toxic to bacterial vectors . We therefore studied an approach for the controlled expression of antigen in Salmonella typhimurium wherein the antigen is not expressed in vitro but is expressed in vivo . A model antigen, beta-galactosidase, was expressed from the trc promoter on one plasmid, while repression was achieved by Lacl expressed in trans from a second plasmid . The second repressor plasmid was incompatible with the expression plasmid encoding beta-galactosidase . Loss by segregation of the repressor plasmid in vitro correlated with increased expression of beta-galactosidase . Oral inoculation of mice with salmonellae containing both plasmids induced serum IgG but not nasal, salivary, or biliary IgA antibody to beta-galactosidase . Serum IgG as well as biliary IgA anti-S . typhimurium antibody, but not salivary or nasal IgA, were also detected . This salmonella vector system for the controlled expression of recombinant antigens may be of value for inducing systemic but not mucosal immunity to antigens that are toxic to bacterial vectors.

Vet Immunol Immunopathol, 1993 Aug, 37(3-4), 217 - 30
Mitogen and antigen induced B and T cell responses of peripheral blood mononuclear cells from the harbour seal (Phoca vitulina); de Swart RL et al.; In vitro assays were developed for studies concerning the functioning of the immune system of the harbour seal (Phoca vitulina) . Proliferative responses of peripheral blood mononuclear cells (PBMC) were measured after stimulation with different concentrations of the mitogens concanavalin A (Con A), pokeweed mitogen (PWM), phytohaemagglutinin (PHA) or lipopolysaccharide from Salmonella typhimurium (LPS) . Con A and PWM induced strong proliferative responses, while PHA and LPS induced comparatively low proliferative responses . Responses of mitogen stimulated PBMC to recombinant human interleukin-2 (rhIL-2) and in vitro immunoglobulin production by mitogen stimulated PBMC were measured to discriminate between stimulation of T cells and B cells . It was found that Con A and PHA stimulate phocine T cells, PWM stimulates both T cells and B cells and LPS predominantly stimulates phocine B cells . Antigen-specific immune responses were measured after immunization of seals with an inactivated rabies vaccine and/or with tetanus toxoid . Antigen-specific proliferation of PBMC and the presence of antigen-specific antibody forming cells were demonstrated for both antigens in the PBMC of immunized animals . The responses measured in vitro correlated well with the development of specific serum antibody titers to these antigens.

Mol Microbiol, 1993 Aug, 9(4), 835 - 46
Isozymes of S-adenosylmethionine synthetase are encoded by tandemly duplicated genes in Escherichia coli; Satischandran C et al.; The sole biosynthetic route to S-adenosylmethionine, the primary biological alkylating agent, is catalysed by S-adenosylmethionine synthetase (ATP:L-methionine S-adenosyltransferase) . In Escherichia coli and Salmonella typhimurium numerous studies have located a structural gene (metK) for this enzyme at 63 min on the chromosomal map . We have now identified a second structural gene for S-adenosylmethionine synthetase in E . coli by DNA hybridization experiments with metK as the probe; we denote this gene as metX . The metX gene is located adjacent to metK with the gene order speA metK metX speC . The metK and metX genes are separated by approximately 0.8 kb . The metK and the metX genes are oriented convergently as indicated by DNA hybridization experiments using sequences from the 5' and 3' ends of metK . The metK gene product is detected immunochemically only in cells growing in minimal media, whereas the metX gene product is detected immunochemically in cells grown in rich media at all growth phases and in stationary phase in minimal media . Mutants in metK or metX were obtained by insertion of a kanamycin resistance element into the coding region of the cloned metK gene (metK::kan) followed by use of homologous recombination to disrupt the chromosomal metK or metX gene . The metK::kan mutant thus prepared does not grow on minimal media but does grow normally on rich media, while the corresponding metX::kan mutant does not grow on rich media although it grows normally on minimal media . These results indicate that metK expression is essential for growth of E . coli on minimal media and metX expression is essential for growth on rich media . Our results demonstrate that AdoMet synthetase has an essential cellular and/or metabolic function . Furthermore, the growth phenotypes, as well as immunochemical studies, demonstrate that the two genes that encode S-adenosylmethionine synthetase isozymes are differentially regulated . The mutations in metK and metX are highly unstable and readily yield kanamycin-resistant cells in which the chromosomal location of the kanamycin-resistance element has changed.

Gesundheitswesen, 1993 Aug-Sep, 55(8-9), 418 - 26
{Epidemiologic pattern of Salmonella enteritidis and Salmonella typhimurium in man--analysis of Salmonella cases in the Brandenburg federal territory}; Kasbohrer A et al.; It is the aim of this study (a study that is part of several investigations suggested and coordinated by the WHO) to highlight the characteristics of transmission of Salmonella in humans . To achieve this, the data collected on the basis of the Federal German Law governing Epidemic Diseases as well as additionally available data (sex, serovar, differentiation between diseased and symptomatic, allocation to the respective place of residence) have been evaluated . In this manner it became possible to determine different patterns of incidence of the presently most frequently occurring salmonella serovars S . enteritidis and S . typhimurium in respect of distribution according to age and sex, as well as the dynamics of distribution and incidence in terms of time . The incidence is particularly remarkable among children, where S . enteritidis is not so frequent as S . typhimurium . There is a distinct trend towards the male sex among children in respect of the infestation . The fact that women are particularly often asymptomatic carriers points to the special nature of the sources and paths of infection . In respect of seasonal dynamics S . typhimurium does not have a specific summer peak, contrary to S . enteritidis, and this too indicates that the mechanisms of transmission or multiplication differ from one another . The study presented here is supplemented by the evaluation of further parameters to support and promote on-target epidemiological investigation into the various problems raised by this and other studies.

Zentralbl Bakteriol, 1993 Aug, 279(3), 336 - 43
A comparison of the efficiency of Rappaport-Vassiliadis, tetrathionate and selenite broths with and without pre-enrichment for the isolation of Salmonella in animal waste biogas plants; Schlundt J et al.; A total of 481 samples of biomass from biogas plants treating slurry and other types of animal waste were examined for the presence of salmonellae by means of five different isolation methods . In 131 samples, Salmonella was isolated by means of one or more methods . A statistical evaluation of the isolation frequencies showed that Rappaport-Vassiliadis broth was significantly better than selenite broth with and without pre-enrichment and tetrathionate broth with pre-enrichment, whereas tetrathionate broth without pre-enrichment was significantly poorer than the other four methods . For each of the thirty different Salmonella serotypes, the isolation frequencies for the five methods are presented . Remarkably, Rappaport-Vassiliadis broth had very high isolation frequencies and tetrathionate broth with and without pre-enrichment had very low isolation frequencies for Salmonella typhimurium as well as Salmonella dublin.

Arzneimittelforschung, 1993 Aug, 43(8), 897 - 903
In vitro and in vivo mutagenicity studies on taurohyodeoxycholic acid; Tripodi AS et al.; The mutagenicity of a new biliary acid, taurohyodeoxycholic acid (THDCA, Io, Praxis, CAS 2958-04-5), was assayed by using 5 different tests . The Ames test (reverse mutation assay on Salmonella typhimurium) and the DNA damage and repair test (in Saccharomyces cerevisiae) allowed to study the genetic THDCA-induced mutations in prokaryotes and eukaryotes (doses of 100, 200 and 400 micrograms/plate or 100, 200 and 400 micrograms/ml, respectively) . In vivo and in vitro chromosomal aberrations were studied by using micronucleus test in mice (doses of 100, 220 and 500 mg/kg in oral study and 50, 100 and 200 mg/kg in subcutaneous study) and human lymphocytes cytogenetic test (doses of 50, 100, 220 and 500 micrograms/ml of THDCA) . At last the host-mediated assay was performed on THDCA-treated mice (following oral or subcutaneous administration) in order to test the potential mutagenic activity of its metabolites on a S . typhimurium strain . The results obtained in these studies showed that THDCA did not induce any signs of promutagenic, mutagenic or clastogenic direct or metabolite-mediated activity.

Berl Munch Tierarztl Wochenschr, 1993 Aug, 106(8), 265 - 9
{The epidemiological analysis of Salmonella typhimurium infections in cattle--results of lysotyping and biochemotyping in the region of East Thuringia from 1974 to 1991}; Jacob WK et al.; 597 strains of Salmonella typhimurium found in samples from cattle in Eastern Thuringia from 1974 to 1991 were investigated to their phage types and biochemotypes . 21 different combined types were found out among which the phage types n.c . 1/72/n . c.(204), 2 b/23 (92) and 1 a/9 (49) dominated at certain times . With the help of two examples it is shown that cases of Salmonellosis with Salmonella typhimurium within one farm occurring one after another can be different infections that are independent from each other . The complex typings are capable to clear up epidemiological connections and therewith support the control of infections and the protection of people's health consequently.

FEMS Microbiol Lett, 1993 Aug 1, 111(2-3), 309 - 14
Dual control by purines and pyrimidines of the expression of the pyrD gene of Salmonella typhimurium; Vial TC et al.; Expression of the Salmonella typhimurium pyrD gene was found to be repressed two-fold when cells were grown in the presence of hypoxanthine . Purine-mediated repression was evident for reporter plasmids containing pyrD-lacZ transcriptional or translational fusions, indicating that regulation was being exercised at the level of transcriptional initiation . In a strain harbouring a purR6::Tn10 mutation inactivating the purine regulon repressor (PurR), expression of pyrD was not repressed by hypoxanthine . Gel retardation experiments provided evidence that PurR binds to a PUR box centered 27 base pairs upstream of the -35 region of the pyrD promoter . Site-directed mutagenesis was used to decrease the similarity of the putative PUR box to the consensus sequence; each mutation eliminated binding of PurR to the mutated DNA in vitro and abolished repression by hypoxanthine in purR+ cells in vivo . Regulation by pyrimidines was unaffected by either of the two PUR box mutations, showing that purine and pyrimidine control of pyrD expression can operate independently.

Ecotoxicol Environ Saf, 1993 Aug, 26(1), 18 - 32
Chemical and mutagenic evaluation of sludge from a large wastewater treatment plant; Ottaviani M et al.; Digested sludges from a wastewater treatment plant were analyzed to assess their level of contamination by some organic (polychlorobiphenyls (PCBs) and chlorinated pesticides) and inorganic (heavy metals) micropollutants and their mutagenicity features . The heavy metal content in none of the samples exceeded the limits set out in EEC Directive 276/86; as far as PCBs are concerned, the sludges analyzed indicated a level of contamination up to two orders of magnitude higher than some Italian agricultural soils . Mutagenicity assays on either crude or fractionated sludge extracts using Salmonella typhimurium tester strains TA98 and TA100 gave negative results, thus suggesting the absence of genotoxic contaminants in the samples investigated.

Mutat Res, 1993 Aug, 292(1), 51 - 61
Mutagenicity of organic extracts from Santiago (Chile) airborne particulate matter; Adonis M et al.; The Ames test has been used to detect the mutagenic activity of organic extracts from Santiago (Chile) airborne particles collected in 1990 and 1991 in one of the monitoring net system stations (MACAM) . The samples were assayed with the strains TA98, TA98-NR, and TA98/1,8-DNP6 of Salmonella typhimurium, in the presence and in the absence of liver S9 fraction obtained from rats treated with Aroclor 1254 . With the strain TA98 all the samples showed a very high mutagenic response either in the presence or in the absence of S9 fraction, suggesting that Santiago airborne particles contain both indirect-acting (polycyclic aromatic hydrocarbons) and direct-acting mutagenic agents . The mutagenicity of Santiago airborne particles was much higher than that reported in studies performed in other countries . Results obtained with the strains TA98-NR and TA98/1,8-DNP6 suggest that the extracts also contain mononitro and dinitroarenes . These nitroarenes have been described as very potent mutagenic agents, that can be generated by photochemical reactions under certain atmospheric conditions, or in the combustion of fuel, especially of diesel motors . The presence of polycyclic aromatic hydrocarbons and nitroarenes in Santiago airborne particles, as well as the high levels of mutagenicity detected, suggest that the inhabitants permanent exposure to these kinds of compounds represents a high risk for human health.

Mutat Res, 1993 Aug, 300(3-4), 265 - 71
The mutagenicity of organotin compounds as environmental pollutants; Hamasaki T et al.; The mutagenicity of 14 organotin compounds which have been reported to be environmental pollutants, their environmental metabolites and inorganic tin (SnCl4) was studied . The experiments were carried out by a modification of the conventional Salmonella assay . Each tested chemical was removed by washing the tested strain with phosphate buffer, before the strain with top agar was poured onto minimal glucose agar . By this method, we were able to estimate the mutagenicity of organotin compounds which had antibacterial activity . It was apparent that mono-n-butyltin oxide, n-butyltin trichloride, di-n-butyltin dichloride, tri-n-butyltin chloride, bis-(tri-n-butyltin)-oxide and dimethyltin dichloride were mutagens on Salmonella typhimurium TA100 and bis-(tri-n-butyltin)-oxide showed the highest mutagenicity . With S . typhimurium TA98, di-n-butyltin dichloride was found to be a mutagen.

Mutat Res, 1993 Aug, 300(3-4), 201 - 6
Anticlastogenic and bio-antimutagenic activity of cultured broth of Saccharomyces cerevisiae 28 on mutagens; Zhang XB et al.; The possible anticlastogenic activity and bio-antimutagenic mechanism of the cultured broth of Saccharomyces cerevisiae 28 were examined using in in vivo and in vitro test systems . In the Ames test with Salmonella typhimurium TA100 (SD-) and in the umu-test with S . typhimurium TA1535/psk1002, the cultured broth of S . cerevisiae 28 showed bio-antimutagenic activity against mutagenicity induced by 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) and 2-amino-3-methylimidazo{4,5-f}quinoline (IQ) . The cultured broth also showed bio-antimutagenic activity towards reverse mutations induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in Escherichia coli B/r WP2 trp-, but not by UV radiation . It is clear that the cultured broth could inhibit base substitution mutations induced by mutagens . Using mitomycin C (MMC) as a mutagen, the micronucleus test (with bone marrow cells of mice) showed anticlastogenic action when the cultured broth was given orally to mice . Micronucleated polychromatic erythrocytes induced by the mutagen were reduced by about 47% by the cultured broth.

Mutat Res, 1993 Aug, 300(3-4), 151 - 4
Antimutagenicity of cyclohexanol towards 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone and N-nitrosodiethylamine in Salmonella typhimurium strain TA100; Espinosa-Aguirre JJ et al.; The ability of cyclohexanol to inhibit the mutagenicity of tobacco-specific nitrosamine 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) and of N-nitrosodiethylamine (NDEA) was tested on Salmonella typhimurium strain TA100 . Cyclohexanol produced a dose-dependent decrease in the number of revertants induced by a single dose of NNK (24 mumoles) or NDEA (59 mumoles) . Nevertheless, this inhibitory effect was not observed with other premutagenic agents such as benzo{a}pyrene and 2-aminoanthracene nor with direct mutagens such as ethyl methanesulfonate and methyl methanesulfonate . These results suggest that cyclohexanol interferes with the 'bioactivation' of the tested nitrosamines in a similar way that other alcohols such as ethanol or isopropanol interfere with N-nitro-sodimethylamine and NDEA metabolism.

J Mol Biol, 1993 Jul 20, 232(2), 555 - 73
Refined structures of the ligand-binding domain of the aspartate receptor from Salmonella typhimurium; Scott WG et al.; The aspartate receptor is a transmembrane-signalling protein that mediates chemotaxis behaviour in bacteria . Aspartate receptors in Salmonella typhimurium and Escherichia coli exist as dimers of two subunits in the presence as well as in the absence of aspartate . We have previously reported the three-dimensional structures of the external ligand-binding domain of the S . typhimurium aspartate receptor with and without bound aspartate . The external or periplasmic region of the aspartate receptor is a dimer of four-alpha-helical bundle subunits; a single aspartate molecule binds to one of two sites residing at the subunit interface, increasing the affinity of the subunits for one another . Here we report the results of a detailed analysis of the aspartate receptor ligand-binding domain structure (residues 25 to 188) . The dimer interface between the twofold related subunits consists primarily of contacts mediated by the side-chains of the N-terminal helix of each four-alpha-helical bundle subunit . The N-terminal helices pack approximately 20 degrees from parallel as an approximate coiled-coil super-secondary structure . We have refined aspartate receptor ligand-binding domain structures in the presence and in the absence of a bound aromatic compound, 1,10-phenanthroline, to 2.2 A and 2.3 A resolution, respectively, as well as crystal structures in the presence of specifically bound Au(I), Hg(II) and Pt(IV) complex ions at 2.4 A, 3.0 A and 3.3 A resolution, respectively . The possible biological relevance of the aromatic ligand-binding site and the metal ion-binding sites is discussed . The dimer of four-alpha-helical bundle subunits composing the periplasmic region of the S . typhimurium aspartate receptor provides a basis for understanding the results of mutational analyses performed on related chemotaxis transmembrane receptors . The crystal structure analysis provides an explanation for the way in which mutations in the E . coli aspartate receptor affect its binding to the periplasmic maltose-binding protein and how mutations in the more distantly related E . coli Trg chemotaxis receptor affect its binding to the periplasmic ribose and glucose-galactose binding proteins.

FEMS Microbiol Lett, 1993 Jul 15, 111(1), 87 - 92
Location of the enterotoxin gene from Salmonella typhimurium and characterization of the gene products; Chary P et al.; The enterotoxin gene (stn) in Salmonella typhimurium (Q1 strain) was confined to an 800 bp ClaI-EcoRI genomic DNA fragment (pCE3) that coded for two polypeptides (25 and 12 kDa) under the control of the T7 RNA polymerase/promoter system . The appearance of the 25 kDa protein corresponded to the enterotoxic activity, as determined by elongation of Chinese hamster ovary (CHO) cells, fluid accumulation in rabbit intestinal loops, and altered vascular permeability in rabbit skin . The stn gene products (STN) caused an elevation of intracellular cAMP in CHO cells . These values were at control levels in stn mutants devoid of enterotoxicity, and the 25-kDa protein concurrently disappeared . The biological activity of the heat-labile enterotoxin was blocked by GM1 ganglioside and neutralized by affinity-purified antibodies made against cholera toxin . The 12 kDa protein however was not correlated with an enterotoxic response.

Gene, 1993 Jul 15, 129(1), 9 - 16
The 92-min region of the Escherichia coli chromosome: location and cloning of the ubiA and alr genes; Lilley PE et al.; A cosmid (pND320) bearing 42.5 kb of Escherichia coli chromosomal DNA, including the genes between xylE and ssb near minute 92 on the linkage map, was isolated by selection for complementation of a dnaB mutation . Known nucleotide (nt) sequences were used to align restriction maps in this region to the physical map of the chromosome (coordinates 4319.5 to 4362 kb), and to locate precisely and define the orientations of 19 genes . Predicted physical linkage of sequenced genes across unsequenced gaps of defined length was confirmed by the nt sequence analysis of fragments subcloned from pND320 . Mutant complementation by plasmids showed that ubiA is located between malM and plsB . A previously sequenced long open reading frame that encodes the C-terminal portion of the E . coli ubiA product (4-hydroxybenzoate polyprenyltransferase, HPTase) shows a high degree of sequence identity with the corresponding segment of yeast HPTase (the COQ2 gene product) . Comparison of homologous regions from E . coli and Salmonella typhimurium was used to locate precisely the gene alr that encodes alanine racemase (ARase) between dnaB and tyrB . Subcloning of alr downstream from tandem bacteriophage lambda promoters produced a plasmid that directed high-level overproduction of a soluble approx . 40-kDa protein with ARase activity.

J Mol Biol, 1993 Jul 5, 232(1), 67 - 78
Glutamate at the site of phosphorylation of nitrogen-regulatory protein NTRC mimics aspartyl-phosphate and activates the protein; Klose KE et al.; The NTRC protein of enteric bacteria is an enhancer-binding protein that activates transcription by the sigma 54-holoenzyme form of RNA polymerase under nitrogen-limiting conditions . In vitro NTRC must be phosphorylated to catalyze ATP hydrolysis and activate transcription . The site of phosphorylation of NTRC from Salmonella typhimurium is Aspartate 54, which lies in the amino-terminal regulatory domain of the protein . We used site-directed mutagenesis to make "conservative" substitutions at residue 54 to alanine, asparagine, and glutamate, and examined the properties of the mutant NTRC proteins in vitro and in vivo . In vitro none of them was detectably phosphorylated, as expected if D54 is, in fact, the sole site of phosphorylation . D54A and D54N did not activate transcription of glnA but, interestingly, D54E activated constitutively . Activation by D54E was partial compared to that by phosphorylated wild-type NTRC . Combining D54A or D54N with S160F, a change in the central domain of NTRC that partially bypasses the requirement for phosphorylation, yielded doubly mutant proteins that were as active as a form carrying S160F alone, indicating that the changes in D54 did not adversely affect the function of the remainder of NTRC . Combining D54E with S160F increased the levels of constitutive ATPase activity and transcriptional activation above those of mutant NTRC proteins carrying either single change alone . We conclude that phosphorylation of aspartate 54 is required to activate NTRC and postulate that the D54E mutation mimics phosphorylation, thereby allowing NTRC to hydrolyze ATP and activate transcription . Phenotypes of mutant strains encoding NTRC proteins with substitutions at D54 indicated that phosphorylation of NTRC at position 54 was necessary for normal growth in the absence of glutamine and that such phosphorylation occurred to some extent even in the absence of NTRB.

J Biol Chem, 1993 Jul 5, 268(19), 14182 - 8
Conserved cysteine residues of histidinol dehydrogenase are not involved in catalysis . Novel chemistry required for enzymatic aldehyde oxidation; Teng H et al.; The 4-electron oxidoreductase L-histidinol dehydrogenase (HDH, EC 1.1.1.23) oxidizes the amino alcohol histidinol to histidine via an aldehyde-level intermediate at a single active site . The enzyme contains two Zn2+ per dimer, and treatment with metal chelators causes a metal-reversible inactivation . NAD-linked aldehyde oxidations, for which glyceraldehyde-3-phosphate dehydrogenase has served as the major paradigm, are thought to proceed via cysteine-based thiohemiacetals . Sequenced forms of HDH contain two conserved cysteine residues, Cys-116 and Cys-153 in the Salmonella typhimurium enzyme, and in previous work we have shown that HDH is inactivated by active site modification of Cys-116 by the reagent 4-nitro-7-chlorobenzadioxazole . Thus, Cys-116 is an excellent candidate for the active site nucleophile in HDH . In the current studies we show that treatment of HDH with the Zn2+ chelator 1,10-phenanthroline exposes Cys-116 to specific modification by iodoacetate, resulting in irreversible loss of activity . Site-specific mutagenesis was used to explore the roles of the conserved cysteine residues . The mutant enzymes C116S, C153S, C116A, and C153A and the double mutant C116,153A were each overproduced and purified to homogeneity . All mutant enzymes showed normal kcat and Km values for catalysis . The double mutant protein was unstable, and the single mutants also lose significant activities over a 3-h period during which wild-type enzyme retains full activity . The C116S mutant, and to a lesser extent the C116A mutant, were sensitive to the presence of EDTA in the assay medium, but the other mutants or wild-type enzyme were not, suggesting that Cys-116 may be near, but probably not liganded to, the bound metal ion . The results clearly indicate that HDH does not use a cysteine-based thiohemiacetal as a catalytic intermediate, requiring a new paradigm for NAD-linked aldehyde oxidation . Some models for the reaction are presented and discussed.

J Biol Chem, 1993 Jul 5, 268(19), 14071 - 80
Sequence and topology of the CorA magnesium transport systems of Salmonella typhimurium and Escherichia coli . Identification of a new class of transport protein; Smith RL et al.; The CorA Mg2+ transport systems of Salmonella typhimurium and Escherichia coli mediate both influx and efflux of Mg2+ . The product of the CorA locus is sufficient for mediation of Mg2+ influx while product(s) of the unlinked CorBCD loci allow CorA to mediate efflux in addition to influx . The nucleotide sequences of the S . typhimurium and E . coli CorA loci have been determined . The locus in each species consists of a single gene expressing a protein with gel molecular masses of 42 kDa (S . typhimurium) and 39 kDa (E . coli) . The predicted amino acid sequences of these proteins are each 316 amino acids in length, are 98% identical, and lack homology to any known protein . Although CorA is an integral membrane protein by biochemical criteria, its predicted amino acid sequence contains 28% charged amino acid . Membrane localization of CorA was shown to be dependent on the Sec pathway in E . coli . Hydropathy analysis predicts two C-terminal hydrophobic sequences of sufficient length to span the membrane bilayer . The membrane topology of CorA was determined by constructing deletion derivatives of CorA and genetically fusing them to BlaM or LacZ cassettes . The enzymatic activities of these hybrid proteins indicate that the N-terminal 235 amino acid residues of the CorA protein are located within the periplasmic space, comprising a single periplasmic domain . The C-terminal region of CorA is composed of three membrane-spanning segments rather than the two suggested by hydropathy plots, thus depositing the C terminus within the cytoplasm . This topology suggests that CorA functions as an oligomer since three membrane loops are most likely insufficient for any sort of membrane pore or channel . Its lack of homology to known proteins and its topology indicate that the CorA Mg2+ transporter represents a new class of membrane transport system.

Mol Microbiol, 1993 Jul, 9(1), 195 - 209
Molecular analysis of two ScrR repressors and of a ScrR-FruR hybrid repressor for sucrose and D-fructose specific regulons from enteric bacteria; Jahreis K et al.; The scr regulon of pUR400 and the chromosomally encoded scr regulon of Klebsiella pneumoniae KAY2026 are both negatively controlled by a specific repressor (ScrR) . As deduced from the nucleotide sequences, both scrR genes encode polypeptides of 334 residues (85.5% identical base pairs, 91.3% identical amino acids), containing an N-terminal helix-turn-helix motif . Comparison with other regulatory proteins revealed 30.6% identical amino acids to FruR, 27.0% to Lacl and 28.1% to GalR . Six scrRs super-repressor mutations define the inducer-binding domain . The scr operator sequences were identified by in vivo titration tests of the sucrose repressor and by in vitro electrophoretic mobility shift assays . D-fructose, an intracellular product of sucrose transport and hydrolysis, and D-fructose 1-phosphate were shown to be molecular inducers of both scr regulons . An active ScrR-FruR hybrid repressor protein was constructed with the N-terminal part of the sucrose repressor of K . pneumoniae and the C-terminal part of the fructose repressor of Salmonella typhimurium LT2 . Gel retardation assays showed that the hybrid protein bound to scr-specific operators, and that D-fructose 1-phosphate, the inducer for FruR, was the only inducer . In vivo, neither the operators of the fru operon nor of the pps operon, the natural targets for FruR, were recognized, but the scr operators were . These data and the data obtained from the super-repressor alleles confirm previous models on the binding of repressors of the Lacl family to their operators.

J Biochem (Tokyo), 1993 Jul, 114(1), 39 - 44
Flagellar growth in a filament-less Salmonella fliD mutant supplemented with purified hook-associated protein 2; Ikeda T et al.; Bacterial flagellum consists of a basal body, a hook, HAP1 (hook-associated protein 1), HAP3, a long helical filament, and a cap (composed of HAP2), all connected in series . The mutant deficient in the HAP2 structural gene (fliD) of Salmonella typhimurium has flagella composed of only hook-HAP1-HAP3 and excretes flagellin monomers into the culture medium . However, when purified HAP2 was added to this mutant, the flagellin stopped leaking out and flagellar filaments grew . Turnover of HAP2 was not necessary for the growth of a filament . Therefore HAP2 facilitates the polymerization of endogenous flagellin, apparently without falling off the filament tip . This experimental system with exogenous HAP2 allowed us to synchronize filament growth; the average rate of filament growth can be estimated by measuring the length of grown filaments at various time periods in electron micrographs . The initial growth rate was about 30 nm/min, which corresponds to one flagellin per second.

Immunology, 1993 Jul, 79(3), 375 - 80
Specific and natural antibody production during Salmonella typhimurium infection in genetically susceptible and resistant mice; Matsiota-Bernard P et al.; Genetically susceptible (C57BL/6) and resistant (CBA) mice were infected with an avirulent strain of Salmonella typhimurium and studied over a 35-day period for the production of antibodies directed against bacterial antigens including lipopolysaccharide (LPS) (specific antibodies) and antibodies directed against self antigens {natural antibodies (NAb)} . Antibodies directed against LPS and self antigens were detected by enzyme immunoassay (EIA) and those directed against other bacterial antigens by immunoblotting . We found that serum natural antibody titres in C57BL/6 and CBA mice were similar and correlated with the bacterial load in the spleen and liver . In C57BL/6 mice, anti-LPS antibodies remained polyreactive and of the IgM isotype . In contrast, CBA mice, after an early increase in polyreactive IgM anti-LPS antibodies, mounted a specific anti-LPS IgG antibody response . The immunoblotting results demonstrated that the IgM polyreactive antibodies in the resistant and susceptible mice recognized bacterial antigens of different molecular weights and that CBA, but not C57BL/6 mice, were able to produce IgG antibodies recognizing bacterial components . Our results suggest that the synthesis of antibodies directed against bacterial antigens and natural antibodies follow, at least partially, distinct pathways, but they do not allow us to determine whether these two antibody populations are produced by the same or distinct B-cell subpopulations.

J Bacteriol, 1993 Jul, 175(14), 4475 - 84
A PhoP-repressed gene promotes Salmonella typhimurium invasion of epithelial cells; Behlau I et al.; The Salmonella typhimurium transcriptional regulators, PhoP/PhoQ, induce phoP-activated gene (pag) expression to promote virulence and intracellular survival within macrophages . This response to the macrophage intracellular environment is simulated by phoP/phoQ constitutive mutations (phenotype PhoPc) that increase the expression of pag genes and repress the synthesis of approximately 20 proteins encoded by phoP-repressed genes (prg genes) (S . I . Miller and J . J . Mekalanos, J . Bacteriol . 172:2485-2490, 1990) . PhoPc bacteria are attenuated for mouse virulence, suggesting that prg genes are virulence genes . We now report the identification of five unlinked prg loci by use of the transposon TnphoA . In general, medium conditions (i.e., starvation) that activate pag expression repress prg expression . However, variable effects on the PhoP regulon were observed when bacteria were grown under different oxygen tensions (pag and prg genes) or exposed to low pH (prg genes), suggesting heterogenous control of the regulon . One prg locus, prgH, was demonstrated to contribute to mouse virulence by both the oral and the intraperitoneal routes . prgH was located at 59 min on the Salmonella chromosome, a region where other genes essential to invasion of epithelial cells are clustered . The prgH locus was highly linked to one invasion locus, hil (C.A . Lee, B.D . Jones, and S . Falkow, Proc . Natl . Acad . Sci . USA 89:1847-1851, 1992), although transcription of prgH was opposite that of the Tn5B50-encoded promoters that result in a hyperinvasive or hil phenotype . Both PrgH and PhoPc mutant S . typhimurium were found to be defective in induction of endocytosis by Madin-Darby canine kidney (MDCK) epithelial cells . The invasion defect of PrgH but not that of PhoPc mutant bacteria was complemented by plasmids containing prgH (hil) DNA . Therefore, two virulence properties of Salmonella species, induction of endocytosis by epithelial cells and survival within macrophages, are oppositely modulated by the PhoP/PhoQ virulence regulators.

J Bacteriol, 1993 Jul, 175(13), 4154 - 64
Spontaneous pmrA mutants of Salmonella typhimurium LT2 define a new two-component regulatory system with a possible role in virulence; Roland KL et al.; We isolated spontaneous mutations (pmrA) in the smooth strain Salmonella typhimurium LT2 that show increased resistance to the cationic antibacterial proteins of human neutrophils and to the drug polymyxin B . The mutation in one strain, JKS5, maps to 93 min on the S . typhimurium chromosome, near the proP gene and the melAB operon . The mutation, designated pmrA505, confers a 1,000-fold increase in resistance to polymyxin B and a 2- to 4-fold increase in resistance to neutrophil proteins . We cloned both the pmrA505 and pmrA+ alleles and found that the pmrA+ gene is partially dominant over pmrA505 . DNA sequence analysis of the pmrA505 clone revealed three open reading frames (ORFs) . The deduced amino acid sequences indicated that ORF1 encodes a 548-amino-acid (aa) protein with a putative membrane-spanning domain and no significant homology to any known protein . ORF2 and ORF3, which encode 222- and 356-aa proteins, respectively, show strong homology with the OmpR-EnvZ family of two-component regulatory systems . ORF2 showed homology with a number of response regulators, including OmpR and PhoP, while ORF3 showed homology to histidine kinase-sensor proteins EnvZ and PhoR . Genetic analysis of the cloned genes suggested that ORF2 contained the pmrA505 mutation . Comparison of the pmrA505 and pmrA+ ORF2 DNA sequences revealed a single G-A transition, which would result in a His-to-Arg substitution at position 81 in the ORF2 mutant protein . We therefore designate ORF2 PmrA and ORF3 PmrB . The function of ORF1 is unknown.

Infect Immun, 1993 Jul, 61(7), 3017 - 25
Mechanism of protective immunity induced by porin-lipopolysaccharide against murine salmonellosis; Muthukkumar S et al.; Investigations were undertaken to characterize the protective immunity induced by porin-lipopolysaccharide (LPS) against Salmonella typhimurium infection in mice . Mice immunized with porin-LPS showed higher levels of antiporin immunoglobulin G than mice which received porin alone . Further, T cells from porin-LPS-immunized mice showed an augmented proliferative response to porin in vitro compared with the response of T cells from porin-injected animals . The passive transfer of anti-LPS antibodies conferred significant protection (17%), while antiporin serum failed to protect mice against lethal challenge, indicating the protective ability of anti-LPS antibodies . However, the transfer of serum obtained from porin-LPS-immunized mice resulted in better protection (30%) than did anti-LPS or antiporin antibodies alone . In contrast to LPS, monophosphoryl lipid A completely failed to induce protection against lethal infection . However, comparable to the effect of LPS, injection of porin with monophosphoryl lipid A enhanced antibody response and the protective ability of porin (81.25%) . The transfer of T cells from porin-LPS-immunized mice provided higher levels of protection (47%) against lethal challenge than did T cells from porin-immunized mice (23%) . The combination of T cells and serum from porin-immunized mice transferred 36% protection . However, a combination of T cells and serum from porin-LPS-immunized mice conferred the highest level of protection (92%), which was reflected by the number of survivors (100%) in the porin-LPS-immunized group . These results demonstrate that besides the protective effect of anti-LPS antibodies, the ability of LPS to augment humoral and cell-mediated immune responses to porin confers effective protection against Salmonella infection.

Plasmid, 1993 Jul, 30(1), 30 - 8
The parVP region of the Salmonella typhimurium virulence plasmid pSLT contains four loci required for incompatibility and partition; Cerin H et al.; A 4332 nt MluI-PstI DNA fragment, earlier termed parVP, carries the incompatibility and partition determinants of the virulence plasmid, pSLT, of Salmonella typhimurium . This fragment was sequenced and the genes and regions responsible for incompatibility and partition phenotypes were identified by reference to previously published work . Incompatibility with pSLT, mediated by the 4332 nt fragment, requires both a homolog of the parS region of the Escherichia coli plasmids P1/P7 and a new incompatibility determinant, termed incR . The two genes identified in the sequence were also close homologs of the parA and parB loci of P1/P7 . Unlike the situation in P1/P7, however, the parA and parB genes appear to be independently transcribed . Effective partition of pJRD158-based plasmids carrying fragments of the sequenced DNA required ParA and the incR region; ParB and parS were not necessary . It appears that pSLT may possess two related partitioning mechanisms, an analog of the recognized ParA/ParB/parS system and another system which requires ParA and incR.

Mutagenesis, 1993 Jul, 8(4), 321 - 7
Synthesis and mutagenicity of a series of nitrated carbazoles and hydroxycarbazoles; Holloway TC et al.; Nitrated derivatives of the aza-arenes carbazole and 2-hydroxycarbazole were synthesized and tested for mutagenicity in the Ames plate-incorporation assay . 3,6-Dinitrocarbazole was the most mutagenic towards Salmonella typhimurium strain TA98 (> 100 revertants/micrograms, 25 revertants/nmol, with or without S9) followed by 2-hydroxy-1,3,6-trinitrocarbazole (24 revertants/micrograms, 7 revertants/nmol without S9) and 2-hydroxy-3-nitrocarbazole (27 revertants/micrograms, 6 revertants/nmol without S9) . 2-Hydroxy-1,3-dinitrocarbazole, 1,6-dinitrocarbazole, 3-nitrocarbazole and 1-nitrocarbazole ranged from moderately to weakly active (7-1 revertants/micrograms, 2-0.3 revertants/nmol without S9) . Carbazole and 2-hydroxycarbazole, and 2-hydroxy-1-nitrocarbazole, were quite inactive . Activity was generally decreased by the presence of S9, except for the dinitrocarbazoles, and was also lower in the variant TA98NR, indicating that mutagenicity was largely dependent on the presence of the 'classical' bacterial nitroreductase . The relative activities of these compounds are consistent with the hypothesis that structural features (orientation of the nitro group relative to the plane and to the long axis of the molecule, and ability to resonance-stabilize the positive charge on the arylnitrenium active electrophile intermediate) are major influences determining mutagenic potency of nitrated compounds.

Mutagenesis, 1993 Jul, 8(4), 307 - 10
The mutagenicity of chemically synthesized metabolites of 16,17-dihydro-15H-cyclopenta{a}phenanthrene and its carcinogenic 11-methyl homologue; Papaparaskeva-Petrides C et al.; Putative synthetic metabolites of the hydrocarbon 16,17-dihydro-15H-cyclopenta{a}phenanthrene and its carcinogenic 11-methyl analogue, namely trans-3,4-dihydroxy-3,4,16,17-tetrahydro-15H- cyclopenta{a}phenanthrene and its 11-methyl derivative, together with the four associated trans-3,4-dihydroxy-syn- and anti-1,2-epoxides, were assayed for mutagenicity in the Ames test with Salmonella typhimurium TA100 with and without microsomal activation . The hydrocarbons were weakly mutagenic and the 3,4-diols were more strongly so, but all required activation to express their mutagenic potential . All four diol-epoxides were much more potent mutagens, even in the absence of activation . This is in accord with the anticipated metabolic activation sequence: hydrocarbons-->3,4-diols-->3,4-diol-1,2-epoxides.

Chem Res Toxicol, 1993 Jul-Aug, 6(4), 445 - 51
Glutathione and N-acetylcysteine inactivations of mutagenic 2(5H)-furanones from the chlorination of humics in water; LaLonde RT et al.; The mutagenic 2(5H)-furanones resulting from the chlorination of lignohumic substances in water disinfection and paper pulp bleaching are known to be inactivated by thiols . The objectives of the present study were to characterize the kinetics of an inactivating reaction, isolate and characterize products, and determine their mutagenicity in relation to the starting, mutagenic 2(5H)-furanones . The Salmonella typhimurium (TA100) mutagenicity of mucochloric acid (MCA) had a mean value of 2800 revertants/mumol from four assays and was twice as potent as the C-5 isopropyl ether of MCA (MCA-IPE), whose mutagenicity was determined in the same four assays . A second-order reaction of MCA with GSH at pH 7 was observed . The major product, making up 70% of the total product mixture, was identified as a 1.5:1 mixture of two diastereomers formed by sulfur displacement of the C-4 Cl atom from MCA . The major diastereomer was isolated from the 1.5:1 mixture . Connectivity of GSH to the MCA moiety in the product was established by 2D long-range coupling NMR and fully coupled 13C NMR . On the basis of circular dichroism, the major diastereomer had the S configuration at the hydroxyl-bearing, C-5 ring carbon . MCA-IPE reacted with GSH and N-acetylcysteine (NAC), giving 1:1 mixtures of two diastereomers, again by displacement of the C-4 Cl atom from MCA . A single diastereomer was isolated from the 1:1 MCA-IPE plus NAC reaction . Its structure, determined by X-ray crystallography, had the 5R,8R configuration and was in agreement with the ross structure deduced from the NMR analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

J Vet Diagn Invest, 1993 Jul, 5(3), 372 - 7
Prevalence of Salmonella in raw meat used in diets of racing greyhounds; Chengappa MM et al.; One hundred twelve samples of commercial raw meat used in greyhound diets were collected and cultured for Salmonella using standard procedures . Fifty (44.64%) of these samples were positive for Salmonella . Salmonella typhimurium was the most frequently isolated serovar (48%), followed by S . newport (12.76%), S . agona (8.51%), and S . muenster (6.38%) . The remaining 10 serovars recovered in this study represented 27.59% of the total Salmonella isolates . In addition, the meat samples were screened for Salmonella using a commercial DNA probe . Of the 106 samples tested, 70 (66.03%) were positive for Salmonella, which indicated that the DNA probe assay was more sensitive than the culture method for screening of Salmonella in raw meat . Antimicrobial susceptibility testing revealed that most of the Salmonella isolates were sensitive to a variety of antimicrobials, particularly amikacin and apramycin, and resistant to some others, such as clindamycin, erythromycin, penicillin, and sulfadimethoxine . The cumulative percentages of susceptibility (MIC50 and MIC90) of the Salmonella isolates were also determined . Most isolates were susceptible (MIC90) to low concentrations of gentamicin (2.0 micrograms/ml), imipenem (< or = 0.25 microgram/ml), and ciprofloxacin (< or = 0.5 microgram/ml) . Marked resistance was found with the other antimicrobial agents . However, the high MIC values found for these isolates would not be achievable in vivo with the normal recommended doses of antimicrobial agents, so their use would not be beneficial . Numerous plasmid patterns were found in 17 randomly selected Salmonella isolates . Eight of the 17 isolates had 2-7 plasmids ranging from 2.4 to 15 kilobases in size . Eight isolates also exhibited large plasmids in the range of 50-60 and 95-105 kilobases.(ABSTRACT TRUNCATED AT 250 WORDS)

J Gen Microbiol, 1993 Jul, 139 ( Pt 7), 1469 - 76
Peptidoglycan synthesis in Salmonella typhimurium 2616; Gally D et al.; HPLC analysis of peptidoglycan synthesis in Salmonella typhimurium strain 2616 has revealed that: (i) there is observable variation in the composition, but no significant variation in the overall degree of cross-linking, of newly synthesized peptidoglycan during the division cycle; (ii) the types of muropeptide that constitute peptidoglycan do not vary over a wide range of growth rates; and (iii) the composition and maturation kinetics of S . typhimurium peptidoglycan are, as expected, similar to those of Escherichia coli . We propose a unitary model of peptidoglycan synthesis, with single-strand incorporation occurring at both side wall and polar sites.

J Gen Microbiol, 1993 Jul, 139 ( Pt 7), 1401 - 7
Organization of the Escherichia coli and Salmonella typhimurium chromosomes between flagellar regions IIIa and IIIb, including a large non-coding region; Raha M et al.; Flagellar regions IIIa and IIIb of the Escherichia coli and Salmonella typhimurium chromosomes (at 40 min and 42-43 min, respectively) has been shown to be separated by DNA unrelated to flagellar function, with region IIIa being immediately followed by a gene, amyA, that encodes a cytoplasmic alpha-amylase . The chromosome between amyA and flagellar region IIIb has now been investigated . The high level of DNA similarity between the E . coli and S . typhimurium sequences that exists in flagellar region IIIa and in amyA continues initially, with three genes of unknown function; in E . coli, there may be a fourth gene . The remainder of the region, up to the start of flagellar region IIIb, lacks any obvious open reading frames, scores poorly on an algorithm for coding probability, has a high A+T content, and is totally dissimilar in the two species . We conclude that it is non-coding . In E . coli this region extends for 2.7 kb and in S . typhimurium for 0.8 kb . These values are unusually large for prokaryotes, where the non-coding regions between operons are generally quite short . The data, which are discussed in the context of a hypothesized disruption of a contiguous ancestral flagellar region, may give new insight into the organization and evolution of the bacterial chromosome.

Eur J Cancer Prev, 1993 Jul, 2(4), 351 - 6
Inhibition of dexamethasone-induced cytochrome P450-mediated mutagenicity and metabolism of aflatoxin B1 by Chinese medicinal herbs; Wong BY et al.; Oldenlandia diffusa (OD) and Scutellaria barbata (SB) have been used in traditional Chinese medicine for treating liver, lung and rectal tumours . We previously showed that they inhibited mutagenesis, DNA binding and metabolism of aflatoxin B1 (AFB1) and benzo(a)pyrene (BaP) bioactivated by Aroclor 1254-induced rat S9 . The purpose of this study was to investigate the effects of OD and SB on the mutagenicity of AFB1 in Salmonella typhimurium TA100 using dexamethasone (DXM)-induced rat hepatic S9, on cytochrome P450-linked aminopyrine N-demethylase (APND) activity in DXM-induced hepatic microsomes and on the metabolism of AFB1 by DXM-induced S9 using high-performance liquid chromatography (HPLC) . The experimental results showed that OD and SB consistently inhibited the mutagenicity of AFB1 bioactivated by either non-induced or DXM-induced S9 . These effects correlated with the inhibition of cytochrome P450-linked APND activity in DXM-induced microsomes and with an inhibition of DXM-induced S9 mediated metabolism of {3H}AFB1 as determined by HPLC . Since DXM treatment has been associated with an induction of the CYP3 enzyme family, these results suggest that OD and SB may possess antimutagenic and antitumorigenic activity towards AFB1 through an inhibition of CYP3-mediated metabolism of AFB1.

Genetics, 1993 Jul, 134(3), 717 - 28
Isogenic strain construction and gene mapping in Candida albicans; Fonzi WA et al.; Genetic manipulation of Candida albicans is constrained by its diploid genome and asexual life cycle . Recessive mutations are not expressed when heterozygous and undesired mutations introduced in the course of random mutagenesis cannot be removed by genetic back-crossing . To circumvent these problems, we developed a genotypic screen that permitted identification of a heterozygous recessive mutation at the URA3 locus . The mutation was introduced by targeted mutagenesis, homologous integration of transforming DNA, to avoid introduction of extraneous mutations . The ura3 mutation was rendered homozygous by a second round of transformation resulting in a Ura- strain otherwise isogenic with the parental clinical isolate . Subsequent mutation of the Ura- strain was achieved by targeted mutagenesis using the URA3 gene as a selectable marker . URA3 selection was used repeatedly for the sequential introduction of mutations by flanking the URA3 gene with direct repeats of the Salmonella typhimurium hisG gene . Spontaneous intrachromosomal recombination between the flanking repeats excised the URA3 gene restoring a Ura- phenotype . These Ura- segregants were selected on 5-fluoroorotic acid-containing medium and used in the next round of mutagenesis . To permit the physical mapping of disrupted genes, the 18-bp recognition sequence of the endonuclease I-SceI was incorporated into the hisG repeats . Site-specific cleavage of the chromosome with I-SceI revealed the position of the integrated sequences.

Nippon Shokakibyo Gakkai Zasshi, 1993 Jul, 90(7), 1547 - 54
{Mutagenicity of the gastric juice in chronic gastritis}; Katayama S; Mutagenicity of the gastric juice in patients with chronic gastritis was determined by reverse mutation test using Salmonella typhimurium TA100 and investigated the relationship between intestinal metaplasia which has been regarded as a precancerous lesion (candidate) of gastric cancer, and atrophy of gastric mucosa . The degree of intestinal metaplasia was quantified in six biopsy specimen for one patient and represented as metaplasia index (MI) . The Ames ratio, mutagenicity, of gastric juice showed no significant relationship with the age of patient . On the other hand, Ames ratio and MI, and the age of patient showed significant relationship, respectively (p < 0.01) . Furthermore, patients with more intensive atrophy tended to show higher Ames ratio . As shown above, mutagenicity of the gastric juice suggested some relationships to the development of intestinal metaplasia and atrophy of gastric mucosa.

Food Chem Toxicol, 1993 Jul, 31(7), 483 - 9
Influence of dietary fat on DNA binding by 2-amino-3,8-dimethylimidazo{4,5-f}quinoxaline (MeIQx) in the mouse liver; Alldrick AJ et al.; Female BALB/c mice were fed either a low (1%)-fat or one of three high-fat diets (containing an additional 25% (w/w) beef fat, hydrogenated vegetable oil or non-hydrogenated vegetable oil) for 4 wk . They were then orally treated with 10 mg 2-amino-3,8-dimethylimidazo{4,5-f}quinoxaline (MeIQx)/kg body weight and killed 6 hr later . Consumption of the hydrogenated vegetable oil was accompanied by increased DNA adduct formation in mice . The abilities of hepatic S-9 preparations from mice fed the various diets to convert MeIQx to an active bacterial mutagen was assessed using Salmonella typhimurium TA98 . Preparations from mice fed the high-fat diets exhibited significantly greater capacity to activate MeIQx than did those from low-fat-fed mice . The greatest increases were seen with S-9 from animals fed either beef fat or hydrogenated vegetable oil.

Carcinogenesis, 1993 Jul, 14(7), 1429 - 34
DNA binding and mutagenicity of aflatoxin B1 catalyzed by isolated rabbit lung cells; Daniels JM et al.; The abilities of different rabbit lung cell types to bioactivate aflatoxin B1 (AFB1) to a DNA-binding and mutagenic metabolite have been examined . Microsomes were prepared from centrifugal elutriation-enriched preparations of isolated rabbit lung cell types . The activation of {3H}AFB1 (5.0 or 200 microM), measured indirectly as covalent binding to calf thymus DNA, was concentrated in microsomes from the non-ciliated bronchiolar epithelial (Clara) cell-rich fractions (13-22 times the activity of whole lung microsomes) . Microsomes from type II cell-rich fractions had minimal activity . Significant correlations were detected between the rates of microsomal DNA binding and the percentages of Clara cells in the fractions . Prior treatment of rabbits with the cytochrome P450 class 1A inducer beta-naphthoflavone had no significant effect on the microsomal activation of AFB1 . In other experiments, intact, enriched isolated rabbit lung cells were incubated with AFB1 (0-1.5 microM) in a modification of the Ames mutagenicity assay, using Salmonella typhimurium strain TA100 . The ability to activate AFB1 to mutagenic metabolite(s) in this system was localized in Clara cell-rich fractions, with no significant activity being detected in other fractions . The results of these studies indicate that the biotransformation of AFB1 to DNA-binding and mutagenic metabolite(s) in rabbit lung is heterogeneous, and that the Clara cell is specifically implicated in this ability.

Carcinogenesis, 1993 Jul, 14(7), 1371 - 6
Human glutathione S-transferase-expressing Salmonella typhimurium tester strains to study the activation/detoxification of mutagenic compounds: studies with halogenated compounds, aromatic amines and aflatoxin B1; Simula TP et al.; We have developed Salmonella typhimurium strains expressing human glutathione S-transferases (GSTs) to establish the role of these enzymes in chemical activation and deactivation . Alpha and pi class GSTs, GSTA1-1 and GSTP1-1, were expressed in Salmonella TA100 using a regulatable tac promoter expression system . The ability of these GST to modulate the mutagenicity of a range of mutagens including ethylene dibromide, ethylene dichloride and methylene dichloride was then investigated . Ethylene dibromide, ethylene dichloride and methylene dichloride were directly mutagenic in the control TA100 strain . The mutagenicity of ethylene dibromide and ethylene dichloride was increased in cells expressing GSTA1-1, but not in cells expressing GSTP1-1 . In contrast, methylene dichloride mutagenicity was unaffected by the presence of either GST . The mutagenicity of 2-aminofluorene, was not altered by the presence of either GST isozyme, while that of N-hydroxy-2-acetylaminofluorene was slightly reduced with both isozymes . The mutagenicity of aflatoxin B1 (AFB1) was marginally decreased in strains expressing GSTP1-1 . When GSTA1-1 expression was maximally induced, however, a more pronounced reduction was observed suggesting a role for GSTA1-1 in AFB1 deactivation . The tester strains described here should be valuable in establishing the specificity of human GST isozymes towards chemical toxins and carcinogens, especially for compounds whose reactive intermediates are short lived.

Carcinogenesis, 1993 Jul, 14(7), 1271 - 8
Roles of different forms of cytochrome P450 in the activation of the promutagen 6-aminochrysene to genotoxic metabolites in human liver microsomes; Yamazaki H et al.; We reported previously that the potent mutagen 6-aminochrysene is catalyzed principally by rat liver microsomal P4501A and P4502B enzymes to reactive metabolites that induce umu gene expression in O-acetyltransferase-over-expressing strain Salmonella typhimurium NM2009; the proposal was made that there are different mechanisms in the formation of reactive N-hydroxylated and diolepoxide metabolites by P450 enzymes (Yamazaki, H . and Shimada, T., Biochem . Pharmacol., 44, 913-920, 1992) . Here we further examined the roles of human liver P450 enzymes and the mechanism of activation of 6-aminochrysene by rat and human P450 enzymes in the Salmonella tester strains . Liver microsomes from 18 different human samples catalyzed activation of 6-aminochrysene more efficiently in S . typhimurium NM2009 than in the original strain of S . typhimurium TA1535/pSK1002 . The rates of 6-aminochrysene activation in 18 human liver samples showed good correlation to the contents of P4502B6 as well as contents of P4503A4 and the respective mono-oxygenase activities catalyzed by P4503A4 . Among purified P450 enzymes examined, P4501A2 as well as P4503A4 were highly active in transforming 6-amino-chrysene to reactive metabolites, suggesting the involvement of different human P450 enzymes in the reaction . Four human samples that contained relatively high levels of particular P450 enzymes in their microsomes were selected and used for further characterization . Liver microsomes from human samples HL-13 and HL-4 that contained the highest levels of P4502B6 and P4503A4 respectively, were sensitive to the respective antibodies raised against monkey P4502B and human P4503A4; the activity in sample HL-16 having the highest level of P4501A2 was inhibited by anti-P4501A2 IgG . alpha-Naphthoflavone enhanced the activation of 6-aminochrysene very significantly in human liver microsomes enriched in P4503A4 and P4502B6 enzymes . Pentachlorophenol, an inhibitor of acetyltransferase activity, suppressed the activation of 6-aminochrysene in liver microsomes from phenobarbital-treated rats and from human samples HL-4, HL-13 and HL-18 but not HL-16 . In contrast, 1,1,1-trichloropropane-2,3-oxide, an inhibitor of epoxide hydrolase activity, enhanced the activation of 6-aminochrysene catalyzed by liver microsomes from beta-naphthoflavone-treated rats and from human samples HL-16 but not HL-4, HL-13 and HL-18 . Inclusion of purified rat epoxide hydrolase to the reconstituted system containing rat and human P4501A enzymes caused a decrease in the rates of 6-aminochrysene activation.(ABSTRACT TRUNCATED AT 400 WORDS)

J Bacteriol, 1993 Jul, 175(13), 4104 - 20
The XbaI-BlnI-CeuI genomic cleavage map of Salmonella typhimurium LT2 determined by double digestion, end labelling, and pulsed-field gel electrophoresis; Liu SL et al.; Endonuclease digestion of the 4,800-kb chromosome of Salmonella typhimurium LT2 yielded 24 XbaI fragments, 12 BlnI fragments, and 7 CeuI fragments, which were separated by pulsed-field gel electrophoresis . The 90-kb plasmid pSLT has one XbaI site and one BlnI site . The locations of the fragments around the circular chromosome and of the digestion sites of the different endonucleases with respect to each other were determined by excision of agarose blocks containing fragments from single digestion, redigestion with a second enzyme, end labelling with 32P by using T7 DNA polymerase, reelectrophoresis, and autoradiography . Forty-three cleavage sites were established on the chromosome, and the fragments and cleavage sites were designated in alphabetical order and numerical order, respectively, around the chromosome . One hundred nine independent Tn10 insertions previously mapped by genetic means were located by pulsed-field gel electrophoresis on the basis of the presence of XbaI and BlnI sites in Tn10 . The genomic cleavage map was divided into 100 units called centisomes; the endonuclease cleavage sites and the genes defined by the positions of Tn10 insertions were located by centisome around the map . There is very good agreement between the genomic cleavage map, defined in centisomes, and the linkage map, defined in minutes . All seven rRNA genes were located on the map; all have the CeuI digestion site, all four with the tRNA gene for glutamate have the XbaI and the BlnI sites, but only four of the seven have the BlnI site in the 16S rRNA (rrs) gene . Their inferred orientation of transcription is the same as in Escherichia coli . A rearrangement of the rrnB and rrnD genes with respect to the arrangement in E . coli, observed earlier by others, has been confirmed . The sites for all three enzymes in the rrn genes are strongly conserved compared with those in E . coli, but the XbaI and BlnI sites outside the rrn genes show very little conservation.

J Indian Med Assoc, 1993 Jul, 91(7), 180 - 1
Leukaemoid reaction in diarrhoeal diseases; Chakraborty AK et al.; There was an outbreak of diarrhoea/dysentery in Naxalbari, North Bengal in August-September of 1992 . Ninety-seven cases were investigated . Bacterial pathogens were isolated from stools of 17 cases and the organisms were Salmonella typhimurium (76%), Salmonella enteritidis (12%) and Shigella dysenteriae type 1(12%) . A leukaemoid reaction was observed in 4 cases (24%) amongst all 17 patients and they were all children.

Zentralbl Veterinarmed B, 1993 Jul, 40(5), 305 - 25
{Serologic diagnosis of avian salmonelloses: adjustment of an ELISA test using antigens adsorbed with the aid of anti-colibacillary sera}; Kles V et al.; A serological ELISA test for diagnosis of avian salmonellosis infections with Salmonella typhimurium or enteritidis has been established . Plates were coated half with a negative antigen and half with a positive antigen . Both negative and positive antigens were adsorbed with a monovalent agglutinant anti Escherichia coli serum prior to being distributed into wells of the plates . Sera from SPF birds and from SPF birds vaccinated and/or inoculated with numerous viruses or bacteria, or sera from conventional birds bacteriologically free of any salmonellosis were tested and the percentage of false positive reactions was inferior than 1% . In groups of birds naturally infected or experimentally inoculated with Salmonella enteritidis or typhimurium the percentages of positive individuals were ranged from 15 to more than 90% according to the doses and routes of inoculation and the delay between contamination and sampling.

Res Microbiol, 1993 Jul-Aug, 144(6), 423 - 33
Effect of mecillinam on peptidoglycan synthesis during the division cycle of Salmonella typhimurium 2616; Licht J et al.; The effects of mecillinam, ampicillin and cephalexin on peptidoglycan synthesis in Salmonella typhimurium 2616 have been studied at equivalent concentrations or "isoactivities" . Using antibiotics at isoactivities allows a direct comparison of the biochemical effects of different antibiotics . When mecillinam was added at different times during the division cycle at a concentration that produced 50% inhibition of peptidoglycan synthesis in an exponential culture over a short period of time, the inhibition of synthesis was greatest in the newborn cells and least in the dividing cells . Antibiotic competition experiments showed that mecillinam preferentially bound to penicillin-binding protein 2 in S . typhimurium 2616 . High performance liquid chromatography analysis of the residual peptidoglycan synthesized in the presence of mecillinam showed an unexpected increase in pentapeptides and a significant increase in cross-linking . Other antibiotics added at equivalent activities did not show an increase in cross-linking.

J Bacteriol, 1993 Jul, 175(13), 4137 - 44
Nucleotide pool-sensitive selection of the transcriptional start site in vivo at the Salmonella typhimurium pyrC and pyrD promoters; Sorensen KI et al.; Expression of the Salmonella typhimurium pyrC and pyrD genes is regulated in response to fluctuations in the intracellular CTP/GTP pool ratio . The repressive mechanism involves the formation of a stable secondary structure (hairpin) at the 5' ends of the transcripts that precludes translational initiation by sequestering sequences required for ribosomal binding . The potential for hairpin formation is controlled through CTP/GTP-modulated selection of the transcriptional start site . Substitution of nucleotides in the region of transcriptional initiation has revealed that selection of the transcriptional start point in vivo depends on the nucleotide context within the initiation region and the nucleoside triphosphate pool ratios . For maximal control in response to CTP/GTP pool ratios, the wild-type CCGG start site motif appears to be optimal . Changing the -35 region in the pyrC promoter to the consensus sequence, or replacement of the pyrC promoter with the lac promoter from Escherichia coli, has served to illustrate that the ability of the RNA polymerase to select the initiation site in response to the intracellular nucleoside triphosphate pools is not promoter specific but is determined by the kinetic properties of the initiating RNA polymerase during the formation of the first phosphodiester bond of the transcript.

Mutat Res, 1993 Jul, 300(2), 99 - 109
Genotoxicity assessment of waste products of aluminum plasma etching with the SOS chromotest; Raabe F et al.; We evaluated 36 characteristic waste products from the plasma etching of aluminum for genotoxicity with the SOS chromotest . The majority of the samples showed genotoxic activity in tester strain Escherichia coli PQ37 without metabolic activation using S9 mix . In the presence of S9, a deactivation of the samples was regularly observed . Comparable studies with the Salmonella/microsome (Ames) test using tester strains Salmonella typhimurium TA98 and TA100 indicated actual mutagenicity of waste products . Gas chromatograms of the organic constituents of all waste products were performed in parallel with the genotoxicity assays . In contrast to the similarity of the peak patterns of all chromatograms, the biological effects of individual waste samples showed large differences . Information on chemical composition and the SOS chromotest results of a representative sample recovered over a period of 2 years is given . For this sample, the influences of sample preparation and cytotoxic matrix effects on the test parameters are also shown.

Biochemistry, 1993 Jun 29, 32(25), 6433 - 42
Kinetic mechanisms of the A and B isozymes of O-acetylserine sulfhydrylase from Salmonella typhimurium LT-2 using the natural and alternative reactants; Tai CH et al.; The resonance-stabilized quinonoid 5-mercapto-2-nitrobenzoate (TNB) is a substrate for O-acetylserine sulfhydrylase-A (OASS-A) and -B (OASS-B), giving rise to the product S-(3-carboxy-4-nitrophenyl)-L-cysteine (S-CNP-cysteine) as confirmed by ultraviolet-visible and 1H NMR spectroscopies . A comparison of the kinetics of OASS-A and OASS-B indicates that the mechanism proceeds predominantly via a bi-bi ping pong kinetic mechanism as suggested by an initial velocity pattern consisting of parallel lines at low concentrations of reactants, but competitive inhibition by both substrates as the reactant concentrations are increased . Thus, in the first half-reaction, O-acetyl-L-serine (OAS) or beta-chloro-L-alanine (BCA) is converted to alpha-aminoacrylate in Schiff base with the active site pyridoxal 5'-phosphate, while in the second half-reaction cysteine (with sulfide as the reactant) or S-CNP-cysteine (with TNB as the reactant) is formed . The ping pong mechanism is corroborated by a qualitative and quantitative analysis of product and dead-end inhibition . Product inhibition by acetate is S-parabolic noncompetitive . These data are consistent with acetate reversing the first half-reaction and producing more free enzyme to which acetate may also bind . Thus, there may be some randomness to the mechanism at high concentrations of the nucleophilic substrate.

J Mol Biol, 1993 Jun 20, 231(4), 1130 - 2
Crystallization and preliminary X-ray data for the A-isozyme of O-acetylserine sulfhydrylase from Salmonella typhimurium; Rao GS et al.; The A-isozyme of O-acetylserine sulfhydrylase, a pyridoxal phosphate-dependent enzyme isolated from Salmonella typhimurium catalyzes the synthesis of L-cysteine from O-acetyl-L-serine and sulfide . The pyridoxal form of the enzyme has been crystallized in two different forms . One form is in the orthorhombic space group P2(1)2(1)2(1) with cell constants a = 144.4 A, b = 96.9 A and c = 54.3 A and contains two monomers each of molecular weight 34,000 per asymmetric unit . The second form is in a hexagonal space group with unit cell dimensions a = b = 115 A, and c = 348 A and contains two 68,000 dimers per asymmetric unit . Complete native enzyme data sets have been collected for both crystal forms using an R-Axis II detector . A search for suitable heavy-atom derivatives is underway . Although both crystal forms diffract X-rays to better than 2.5 A, the orthorhombic form is more suited to a detailed structural analysis due to the extended lifetime in the X-ray beam and the relative size of the unit cell.

Epidemiol Infect, 1993 Jun, 110(3), 553 - 65
Epidemiology of Salmonella typhimurium: ribosomal DNA analysis of strains from human and animal sources; Nastasi A et al.; Salmonella typhimurium is the most frequently identified serovar of Salmonella in Italy . This serovar is characterized by the widespread dissemination among human and non-human sources of phenotypically and genetically well-differentiated clones . In this study 457 strains of S . typhimurium isolated in Italy in the years 1982-91 from human and animal sources were submitted to characterization by the rDNA fingerprinting technique . Application of this typing method, after digestion of chromosomal DNA with HincII endonuclease, confirmed the greatest genetic differentiation of clones of S . typhimurium, allowing reliable identification of 45 rDNA patterns linked into 9 major clusters . rDNA pattern clusters or ribotypes specific to man were not recognized, whereas some rDNA patterns were characteristically related to ducks, pigeons and pet birds . The ribotyping results for isolates from animal hosts suggest that pig and cattle are the main source of human infection.

Food Chem Toxicol, 1993 Jun, 31(6), 439 - 42
Dose-dependent genotoxic effect of pan masala and areca nut in the Salmonella typhimurium assay; Polasa K et al.; Aqueous extracts of different brands of pan masala and scented supari were tested for mutagenicity by the Salmonella typhimurium assay using tester strains TA98 and TA100 . These extracts were found to be mutagenic to both tester strains . The mutagenic effects of pan masala and scented supari extracts were similar to that produced by areca nut extract . The addition of 500 ppm saccharin to the supari extracts did not alter the mutagenic response.

J Bacteriol, 1993 Jun, 175(12), 3897 - 9
The Salmonella typhimurium uracil-sensitive mutation use is in argU and encodes a minor arginine tRNA; Lu CD et al.; The use gene of Salmonella typhimurium was previously identified by a mutation conferring sensitivity to uracil in glucose minimal medium . The use gene was cloned and identified as an allele of argU encoding a tRNA for a minor arginine codon (CGG) . The uracil-sensitive phenotype was shown to result from a base substitution in the anticodon stem of this tRNA.

J Bacteriol, 1993 Jun, 175(12), 3744 - 8
Growth rate paradox of Salmonella typhimurium within host macrophages; Abshire KZ et al.; The growth rate of Salmonella typhimurium U937 within host macrophages was estimated by two independent methods . The extent to which ribosomal protein L12 is acetylated to produce ribosomal protein L7 changes markedly with the growth rate . By this measure, the intracellular bacteria appeared to be growing rapidly . Measurements of viable bacteria, however, indicated that the bacteria were growing slowly . A solution of this apparent growth rate paradox was sought by treating U937 cells infected with S . typhimurium X3306 with ampicillin or chloramphenicol to help determine the number of bacteria that were actively growing and dividing in the intracellular condition . Use of these antibiotics showed that by 2 h after invasion, the intracellular bacteria consisted of at least two populations, one static and the other rapidly dividing . This finding implies that previously described changes in the gene expression of S . typhimurium are important for the survival and/or multiplication of the bacteria within the macrophage.

J Bacteriol, 1993 Jun, 175(12), 3734 - 43
Analysis of proteins synthesized by Salmonella typhimurium during growth within a host macrophage; Abshire KZ et al.; Salmonella typhimurium is a facultative intracellular pathogen, able both to invade and to survive within eukaryotic cells and to grow in various extracellular environments . To compare the bacterial responses to these disparate environments and to shed light on the nature of the intracellular environment, we have examined the pattern of protein synthesis by two-dimensional polyacrylamide gel electrophoresis . The levels of approximately 40 proteins were observed to increase during growth within macrophage-like U937 cells, while approximately 100 proteins exhibited levels that were repressed relative to those of an extracellular control culture . To aid in the interpretation of these results, the patterns of proteins made by S . typhimurium exposed to various environmental conditions in the laboratory were determined . The intracellular protein pattern was then compared with each of these benchmark protein patterns . This analysis revealed that, as expected, the intracellular environment appears to impose numerous stresses on the bacteria, but unexpectedly, the macrophage-induced response was not a simple sum of the individual stress responses displayed during extracellular growth.

Carcinogenesis, 1993 Jun, 14(6), 1189 - 93
Examination of alpha-carbonyl derivatives of nitrosodimethylamine and ethylnitrosomethylamine as putative proximate carcinogens; Elespuru RK et al.; Metabolites produced by enzymic oxidation are believed to be responsible for the mutagenicity and carcinogenicity of N-nitrosamines . Although alpha-hydroxy compounds are often considered, a related and more stable oxidation product, the alpha-carbonyl compound, was studied here . The alpha-carbonyl derivatives of nitrosodimethylamine (NDMA) and ethylnitrosomethylamine (oxidized at either the methyl or the ethyl group) were synthesized . The derivatives were methylnitrosoformamide (MNFA), ethylnitrosoformamide (ENFA) and methylnitrosoacetamide (MNAA) . These compounds were then studied as potential toxic, mutagenic and carcinogenic intermediates . All three compounds were very potent directly acting mutagens to Salmonella typhimurium TA1535 . Mutational Fingerprints in Escherichia coli of MNFA and ENFA (but not MNAA) matched those produced by SN1-type methylating and ethylating compounds respectively . The latter results indicate that the two alkylnitrosoformamides could be intermediates in the mutagenicity of the parent nitrosamines . In animal studies the putative metabolite MNFA was more acutely toxic than NDMA in F344 rats . In chronic experiments with MNFA in F344 rats and Syrian golden hamsters, tumors of the forestomach were induced by oral administration in most animals (except female hamsters) within 8 months . The properties of these oxidized derivatives of N-nitrosamines are consistent with expectations for proximate carcinogenic intermediates.

Carcinogenesis, 1993 Jun, 14(6), 1125 - 31
Genotoxic effects of cyclopenta-fused polycyclic aromatic hydrocarbons in isolated rat hepatocytes and rabbit lung cells; Holme JA et al.; Benz{j}aceanthrylene (B{j}A) and benz{l}aceanthrylene (B{l}A), two isomeric cyclopenta polycyclic aromatic hydrocarbons (CP-PAH) structurally related to 3-methylcholanthrene, were studied with respect to their genotoxic effects in isolated liver and lung cells . Both compounds were found to cause DNA adducts measured by the 32P-postlabelling technique . The level of DNA-adducts in rat hepatocytes exposed to 30 micrograms/ml B{l}A and B{j}A for 4 h were 46.5 +/- 22.0 and 8.3 +/- 5.1 fmol/micrograms DNA respectively . Using butanol extractions, the major and one of the minor B{j}A adducts co-chromatographed with B{j}A-1,2-oxide adducts of 2'-deoxyadenosine and 2'-deoxyguanosine . Thus, oxidation at the cyclopenta-ring of B{j}A appears to be an important activation pathway . In hepatocytes, 3-30 micrograms/ml of B{j}A and B{l}A induced DNA damage and repair measured both as increased alkaline elution of DNA and as increased incorporation of {3H}TdR in the DNA . B{l}A was somewhat more potent than B{j}A in inducing DNA repair . Reactive CP-PAH intermediates formed in the hepatocytes caused mutations in Salmonella typhimurium TA98 upon co-incubation . DNA adducts were also observed in isolated rabbit lung cells exposed to 30 micrograms/ml B{l}A or B{j}A for 2 h . A total of 14.5 +/- 6.9, 2.9 +/- 2.1 and 0.2 +/- 0.6 fmol B{l}A adducts/micrograms DNA were observed in Clara cells, type II pneumocytes and alveolar macrophages respectively . The main B{l}A adduct observed in the liver cells was not found in the lung cells . On the other hand, the levels of B{j}A adducts in the lung cells were in the range 4-14% of that found in liver cells, and no major differences between the various lung cells were observed . Neither B{l}A nor B{j}A induced DNA damage measured by alkaline elution in the lung cells, indicating that these adducts are not alkali labile.

Biochemistry, 1993 Jun 1, 32(21), 5566 - 75
Mutagenic analysis of the interior packing of an alpha/beta barrel protein . Effects on the stabilities and rates of interconversion of the native and partially folded forms of the alpha subunit of tryptophan synthase; Tsuji T et al.; A series of single and double amino acid replacements in four beta strands of the alpha subunit of tryptophan synthase from Salmonella typhimurium, and alpha/beta barrel protein, was made to study the interior packing of the barrel and to clarify its folding mechanism . The urea-induced unfolding of the alpha subunit is thought to involve a stable intermediate in which the amino folding unit (residues 1-188; helices 0-5, strands 1-6) remains folded while the carboxy folding unit (residues 189-268; helices 6-8, strands 7-8) becomes disordered {Beasty, A . M., & Matthews, C . R . (1985) Biochemistry 24, 3547; Miles, E . W., Yutani, K., & Ogasahara, K . (1982) Biochemistry 21, 2586} . Mutations in strands 1 (A18G and A18V), 6 (Y175Q), 7 (L209V), and 8 (G230A, G230V, and I232V) at the interface between these two folding units show that the effects on the stabilities of the native and intermediate conformations critically depend on the site of the replacement . Although all of these mutations decrease the stability of the native conformation, only the replacements in strand 6, Y175Q, and possibly strand 8, I232V, also perturb the intermediate . Comparisons of the effects of three pairs of double mutants with the effects of the constituent single mutants on stability show that strands 6 and 7 interact in both the intermediate and native conformations, while strands 1 and 8 interact only in the native conformation . Kinetic studies of unfolding indicate that the interactions which occur in the native conformation arise in the preceding transition state . These results demonstrate that the carboxy folding unit adopts an organized structure in the intermediate, contrary to our previous interpretation . The general implication is that the state of folding of one segment of a protein can depend on the presence of another, more stable element of structure.

Virology, 1993 Jun, 194(2), 564 - 9
Isolation of a phospholipid-free protein shell of bacteriophage PRD1, an Escherichia coli virus with an internal membrane; Luo C et al.; PRD1 is a double-stranded DNA virus infecting Escherichia coli and Salmonella typhimurium . It has an icosahedral outer protein capsid which encloses the viral membrane, inside of which resides the phage genome . In this investigation we demonstrate the detergent resistance of the intact virus particles . The membrane of empty DNA-free particles, however, is very sensitive to detergent action . We assume that their sensitivity is due to the access of detergents through a portal structure to the virus interior . Using the anionic detergent sodium dodecyl sulfate, it is possible to obtain a shell structure composed of the major coat protein P3 alone . The treatment of empty particles with the milder nonionic detergents n-octyl beta-D-glucopyranoside and Triton X-100 yielded P3 particles which retained the membrane-associated proteins P7 and P11 . Deoxycholic acid treatment yielded shells of intermediate composition between those obtained with the nonionic detergents and sodium dodecyl sulfate.

Virology, 1993 Jun, 194(2), 557 - 63
Dissociation of the lipid-containing bacteriophage PRD1: effects of heat, pH, and sodium dodecyl sulfate; Caldentey J et al.; The double-stranded DNA bacteriophage PRD1 replicates in Escherichia coli and Salmonella typhimurium . It has an outer protein coat surrounding a membrane . The phage lipids are derived from the host, but the membrane proteins are of phage origin . In this investigation we studied the effects of heat, pH, and sodium dodecyl sulfate on the integrity of phage particles . Heat and high pH result in the release of the main coat protein, P3, as trimers, whereas treatment of phage particles with detergent results in the solubilization of the membrane . Our results enable a distinction to be made between the phage structural proteins that are embedded in the lipid bilayer and those that appear to be more loosely associated with the membrane.

J Bacteriol, 1993 Jun, 175(11), 3468 - 79
Flagellin gene transcription in Bordetella bronchiseptica is regulated by the BvgAS virulence control system; Akerley BJ et al.; The products of the bvgAS locus activate expression of a majority of the known Bordetella virulence factors but also exert negative control over a class of genes called vrg genes (bvg-repressed genes) . BvgAS negatively controls the production of flagella and the phenotype of motility in Bordetella bronchiseptica . In this study flaA, the flagellin gene, was cloned and characterized to facilitate studies of this negative control pathway . An internal flaA probe detected hybridizing sequences on genomic Southern blots of Bordetella pertussis, Bordetella parapertussis, and Bordetella avium, although B . pertussis and B . parapertussis are nonmotile . FlaA is similar to the FliC flagellins of Salmonella typhimurium and Escherichia coli, and flaA complemented an E . coli flagellin mutant . Insertional inactivation of the chromosomal flaA locus eliminated motility, which was restored by complementation with the wild-type locus . Analysis of flaA mRNA production by Northern (RNA) blotting and primer extension indicated that negative regulation by BvgAS occurs at the level of transcription . The transcriptional start site of flaA mapped near a consensus site for the alternative sigma factor, sigma F, encoded by fliA in E . coli and S . typhimurium . Consistent with a role for a fliA analog in B . bronchiseptica, transcriptional activation of a flaA-lacZ fusion in E . coli required fliA and a flaA-linked locus designated frl.frl also efficiently complemented mutations in the flagellar master regulatory locus, flhDC, of E . coli . Our analysis of the motility phenotype of B . bronchiseptica suggests that the Bordetella virulence control system mediates transcriptional control of flaA through a regulatory hierarchy that includes the frl locus and an alternative sigma factor.

J Bacteriol, 1993 Jun, 175(11), 3401 - 7
The Escherichia coli K-12 "wild types" W3110 and MG1655 have an rph frameshift mutation that leads to pyrimidine starvation due to low pyrE expression levels; Jensen KF; The widely used and closely related Escherichia coli "wild types" W3110 and MG1655, as well as their common ancestor W1485, starve for pyrimidine in minimal medium because of a suboptimal content of orotate phosphoribosyltransferase, which is encoded by the pyrE gene . This conclusion was based on the findings that (i) the strains grew 10 to 15% more slowly in pyrimidine-free medium than in medium containing uracil; (ii) their levels of aspartate transcarbamylase were highly derepressed, as is characteristic for pyrimidine starvation conditions; and (iii) their levels of orotate phosphoribosyltransferase were low . After introduction of a plasmid carrying the rph-pyrE operon from strain HfrH, the growth rates were no longer stimulated by uracil and the levels of aspartate transcarbamylase were low and similar to the levels observed for other strains of E . coli K-12, E . coli B, and Salmonella typhimurium . To identify the mutation responsible for these phenotypes, the rph-pyrE operon of W3110 was cloned in pBR322 from Kohara bacteriophage lambda 2A6 . DNA sequencing revealed that a GC base pair was missing near the end of the rph gene of W3110 . This one-base-pair deletion results in a frame shift of translation over the last 15 codons and reduces the size of the rph gene product by 10 amino acid residues relative to the size of RNase PH of other E . coli strains, as confirmed by analysis of protein synthesis in minicells . The truncated protein lacks RNase PH activity, and the premature translation stop in the rph cistron explains the low levels of orotate phosphoribosyltransferase in W3110, since close coupling between transcription and translation is needed to support optimal levels of transcription past the intercistronic pyrE attenuator.

J Bacteriol, 1993 Jun, 175(11), 3317 - 26
Analysis of mutants of Salmonella typhimurium defective in the synthesis of the nucleotide loop of cobalamin; O'Toole GA et al.; The CobIII region of the cobalamin (CBL) biosynthetic (cob) operon of Salmonella typhimurium encodes functions necessary for the synthesis of the nucleotide loop of CBL and comprises three genes, designated cobU, cobS, and cobT (26) . Complementation studies identified two classes of CobIII mutants: (i) 34 mutants were complemented by a plasmid carrying the cobU+ gene, and (ii) 27 mutants were complemented by a plasmid carrying the cobS+ gene; none of the mutants tested was complemented by the cobT+ clone, a result suggesting that no cobT mutations were isolated . These data were consistent with those of complementation studies done with F' cobUST plasmids, which also suggested that the CobIII region comprises two complementation groups . A plasmid carrying cobUS+ was sufficient to complement a deletion of the entire CobIII region, a result suggesting that CobT was not required for CBL biosynthesis . Nutritional studies done with synthetic putative intermediates of the CobIII pathway were performed to further classify cobIII mutants . A subset of cobU mutants were found to be responsive to exogenous dicyano-cobinamide-GDP, while cobS mutants were found to be responsive only to CBL . These results are consistent with the adenosyl-cobinamide kinase-GTP:adenosyl-cobinamide-phosphate guanylyltransferase and CBL synthase activities proposed for CobU and CobS, respectively . The cobIII genes under the control of the T7 promoter were overexpressed, and the resulting polypeptides were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Three polypeptides with apparent molecular masses of 22, 26 and 39 kDa, consistent with the predicted masses for CobU, CobS, and CobT, respectively, were detected.

Infect Immun, 1993 Jun, 61(6), 2602 - 10
Sequence analysis of scrA and scrB from Streptococcus sobrinus 6715; Chen YY et al.; The complete nucleotide sequences of Streptococcus sobrinus 6715 scrA and scrB, which encode sucrose-specific enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system and sucrose-6-phosphate hydrolase, respectively, have been determined . These two genes were transcribed divergently, and the initiation codons of the two open reading frames were 192 bp apart . The transcriptional initiation sites were determined by primer extension analysis, and the putative promoter regions of these two genes overlapped partially . The gene encoding enzyme IIScr, scrA, contained 1,896 nucleotides, and the molecular mass of the predicted protein was 66,529 Da . The hydropathy plot of the predicted amino acid sequence indicated that enzyme IIScr was a relatively hydrophobic protein . The gene encoding sucrose-6-phosphate hydrolase, scrB, contained 1,437 nucleotides . The molecular mass of the predicted protein was 54,501 Da, and the encoded enzyme was hydrophilic . The predicted amino acid sequences of the two open reading frames exhibited approximately 45 and 70% identity with those encoded by scrA and scrB, respectively, from Streptococcus mutans GS5 . Homology also was observed between the N-terminal region of the S . sobrinus 6715 enzyme IIScr and other enzyme IIs specific for the glucopyranoside molecule, all of which generate glucopyranoside-6-phosphate during translocation and phosphorylation of the respective substrates . The sequence of the C-terminal domain of the S . sobrinus 6715 enzyme IIScr shared significant homology with enzyme IIIGlc from Escherichia coli and Salmonella typhimurium and with the C-terminal domain of enzyme IIBgl from E . coli, indicating that the two functional domains, enzyme IIScr and enzyme IIIScr, were covalently linked as a single polypeptide in S . sobrinus 6715 . The deduced amino acid sequence of the gene product of S . sobrinus scrB shared strong homology with sucrase from Bacillus subtilis, Klebsiella pneumoniae, and Vibrio alginolyticus, suggesting conservation based on the physiological roles of these proteins.

Microb Pathog, 1993 Jun, 14(6), 473 - 80
Effect of late administration of anti-TNF alpha antibodies on a Salmonella infection in the mouse model; Mastroeni P et al.; The effect of late administration of anti-TNF alpha antibodies on the course of a Salmonella infection in mice was evaluated . Administration of anti-TNF alpha antiserum as late as day 5 after challenge enhanced a sublethal primary infection with the virulent Salmonella typhimurium C5 in innately resistant (Ityr) A/J mice by preventing the suppression of exponential bacterial growth in the reticuloendothelial system (RES) (plateau phase) . When the anti-TNF alpha treatment was started well after the establishment of the plateau (day 7) a prompt relapse of the infection occurred, with the rapid resurgence of bacterial growth in the reticuloendothelial system leading to the death of the animals . In contrast, late administration of the antiserum did not affect the clearance from the tissues of an avirulent temperature-sensitive mutant of S . typhimurium C5 (C5TS) . Innately susceptible (Itys) BALB/c mice immunized with the SL3261 aroA live vaccine acquire solid long-lasting protection from oral challenge with the virulent C5 strain, suppressing growth of the challenge in the RES . Administration of anti-TNF alpha antibodies on day 8 of a secondary oral infection with strain C5 abrogated vaccine-induced protection, with a progressive increase of bacterial numbers in the RES leading to the death of the animals . The results indicate that TNF alpha is constantly required for the control of virulent salmonellae in the RES, both in a sublethal primary infection in innately resistant mice and also in a secondary infection in innately susceptible mice immunized with a live vaccine . TNF alpha may not be essential for bacterial clearance of avirulent organisms from the tissues.

Immunobiology, 1993 Jun, 188(1-2), 1 - 12
Cross-binding activity and protective capacity of monoclonal antibodies to lipid A; Mitov I et al.; Six hybridoma clones (1M, 4M, 9M, 11M, 18M and 31G), secreting monoclonal antibodies (mAbs) against lipid A were obtained after fusion between cells of mouse myeloma line X63-Ag8.653 and spleen cells from BALB/c mice immunized with acid treated Salmonella minnesota bacteria coated with additional free lipid A . The specificity and cross-binding activity of the mAbs were characterized in ELISA by using synthetic lipid A analogs as well as different lipid A and lipopolysaccharides (LPS) extracted from R- and S-form bacteria . It was found that the antibodies recognize epitopes in which phosphate groups, especially those at the C4' position of the glucosamine backbone of lipid A, were present . These epitopes were accessible also for the antibodies in purified intact LPS . By using a set of core glycolipids with increasing completion of the core region of the molecule and S-LPSs it was shown that the mAbs cross-reacted with a variety of R- and S-form LPS . The binding activity decreased with increasing length of the polysaccharide chain . The mAb did not prevent ultimate lethality of mice challenged with Klebsiella pneumoniae B and Salmonella typhimurium C5 . However a delay of mortality rate of mice pretreated with antibodies 18M and 31G and infected with K . pneumoniae was seen.

J Gen Microbiol, 1993 Jun, 139 ( Pt 6), 1133 - 40
Genotypes and phylogenetic relationships of Salmonella typhimurium are defined by molecular fingerprinting of IS200 and 16S rrn loci; Stanley J et al.; Molecular fingerprints of chromosomal genotypes in Salmonella typhimurium were generated by analysis of variation at the 16S rrn gene loci and the sites of the insertion sequence IS200 . Genetic and reference strains of S . typhimurium were compared with clinical phage type strains from cases of human salmonellosis . Three 16S rrn profiles, one of which was predominant, were found . The copy number of the Salmonella-specific insertion sequence IS200 varied from 6 to 12, and all insertions were chromosomal . Three of the insertion sites shared by all strains were serovar-specific for S . typhimurium . Thirteen distinct profiles of IS200 were detected, providing a high level of intraserovar strain discrimination . Profiles were generally more conserved among genetic and reference strains; representatives of clinical phage type strains, which are recent human isolates, showed much greater diversity of IS200 profiles . Irrespective of their origin, strains could be assigned to IS200 profile groups, phylogenetically related lines identified by combinations of conserved insertion sites . Hybridization profiles of this mobile element are markers of intermediate and short-term evolution in S . typhimurium . They provide a fingerprinting scheme for the purposes of genetics, and delineate a molecular typing scheme for the purposes of epidemiology.

Int J Androl, 1993 Jun, 16(3), 235 - 43
Alterations in testicular function after endotoxin injection in the boar; Wallgren M et al.; Twelve mature boars were injected intravenously with endotoxin from Salmonella typhimurium . Blood plasma was analysed for 15-ketodihydro-PGF2 alpha, LH and testosterone . The boars were castrated at various times after endotoxin administration and the testes examined by light and electron microscopy . The levels of 15-ketodihydro-PGF2 alpha and LH rose immediately after endotoxin injection followed by an increase in testosterone levels, and in five boars a second increase in LH levels was observed . Morphological examination of the testes revealed infiltration of polymorphonuclear neutrophils (PMN) into the testicular interstitium . The Leydig and myoid cells showed morphological changes . Alterations were also present in the seminiferous epithelium among Sertoli cells, spermatocytes and spermatids . The results indicate that endotoxins can exert a negative effect on testicular function in the boar . Except for the initial increase in LH levels, which is not clearly understood, the hormonal changes are thought to be mediated by alterations in Leydig cell function . The alterations further support previous findings in the boar, and indicate that there is short-term, moderate damage to the seminiferous epithelium following endotoxin injection.

Int J Food Microbiol, 1993 Jun 1, 18(4), 271 - 8
Heat and acid sensitivity of motile Aeromonas: a comparison with other food-poisoning bacteria; Nishikawa Y et al.; The present study was undertaken to compare the heat and acid sensitivity of aeromonads with those of other food-poisoning bacteria . It became obvious that aeromonads were more sensitive to heat than Escherichia coli O157:H7, Staphylococcus aureus, and Salmonella typhimurium . Aeromonads were killed in peptone water within 2 min at 55 degrees C, while the other bacteria survived heating at 55 degrees C for more than 15 min . Aeromonas cells were also less resistant to heat in hamburger steaks . These findings suggest that Aeromonas infection can easily be prevented by heat treatment, although correct handling of food is required to avoid recontamination since aeromonads are very common in various kinds of food . E . coli, S . aureus and S . typhimurium cells survived in buffer at pH 3.2 and in foods seasoned with vinegar . By contrast, Aeromonas cells were found to be highly sensitive to acid . However, the resistance of Aeromonas to acid may be sufficient to allow it to infect the gastrointestinal tract since Vibrio parahaemolyticus, which causes numerous outbreaks of food-poisoning every year in Japan, was susceptible to acid to the same extent as Aeromonas.

Appl Environ Microbiol, 1993 Jun, 59(6), 1842 - 7
Acid adaptation induces cross-protection against environmental stresses in Salmonella typhimurium; Leyer GJ et al.; The relationship of acid adaptation to tolerance of other environmental stresses was examined in Salmonella typhimurium . S . typhimurium was adapted to acid by exposing the cells to mildly acidic conditions (pH 5.8) for one to two cell doublings . Acid-adapted cells were found to have increased tolerance towards various stresses including heat, salt, an activated lactoperoxidase system, and the surface-active agents crystal violet and polymyxin B . Acid adaptation increased cell surface hydrophobicity . Specific outer membrane proteins were induced by acid adaptation, but the lipopolysaccharide component appeared to be unaltered . These results show that acid adaptation alters cellular resistance to a variety of environmental stresses . The mechanism of acid-induced cross-protection involved changes in cell surface properties in addition to the known enhancement of intracellular pH homeostasis.

Poult Sci, 1993 Jun, 72(6), 1164 - 8
Development of defined cultures of indigenous cecal bacteria to control salmonellosis in broiler chicks; Corrier DE et al.; An in vitro continuous-flow (CF) culture system was utilized to isolate and maintain a defined mixed culture of indigenous cecal bacteria from adult broilers . The protective effects of the defined CF culture and dietary lactose on Salmonella typhimurium colonization were evaluated in broiler chicks . The CF culture was administered to chicks by crop gavage on the day of hatch . Lactose was provided as 5% (wt/wt) of the feed ration . The chicks were challenged orally with 10(4) S . typhimurium at 3 days of age and evaluated for Salmonella colonization 7 days after challenge . The experiment was repeated in six separate trials using newly hatched chicks and CF culture that was maintained in continuous steady-state conditions from 42 to 190 days . Compared with controls, the mean number of S . typhimurium in the cecal contents of the chicks given CF culture and dietary lactose decreased significantly (P < .01) by 4.2 log10 units . Similarly, the numbers of Salmonella cecal culture-positive chicks was significantly decreased (P < .01) by 55% in the chicks given CF culture and lactose . The results indicated that a defined culture of indigenous cecal bacteria isolated and maintained in CF culture, together with dietary lactose, effectively controlled S . typhimurium cecal colonization in newly hatched broiler chicks.

Mol Gen Genet, 1993 Jun, 239(3), 378 - 92
Homology between the HrpO protein of Pseudomonas solanacearum and bacterial proteins implicated in a signal peptide-independent secretion mechanism; Gough CL et al.; A region of approximately 22 kb of DNA defines the large hrp gene cluster of strain GMI1000 of Pseudomonas solanacearum . The majority of mutants that map to this region have lost the ability to induce disease symptoms on tomato plants and are no longer able to elicit a hypersensitive reaction (HR) on tobacco, a non-host plant . In this study we present the complementation analysis and nucleotide sequence of a 4772 bp region of this hrp gene cluster . Three complete open reading frames (ORFs) are predicted within this region . The corresponding putative proteins, HrpN, HrpO and HpaP, have predicted sizes of 357, 690 and 197 amino acids, respectively, and predicted molecular weights of 38,607, 73,990 and 21,959 dalton, respectively . HrpN and HrpO are both predicted to be hydrophobic proteins with potential membrane-spanning domains and HpaP is rich in proline residues . A mutation in hpaP (for hrp associated) does not affect the HR on tobacco or the disease on tomato plants . None of the proteins is predicted to have an N-terminal signal sequence, which would have indicated that the proteins are exported . Considerable sequence similarities were found between HrpO and eight known or predicted prokaryotic proteins: LcrD of Yersinia pestis and Y . enterocolitica, FlbF of Caulobacter crescentus, FlhA of Bacillus subtilis, MxiA and VirH of Shigella flexneri, InvA of Salmonella typhimurium and HrpC2 of Xanthomonas campestris pv . vesicatoria . These homologies suggest that certain hrp genes of phytopathogenic bacteria code for components of a secretory system, which is related to the systems for secretion of flagellar proteins, Ipa proteins of Shigella flexneri and the Yersinia Yop proteins . Furthermore, these homologous proteins have the common feature of being implicated in a distinct secretory mechanism, which does not require the cleavage of a signal peptide . The sequence similarity between HrpO and HrpC2 is particularly high (66% identity and 81% similarity) and the amino acid sequence comparison between these two proteins presented here reveals the first such sequence similarity to be shown between Hrp proteins of P . solanacearum and X . campestris . An efflux of plant electrolytes was found to be associated with the interactions between P . solanacearum and both tomato and tobacco leaves . This phenomenon may be part of the mechanism by which hrp gene products control and determine plant-bacterial interactions, since hrpO mutants induced levels of leakage which were significantly lower than those induced by the wild type on each plant.

J Leukoc Biol, 1993 Jun, 53(6), 691 - 6
Enhancement in vitro of the low interferon-gamma production of leukocytes from human newborn infants; McKenzie SE et al.; Interferon-gamma (IFN-gamma), a lymphokine produced by lymphocytes with the help of monocytes, is essential for host resistance to intracellular pathogens . Leukocytes from normal term newborn infants cannot produce IFN-gamma in vitro in response to stimulation by antigen or mitogens in vitro or in vivo . We investigated the production of IFN-gamma in vitro using endotoxin from Salmonella typhimurium as a stimulus . In contrast to those from adults, mononuclear cells derived from the cord blood of newborn infants did not produce IFN-gamma in response to this endotoxin . We investigated the contribution of the functional immaturity of cord blood monocytes to this relative inability to produce IFN-gamma . Aging of the monocytes for 2 weeks in vitro or treatment of freshly isolated cord blood monocytes with conditioned medium (from cultures of mononuclear cells from healthy adults) greatly enhanced IFN-gamma production stimulated by endotoxin . Furthermore, recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), or IFN-gamma was able to substitute in part for the conditioned medium from adult cells . Thus correction of the functional immaturity of monocytes derived from newborn infants can result in enhanced production of IFN-gamma in vitro.

Wei Sheng Wu Xue Bao, 1993 Jun, 33(3), 219 - 26
{Studies on the mechanisms of resistance to trimethoprim in Salmonella typhimurium}; Hu Y; The mechanisms of resistance to trimethoprim (TMP) in the clinical isolates of Salmonella typhimurium were studied . The experimental results indicated that the frequency of TMP resistance of 50 strains of Salmonella typhimurium was 76% . Seven of the resistant strains were highly resistant to TMP and four of them contained different plasmids which could transfer in the same species and between different ones, and could be eliminated with 8% of SDS . The results of comparing the activities and characteristics of dihydrofolate reductase (DHFR) in crude extracts from seven resistant strains and those from controls suggested that overproduction of the chromosomal DHFR was the resistance mechanisms in three plasmid-free strains and the levels of DHFR activity of the strains was increased during prolonged exposure to TMP in vitro . However resistance to TMP of the plasmid-containing strains resulted from production of plasmid-mediated TMP resistant type Ia DHFR and a new type DHFR which has not been reported up to now . The present work provided the theoretical basis for clinical use of TMP and attempts to suppress development and spread of duge resistance of pathogenic bacteria.

Chin Med J (Engl), 1993 Jun, 106(6), 423 - 7
Molecular epidemiologic study of an outbreak of Salmonella typhimurium infection at a newborn nursery; Wu SX et al.; Plasmid analysis, restriction endonuclease analysis, antimicrobial susceptibility testing, biotyping, phage typing and outer membrane protein electrophoresis were used to study an outbreak of Salmonella typhimurium infection at a newborn nursery . Seven out of the 12 neonates had positive blood cultures for S . typhimurium, and 2 of them died of severe sepsis . Thirty epidemic strains of S . typhimurium belonging to phage type 12 had the same plasmid profiles (98.0, 6.7 and 3.8 Kb) and identical restriction digest patterns (23.0, 20.4, 15.0, 9.6, 8.2, 7.4, 5.8, 4.3, 3.8, 2.0 and 1.8 Kb) which were different from those of the 2 non-epidemic strains . Laboratory data suggested that the source of the infection was the index patient's mother who had a slight diarrhea; the mode of transmission was most likely due to the transfer of organisms from infant to infant by the contaminated hands of nurses during milk feeding.

Vet Microbiol, 1993 Jun, 35(3-4), 313 - 9
Bacterial resistance monitoring of salmonellas isolated from animals, national experience of surveillance schemes in the United Kingdom; Wray C et al.; Antimicrobial resistance has been monitored in salmonellas isolated from animals in England and Wales since 1970 . The current trends are indicated by comparing the results for the years 1981, 1989 and 1990 . Seventy-six per cent of all salmonella isolations are still sensitive to all 16 antimicrobials used for testing . Most antimicrobial resistance is encountered in bovine isolations of Salmonella typhimurium especially phage type DT204C . This phage type, which was initially resistant to at least seven antimicrobials, has however become more susceptible in recent years . Ninety-eight per cent of S . dublin strains from cattle are still sensitive to all the antimicrobials used for testing . Although the number of porcine salmonella isolations is small, many show antimicrobial resistance especially to tetracyclines . A large increase in the number of salmonellas isolated from poultry has occurred in recent years and 75% of these strains are sensitive to all the antibiotics used for testing . Although there has been a slight decrease in the percentage of S . enteritidis strains showing susceptibility 87% of isolations are still sensitive . The emergence of resistance to the newer antimicrobials trimethoprim, apramycin and fluorquinolones has been studied and data presented . The results are discussed with regards to the choice of techniques, bacteria monitored and future surveillance programmes in relation to the veterinary use of antimicrobials.

Genomics, 1993 Jun, 16(3), 655 - 63
High-resolution linkage map in the vicinity of the host resistance locus Bcg; Malo D et al.; The mouse chromosome 1 locus Bcg determines natural resistance/susceptibility of inbred mouse strains to infection with antigenically unrelated intracellular parasites, including several Mycobacterium species, Salmonella typhimurium, and Leishmania donovani . In our effort to clone Bcg, we have constructed a high-resolution genetic linkage map in the vicinity of the gene . We have developed eight new highly polymorphic markers (simple sequence repeats) corresponding to cloned genes (Vil, Inha, Des), microdissected chromosome 1 anonymous probes (lambda Mm1C136, lambda Mm1C163, lambda Mm1C165), or novel DNA markers from the region obtained by chromosome walking (D1Mcg101 and D1Mcg105) . We have followed the cosegregation of these markers with respect to Bcg in a novel panel of 1000 (C57L/J x C57BL/6J) x C57BL/6J segregating backcross mice . Additional segregation analyses were carried out in preexisting panels of intra- and interspecific backcross mice and recombinant inbred strains . Three of these markers were found to be very tightly linked to Bcg: lambda Mm1C165 did not show recombination with Bcg in 1424 meioses analyzed, while D1Mcg105 and lambda Mm1C136 were located 0.1 cM proximal and 0.2 cM distal to Bcg, respectively . This analysis enabled us to define further the proximal and distal boundaries of the Bcg interval: the proximal limit was defined by a single crossover occurring between D1Mcg105 and Bcg/lambda Mm1C165/Vil, and the distal limit by 1 cross-over between Bcg/lambda Mm1C165/Vil and lambda Mm1C136 in 1683 and 575 informative meioses, respectively, for a maximal interval of 0.3 cM.(ABSTRACT TRUNCATED AT 250 WORDS)

Proc Natl Acad Sci U S A, 1993 Jun 1, 90(11), 4996 - 5000
Feedback inhibition of fully unadenylylated glutamine synthetase from Salmonella typhimurium by glycine, alanine, and serine; Liaw SH et al.; Bacterial glutamine synthetase (GS; EC 6.3.1.2) was previously shown to be inhibited by nine end products of glutamine metabolism . Here we present four crystal structures of GS, complexed with the substrate Glu and with each of three feedback inhibitors . The GS of the present study is from Salmonella typhimurium, with Mn2+ ions bound, and is fully unadenylylated . From Fourier difference maps, we find that L-serine, L-alanine, and glycine bind at the site of the substrate L-glutamate . In our model, these four amino acids bind with the atoms they share in common (the "main chain" +NH3-CH-COO-) in the same positions . Thus on the basis of our x-ray work, glycine, alanine, and serine appear to inhibit GS-Mn by competing with the substrate glutamate for the active site.

Mutat Res, 1993 Jun, 287(2), 261 - 74
Antimutagenic effects of flavonoids, chalcones and structurally related compounds on the activity of 2-amino-3-methylimidazo{4,5-f}quinoline (IQ) and other heterocyclic amine mutagens from cooked food; Edenharder R et al.; Sixty-four flavonoids were tested for their antimutagenic potencies with respect to IQ in Salmonella typhimurium TA98 and in part also towards MeIQ, MeIQx, Trp-P-2, and Glu-P-1 and in S . typhimurium TA100 . Antimutagenic potencies were quantified by the inhibitory dose for 50% reduction of mutagenic activity (ID50) . A carbonyl function at C-4 of the flavane nucleus seems to be essential for antimutagenicity: two flavanols and four anthocyanidines were inactive . Again, five isoflavons, except biochanin A, were inactive . Within the other groups of 21 flavones, 16 flavonols and 16 flavanones the parent compounds flavone, flavonol, and flavanone possessed the highest antimutagenic potencies (ID50: 4.1, 2.5, 5.5 nmoles) . Increasing polarity by introduction of hydroxyl functions reduced antimutagenic potency . Reducing polarity of hydroxy flavonoids by methyl etherification, however, increased antimutagenic potency again . 6-Hydroxy- and 2'-hydroxy substituted flavonoids were considerably less potent antimutagens . Of 11 flavonoid glycosides tested all compounds except apigenin- and luteolin-7-glucoside (ID50:74, 115 nmoles) were inactive or only weakly antimutagenic . Rings C and A of the nucleus were not essential for antimutagenicity: chalcone and three derivatives were nearly as active as comparable flavones while antimutagenicity of benzylidenacetone was considerably reduced (ID50: 95 nmoles) . Cinnamylaldehyde and cinnamoates, however, were inactive . A planar structure in the vicinity of the carbonyl group may also be important for antimutagenicity . Flavanones were less potent antimutagens than the corresponding flavones, but dihydrochalcones and 14 structurally related saturated aromatic carbonyl compounds were inactive . Fisetin and 6-hydroxyflavone were competitive inhibitors, but luteolin was a mixed type inhibitor . The inhibition mechanisms of flavone, kaempferol, morin, flavanone, and 2'-hydroxyflavanone were concentration dependent, being competitive at low concentrations and mixed or non-competitive (2'-hydroxyflavanone) at concentrations about the ID50 value . No fundamental differences between the two tester strains and no clear influence of mutagen structure on antimutagenic potency could be detected.

Mutat Res, 1993 Jun, 287(2), 235 - 41
On the mutagenicity of MX compounds; Tuppurainen K et al.; Electronic properties of chlorofuranones including MX, one of the strongest bacterial mutagens, were studied using the semi-empirical AM1 method to elucidate the key features related to the high mutagenic activity of MX . The electronic structures of MX and guanine (the target base of the Salmonella typhimurium TA100 tester strain) are rationalized with the frontier electron theory . Hydrophobic properties of the MX family are examined . Based on these results and our previous QSAR model a possible mechanism for the MX-DNA interaction is proposed.

Mutat Res, 1993 Jun, 287(2), 181 - 90
Activation and metabolism of benz{j}aceanthrylene-9,10-dihydrodiol, the precursor to bay-region metabolism of the genotoxic cyclopenta-PAH benz{j}aceanthrylene; Newcomb KO et al.; Benz{j}aceanthrylene, a cyclopentafused polycylic aromatic hydrocarbon produced in combustion emissions, possesses a bay region and an etheno bridge which may both contribute to the overall genotoxicity of the compound . In order to assess the role of activation at the bay region, the precursor epoxide benz{j}aceanthrylene 9,10-oxide, its dehydration product 10-hydroxybenz{j}aceanthrylene, the key dihydrodiol 9,10-dihydroxy-9,10-dihydrobenz{j}aceanthrylene and the bay-region diol-epoxide 7,8-epoxy-9,10-dihydroxy-7,8,9,10- tetrahydrobenz{j}aceanthrylene were evaluated in the bacterial histidine-reversion plate incorporation assay (Ames assay) with Salmonella typhimurium strain TA98 . The diol-epoxide alone showed direct-acting mutagenicity (10 revertants per nmole), which was decreased by addition of exogenous metabolic activation (Aroclor 1254-treated rat-liver S9), whereas all the other compounds tested were activated by increasing concentrations of S9 . The potency of the diol-epoxide was not sufficient to account for the activity of the parent compound . Identification by proton nuclear magnetic resonance and mass spectrometry of the major products of further metabolism by Aroclor 1254-treated rat-liver S9 of the bay region precursor dihydrodiol 9,10-dihydroxy-9,10-dihydrobenz{j}aceanthrylene indicated that oxidation occurred predominantly at the etheno bridge, to give 9,10-dihydroxy-9,10-dihydrobenz{j}aceanthrylene-2(1H)-one, arising by (non-enzymic) rearrangement of the etheno bridge epoxide and the tetrol 1,2,9,10-tetrahydroxy-1,2,9,10- tetrahydrobenz{j}aceanthrylene . The bay region tetrol 7,8,9,10-tetrahydroxy-7,8,9,10-tetrahydrobenz{j} aceanthrylene was observed, implying further bay-region metabolism; re-aromatization of the benzo ring to benz{j}aceanthrylene-9,10-diol also occurred . Thus oxidation at the etheno bridge accounts for the majority of the activity of benz{j}aceanthrylene and its derivatives when Aroclor 1254-treated rat-liver S9 is used for exogenous metabolic activation.

Mutat Res, 1993 Jun, 287(2), 165 - 79
Photobiological activities of 1,6-dioxapyrene in pro- and eukaryotic cells; Averbeck D et al.; The photobiological effect of a new pyrene derivative, 1,6-dioxapyrene (1,6-DP), was studied in Salmonella typhimurium (strain TA100) and in the diploid strain D7 of the yeast Saccharomyces cerevisiae . In Salmonella, 1,6-DP shows little mutagenicity in the dark in comparison to benzo{a}pyrene (B{a}P) . This mutagenic activity decreases in the presence of liver S9 homogenates from Aroclor induced XVIInc/Z mice . However, in combination with 365 nm (UVA) radiation and in the absence of S9 mix, 1,6-DP behaves as an effective photodynamic compound inducing lethal and mutagenic effects in both organisms . In yeast, its activity, like that of B{a}P, is highly dependent on the presence of oxygen . For the same incident dose of UVA, 1,6-DP is, however, at least 6 times more effective than B{a}P in inducing cytotoxic and mutagenic effects . At equitoxic doses, 1,6-DP is as photomutagenic as B{a}P, suggesting that in both cases mutagenicity is due to similar mechanisms . Spectrophotometric measurements indicate physical interaction of 1,6-DP with DNA in the dark . Laser flash photolysis experiments show that 1,6-DP generates singlet oxygen with a quantum yield of 0.17 . In vitro 1,6-DP produces oxidative damage to guanine bases specific for singlet oxygen mediated reactions . Alkaline step elution analysis of 1,6-DP plus UVA treated yeast cells indicates a decrease in average molecular weights in DNA and an induction of single strand breaks (ssb) originating from alkali labile sites . This effect is enhanced by D2O and is thus likely to be due to the production of singlet oxygen . The strand breaks appear to differ from those induced by gamma-rays because little, if any, repair of these ssb occurs during 30 min of post-treatment incubation in complete growth medium . These results suggest that the photobiological effects of 1,6-DP are due to oxidative damage in DNA mostly induced by singlet oxygen.

EMBO J, 1993 Jun, 12(6), 2559 - 66
Functional tRNAs with altered 3' ends; O'Connor M et al.; The CCA trinucleotide is a universally conserved feature of the 3' end of tRNAs, where it serves as the site of amino acid attachment . Despite this extreme conservation, we have isolated functional mutants of tRNA(His) and tRNA(Val1) with altered CCA ends . A mutant that leads to de-repression of the histidine biosynthetic operon in Salmonella typhimurium has been characterized and found to have the CCA end of the sole tRNA(His) species mutated to UCA . However, constructed mutants of tRNA(His) with ACA or GCA ends appeared to be nonfunctional in vivo . Mutants of Escherichia coli tRNA(Val1) with GCA or ACA ends were isolated on the basis of their ability to promote frameshifting at a specific sequence . These same tRNA(Val1) mutants also caused read-through of stop codons that were one, or in some instances two, codons downstream of the valine codon decoded by the mutant tRNA . A startling implication of these data is that disruption of interactions between the CCA end of the tRNA and the large ribosomal subunit promotes these aberrant codon-anticodon interactions on the small ribosomal subunit.

Mutat Res, 1993 Jun, 291(3), 171 - 80
Specificity and sensitivity of Salmonella typhimurium YG1041 and YG1042 strains possessing elevated levels of both nitroreductase and acetyltransferase activity; Hagiwara Y et al.; Acetyltransferase and nitroreductase are enzymes involved in the intracellular metabolic activation of nitroarenes and/or aromatic amines in Salmonella typhimurium . The plasmid carrying both the acetyltransferase and nitroreductase genes was introduced into S . typhimurium TA98 and TA100 . The resulting strains, YG1041 and YG1042, respectively, showed high levels of both enzyme activities and were more sensitive to the mutagenic action of some nitro-aromatic compounds such as 2-nitrofluorene, 1-nitropyrene and p-nitrophenetole than did the sensitive strains previously established in this laboratory or the conventional strains . These results indicate that the new strains permit the very efficient detection of the mutagenicity of nitroarenes in the environment.

Mutat Res, 1993 Jun, 302(2), 109 - 17
Antimutagenic activity of extracts of leaves of four common edible vegetable plants in Nigeria (west Africa); Obaseiki-Ebor EE et al.; Organic solvent extracts of leaves of 4 common edible vegetable plants--Bryophyllum pinnatum, Dialium guincense, Ocimum gratissimum and Vernonia amygdalina--had inhibitory activity for His- to His+ reverse-mutations induced by ethyl methanesulfonate acting on Salmonella typhimurium TA100 . The concentrated ethyl acetate, methanol and petroleum ether extracts were heat-stable when dissolved in dimethyl sulfoxide . The Bryophyllum ethyl acetate extract was fractionated into alkaloidal/water-soluble, acids, polar lipid and non-polar lipid fractions . The polar and non-polar lipid fractions inhibited reversion mutations induced by ethyl methanesulfonate acting on TA100 or TA102, and were also active against reversions induced by 4-nitro-O-phenylenediamine and 2-aminofluorene in TA98 . The alkaloidal/water-soluble and the acid fractions had no appreciable antimutagenic activities.

Mutat Res, 1993 Jun, 300(1), 1 - 3
Studies on antimutagenic effects of guava (Psidium guajava) in Salmonella typhimurium; Grover IS et al.; The water and chloroform extracts of guava were tested for their antimutagenicity . The water extract was effective in inactivating the mutagenicity of direct-acting mutagens, e.g., 4-nitro-o-phenylenediamine, sodium azide, and the S9-dependent mutagen, 2-aminofluorene, in the tester strains of Salmonella typhimurium . The chloroform extract was inactive . Autoclaving of the water extract for 15 min did not reduced its activity appreciably . The enhanced inhibitory activity of the extracts on pre-incubation suggests the possibility of desmutagens in the extracts . Besides ascorbic acid and citric acid, the major constituents of the extracts, the role of other antimutagenic factors in the extracts cannot be ruled out.

J Biol Chem, 1993 May 25, 268(15), 11348 - 55
Three-dimensional structures of the periplasmic lysine/arginine/ornithine-binding protein with and without a ligand; Oh BH et al.; Many proteins exhibit a large-scale movement of rigid globular domains . Among these, bacterial periplasmic binding proteins involved in substrate transport, or transport and chemotaxis, can be used as prototypes for understanding the mechanism of the movement . Such movements have been found to be associated with specific functions, such as substrate binding, catalysis, and recognition by other biomolecules . We have determined the three-dimensional structures of the lysine/arginine/ornithine-binding protein (LAO) from Salmonella typhimurium with and without lysine by x-ray crystallographic methods at 1.8- and 1.9-A resolution, respectively . The structures are composed of two lobes held together by two short connecting strands . The two lobes are far apart in the unliganded structure, but in contact with each other in the lysine-liganded structure . The large movement of the lobes is a consequence of a 52 degrees rotation of a single backbone torsion angle in the first connecting strand and of distributed smaller changes of three backbone torsion angles of the second connecting strand . The absence of contact between the lysine and the connecting strands suggests that the ligand does not induce the conformational change directly . We instead propose that the unliganded protein undergoes a dynamic change between an "open" and a "closed" conformation and that the role of the ligand is to stabilize the closed conformation . We discuss the nature of a surface area which might be recognized by the membrane-bound complex of these amino acids transport systems.

Eur J Biochem, 1993 May 15, 214(1), 251 - 8
Molecular cloning, sequence analysis and expression of the gene for catalase-peroxidase (cpeA) from the photosynthetic bacterium Rhodobacter capsulatus B10; Forkl H et al.; The gene encoding catalase-peroxidase was cloned from chromosomal DNA of Rhodobacter capsulatus B10 . The nucleotide sequence of a 3.7-kb SacI-HindIII fragment, containing the catalase-peroxidase gene (cpeA) and its flanking regions were determined . A 1728-bp open reading frame, coding for 576 amino acid residues (molecular mass 61516 Da) of the enzyme, was observed . A Shine-Dalgarno sequence was found 5 bp upstream from the translational start site . The deduced amino acid sequence coincides with that of the amino terminus and of four peptides derived from trypsin digestion of the purified catalase-peroxidase of R . capsulatus B10 . The amino acid sequence of R . capsulatus catalase-peroxidase shows interesting similarities to the amino acid sequences of the hydroperoxidases of Escherichia coli (42.7%) and Salmonella typhimurium (39.9%), the peroxidase of Bacillus stearothermophilus (32.1%) and the catalase-peroxidase of Mycobacterium intracellulare (42.2%) . As shown by a cpeA::lacZ fusion in trans in R . capsulatus, the expression of the catalase-peroxidase gene is regulated by oxygen . The promoter of the cpeA gene was localized within 320 bp upstream of the ATG start codon.

Gene, 1993 May 15, 127(1), 105 - 10
Sequence of the Salmonella typhimurium StyLT1 restriction-modification genes: homologies with EcoP1 and EcoP15 type-III R-M systems and presence of helicase domains; Dartois V et al.; The StyLT1 restriction-modification (R-M) system of Salmonella typhimurium has recently been suggested to belong to the type-III R-M systems {De Backer and Colson, Gene 97 (1991) 103-107} . The nucleotide sequences of StyLT1 mod and res have been determined . Two closely adjacent open reading frames were found 12 bp apart with coding capacities of 651 (Mod) and 982 (Res) amino acids (aa), respectively . The genes, lying in the same direction of transcription in the mod-res order, are transcribed as distinct units . The deduced aa sequences reveal homologies with known type-III enzymes from the Escherichia coli P1 prophage, E . coli P15 plasmid and Bacillus cereus chromosome . In addition, the StyLT1 restriction endonuclease (ENase), like other type-I and type-III ENases, contains sequence motifs characteristic of superfamily-II helicases, which may be involved in DNA unwinding at the cleavage site.

FEMS Microbiol Lett, 1993 May 15, 109(2-3), 225 - 9
Characterization of a small cryptic plasmid from Salmonella enteritidis that affects the growth of Escherichia coli; Ibanez M et al.; We examined the plasmid content of 25 clinical isolates of Salmonella enteritidis, and detected the presence of small plasmids (3-5.3 kb) in 9 of them, alone, or in addition to the large, so-called virulence plasmid . A 5.3-kb plasmid isolated as unique extrachromosomal DNA from a strain responsible for a high-mortality outbreak was characterized by restriction mapping and cloning . The plasmid replicon was localized in a 1.7-kb fragment, that hybridized with three of the small plasmids detected in S . enteritidis, and with another small plasmid from Salmonella typhimurium . A strain of Escherichia coli carrying this plasmid, or a cloned 3.7-kb PvuII restriction fragment, showed a slower growth rate, especially in minimal medium, as well as a noticeable increase in DNA methyltransferase activity.

FEMS Microbiol Lett, 1993 May 15, 109(2-3), 179 - 84
Isolation of a cytotoxin from L-form Salmonella typhimurium; Kita E et al.; A cytotoxic protein was isolated from the sodium dodecyl sulphate (SDS)-solubilized extract of the stable L forms of Salmonella typhimurium by ion-retardation chromatography, ion-exchange chromatography, isoelectric focusing and gel filtration . The purified toxin, with a molecular mass of 32 kDa and with isoelectric point of 6.4, was thermolabile and trypsin-sensitive . Against mouse macrophages, its cytolytic effect was detectable in vitro at concentrations higher than 0.7 micrograms/ml, with a complete lysis obtained at 5 micrograms/ml . In contrast, it stimulated C3H/HeJ macrophages in the dose range of 0.1-0.5 micrograms/ml to allow the cell to respond to endotoxin, resulting in the significant production of tumor necrosis factor alpha . By Northern blot analysis, this effect was detectable at a dose as low as 0.01 micrograms/ml . These findings suggest that the transformation of bacillary S . typhimurium into L forms in vivo may induce alterations in host resistance against murine typhoid.

J Biol Chem, 1993 May 5, 268(13), 9787 - 92
The three-dimensional structure of the ligand-binding domain of a wild-type bacterial chemotaxis receptor . Structural comparison to the cross-linked mutant forms and conformational changes upon ligand binding; Yeh JI et al.; The three-dimensional structures of the ligand-binding domain of the wild-type Salmonella typhimurium aspartate receptor have been determined in the absence (apo) and presence of bound aspartate (complex) and compared to a cross-linked mutant containing a cysteine at position 36 which does not change signaling behavior of the intact receptor . The structures of the wild-type forms were determined in order to assess the effects of cross-linking on the structure and its influence on conformational changes upon ligand binding . As in the case of the cross-linked mutant receptor, the non-cross-linked ligand-binding domain is dimeric and is composed of 4-alpha-helical bundle monomer subunits related by a crystallographic 2-fold axis in the unbound form and by a non-crystallographic axis in the aspartate-bound form . A comparative study between the non-cross-linked and cross-linked structures has led to the following observations: 1) The long N-terminal helices of the individual subunits in the cross-linked structures are bent toward each other to accommodate the disulfide bond . 2) The rest of the subunit conformation is very similar to that of the wild-type . 3) The intersubunit angle of the cross-linked apo structure is larger by about 13 degrees when compared to the wild-type apo structure . 4) The nature and magnitude of the aspartate-induced conformational changes in the non-cross-linked wild-type structures are very similar to those of the cross-linked structures.

J Biol Chem, 1993 May 5, 268(13), 9473 - 7
The MglA component of the binding protein-dependent galactose transport system of Salmonella typhimurium is a galactose-stimulated ATPase; Richarme G et al.; Binding protein-dependent transport systems mediate the accumulation of several ions, sugars, amino acids, and peptides in Gram-negative bacteria by using the energy of ATP hydrolysis and belong to a superfamily of membrane proteins which extends to eukaryotic cells and includes the multidrug resistance P-glycoprotein and the cystic fibrosis transmembrane conductance regulator . The binding protein-dependent galactose transport system of Salmonella typhimurium comprises four proteins which have been characterized previously by molecular cloning experiments (51,000-dalton MglA protein, with a stable proteolytic product of 38,000 daltons, 33,000-dalton MglB protein, 29,000-dalton MglC protein, 21,000-dalton MglE protein) . By using a MglA hyperproducing strain, we have purified a galactose-stimulated ATPase which shows a single band in polyacrylamide gels under nondenaturing conditions and shows three bands at 51,000, 38,000, and 15,000 daltons on sodium dodecyl sulfate-polyacrylamide gels (our results suggest that the bands at 38,000 and 15,000 daltons represent proteolytic products of the 51,000-dalton protein) . The ATPase activity coincides with the purified protein during the two last chromatographic steps of the purification procedure, and it cannot be isolated from a strain which does not contain the mglA gene . The MglA ATPase is stimulated 3-fold by galactose and hydrolyzes ATP to ADP and Pi (Km ATP = 60 microM, Ka galactose = 0.3 mM, Vmax = 140 nmol/min/mg of protein) . The gamma-phosphate of ATP is transferred neither to galactose nor to the protein itself . Vanadate, N-ethylmaleimide and 5-methoxyindole-2-carboxylic acid, a specific inhibitor of binding protein-dependent transport systems, inhibit the MglA ATPase.

Appl Environ Microbiol, 1993 May, 59(5), 1383 - 90
Further studies on the feasibility of one-day Salmonella detection by enzyme-linked immunosorbent assay; Wyatt GM et al.; A model system previously developed for the rapid detection of Salmonella typhimurium in foods was improved and extended to many other Salmonella serotypes . The original protocol, which consisted of an overnight nonselective culture followed by a specific enzyme-linked immunosorbent assay (ELISA), was modified and improved . A sandwich ELISA which used polyclonal antibodies for the capture stage and a cocktail of monoclonal antibodies for the detector stage was developed . The assay recognized a wide range of Salmonella serotypes; S . enteritidis, the most important serotype in the United Kingdom had a detection limit in the ELISA of about 4 x 10(2) cells ml-1 . The cultural stage prior to the ELISA was either a single nonselective broth (incubated for 28 h) or a preenrichment broth (incubated for 7 h) plus a selective broth (incubated for 21 h) . Antibodies which bind to cells grown in the unfavorable conditions of a selective medium were selected . It was concluded that, in the future, the shortened protocols for the detection of Salmonella spp . in foods described here will be of considerable value.

Appl Environ Microbiol, 1993 May, 59(5), 1342 - 6
Rapid detection of salmonellae in poultry with the magnetic immuno-polymerase chain reaction assay; Fluit AC et al.; Rapid detection of salmonellae in chicken meat was accomplished by using the magnetic immuno-polymerase chain reaction assay (MIPA) . A direct polymerase chain reaction assay performed with chicken meat spiked with Salmonella typhimurium resulted in poor sensitivity (approximately 10(7) CFU/g of meat) . The use of immunoseparation with a Salmonella serogroup B-specific monoclonal antibody improved the sensitivity, but enrichment was required for the detection of low levels of contamination . Enrichment for 6 h in either buffered peptone water, lactose broth containing tergitol-7, or selenite-cystine broth resulted in the detection of an initial inoculum of 100 CFU per g of meat . Enrichment of the salmonellae present on 25 g of spiked chicken meat for 24 h in either buffered peptone water or selenite-cystine broth before detection by the MIPA yielded a detection limit of approximately 0.1 CFU/g of meat . A detection limit of approximately 1 CFU/g of meat was obtained when the spiked meat was stored at -20 degrees C before enrichment for 24 h and analysis with the MIPA . Although the MIPA was developed for S . typhimurium, a MIPA in which a panel of six monoclonal antibodies specific for Salmonella serogroups A through E was used detected the presence of 0.1 CFU of Salmonella enteritidis per g of chicken meat . These data indicate that the method is applicable to other commonly isolated serotypes.

Mol Gen Genet, 1993 May, 239(1-2), 97 - 108
The yeast gene MSH3 defines a new class of eukaryotic MutS homologues; New L et al.; We have identified a gene in Saccharomyces cerevisiae, MSH3, whose predicted protein product shares extensive sequence similarity with bacterial proteins involved in DNA mismatch repair as well as with the predicted protein product of the Rep-3 gene of mouse . MSH3 was obtained by performing a polymerase chain reaction on yeast genomic DNA using degenerate oligonucleotide primers designed to anneal with the most conserved regions of a gene that would be homologous to Rep-3 and Salmonella typhimurium mutS . MSH3 seems to play some role in DNA mismatch repair, inasmuch as its inactivation results in an increase in reversion rates of two different mutations and also causes an increase in postmeiotic segregation . However, the effect of MSH3 disruption on reversion rates and postmeiotic segregation appears to be much less than that of previously characterized yeast DNA mismatch repair genes . Alignment of the MSH3 sequence with all of the known MutS homologues suggests that its primary function may be different from the role of MutS in repair of replication errors . MSH3 appears to be more closely related to the mouse Rep-3 gene and other similar eukaryotic mutS homologues than to the yeast gene MSH2 and other mutS homologues that are involved in replication repair . We suggest that the primary function of MSH3 may be more closely related to one of the other known functions of mutS, such as its role in preventing recombination between non-identical sequences.

Food Chem Toxicol, 1993 May, 31(5), 323 - 9
Mutagenic activity of peptides and the artificial sweetener aspartame after nitrosation; Shephard SE et al.; Naturally occurring dipeptides, cholecystokinine (CCK, a tetrapeptide hormone) and the artificial sweetener aspartame were nitrosated for 10-30 min with 40 mM-nitrite (pH 3.5, 37 degrees C), and the resultant products examined for mutagenicity in Salmonella typhimurium TA100 . Specific mutagenicities (net revertants per mumol precursor) spanned four orders of magnitude, with CCK being the most potent precursor (4700 revertants/mumol) followed by tryptophyl-tryptophan (Trp-Trp; 1000 revertants/mumol) . Aspartame and glycyl-Trp (Gly-Trp) had intermediate activity (300 revertants/mumol), while Gly-Gly and methionyl-methionine were only weakly mutagenic (20 and 12 revertants/mumol, respectively) . The dipeptides of aspartic acid, phenylalanine and tyrosine had no detectable mutagenicity (limits of detection 0.5, 40 and 5 revertants/mumol, respectively) . Kinetic studies with aspartame and Gly-Trp suggested that the mutagenic products arose primarily from nitrosation of the primary amine rather than the amide or indole group . The mutagenicities of nitrosated aspartame and Gly-Trp were higher in TA100 than in TA98, and higher without than with enzymatic activation (S-9 mix) in both strains . The time-course study of Trp-Trp nitrosation showed the production of at least two mutagens: a potent but unstable mutagenicity was seen at very short nitrosation times and a more stable but weaker effect was obtained after more than 60 min of nitrosation . Not only the absolute specific mutagenicity but also the nitrite dependence of the nitrosation reaction and the stability of the nitroso product must be taken into account in determining the risk posed by endogenous nitrosation of foods in the human stomach . Under stomach conditions, nitrosation of the side-chains of certain Trp peptides would be expected to contribute more to the endogenous burden of nitrosated products than nitrosation of aspartame or Gly peptides.

J Bacteriol, 1993 May, 175(10), 3131 - 8
Genetic and biochemical analysis of Salmonella typhimurium FliI, a flagellar protein related to the catalytic subunit of the F0F1 ATPase and to virulence proteins of mammalian and plant pathogens; Dreyfus G et al.; FliI is a Salmonella typhimurium protein that is needed for flagellar assembly and may be involved in a specialized protein export pathway that proceeds without signal peptide cleavage . FliI shows extensive sequence similarity to the catalytic beta subunit of the F0F1 ATPase (A . P . Volger, M . Homma, V . M . Irikura, and R . M . Macnab, J . Bacteriol . 173:3564-3572, 1991) . It is even more similar to the Spa47 protein of Shigella flexneri (M . M . Venkatesan, J . M . Buysse, and E . V . Oaks, J . Bacteriol . 174:1990-2001, 1992) and the HrpB6 protein of Xanthomonas campestris (S . Fenselau, I . Balbo, and U . Bonas, Mol . Plant-Microbe Interact . 5:390-396, 1992), which are believed to play a role in the export of virulence proteins . Site-directed mutagenesis of residues in FliI that correspond to catalytically important residues in the F1 beta subunit resulted in loss of flagellation, supporting the hypothesis that FliI is an ATPase . FliI was overproduced and purified almost to homogeneity . It demonstrated ATP binding but not hydrolysis . An antibody raised against FliI permitted detection of the protein in wild-type cells and an estimate of about 1,500 subunits per cell . An antibody directed against the F1 beta subunit of Escherichia coli cross-reacted with FliI, confirming that the proteins are structurally related . The relationship between three proteins involved in flagellar assembly (FliI, FlhA, and FliP) and homologs in a variety of virulence systems is discussed.

J Bacteriol, 1993 May, 175(10), 3121 - 30
Synthesis of peptidoglycan and membrane during the division cycle of rod-shaped, gram-negative bacteria; Gally D et al.; A modified procedure for determining the pattern of peptidoglycan synthesis during the division cycle has allowed the measurement of the rate of side wall synthesis during the division cycle without the contribution due to pole formation . As predicted by a model proposing that the surface growth of the cell is regulated by mass increase, we find a decrease in side wall synthesis in the latter half of the division cycle . This supports the proposal that, upon invagination, pole growth accommodates a significant proportion of the increasing cell mass and that residual side wall growth occurs in response to the residual mass increase not accommodated by pole volume . The observed side wall synthesis patterns support the proposal that mass increase is a major, and possibly sole, regulator of bacterial surface increase . Membrane synthesis during the division cycle of the gram-negative, rod-shaped bacteria Escherichia coli and Salmonella typhimurium has also been measured with similar methods . The rate of membrane synthesis--measured by incorporation of radioactive glycerol or palmitate relative to simultaneous labeling with radioactive leucine--exhibits the same pattern as peptidoglycan synthesis . The results are compatible with a model of cell surface growth containing the following elements . (i) During the period of the division cycle prior to invagination, growth of the cell occurs predominantly in the side wall and the cell grows only in length . (ii) When invagination begins, pole growth accommodates some cytoplasmic increase, leading to a concomitant decrease in side wall synthesis . (iii) Surface synthesis increases relative to mass synthesis during the last part of the division cycle because of pole formation . It is proposed here that membrane synthesis passively follows the pattern of peptidoglycan synthesis during the division cycle.

Tijdschr Diergeneeskd, 1993 May 1, 118(9), 301 - 2
{Consequences of a Salmonella typhimurium infection on a veal calf farm}; Bosch JC et al.; An outbreak of Salmonella typhimurium in veal calves is described . The pattern of resistance to antibiotics was unique in the Netherlands at the time of the outbreak . Other factors besides antibiotic resistance also played a role in the outbreak, resulting in a mortality exceeding 90%.

Proc Natl Acad Sci U S A, 1993 May 1, 90(9), 4295 - 8
PutA protein, a membrane-associated flavin dehydrogenase, acts as a redox-dependent transcriptional regulator; Ostrovsky de Spicer P et al.; The proline utilization (put) operon of Salmonella typhimurium is transcriptionally repressed by PutA protein in the absence of proline . PutA protein also carries out the enzymatic steps in proline catabolism . These two roles require different cellular localizations of PutA . Catabolism of proline requires PutA to associate with the membrane because reoxidation of the FAD cofactor in PutA needs the presence of an electron acceptor . Repression of the put operon requires PutA to bind to the put control-region DNA in the cytoplasm . The presence of proline, the inducer, is necessary but not sufficient for PutA to discriminate between its roles as an enzyme or as a repressor . Two conditions that prevent PutA protein binding to the put control region are (i) when proline and an electron acceptor or the cytoplasmic membrane are present or (ii) when PutA is reduced by dithionite . These two conditions increase the relative hydrophobicity of PutA protein, favoring membrane association and therefore enzymatic activity.

J Bacteriol, 1993 May, 175(9), 2534 - 40
Cloning and characterization of the Escherichia coli K-12 rfa-2 (rfaC) gene, a gene required for lipopolysaccharide inner core synthesis; Chen L et al.; A genetically defined mutation, designated rfa-2, results in altered lipopolysaccharide (LPS) biosynthesis . rfa-2 mutants produce a core-defective LPS that contains lipid A and a single sugar moiety, 2-keto-3-deoxyoctulosonic acid, in the LPS core region . Such LPS core-defective or deep-rough (R) mutant structures were previously designated chemotype Re . Phenotypically, rfa-2 mutants exhibit increased permeability to a number of hydrophilic and hydrophobic agents . By restriction analyses and complementation studies, we clearly defined the rfa-2 gene on a 1,056-bp AluI-DraI fragment . The rfa-2 gene and the flanking rfa locus regions were completely sequenced . Additionally, the location of the rfa-2 gene on the physical map of the Escherichia coli chromosome was determined . The rfa-2 gene encodes a 36,000-dalton polypeptide in an in vivo expression system . N-terminal analysis of the purified rfa-2 gene product confirmed the first 24 amino acid residues as deduced from the nucleotide sequence of the rfa-2 gene coding region . By interspecies complementation, a Salmonella typhimurium rfaC mutant (LPS chemotype Re) is transformed with the E . coli rfa-2+ gene, and the transformant is characterized by wild-type sensitivity to novobiocin (i.e., uninhibited growth at 600 micrograms of novobiocin per ml) and restoration of the ability to synthesize wild-type LPS structures . On the basis of the identity and significant similarity of the rfa-2 gene sequence and its product to the recently defined (D . M . Sirisena, K . A . Brozek, P . R . MacLachlan, K . E . Sanderson, and C . R . H . Raetz, J . Biol . Chem . 267:18874-18884, 1992), the S . typhimurium rfaC gene sequence and its product (heptosyltransferase 1), the E . coli K-12 rfa-2 locus will be designated rfaC.

Infect Immun, 1993 May, 61(5), 2122 - 30
Abrogation of suppression of delayed hypersensitivity induced by Candida albicans-derived mannan by treatment with monophosphoryl lipid A; Domer JE et al.; Monophosphoryl lipid A (MLA), derived either from Salmonella minnesota or Salmonella typhimurium, was tested for its ability to alter Candida albicans mannan (MAN)-specific suppression . Since we showed previously that naive mice injected intravenously (i.v.) with MAN developed suppressor T cells capable of down-regulating delayed-type hypersensitivity when transferred to immunized recipients, MLA was tested for its ability to influence suppressor activity in the donors of suppressor cells . T-lymphocyte-enriched suspensions from donor mice treated with MLA, especially that derived from S . typhimurium, 2 or 3 days after the injection of MAN lost the ability to suppress delayed-type hypersensitivity when transferred to immunized mice . Transferable suppressor activity was reduced but not always completely abrogated when donor animals were treated with MLA 1 day following the administration of MAN . In several experiments, S . minnesota MLA also abrogated activity, but it was not effective in other transfer experiments . In a different type of experiment, MLA was given to immunized mice which had been suppressed directly with MAN . Mice were immunized, either by the introduction of C . albicans intragastrically followed by inoculation intradermally (i.d.) or by two i.d . inoculations, and MAN-specific suppressor cells were induced in such animals by the i.v . injection of MAN 1 day before the first or second i.d . inoculation in animals given intragastric plus i.d . inoculations and those given two i.d . inoculations, respectively . MLA was administered to such mice prior to the i.v . injection of MAN, on the same day, or 1 to 4 days thereafter . S . typhimurium MLA, especially when given to mice 2 days following the administration of MAN, caused a partial abrogation of suppressor activity . Overall, however, MLA, at 5 to 100 micrograms, had variable and minimal effects on suppressor activity in immunized mice suppressed by the i.v . administration of MAN . In summary, MLA is clearly capable of abrogating MAN-induced suppression when given to nonimmunized animals in which MAN-specific suppressor cells had been induced, but its efficacy in immunized animals suppressed by the i.v . administration of MAN was marginal.

Infect Immun, 1993 May, 61(5), 1859 - 66
Immune responses to Streptococcus sobrinus surface protein antigen A expressed by recombinant Salmonella typhimurium; Doggett TA et al.; In this study, we used a vaccine strain of Salmonella typhimurium to express antigenic determinants of the SpaA antigen of Streptococcus sobrinus, which is involved in the caries-forming process . We cloned either a single repeat (pYA2901) or three tandem repeats (pYA2905) of the 0.48-kb fragment of the spaA gene, which codes for an important component of the SpaA protein, plus a 1.2-kb minor antigenic determinant and measured the resulting immune responses to SpaA in orally immunized BALB/c mice . The single or triple repeat of the spaA gene fragment was inserted into the Asd+ vector pYA292 and was transformed into the S . typhimurium delta cya delta crp vaccine strain chi 4072 containing delta asd in the chromosome . Female BALB/c mice were then orally immunized with two doses of the S . typhimurium containing either of the two SpaA constructs, and the immune responses to the expressed SpaA protein were assessed . Significant serum immunoglobulin G (IgG) anti-SpaA titers were detected in mice immunized with chi 4072(pYA2905) but not chi 4072(pYA2901) . Salivary anti-SpaA IgA titers were minimal and were only detected in mice immunized with S . typhimurium expressing the SpaA encoded by pYA2905 . Intestinal anti-SpaA IgA titers, however, were detected in both groups of mice, particularly in mice immunized with chi 4072(pYA2905) . An oral booster 26 weeks after the initial series of immunizations resulted in increased serum IgG titers in both chi 4072(pYA2901)- and chi 4072(pYA2905)-immunized animals, particularly in the chi 4072(pYA2905)-immunized animals . No anamnestic IgA response was detected in the saliva following the booster immunization.

J Immunol, 1993 May 1, 150(9), 3973 - 81
Salmonella typhimurium induces IFN-gamma production in murine splenocytes . Role of natural killer cells and macrophages; Ramarathinam L et al.; IFN-gamma is a cytokine known to play an important role in host defense against Salmonella typhimurium . The lymphoid cells required for in vitro production of IFN-gamma after S . typhimurium stimulation of mouse spleen cells was investigated . Spleen cells depleted of cells bearing NK1.1, asialo GM1, Thy 1.2, or CD5 resulted in a significant reduction in IFN-gamma production after stimulation with S . typhimurium . In contrast, Con A-induced IFN-gamma production was only slightly reduced after depletion of NK1.1- or asialo GM1-bearing cells . Spleen cells from SCID mice produced elevated levels of IFN-gamma after stimulation with S . typhimurium . IFN-gamma production by SCID spleen cells was dependent upon asialo GM1+ T cells, suggesting that NK cells were the cells producing IFN-gamma in response to S . typhimurium . Splenic adherent cells were required for optimal IFN-gamma production . However, direct contact between the adherent and nylon wool nonadherent (NWNA) cell populations was not essential . IFN-gamma production was observed when the adherent and NWNA cell populations were physically separated or when supernatant from S . typhimurium-stimulated adherent cells was added to NWNA cells . Optimal IFN-gamma production was dependent on the presence of TNF-alpha, inasmuch as addition of antibody to TNF-alpha to spleen cell or NWNA cell cultures significantly reduced IFN-gamma production . However, addition of rTNF-alpha did not induce IFN-gamma production by NWNA cells . These findings document the existence of a T-independent mechanism for early IFN-gamma production in response to S . typhimurium, and show that TNF-alpha is necessary but not sufficient for the production of IFN-gamma.

J Immunol, 1993 May 1, 150(9), 3965 - 72
Ity influences the production of IFN-gamma by murine splenocytes stimulated in vitro with Salmonella typhimurium; Ramarathinam L et al.; The Ity-Lsh-Bcg genetic locus in the mouse has been documented to confer innate resistance to at least three intracellular pathogens: Salmonella typhimurium, Leishmania donovani, and Mycobacterium . Expression of the resistance gene(s) results in a slower net growth of these pathogens in the reticuloendothelial system early postinfection . Although it is clear that the resident macrophages in resistant mice are functionally superior with regard to antimicrobial activity, the exact mechanism(s) underlying the control exerted by this gene is not understood . Using S . typhimurium infection as a model, we have examined the influence of this resistance gene(s) on the production of IFN-gamma, a cytokine known to play an important role in host-defense against several intracellular pathogens . We compared IFN-gamma production by splenocytes from resistant (Ity(r)) and sensitive (Ity(s)) inbred mouse strains after stimulation in vitro with S . typhimurium . Spleen cells from Ity(r) mouse strains produced significantly higher levels of IFN-gamma when compared to spleen cells obtained from Ity(s) mouse strains . Enhanced IFN-gamma production was not a generalized response to bacteria . Listeria monocytogenes induced comparable levels of IFN-gamma production from both Ity(r) (CBA/J) and Ity(s) (C57BL/6) mice . Splenocytes from Ity congenic mouse strains displayed similar differences in the level of IFN-gamma produced after S . typhimurium stimulation, with spleen cells from the Ity(r) strain producing significantly higher levels of IFN-gamma when compared to spleen cells from the Ity(s) strain . A requirement for adherent cells and/or adherent cell-derived factors has been documented for IFN-gamma production by S . typhimurium-stimulated splenocytes . Interestingly, supernatant from adherent cells obtained from Ity(r) mouse strains was found to induce the production of significantly higher levels of IFN-gamma when compared to adherent cell supernatant from Ity(s) strains . Nylon wool nonadherent cells from Ity(s) mouse strains produced high levels of IFN-gamma when exposed to supernatants obtained from adherent cells of Ity(r) mouse strains . In contrast, nylon wool nonadherent cells from Ity(r) mouse strains produced reduced levels of IFN-gamma when exposed to supernatant obtained from adherent cells of Ity(s) mouse strains . Thus, modulation of IFN-gamma production appears to be a function of the Ity(r) gene(s) . This study documents for the first time that the Ity locus may play a role in controlling resistance to Salmonella infection by regulating IFN-gamma production by NK cells.

J Gen Microbiol, 1993 May, 139 ( Pt 5), 1027 - 31
Regulation of methylthioribose kinase by methionine in Klebsiella pneumoniae; Tower PA et al.; 5-Methylthioribose (MTR) kinase catalyses a key step in the recycling of methionine from 5'-methylthioadenosine, a co-product of polyamine biosynthesis, in Klebsiella pneumoniae . In defined medium lacking methionine, K . pneumoniae exhibits abundant MTR kinase activity . When the bacterium is transferred to a medium containing 10 mM-methionine, the specific activity of MTR kinase decreases in a fashion consistent with repression of new enzyme synthesis and dilution of existing enzyme by cell division . The specific activity of methionine synthase decreases to a similar degree under the same conditions . In Escherichia coli and Salmonella typhimurium, the gene for methionine synthase is co-ordinately controlled as part of the methionine regulon . Taken together, our results indicate that a methionine regulon may function in K . pneumoniae and that expression of MTR kinase may be under its control.

Genetika, 1993 May, 29(5), 760 - 7
{Antimutagenic action of superoxide dismutase on sodium azide- and nitrosoguanidine-induced mutagenesis in Salmonella typhimurium TA 1535}; Vorob'eva LI et al.; It was shown that superoxide dismutase (SOD) decreased the mutagenic action of sodium azide (NaN3) and N-methyl-N'-nitro-N-nitrosoguanidine in Salmonella typhimurium TA1535 . Catalase and quenchers of hydroxyl radicals showed, unlike SOD no effect on the mutagenicity of NaN3 . Cell extract from propionic acid bacteria also possessed the antimutagenic activity, only partially depending on the SOD activity.

Chem Res Toxicol, 1993 May-Jun, 6(3), 310 - 2
The importance of hydrophobicity in the mutagenicity of methanesulfonic acid esters with Salmonella typhimurium TA100; Debnath AK et al.; The analysis of the mutagenic activity of a series of 15 methanesulfonate esters with Salmonella typhimurium TA100 shows that it can be correlated with the following equation: log TA100 = 1.10 log P + 0.73 log MMI -2.53, where MMI is the relative rate of reaction of the esters with N-methyl-2-mercaptoimidazole and P is the octanol/water partition coefficient . The results are compared with a number of other structure-activity studies on mutagenesis by a variety of types of chemicals.

J Bacteriol, 1993 May, 175(10), 3089 - 95
Binding of an Escherichia coli double-stranded DNA virus PRD1 to a receptor coded by an IncP-type plasmid; Kotilainen MM et al.; IncP plasmid RP1 Tra regions are needed to assemble the receptor for lipid-containing double-stranded DNA bacteriophage PRD1 on the cell surface . Using radioactively labeled phage and electron microscopic techniques, we showed that the surfaces of Salmonella typhimurium(RP1) and Escherichia coli(RP1) cells contained approximately 50 and 20 PRD1 binding sites, respectively . Expression of the receptor was growth phase dependent and was highest at late logarithmic or early stationary phase . The PRD1-resistant RP1 transposon mutants isolated were all Tra-, and the transposons were located in both the Tra1 and Tra2 regions.

Microb Pathog, 1993 May, 14(5), 369 - 79
Delayed (footpad) hypersensitivity and Arthus reactivity using protein-rich antigens and LPS in mice immunized with live attenuated aroA Salmonella vaccines; Mastroeni P et al.; Footpad reactions to protein-rich salmonella extracts and lipopolysaccharide (LPS) were studied in BALB/c mice 2 and 8 months after immunization with the Salmonella typhimurium SL3261 aroA live vaccine . T-cell depletion in vivo and adoptive serum transfer showed that protein-rich antigens induced T-cell dependent delayed hypersensitivity reactions, whereas LPS only elicited Arthus reactions . The footpad reactions to crude protein extracts were not always T-cell mediated, but depended on the nature and the dose of the antigen . Selective depletion of CD4+ T cells alone had a greater effect than depletion of CD8+ T cells alone, but neither was as marked as simultaneous depletion of both CD4+ and CD8+ T cells, which abolished the delayed type hypersensitivity (DTH) response . Crude protein-rich extracts subjected to alkaline hydrolysis (which removes some ester-linked fatty acids and causes disaggregation of LPS resulting in decreased toxicity while conserving O-specificity) still gave positive T-cell dependent reactions, but with reduced T-cell independent reactivity . Purified phenol-water LPS (2.5 micrograms) produced Arthus reactivity which could be confused with DTH . LPS induced positive reactions which still occurred in T-cell depleted mice and were transferable by immune serum . Arthus reactions did not occur when using alkali-treated LPS, which showed reduced complement fixation in vitro when using serum from immunized mice . The results indicate that footpad testing using salmonella antigens containing LPS elicit DTH but can also produce toxic reactions, some of which are T-cell independent and not necessarily a true measure of DTH . Arthus reactivity to LPS can be confused with DTH . Alkaline hydrolysis of the antigens can eliminate non-specific reactogenicity while retaining the ability of the (protein-rich) antigen to elicit a true T-cell dependent footpad response, which requires the participation of both CD4+ and CD8+ T cells.

Mol Microbiol, 1993 May, 8(5), 843 - 55
Identification of cpsD, a gene essential for type III capsule expression in group B streptococci; Rubens CE et al.; We showed previously that a mutant strain of group B Streptococcus (GBS) defective in capsule production was avirulent . This study describes the derivation of an unencapsulated mutant from a highly encapsulated wild-type strain of type III GBS, COH1, by transposon mutagenesis with Tn916 delta E . The mutant, COH1-13, was sensitive to phagocytic killing by human leukocytes in vitro and was relatively avirulent in a neonatal rat sepsis model compared with the wild-type strain . No capsular polysaccharide was evident in the cytoplasm or on the cell surface of the mutant strain . The Tn916 delta E insertion site in COH1-13 was mapped to the same chromosomal location as the Tn916 insertion site in the unencapsulated type III mutant COH31-15 reported previously . Nucleotide sequencing of DNA flanking the insertion site in COH1-13 revealed an open reading frame, designated cpsD, with significant homology to the rfbP gene of Salmonella typhimurium . RfbP encodes a galactosyl transferase enzyme that catalyses the transfer of galactose to undecaprenol phosphate, the initial step in O-polysaccharide synthesis . A particulate fraction of a lysate of wild-type strain GBS COH1 mediated the transfer of galactose from UDP-galactose to an endogenous acceptor . The galactose-acceptor complex partitioned into organic solvents, suggesting it is lipid in nature or membrane-associated . Galactosyl transferase activity was significantly reduced in the unencapsulated mutant strain COH1-13 . These results, together with the similarity in deduced amino acid sequence between cpsD and rfbP suggest that cpsD encodes a galactosyl transferase essential for assembly of the GBS type III capsular polysaccharide.

Photochem Photobiol, 1993 May, 57(5), 814 - 8
UV-A-mediated activity of p-methoxymethylcinnamate; Ashwood-Smith M et al.; Methyl esters of hydroxycinnamic acids are photobiologically active . Cis(Z) and trans(E) p-methoxymethyl-cinnamate photosensitize Escherichia coli and Chinese hamster ovary cells . They also produce sister chromatid exchanges . Photosensitization is oxygen independent, and the cinnamates are not genetically active in the absence of light in the Ames Salmonella typhimurium test.

J Gen Microbiol, 1993 May, 139 ( Pt 5), 1093 - 103
Cloning and analysis of the leuB gene of Leptospira interrogans serovar pomona; Ding M et al.; The leuB gene of Leptospira interrogans serovar pomona strain kenniwicki has been cloned on a 9.5 kb plasmid, pWVL1, by complementation of Escherichia coli leuB mutants . Subcloning and Tn5 mutagenesis showed that the region required for complementation was approximately 1.2 kb in length . Enzyme assays showed that the product of the cloned gene was a beta-isopropylmalate dehydrogenase . Defects in the leuA, leuC and leuD genes of E . coli were not complemented by pWVL1 . The nucleotide sequence of the leuB-complementing region and surrounding DNA has been determined . Three open reading frames were found which encode proteins of 40.9, 38.8 and 15 kDa . Analysis of subclones containing nucleotide deletions of varying sizes showed that only the 38.8 kDa protein was necessary to obtain complementation of E . coli leuB mutations . The PIR data base was searched and the enzyme 3-isopropylmalate dehydrogenase from six different micro-organisms was found to share significant amino acid sequence similarity (43-57%) with the 38.8 kDa L . interrogans leuB gene product . The organization of the leucine biosynthetic genes in L . interrogans differs from that found in E . coli, Salmonella typhimurium and Bacillus subtilis.

Br Vet J, 1993 May-Jun, 149(3), 225 - 34
The humoral and cell mediated immune response of young chicks to Salmonella typhimurium and S . Kedougou; Brito JR et al.; Day old chicks were inoculated with either Salmonella typhimurium or S . Kedougou as representative examples of an invasive and a non-invasive strain respectively . The titres of IgA, IgG and IgM antibodies were determined, using an enzyme-linked immunosorbent assay (ELISA) in the bile, serum and homogenates of intestinal mucosa each week up to 5 weeks of age . Statistically significant increases in antibodies were detected in the bile (IgA and IgG) and the intestinal mucosa and serum (IgG and IgM) 1 week after the birds were inoculated with S . typhimurium . In contrast, only a limited response was recorded for S . kedougou 4 weeks after challenge commenced . The birds appeared capable of eliminating systemic infection with S . typhimurium as they grew older although a cell mediated immune response was demonstrated in less than half the birds aged 4 or 5 weeks . The demonstration of only a limited serological response to S . kedougou indicates that serological testing may have a limited role in monitoring flocks for infection with non-invasive serotypes.

Mutagenesis, 1993 May, 8(3), 221 - 9
Induction of micronucleated erythrocytes by recombinant human erythropoietin; Yajima N et al.; Induction of micronucleated erythrocytes by a recombinant human erythropoietin (rhEPO) was examined using both in vitro and in vivo test systems . A small, significant and dose-related increase in the frequency of micronucleated polychromatic erythrocytes (MNPCE) in bone marrow of mice administered i.p . with 12,500-50,000 IU/kg rhEPO was induced at 48 h sampling time . A clear positive dose--response relationship and significant increase in the frequency of micronucleated reticulocytes (MNRET) in peripheral blood of mice administered i.p . with 400-50,000 IU/kg rhEPO was noted at 48, 72 and 96 h sampling times . Conversely, in bacterial reverse mutation tests, no noticeable increase of auxotrophic revertants was observed in Salmonella typhimurium, TA100, TA98, TA1535, TA1537, or Escherichia coli, WP2 uvrA-, by treatment with 188-6000 IU/plate of rhEPO, with or without S9 mix . Furthermore, rhEPO at 750-6000 IU/ml did not induce chromosomal aberrations in vitro in CHL cells or human peripheral blood lymphocytes in a direct method nor in a metabolic activation method . Moreover, chromosomal aberrations were not detected in bone marrow cells of CD-1 male mice, even at high rhEPO concentrations (100,000 IU/kg) in vivo . Consequently, it was concluded that errors in the process of enucleation or differentiation of the erythrocytes should be equally considered as possible mechanisms for the increased frequencies of MNPCE and MNRET alongside induction of DNA damage or errors in the process of DNA repair.

Mutagenesis, 1993 May, 8(3), 183 - 8
Synthesis of a series of 5-nitro-(benzimidazoles and indoles) as novel antimycotics and evaluation as genotoxins in the Ames test; Hrelia P et al.; Nitrobenzimidazole and nitroindole derivatives, related to oxiconazole and characterized by an oxyiminic function, have been synthesized as novel antimycotics and their mutagenic activity tested in Salmonella typhimurium strains TA100 and TA98 with and without an exogenous metabolizing system . TA98NR and TA98/1,8-DNP6 strains were employed to identify a specific metabolic reaction which governs the mutagenic potency . Active compounds are weak direct-acting mutagens . Only derivatives bearing a nitro group on the phenyl ring linked to the oxyiminic function and lacking halogenated substituents show mutagenic activity . Metabolism by bacterial enzyme systems is important to the expression of genotoxicity . The reductive activation of nitrobenzimidazoles and nitroindoles carried out by the 'classical' nitroreductase of Salmonella, which is defective in TA98NR, is required of mutagenicity . Similarly, the O-acetyltransferase defective in TA98/1,8-DNP6 is required for the efficient production of the ultimate electrophilic nitrogen species, which react with DNA . The role of bacterial metabolism in mutation induction needs careful consideration to assess the potential risk to humans from nitrobenzimidazole and nitroindole antimycotics.

Chem Res Toxicol, 1993 May-Jun, 6(3), 317 - 27
Construction of a vector for site-specific frameshift mutagenesis containing the mutable hotspot of Salmonella typhimurium TA98 on an M13 bacteriophage; Benamira M et al.; Frameshift mutations demonstrate a high degree of sequence specificity . In order to provide a vector for site-specific frameshift mutagenesis experiments, a recombinant M13 phage (M13MB102) was constructed by substitution of 27 base pairs of the Salmonella typhimurium hisD3052 sequence for 27 base pairs of the polylinker region of M13mp19 . The inserted sequence contains most of the hotspot for frameshift mutations in hisD3052 and its derivative strain TA98 . Structural elements of the insert include reiterated bases, direct repeats, and palindromes, and four unique restriction endonuclease cleavage sites . The recombinant phage produced blue plaques when grown in Escherichia coli strain JM105 on X-Gal indicator plates and exhibited a spontaneous mutation frequency similar to that of M13mp19 . Methodology is described for preparation, isolation, and purification of closed circular duplex M13MB102 genomes containing an adduct between the SphI and BssHII cleavage sites in the (-)-strand and uracil residues in the (+)-strand . The latter modification decreases replication of the (+)-strand by 4 orders of magnitude and maximizes use of the adducted (-)-strand for in vivo replication . The structure of M13MB102 and the procedures described for introducing adducts at defined positions in its hisD3052 insert provide a convenient approach for evaluating the potential of individual carcinogen-DNA adducts to induce frameshift mutations.

Chem Res Toxicol, 1993 May-Jun, 6(3), 294 - 301
Genotoxicity of neurotoxic triaryl phosphates: identification of DNA adducts of the ultimate metabolites, saligenin phosphates; Mentzschel A et al.; 2-Phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide is an electrophilic and neurotoxic metabolite of o-tolyl phosphates . We have investigated the genotoxicity of this saligenin phosphate and the structure of adducts formed by incubation of 2-phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide with nucleosides and DNA . o-Tolyl phosphate was mutagenic in the Ames test (695 revertants/mumol, Salmonella typhimurium TA 100) only with metabolic activation . 2-Phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide, which is a cyclization product similar to those expected from o-tolyl phosphate, was a potent mutagen in bacteria (1452 revertants/mumol, S . typhimurium TA 100) which did not require metabolic activation . Incubation of 2-phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide with guanosine, deoxycytidine, and deoxyadenosine resulted in formation of guanosine, deoxyuridine, and adenine adducts . These were identified as N-2-(o-hydroxybenzyl)guanosine, N-3-(o-hydroxybenzyl)deoxyuridine, N-1-(o-hydroxybenzyl)adenine, and N-3-(o-hydroxybenzyl)adenine by 1H-NMR spectroscopy, thermospray mass spectrometry, and pH-dependent electronic spectrometry . The deoxyuridine adduct is formed by an alkylation at N-3 of deoxycytidine followed by conversion of the adjacent exocyclic imino group to carbonyl (hydrolytic deamination) . The formation of N-2-(o-hydroxybenzyl)-deoxyguanosine, N-3-(o-hydroxybenzyl)deoxyuridine, and N-1-(o-hydroxybenzyl)deoxyadenosine was also demonstrated when 2-phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide was incubated with calf thymus DNA . Adducts formed with nucleosides in calf thymus DNA reacted with 2-phenoxy-4H-1,3,2-benzodioxaphosphorin 2-oxide in vitro were detected by the 32P-postlabeling technique and identified by comparison with synthetic references . DNA adducts are formed by an o-hydroxybenzylation from cyclic phosphoranes derived from o-alkyl-substituted triaryl phosphates.(ABSTRACT TRUNCATED AT 250 WORDS)

East Afr Med J, 1993 May, 70(5), 255 - 8
Acquired tetracycline resistance genes in nosocomial Salmonella typhimurium infection in a Kenyan hospital; Kariuki S et al.; Tetracyclines have been among the most widely used antibiotics worldwide . Plasmid-mediated tetracycline resistance among hospital strains of bacteria has continued to rise and of major concern has been the transfer of resistance to pathogenic organisms . Bacteraemia due to hospital acquired S . typhimurium has been a major cause of morbidity at Kenyatta National Hospital (KNH), hence the need to study drug susceptibility pattern of this organism . This study also characterized the tetracycline resistance genes using oligonucleotide probes . Ninety seven S . typhimurium strains isolated from patients at KNH were used . Agar dilution method was used to determine minimum inhibitory concentration (MIC) . Plasmids were isolated from each strain and the different plasmid profiles were grouped by their molecular weights into 6 patterns . Out of 97, 87 (88%) strains were resistant . MIC ranged from 1 microgram/ml to 128 micrograms/ml . Genes encoding for tetracycline resistance were located on plasmids of molecular weights 65 MDa, 5.2 or both . Plasmid-encoded antimicrobial resistance is likely to spread to other pathogenic organisms, reduce our ability to treat the infection and increase the cost and duration of treatment.

Mol Microbiol, 1993 May, 8(3), 543 - 58
Nucleotide sequence of a 13.9 kb segment of the 90 kb virulence plasmid of Salmonella typhimurium: the presence of fimbrial biosynthetic genes; Friedrich MJ et al.; The 90kb plasmid resident in Salmonella typhimurium confers increased virulence in mice by promoting the spread of infection after invasion of the intestinal epithelium . The nucleotide sequence of a 13.9kb segment of this plasmid known to encode an outer membrane protein related in sequence to components of fimbrial biosynthesis in enteric bacteria was determined . This cloned segment between the repB and repC replicon regions programmed expression of abundant surface fimbriae in Escherichia coli and S . typhimurium cells . A 7kb region contained seven open reading frames, the protein products of five of which were related in sequence to regulatory, structural, and assembly proteins of adherence fimbriae/pili, such as the P and K88 pili . These five genes and two adjacent ones which were not markedly related to proteins in the data bases comprise the pef (plasmid-encoded fimbriae) locus . Transposon TnphoA insertions in four genes in the pef locus (pefA, pefC, orf5 and orf6) resulted in active PhoA fusions and blocked or reduced the surface presentation of fimbriae, indicating that the proteins encoded by these four genes are translocated at least across the cytoplasmic membrane and contribute to formation of the fimbrial structure . The differences in genetic organization and protein sequence relatedness from other fimbrial gene clusters suggest that the pef locus might encode a novel type of fimbria . Between the pef and the repB loci, there were five open reading frames, one of which (orf8) gave rise to active PhoA fusions but was not necessary for fimbrial expression . Two of the other proteins were homologous to transcription regulatory proteins and a third was the rck gene, which encodes an outer membrane protein that confers complement resistance to serum-sensitive hosts.

Mutat Res, 1993 May, 302(1), 45 - 52
Prostaglandin H synthase-dependent formation of the direct-acting mutagen 2-nitro-3-methylimidazo{4,5-f}quinoline (nitro-IQ) from IQ; Morrison LD et al.; The mutagenic effects of 2-amino-3-methylimidazo{4,5-f}quinoline (IQ) following activation by ram seminal vesicle microsomes (RSVM, a source of prostaglandin H synthase, PHS) were studied in Salmonella typhimurium tester strains possessing elevated levels of acetyl-CoA: arylamine N-acetyltransferase (NAT) . The metabolites formed by RSVM were extracted and fractionated by high pressure liquid chromatography (HPLC) . One isolable product accounted for most of the direct-acting mutagenicity observed in the extracts . The metabolite was identified as 2-nitro-3-methylimidazo{4,5-f}quinoline (nitro-IQ) . Since nitro-IQ is a potent direct-acting mutagen, its role in IQ genotoxicity warrants further study.

Mutat Res, 1993 May, 299(3-4), 135 - 45
DNA damage induced by photosensitizers in cellular and cell-free systems; Epe B et al.; The specific recognition of DNA modifications by repair endonucleases was used to characterize the DNA damage induced by photosensitizers in the presence of visible light . Under cell-free conditions, chemically unrelated photosensitizers (methylene blue, acridine orange, proflavin, riboflavin, hematoporphyrin) induce the same type of DNA damage . It is characterized by a high number of base modifications sensitive to the repair endonuclease FPG protein (formamidopyrimidine-DNA glycosylase), while both the number of DNA strand breaks and the number of sites of base loss (sensitive to exonuclease III or endonuclease IV) is low . Therefore the damage is markedly different from that induced by hydroxyl radicals . Mechanistically, the generation of the base modifications sensitive to FPG protein involves singlet oxygen in some, but possibly not all cases, as substituting D2O for H2O increases the reaction yield six-fold in the case of methylene blue, but only 1.4-fold in the case of acridine orange . In plasmids from Salmonella typhimurium strains treated with methylene blue or acridine orange plus light and from Escherichia coli strains treated with acridine orange or proflavin plus light, the same type of damage was observed as under cell-free conditions . In L1210 mouse leukemia cells exposed to acridine orange plus light, the numbers of modifications sensitive to FPG protein and exonuclease III were quantified, in addition to strand breaks, by a modified alkaline elution assay . Again, the number of base modifications sensitive to FPG protein was found to be several-fold higher than the number of strand breaks and sites of base loss . It has to be concluded that the DNA damage in the intact cells is not mediated by hydroxyl radicals or cellular nucleases, but by the same mechanism as operates under cell-free conditions with these agents.

J Biol Chem, 1993 Apr 25, 268(12), 8727 - 34
Lysine 87 in the beta subunit of tryptophan synthase that forms an internal aldimine with pyridoxal phosphate serves critical roles in transimination, catalysis, and product release; Lu Z et al.; This study provides valuable insights into the functions of the lysine residue that forms an internal aldimine with pyridoxal phosphate in the beta subunit of tryptophan synthase from Salmonella typhimurium . Our spectroscopic and kinetic studies demonstrate that a mutant alpha 2 beta 2 complex having beta subunit lysine 87 replaced by threonine forms external aldimines with several amino acids including L-serine, beta-chloro-1-alanine, L-tryptophan, and D-tryptophan . Because the rates of aldimine formation are very slow, we conclude that one role of lysine 87 in the wild type enzyme is to facilitate formation of external aldimines by transimination . Lysine 87 is an essential catalytic residue because the mutant alpha 2 beta 2 complex has no measurable activity in reactions catalyzed by the beta subunit and does not convert external aldimines to products . The mutant enzyme carries out two slow partial beta-elimination reactions: the conversion of beta-chloro-L-alanine and L-serine to enzyme-bound aminoacrylate . The reaction with L-serine is catalyzed by ammonia, which partially replaces the deleted epsilon-amino group . Lysine 87 is important for substrate and product release because L-serine, L-tryptophan, and aminoacrylate dissociate very slowly from the mutant alpha 2 beta 2 complex . Our ability to prepare very stable derivatives of the mutant alpha 2 beta 2 complex containing tightly bound aldimines with a substrate, a product, or a reaction intermediate provides valuable materials for ongoing x-ray crystallographic investigations and future kinetic analyses of the allosteric activation of the alpha subunit by beta subunit ligands.

Nucleic Acids Res, 1993 Apr 25, 21(8), 1805 - 9
Molecular characterization of the Salmonella typhimurium parE gene; Springer AL et al.; The DNA sequence of the wild type S . typhimurium parE gene was determined . The predicted protein has 96.7% amino acid identity with the ParE protein of E.coli, but is 29 amino acids longer, due to an additional basepair in the 3' end of the S . typhimurium gene . Subclones of the S . typhimurium parE gene localized the sites of four heat sensitive mutations within parE . The parE206 and parE374 mutations are identical (Val67-Met) and lie in a highly conserved region corresponding to the ATP binding pocket of GyrB . Two additional heat sensitive mutations were sequenced and predict the following amino acid substitutions: parE377 (Gly399-Ser) and parE493 (Thr583-Pro) . All of the heat sensitive mutations lie in regions with strong amino acid homology to GyrB.

J Mol Biol, 1993 Apr 20, 230(4), 1304 - 8
Primary structure and crystallization of orotate phosphoribosyltransferase from Salmonella typhimurium; Scapin G et al.; Orotate phosphoribosyltransferase (OPRTase; EC 2.4.2.10) catalyzes phosphoribosyl group transfer between alpha-D-5-phosphoribosyl-1-pyrophosphate and orotate to form orotidine-5'-monophosphate and pyrophosphate, the nucleotide-forming step in pyrimidine biosynthesis . It is one of ten PRTases that perform vital roles in de novo and salvage pathways for purine, pyrimidine and pyridine nucleotides . Although the PRTases are important drug targets, they are poorly understood mechanistically, and no three-dimensional structures exist . Here, we report the complete sequence of the Salmonella typhimurium pyrE gene and the deduced sequence of the OPRTase gene product . OPRTase forms tetragonal crystals from polyethylene glycol solutions; these crystals diffract to better than 2 A resolution, and are stable to radiation damage . The space group is P4(1)2(1)2 (or P4(3)2(1)2) with unit cell dimensions of a = b = 48.5 A, c = 210.5 A, and alpha = beta = gamma = 90 degrees . A crystalline form of the selenomethionine derivative of the protein is also reported.

J Mol Biol, 1993 Apr 5, 230(3), 800 - 11
Structure of the maltodextrin-uptake locus of Streptococcus pneumoniae . Correlation to the Escherichia coli maltose regulon; Puyet A et al.; The mechanism of induction of the maltose/maltodextrin regulon of the Gram-positive bacterium Streptococcus pneumoniae seems to be different to the positively controlled maltose regulons of the enteric bacteria Escherichia coli, Klebsiella pneumoniae and Salmonella typhimurium . In this work, we report on the structure of the S . pneumoniae genes involved in maltodextrin uptake malX, malC and malD . Comparisons of the amino acid sequences encoded by these genes indicate that they are homologous to the E . coli MalE periplasmic maltose binding protein and the two maltose permeases MalG and MalF . The analysis of transcription start points indicates that malXCD could be transcribed from a single consensus promoter sequence . Northern analysis of the mRNA molecules pertaining to this region reveals that the transcript encompassing all these three genes is apparently cleaved at a large putative mRNA secondary structure, yielding two mRNA molecules . The smaller of these molecules would include only the malX gene while a larger fragment spans through malC and malD . The processing of mRNA has not been reported in the Gram-negative maltose regulons, and may suggest either a less evolved or a divergent system for the control of gene expression of this regulon in S . pneumoniae.

J Biol Chem, 1993 Apr 5, 268(10), 7298 - 314
Toxicity of Bordetella avium beta-cystathionase toward MC3T3-E1 osteogenic cells; Gentry-Weeks CR et al.; Bordetella avium is the etiological agent of an upper respiratory disease in birds which, symptomatically and pathologically, resembles bordetellosis in humans . Studies of the virulence of this organism revealed a novel cytotoxic protein, designated osteotoxin, that was lethal for MC3T3-E1 osteogenic cells, fetal bovine trabecular cells, UMR106-01(BSP) rat osteosarcoma cells, and embryonic bovine tracheal cells . The osteotoxin lacked dermonecrotic toxin activity, exhibited no cross-reactivity with antibody against B . avium dermonecrotic toxin, and was non-proteolytic . Osteotoxin (M(r) approximately 80,000 by gel filtration, pI 5.4) was purified to electrophoretic homogeneity from B . avium 197 . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and spectrophotometric analyses showed that the native protein was a homodimer and that each of the non-covalently linked subunits (M(r) approximately 41,000) contained one molecule of pyridoxal 5'-phosphate . Microsequencing of the first 32 amino acids from the NH2 terminus allowed the synthesis of two oligonucleotide probes, which, together with polyclonal antibody to the purified protein, facilitated cloning, sequencing, and expression of the osteotoxin gene product in Escherichia coli . The open reading frame encodes a polypeptide of 396 amino acid residues (M(r) = 42,606, calculated pI 5.9), whose sequence exhibits approximately 38% identity (approximately 60% similarity) to pyridoxal 5'-phosphate-dependent beta-cystathionase(s) from E . coli, Salmonella typhimurium, and rat liver . The characteristic motif, TKYXXGHSD, associated with binding the cofactor in these enzymes is also present in osteotoxin . Physicochemical and enzymatic analyses established the coidentity of osteotoxin with beta-cystathionase . The region upstream of the beta-cystathionase (metC) gene in B . avium 197 lacked regulatory sequences ("Met boxes") described for metC in enteric species, and enzyme production was not repressed by methionine . Incubation of MC3T3-E1 osteogenic cells in medium containing L-{35S}cystine and purified beta-cystathionase resulted in 35S-labeling of the enzyme and at least one major MC3T3-E1 cell protein (M(r) approximately 50,000) . cytotoxicity can be attributed to: 1) beta-cystathionase-catalyzed cleavage of L-cystine in the medium and formation of reactive sulfane-containing derivative(s), and 2) transfer of sulfane sulfur to metabolically sensitive or structurally important proteins in the osteogenic cells.

Arch Oral Biol, 1993 Apr, 38(4), 309 - 17
Longitudinal evaluation of peripheral blood monocyte secretory function in periodontitis-resistant and periodontitis-susceptible patients; Payne JB et al.; The purpose of this investigation was to evaluate lipopolysaccharide (LPS)-stimulated monocyte secretory responses longitudinally in patients with generalized severe chronic adult periodontitis (periodontitis-susceptible) and controls with gingivitis (periodontitis-resistant) . In addition, the expression of constitutive (Leu-M3) and LPS-inducible (Mo3e) antigens on monocytes isolated from these two groups was examined . Monocyte secretory function was assessed longitudinally; the effect of periodontal therapy in the susceptible patients was examined by comparing monocyte function before and after their treatment . Peripheral blood monocytes were isolated by counterflow centrifugal elutriation and treated with control medium or media containing 1 microgram/ml of Salmonella typhimurium LPS or Prevotella intermedia LPS with or without human recombinant interferon (IFN)-gamma pretreatment . Prostaglandin E2, F2 alpha and thromboxane B2 were quantified in culture samples by gas chromatography-mass spectrometry (GC-MS) and interleukin-1 beta was quantified by enzyme-linked immunosorbent assay . Leu-M3 and Mo3e antigen expression was assessed by FACScan . Three major findings were made . First, LPS-stimulated IL-1 beta release by monocytes from susceptible patients was depressed relative to that in resistant patients at the initial donation . After periodontal therapy, there was virtually identical IL-1 beta release in LPS-stimulated cultures from both groups . However, in susceptible patients IL-beta release was diminished after periodontal therapy in cultures pretreated with IFN-gamma . Second, there was a significant drift in monocyte secretion of prostaglandin E2 in samples from the resistant patients between the first two donations and the third donation . PGE2 release did not differ between groups at the initial donation, although there was a depression in PGE2 release in the susceptible group at the final donation when IFN-gamma was followed by S . typhimurium LPS.(ABSTRACT TRUNCATED AT 250 WORDS)

Ethiop Med J, 1993 Apr, 31(2), 91 - 8
Fate of Salmonella enteritidis and Salmonella typhimurium during the fermentation of ergo, a traditional Ethiopian sour milk; Ashenafi M; The growth potential of Salmonella enteritidis and Salmonella typhimurium in milk in smoked and non-smoked containers and their inhibition by lactic acid bacteria was determined . In the absence of lactic acid bacteria, both Salmonella strains could grow to the level of 10(8) cfu/ml within 12 h . Smoking of containers significantly retarded Salmonella growth only until 12 h . Growth of lactic acid bacteria in souring milk retarded growth of Salmonella strains and complete inhibition was observed between 48 and 60 h at pH and titratable acidity values of 3.7 and 0.70%, respectively . The synergistic effect of lower pH, acids and smoking was important in the complete inhibition of the test organisms . Although ergo was preferably consumed after 24 h of fermentation, the Salmonella strains were not completely inhibited at this time . Thus, traditional making of ergo by the natural fermentation of raw milk could be hazardous to health . The use of a three-day old ergo as a starter for boiled milk is recommended to ensure the wholesomeness of ergo.

Chem Pharm Bull (Tokyo), 1993 Apr, 41(4), 649 - 53
Modification of the amino group of guanosine by methylglyoxal and other alpha-ketoaldehydes in the presence of hydrogen peroxide; Nukaya H et al.; Methylglyoxal is directly mutagenic to Salmonella typhimurium TA100 and its mutagenicity is markedly enhanced in the presence of hydrogen peroxide . We found that methylglyoxal in phosphate buffer was decomposed easily by hydrogen peroxide at room temperature to yield acetic acid and formic acid as major products and diacetyl as a minor product; acetyl radical was detected in the solution by ESR spectroscopy by the use of a spin-trapping reagent, 5,5-dimethyl-1-pyrroline N-oxide . Furthermore, guanosine was converted into N2-acetylguanosine by a combination of methylglyoxal and hydrogen peroxide in 0.1 M phosphate buffers (pH 6.1 to 7.4) . This acetylation may be related to the enhancement of methylglyoxal mutagenicity by hydrogen peroxide . Other alpha-ketoaldehydes such as glyoxal and phenylglyoxal also yielded the corresponding acids and alpha-dicarbonyls upon reaction with hydrogen peroxide under the same conditions as above . These acids would have been produced through Baeyer-Villiger reaction or coupling of acyl radical with hydroxy radical, and dicarbonyls by dimerization of acyl radicals . In addition, when phenylglyoxal was used, the generation of benzoyl radical and the conversion of guanosine to N2-benzoylguanosine were observed . However, it remains to be established whether the generation of acyl radicals is directly involved in the N-2 acylation of guanosine.

Antimicrob Agents Chemother, 1993 Apr, 37(4), 662 - 6
Ciprofloxacin resistance in clinical isolates of Salmonella typhimurium obtained from two patients; Piddock LJ et al.; Two patients (patients A and B) infected with Salmonella typhimurium failed ciprofloxacin therapy, and the posttherapy isolates had reduced susceptibilities to quinolones; 6 of 11 isolates from patient B were also cross-resistant to chemically unrelated agents . No transferable resistance, chloramphenicol-acetylating enzymes, or beta-lactamases were detected . For 13 of 14 isolates, the concentrations of ciprofloxacin that inhibited DNA synthesis by 50% were similar to the MICs, suggesting a mutation in gyrA . Insertion of pNJR3-2 (gyrA) in the posttherapy isolate from patient A and 5 of 11 of the posttherapy isolates from patient B resulted in lower quinolone MICs, also suggesting that resistance was due to a mutation in gyrA . Three of the five isolates also had reduced levels of accumulation of quinolones . All six cross-resistant isolates from patient B had reduced levels of accumulation of quinolones, but only one isolate had increased susceptibility when pNJR3-2 was inserted . Despite the lack of OmpF seen in five isolates from patient B, there was no correlation with decreased levels of quinolone accumulation . All isolates had identical smooth lipopolysaccharide profiles . The mechanism of apparently reduced accumulation has yet to be determined.

FEMS Microbiol Lett, 1993 Apr 1, 108(2), 145 - 50
MetJ-mediated regulation of the Salmonella typhimurium metE and metR genes occurs through a common operator region; Wu WF et al.; In Salmonella typhimurium the metE and metR promoters overlap and are divergently transcribed . Three tandem repeats of an 8 bp sequence defined previously as the metE operator site for MetJ-mediated repression also overlap the -35 region of the metR promoter . Starting with a metE-lacZ.metR-galK double gene fusion, site-directed mutagenesis was used to change nucleotides in each of the repeat units from the consensus sequence . Each mutation, along with the wild-type metE-lacZ.metR-galK gene fusion, was cloned into phage lambda gt2 . Regulation of the metE and metR genes was examined by measuring beta-galactosidase and galactokinase levels in Escherichia coli strains lysogenized with phage carrying the wild-type and mutant fusions . Mutations in each of the 8 bp repeat units disrupt MetJ-mediated repression for both the metE-lacZ and metR-galK gene fusions, suggesting that the metE and metR genes share a common operator site for the MetJ repressor.

Poult Sci, 1993 Apr, 72(4), 752 - 4
Research note: differential chemotaxis of Physarum polycephalum to Salmonella gallinarum and Salmonella pullorum; Moore RW et al.; The chemotactic and chemotropic responses of the plasmodial stage of the slime mold Physarum polycephalum were used to distinguish Salmonella gallinarum and Salmonella pullorum from 10 Salmonella serovars that are commonly isolated from domestic poultry . Utilizing an in vitro plasmodium agar plate assay method, P . polycephalum was attracted to S . gallinarum and S . pullorum, but the organism was repelled by Salmonella derby, Salmonella dublin, Salmonella enteritidis, Salmonella heidelberg, Salmonella minnesota, Salmonella montevideo, Salmonella newington, Salmonella newport, Salmonella reading, and Salmonella typhimurium.

Poult Sci, 1993 Apr, 72(4), 628 - 35
Comparison of probiotics maintained by in vivo passage through laying hens and broilers; Ziprin RL et al.; Cecal colonization by salmonellae may be greatly reduced by inoculating chickens with normal cecal microflora, a phenomenon known as competitive exclusion . Unfortunately, it has not been possible to reliably store active cecal microflora over long time periods, and it is difficult to obtain consistent experimental results with different batches of microflora . In order to overcome these problems, the present authors have maintained active cecal flora through a 2-yr period by in vivo passage through both broiler chicks and layers that were fed a diet containing 5% lactose . Competitive exclusion cultures maintained in laying hens worked as well as cultures from broilers . Cecal microflora from either source excluded both nonlactose-fermenting and lactose-fermenting Salmonella strains . Colonization by both types of Salmonella was reduced even when the competitive exclusion organisms were given as late as 3 days after oral challenge inoculation with Salmonella typhimurium.

Epidemiol Infect, 1993 Apr, 110(2), 239 - 46
A large outbreak of human salmonellosis traced to a local pig farm; Maguire HC et al.; An outbreak of Salmonella typhimurium definitive type (DT) 193 affecting 206 persons occurred in July and August 1989 in a small town in northern England . A descriptive study suggested that cold meats including pork from a butcher's shop in the town were vehicles of infection . An analytical study of a cohort attending a function in the town showed a significant association between illness and consumption of cold roast pork supplied by the butcher's shop (P = 0.00000004) . S . typhimurium DT 193 with the same antibiotic resistance pattern (to ampicillin, streptomycin, sulphonamides and tetracyclines) as the outbreak strain, and possessing a single plasmid of 80 MDa was isolated from samples of meat bought from the shop and implicated in illness, and from samples of pig faeces taken from the farm supplying the shop . It was concluded that inadequate processing of infected pork meat at the shop may have contributed to this outbreak but that cross contamination also played an important part in transmission . Control measures included a temporary closure of the shop and subsequent implementation of a detailed protocol for meat processing and monitoring of all procedures at the shop.

Carcinogenesis, 1993 Apr, 14(4), 599 - 602
Sulfotransferase-mediated mutagenicity of 1-hydroxymethylpyrene and 4H-cyclopenta{def}chrysen-4-ol and its enhancement by chloride anions; Glatt H et al.; 1-Hydroxymethylpyrene (HMP), a primary benzylic alcohol, and 4H-cyclopenta{def}chrysen-4-ol (OH-CPC), a secondary benzylic alcohol, were investigated for mutagenicity in Salmonella typhimurium (reversion of the his- strain TA98) in the presence of various xenobiotic-metabolizing systems . In the direct test, HMP was inactive and OH-CPC was very weakly active . In the presence of NADPH-fortified postmitochondrial fraction from rat liver (S9/NADPH), no activation of OH-CPC was observed, whereas strong mutagenic effects were elicited by HMP . In the presence of cytosol and 3'-phosphoadenosine-5'-phosphosulfate (PAPS), both alcohols were activated to potent mutagens . For equal mutagenic effects, approximately 650-fold lower concentrations of HMP were required in the cytosol/PAPS-mediated assay than in the S9/NADPH-mediated assay . The cytosol/PAPS-mediated mutagenicity of both alcohols was 3- to 4-fold enhanced, when KCl (125 mM) was present during the exposure . The authentic chloromethylarenes, 1-chloromethylpyrene and 4-chloro-4H-cyclopenta{def}chrysene, showed very strong direct mutagenicity . These results, taken together with previous findings, indicate that both primary and secondary benzylic alcohols derived from polycyclic aromatic hydrocarbons may be activated by sulfotransferases to electrophilic sulfuric acid esters, and by subsequent substitution reaction to further active species such as benzylic chlorides.

J Bacteriol, 1993 Apr, 175(7), 1981 - 7
The acid tolerance response of Salmonella typhimurium involves transient synthesis of key acid shock proteins; Foster JW; Although Salmonella typhimurium prefers neutral-pH environments, it can adapt to survive conditions of severe low-pH stress (pH 3.3) . The process, termed the acid tolerance response (ATR), includes two distinct stages . The first stage, called pre-acid shock, is induced at pH 5.8 and involves the production of an inducible pH homeostasis system functional at external pH values below 4.0 . The second stage occurs following an acid shock shift to pH 4.5 or below and is called the post-acid shock stage . During this stage of the ATR, 43 acid shock proteins (ASPs) are synthesized . The present data reveal that several ASPs important for pH 3.3 acid tolerance are only transiently produced . Their disappearance after 30 to 40 min of pH 4.4 acid shock coincides with an inability to survive subsequent pH 3.3 acid challenge . Clearly, an essential feature of inducible acid tolerance is an ability to synthesize these key ASPs . The pre-acid shock stage, with its inducible pH homeostasis system, offers the cell an enhanced ability to synthesize ASPs following rapid shifts to conditions below pH 4.0, an external pH that normally prevents ASP synthesis . The data also address possible signals for ASP synthesis . The inducing signal for 22 ASPs appears to be internal acidification, while external pH serves to induce 13 others . Of the 14 transient ASPs, 10 are induced in response to changes in internal pH . Mutations in the fur (ferric uptake regulator) locus that produce an Atr- acid-sensitive phenotype also eliminate induction of six transiently induced ASPs.

Infect Immun, 1993 Apr, 61(4), 1544 - 6
Prolonged inhibition of bacterial protein synthesis abolishes Salmonella invasion; MacBeth KJ et al.; We have found that prolonged inhibition of bacterial protein synthesis abolishes the ability of Salmonella typhimurium to enter HEp-2 cells . Our results suggest that an essential invasion factor has a functional half-life that is seen as a gradual loss of invasiveness in the absence of protein synthesis . Therefore, Salmonella invasiveness appears to be a transient phenotype that is lost unless protein synthesis is maintained . This finding may explain why salmonellae grown to stationary phase lose their ability to enter cultured cells . In addition, a short-lived capacity to enter cells may be important during infection so that bacterial invasiveness is limited to certain times and host sites during pathogenesis.

Toxicol Lett, 1993 Apr, 67(1-3), 41 - 55
Genotoxicity studies of benzofuran dioxetanes and epoxides with isolated DNA, bacteria and mammalian cells; Adam W et al.; 1,2-Dioxetanes, very reactive and high energy molecules, are involved as labile intermediates in dioxygenase-activated aerobic metabolism and in physiological processes . Various toxicological tests reveal that dioxetanes are indeed genotoxic . In supercoiled DNA of bacteriophage PM2 they induce endonuclease-sensitive sites, most of them are FPG protein-sensitive base modifications (8-hydroxyguanine, formamidopyrimidines) . Pyrimidine dimers and sites of base loss (AP sites) which were probed by UV endonuclease and exonuclease III are minor lesions in this system . While the alkyl-substituted dioxetanes do not show any significant mutagenic activity in different Salmonella typhimurium strains, heteroarene dioxetranes such any significant mutagenic activity in different Salmonella typhimurium strains, heteroarene dioxetanes such as benzofuran and furocoumarin dioxetanes are strongly mutagenic in S . typhimurium strain TA100 . DNA adducts formed with an intermediary alkylating agent appear to be responsible for the mutagenic activity of benzofuran dioxetane . We assume that the benzofuran epoxides, generated in situ from benzofuran dioxetanes by deoxygenation are the ultimate mutagens of the latter, since benzofuran epoxides are highly mutagenic in the S . typhimurium strain TA100 and they form DNA adducts, as detected by the 32P-postlabelling technique . Our results imply that the type of DNA damage promoted by dioxetanes is dependent on the structural feature of dioxetanes . Furthermore, the direct photochemical DNA damage by energy transfer, i.e., pyrimidine dimers, plays a minor role in the genotoxicity of dioxetanes . Instead, photooxidation dominates in isolated DNA, while radical damage and alkylation prevail in the cellular system.

Protein Sci, 1993 Apr, 2(4), 559 - 66
Three-dimensional structural model of the serine receptor ligand-binding domain; Jeffery CJ et al.; Computer-based homology modeling techniques were used to construct a three-dimensional model of the Escherichia coli serine receptor ligand-binding domain based on the crystal structure of the Salmonella typhimurium aspartate receptor and the sequence homology between the two receptors . Residues that have been found in mutagenesis studies to be necessary for serine binding are located in a proposed serine-binding site . Several other mutations that affect swimming behavior require relatively small shifts in alpha-carbon positions in the model to give a minimized structure, suggesting that small changes in receptor conformation can affect the signaling state of the receptor.

FEMS Microbiol Lett, 1993 Apr 1, 108(2), 183 - 7
Porins and lipopolysaccharide of Escherichia coli ATCC 25922 and isogenic rough mutants; Rivera M et al.; The lipopolysaccharide and porin profile of Escherichia coli ATCC 25922, a smooth strain commonly used in antibiotic susceptibility testing, and five isogenic rough mutants was examined . The lipopolysaccharide of the parent strain had the characteristic ladder pattern on polyacrylamide gels, while that of the mutants appeared similar to chemotypes Ra and Rc of Salmonella typhimurium with some changes in chemical composition . Of the porins, OmpC appeared markedly reduced in the parent strain while OmpF appeared markedly reduced in the mutants . In addition, a new outer-membrane protein of size intermediate to that of OmpC and OmpF was detected in all mutants . Neither parent nor mutants were susceptible to the LPS core-specific P1 phage or the porin-specific PA2 and K20 phages.

Avian Dis, 1993 Apr-Jun, 37(2), 528 - 35
Effect of mixed cecal microflora maintained in continuous culture and of dietary lactose on Salmonella typhimurium colonization in broiler chicks; Nisbet DJ et al.; Mixed cecal microflora obtained from a mature chicken were maintained in vitro in continuous-flow (CF) culture . The effect of the CF culture and dietary lactose on Salmonella typhimurium cecal colonization in broiler chicks was evaluated . When averaged across four replicates, chicks treated with the culture alone (1.75 log10 decrease) or with 5% dietary lactose alone (2.98 log10 decrease) were protected against S . typhimurium . Optimum protection against S . typhimurium was observed when birds were treated with the culture in combination with dietary lactose (4.27 log10 decrease) . Dietary lactose resulted in reduced cecal pH . A large increase in cecal propionic acid was observed in the birds given the CF culture . A significant correlation (P < 0.001) was observed between the cecal concentration of undissociated propionic acid and protection against S . typhimurium colonization (r = -0.78) . The results indicated that indigenous cecal flora that protect against Salmonella colonization can be maintained without loss of efficacy in CF culture.

Avian Dis, 1993 Apr-Jun, 37(2), 396 - 8
Effect of short-chain fatty acids on the growth of Salmonella typhimurium in an in vitro system; McHan F et al.; Previous studies have revealed a reduction of cecal Salmonella carriage from feeding either carbohydrate or short-chain fatty acids (SCFAs) . This in vitro study presents a profile of the relative SCFA content of the ceca when chicks are fed an unmedicated diet with 2.5% carbohydrate . Subsequent incorporation of these acids into culture medium was used to demonstrate their antagonistic activity toward in vitro growth of Salmonella typhimurium . Commonly found concentrations of SCFAs based upon the above findings reduced in vitro Salmonella growth by at least 50%, and 10 x concentrations inhibited growth more than 80% . An explanation of the mechanism(s) involved in growth reduction is offered.

Avian Dis, 1993 Apr-Jun, 37(2), 265 - 73
Virulence of Salmonella typhimurium mutants for White Leghorn chicks; Porter SB et al.; A strain of Salmonella typhimurium that is highly virulent for 1-day-old white leghorn chicks was genetically modified by deletion (delta) of the adenylate cyclase (cya) and cyclic AMP receptor protein (crp) genes or by removal (curing) of the 91-kilobase virulence plasmid . These mutants were then compared with the wild-type S . typhimurium strain for virulence in 1-day-old chicks and for their ability to colonize chicks of various ages . The plasmid-cured mutant showed a slight reduction in virulence, whereas the delta cya delta crp mutant was completely avirulent . The wild-type strain and both mutant strains were capable of colonizing various organs within the chicks . At all time points, the delta cya delta crp strain colonized chicks at lower levels than the wild-type strain . Titers of the plasmid-cured strain increased more slowly in visceral organs than did those of the wild type.

What Is Bioreactor?, What Is Functional Genomics?, What Is MIC?, What Is Rhizobia?, What Is Biofilm?, e, Microorganism, i, Bacteria, c, Bacteriology, e, Bacterium, r, Microbe, r, Antibiotics, c, Pseudomonas aeruginosa, e, Bacteria, c, Streptococcal, s, Bacteria, n, Fermentations, i, Enterobacters, r, Escherichia coli, i, Pseudomonas aeruginosa, n, Multidrug resistant, s, Staphylococcus, s, Escherichia coli, e, Salmonella typhimurium, o, Escherichia coli, i, Microorganisms, o, Saccharomyces yeast, e, Escherichia coli, a, Bacteriological, e, Antibiotics, e, S. cerevisiae, r, Eubacter

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005