Microbiology, 1998 Mar, 144 ( Pt 3), 655 - 64 Magnesium transport in Salmonella typhimurium: biphasic magnesium and time dependence of the transcription of the mgtA and mgtCB loci; Tao T et al.; Salmonella typhimurium has three distinct Mg2+ transport systems, the constitutive high-capacity CorA transporter and two P-type ATPases, MgtA and MgtB, whose transcription is repressed by normal concentrations of Mg2+ in the growth medium . The latter Mg(2+)-transporting ATPase is part of a two-gene operon, mgtCB, with mgtC encoding a 23 kDa protein of unknown function . Transcriptional regulation using fusions of the promoter regions of mgtA and mgtCB to luxAB showed a biphasic time and Mg2+ concentration dependence . Between 1 and 6 h after transfer to nitrogen minimal medium containing defined concentrations of Mg2+, transcription increased about 200-fold for mgtCB and up to 400-fold for mgtA, each with a half-maximal dependence on Mg2+ of 0.5 mM . Continued incubation revealed a second phase of increased transcription, up to 2000-fold for mgtCB and up to 10,000-fold for mgtA . This secondary increase occurred between 6 and 9 h after transfer to defined medium for mgtCB but between 12 and 24 h for mgtA and had a distinct half-maximal dependence for Mg2+ of 0.01 mM . A concomitant increase of at least 1000-fold in uptake of cation was seen between 8 and 24 h incubation with either system, showing that the transcriptional increase was followed by functional incorporation of large amounts of the newly synthesized transporter into the membrane . Regulation of transcription by Mg2+ was not dependent on a functional stationary-phase sigma factor encoded by rpoS, but it was dependent on the presence of a functional phoPQ two-component regulatory system . Whereas mgtCB was completely dependent on regulation via phoPQ, the secondary late Mg(2+)-dependent phase of mgtA transcription was still evident in strains carrying a mutation in either phoP or phoQ, albeit substantially diminished . Several divalent cations blocked the early phase of the increase in transcription elicited by the decrease in Mg2+ concentration, including cations that inhibit Mg2+ uptake (Co2+, Ni2+ and Mn2+) and those which do not (Ca2+ and Zn2+) . In contrast, the second later phase of the transcriptional increase was not well blocked by any cation except those which inhibit uptake . Overall, the data suggest that at least two distinct mechanisms for transcriptional regulation of the mgtA and mgtCB loci exist. J Biol Chem, 1998 Mar 6, 273(10), 5572 - 6 ApbA, the ketopantoate reductase enzyme of Salmonella typhimurium is required for the synthesis of thiamine via the alternative pyrimidine biosynthetic pathway; Frodyma ME et al.; The apbA gene of Salmonella typhimurium was shown to encode ketopantoic acid reductase . ApbA was purified from crude cell-free extracts to greater than 95% homogeneity after two chromatographic steps . N-terminal amino acid sequencing (first 15 amino acids) and Western blot analysis confirmed the isolated protein was ApbA . The functional protein was a monomer with a molecular mass of 31.1 kDa . Optimal reaction conditions for the reduction of ketopantoic acid were established at a pH of 6.25, and a temperature of 42 degreesC . The preferred electron source was NADPH, and the apparent Km constants of the enzyme for NADPH and ketopantoic acid were determined to be 0.776 +/- 0.09 mM and 0.742 +/- 0.01 mM, respectively . The homogeneous enzyme had a specific activity of 64.3. Proc Natl Acad Sci U S A, 1998 Mar 3, 95(5), 2574 - 9 A substrate of the centisome 63 type III protein secretion system of Salmonella typhimurium is encoded by a cryptic bacteriophage; Hardt WD et al.; Salmonella enterica has evolved a type III protein secretion system that allows these enteropathogens to translocate effector molecules directly into the host cell cytoplasm . These effectors mediate a variety of responses, including cytoskeletal rearrangements, cytokine production, and in certain cells, the induction of apoptosis . We report here the characterization of a substrate of this secretion system in S . enterica serovar typhimurium (Salmonella typhimurium) that is homologous to the SopE protein of Salmonella dublin implicated in bacterial entry into cultured epithelial cells . The sopE locus is located within a cluster of genes that encode tail and tail fiber proteins of a cryptic P2-like prophage, outside of the centisome 63 pathogenicity island that encodes the invasion-associated type III secretion system . Southern hybridization analysis revealed that sopE is present in only a subset of S . enterica serovars and that the flanking bacteriophage genes are also highly polymorphic . Encoding effector proteins that are delivered through type III secretion systems in highly mobile genetic elements may allow pathogens to adapt rapidly by facilitating the assembly of an appropriate set of effector proteins required for successful replication in a new environment. J Trauma, 1998 Mar, 44(3), 517 - 22 Growth hormone improves the resistance of thermally injured mice infected with herpes simplex virus type 1; Takagi K et al.; BACKGROUND: Growth hormone (GH) has been shown to promote wound healing and to improve protein metabolism in burned patients . Through immunomodulation, GH has also protected rats infected with Salmonella typhimurium and mice infected with Escherichia coli . In spite of advances in the management of patient care for those with thermal injuries, high mortality rates of burned patients as a result of infections are of special concern . An improvement in the resistance of burned patients to certain infections will make the beneficial role of GH very clear . In this study, therefore, the immunomodulating effects of recombinant human GH (rhGH) in thermally injured mice exposed to opportunistic herpesvirus infections were investigated . METHODS: (1) Burned mice, exposed to herpes simplex virus type 1 (HSV-1), were treated subcutaneously with rhGH (4 mg/kg) and observed for 21 days to determine the protective antiviral effect of rhGH . (2) Because of reports describing a lack of interferon-gamma (IFN-gamma) responsiveness in burned mice, the IFN-gamma-producing ability of the splenic mononuclear cells (SMNC) from burned mice treated with rhGH was examined . (3) Because the generation of burn-associated suppressor macrophages that can inhibit the IFN-gamma production by SMNC has been previously described, the suppressor cell activities of macrophages from burned mice treated with rhGH were examined . RESULTS: After exposure to lethal amounts of HSV-1, mice treated with rhGH displayed a reduced mortality rate compared with control mice treated with saline . SMNC from burned mice treated with rhGH produced IFN-gamma, whereas this cytokine was not produced by SMNC from burned mice treated with saline . Also, an inhibition of the generation of burn-associated suppressor macrophages was displayed in burned mice treated with rhGH . CONCLUSION: Exogenous administration of rhGH caused an improvement in the resistance of burned mice to HSV-1 infection . In burned mice treated with rhGH, the impaired IFN-gamma responsiveness was restored and the generation of burn-associated suppressor macrophages was inhibited . IFN-gamma, a typical antiviral cytokine induced by rhGH through the regulation of the suppressor macrophage generation, may therefore play a role in the protection of burned mice infected with a lethal amount of HSV-1. Infect Immun, 1998 Apr, 66(4), 1806 - 11 Trafficking of porin-deficient Salmonella typhimurium mutants inside HeLa cells: ompR and envZ mutants are defective for the formation of Salmonella-induced filaments; Mills SD et al.; Outer membrane porin genes of Salmonella typhimurium, including ompC, ompF, and tppB, are regulated by the products of ompB, a two-component regulatory locus encoding OmpR and EnvZ . S . typhimurium ompR mutants are attenuated in mice, but to date no one has studied the intracellular trafficking of S . typhimurium porin-deficient mutants . In this study, isogenic transposon mutants of S . typhimurium with insertions in ompR, envZ, ompF, ompC, ompD, osmZ, and tppB were compared with wild-type SL1344 for trafficking in the human epithelial cell line HeLa . We found that ompR and envZ mutants were reduced or completely inhibited for the formation of Salmonella-induced filaments (Sifs) . This result was confirmed with an ompB deletion mutant . Sifs are tubular structures containing lysosomal glycoprotein which are induced specifically by intracellular Salmonella . Genetic analysis showed that the ompR mutation could be complemented in trans by cloned ompR to restore its ability to induce Sifs . In contrast, mutations in the known ompR-regulated genes ompF, ompC, and tppB (as well as the ompR-independent porin gene, ompD) had no effect on Sif formation relative to that of wild-type SL1344, thus indicating that OmpR does not exert its role on these genes to induce Sif formation . The omp mutants studied were able to invade and replicate in HeLa cells at levels comparable to those in wild-type SL1344 . We conclude that OmpR and EnvZ appear to regulate Sif formation triggered by intracellular S . typhimurium. Infect Immun, 1998 Apr, 66(4), 1432 - 8 Mutation of invH, but not stn, reduces Salmonella-induced enteritis in cattle; Watson PR et al.; The induction of secretory and inflammatory responses in calves by Salmonella typhimurium and Salmonella dublin strains was compared, and the effects of mutations in the invH and stn genes were assessed . S . typhimurium induced greater secretory and inflammatory responses than S . dublin in bovine ileal loops, despite the fact that these serotypes were recovered from bovine ileal mucosa in comparable numbers (P . R . Watson, S . M . Paulin, A . P . Bland, P . W . Jones, and T . S . Wallis, Infect . Immun . 63:2743-2754, 1995) . These results implicate serotype-specific factors other than, or in addition to, intestinal invasion in the induction of enteritis . The secretory and inflammatory responses induced by S . typhimurium and S . dublin in bovine ligated ileal loops were not significantly altered by mutation of stn, which suggests that stn does not have a major role in Salmonella-induced enteritis . The invH mutation significantly reduced the secretory and inflammatory responses induced in bovine ileal loops, and this correlated with a reduction in the severity of enteritis following oral inoculation of calves . The attenuation associated with the invH mutation did not appear to be due to an increased susceptibility to the innate host defense mechanisms, because the resistance of S . typhimurium to the bactericidal action of either bovine polymorphonuclear leukocytes or bovine serum was not significantly altered . However, lysis of macrophages following infection with S . typhimurium was significantly reduced by the invH mutation . The invH mutation prevented the normal secretion of several proteins, including SipC, by S . typhimurium, indicating that the function of the inv-spa-encoded type III protein secretion system was disrupted . Taken together, these observations implicate inv-spa-dependent effectors in mediation of Salmonella-induced enteritis in cattle . Clearly, however, other undefined serotype-specific virulence factors are also involved in Salmonella-induced enteritis. Microbiol Immunol, 1998, 42(1), 1 - 5 Immunobiology of lipopolysaccharide (LPS) and LPS-derived immunoconjugates vaccinate mice against Salmonella typhimurium; Tiwari RP et al.; The immunobiology of lipopolysaccharide (LPS) of Salmonella typhimurium LT2-71 was studied in its native, modified and conjugated states using mice as the experimental model . An alkali-treated detoxified fraction of LPS (D-LPS) was found to be not only non-toxic but also equally immunogenic, like LPS . In addition D-LPS alone or conjugated with enterotoxin or hemolysin was also non-pyrogenic and non-indurogenic . The immunoprophylactic activity of D-LPS conjugates to a 100 ID50 challenge dose of S . typhimurium was also higher than that of detoxified LPS or native LPS. Biochemistry, 1998 Mar 10, 37(10), 3491 - 8 A single amino acid substitution in the human and a bacterial hypoxanthine phosphoribosyltransferase modulates specificity for the binding of guanine; Lee CC et al.; Early studies involving purine salvage in Salmonella typhimurium resulted in the isolation and identification of a mutant strain possessing a genetically modified hypoxanthine phosphoribosyl-transferase (HPRT) with enhanced substrate specificity for guanine {Benson, C . E., and Gots, J . S . (1975) J . Bacteriol . 121, 77-82} . To explore the molecular basis for this altered substrate specificity in the mutant hpt gene product, degenerate oligonucleotide primers, designed according to the N- and C-termini of the HPRT of Escherichia coli, were used in polymerase chain reactions to amplify both the mutant and wild-type S . typhimurium hpt genes from genomic DNA . Analysis of the deduced amino acid sequences revealed that a single base mutation resulted in the encoding of a Thr in the mutant HPRT, instead of an Ile found in the wild-type enzyme, at a position analogous to position 192 (Leu-192) of the human HPRT . Comparison of kinetic data for purified recombinant mutant and wild-type HPRTs showed no difference in the overall catalytic efficiency (kcat/K(m)) with hypoxanthine as substrate, but with guanine, the mutant enzyme exhibited a more than 50-fold higher kcat/K(m) largely as a result of a decrease of nearly 2 orders of magnitude in K(m) . Involvement in substrate binding of the cognate amino acid at position 192 in the human HPRT was investigated using site-directed mutagenesis . Mutation of Leu-192 to Thr did not significantly alter kcat/K(m) values for hypoxanthine and guanine compared to wild-type, and replacement of Leu-192 with Ile had no significant change in kinetics for either hypoxanthine or PRPP . However, this Ile substitution resulted in an over 15-fold decrease in the kcat/K(m) for guanine due to a greater than 15-fold increase in K(m) . These results demonstrate that a single active site amino acid substitution in HPRTs can significantly alter the specificity for binding guanine. Poult Sci, 1998 Mar, 77(3), 394 - 9 Effect of Salmonella in young chicks on competitive exclusion treatment; Bailey JS et al.; The potential of Salmonella contamination in hatching cabinets to 1) generate seeder chicks and 2) interfere with the efficacy of competitive exclusion (CE) treatments was investigated in six experiments . Hatchery-generated seeder: chicks were produced by hatching in the same hatcher with inoculated eggs (immersed in a 1.0 x 10(5) cfu/mL suspension of Salmonella typhimurium or inoculated with 10(5) to 10(6) cells on the air cell membrane) . Salmonella spread through the hatching cabinet to chicks hatching from uninoculated eggs in adjacent trays . In two experiments, Salmonella was isolated from 31 and 100% of the chick rinses after the birds were held in groups for 3 d in isolation units . When these hatchery-generated seeder chicks were stocked in floor pens at a 1:10 ratio with uncontaminated contact chicks, the pen environment became contaminated to the extent that greater than 50% of the contact chicks became contaminated . In two experiments with only one to three inoculated eggs per 200 egg hatching cabinet, 98% of uninoculated chicks were intestinally colonized with Salmonella after the birds were held 1 wk in isolation cabinets . This hatchery-acquired Salmonella substantially reduced the effectiveness of subsequent CE treatments to prevent Salmonella colonization of the young chicks . These studies demonstrate that control of Salmonella in hatching cabinets is critically important for control of Salmonella in broilers. Antimicrob Agents Chemother, 1998 Mar, 42(3), 571 - 8 Effects of bicyclomycin on RNA- and ATP-binding activities of transcription termination factor Rho; Carrano L et al.; Bicyclomycin is a commercially important antibiotic that has been shown to be effective against many gram-negative bacteria . Genetic and biochemical evidence indicates that the antibiotic interferes with RNA metabolism in Escherichia coli by inhibiting the activity of transcription termination factor Rho . However, the precise mechanism of inhibition is not completely known . In this study we have used in vitro transcription assays to analyze the effects of bicyclomycin on the termination step of transcription . The Rho-dependent transcription termination region located within the hisG cistron of Salmonella typhimurium has been used as an experimental system . The possible interference of the antibiotic with the various functions of factor Rho, such as RNA binding at the primary site, ATP binding, and hexamer formation, has been investigated by RNA gel mobility shift, photochemical cross-linking, and gel filtration experiments . The results of these studies demonstrate that bicyclomycin does not interfere with the binding of Rho to the loading site on nascent RNA . Binding of the factor to ATP is not impeded, on the contrary, the antibiotic appears to decrease the apparent equilibrium dissociation constant for ATP in photochemical cross-linking experiments . The available evidence suggests that this decrease might be due to an interference with the correct positioning of ATP within the nucleotide-binding pocket leading b an inherent block of ATP hydrolysis . Possibly, as a consequence of this interference, the antibiotic also prevents ATP-dependent stabilization of Rho hexamers. Microb Pathog, 1998 Jan, 24(1), 47 - 55 Mouse susceptibility to infection by the Salmonella abortusovis vaccine strain Rv6 is controlled by the Ity/Nramp 1 gene and influences the antibody but not the complement responses; Gautier AV et al.; Early growth of Salmonella typhimurium in spleen and liver of mice is controlled by the mouse chromosome 1 locus Ity/Nramp 1 . Genetic control of resistance to the attenuated vaccine strain Rv6 of Salmonella abortusovis was studied in mice infected by the intravenous route . Comparison of kinetics of bacterial colonization of spleen and liver in two congenic BALB/c-susceptible (Itys) and -resistant (Ityr) mouse lines showed that BALB/c mice (Itys) were significantly more susceptible to infection than C.D2 mice (Ityr) suggesting that infection by this vaccine strain is controlled by a gene which is close or identical to Ity/Nramp 1 . Congenic mice also differed in their anti-Salmonella antibody response, measured by ELISA: susceptible mice had a significantly higher antibody level than resistant mice, whatever the immunoglobulin isotype (IgM, IgG1, IgG2a, IgG3, IgA, and total immunoglobulins) . The two congenic BALB/c mouse lines had equal serum C3c levels in response to infection . However, we observed a highly significant difference according to the sex of mice, suggesting a role of sex hormones in the regulation of the level of some complement factors . These results, obtained with congenic mice, strongly suggest that the Ity/Nramp 1 locus controls susceptibility to infection by the S . abortusovis vaccine strain Rv6 and influences the antibody response . Biochem Biophys Res Commun, 1998 Mar 17, 244(2), 428 - 33 Insertional mutagenesis in the tailspike protein of bacteriophage P22; Carbonell X et al.; The tailspike protein (TSP) of bacteriophage P22 is a homotrimeric multifunctional protein responsible for recognition and hydrolysis of Salmonella typhimurium host receptors . Once properly folded, TSP shows an unusual stability to temperature and detergent denaturation, prompting the analysis of TSP as a framework for the positioning of heterologous protein segments . We have explored the flexibility of inner sites and both amino and carboxy termini to accommodate foreign peptides for phage display . In the examined inner sites, TSP is extremely sensitive to minor sequence modifications, the folding intermediates being rapidly degraded . However, both the amino and carboxy termini are tolerant to peptide fusions, rendering stable and functional chimeric proteins . Surprisingly, the amino terminus, which connects the tail to the neck structure, can accept large peptide fusions, and the foreign amino acid stretches are solvent-exposed and highly antigenic on assembled, infectious virus particles. Plasmid, 1998, 39(2), 134 - 40 A 3.3-kb plasmid of enterohemorrhagic Escherichia coli O157:H7 is closely related to the core region of the Salmonella typhimurium antibiotic resistance plasmid NTP16; Haarmann C et al.; A restriction enzyme map was constructed of a 3.3-kb plasmid derived from enterohemorrhagic Escherichia coli O157:H7 strain 4821 using the enzymes SmaI, HpaI, FokI, HaeIII, StyI, RsaI, Bg/II, ClaI, and EcoRV . The molecular size of p4821 was 3307 bp, determined by nucleotide sequencing . Homology searches in the EMBL database library revealed that the nucleotide sequence of P4821 is similar (> 98%) to the core region of the antibiotic resistance plasmid NTP16 of Salmonella typhimurium strains . Nucleotide sequence analysis showed that p4821 contains all the information necessary for its replication, stability, and mobilization . However, the lack of tra genes indicates that the plasmid is nonconjugative . A possible transposon insertion site was found in p4821 but two such sites were found at the boundaries between the core region and the antibiotic resistance encoding transposons in plasmid NTP16 . Using a hybridization assay, we determined that of 50 E . coli O157:H7 strains isolated in 1996 in Wurzburg, Germany from patients with diarrhea and hemolytic-uremic syndrome, 4 strains (8%) contained plasmid p4821-specific sequences. J Antimicrob Chemother, 1998 Jan, 41(1), 119 - 21 A plasmid-mediated beta-lactamase conferring resistance to cefotaxime in a Salmonella typhimurium clone found in St Petersburg, Russia; Gazouli M et al.; Four clinical and two environmental isolates of cefotaxime-resistant Salmonella typhimurium strains collected in St Petersburg, Russia, were studied . Molecular typing demonstrated that they constituted a single clone . The isolates expressed a cefotaxime-hydrolysing beta-lactamase with a pl of 8.4 . The enzyme was encoded by a 12 kb plasmid which did not hybridize with TEM, SHV or ampC probes. Mutat Res, 1998 Jan 13, 412(1), 99 - 107 Mutagenicity of isomeric alkanediazotates, precursors for ultimate alkylating species of carcinogenic N-nitroso compounds; Ukawa-Ishikawa S et al.; Alkanediazohydroxides are common key intermediates in carcinogenesis and mutagenesis of N-nitroso compounds, which are widely found in human environment . Mutagenicity of (E)- and (Z)-potassium alkanediazotates, as precursors of corresponding alkanediazohydroxides were evaluated to investigate the effect of geometric isomerism and also the effect of alkyl groups on their biological activity . Mutagenicity of N-nitroso-N-alkylureas which spontaneously produce alkanediazohydroxides after non-enzymatic hydrolysis were also tested in comparison to that of the corresponding diazotates and other activated chemical species of N-nitrosamines . When the mutagenicity was assayed in three microbial strains, Salmonella typhimurium TA1535, and Escherichia coli WP2 and WP2 uvrA, the order of mutagenic potency of the compounds with the same alkyl group was as follows; (E)-diazotates > (Z)-diazotates > nitrosoureas . The effect of alkyl groups on the mutagenic potency was different in Salmonella strain and in E . coli strains, and this result could be explained by the efficiency of O6-alkylguanine-DNA alkyltransferase . In each bacterial strain, this effect of alkyl groups was similar in mutagenicity induced by (E)- and (Z)-diazotates, N-nitroso-N-alkylureas and other activated N-nitrosodialkylamines such as alpha-hydroxy nitrosamines . The geometrical isomerism affected the mutagenicity of (E)- and (Z)-potassium alkanediazotates, and the result suggested that alkanediazohydroxides react through diazonium ions in a cage rather than through free alkyldiazonium ions which have no geometrical isomerism . Our results confirmed that (E)-potassium alkanediazotates, (Z)-potassium alkanediazotates and N-nitroso-N-alkylureas all decomposed through diazohydroxides, and that alkanediazohydroxides are the active alkylating species of N-nitroso compounds, and also that the geometrical isomerism is important for carcinogenic N-nitroso compounds to show their biological activity. Mutat Res, 1998 Jan 13, 412(1), 17 - 31 Comparisons on chemically-induced mutation among four bacterial strains, Salmonella typhimurium TA102 and TA2638, and Escherichia coli WP2/pKM101 and WP2 uvrA/pKM101: collaborative study II; Watanabe K et al.; A collaborative study of chemically-induced mutation was performed using the four bacterial strains Salmonella typhimurium TA102 and TA2638, and Escherichia coli WP2/pKM101 and WP2 uvrA/pKM101, used in the previous successive studies in order to compare the specific spectrum of response to chemicals among the four strains and to determine the usefulness (sensitivity) of each strain . Twenty-two laboratories participated in this study . Following the previous study, 28 compounds were selected consisting of oxidative agents or crosslinking agents . These compounds were tested for mutagenicity using the plate incorporation method with or without metabolic activation . The tests were performed in two laboratories per chemical . The number of chemicals which showed positive results were 18, 17, 16 and 14 with WP2 uvrA/pKM101, TA2638, TA102 and WP2/pKM101, respectively . In all four strains 19/28 chemicals (68%) were either positive or negative while the remaining 9 chemicals showed a heterogeneous response . With TA102 and TA2638 89% (25/28) of the test chemicals showed the same response, while 75-79% (21-22/28) showed the same response in these two S . typhimurium strains and in WP2 uvrA/pKM101 . In conclusion, WP2/pKM101 strain showed the least sensitivity to the chemicals used . Among the other three strains, WP2 uvrA/pKM101 was the most sensitive strain, although the difference in the number of chemicals which showed positive results was minor . Concerning the spectrum of response to chemicals, it is apparent that TA102 and TA2638 have almost the same sensitivity, but some chemicals induce different responses between the two S . typhimurium strains and WP2 uvrA/pKM101. FEMS Microbiol Lett, 1998 Feb 15, 159(2), 179 - 85 RNase III deficient Salmonella typhimurium LT2 contains intervening sequences (IVSs) in its 23S rRNA; Mattatall NR et al.; Salmonella typhimurium LT2 contains intervening sequences (IVSs) of 90-110 nt within all its 23S rRNA that are cleaved out by RNase III, resulting in rRNA fragmentation . In order to determine the functionality of 23S rRNA that contains unexcised IVSs, we constructed an S . typhimurium RNase III (rnc) deficient strain by transducing a mini-Tn10 (rnc-14::Tn10) from Escherichia coli K-12 . The resulting strain of S . typhimurium was viable, contained IVSs within all of its 23S rRNA, and showed a growth reduction similar to that observed for the RNase III deficient strain of E . coli . These results indicate that ribosomes containing 23S rRNA in which IVSs are not excised are functional in translation, and make it unlikely that RNase III excision of IVSs from strain LT2 23S rRNA is dictated by a selective pressure to uphold the functional integrity of ribosomes. SAR QSAR Environ Res, 1997, 7(1-4), 281 - 6 A plausible mechanism for the mutagenic activity (Salmonella typhimurium TA100) of MX compounds: a formation of CG-CG(+)-CG radical cation by one-electron reduction; Tuppurainen K; Combining our previous QSAR work with recent high-level quantum mechanical calculations, a plausible mechanism for the mutagenic activity of halogenated furanones (so called MX compounds) in Salmonella typhimurium TA100 tester strain is proposed . The mechanism involves one-electron reduction as a key step and it seems reasonable to suggest that the mutagenicity of these direct-acting compounds may be a purely thermodynamic phenomenon, rather than the result of site-specific binding or adduct formation . Overall, the proposed model is consistent with the most experimental findings. J Leukoc Biol, 1998 Mar, 63(3), 297 - 304 Salmonella infections in the absence of the major histocompatibility complex II; Chapes SK et al.; We examined the pathogenesis of the facultative intracellular bacterium, Salmonella typhimurium in MHCII-/-, C2D knock-out mice, and wild-type C57BL/6J mice . The MHCII knock-out shortened the kinetics of animal death and reduced the dose of S . typhimurium needed to kill mice . We measured the physiological and cytokine responses of both mouse strains after S . typhimurium injection . Animal weight loss, spleen weights, liver weights, thymus weights, and serum corticosterone concentrations were comparable after injection with several doses of bacteria . The only physiological differences observed between the two strains were observed 3 days after injection of the highest dose of bacteria tested . Serum concentrations of tumor necrosis factor alpha, interleukin-2, and interleukin-6 increased in a dose-dependent fashion irrespective of mouse MHCII expression . Therefore, even in the absence of MHCII, mice are able to mount relatively normal physiological and immunological responses . Consistent with these normal responses, an increased percentage of MHCII-/- mice, primed with a low dose of bacteria 13 days earlier, were able to survive a lethal challenge of Salmonella compared with unprimed controls . Lastly, C2D mice had significantly higher serum interleukin-10 concentrations than C57BL/6J mice 48 h after infection with all doses of S . typhimurium . C2D macrophages also secreted significantly more IL-10 and less NO and O2- after lipopolysaccharide or phorbol ester stimulation in vitro than wild-type macrophages. Microb Pathog, 1997 Nov, 23(5), 311 - 6 Salmonella typhimurium aroB mutants are attentuated in BALB/c mice; Gunel-Ozcan A et al.; The aroB gene of Salmonella typhimurium, encoding dehydroquinate synthase, has been cloned into pUC19 and the DNA sequence determined . The aroB gene was isolated from a cosmid gene bank by complementation of an Escherichia coli aroB mutant and screening by Southern blot analysis . The nucleotide sequence of the S . typhimurium aroB gene revealed the presence of an open reading frame, encoding a protein of 362 amino acids with a calculated molecular mass of 38696 Daltons . The amino acid sequence of S . typhimurium dehydroquinate synthase is nearly identical to the E . coli homologue and shows high homology with other aroB gene products from other organisms . Subsequently, a stable insertional mutation in aroB was introduced into the wild-type S . typhimurium C5 strain . This mutant was auxotrophic for aromatic compounds . Infection of BALB/c mice with this mutant demonstrated attenuation comparable to other S . typhimurium mutants unable to biosynthesize aromatic amino acids . Int J Food Microbiol, 1997 Sep 16, 38(2-3), 181 - 9 Detection and quantification of poultry probiotic bacteria in mixed culture using monoclonal antibodies in an enzyme-linked immunosorbent assay; Durant JA et al.; Murine monoclonal antibodies were used in an enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of selected probiotic bacteria present in a continuous-flow competitive exclusion culture known to be effective at reducing chicken cecal and crop colonization by Salmonella typhimurium . Veillonella, Enterococcus avium and S . typhimurium were grown anaerobically in batch culture of Viande Levure broth in pure culture and mixed culture . The mixed cultures produced significantly more acetate and propionate than any of the pure cultures with acetate and propionate being the predominant volatile fatty acids . The association in mixed culture resulted in a significant increase in cell numbers compared to the respective pure cultures . The ELISA was capable of detecting 10(4) cells per ml of the bacteria . The plots of cell numbers determined by the ELISA versus direct plating increased in accordance with increases in cell numbers with r2 values of 0.950, 0.922 and 0.940 for the pure culture incubations and 0.901, 0.924 and 0.905 in the mixed culture incubation for E . avium, S . typhimurium and Veillonella, respectively . The results indicate that the monoclonal antibodies can be used to quantitatively assay individual probiotic bacterial species grown in a mixed culture incubation. Carcinogenesis, 1998 Feb, 19(2), 359 - 63 Characterization of the carcinogen 2-amino-3,8-dimethylimidazo{4,5-f}quinoxaline in cooking aerosols under domestic conditions; Yang CC et al.; Lung cancer in women is the leading cause of cancer death in Taiwan . Most Chinese women are non-smokers and 60% of female lung cancer patients have adenocarcinomas . Epidemiological data indicate that the incidence of lung cancer among Chinese women may be correlated with cooking fumes . However, the carcinogenic compound(s) in cooking fume aerosols is not defined . In the present study, the cooking aerosols from Chinese stir-frying of fish were prepared under domestic conditions . To determine the mutagenic compounds in the cooking aerosol, mutagens were purified by two steps of high-performance liquid chromatography (HPLC), and their mutagenicity was monitored with Salmonella typhimurium TA98 . The mutagen was eluted as a single peak . The chemical structure of the mutagenic fraction of cooking aerosol from frying of fish was characterized by UV spectra and electrospray mass spectrometry . The bacterial indirect-acting mutagenic compound in the cooking aerosol extract was determined to be 2-amino-3,8-dimethylimidazo{4,5-f}quinoxaline (MeIQx) . An amount of 0.25 ng MeIQx/g of meat per min was estimated based on the mutagenic response . These data indicated that significant amounts of MeIQx (268.1 ng/Chinese dish of frying fish) were present in cooking aerosol in a short time . Chinese women spend approximately 1 h preparing meals everyday, thus, they may be exposed to significant amounts of MeIQx from cooking aerosols in the kitchen. J Bacteriol, 1998 Mar, 180(5), 1185 - 93 Salmonella typhimurium encodes an SdiA homolog, a putative quorum sensor of the LuxR family, that regulates genes on the virulence plasmid; Ahmer BM et al.; Quorum sensing is a phenomenon in which bacteria sense and respond to their own population density by releasing and sensing pheromones . In gram-negative bacteria, quorum sensing is often performed by the LuxR family of transcriptional regulators, which affect phenotypes as diverse as conjugation, bioluminescence, and virulence gene expression . The gene encoding one LuxR family member, named sdiA (suppressor of cell division inhibition), is present in the Escherichia coli genome . In this report, we have cloned the Salmonella typhimurium homolog of SdiA and performed a systematic screen for sdiA-regulated genes . A 4.4-kb fragment encoding the S . typhimurium sdiA gene was sequenced and found to encode the 3' end of YecC (homologous to amino acid transporters of the ABC family), all of SdiA and SirA (Salmonella invasion regulator), and the 5' end of UvrC . This gene organization is conserved between E . coli and S . typhimurium . We determined that the S . typhimurium sdiA gene was able to weakly complement the E . coli sdiA gene for activation of ftsQAZ at promoter 2 and for suppression of filamentation caused by an ftsZ(Ts) allele . To better understand the function of sdiA in S . typhimurium, we screened 10,000 random lacZY transcriptional fusions (MudJ transposon mutations) for regulation by sdiA . Ten positively regulated fusions were isolated . Seven of the fusions were within an apparent operon containing ORF8, ORF9, rck (resistance to complement killing), and ORF11 of the S . typhimurium virulence plasmid . The three ORFs have now been named srgA, srgB, and srgC (for sdiA-regulated gene), respectively . The DNA sequence adjacent to the remaining three fusions shared no similarity with previously described genes. J Bacteriol, 1998 Mar, 180(5), 1166 - 73 The nac (nitrogen assimilation control) gene from Escherichia coli; Muse WB et al.; The nitrogen assimilation control gene, nac, was detected in Escherichia coli but not in Salmonella typhimurium by Southern blotting, using a probe from the Klebsiella aerogenes nac (nacK) gene . The E . coli nac gene (nacE) was isolated from a cosmid clone by complementation of a nac mutation in K . aerogenes . nacE was fully functional in this complementation assay . DNA sequence analysis showed considerable divergence between nacE and nacK, with a predicted amino acid sequence identity of only 79% and most of the divergence in the C-terminal half of the protein sequence . The total predicted size of NAC(E) is 305 amino acids, the same as for NAC(K) . A null mutation, nac-28, was generated by reverse genetics . Mutants bearing nac-28 have a variety of phenotypes related to nitrogen metabolism, including slower growth on cytosine, faster growth on arginine, and suppression of the failure of an Ntr-constitutive mutant to grow with serine as sole nitrogen source . In addition to a loss of nitrogen regulation of histidase formation, nac-28 mutants also showed a loss of a weak repression of glutamate dehydrogenase formation . This repression was unexpected because it is balanced by a NAC-independent activation of glutamate dehydrogenase formation during nitrogen-limited growth . Attempts to purify NAC(E) by using methods established for NAC(K) failed, and NAC(E) appears to be degraded with a half-life at 30 degrees C as short as 15 min during inhibition of protein synthesis. Microbiology, 1998 Feb, 144 ( Pt 2), 385 - 90 Permeabilizing action of polyethyleneimine on Salmonella typhimurium involves disruption of the outer membrane and interactions with lipopolysaccharide; Helander IM et al.; Polyethyleneimine (PEI), a polycationic polymer substance used in various bioprocesses as a flocculating agent and to immobilize enzymes, was recently shown to make Gram-negative bacteria permeable to hydrophobic antibiotics and to detergents . Because this suggests impairment of the protective function of the outer membrane (OM), the effect of PEI on the ultrastructure of Salmonella typhimurium was investigated . Massive alterations in the OM of PEI-treated and thin-sectioned bacteria were observed by electron microscopy . Vesicular structures were seen on the surface of the OM, but no liberation of the membrane or its fragments was evident . Since a potential mechanism for the action of PEI could be its binding to anionic LPSs on the OM surface, the interaction of PEI with isolated LPSs was assayed in vitro . The solubility of smooth-type LPSs of Salmonella, regardless of the sugar composition of their O-specific chains, was not affected by PEI, nor was that of Ra-LPS (lacking O-specific chains but having a complete core oligosaccharide) . PEI strongly decreased the solubility of rough-type LPSs of the chemotypes Rb2 and Re, whereas it had only a weak effect on the abnormally cationic Rb2-type pmrA mutant LPS, suggesting that the negative charge to mass ratio of LPS plays a critical role in the interaction. Parasitol Res, 1998, 84(2), 117 - 22 A Taenia solium metacestode factor nonspecifically inhibits cytokine production; Arechavaleta F et al.; Studies of the immune response in chronic helminth infections suggest that parasites modulate the host's immune response . Taenia solium metacestodes, in particular, produce molecules that down-regulate cell-mediated immunity . We have described a small RNA peptide termed metacestode factor (MF) that depresses the murine immune response to Salmonella typhimurium antigens . MF inhibits mitogen-induced proliferation, humoral and cellular responses to metacestode antigens, and inflammation surrounding metacestodes implanted subcutaneously in mice . To assess the effects of MF on cytokine production we stimulated murine spleen cells in vitro with concanavalin A and measured cytokine concentrations in the culture supernatants by enzyme-linked immunosorbent assay . When cultured with MF, the cells showed significantly decreased production of interleukin 2 (IL-2), interferon-gamma (IFN-gamma), and IL-4 as compared with mitogen alone . Exogenous rIL-2 and rIL-4 largely restored the proliferative response (85% and 71% of control cells, respectively) . MF also decreased production of tumor necrosis factor-alpha (TNF-alpha) by macrophages stimulated with lipopolysaccharide and IFN-gamma . The TNF-alpha concentration was inversely correlated with the MF concentration . Experiments using spleen cells from mice treated with MF also showed a significant reduction in IL-4 concentration . These results suggest that MF inhibits cytokine production without regard to cell type or cytokine . This may explain the function of this molecule as an inhibitor of the host inflammatory and immune responses. Vaccine, 1998 Mar, 16(5), 522 - 9 Construction and characterisation of Salmonella typhimurium aroA simultaneously expressing the five pertussis toxin subunits; Dalla Pozza T et al.; Pertussis toxin (PT) is a major protective antigen of Bordetella pertussis, the causative agent of whooping cough . Current vaccines against whooping cough are administered parenterally, and generate a systemic immune response . An alternative to this approach is to stimulate mucosal and systemic immune responses by oral immunisation with live vaccine strains of Salmonella spp . We have individually expressed the five PT subunits in Salmonella typhimurium aroA and subsequently expressed a synthetic pertussis toxin operon (pDP16), via the use of a temperature inducible expression system . In S . typhimurium aroA containing individual subunit expression plasmids the S5 subunit was found to be completely processed, while all other subunits were partially processed . In S . typhimurium aroA (pDP16) S5 was completely processed, S1 was partially processed, whilst S2, S3 and S4 remained unprocessed . Induction of the synthetic PT operon decreased the in vitro invasiveness of S . typhimurium aroA, compared to vector-only and plasmid-less strains . Following oral immunisation of Balb/c mice with S . typhimurium aroA (pDP16), statistically significant IgG (p < 0.05) specific to PT was detected in the serum of mice . Nonetheless, despite a statistically significant anti-PT serum antibody response in immunised mice, vaccination with S . typhimurium aroA (pDP16) failed to protect mice from virulent challenge. Vaccine, 1998 Mar, 16(5), 460 - 71 Protection of mice against gastric colonization by Helicobacter pylori by single oral dose immunization with attenuated Salmonella typhimurium producing urease subunits A and B; Gomez-Duarte OG et al.; Helicobacter pylori is a Gram-negative bacterial pathogen associated with gastritis, peptic ulceration, and gastric carcinoma . The bacteria express a strong urease activity which is known to be essential for colonization of gnotobiotic pigs and nude mice . UreA and UreB, two structural subunits of the active enzyme, were expressed in the attenuated Salmonella typhimurium live vaccine SL3261 strain . Evaluation of protection against H . pylori was performed in Balb/c mice by oral immunization with a single dose of the vaccine strain . Five weeks after immunization, mice were challenged orally three times with a mouse-adapted H . pylori wild type strain and, six weeks later, mice were sacrificed to determine H . pylori infection by detection of urease activity from the antral region of the mouse stomachs . In several independent experiments, we observed 100% infection with H . pylori in the non-immunized mice and no infection (100% protection) in the mice immunized with S . typhimurium expressing recombinant UreA and UreB . Specific humoral and mucosal antibody responses against UreA and UreB were observed in mice immunized as indicated by western blots and ELISA assays . These data shows that oral immunization of mice with urease subunits delivered by an attenuated Salmonella strain induced a specific immune response and protected mice against H . pylori colonization . Single oral dose immunization with UreA and UreB delivered by a live Salmonella vaccine vector appears to be an attractive candidate for human vaccination against H . pylori infection . In addition, this model will aid to elucidate the effective protection mechanisms against H . pylori in the gastric mucosa. Mutagenesis, 1998 Jan, 13(1), 19 - 26 The effectiveness of Salmonella strains TA100, TA102 and TA104 for detecting mutagenicity of some aldehydes and peroxides; Dillon D et al.; Several aldehydes and peroxides were tested for mutagenicity using Salmonella typhimurium tester strains TA97a, TA100, TA102 and TA104, in the presence and absence of Aroclor-induced liver S9 mix from F344 rats and B6C3F1 mice, in either preincubation or vapour phase protocols . Some chemicals were tested in additional Salmonella strains . Benzaldehyde, butyraldehyde, benzoyl peroxide, 4-chlorobenzaldehyde, isobutyraldehyde, propionaldehyde and veratraldehyde were non-mutagenic . Acetaldehyde and dicumyl peroxide gave inconsistent results and furfural gave equivocal responses in TA100 and TA104 . Cumene hydroperoxide, formaldehyde and glutaraldehyde were mutagenic in TA100, TA102 and TA104 . trans-Cinnamaldehyde exhibited a weak mutagenic response in TA100 with mouse liver S9 only . 2,4,5-Trimethoxybenzaldehyde was mutagenic only in strain TA1538 with rat liver S9 . With the exception of butanone peroxide, which was mutagenic only in TA104, all chemicals mutagenic in strains TA102 and/or TA104 were also mutagenic in TA100 . The data do not, therefore, support the preferential use of strains TA102 and TA104 for screening aldehydes and peroxides for mutagenicity . For a number of these chemicals the advantages of using TA102 or TA104 was in the increased responses compared with those obtained with TA100 . Two of the four peroxides were mutagenic and one of these was mutagenic only with TA104 . This suggests that strains TA102 and TA104 be used if peroxides are not mutagenic in TA100 or TA97. Eur J Biochem, 1998 Feb 1, 251(3), 935 - 45 Purification and characterisation of NADH oxidase from Thermus aquaticus YT-1 and evidence that it functions in a peroxide-reduction system; Toomey D et al.; A thermostable enzyme previously identified as an NADH oxidase has been purified from Thermus aquaticus YT-1 by chromatography on DEAE-cellulose and AMP-Sepharose . The enzyme is dimeric with subunits of 54 kDa and one molecule FAD/subunit . The FAD is tightly bound, but it can be removed reversibly by hydrophobic chromatography at low pH . The blue flavin semiquinone is stabilised during photo-chemical reduction of the enzyme . Chemical reduction by static titration with dithionite ion showed that the enzyme requires about 5 mol dithionite/mol FAD for full reduction, and that reduction occurs in four phases . Reduction by the substrate NADH is incomplete, with the formation of a new long-wavelength absorption underlying the semiquinone absorption . Amino acid sequencing showed that the T aquaticus enzyme resembles other microbial flavoenzymes that function in two-enzyme systems for the reduction of peroxides, and which contain two redox-active disulphide groups in addition to the flavin . The enzyme catalyses the reduction of O2, ferricyanide ion, 2,6-dichloroindophenol, and 5,5'dithiobis(2,2'-dinitrobenzoate), and of cumene hydroperoxide in the presence of the small protein component (AhpC) of the peroxide-reducing system of Salmonella typhimurium . The reduction of O2 is slow in the absence of exogenous flavin while dye reduction is fast, suggesting that the free flavin that is added to the usual assay for T . aquaticus NADH oxidase functions by mediating electron transfer from enzyme-bound reduced flavin to O2 . The physiological function of the enzyme is probably in peroxide reduction with a small protein analogous to AhpC as the natural electron acceptor. East Afr Med J, 1997 Jun, 74(6), 353 - 6 Laboratory investigations in the diagnosis of septicaemia and malaria; Dougle ML et al.; During a three month prospective study, 229 in-patients with fever, admitted to St . Mary's Hospital, Mumias, were examined for bacterial and malarial causes of fever . Blood cultures taken from patients appeared to contain true pathogens in 51 (22%) cases . Nine different bacterial species were identified from positive blood cultures of which four predominated: Salmonella typhi (46%), Streptococcus pneumoniae (19%), Salmonella enteritidis (12%), Salmonella typhimurium (8%) . S . enteritidis and S . typhimurium isolates were mostly multi-antibiotic resistant, compared to S . typhi isolates which were relatively susceptible to the antibiotics used in the hospital . Only 70% of the S . pneumoniae isolates were susceptible to penicillin . Among 227 patients in whom a thick blood-film for malaria parasites and HIV serology were performed, only 25 (11%) revealed malaria parasites . HIV-1 antibodies were detected in 51 (22%) patients . Without appropriate laboratory examinations, the majority of the diagnoses would have been missed and no optimal treatment would have been administered . This may increase resistance to antimalarials and antibioticsPIP: In developing countries, the presence of infectious diseases is diagnosed mainly on the basis of clinical symptoms and basic laboratory investigations . Such diagnosis, however, can lead to incorrect or overtreatment, resulting in an increased resistance to antibiotics . This study was conducted to characterize bacterial causes of community-acquired sepsis, in relation to malaria and HIV prevalence, and to assess whether isolated bacteria were susceptible to commonly used antibiotics . During the 3-month prospective study, 229 inpatients with fever admitted to St . Mary's Hospital, Mumias, were examined for bacterial and malarial causes of fever . Blood cultures taken from patients seemed to contain true pathogens in 51 (22%) cases . 9 different bacterial species were identified from positive blood cultures, of which the following predominated: Salmonella typhi (46%), Streptococcus pneumoniae (19%), Salmonella enteritidis (12%), and Salmonella typhimurium (8%) . The S . enteritidis and S . typhimurium isolates were mainly multi-antibiotic resistant, compared to the S . typhi isolates which were relatively susceptible to the antibiotics used in the hospital . 70% of the S . pneumoniae isolates were susceptible to penicillin . Among the 227 patients upon whom a thick blood-film for malaria parasites and HIV serology were performed, 25 (11%) had malaria parasites . HIV-1 antibodies were detected in 51 (22%) patients . These study findings show how the culture of 1 blood sample and the performance of simple microbiological studies can facilitate the initiation of appropriate treatment with antibiotics . Teratog Carcinog Mutagen, 1997-98, 17(6), 305 - 12 Potent suppressive activity of nonpolyphenolic fraction of green tea (Camellia sinensis) against genotoxin-induced umu C gene expression in Salmonella typhimurium (TA 1535/pSK 1002), tumor promotor-dependent ornithine decarboxylase induction of BALB/c 3T3 fibroblast cells, and chemically induced mouse skin tumorigenesis; Okai Y et al.; Many experimental studies for anticarcinogenic activity of green tea (Camellia sinensis) and tea-derived polyphenols have been carried out . However, the anticarcinogenic activity of the nonpolyphenolic fraction of green tea has been poorly elucidated . To study this problem, the effect of the nonpolyphenolic fraction of green tea leaves was analyzed using in vitro and in vivo experiments associated with tumor initiation and promotion as follows: 1) The nonpolyphenolic fraction caused a strong suppressive effect on umu C gene expression in Salmonella typhimurium (TA 1535/pSK 1002) induced by genotoxic substances such as 2-amino-6-methyldipirido{1,2-a:3',2'-d}imidazole (Glu-P-1) and 2-aminoanthracene (2-AA) in the presence of a hepatic metabolizing enzyme mixture . 2) The same fraction showed a dose-dependent inhibition of ornithine decarboxylase (ODC) in BALB/c 3T3 fibroblasts induced by a tumor promotor, 12-O-tetradecanoylphorbol-13-acetate (TPA) . 3) The same fraction also exhibited a significant suppression against mouse skin tumorigenesis induced by 7,12-dimethylbenz{a}anthracene (DMBA) (initiator) and TPA (promotor) through inhibition at both stages of tumor initiation and promotion . These results suggest that the nonpolyphenolic fraction of green tea has a potent suppressing activity against carcinogenesis associated with tumor initiation and promotion. Mol Microbiol, 1998 Jan, 27(2), 359 - 68 The Salmonella typhimurium tyrosine phosphatase SptP is translocated into host cells and disrupts the actin cytoskeleton; Fu Y et al.; The Salmonella typhimurium protein tyrosine phosphatase SptP is a target of the centisome 63 type III protein secrtion system . This system is essential for the interaction of these bacteria with host cells . We have shown here by a combination of biochemical and microscopy techniques that S . typhimurium directs the translocation of SptP into cultured epithelial cells . Translocation requires the function of the secreted proteins, SipB, SipC and SipD, as strains carrying mutations in any of the genes encoding these proteins fail to translocate SptP . Microinjection of purified GST-SptP into cultured cells results in the disruption of the actin cytoskeleton and the disappearance of stress fibres . These changes are reversible, as microinjected cells regain the normal appearance of their actin cytoskeleton upon prolonged incubation . Microinjection of the catalytically active GST-SptP(C481S) protein results in changes similar to those induced by the wild-type toxin . Furthermore, microinjection of a fusion protein between GST and the first 285 amino acids of SptP also leads to identical disruption of the host cell actin cytoskeleton, indicating that the amino-terminal half of SptP is sufficient to mediate this effect . However, microinjection of a fusion protein between GST and the last 259 amino acids of SptP also disrupted the normal appearance of the cytoskeleton . These results support the hypothesis that SptP is an effector protein arranged in modular domains that may co-operate with each other to exert relate functions. Genomics, 1998 Jan 15, 47(2), 180 - 6 Mapping of genetic modulators of natural resistance to infection with Salmonella typhimurium in wild-derived mice; Sebastiani G et al.; Despite antibiotic therapy and vaccination programs, microbial diseases continue to be the leading cause of morbidity and mortality worldwide . The genetic basis of the host response to infection is complex, and its understanding has been facilitated through the study of mouse models of human infectious diseases . Genetic variation in resistance of mice to infection with Salmonella typhimurium has been recognized for over 50 years and shown to be a multifactorial trait . We have studied the genetic basis of resistance or susceptibility to infection with S . typhimurium in the wild-derived inbred mouse Mus musculus molossinus (MOLF/Ei) . MOLF/Ei mice are extremely susceptible to infection with S . typhimurium despite the presence of resistance alleles at Nramp1 and Lps . To identify genes that modulate the expression of natural resistance or susceptibility to infection with S . typhimurium in MOLF/Ei, we have performed a genome-wide study using an F2 intercross between C56BL/6J and MOLF/Ei inbred mice . We have mapped three QTLs that significantly affect survival time following lethal infection with S . typhimurium . The Salmonella-resistant phenotype was linked to Nramp1 on proximal chromosome 1 (maximum lod score of 18.8 at D1Mcg4) and to a newly mapped region on mouse chromosome 11 (maximum lod score of 7.0 at D11Mit5) . The third QTL conferred recessive susceptibility and was located on mouse chromosome 1, approximately 25 cM distal to Nramp1 (maximum lod score of 4.8 at D1Mit100). J Biol Chem, 1998 Jan 30, 273(5), 2684 - 91 CobD, a novel enzyme with L-threonine-O-3-phosphate decarboxylase activity, is responsible for the synthesis of (R)-1-amino-2-propanol O-2-phosphate, a proposed new intermediate in cobalamin biosynthesis in Salmonella typhimurium LT2; Brushaber KR et al.; The cobD gene of Salmonella typhimurium LT2 has been cloned, sequenced, and overexpressed . The overexpressed protein had a molecular mass of approximately 40 kDa, in agreement with the mass predicted by the deduced amino acid sequence (40.8 kDa) . Computer analysis of the deduced amino acid sequence of CobD identified a consensus pyridoxal phosphate-binding motif . The role of CobD in cobalamin biosynthesis in this bacterium has been established . CobD was shown to decarboxylate L-threonine O-3-phosphate to yield (R)-1-amino-2-propanol O-2-phosphate . We propose that the latter is a substrate in the reaction catalyzed by the CbiB enzyme proposed to be responsible for the conversion of adenosylcobyric acid to adenosylcobinamide and that the product of the reaction is adenosylcobinamide phosphate, not adenosylcobinamide as previously thought . The implications of these findings are discussed in light of the demonstrated kinase activity of the CobU enzyme (O'Toole, G . A., and Escalante-Semerena, J . C . (1995) J . Biol . Chem . 270, 23560-23569) responsible for the conversion of adenosylcobinamide to adenosylcobinamide phosphate . These findings shed light on the strategy used by this bacterium for the assimilation of exogenous unphosphorylated cobinamide from its environment . To our knowledge, CobD is the first enzyme reported to have L-threonine-O-3-phosphate decarboxylase activity, and computer analysis of its amino acid sequence suggests that it may be a member of a new class of pyridoxal phosphate-dependent decarboxylases. Nucleic Acids Res, 1997 Dec 15, 25(24), 4994 - 5002 Information analysis of Fis binding sites; Hengen PN et al.; Originally discovered in the bacteriophage Mu DNA inversion system gin, Fis (Factor for Inversion Stimulation) regulates many genetic systems . To determine the base frequency conservation required for Fis to locate its binding sites, we collected a set of 60 experimentally defined wild-type Fis DNA binding sequences . The sequence logo for Fis binding sites showed the significance and likely kinds of base contacts, and these are consistent with available experimental data . Scanning with an information theory based weight matrix within fis, nrd, tgt/sec and gin revealed Fis sites not previously identified, but for which there are published footprinting and biochemical data . DNA mobility shift experiments showed that a site predicted to be 11 bases from the proximal Salmonella typhimurium hin site and a site predicted to be 7 bases from the proximal P1 cin site are bound by Fis in vitro . Two predicted sites separated by 11 bp found within the nrd promoter region, and one in the tgt/sec promoter, were also confirmed by gel shift analysis . A sequence in aldB previously reported to be a Fis site, for which information theory predicts no site, did not shift . These results demonstrate that information analysis is useful for predicting Fis DNA binding. Chem Res Toxicol, 1998 Jan, 11(1), 70 - 4 Metabolic activation of aromatic amine mutagens by simultaneous expression of human cytochrome P450 1A2, NADPH-cytochrome P450 reductase, and N-acetyltransferase in Escherichia coli; Josephy PD et al.; We describe the construction of a new strain of Escherichia coli designed to bioactivate aromatic amines and to detect their mutagenicity with high sensitivity . Strain DJ4309 bears two plasmids, a pACYC184-derived plasmid which expresses Salmonella typhimurium acetyl CoA:arylamine N-acetyltransferase (NAT) and a pBR322-derived plasmid which expresses human cytochrome P450 1A2 and NADPH-cytochrome P450 reductase . The combined actions of these enzymes convert aromatic amines into reactive, mutagenic N-acetoxy esters . The strain also carries a mutated copy of the lacZ gene (on an F' factor) which reverts to the wild-type gene by a -(GpC) frameshift mutation . Strain DJ4309 expresses high levels of NAT and cytochrome P450 1A2 and is very sensitive to mutagenesis induced by representative aromatic amines . Mutagenicity of 2-aminoanthracene in strain DJ4309 is higher than can be obtained by rat liver homogenate 9000g supernatant (S9) activation in the parent strain lacking the P450 expression vector . Strain DJ4309 provides a useful system for detecting mutagenic aromatic amines and for studying their metabolism by human P450 1A2. Pharmazie, 1998 Jan, 53(1), 60 - 2 Pharmacodynamic parameters of gentamicin and their effect on biological properties of gram-negative bacteria; Majtanova L et al.; The in vitro postantibiotic effect (PAE) and the postantibiotic effect of subinhibitory concentrations (PA SME) of gentamicin were investigated on clinical isolates of Salmonella typhimurium, Salmonella enteritidis and Pseudomonas aeruginosa . The PAE was induced by 2 x MIC and 4 x MIC of gentamicin for 0.5 h . The PA SME were studied by the addition of 0.1, 0.2 and 0.3 x MIC during the postantibiotic phase of the bacteria . The S . enteritidis strain did no regrow after affecting of supra-subinhibitory concentrations for 24 h with exception of the concentration 2 x MIC + 0.1 x MIC . The PAEs against P . aeruginosa were nearly identical for all the suprainhibitory concentrations tested (4.4-4.6 h) and no regrowth after PA SME was observed . The studied pharmacodynamic parameters decreased the surface hydrophobicity of Salmonella sp., mainly of S . enteritidis, evaluated by the ability to bind Congo red and by the aggregation in solutions of ammonium sulphate (SAT) . Gentamicin in suprainhibitory concentrations expressively decreased the phospholipase C production--an important virulence factor of P . aeruginosa. J Bacteriol, 1998 Feb, 180(4), 885 - 91 The apbE gene encodes a lipoprotein involved in thiamine synthesis in Salmonella typhimurium; Beck BJ et al.; Thiamine pyrophosphate is an essential cofactor that is synthesized de novo in Salmonella typhimurium . The biochemical steps and gene products involved in the conversion of aminoimidazole ribotide (AIR), a purine intermediate, to the 4-amino-5-hydroxymethyl-2-methyl pyrimidine (HMP) moiety of thiamine have yet to be elucidated . We have isolated mutations in a new locus (Escherichia coli open reading frame designation yojK) at 49 min on the S . typhimurium chromosome . Two significant phenotypes associated with lesions in this locus (apbE) were identified . First, apbE purF double mutants require thiamine, specifically the HMP moiety . Second, in the presence of adenine, apbE single mutants require thiamine, specifically both the HMP and the thiazole moieties . Together, the phenotypes associated with apbE mutants suggest that flux through the purine pathway has a role in regulating synthesis of the thiazole moiety of thiamine and are consistent with ApbE being involved in the conversion of AIR to HMP . The product of the apbE gene was found to be a 36-kDa membrane-associated lipoprotein, making it the second membrane protein implicated in thiamine synthesis. J Bacteriol, 1998 Feb, 180(4), 840 - 5 tRNA(Arg) (fimU) and expression of SEF14 and SEF21 in Salmonella enteritidis; Clouthier SC et al.; A Tn10 insertion affecting SEF14 fimbrial synthesis in Salmonella enteritidis was located 13 bp upstream of a gene designated fimU . The 77-bp DNA sequence of fimU from S . enteritidis was identical to that of fimU encoding tRNA(Arg) (UCU) from Salmonella typhimurium and 96% identical to that of the Escherichia coli argU homolog . Furthermore, the open reading frame adjacent to and overlapping the 3' end of fimU was similar to the prophage DLP12 integrase gene . The fimU-encoded transcript comigrated with total cellular tRNA and was predicted to form a tRNA-like cloverleaf structure containing the arginine anticodon UCU . Thus, fimU encoded a tRNA(Arg) specific for the rare codon AGA . fimU mapped to the SEF21 fim operon located 15 C's from the sef14 gene cluster . Although fimU was located within the SEF21 fim gene cluster, the fimU Tn10 insertion mutant of S . enteritidis was found to be defective in SEF14 as well as SEF21 (type 1) fimbria production . SEF17 and SEF18 fimbria production was not affected . Complementation of this mutant with plasmid-borne fimU restored normal production of the fimbrins SefA and FimA as well as their respective fimbriae SEF14 and SEF21 . This is the first description of tRNA simultaneously controlling the production of two distinct fimbriae. Aliment Pharmacol Ther, 1997 Dec, 11 Suppl 3, 57 - 62; discussion 62-3 Review article: Pathobiology of neutrophil interactions with intestinal epithelia; Madara JL; Neutrophil-epithelial interactions were modelled using polarized T84 cells and ligands were identified through observations of beta2-integrin dependence in patients with chronic granulomatious disease . Interactions between neutrophils and the apical membrane of crypt cells were analysed using HPLC and an in vitro model with T84 monolayers colonized by Salmonella typhimurium was used to assess neutrophil movement across the epithelium . The decline in transepithelial resistance following movement of neutrophils across the epithelial monolayer may have been due to an interaction between neutrophils and ligand ICAM-1 in which the neutrophils move along the paracellular pathway of epithelial cells . Cell surface polarity may influence these neutrophil-epithelial interactions which influence Cl secretion . These studies revealed that only strains produced in vivo were able to induce neutrophil transmigration in the in vitro model and may be indicative of new progressive therapies for inflammatory bowel disease. Mutat Res, 1997 Dec 12, 395(2-3), 223 - 7 Base-pair mutation caused by four nitro-group containing aromatic amines in Salmonella typhimurium TA100, TA104, TA4001 and TA4006; Chen SC et al.; Among p-phenylenediamine, benzidine and the analogues we previously tested, only the nitro-group containing 2-nitro-p-phenylenediamine, 3-nitro-o-phenylenediamine, 4-nitro-o-phenylenediamine and 4,4'-dinitro-2-biphenylamine caused base-pair reversion in the histidine locus of Salmonella typhimurium TA100 . In order to determine the types of mutations involved, such as transversion or transition, these four nitro-group containing compounds were tested with S . typhimurium strains TA100, TA104, TA4001 and TA4006 . Dose-mutagenicity relationships occurred with TA100 and TA104 . However, the majority of revertants from TA100 and TA104 were insensitive to inhibition by histidine analogue, DL-1,2,4-triazole-3-alanine . These results suggested that the occurrence of histidine revertants was predominantly induced by base-pair (point) mutations and not by suppressor gene mutations . The CG-TA transition and CG-AT transversion are the major types of mutation induced by all these compounds in TA100 . The TA-AT transversion also contributed to the mutagenicity of 4-nitro-o-phenylenediamine and 4,4'-dinitro-2-biphenylamine in TA104 . These nitro-group containing compounds showed no mutagenicity in TA4001, but induced CG-GC transversion in TA4006. Mutat Res, 1997 Dec 12, 395(2-3), 159 - 71 Dialkylquinoneimine metabolites of chloroacetanilide herbicides induce sister chromatid exchanges in cultured human lymphocytes; Hill AB et al.; Some of the most widely-used herbicides are the chloroacetanilides exemplified by alachlor and butachlor (derived from 2,6-diethylaniline) and metolachlor and acetochlor (synthesized from 2-ethyl-6-methylaniline) . This investigation tests the hypothesis that the previously-observed oncogenicity of these herbicides is due to genotoxic intermediates such as diethylbenzoquinoneimine, a purported alachlor metabolite . Syntheses are reported here for the corresponding 2,6-dialkylbenzoquinoneimines, selected chloroacetyldialkylbenzoquinoneimines and several other candidate or known metabolites . The possible mutagenicity of diethylbenzoquinoneimine was tested in Salmonella typhimurium strains TA98 and TA100 with a weakly-positive response in the TA100 strain indicating induction of base-pair substitution mutations . The frequency of sister chromatid exchange (SCE) in Chinese hamster ovary cells was increased by alachlor at 10 microM and diethylaniline but not ethylmethylaniline at 30 and 3 microM . Isolated and cultured peripheral lymphocytes (mostly T cells) were used from two human donors to study the effects of the chloroacetanilides and their metabolites on primary human cells . In tests at 10 microM, the SCE frequency was increased by alachlor and possibly acetochlor but not by butachlor, metolachlor, dimethachlor (a 2,6-dimethyl analog) and dimethenamid (an analog based on 2,4-dimethyl-3-thienylamine) . At 0.3 microM in cultured human lymphocytes, alachlor, the corresponding chloroacetanilide (N-dealkyl-alachlor) and aniline metabolites (and their 4-hydroxy derivatives), and diethylbenzoquinone were inactive or active in only one of the two donors whereas at 0.1-0.3 microM the SCE ratio for treated cells divided by the controls was always higher for diethylbenzoquinoneimine than for ethylmethyl- and dimethylbenzoquinoneimines . All the tested compounds were toxic to lymphocytes, but the depression of the mitotic index and increased duration of the cell cycle were not directly linked with SCE induction . Previous investigations have suggested that chloroacetanilide herbicides such as alachlor derived from 2,6-dialkylanilines are metabolized to 2,6-dialkylbenzoquinoneimines and the present study provides the first direct evidence that these metabolites are genotoxic in human lymphocytes. Mutat Res, 1997 Dec 5, 395(1), 47 - 56 DNA strand-breaking activity and mutagenicity of 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP), a Maillard reaction product of glucose and glycine; Hiramoto K et al.; Aqueous solution of glucose and glycine was heated under reflux for 4 h and extracted with ethyl acetate . Reversed phase HPLC of the extract revealed a new DNA strand-breaking substance, which was purified by repeated HPLC and identified as 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP) . DDMP induced DNA strand breaking in a dose- and time-dependent manner . It was active to break DNA strands at pH 7.4 and 9.4 . Its pyranone skeleton was destroyed at the pH values . DNA strand breaking by DDMP was inhibited by superoxide dismutase, catalase, scavengers for hydroxyl radical, spin trapping agents and metal chelators, and the breaking was enhanced by Fe(III) ion . A mixture of DDMP and a spin trap DMPO gave electron spin resonance signals of a spin adduct DMPO-OH, indicating generation of hydroxyl radical . DDMP was found to be mutagenic to Salmonella typhimurium TA100 without metabolic activation . These results show DDMP generated active oxygen species to cause DNA strand breaking and mutagenesis. Mutat Res, 1997 Dec 5, 395(1), 1 - 7 Co-mutagenicity of glyco- and tauro-deoxycholic acids in the Ames test; Shibuya N et al.; Mutagenicity and co-mutagenicity of glyco- and tauro-deoxycholic acids (GDCA and TDCA), which are abundant in human bile, were examined by the Ames test . The two chemicals were not mutagenic for themselves to Salmonella typhimurium TA98 and TA100, with and without S9 mix . They enhanced, however, the mutagenic activities of the pro-mutagens, 2-aminoanthracene (2AA) and benzo{a}pyrene (BaP), for both TA98 and TA100 with S9 mix . They were more co-mutagenic for the pro-mutagens on TA98 than on TA100 . On TA98, the mutagenic activities of 2AA with GDCA (5 mumol/plate) and with TDCA (5 mumol/plate) were 9.7-fold and 11.8-fold as high as that of the corresponding control (2AA only), respectively . BaP with GDCA (2.5 mumol/plate) and with TDCA (2.5 mumol/plate) showed 2.8-fold and 3.0-fold increases over the corresponding control level (BaP only), respectively . It is hence concluded that GDCA and TDCA may enhance the activity of some mutagens existing in bile. Eur J Immunol, 1997 Dec, 27(12), 3456 - 60 Bacterial invasion induces interleukin-7 receptor expression in colonic epithelial cell line, T84; Yamada K et al.; The intestinal epithelial layer forms the interface between the external and the internal environments of a host . Since the interleukin-7 (IL-7) and IL-7 receptor (IL-7R) signaling pathway has been shown to play an important role in the mucosal immune system, we studied the expression of IL-7R in T84, a colonic epithelial cell line, after cells were infected with several types of enteropathogenic bacteria, including Salmonella typhimurium, enteropathogenic Escherichia coli and enteroinvasive Escherichia coli . Bacterial invasion induced IL-7R expression in T84 assessed by a semi-quantitative reverse transcription-polymerase chain reaction technique and flow cytometry . The inhibition of bacterial invasion by cytochalasin D, a specific inhibitor of actin polymerization, led to a reduction in the expression of IL-7R . These data indicate that bacterial invasion into intestinal epithelial cells is likely to be an essential process in the induction of IL-7R . The communication between the epithelium and mucosal lymphocytes which is mediated via IL-7 and IL-7R may be involved in the modulation of the mucosal inflammation which occurs in bacterial infection. J Clin Invest, 1998 Jan 1, 101(1), 263 - 72 Expression of arthritis-causing HLA-B27 on Hela cells promotes induction of c-fos in response to in vitro invasion by Salmonella typhimurium; Ikawa T et al.; HLA-B27 confers a very strong genetic predisposition to development of a reactive arthritis after infection by bacteria such as Salmonella typhimurium . This study examines the role of HLA-B27 in the initiation of the earliest host activities after exposure to Salmonella, namely activation of the immediate early genes in the epithelial cells . Our major finding is that in Hela cells, the expression of c-fos was induced by Salmonella invasion only when the cells expressed the transfected HLA-B27 gene, but not the HLA-A1 gene or a truncated HLA-B27 gene lacking the exons encoding the cytoplasmic domain . C-fos is potentially capable of complexing with members of the c-jun family to become the AP-1 transcription complex . Parallel to c-fos expression, we found that only with the HLA-B27 transfectant was there expression of AP-1 . AP-1 potentially controls the expression of a large number of genes . On screening a panel of proinflammatory molecules, we found that Salmonella invasion induced expression of monocyte chemoattractant protein-1 in the HLA-B27 cells . Since each of these separate positive findings belong to the same cascade of events after cell activation, together they reinforce the hypothesis that HLA-B27 plays a modulatory role in the early signal transduction events induced by Salmonella invasion . This hypothesis adds another item to the list of allele-specific activities carried out by HLA class I molecules . If similar activation also occurs in the joints, it may play a major role in arthritis. Zh Mikrobiol Epidemiol Immunobiol, 1997 Sep-Oct, (5), 120 - 3 {The immunosuppressive action of different fractions of Salmonella typhimurium lipopolysaccharide}; Borisova EV et al.; The suppressing activity of lipopolysaccharide (LPS) of the filtrates of genetically related S . typhimurium 415 and 415/307 cultures with respect to delayed hypersensitivity to nonbacterial antigen in mice was studied . In LPS of the virulent strain two immunosuppressing fractions differing in their molecular weight were detected by the method of gel filtration . One of them was detected in LPS of the genetically related nonplasmid clone of S . typhimurium 415/307 . The possibility of the chemical inactivation and reactivation of the immunosuppressing properties of LPS was shown. Proc Natl Acad Sci U S A, 1997 Dec 9, 94(25), 13997 - 4001 Periplasmic superoxide dismutase protects Salmonella from products of phagocyte NADPH-oxidase and nitric oxide synthase; De Groote MA et al.; Superoxide dismutase (SOD) catalyzes the conversion of superoxide radical to hydrogen peroxide . Periplasmic localization of bacterial Cu,Zn-SOD has suggested a role of this enzyme in defense against extracellular phagocyte-derived reactive oxygen species . Sequence analysis of regions flanking the Salmonella typhimurium sodC gene encoding Cu,Zn-SOD demonstrates significant homology to lambda phage proteins, reflecting possible bacteriophage-mediated horizontal gene transfer of this determinant among pathogenic bacteria . Salmonella deficient in Cu,Zn-SOD has reduced survival in macrophages and attenuated virulence in mice, which can be restored by abrogation of either the phagocyte respiratory burst or inducible nitric oxide synthase . Moreover, a sodC mutant is extremely susceptible to the combination of superoxide and nitric oxide . These observations suggest that SOD protects periplasmic or inner membrane targets by diverting superoxide and limiting peroxynitrite formation, and they demonstrate the ability of the respiratory burst and nitric oxide synthase to synergistically kill microbial pathogens in vivo. Chem Res Toxicol, 1997 Oct, 10(10), 1180 - 5 Identification of adenine adducts formed in reaction of calf thymus DNA with mutagenic chlorohydroxyfuranones found in drinking water; Le Curieux F et al.; Calf thymus DNA was reacted with the extremely potent bacterial mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) and the structurally related compounds 3,4-dichloro-5-hydroxy-2(5H)-furanone (MCA) and 3-chloro-4-methyl-5-hydroxy-2(5H)-furanone (MCF) . The chromatograms of the HPLC analyses of the DNA hydrolysates showed peaks that represented adducted base moieties . It was possible to establish the structures of the adducts by comparing UV spectra and chromatographical properties of the DNA adducts with known adenosine and 2'-deoxyadenosine adducts . The DNA adduct produced by MX was identified as 3-(2'-deoxyribofuranosyl-N6-adenosinyl)propenal (M1A-dR) . It was calculated that 1 nucleotide/10(5) nucleotides was converted to M1A-dR . The same adduct was formed also in the reaction of MX with 2'-deoxyadenosine (yield 0.01%) . The M1A-dR adduct may play a role in the mutational events induced by MX in Salmonella typhimurium strain TP2428 . The adducts produced in the reactions of MCA and MCF with DNA were identified as 3-(2'-deoxyribofuranosyl)-7-formylimidazo{2,1-i}purine (epsilon cA-dR) and 4-(2'-deoxyribofuranosyl-N6-adenosinyl)-3-formyl-3-butenoic acid (fbaA-dR), respectively . The yield of epsilon cA-dR was 5 adducts/ 10(6) nucleotides and of fbaA-dR 4 adducts/10(5) nucleotides . The biological significance of these adducts is unknown. Chem Res Toxicol, 1997 Oct, 10(10), 1061 - 6 Isolation and chemical-structural determination of a novel aromatic amine mutagen in water from the Nishitakase River in Kyoto; Nukaya H et al.; Water samples from the Nishitakase River in Kyoto, Japan, especially taken at sites below sewage plants, show significantly high mutagenicity in the Ames test . In the present study, mutagens in the river water were adsorbed to 24 g of blue rayon, extracted, and separated by HPLC on ODS columns . Five mutagenic compounds (I-V) were isolated, and they accounted for 21%, 17%, 11%, 12%, and 6%, respectively, of the total mutagenicity of the blue rayon-adsorbed materials . With compound I obtained from adsorbate to 24 g of blue rayon as a marker, a large quantity (1.1 mg) of mutagenic compound I was isolated by Sephadex LH-20 column chromatography and HPLC on ODS columns from material adsorbed to 27 kg of blue cotton . X-ray crystal analysis was carried out with the debrominated derivative of compound I . Based on this X-ray crystallography data and the UV, mass, and 1H-NMR spectra of both the derivative and compound I, the structure of compound I was determined to be 2-{2-(acetylamino)-4-{bis(2-methoxyethyl)amino}-5-methoxyphenyl}-5-amino - 7-bromo-4-chloro-2H-benzotriazole (PBTA-1) . PBTA-1 is a newly identified potent mutagen, inducing 1,200,000 revertants of Salmonella typhimurium YG1024 per microgram in the presence of S9 mix. J Bacteriol, 1998 Feb, 180(3), 722 - 31 Curli fibers are highly conserved between Salmonella typhimurium and Escherichia coli with respect to operon structure and regulation; Romling U et al.; Mouse-virulent Salmonella typhimurium strains SR-11 and ATCC 14028-1s express curli fibers, thin aggregative fibers, at ambient temperature on plates as judged by Western blot analysis and electron microscopy . Concomitantly with curli expression, cells develop a rough and dry colony morphology and bind the dye Congo red (called the rdar morphotype) . Cloning and characterization of the two divergently transcribed operons required for curli biogenesis, csgBA(C) and csgDEFG, from S . typhimurium SR-11 revealed the same gene order and flanking genes as in Escherichia coli . The divergence of the curli region between S . typhimurium and E . coli at the nucleotide level is above average (22.4%) . However, a high level of conservation at the protein level, which ranged from 86% amino acid homology for the fiber subunit CsgA to 99% homology for the lipoprotein CsgG, implies functional constraints on the gene products . Consequently, S . typhimurium genes on low-copy-number plasmids were able to complement respective E . coli mutants, although not always to wild-type levels . rpoS and ompR are required for transcriptional activation of (at least) the csgD promoter . The high degree of conservation at the protein level and the identical regulation patterns in E . coli and S . typhimurium suggest similar roles of curli fibers in the same ecological niche in the two species. J Bacteriol, 1998 Feb, 180(3), 626 - 33 A promoter relay mechanism for sequential gene activation; Fang M et al.; The effect of DNA supercoiling on gene expression is dependent not only on specific genes but also on the sequence context of the genes . This position-dependent supercoiling effect on gene activation is best illustrated in the study of the suppression of the leu-500 mutation of the leuABCD operon in a Salmonella typhimurium topA mutant . In this communication, we report a novel promoter relay mechanism whereby several genes are sequentially expressed in a position-dependent manner: the ilvIH promoter (pilvIH) activates a cryptic leuO promoter (pleuO) located between the two divergently arrayed ilvIH and leu-500 promoters . Both the cis-acting pleuO activity and the trans-acting LeuO protein are necessary for subsequent activation of the leu-500 promoter (pleu-500) . Furthermore, pleuO can be functionally replaced with the inducible tac promoter (ptac) for leu-500 activation, suggesting that transcription-driven DNA supercoiling underlies the relay mechanism . This is the first example of several related genes communicating via a promoter relay mechanism for their coordinated expression. J Bacteriol, 1998 Feb, 180(3), 571 - 7 Genetic analysis, using P22 challenge phage, of the nitrogen activator protein DNA-binding site in the Klebsiella aerogenes put operon; Chen LM et al.; The nac gene product is a LysR regulatory protein required for nitrogen regulation of several operons from Klebsiella aerogenes and Escherichia coli . We used P22 challenge phage carrying the put control region from K . aerogenes to identify the nucleotide residues important for nitrogen assimilation control protein (NAC) binding in vivo . Mutations in an asymmetric 30-bp region prevented DNA binding by NAC . Gel retardation experiments confirmed that NAC specifically binds to this sequence in vitro, but NAC does not bind to the corresponding region from the put operon of Salmonella typhimurium, which is not regulated by NAC. J Bacteriol, 1998 Feb, 180(3), 563 - 70 Alanine catabolism in Klebsiella aerogenes: molecular characterization of the dadAB operon and its regulation by the nitrogen assimilation control protein; Janes BK et al.; Klebsiella aerogenes strains with reduced levels of D-amino acid dehydrogenase not only fail to use alanine as a growth substrate but also become sensitive to alanine in minimal media supplemented with glucose and ammonium . The inability of these mutant strains to catabolize the alanine provided in the medium interferes with both pathways of glutamate production . Alanine derepresses the nitrogen regulatory system (Ntr), which in turn represses glutamate dehydrogenase, one pathway of glutamate production . Alanine also inhibits the enzyme glutamine synthetase, the first enzyme in the other pathway of glutamate production . Therefore, in the presence of alanine, strains with mutations in dadA (the gene that codes for a subunit of the dehydrogenase) exhibit a glutamate auxotrophy when ammonium is the sole source of nitrogen . The alanine catabolic operon of Klebsiella aerogenes, dadAB, was cloned, and its DNA sequence was determined . The clone complemented the alanine defects of dadA strains . The operon has a high similarity to the dadAB operon of Salmonella typhimurium and the dadAX operon of Escherichia coli, each of which codes for the smaller subunit of D-amino acid dehydrogenase and the catabolic alanine racemase . Unlike the cases for E . coli and S . typhimurium, the dad operon of K . aerogenes is activated by the Ntr system, mediated in this case by the nitrogen assimilation control protein (NAC) . A sequence matching the DNA consensus for NAC-binding sites is located centered at position -44 with respect to the start of transcription . The promoter of this operon also contains consensus binding sites for the catabolite activator protein and the leucine-responsive regulatory protein. Avian Dis, 1997 Oct-Dec, 41(4), 783 - 91 Efficacy of a live avirulent Salmonella typhimurium vaccine in preventing colonization and invasion of laying hens by Salmonella typhimurium and Salmonella enteritidis; Hassan JO et al.; An avirulent live delta cya delta crp Salmonella typhimurium strain chi 3985 that precludes colonization and invasion of chickens by homologous and heterologous Salmonella serotypes was evaluated for its long-term protection efficacy . Chickens vaccinated orally at 2 and 4 wk of age were assessed for protection against oral challenge with wild-type S . typhimurium and Salmonella enteritidis strains at 3, 6, 9, and 12 mo of age . A comparison of Salmonella isolation from vaccinated and nonvaccinated layers after challenge with S . typhimurium or S . enteritidis showed that delta cya delta crp S . typhimurium chi 3985 induced excellent protection against intestinal, visceral, reproductive tract, and egg colonization, invasion, and/or contamination by Salmonella . The duration of protection lasted for 11 mo after vaccination, at which time the experiment was terminated . S . enteritidis and S . typhimurium were isolated from the yolk, albumen, and shells of eggs laid by nonvaccinated chickens challenged with Salmonella . S . typhimurium caused pathological lesions in nonvaccinated chickens, whereas vaccinated and nonvaccinated chickens challenged with S . enteritidis showed no pathological lesion in the visceral and reproductive organs . Vaccination with chi 3985 prevented transmission of S . typhimurium or S . enteritidis into eggs laid by vaccinated layers with no effect on egg production . To our knowledge, this is the first publication confirming that vaccination with live avirulent Salmonella can induce long-term protection against Salmonella infection in layers. Infect Immun, 1998 Feb, 66(2), 856 - 61 Cell-specific proteins synthesized by Salmonella typhimurium; Burns-Keliher L et al.; Studies of the proteins synthesized by Salmonella typhimurium during growth within tissue culture cells have previously focused on a single cell type . In the present study we examine the different protein patterns exhibited by S . typhimurium during growth within three different cell types relevant to those it would encounter throughout the course of a natural infection, including intestinal epithelial cells (Intestine-407), macrophages (J774.A, rat bone marrow-derived macrophages, and mouse bone marrow-derived macrophages), and liver cells (NMuLi) . Side-by-side comparisons reveal that S . typhimurium responds to these different cellular environments with specific patterns of protein synthesis unique to each cell type . The numbers of proteins detected in each cell line are as follows: 142 proteins in Intestine-407, of which 58 appear to be unique to growth within this cell line; 413 proteins in J774.A, of which 157 appear to be unique; 260 proteins in rat bone marrow-derived macrophages, of which 40 appear to be unique; 336 proteins in mouse bone marrow-derived macrophages, of which 113 appear to be unique; and 183 proteins in NMuLi, of which 91 appear to be unique. Infect Immun, 1998 Feb, 66(2), 732 - 40 Comparison of the abilities of different attenuated Salmonella typhimurium strains to elicit humoral immune responses against a heterologous antigen; Dunstan SJ et al.; We compared the abilities of different Salmonella enterica var . Typhimurium (S . typhimurium) strains harboring mutations in the genes aroA, aroAD, purA, ompR, htrA, and cya crp to present the heterologous antigen, C fragment of tetanus toxin, to the mouse immune system . Plasmid pTETtac4, encoding C fragment, was transferred into the various S . typhimurium mutants, and the levels of antigen expression were found to be equivalent . After primary oral immunization of BALB/c mice, all attenuated strains were capable of penetrating the gut epithelium and colonizing the Peyer's patches and spleens of mice . Of all strains compared, the delta purA mutant colonized and persisted in the Peyer's patches at the lowest level, whereas the delta htrA mutant colonized and persisted in the spleen at the lowest level . The level of specific antibody elicited by the different strains against either S . typhimurium lipopolysaccharide or tetanus toxoid was strain dependent and did not directly correlate to the mutants' ability to colonize the spleen . The level of immunoglobulin G1 (IgG1) and IgG2a antibody specific for tetanus toxoid was determined in mice immunized with four S . typhimurium mutants . The level of antigen-specific IgG1 and IgG2a was significantly lower in animals immunized with S . typhimurium delta purA . Antigen-specific T-cell proliferation assays indicated a degree of variability in the capacity of some strains to elicit T cells to the heterologous antigen . Cytokine profiles (gamma interferon and interleukin-5) revealed that the four S . typhimurium mutants tested induced a Th1-type immune response . Mice were challenged with a lethal dose of tetanus toxin 96 days after oral immunization . With the exception of the S . typhimurium delta purA mutant, all strains elicited a protective immune response . These data indicate that the level of total Ig specific for the carried antigen, C fragment, does not correlate with the relative invasiveness of the vector, but it is determined by the carrier mutation and the background of the S . typhimurium strain. Infect Immun, 1998 Feb, 66(2), 724 - 31 Inoculum composition and Salmonella pathogenicity island 1 regulate M-cell invasion and epithelial destruction by Salmonella typhimurium; Clark MA et al.; In the mouse model of Salmonella typhimurium infection, the specialized antigen-sampling intestinal M cells are the primary route of Salmonella invasion during the early stages of infection . Under certain experimental conditions, M-cell invasion is accompanied by M-cell destruction and loss of adjacent regions of the follicle-associated epithelium (FAE), although the conditions responsible for expression of the cytotoxic phenotype in a proportion of previous studies have not been defined . In the present study, we have demonstrated that the cytotoxic effect exerted by wild-type S . typhimurium on mouse Peyer's patch FAE is dependent on the inoculum composition . We have also demonstrated that the extent of FAE destruction correlates with the extent of M-cell invasion . Bacteria inoculated in Luria-Bertani (LB) broth induce extensive FAE loss and exhibit efficient M-cell invasion, whereas bacteria inoculated in phosphate-buffered saline fail to induce significant FAE disruption and invade M cells at significantly lower levels . Similarly, inoculation in LB significantly enhances invasion of Madin-Darby canine kidney cells by wild-type S . typhimurium . Mutants defective for expression of invA, a component of Salmonella pathogenicity island 1 which is vital for efficient invasion of cultured cells, fail to induce FAE destruction and, when inoculated in LB, are attenuated for M-cell invasion . Variation in inv gene expression is, therefore, one possible mechanism by which inoculate composition may regulate the virulence of wild-type S . typhimurium . Our findings suggest that the composition of the gut luminal contents may be critical in determining the outcome of naturally acquired Salmonella infections and that both vaccine formulation and dietary status of vaccine recipients may significantly affect the efficacy and safety of live Salmonella oral vaccine delivery systems. Infect Immun, 1998 Feb, 66(2), 581 - 6 Mice are protected from Helicobacter pylori infection by nasal immunization with attenuated Salmonella typhimurium phoPc expressing urease A and B subunits; Corthesy-Theulaz IE et al.; Live Salmonella typhimurium phoPc bacteria were tested as mucosal vaccine vectors to deliver Helicobacter pylori antigens . The genes encoding the A and B subunits of H . pylori urease were introduced into S . typhimurium phoPc and expressed under the control of a constitutive tac promoter (tac-ureAB) or a two-phase T7 expression system (cT7-ureAB) . Both recombinant Salmonella strains expressed the two urease subunits in vitro and were used to nasally immunize BALB/c mice . The plasmid carrying cT7-ureAB was stably inherited by bacteria growing or persisting in the spleen, lungs, mesenteric or cervical lymph nodes, and Peyer's patches of immunized mice, while the plasmid carrying tac-ureAB was rapidly lost . Spleen and Peyer's patch CD4+ lymphocytes from mice immunized with S . typhimurium phopc cT7-ureAB proliferated in vitro in response to urease, whereas cells from mice given S . typhimurium phoPc alone did not . Splenic CD4+ cells from mice immunized with phoPc cT7-ureAB secreted gamma interferon and interleukin 10, while Peyer's patch CD4+ cells did not secrete either cytokine . Specific H . pylori anti-urease immunoglobulin G1 (IgG1) and IgG2A antibodies were detected following immunization, confirming that both Th1- and Th2-type immune responses were generated by the live vaccine . Sixty percent of the mice (9 of 15) immunized with S . typhimurium phoPc cT7-ureAB were found to be resistant to infection by H . pylori, while all mice immunized with phoPc tac-ureAB (15 of 15) or phoPc (15 of 15) were infected . Our data demonstrate that H . pylori urease delivered nasally by using a vaccine strain of S . typhimurium can trigger Th1- and Th2-type responses and induce protective immunity against Helicobacter infection. Carcinogenesis, 1997 Dec, 18(12), 2429 - 33 Mutation and formation of methyl- and hydroxylguanine adducts in DNA caused by N-nitrosodimethylamine and N-nitrosodiethylamine with UVA irradiation; Arimoto-Kobayashi S et al.; Previously we reported that when Escherichia coli was treated with N-nitrosodialkylamine under irradiation with near UV light, mutagenesis of the bacteria took place: there was no requirement for metabolic activation . We have now studied the spectra of mutations caused by N-nitrosodimethylamine (NDMA) and N-nitrosodiethylamine (NDEA) with UVA (320-400 nm) irradiation, using standard tester strains for identifying types of mutations . Induced mutations by NDMA + UVA were the transition GC-->AT and transversions GC-->CG, GC-->TA and AT-->TA . NDEA + UVA induced mainly the GC-->CG transversion . In both cases no frameshift mutations were observed . When O6-alkylguanine-DNA alkyltransferase-deficient strains of E . coli and Salmonella typhimurium were used, the mutation levels with both NDMA + UVA and NDEA + UVA became remarkably higher than those observed with the proficient strains . We measured the O6-methylguanine (O6-meG) level in calf thymus DNA treated with NDMA + UVA . The O6-meG level was increased as a function of NDMA concentration and irradiation time . We also detected N7-methylguanine in DNA treated with NDMA + UVA . In our previous work we found formation of 8-oxodeoxyguanosine (8-oxodG) in DNA treated with N-nitrosomorpholine + UVA . The 8-oxodG/dG ratio in DNA treated with NDMA + UVA increased up to 42-fold over that of the untreated control and that in DNA treated with NDEA + UVA increased up to 67-fold . 8-OxodG formation was not affected by replacing H2O in the reaction mixture with D2O, suggesting that singlet oxygen is not the rate limiting factor in this photoactivation . We conclude that both alkylation and oxidation are involved in mutations induced by NDMA + UVA and NDEA + UVA. Arzneimittelforschung, 1997 Dec, 47(12), 1398 - 402 Organogermanium compounds as inhibitors of the activity of direct acting mutagens in Salmonella typhimurium; Schimmer O et al.; The organogermanium compounds bis(D,L-lactato)germanium(IV), bis(L-lactato)germanium(IV), bis (thiolactato)germanium(IV) and bis(thioglycolato)germanium(IV) were tested for their antimutagenic activity in Salmonella typhimurium strains TA98 and TA100 . Each compound showed moderate activity against the mutagenic effect of nitroaromatic compounds and weak effects against the mutagenic activity of ethylmethane sulfonate . No inhibition of mutagenicity was observed against the indirect acting promutagens benzo(a)pyrene and 2-aminoanthracene . The compounds differed only quantitatively in their antimutagenicity spectrum . It is concluded from these results that an intracellular mechanism is involved in the inhibition of ethylmethane sulfonate-induced mutagenicity . The effect is probably produced, at least partially, at the level of DNA repair . Frameshift mutations seem to be prevented with higher efficiency than base pair substitutions. J Appl Microbiol, 1997 Dec, 83(6), 699 - 706 Determination of clonal relationships of Salmonella typhimurium by numerical analysis of macrorestriction profiles; On SL et al.; The use of numerical analysis of macrorestriction profiles resolved by pulsed-field gel electrophoresis (PFGE) for identifying epidemiological and clonal relationships between strains of Salmonella typhimurium was investigated . A cluster analysis based on Xba1 macrorestriction profiles of 40 strains was compared to biotype, phage-type and ribotype data on each isolate to validate the integrity and significance of the results . A significant correlation between these data was noted, suggesting that evolutionary divergence, of both recent and a more distant nature, may be detected . Moreover, two principal clonal lines of Salm . typhimurium appear to be prevalent in Denmark, of which one is comparatively stable with respect to phenotype and genotype, while the other is relatively diverse in these respects . We conclude that numerical analysis of macrorestriction profiles is a useful means of evaluating interstrain relationships between Salm . typhimurium strains and has practical applications for both short- and long-term epidermiological surveillance purposes. Folia Microbiol (Praha), 1997, 42(4), 401 - 2 Mutagenicity of 4-nitroquinoline n-oxide after its complexation with copper (II) 2-chlorophenoxyacetate; Blahova M et al.; Using the AMes test with Salmonella typhimurium strains TA97, TA100 and TA102, it was found that the mutagenicity of 4-nitroquinoline-N-oxide was much lower than that of the tetrakis{mu-2-chlorophenoxyacetato(1-)-O,O{bis(4-nitro-quinoline- N-oxide-O)dicopper(II) complex. Folia Microbiol (Praha), 1997, 42(4), 327 - 32 Postantibiotic effects and postantibiotic sub-MIC effects of ciprofloxacin, pefloxacin and amikacin on the biological properties of Salmonella strains; Majtan V et al.; The postantibiotic effect (PAE) and the postantibiotic sub-MIC effect (PASME) of ciprofloxacin, pefloxacin and amikacin were studied for Salmonella typhimurium and S . enteritidis strains . PAE was induced by 2 x and 4 x MIC of antibiotics studied for 0.5 h . After PAE and PASME their effect on prophage induction of a lysogenic S . typhimurium strain and on Congo red binding for both strains as a marker of their surface hydrophobicity was examined . The longest PAE was found after treatment with ciprofloxacin, higher values being observed with S . typhimurium . PAEs of pefloxacin and amikacin were much lower, except for the suprainhibitory concentration 4 x MIC of amikacin with S . enteritidis (6.9h) . PASMEs of ciprofloxacin did not allow any regrowth of either strain . For other antibiotics the PASMEs were different while concentrations of 2 x MIC + 0.2 x MIC and 0.3 x MIC, and of 4 x MIC + 0.1 x MIC, 0.2 x MIC and 0.3 x MIC of amikacin did not allow any regrowth of S . enteritidis . PAEs of the antibiotics tested did not affect the Congo red binding by both Salmonella strains, but the PAEs of ciprofloxacin and pefloxacin expressively induced a prophage of lysogenic S . typhimurium strain . We noted the influence of Congo red binding after applying 4 x MIC + 0.1 x MIC, 0.2 x MIC and 0.3 x MIC of amikacin for S . typhimurium and 2 x MIC + 0.1 x MIC for S . enteritidis. Biophys J, 1998 Jan, 74(1), 436 - 43 Kinetic analysis of the growth rate of the flagellar hook in Salmonella typhimurium by the population balance method; Koroyasu S et al.; The growth rate of flagellar hooks in Salmonella typhimurium was analyzed by computer-aided simulation of the length distributions of mutant hooks of uncontrolled length (polyhooks) . The wild-type hook has a relatively well-controlled length, with an average of 55 nm and a standard deviation of 6 nm . Mutations in the fliK gene give rise to polyhooks . A histogram of the lengths of polyhooks from a fliK mutant shows a peak at 55 nm with a long monotonic tail extending out to 1 microm . To analyze the growth rate, we employed the population balance method . Regression analysis showed that the histogram could fit a combination of two theoretical curves . In the first phase of growth, the hook starts with a very fast growth rate (40 nm/min), and then the rate exponentially slows until the length reaches 55 nm . In the second phase of growth, where the hook length is over 55 nm, the hook grows at a constant rate of 8 nm/min . Second mutations in either the fliK or flhB genes, as found in pseudorevertants from fliK mutants, give rise to polyhook filaments (phf) . The ratio between the numbers of hooks with and without filament was 6:4 . The calculated probability of filament attachment to polyhooks was low so that the proportion of hooks that start filament growth was only 2% per minute . The lengths of polyhooks with and without filaments were measured . A histogram of hook length in phf's was the same as that for polyhooks in single-site fliK mutants, against the expectation that the distribution would shift to a shorter average . The role of FliK in hook length control is discussed. Antimicrob Agents Chemother, 1998 Jan, 42(1), 45 - 52 Antibacterial efficacy against an in vivo Salmonella typhimurium infection model and pharmacokinetics of a liposomal ciprofloxacin formulation; Webb MS et al.; The fluoroquinolone antibiotic ciprofloxacin has been encapsulated into large unilamellar vesicles (LUV) at efficiencies approaching 100% . Drug accumulation proceeded in response to a transmembrane gradient of methylammonium sulfate and occurred concomitantly with the efflux of methylamine . A mechanism for the encapsulation process is described . LUV composed of dipalmitoylphosphatidylcholine-cholesterol (DPPC/chol), distearoylphosphatidylcholine-cholesterol (DSPC/chol), or sphingomyelin-cholesterol (SM/chol) increased the circulation lifetime of ciprofloxacin after intravenous (i.v.) administration by > 15-fold . The retention of ciprofloxacin in liposomes in the circulation decreased in the sequence SM/chol > DSPC/chol > DPPC/chol . Increased circulation lifetimes were associated with enhanced delivery of the drug to the livers, spleens, kidneys, and lungs of mice . Encapsulation of ciprofloxacin also conferred significant increases in the longevity of the drug in the plasma after intraperitoneal administration and in the lungs after intratracheal administration in comparison to free ciprofloxacin . The efficacy of a single i.v . administration of an SM/chol formulation of ciprofloxacin was measured in a Salmonella typhimurium infection model . At 20 mg of ciprofloxacin per kg of body weight, the encapsulated formulation resulted in 10(3)- to 10(4)-fold fewer viable bacteria in the livers and spleens of infected mice than was observed for animals treated with free ciprofloxacin . These results show the utility of liposomal encapsulation of ciprofloxacin in improving the pharmacokinetics, biodistribution, and antibacterial efficacy of the antibiotic . In addition, these formulations are well suited for i.v., intraperitoneal, and intratracheal or aerosol administration. Genetika, 1997 Sep, 33(9), 1310 - 2 {Genotoxic effects of tonarol}; Karamova NS et al.; Genotoxic effects of 2,6-di-tret-butyl-4-methylphenol (tonarol) were studied using four test systems: the Ames test, the SOS chromotest, the cytogenetic test with rootlets of onion (Allium cepa), and the in vivo micronucleus test . Tonarol did not affect gene mutation induction in Salmonella typhimurium tester strains, the SOS response in the Escherichia coli strain PQ37, chromosomal aberrations in cells of onion (Allium cepa) rootlets, and micronuclei in erythrocytes of peripheral blood of CBA x C5713 L/G mice . Tonarol induced cell division in A. Genetika, 1997 Sep, 33(9), 1215 - 20 {Analysis of the dependence of the frequency of emergence of His+-reversions in Salmonella typhimurium on the density of the cell population}; Babynin EV et al.; The dependence of the appearance of mutants on the number of viable cells plated on a selective medium was analyzed with the spontaneous His+ reversion system in the histidine auxotroph of Salmonella typhimurium strain BA13 . The frequency of spontaneous His+ revertants was shown to be inversely proportional to the number of plated cells . Evaluation of residual culture growth on a histidine-deficient medium suggests that this factor cannot be the reason behind the inverse dependence . The results of experiments involving previously added His+ revertants showed that the inverse relationship between mutant frequency and population density is not connected with the inhibition of preadaptive revertant growth by nonmutant cells . Moreover, an inhibitory effect of the culture medium on the frequency of spontaneous His+ revertants upon histidine starvation was detected . On the basis of these results, it was assumed that the His+ reversion generated by histidine starvation is suppressed by metabolites of starved cells. Acta Pol Pharm, 1996 Sep-Oct, 53(5), 357 - 9 Characteristics of mutagenesis by bleomycin and adriamycin in Salmonella typhimurium: action of catalase; Ejchart A et al.; The influence of catalase activity in adriamycin and bleomycin mutagenesis was investigated in Salmonella typhimurium TA98 and TA102, respectively . The activity of catalase in bacterial cells was inhibited by sodium azide . Mutagenicity of both drugs was not changed in bacterial cells with depressed catalase activity. J Biol Chem, 1997 Dec 26, 272(52), 32878 - 88 Cysteine and disulfide scanning reveals a regulatory alpha-helix in the cytoplasmic domain of the aspartate receptor; Danielson MA et al.; The transmembrane, homodimeric aspartate receptor of Escherichia coli and Salmonella typhimurium controls the chemotactic response to aspartate, an attractant, by regulating the activity of a cytoplasmic histidine kinase . The cytoplasmic domain of the receptor plays a central role in both kinase regulation and sensory adaptation, although its structure and regulatory mechanisms are unknown . The present study utilizes cysteine and disulfide scanning to probe residues Leu-250 through Gln-309, a region that contains the first of two adaptive methylation segments within the cytoplasmic domain . Following the introduction of consecutive cysteine residues by scanning mutagenesis, the measurement of sulfhydryl chemical reactivities reveals an alpha-helical pattern of exposed and buried positions spanning residues 270-309 . This detected helix, termed the "first methylation helix," is strongly amphiphilic; its exposed face is highly anionic and possesses three methylation sites, while its buried face is hydrophobic . In vivo and in vitro assays of receptor function indicate that inhibitory cysteine substitutions are most prevalent on the buried face of the first methylation helix, demonstrating that this face is involved in a critical packing interaction . The buried face is further analyzed by disulfide scanning, which reveals three "lock-on" disulfides that covalently trap the receptor in its kinase-activating state . Each of the lock-on disulfides cross-links the buried faces of the two symmetric first methylation helices of the dimer, placing these helices in direct contact at the subunit interface . Comparative sequence analysis of 56 related receptors suggests that the identified helix is a conserved feature of this large receptor family, wherein it is likely to play a general role in adaptation and kinase regulation . Interestingly, the rapid rates and promiscuous nature of disulfide formation reactions within the scanned region reveal that the cytoplasmic domain of the full-length, membrane-bound receptor has a highly dynamic structure . Overall, the results demonstrate that cysteine and disulfide scanning can identify secondary structure elements and functionally important packing interfaces, even in proteins that are inaccessible to other structural methods. J Biol Chem, 1998 Jan 16, 273(3), 1727 - 32 Overexpression of the Saccharomyces cerevisiae magnesium transport system confers resistance to aluminum ion; MacDiarmid CW et al.; Ionic aluminum (Al3+) is toxic to plants, microbes, fish, and animals, but the mechanism of its toxicity is unknown . We describe the isolation of two yeast genes (ALR1 and ALR2) which confer increased tolerance to Al3+ and Ga3+ ions when overexpressed while increasing strain sensitivity to Zn2+, Mn2+, Ni2+, Cu2+, Ca2+, and La3+ ions . The Alr proteins are homologous to the Salmonella typhimurium CorA protein, a bacterial Mg2+ and Co2+ transport system located in the periplasmic membrane . Yeast strains lacking ALR gene activity required additional Mg2+ for growth, and expression of either ALR1 or ALR2 corrected the Mg(2+)-requiring phenotype . The results suggest that the ALR genes encode the yeast uptake system for Mg2+ and other divalent cations . This hypothesis was supported by evidence that 57Co2+ accumulation was elevated in ALR-overexpressing strains and reduced in strains lacking ALR expression . ALR overexpression also overcame the inhibition of Co2+ uptake by Al3+ ions . The results indicate that aluminum toxicity to yeast occurs as a consequence of reduced Mg2+ influx via the Alr proteins . The molecular identification of the yeast Mg2+ transport system should lead to a better understanding of the regulation of Mg2+ homeostasis in eukaryote cells. J Bacteriol, 1998 Jan, 180(2), 303 - 16 Differential regulation of multiple flagellins in Vibrio cholerae; Klose KE et al.; Vibrio cholerae, the causative agent of the human diarrheal disease cholera, is a motile bacterium with a single polar flagellum . Motility has been implicated as a virulence determinant in some animal models of cholera, but the relationship between motility and virulence has not yet been clearly defined . We have begun to define the regulatory circuitry controlling motility . We have identified five V . cholerae flagellin genes, arranged in two chromosomal loci, flaAC and flaEDB; all