Indian Pediatr, 1995 Aug, 32(8), 881 - 5 An outbreak of multidrug resistant . Salmonella typhimurium in a nursery; Kumar A et al.; A nursery epidemic caused by multidrug resistant Salmonella typhimurium is reported . In total, 21 infants developed symptomatic illness; of these, 17 had septicemia (7 blood culture positive) and 4 had diarrhea alone . Asymptomatic carrier state was identified in 13 infants . Male sex and birth asphyxia increased the risk for symptomatic illness . Fever, lethargy, and diarrhea were the most common clinical features . Amongst the septicemic infants there was no difference in clinical profile whether the blood culture was positive or negative for S . typhimurium . In the symptomatic group, S . typhimurium was isolated from feces in 19 cases and from blood in 7 cases . In both symptomatic and asymptomatic infants, all isolates of S . typhimurium, whether obtained from feces and/or from blood, were resistant to ampicillin, chloramphenicol, and trimethoprim, and a significant number (almost one-fifth) of them also showed resistance to third generation cephalosporins . More than 90% of isolates were sensitive to aminoglycosides and ciprofloxacin . On a combination of third generation cephalosporin (cefotaxime or ceftriaxone) and amikacin, 17 (81%) infants recovered, 2 succumbed to their illness, and 2 failed to improve and required ciprofloxacin . The origin of epidemic was traced to a carrier staff nurse working in nursery. Mol Microbiol, 1995 Aug, 17(3), 523 - 31 Salmonella typhimurium responses to a bactericidal protein from human neutrophils; Qi SY et al.; Bactericidal/permeability-increasing protein {BPI} is a cationic antimicrobial protein from neutrophils that specifically binds to the surfaces of Gram-negative bacteria via the lipid A component of lipopolysaccharide . To obtain information about the responses of Salmonella typhimurium to cell-surface damage by BPI, two-dimensional gel electrophoresis and N-terminal microsequencing were used to identify proteins that were induced or repressed following BPI treatment . The majority of the affected proteins are involved in central metabolic processes . Upon addition of BPI, the beta-subunit of the F1 portion of Escherichia coli ATP synthase was repressed threefold whereas six proteins were induced up to 11-fold . Three of the latter were identified as lipoamide dehydrogenase, enoyl-acyl carrier protein reductase, and the heat-shock protein HtpG . Additionally, a novel protein, BipA, was identified that is induced over sevenfold by BPI; sequence analysis suggests that it belongs to the GTPase superfamily and interacts with ribosomes . A conserved direct-repeat motif is present in the regulatory regions of several BPI-inducible genes, including the bipA gene . Only one of the BPI-responsive proteins was induced when cells were treated with polymyxin B, which also binds to lipid A . We therefore conclude that BPI and polymyxin B affect different global regulatory networks in S . typhimurium even though they bind with high affinity to the same cell-surface component. Cent Eur J Public Health, 1995 Aug, 3(3), 161 - 2 Salmonella phage types distribution in the Czech Republic in 1991-1994; Karpiskova R et al.; The Salmonella enteritidis and Salmonella typhimurium phage types in the Czech Republic during the monitored period 1991-1994 are described . The total number of 2318 strains were examined . From 652 Salmonella typhimurium strains 24 various phage types were identified . PT 104 was predominating in both human and non-human strains . 1666 of Salmonella enteritidis strains were identified and were representative of 14 different phage types . PT 8 was the most common type in humans (91.7%) and in animals (79.1%). Am J Vet Res, 1995 Aug, 56(8), 1012 - 8 Tumor necrosis factor-alpha production in swine after oral or respiratory challenge exposure with live Salmonella typhimurium or Salmonella choleraesuis; Stabel TJ et al.; A series of experiments was conducted to document tumor necrosis factor-alpha (TNF) activity in serum of swine after inoculation with Salmonella spp endotoxin and after oral or respiratory tract challenge exposure with live Salmonella spp . For experiment 1, a potentially lethal dose of S typhimurium endotoxin (25 micrograms/kg of body weight) was administered IV, and serum TNF activity was measured . High TNF (approx 700 IU/ml) activity at 1 to 2 hours after administration of the inoculum was associated with death, whereas lower TNF (approx 30 IU/ml) activity was associated with a general prolonged state of shock . For experiment 2, pigs were administered a nonlethal dose (5 micrograms/kg, IV) of either S typhimurium or S choleraesuis endotoxin . Difference in the ability to induce porcine serum TNF activity was not observed between strains . During experiment 3, pigs were inoculated with 10(4) colony-forming units of S typhimurium chi 4232 either orally by gelatin capsule (GC) or by intranasal (IN) instillation . A late serum TNF response (17 IU/ml) was measured at 6 weeks after IN inoculation . A serum TNF response was not detected in GC-inoculated pigs . All tissues and feces were test-negative for S typhimurium prior to the 6-week TNF response . Serum TNF activity may be related to clearance of S typhimurium after respiratory tract exposure, but it is not important to or indicative of clearance of orally presented S typhimurium in swine . During experiment 4, pigs were inoculated with 10(6) colony-forming units of S typhimurium chi 4232 similarly as for experiment 3.(ABSTRACT TRUNCATED AT 250 WORDS) J Bacteriol, 1995 Aug, 177(16), 4628 - 37 Fur regulon of Salmonella typhimurium: identification of new iron-regulated genes; Tsolis RM et al.; In order to identify genes belonging to the Fur regulon of Salmonella typhimurium, a bank of 10,000 independent S . typhimurium MudJ insertion mutants was screened for lacZ fusions regulated by the iron response regulator Fur . In parallel, a plasmid gene bank of S . typhimurium consisting of 10,000 independent clones was screened for Fur-regulated promoters or iron binding proteins by the Fur titration assay (FURTA) . Fur-regulated MudJ insertions and Fur-regulated promoters were mapped . In addition, iron-regulated promoter activities of transcriptional fusions from MudJ insertions and FURTA-positive clones were quantified . The nucleotide sequences of 11 FURTA-positive plasmids and of short fragments of DNA flanking three MudJ insertions were determined . By these methods we identified 14 Fur-regulated genes of S . typhimurium . For 11 of these genes, Fur-regulated homologs have been described in Escherichia coli or Yersinia enterocolitica, including fhuA,fhuB,fepA,fes,fepD,p43,entB,fur ,foxA,hemP, and fhuE . In addition, we identified three genes with homologs in other bacteria which have not previously been shown to be Fur regulated. J Bacteriol, 1995 Aug, 177(15), 4524 - 7 Involvement of cysB and cysE genes in the sensitivity of Salmonella typhimurium to mecillinam; Oppezzo OJ et al.; cysB and cysE strains were obtained as spontaneous mecillinam-resistant mutants of Salmonella typhimurium . The resistance to mecillinam was caused by the cys mutations which also conferred tolerance to lethal cell shape mutations . Most, but not all, cysB and cysE mutations from other origins displayed the same behavior . Resistance was abolished by O- and N-acetylserine in cysE mutants; by thiosulfate, sulfite, and sulfide in cysB mutants; and by cysteine in both types of mutants . It is concluded that an event involved in mecillinam action requires the inducer and the activator protein of the cysteine regulon. J Bacteriol, 1995 Aug, 177(15), 4508 - 13 Fine-structure deletion analysis of the transcriptional silencer of the proU operon of Salmonella typhimurium; Fletcher SA et al.; Transcriptional control of the osmotically regulated proU operon of Salmonella typhimurium is mediated in part by a transcriptional silencer downstream from the promoter (D.G . Overdier and L.N . Csonka, Proc . Natl . Acad . Sci . USA 89:3140-3144, 1992) . We carried out a fine-structure deletion analysis to determine the structure and the position of the silencer, which demonstrated that this regulatory element is located between nucleotide positions +73 to +274 downstream from the transcription start site . The silencer appears to be made up of a number of components which have cumulative negative regulatory effects . Deletions or insertions of short nucleotide sequences (< 40 bp) between the proU promoter and the silencer do not disrupt repression exerted by the silencer, but long insertions (> or = 0.8 kbp) result in a high level of expression from the proU promoter, similar to that imparted by deletion of the entire silencer . The general DNA-binding protein H-NS is required for the full range of repression of the proU operon in media of low osmolality . Although in the presence of the silencer hns mutations increased basal expression from the proU promoter three- to sixfold, in the absence of the silencer they did not result in a substantial increase in basal expression from the proU promoter . Furthermore, deletion of the silencer in hns+ background was up to 10-fold more effective in increasing basal expression from the proU promoter than the hns mutations . These results indicate that osmotic control of the proU operon is dependent of some factor besides H-NS . We propose that the transcriptional regulation of this operon is effected in media of low osmolality by a protein which makes the promoter inaccessible to RNA polymerase by forming a complex containing the proU promoter and silencer. J Bacteriol, 1995 Aug, 177(15), 4297 - 302 Salmonella typhimurium pgtB mutants conferring constitutive expression of phosphoglycerate transporter pgtP independent of pgtC; Niu S et al.; PgtC is one of the three components of the atypical "two-component" pgt regulatory system . To investigate whether functional PgtC required for the induction of pgtP expression could be bypassed in the signal transduction process, we sought, and succeeded in isolating, intergenic suppressors arising in the low-copy mini-F plasmid, pSJ11, bearing the entire pgt system except for a 168-bp deletion near the end of the pgtC gene . By transport assays, these suppressors were found to confer constitutive pgtP expression . Intriguingly, all five mutations reside near the 5' end of the pgtB gene, at codons 19 and 21 . One mutation alters Arg-19 to Gln, two alter Ala-21 to Thr, one alters Ala-21 to Val, and one alters Ala-21 to Ile . Appropriate strains in which the pgtP promoter was fused to lacZ and which bore the pgtB mutations with and without mutations in pgtC and pgtA genes were constructed, and the epistatic relationships of the wild-type pgtC allele, a mutant pgtA allele, and an essentially total deletion of pgtC to the constitutive pgtB mutations were determined . In the mutant strains bearing the Ala-21 --> Ile and Ala-21 --> Val substitutions, the level of constitutive pgtP-lacZ reporter expression was not affected by the presence of the wild-type pgtC allele, nor was it affected by the total absence of PgtC in the case of the Ala-21 --> Val alteration examined; however, in the mutant strains bearing the Ala-21 --> Thr and the Arg-19 --> Gln substitutions, the extent of constitutive pgtP-lacZ reporter expression was markedly enhanced by the presence of wild-type pgtC allele and, in the case of the Arg-19 -->Gln change examined, by the total absence of PgtC as well . These results indicate that PgtC contains no domain necessary for the kinase activity; that PgtB can be activated in the absence of PgtC mutational alterations of the protein itself; and that PgtB and PgtC interact in the signaling process, with PgtC functioning to activate and modulate the kinase activity of Pgtb . In all strains, the replacement of the wild type pgtA allele with a mutant pgtA allele completely abolished expression of the pgtP-lacZ reporter, indicating that functional pgtA is essential for the constitutivity . His-457 of PgtB, a potential site of autophosphorylation, is also required for the constitutivity because its change to Val drastically reduced pgtP-lacZ reporter expression . The structural basis for the activation of the altered PgtB is discussed in terms of putative structure of PgtB in the membrane. J Infect Dis, 1995 Aug, 172(2), 490 - 6 Deferoxamine B but not deferoxamine G1 inhibits cytokine production in murine bone marrow macrophages; Autenrieth IB et al.; The iron chelator deferoxamine (DFO) B enhances virulence of Yersinia enterocolitica and modulates cellular immune responses . Since cytokines mediate effector mechanisms in resolution of yersiniae from infected tissues, the impact of DFO B and DFO G1 on cytokine production by murine bone marrow macrophages (BMM) was investigated . BMM were stimulated with lipopolysaccharide (LPS) of Salmonella typhimurium or infected with Y . enterocolitica . DFO B inhibited interleukin (IL)-6, IL-12, and tumor necrosis factor (TNF)-alpha mRNA production 4-fold (shown by semiquantitative reverse transcription polymerase chain reaction) . TNF-alpha and IL-6 protein production was reduced 50% by DFO B . In contrast, DFO G1 had no effect on cytokine production . Moreover, cytokine production by Yersinia-infected BMM was decreased by plasmid-encoded Yersinia proteins . Thus, plasmid-cured strains induced higher cytokine responses in BMM than did the wild type strain . These results suggest that DFO B acts in a bimodal fashion in yersiniosis: iron supply to the pathogen and immunosuppression of the host. Infect Immun, 1995 Aug, 63(8), 3196 - 8 Exogenous tumor necrosis factor alpha and interleukin-1 alpha increase resistance to Salmonella typhimurium: efficacy is influenced by the Ity and Lps loci; Morrissey PJ et al.; Interleukin-1 alpha (IL-1 alpha) or tumor necrosis factor alpha (TNF-alpha) administered prior to infection with Salmonella typhimurium increases survival in mice that are Ityr, not in susceptible Lpsd or Itys mice . Combined IL-1 alpha and TNF-alpha pretreatment results in greater survival than that seen with either cytokine alone in Ityr mice . Treatment after infection with TNF-alpha and/or IL-1 alpha increases the mean time to death but not the survival fraction of Lpsd mice and was ineffective in either Ityr or Itys mice. Infect Immun, 1995 Aug, 63(8), 2859 - 66 A novel synthetic lipid A analog with low endotoxicity, DT-5461, prevents lethal endotoxemia; Sato K et al.; Bacterial endotoxin (lipopolysaccharide {LPS}) causes severe damage to the host organism as a result of excessive release of inflammatory cytokines, including interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha), from mononuclear phagocytes during gram-negative bacterial infection . We evaluated the ability of a novel synthetic lipid A analog with low endotoxicity, DT-5461, to antagonize LPS-induced IL-1 and TNF-alpha production in cells of monocyte/macrophage lineage and examined the protective effect of DT-5461 against lethal endotoxic shock in mice . The IL-1- or TNF-alpha-inducing activity of DT-5461 is 100,000 to 10,000 times less active than that of Escherichia coli LPS (EcLPS) or synthetic lipid A . DT-5461 significantly inhibited EcLPS-induced IL-1 and TNF-alpha release when murine peritoneal macrophages were incubated with DT-5461 2 h prior to EcLPS stimulation at the same concentration (1 microgram/ml) . The antagonistic effect of DT-5461 on the production of IL-1 and TNF-alpha induced by EcLPS occurred in a concentration-dependent manner . DT-5461 also inhibited IL-1 and TNF-alpha induction when murine peritoneal macrophages were stimulated by LPS from Salmonella typhimurium or synthetic lipid A, as well as by EcLPS, but not by muramyl dipeptides . This indicated that DT-5461 specifically antagonized the action of LPS . DT-5461 also antagonized EcLPS-mediated activation of human peripheral blood monocytes . DT-5461 blocked the binding of fluorescein isothiocyanate-labelled LPS to murine peritoneal macrophages as well as it did the binding of EcLPS and synthetic lipid A, i.e., in a concentration-dependent fashion . Injection of DT-5461 2 h before EcLPS challenge prevented the production of serum IL-1 and TNF-alpha in D-galactosamine-treated mice . Furthermore, this treatment modality protected mice against LPS-induced lethal toxicity . This study suggests that DT-5461 possesses a potent LPS antagonistic effect and may be useful in a protective strategy against lethal endotoxemia caused by gram-negative bacterial infection. Mutat Res, 1995 Aug, 335(1), 21 - 6 Modifying role of trace elements on the mutagenicity of benzo{a}pyrene; Olson B et al.; Benzo{a}pyrene (BaP) is a polycyclic aromatic hydrocarbon that is found in tobacco smoke and various environmental contaminants and has been shown to be carcinogenic and mutagenic in animal and cell culture studies, respectively . Research studies suggest that various nutritional factors such as the antioxidant vitamins and selenium are very promising as potential anticarcinogenic agents . Moreover, some evidence exists showing that both iron and germanium, at specific dosage levels, may possess antimutagenic potential . This study examined the influence of ferrous sulfate and germanium oxide, independently, upon the mutagenic potential of BaP in the Ames test . Four test strains of Salmonella typhimurium were exposed to BaP (15 micrograms/plate) in the presence of different dosage levels of iron (0-1000 micrograms/plate) and germanium (0-600 micrograms/plate) . In the case of iron, it was observed that, depending upon the strain tested, iron reduced BaP's mutagenicity . In strain TA98, this was a significant effect at 100 micrograms/plate and higher . In strains TA97a and TA100, iron concentrations had to reach 250 micrograms/plate or higher to produce significant effects . Iron was much less effective in reducing BaP mutagenicity in strain TA102 . In general, germanium was not as effective in reducing the mutagenic potential of BaP . Only in the case of the highest concentrations tested (400 and 600 micrograms/plate) was any effect noted, and this in only three of the four strains evaluated. J Appl Bacteriol, 1995 Aug, 79(2), 128 - 34 The effect of transient temperatures on the growth of Salmonella typhimurium LT2 . II: Excursions outside the growth region; Mitchell GA et al.; The effect of fluctuating temperatures on microbial growth is important in the passage of foods from production to consumption . Suspensions of Salmonella typhimurium have been subjected to sinusoidally time-varying temperatures of periods from 60 to 240 min between 4 degrees and 22 degrees C, that is within and below the growth temperature range . The suspensions were prepared with two concentrations of sodium chloride and adjusted to two different values of pH . The change in the numbers of viable bacteria was measured with time and the experimental growth curves and average generation times compared with predictions based on isothermal growth data . Generally, the experimental average generation times exceeded the predictions by not more than 10% . In enumerating viable bacteria in the suspensions containing 3.5% (w/v) sodium chloride it was necessary to use sodium chloride in the diluent and recovery medium in order to recover the bacteria quantitatively. Genetika, 1995 Aug, 31(8), 1073 - 8 {Isolation and characteristics of Salmonella typhimurium mutants with a disrupted process of generating nonculturable forms}; Romanova IuM et al.; A laboratory model of the induction of nonculturable forms in Salmonella typhimurium has been developed . Mutants of S . typhimurium were obtained using insertion mutagenesis via the TnPhoA transposon . These mutants were impaired in the cell transition from the vegetative to the nonculturable state assayed in this model . Mutants have various phenotypes and are located in different regions of the chromosome, as shown by the data obtained using pulsed-field electrophoresis of genomic DNA. Microbiology, 1995 Aug, 141 ( Pt 8), 1937 - 45 I-CeuI recognition sites in the rrn operons of the Bacillus subtilis 168 chromosome: inherent landmarks for genome analysis; Toda T et al.; The Bacillus subtilis 168 circular chromosome yielded ten fragments on I-CeuI endonuclease digestion . I-CeuI recognizes a 26 bp sequence that is located within the gene encoding the 23S subunit of the rRNA in Chlamydomonas eugametos, Escherichia coli and Salmonella typhimurium . The precise locations of the I-CeuI sites of the B . subtilis chromosome were determined on a NotI-SfiI physical map by (i) double digestion analyses with I-CeuI and SfiI, (ii) comparison of mutant strains lacking a specific rrn operon, (iii) using an I-CeuI linking clone and (iv) analysis of nucleotide sequence data of some rrn operons . In conclusion, all the I-CeuI sites were located within the B . subtilis rrn operons and the I-CeuI sites were conserved in all the B . subtilis 168 derivatives tested . Thus, variations in size of the I-CeuI fragments must be due to genome alterations . A B . subtilis 168 strain was investigated with I-CeuI . We demonstrated that the aberrant structure was the outcome of the inversion of an approximately 1700 kb DNA segment. J Bacteriol, 1995 Aug, 177(15), 4364 - 71 Transcriptional autoregulation of the Salmonella typhimurium phoPQ operon; Soncini FC et al.; The Salmonella typhimurium PhoP-PhoQ two-component regulatory system controls the expression of several genes, some of which are necessary for virulence . During a screening for PhoP-regulated genes, we identified the phoPQ operon as a PhoP-activated locus . beta-Galactosidase activity originating from phoPQ-lac transcriptional fusions required the presence of both the transcriptional regulator PhoP and its cognate sensor-kinase PhoQ . At low concentrations, PhoQ stimulated expression of phoPQ-lac transcriptional fusions . However, larger amounts of PhoQ protein without a concomitant increase in PhoP failed to activate phoPQ-lac fusions . Two different transcripts are produced from the phoPQ operon during exponential growth . These transcripts define two promoters: phoPp1, which requires both PhoP and PhoQ for activity and which is environmentally regulated, and phoPp2, which remains active in the absence of PhoP and PhoQ but which is slightly stimulated by these proteins . The pattern of transcriptional autoregulation was also observed at the protein level with anti-PhoP antibodies . In sum, autoregulation of the phoPQ operon provides several levels of control for the PhoP-PhoQ regulon . First, environmental signals would stimulate PhoQ to phosphorylate the PhoP protein that is produced at basal levels from the PhoP-PhoQ-independent promoter . Then, phospho-PhoP would activate transcription of phoPp1, resulting in larger amounts of PhoP and PhoQ and increased expression of PhoP-activated genes . A return to basal levels could be mediated by a posttranscriptional mechanism by which translation of the mRNA produced from phoPp1 is inhibited. Infect Immun, 1995 Aug, 63(8), 2818 - 25 Clearance of Pseudomonas aeruginosa from the murine gastrointestinal tract is effectively mediated by O-antigen-specific circulating antibodies; Pier GB et al.; The colonization of mucosal surfaces by Pseudomonas aeruginosa can lead to local or disseminated disease . Secretory immunoglobulin A (IgA) has been assumed to be responsible for preventing mucosal colonization by interfering with the binding of bacterial ligands to epithelial surface receptors . However, the efficacy of this mechanism of immunity derives little actual support from in vivo experiments . In an investigation of the role of local and systemic immunization strategies in reducing colonization of the gastrointestinal tract of mice by P . aeruginosa, the bacterial antigens that were potential targets for immune effectors promoting mucosal clearance were identified . Levels of gastrointestinal colonization were reduced when immunity to homologous O antigens, but not that to pili or flagella, was elicited . Oral vaccination with attenuated Salmonella typhimurium expressing P . aeruginosa serogroup O11 antigen elicited mucosal and serum IgA antibodies and serum IgG antibodies specific for the recombinant antigen . Oral challenge of immunized mice with P . aeruginosa serogroup O11 demonstrated protection against gastrointestinal colonization . Intraperitoneal immunization with a serogroup O11 high-molecular-weight O-polysaccharide antigen elicited only serum IgG and IgM antibodies yet was as effective as oral vaccination in protecting mice against gastrointestinal colonization . This finding was confirmed by the demonstration that intraperitoneal immunization with purified lipopolysaccharide was also protective against mucosal surface colonization . These results call into question the need for local immune effectors, particularly secretory IgA, directed at bacterial ligands for epithelial surface components, in protecting a mucosal surface from bacterial challenge. Curr Opin Immunol, 1995 Aug, 7(4), 474 - 8 Entry of microbes into the host: using M cells to break the mucosal barrier; Jones B et al.; Enteric microbial pathogens interact with the gut epithelium to establish infection . Recently, it has become clear that many microorganisms that colonize or traverse the intestinal mucosa do so via the specialized M cells . Recent work has shown that Shigella flexneri and Salmonella typhimurium specifically target M cells to initiate infection of the host. Biol Reprod, 1995 Aug, 53(2), 462 - 71 Oral immunization with attenuated Salmonella expressing human sperm antigen induces antibodies in serum and the reproductive tract; Srinivasan J et al.; Induction of immune responses in the reproductive tract will be crucial for a functional gamete antigen-based antifertility vaccine . Here we describe the construction and development of an avirulent Salmonella as an oral vaccine delivery vector to elicit sperm-specific immune responses in reproductive tract secretions . A cDNA sequence encoding the human sperm antigen SP10 was cloned on an asd+vector and expressed to a high level in an avirulent delta cya, delta crp, and delta asd vaccine strain of Salmonella typhimurium . Oral immunization of female BALB/c mice with this recombinant Salmonella elicited high-titer anti-SP10 IgG antibodies in serum and IgA antibodies in vaginal secretions . Anti-SP10 antibody titers could be increased by secondary and tertiary oral administrations of the recombinant Salmonella . Induction of sperm-specific antibodies in the reproductive tract following oral administration of a recombinant Salmonella could lead to the development of a simple, safe, efficient, and easy-to-use antifertility vaccine. AIDS Res Hum Retroviruses, 1995 Aug, 11(8), 909 - 20 Highly attenuated HIV type 2 recombinant poxviruses, but not HIV-2 recombinant Salmonella vaccines, induce long-lasting protection in rhesus macaques; Franchini G et al.; Immunization schemes employing priming with vector-based vaccine candidates followed by subunit booster administrations have been explored and shown to have merit in the human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus systems . In this study, we have assessed the priming capacity of highly attenuated poxvirus vector (NYVAC and ALVAC)-based HIV-2 recombinants, as well as Salmonella typhimurium HIV-2 recombinants in rhesus macaques . ALVAC- and NYVAC-based vaccine candidates expressing the HIV-2 gag, pol, and env genes or NYVAC-based recombinants expressing either gp160 or gp120 were used to immunize rhesus macaques in combination protocols with alum-adjuvanted HIV-2 rgp160 . Following intravenous challenge exposure with 100 infectious doses of the HIV-2SBL6669 parental virus genotype mixture, seven of eight animals were protected from infection . The seven protected animals were rechallenged 6 months postprimary challenge, without additional booster inoculations, with the same dose of the HIV-2SBL6669 parental virus . Five of the seven animals remained protected against HIV-2 infection at 6 months following the second challenge . In contrast, oral immunization with recombinant Salmonella expressing the HIV-2 gag and the gp120 portion of the envelope either alone or in combination with alum-adjuvanted rgp160 failed to confer protection . These results suggest that the NYVAC- and ALVAC-based recombinants may confer long-lasting protection and that these two highly attenuated poxvirus vaccine vectors may represent promising candidates for developing an acquired immunodeficiency syndrome vaccine. Biochemistry, 1995 Jul 25, 34(29), 9466 - 76 Monovalent metal ions play an essential role in catalysis and intersubunit communication in the tryptophan synthase bienzyme complex; Woehl EU et al.; This investigation shows that the alpha 2 beta 2 tryptophan synthase bienzyme complex from Salmonella typhimurium is subject to monovalent metal ion activation . The effects of the monovalent metal ions Na+ and K+ were investigated using rapid scanning stopped-flow (RSSF), single-wavelength stopped-flow (SWSF), and steady-state techniques . RSSF measurements of individual steps in the reaction of L-serine and indole to give L-trytophan (the beta-reaction) as well as the reaction of 3-indole-D-glycerol 3'-phosphate (IGP) with L-serine (the alpha beta-reaction) demonstrate that monovalent metal ions such as Na+ and K+ change the distribution of intermediates in both the transient and steady states . Therefore the metal ion effect alters relative ground-state energies and the relative positions of ground- and transition-state energies . The RSSF spectra and SWSF time courses show that the turnover of indole is significantly reduced in the absence of either Na+ or K+ . The alpha-aminoacrylate Schiff base species, E(A-A), is in a less active state in the absence of monovalent metal ions . Na+ decreases the steady-state rate of IGP cleavage (the alpha-reaction) to about 30% of the value obtained in the absence of metal ions . Steady-state investigations show that in the absence of monovalent metal ions the alpha- and alpha beta-reactions have the same activity . Na+ binding gives a 30-fold stimulation of the alpha-reaction when the beta-site is in the E(A-A) form.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1995 Jul 25, 34(29), 9459 - 65 Monovalent cations affect dynamic and functional properties of the tryptophan synthase alpha 2 beta 2 complex; Peracchi A et al.; Monovalent cations affect both conformational and catalytic properties of the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium . Their influence on the dynamic properties of the enzyme was probed by monitoring the phosphorescence decay of the unique Trp-177 beta, a residue located near the beta-active site, at the interface between alpha- and beta-subunits . In the presence of either Li+, Na+, Cs+, or NH4+, the phosphorescence decay is biphasic and the average lifetime increases indicating a decrease in the flexibility of the N-terminal domain of the beta-subunit . Since amplitudes but not lifetimes are affected, cations appear to shift the equilibrium between preexisting enzyme conformations . The effect on the reaction between indole and L-serine was studied by steady state kinetic methods at room temperature . We found that cations: (i) bind to the L-serine--enzyme derivatives with an apparent dissociation constant, measured as the concentration of cation corresponding to one-half of the maximal activity, that is in the millimolar range and decreases with ion size; (ii) increase kcat with the order of efficacy Cs+ > K+ > Li+ > Na+; (iii) decrease KM for indole, Na+ being the most effective and causing a 30-fold decrease; and (iv) cause an increase of the kcat/KM ratio by 20-40-fold . The influence on the equilibrium distribution between the external aldimine and the alpha-aminoacrylate, intermediates in the reaction of L-serine with the beta-subunits of the enzyme, was found to be cation-specific.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1995 Jul 25, 34(29), 9403 - 12 An expanded two-state model accounts for homotropic cooperativity in biosynthetic threonine deaminase from Escherichia coli; Eisenstein E et al.; The linkage between substrate and regulatory effector binding to separate sites on allosteric enzymes results in shifts in their sigmoidal kinetics to regulate metabolism . Control of branched chain amino acid biosynthesis in Escherichia coli occurs in part through shifts in the sigmoidal dependence of alpha-ketobutyrate production promoted by isoleucine and valine binding to biosynthetic threonine deaminase . The structural similarity of threonine, valine, and isoleucine have given rise to suggestions that there may be competition among different ligands for the same sites on this tetrameric enzyme, resulting in a complex pattern of regulation . In an effort to provide a coherent interpretation of the cooperative association of ligands to the active sites and to the effector sites of threonine deaminase, binding studies using single amino acid variants were undertaken . A previously-isolated, feedback-resistant mutant identified in Salmonella typhimurium, ilvA219, has been cloned and sequenced . The phenotype is attributable to a single amino acid substitution in the regulatory domain of the enzyme in which leucine at position 447 is substituted with phenylalanine . The mutant exhibits hyperbolic saturation curves in both ligand binding and steady-state kinetics . These results, in addition to calorimetric and spectroscopic measurements of isoleucine and valine binding, indicate that the low affinity (T) state is destabilized in the mutant and that it exists predominantly in the high affinity (R) conformation in the absence of ligands, providing an explanation for its resistance to isoleucine . Chemical and spectroscopic analyses of another mutant, in which alanine has replaced an essential lysine at position 62 that forms a Schiff base with pyridoxal phosphate, indicate that the cofactor is complexed to exogenous threonine and is therefore unable to bind additional amino acids at the active sites . Isoleucine and valine binding to this inactive, active site-saturated enzyme revealed that it too was stabilized in the R state, yielding binding constants in excellent agreement with the leucine to phenylalanine mutant . The lysine to alanine mutant was further utilized to demonstrate that both threonine and 2-aminobutyrate bind with stronger affinity to the regulatory sites than to the active sites . A direct consequence of these results is that substrates and analogs have a synergistic effect on the allosteric transition since, in effect, they act as both homotropic and heterotropic effectors.(ABSTRACT TRUNCATED AT 250 WORDS) Science, 1995 Jul 21, 269(5222), 400 - 3 Simultaneous identification of bacterial virulence genes by negative selection; Hensel M et al.; An insertional mutagenesis system that uses transposons carrying unique DNA sequence tags was developed for the isolation of bacterial virulence genes . The tags from a mixed population of bacterial mutants representing the inoculum and bacteria recovered from infected hosts were detected by amplification, radiolabeling, and hybridization analysis . When applied to a murine model of typhoid fever caused by Salmonella typhimurium, mutants with attenuated virulence were revealed by use of tags that were present in the inoculum but not in bacteria recovered from infected mice . This approach resulted in the identification of new virulence genes, some of which are related to, but functionally distinct from, the inv/spa family of S . typhimurium. J Mol Biol, 1995 Jul 21, 250(4), 392 - 406 carP, involved in pyrimidine regulation of the Escherichia coli carbamoylphosphate synthetase operon encodes a sequence-specific DNA-binding protein identical to XerB and PepA, also required for resolution of ColEI multimers; Charlier D et al.; The carP gene involved in pyrimidine-specific regulation of the upstream P1 promoter of the Escherichia coli carAB operon has been cloned in vivo on a mini-Mu replicon, sequenced and shown to be identical to the xerB (pepA) gene encoding aminopeptidase A, a protein also involved in the Xer-mediated site-specific recombination at ColEI cer . The trans-dominant allele carP6 was cloned as well and shown to bear a single G-->A transition that converts the TGG codon (Trp473) into a TAG amber stop codon . The truncated mutant protein, missing the 31 C-terminal amino acid residues, was shown to be partially active; in the multicopy state the carP6 allele can restore pyrimidine repressibility of the carAB promoter P1 . The trans-dominant character of the single copy carP6 allele was found to be suppressed in the presence of multiple copies of the wild-type gene . The carP (pepA) control region was sequenced and transcription shown to be initiated at three promoters, the most upstream one of which was shown to be subject to negative autoregulation . The aminopeptidase activity of CarP (PepA) was found to be dispensable for its role in pyrimidine-mediated repression of carAB transcription . CarP (PepA) was shown to be a sequence-specific DNA-binding protein that does not require, at least not in vitro, any pyrimidine cofactor to bind to the DNA . Mobility-shift and DNase I footprinting experiments have revealed a specific binding of purified CarP (PepA) to two sites in each one of the control regions of the E . coli and Salmonella typhimurium carAB operons and to a single site in the carP (pepA) control region . We propose that integration host factor and CarP/PepA-induced structural modifications in the carAB control region cause conformational changes required to assemble a pyrimidine-specific nucleo-protein regulatory complex. J Mol Biol, 1995 Jul 21, 250(4), 383 - 91 Pyrimidine regulation of the Escherichia coli and Salmonella typhimurium carAB operons: CarP and integration host factor (IHF) modulate the methylation status of a GATC site present in the control region; Charlier D et al.; By measuring the protection against Dam methylase modification of a GATC sequence located 106 bp upstream of the startpoint of promoter P1 in the control region of the carAB operon (encoding carbamoylphosphate synthetase) we have obtained evidence for a direct correlation between the degree of in vivo occupancy of a specific regulatory target site and the repressibility of the P1 promoter by pyrimidine residues . A high uridine nucleotide pool as well as binding of the carP (alias xerB/pepA) gene product and of the integration host factor (IHF) to the carAB control region are prerequisites to observe this in vivo protection . Purified CarP binds in vitro to the carAB control region and protects against DNase I two approximately 25 bp long stretches, one of which is located just downstream of the GATC sequence . Mutations in this site strongly impair the pyrimidine regulation of the P1 promoter and the interference with Dam methylase modification . These processes are also strongly impaired in the absence of integration host factor and in mutants affected in the IHF site located some 200 bp upstream of this Dam methylase modification site . IHF therefore exerts at least part of its antagonistic effects on P1, i.e . increased expression in minimal medium but increased repression in the presence of pyrimidine residues, indirectly by influencing the formation or the stability of a particular protein-DNA complex . Furthermore, we demonstrate that the distance separating the IHF and Dam methylase target sites is crucial for the in vivo protection and for pyrimidine-mediated regulation of the promoter expression . Mutations altering this distance result in severe reductions of the degree of in vivo protection and, concomitantly, of the repressibility by pyrimidine residues of promoter P1 activity in a way indicative of the formation of a complex nucleoprotein structure . Since neither IHF nor CarP require pyrimidine residues to bind to the carAB control region, at least not in vitro, it is tempting to suggest that IHF and CarP-induced bending and looping provide changes in DNA topology that are required for assembling a specific pyrimidine-dependent nucleoprotein complex that modulates P1 activity. Cancer Lett, 1995 Jul 20, 94(1), 33 - 40 Chemoprotective properties of chlorophyllin against vinyl carbamate, p-nitrophenyl vinyl ether and their electrophilic epoxides; Park KK et al.; Chlorophyllin (CHL), a water-soluble sodium and copper derivative of chlorophyll, has been shown to be a strong antimutagen in several test systems, but its mechanism of antimutagenic action is largely unknown . In the present study, we have found the protective properties of CHL against vinyl carbamate, p-nitrophenyl vinyl ether and their electrophilic epoxides . CHL exhibited dose-related inhibition of his+ reversion in Salmonella typhimurium TA 1535 induced by these mutagens . Formation of DNA adducts from vinyl carbamate epoxide (VCO) and 2'-(4-nitrophenoxy)oxirane (NPO) was also markedly attenuated in the presence of CHL . Oral administration of CHL prior to the topical application of each of the above carcinogens resulted in significant reduction in both incidence and multiplicity of skin tumors in mice . The effective protection by CHL against VCO and NPO suggest that its formation of inactive complexes with these carcinogens is mediated by mechanisms other than pi-pi interactions. FEMS Microbiol Lett, 1995 Jul 15, 130(1), 25 - 30 Production and identification of recombinant proteins of Salmonella typhimurium and their use in detection of antibodies in experimentally challenged animals; Kwang J et al.; Antibodies to experimental Salmonella typhimurium challenge in cattle and sheep were assessed by 15 recombinant flagellum proteins . The 15 DNA fragments selected for gene expression were derived from external flagellin, hook, hook-associated protein, and basal body gene domains . Our efforts were focused on characterizing the humoral immune response of Salmonella infected and vaccinated animals and identifying immunodominant antigenic determinants . This communication reports that the 159-261 amino acids of external flagellum (FliCi-1), 285-331 amino acids of hook protein (FlgE-2), and 309-391 amino acids and 440-537 amino acids of hook-associated protein (FlgK-1 and -2) appeared to be the most immunoreactive proteins and were recognized by all of the experimental animal sera tested in this study. J Mol Biol, 1995 Jul 7, 250(2), 276 - 90 Crystal structure of the catalytic domain of the chemotaxis receptor methylesterase, CheB; West AH et al.; Signaling activity of bacterial chemotaxis transmembrane receptors is modulated by reversible covalent modification of specific receptor glutamate residues . The level of receptor methylation results from the activities of a specific S-adenosylmethionine-dependent methyltransferase, CheR, and the CheB methylesterase, which catalyzes hydrolysis of receptor glutamine or methylglutamate side-chains to glutamic acid . The CheB methylesterase belongs to a large family of response regulator proteins in which N-terminal regulatory domains control the activities of C-terminal effector domains . The crystal structure of the catalytic domain of the Salmonella typhimurium CheB methylesterase has been determined at 1.75 A resolution . The domain has a modified, doubly wound alpha/beta fold in which one of the helices is replaced by an anti-parallel beta-hairpin . Previous biochemical and mutagenesis data, suggest that the methylester hydrolysis catalyzed by CheB proceeds through a mechanism involving a serine nucleophile . The methylesterase active site is tentatively identified as a cleft at the C-terminal edge of the beta-sheet containing residues Ser164, His190 and Asp286 . The three-dimensional fold, and the arrangement of residues within the catalytic triad distinguishes the CheB methylesterase from any previously described serine protease or serine hydrolase. J Mol Biol, 1995 Jul 7, 250(2), 123 - 7 Differential codon usage for conserved amino acids: evidence that the serine codons TCN were primordial; Diaz-Lazcoz Y et al.; The availability of specialized sequence databanks for Escherichia coli, Saccharomyces cerevisiae and Bacillus subtilis made it possible to build a set of 105 protein-coding genes that are homologous in these three species . An analysis of the triplets at both the nucleotide and amino acid level revealed that the codon bias of some amino acids are significantly higher at conserved rather than at non-conserved positions . Comparisons of homologous genes in E . coli and Salmonella typhimurium, and in S . cerevisiae and Drosophila melanogaster, led to the same conclusion . A special case was made for serine in E . coli, whose major codon is AGC for non-conserved and TCC for conserved residues . We interpret this observation as evidence that the primordial codons for serine were TCN, while codons AGY appeared later . This conclusion is substantiated by an analysis of the codon usage of catalytic serine residues in ancient, ubiquitous and essential proteins (ATP synthases and topoisomerases) . It is shown that in these proteins the proportion of the catalytic serine residues coded by TCN is significantly higher than the one expected from the overall codon usage of serine residues. J Biol Chem, 1995 Jul 7, 270(27), 16097 - 106 Structure/function analysis of the periplasmic histidine-binding protein . Mutations decreasing ligand binding alter the properties of the conformational change and of the closed form; Wolf A et al.; The periplasmic histidine-binding protein, HisJ, is a receptor for the histidine permease of Salmonella typhimurium . Receptors of this type are composed of two lobes that are far apart in the unliganded structure (open conformation) and drawn close together in the liganded structure (closed conformation) . The binding of the ligand, in a cleft between the lobes, stabilizes the closed conformation . Such receptors have several functions in transport: interaction with the membrane-bound complex, transmission of a transmembrane signal to hydrolyze ATP, and receiving a signal to open the lobes and release the ligand . In this study the mechanism of action of HisJ was further investigated using mutant proteins defective in ligand binding activity and closed form-specific monoclonal antibodies (Wolf, A., Shaw, E . W., Nikaido, K., and Ames G . F.-L . (1994) J . Biol . Chem . 269, 23051-23058) . Y14H is defective in stabilization of the closed form, does not assume the closed empty form, and assumes an altered closed liganded form . T121A and G119R are similar to Y14H, but assume a normal closed liganded form . S72P binds the ligand to the open form, but does not assume a recognizable closed form . S92F is defective in the ability to undergo conformational change and to stabilize the closed form . All other mutant proteins appear to fall within one of these four categories . The biochemical characterization of these mutant proteins agrees with the structural analysis of the protein . We suggest that mutant proteins that do not assume the normal closed form, in addition to their defect in ligand binding, fail to interact with the membrane-bound complex and/or to transmit transmembrane signals. Gene, 1995 Jul 4, 160(1), 123 - 8 A single tyrosine differentiates active and inactive Trypanosoma cruzi trans-sialidases; Cremona ML et al.; Several genes encode members of the Trypanosoma cruzi (Tc) trans-sialidase (TS) family . These proteins contain an enzymatic domain on the N terminus, the only one required for TS activity, and an antigenic domain (SAPA (shed acute phase antigen) amino acid (aa) repeats) on the C terminus . Only some members of this glycoprotein family are enzymatically active . The complete sequence of two clones encoding the enzymatic domain of active and inactive protein from each of two Tc strains has now been obtained . Comparison of these sequences showed a limited divergence among them: 20 out of the 642 deduced aa in the enzymatic domain were found to differ . From these 20 aa, only one was found to be essential for enzymatic activity . A Tyr342 residue is deduced in both active proteins while a His342 is present in both inactive ones . This naturally occurring Tyr342-->His substitution completely abolished the TS activity . In addition to Tyr342, a second deduced aa, Pro231, was found to be necessary for full enzymatic TS activity; a Pro231-->Ala change rendered the TS protein partially active . Fourteen aa residues, including Tyr342, out of the 16 aa in the active site of a sialidase from Salmonella typhimurium are present at the same or very similar positions in the Tc TS. Proc Natl Acad Sci U S A, 1995 Jul 3, 92(14), 6399 - 403 Genetic and redox determinants of nitric oxide cytotoxicity in a Salmonella typhimurium model; De Groote MA et al.; Paradoxically, nitric oxide (NO) has been found to exhibit cytotoxic, antiproliferative, or cytoprotective activity under different conditions . We have utilized Salmonella mutants deficient in antioxidant defenses or peptide transport to gain insights into NO actions . Comparison of three NO donor compounds reveals distinct and independent cellular responses associated with specific redox forms of NO . The peroxynitrite (OONO-) generator 3-morpholinosydnonimine hydrochloride mediates oxygen-dependent Salmonella killing, whereas S-nitrosoglutathione (GSNO) causes oxygen-independent cytostasis, and the NO . donor diethylenetriamine-nitric oxide adduct has no antibacterial activity . GSNO has the greatest activity for stationary cells, a characteristic relevant to latent or intracellular pathogens . Moreover, the cytostatic activity of GSNO may best correlate with antiproliferative or antimicrobial effects of NO, which are unassociated with overt cell injury . dpp mutants defective in active dipeptide transport are resistant to GSNO, implicating heterolytic NO+ transfer rather than homolytic NO . release in the mechanism of cytostasis . This transport system may provide a specific pathway for GSNO-mediated signaling in biological systems . The redox state and associated carrier molecules are critical determinants of NO activity. Rev Latinoam Microbiol, 1995 Jul-Sep, 37(3), 227 - 36 Kdp-like system in Salmonella typhimurium LT-2; Garcia-Cuellar C et al.; Salmonella typhimurium LT-2, as Escherichia coli K12, was able to grow in a potassium concentration-dependent manner, down to a very low concentration (< 5 microM) . Its metabolic swelling also was {K+}-dependent . When the cells were subjected to hyperosmotic shock, this ion was uptaken rapidly, probably due to a K(+)-high affinity transport-system, similar to the E . coli Kdp system . The shrinkage in presence of 0.6 M NaCl, however, was more noticeable in S . typhimurium, which expressed a smaller level of intracellular K+ than E . coli . The genetic locus responsible for the ability of S . typhimurium to grow in low {K+}, was mapped in nitrosoguanidine mutants and localized around min 18, close to the gal operon . This asseveration was confirmed by experiments of reversion, conjugation, and transduction . The mutants required considerably more {K+} to grow and to swell than the parental strain; in addition, below 1 mM {K+}, they showed less internal {K+}. Rev Inst Med Trop Sao Paulo, 1995 Jul-Aug, 37(4), 297 - 302 Lysotypes and plasmidial profile of Salmonella serovar typhimurium isolated from children with enteric processes in the cities of Rio de Janeiro, RJ, and Salvador, BA - Brazil; Asensi MD et al.; The lysotypes, plasmidial profiles, and profiles of resistance to antimicrobial agents were determined in 111 Salmonella Typhimurium strains isolated from feces and blood of children treated in Rio de Janeiro and in Salvador . Six distinct lysotypes (19, 41, 97, 105, 120 and 193) were recognized, with a predominance of lysotype 193 (59.7%) in Rio de Janeiro and of phage type 105 (38.4) in Salvador . Approximately 86.7% of the lysotype 193 strains presented multiple resistance to more than six antimicrobial agents, whereas 93% of lysotype 105 strains were fully susceptible . More than 90% of the strains presented plasmids distributed into 36 different profiles in Rio de Janeiro and into 10 profiles in Salvador . A 40 MDa plasmid was the most frequent (47%) in the strains from Rio de Janeiro, whereas a 61 MDa plasmid predominated (14.5%) in Salvador . Combined analysis of plasmid profile and classification into lysotypes (especially those belonging to types 105 and 103, proved to be more discriminatory than the other methods applied). Avian Dis, 1995 Jul-Sep, 39(3), 627 - 30 Egg dipping in hydrogen peroxide solution to eliminate Salmonella typhimurium from eggshell membranes; Padron M; The effectiveness of egg dipping in a 6% hydrogen peroxide solution to eliminate Salmonella typhimurium from eggshell membranes was evaluated . The first step was to assess the water uptake from broiler hatching eggs dipped in tap water once or twice using a positive-pressure differential method in order to determine the best method to use in the disinfection trials . Double dipping increased water uptake by 86% more than single dipping . Dipping S . typhimurium-contaminated eggs twice in a 6% hydrogen peroxide solution reduced the average number of organisms in eggshell membranes by 95% and the number of S . typhimurium-positive eggs by 55% compared with the infected untreated group . Dipping the eggs in a 6% hydrogen peroxide solution did not adversely affect hatchability. Mikrobiol Z, 1995 Jul-Aug, 57(4), 66 - 72 {The evaluation of the mutagenic activity of gaseous particles in the atmospheric air by using a microbiological test}; Dugan AM et al.; Total mutagen activity of a gas component in chemical pollutions of the atmospheric air in a number of industrially developed towns of Ukraine has been studied and assessed in a microbiological test on Salmonella typhimurium . The towns were chosen proceeding from the specificity of industry: metallurgical industry (Mariupol, Zaporozhye, Donetsk, Krivoi Rog, Makeevka); chemical industry (Cherkassy, Chernigov; Kremenchug, Severodonetsk, Lisichansk, Gorlovka, Rovno, Sumy) and conditionally control ones (Simferopol, Sevastopol, Nikolaev, Poltava, Zhitomir) . The air samples, 100 m3, have been taken in each town weekly during a month by special absorbers of Polysorb-2 type . The extraction of chemical matters from absorbers was carried out by traditional methods . Chemical matters were dissolved in dimethyl sulphoxide and tested for its ability to induce the gene mutations . The studies have shown that the atmospheric air samples from the group of "metallurgical" towns prove the mutagen activity, classified as the "middle" one (the number of revertant colonies in the experiment exceeded the control 10.3 to 22.2 times) . The mutagenicity of "chemical" towns was on the level of "middle" and "weak", that of conditionally control ones was on the level of "weak" only. Infect Immun, 1995 Jul, 63(7), 2770 - 2 Salmonella typhimurium displays normal invasion of mice with defective epidermal growth factor receptors; McNeil A et al.; The role of the epidermal growth factor (EGF) receptor in cell invasion by Salmonella typhimurium was examined in vitro and in vivo by using waved-2 mice which express an EGF receptor with reduced kinase activity . S . typhimurium invaded fibroblasts from waved-2 mice as efficiently as fibroblasts from wild-type control animals . In vivo, S . typhimurium both invaded the gastrointestinal tract and penetrated through to the spleen of waved-2 mice . Our studies suggest that the EGF receptor has only a limited role, if any, in cell invasion by S . typhimurium. Infect Immun, 1995 Jul, 63(7), 2743 - 54 Characterization of intestinal invasion by Salmonella typhimurium and Salmonella dublin and effect of a mutation in the invH gene; Watson PR et al.; The relative levels of invasiveness of two bovine isolates each of Salmonella typhimurium and Salmonella dublin and of invH mutants of S . typhimurium were determined in MDCK and Int 407 cultured-cell assays and in bovine ileal loops . S . dublin was found to be significantly less invasive in cultured cells than S . typhimurium, but this difference was not observed in bovine intestines . The invH mutants exhibited a significant reduction in invasion in both cultured cells and bovine intestines . The invasive phenotypes of the strains were confirmed by fluorescent microscopy and scanning and transmission electron microscopy . The wild-type strains were observed in the laminae propriae of the intestinal villi, while in contrast the invH mutants were generally associated with the enterocyte layer . The degree of damage in the bovine ileum was related to the magnitude of the invasion . There was no difference in the amount of S . typhimurium or S . dublin recovered from the bovine ileum either with or without Peyer's patches 3 h after inoculation of the loop. Infect Immun, 1995 Jul, 63(7), 2564 - 9 Influence of preimmunization with tetanus toxoid on immune responses to tetanus toxin fragment C-guest antigen fusions in a Salmonella vaccine carrier; Chabalgoity JA et al.; We have previously described a new system for the delivery of recombinant antigens in live Salmonella vaccines as genetic fusions to the C terminus of fragment C of tetanus toxin (TetC) driven by the anaerobically inducible nirB promoter . It has been reported that preimmunization with tetanus toxoid (TT) can suppress the antibody response to peptides chemically coupled to TT (epitope-specific suppression) in both animals and humans, which could interfere with efficacy of the Salmonella-TetC delivery system . We report that preimmunization of BALB/c mice with TT in alum did not suppress the response to either of two protective antigens of Schistosoma mansoni, the full-length S . mansoni P28 glutathione S-transferase (P28) and a construct consisting of eight tandem copies of the protective peptide comprising amino acids 115 to 131 of P28 . The guest antigens were expressed in the aroA Salmonella typhimurium SL3261 vaccine strain as fusions to TetC . Preimmunization with TT 10 weeks before administration of the recombinant salmonellae did not alter the antibody response to the full-length P28, whereas the response to the peptide comprising amino acids 115 to 131 was increased by preimmunization with TT, with the increase seen mainly in the immunoglobulin G1 isotype . The antitetanus response was increased by preimmunization with TT in all groups receiving salmonellae expressing TetC . The results could be important when one is considering the use of the Salmonella-TetC delivery system in populations preimmunized with TT. Trop Doct, 1995 Jul, 25(3), 104 - 6 Multidrug resistant non-typhoidal Salmonella infections; Udgaonkar US et al.; Over a period of 2 years, 28 patients admitted to Government General Hospital (GGH), Sangli (which is attached to Government Medical College (GMC), Miraj) yielded multi-drug resistant non-typhoidal salmonellae from their clinical material . The pediatric age group predominated in the study, accounting for 93% of cases . Salmonella typhimurium was the main isolate (86%), the other being Salmonella newport (14%) . Gastroenteritis was the commonest presentation . Septicaemia was seen with 100% mortality, in infants below 1 month of age . Two cases of meningitis were also seen. J Invertebr Pathol, 1995 Jul, 66(1), 68 - 71 Safety testing of Bacillus thuringiensis preparations, including thuringiensin, using the Salmonella assay; Carlberg G et al.; The aim of this study was to test genotoxicity aspects of the safety of two strains of Bacillus thuringiensis . The strains were of serotype H-1, producing thuringiensin, toxic to flies, and serotype H-14, producing endotoxin, toxic to mosquitoes, but not thuringiensin . Four preparations were tested: Tenfold concentrated cell-free culture media of serotypes H-1 and H-14, prepurified thuringiensin, and purified endotoxin . No increases in revertant colony numbers of the tester strains of Salmonella typhimurium TA98 or TA100 were observed at the dose levels used with or without metabolic activation . Infrequent slight increases in revertant colony numbers in strains TA98 and TA1538 at a very high test dose of 50 mg/plate, both in the presence and absence of thuringiensin, were probably caused by histidine present in the growth medium of B . thuringiensis . Furthermore, the effects were at most slightly dose related and not reproducible, and therefore the preparations can be considered nonmutagenic. Appl Environ Microbiol, 1995 Jul, 61(7), 2614 - 9 A rapid, direct method for enumerating respiring enterohemorrhagic Escherichia coli O157:H7 in water; Pyle BH et al.; Simple, rapid methods for the detection and enumeration of specific bacteria in water and wastewater are needed . We have combined incubation using cyanoditolyl tetrazolium chloride (CTC) to detect respiratory activity with a modified fluorescent-antibody (FA) technique, for the enumeration of specific viable bacteria . Bacteria in suspensions were captured by filtration on nonfluorescent polycarbonate membranes that were then incubated on absorbent pads saturated with CTC medium . A specific antibody conjugated with fluorescein isothiocyanate was reacted with the cells on the membrane filter . The membrane filters were mounted for examination by epifluorescence microscopy with optical filters designed to permit concurrent visualization of fluorescent red-orange CTC-formazan crystals in respiring cells which were also stained with the specific FA . Experiments with Escherichia coli O157:H7 indicated that both respiratory activity and specific FA staining could be detected in logarithmic- or stationary-phase cultures, as well as in cells suspended in M9 medium or reverse-osmosis water . Following incubation without added nutrients in M9 medium or unsterile reverse-osmosis water, the E . coli O157:H7 populations increased, although lower proportions of the organisms reduced CTC . Numbers of CTC-positive, FA-positive cells compared with R2A agar plate counts gave a strong linear regression (R = 0.997) . Differences in injury did not appear to affect CTC reduction . The procedure, which can be completed within 3 to 4 h, has also been performed successfully with Salmonella typhimurium and Klebsiella pneumoniae. Appl Environ Microbiol, 1995 Jul, 61(7), 2521 - 6 Flow cytometric assessment of Escherichia coli and Salmonella typhimurium starvation-survival in seawater using rhodamine 123, propidium iodide, and oxonol; Lopez-Amoros R et al.; The use of flow cytometry in microbiology allows rapid characterization of cells from a nonhomogeneous population . A method based on flow cytometry to assess the effects of lethal agents and the bacterial survival in starved cultures through the use of membrane potential-sensitive dyes and a nucleic acid marker is presented . The use of propidium iodide, rhodamine, and oxonol has facilitated the differentiation of cells of Escherichia coli and Salmonella typhimurium of various states of vitality following various treatments (heat, sonication, electroporation, and incubation with gramicidin) and during starvation in artificial seawater . The fluorescence intensity is directly correlated with viable cell counts for rhodamine 123 labelling, whereas oxonol and propidium iodide labelling is inversely correlated with viable counts . The distribution of rhodamine and oxonol uptake during starvation-survival clearly indicates that single-species starved bacteria are heterogeneous populations, and flow cytometry can be a fundamental tool for quantifying this heterogeneity. J Leukoc Biol, 1995 Jul, 58(1), 32 - 9 Dietary modulation of Kupffer cell and splenocyte function during a Salmonella typhimurium challenge in mice; Eicher SD et al.; Oils from cold-water fish are rich in (n-3) polyunsaturated fatty acids, in particular eicosapentaenoic acid (20:5) and docosahexaenoic acid (22:6) . Although those fatty acids are beneficial in the prevention of cardiac disease and have anti-inflammatory properties, they can also decrease survival rates of mice during challenges with food-borne pathogens . This study was designed to determine dietary fat effects on Kupffer cells and splenocytes during a Salmonella typhimurium challenge . Mice were fed a low corn oil diet (3%, control), high corn oil diet (20%, HCO), or a menhaden fish oil diet (17% + 3% corn oil, FO) for 28 days and then orally given 3.1 x 10(8) colony-forming units of S . typhimurium . Kupffer cells and splenocytes were separated immediately prior to and on days 6, 10, and 14 postchallenge . Fish oil decreased Kupffer cell phagocytosis and oxidative burst early in the infection and adhesion molecule (CD18) expression at the end of the infection . In splenocytes, fish oil affected Ia expression prior to and late in the infection and depressed CD18 expression late in the infection . These data suggest that the diet affected Kupffer cells most early in the infection but affected splenocytes primarily later in the infection . Therefore, because the greatest death rate during an S . typhimurium infection occurs early, the reduced function of the Kupffer cells is probably a major factor. J Toxicol Environ Health, 1995 Jul, 45(3), 337 - 47 Mouse bone marrow micronucleus assay: relationships with in vitro mutagenicity and rodent carcinogenicity; Benigni R; In this article, the relationship was studied between the in vivo mutagenicity assay of mouse bone marrow micronucleus (MIC), and four in vitro assays: Salmonella typhimurium, chromosomal aberrations in Chinese hamster ovary (CHO) cells, sister chromatid exchanges (SCE) in CHO cells, and mutation in mouse lymphoma L5178Y cells . A comparison with the rodent carcinogenicity data was also undertaken . The MIC data on 49 chemicals were generated by Shelby et al . (1993) . The MIC assay system employed three daily exposures by intraperitoneal injection; bone marrow samples were obtained 24 h following the final exposure . A preliminary analysis indicated that the 49 chemicals selected by Shelby et al . (1993) are a representative subset of the National Toxicology Program database . This study showed that MIC has a number of particular characteristics that are not shared by other biological systems . MIC is basically different from rodent carcinogenicity, despite being an in vivo system . At the same time, it responds to the chemicals in a different way from that of the in vitro genotoxicity assays . These in vitro assays mainly differ from each other in their different sensitivities to the genotoxins: MIC gives just a few positives (limited sensitivity), but, at the same time, some of these positives are detected only by the most sensitive assays, like the mouse lymphoma or SCE assays . In terms of risk assessment, MIC does not complement Salmonella for predicting chemical carcinogenicity, and would be better used to verify if the in vitro positive chemicals are able to exert their genotoxic potential in vivo. J Bacteriol, 1995 Jul, 177(14), 3985 - 91 Role of methylation in aerotaxis in Bacillus subtilis; Wong LS et al.; Taxis to oxygen (aerotaxis) in Bacillus subtilis was characterized in a capillary assay and in a temporal assay in which the concentration of oxygen in a flow chamber was changed abruptly . A strong aerophilic response was present, but there was no aerophobic response to high concentrations of oxygen . Adaptation to a step increase in oxygen concentration was impaired when B . subtilis cells were depleted of methionine to prevent methylation of the methyl-accepting chemotaxis proteins . There was a transient increase in methanol release when wild-type B . subtilis, but not a cheR mutant that was deficient in methyltransferase activity, was stimulated by a step increase or a step decrease in oxygen concentration . The methanol released was quantitatively correlated with demethylation of methyl-accepting chemotaxis proteins . This indicated that methylation is involved in aerotaxis in B . subtilis in contrast to aerotaxis in Escherichia coli and Salmonella typhimurium, which is methylation independent. J Bacteriol, 1995 Jul, 177(14), 3965 - 71 Homologs of the Shigella IpaB and IpaC invasins are required for Salmonella typhimurium entry into cultured epithelial cells; Kaniga K et al.; Entry into host cells is an essential feature in the pathogenicity of Salmonella spp . The inv locus of Salmonella typhimurium encodes several proteins which are components of a type III protein secretion system required for these organisms to gain access to host cells . We report here the identification of several proteins whose secretion into the culture supernatant of S . typhimurium is dependent on the function of the inv-encoded translocation apparatus . Nucleotide sequence analysis of the genes encoding two of these secreted proteins, SipB and SipC, indicated that they are homologous to the Shigella sp . invasins IpaB and IpaC, respectively . An additional gene was identified, sicA, which encodes a protein homologous to IpgC, a Shigella protein that serves as a molecular chaperone for the invasins IpaB and IpaC . Nonpolar mutations in sicA, sipB, and sipC rendered S . typhimurium unable to enter cultured epithelial cells, indicating that these genes are required for bacterial internalization. Mutat Res, 1995 Jul, 329(2), 205 - 12 Emodin inhibits the mutagenicity and DNA adducts induced by 1-nitropyrene; Su HY et al.; Polygonum cuspidatum S . (PC) is frequently used as a laxative and an anticancer drug in Chinese medicine . The inhibitory effect of this herb and its component, emodin, on the direct-acting mutagenicity of 1-nitropyrene (1-NP) was examined using the Ames/microsomal test with Salmonella typhimurium TA98 and the genotoxicity of 1-NP was evaluated using the SOS chromotest with E . coli PQ37 . Emodin and water extracts of PC markedly decreased the mutagenicity of 1-NP in a dose-dependent manner in both assay systems . Furthermore, emodin and the extracts of PC significantly inhibited the formation of 1-NP DNA adducts in S . typhimurium TA98 in the 32P-postlabeling study . The results suggest that PC extracts and emodin act as blocking and/or suppressing agents to reduce the direct-acting mutagenicity of 1-NP. Eur J Pharmacol, 1995 Jul 1, 293(2), 173 - 81 Activation of benzylic alcohols to mutagens by rat and human sulfotransferases expressed in Escherichia coli; Glatt H et al.; Human hydroxysteroid sulfotransferase, human phenol-sulfating form of phenol sulfotransferase, rat hydroxysteroid sulfotransferase a and rat phenol sulfotransferase IV were expressed in Escherichia coli . Cytosol preparations of transformed bacteria were used as activating systems in mutagenicity tests with Salmonella typhimurium TA98 . All test compounds, 1-hydroxymethylpyrene, 2-hydroxymethylpyrene, 1-(1-pyrenyl)ethanol, 9-hydroxymethylanthracene, 7-hydroxymethyl-12-methylbenz{a}anthracene and 4H-cyclopenta{def}chrysen-4-ol, were activated by both hydroxysteroid sulfotransferases investigated . However, 1-(1-pyrenyl)ethanol was 67-fold more efficiently activated by the human enzyme, whereas 7-hydroxymethyl-12-methylbenz{a}anthracene was 27-fold more efficiently activated by the rat enzyme . The phenol sulfotransferases showed relatively low activities with the benzylic alcohols investigated . The only exception was 4H-cyclopenta{def}chrysen-4-ol, which was activated efficiently by rat phenol sulfotransferase IV . We had previously tested the ability of rat and human hepatic cytosol preparations to activate the same compounds . The results of a statistical analysis suggest that the activities of human hydroxysteroid sulfotransferase, rat hydroxysteroid sulfotransferase a and phenol sulfotransferase IV can account for a substantial portion of the activation of benzylic alcohols in human, female rat and male rat liver, respectively. Clin Microbiol Rev, 1995 Jul, 8(3), 336 - 56 Recovery of uncommon bacteria from blood: association with neoplastic disease; Beebe JL et al.; Table 6 is a summary of the organisms discussed with a listing of the environmental source, the endogenous source, the predisposing factors including neoplasms, and the postulated mechanisms by which the organism can gain access to the circulation . The evidence considered indicates that the entrance of one of these microorganisms into the bloodstream of a human being depends on the presence of multiplicity of predisposing factors . In the majority of cases of bacteremia due to one of these unusual organisms, two or more predisposing factors are present . Certain predisposing factors, such as cancer chemotherapy or intravenous catheterization, often provide a barrier break, while others, such as liver disease, may render the host immune system less capable of clearing organisms from the circulation . For organisms such as Campy-lobacter, Listeria, and Salmonella spp., attributes that allow the invasion of a healthy host are present and seem to be enhanced by the simultaneous presence of a predisposing condition, such as liver disease, in the host . Although somewhat fragmentary, a number of individual case reports describe bacteremia due to one of these organisms occurring weeks to years after surgery and after other therapeutic measures had effected a supposed cure of a cancer . It may be speculated that cancer patients, even after a cure, are still susceptible to bloodstream invasion by one of the aforementioned organisms by virtue of the presence of one or more predisposing metabolic, physiologic, or immunologic factors, even though these factors may be cryptic . The predominance of hematologic malignancies among cases of bacteremia due to these unusual organisms is also apparent . Although, as pointed out by Keusch (169), the reduction in the performance of immune function in hematologic malignancies compared with solid tumors is likely to be responsible, other associations of certain organisms with specific neoplasms warrant further examination . The frequency of bloodstream infections of Salmonella typhimurium and Capno-cytophaga canimorsus in Hodgkin's disease patients seems likely due to a particular mechanism which infection by these species is favored . The specific nature of these mechanisms remains to be determined . The recovery of any unusual bacterium from blood should warrant a careful consideration of the possibility of underlying disease, especially cancer . Microbiologists should advise clinicians of the unusual nature of the identified organism and provide the counsel that certain neoplastic processes, often accompanied by neutropenia, render the human host susceptible to invasion by almost any bacterium . The recovery of such organisms as C . septicum or S . bovis should prompt the clinician to aggressively seek to identify an occult neoplasm if one has not yet been diagnosed. Microbiology, 1995 Jul, 141 ( Pt 7), 1715 - 22 Functional analysis of the flagellar genes in the fliD operon of Salmonella typhimurium; Yokoseki T et al.; The fliD genes of Salmonella typhimurium and Escherichia coli encode the filament-cap protein of the flagellar apparatus, which facilitates the polymerization of endogenous flagellin at the tips of the growing filaments . Previous sequence analysis of this operon in both organisms has revealed that the fliD gene constitutes an operon together with two additional genes, fliS and fliT . Based on the gene-disruption experiment in E . coli, both the fliS and fliT genes have been postulated to be necessary for flagellation . In the present study, we constructed S . typhimurium mutants in which either fliS or fliT on the chromosome was specifically disrupted . Both mutants were found to produce functional flagella, indicating that these genes are dispensable for motility development in S . typhimurium . However, flagellar filaments produced by the fliS mutant were much shorter than those produced by the wild-type strain . This indicates that the fliS mutation affects the elongation step of filament assembly . The excretion efficiency of flagellin was examined in the fliD-mutant background, where the exported flagellin molecules cannot assemble onto the hooks, resulting in their excretion into the culture media . We found that the amount of flagellin excreted was much reduced by the fliS mutation . Based on these results, we conclude that FliS facilitates the export of flagellin through the flagellum-specific export pathway. Microbiology, 1995 Jul, 141 ( Pt 7), 1655 - 61 A homologue to the Escherichia coli alkyl hydroperoxide reductase AhpC is induced by osmotic upshock in Staphylococcus aureus; Armstrong-Buisseret L et al.; Four major proteins are induced in Staphylococcus aureus in response to hyperosmotic shock caused by the presence of two different osmolytes, sucrose and NaCl . The gene encoding one of these proteins was isolated using a novel PCR procedure . The derived protein sequence shows extensive similarity to a subunit of alkyl hydroperoxide reductase (AhpC) from both Escherichia coli and Salmonella typhimurium . Exposure of S . aureus to varying concentrations of H202 did not result in the detectable induction of AhpC. Turk J Pediatr, 1995 Jul-Sep, 37(3), 229 - 33 Prognostic factors in Salmonella typhimurium septicemia . A 10-year retrospective study; Secmeer G et al.; In this study, 74 S.typhimurium septicemia cases were evaluated retrospectively from their records, and the age and sex distribution, presence of underlying disease, signs and symptoms, complete blood count, liver function tests and case fatality rate were documented and prognostic factors determined . It has been shown that S.typhimurium is the most common strain causing Salmonella septicemia, which is more fatal in the newborn period and in the presence of an associated disease, while hemoglobin and leukocyte counts do not play an important role in the prognosis . In Salmonella septicemia, congenital heart disease was the second-most common associated disease, which may be attributed to probable underlying immunodeficiency. Antimicrob Agents Chemother, 1995 Jul, 39(7), 1621 - 3 Salmonella typhimurium gyrA mutations associated with fluoroquinolone resistance; Reyna F et al.; Spontaneous quinolone-resistant mutants obtained from Salmonella typhimurium Su694 were screened for mutations by direct DNA sequencing of an amplified PCR gyrA fragment . Substitutions Ser-83-->Phe (Ser83Phe), Ser83Tyr, Asp87Tyr, and Asp87Asn and double mutation Ala67Pro-Gly81Ser, which resulted in decreased sensitivities to ciprofloxacin, enoxacin, pefloxacin, norfloxacin, ofloxacin, and nalidixic acid, were found . The levels of resistance to quinolones for each mutant were determined. Mutagenesis, 1995 Jul, 10(4), 357 - 64 Biotransformation of genotoxic agents in marine sponges . Mechanisms and modulation; De Flora S et al.; Marine sponges do not appear to suffer from neoplastic diseases, in spite of possible high exposures resulting from their nature as sessile bottom filter feeders which pump large volumes of sea water . The assessment of several parameters related to the biotransformation of mutagens/carcinogens showed that the metabolic machinery of sponge medulla cells is mainly oriented towards detoxification, with some differences depending on species (Geodia cydonium or Tethya aurantium) . Glutathione (GSH) levels were unexpectedly high in these cells, especially in Geodia, in which the concentration of this tripeptide was more than twice that measured in liver preparations from untreated rats, at least when related to the protein content . The oxidoreductive enzyme activities involved in the glutathione cycle were balanced in such a way as to favour a high GSH: oxidized glutathione (GSSG) ratio . GSH S-transferase activity was conversely rather low, compared to that of rat liver, and the dehydrogenases involved in the hexose monophosphate shunt were high in Tethya but low in Geodia . The metabolism of mutagens was investigated by using the Salmonella typhimurium his- strains TA100, TA98 and YG1024 . Sponge S12 fractions failed to activate aflatoxin B1, benzo{a}pyrene and the two heterocyclic amines 3-amino-1-methyl-5H-pyrido{4,3-b}indole and 2-amino-3,4-dimethyl-imidazo{4,5-f}quinoline . Although far less efficiently than untreated rat liver S12 fractions, Geodia and especially Tethya preparations weakly activated the three aromatic amines 2-acetyl-aminofluorene, 2-aminofluorene and 2-aminoanthracene . On the other hand, sponge S12 fractions were remarkably efficient in decreasing the mutagenic potency of the direct-acting mutagens 4-nitroquinoline 1-oxide and sodium dichromate.(ABSTRACT TRUNCATED AT 250 WORDS) Mutagenesis, 1995 Jul, 10(4), 343 - 51 Genotoxicity of 17 gyrase- and four mammalian topoisomerase II-poisons in prokaryotic and eukaryotic test systems; Albertini S et al.; The genotoxic potency of certain classes of topoisomerase II poisons is correlated with their affinity to the topoisomerase protein rather than with the presence of 'classical' structural alerts for DNA reactivity: bacterial topoisomerase II poisons (specifically named gyrase inhibitors) are highly genotoxic in prokaryotic systems; mammalian topoisomerase II poisons are potent mutagens/clastogens in eukaryotic systems . Studies with bacterial, lower eukaryotic and mammalian genotoxicity tests were performed to draw structure-activity conclusions and address risk-benefit considerations for the class of quinolone gyrase inhibitors . All 17 gyrase inhibitors investigated in this study showed genotoxic activity in Salmonella typhimurium strain TA102 and the SOS test . The genotoxic and the toxic activities increased in a highly parallel fashion from the parent compounds, nalidixic acid and oxolinic acid, to the new generation fluoroquinolones . Generally, the most potent fluoroquinolones also show clear-cut positive effects in eukaryotic test systems, although at concentrations 100-1000-fold higher than those effective in bacteria and also 100-1000-fold higher than the minimal genotoxic concentrations of antitumour topoisomerase II inhibitors (ellipticine, teniposide, mAMSA) used as reference compounds . However, subtle structural modifications of the quinolones can strongly diminish the preferential genotoxicity in the prokaryotic test systems. Mutagenesis, 1995 Jul, 10(4), 333 - 41 Use of the SOS chromotest, the Ames-fluctuation test and the newt micronucleus test to study the genotoxicity of four trihalomethanes; Le Curieux F et al.; Three short-term assays (the SOS chromotest, the Ames-fluctuation test and the newt micronucleus test) were carried out to evaluate the genotoxicity of four trihalomethanes (chloroform, bromodichloromethane, chlorodibromomethane and bromoform) . With the SOS chromotest, all the chemicals studied except chloroform were found to induce primary DNA damage in Escherichia coli PQ37 . In the Ames-fluctuation test, only bromoform showed mutagenic activity on Salmonella typhimurium strain TA100 . The newt micronucleus assay detected a clastogenic effect on the peripheral blood erythrocytes of Pleurodeles waltl larvae for bromodichloromethane and bromoform . It appeared that the presence of bromine substituent(s) generally led to significant genotoxic activity . Moreover, the use of the metabolic system significantly increased the genotoxicity of the brominated trihalomethanes in the SOS chromotest . Unlike previous investigations in which the SOS chromotest was always the least interesting assay, this study exhibited the good efficiency of this in vitro test on E.coli for the detection of trihalomethanes with bromine substituents. Mutagenesis, 1995 Jul, 10(4), 321 - 3 Lack of uniformity in the mutational spectra of chlorohydroxyfuranones in Salmonella typhimurium strain TA100; Hyttinen JM et al.; The mutational specificity of three chlorohydroxyfuranones found in chlorinated drinking water, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), 3-chloro-4(chloromethyl)-5-hydroxy-2(5H)-furanone (CMCF) and 3,4-dichloro-5-hydroxy-2(5H)-furanone (mucochloric acid, MCA), was examined in Salmonella typhimurium strain TA100 . DNA colony-hybridization of TA100 revertants showed that MX and CMCF both induced predominantly G:C-->T:A transversions (87 and 75% of total, respectively) with a 3:1 preference for the second position of the hisG46 (CCC) target codon . By contrast, MCA produced primarily G:C-->A:T transitions (66% of the total) with a 4:1 preference for the second position of the CCC codon . The mutational specificity of MCA is consistent with the idea that chloroacetaldehyde, a degradation product of MCA, is responsible for the observed mutations . The chemical mechanism by which either MX or CMCF induces G:C-->T:A transversions remains unknown. Mol Microbiol, 1995 Jul, 17(1), 49 - 56 Co-ordinate, temperature-sensitive regulation of the three Yersinia enterocolitica flagellin genes; Kapatral V et al.; Yersinia enterocolitica cells, when cultured at 30 degrees C or below, are flagellated and motile . Cells cultured at 37 degrees C or above lack flagella and are non-motile . To identify flagellin genes that are a target of this temperature-dependent regulation, a library of Y . enterocolitica genomic inserts in a phage lambda vector was probed with the Salmonella typhimurium fliC (flagellin) gene . A DNA fragment subcloned from a recombinant phage which hybridizes with the probe complements a non-motile S . typhimurium fliC-fljB- (flagellin-minus) mutant . DNA sequence analysis shows that Y . enterocolitica contains three tandem flagellin genes, designated fleA, fleB and fleC . All three genes are co-ordinately transcribed at low, but not high, temperature from fliA-dependent (sigma F) promoters . Flagellin transcription arrests rapidly after upshift to 37 degrees C (host temperature) . In contrast, flagellin transcription resumes only after several generations when cells cultured at 37 degrees C are downshifted to 28 degrees C. Mol Microbiol, 1995 Jul, 17(1), 169 - 81 PhoP/PhoQ transcriptional repression of Salmonella typhimurium invasion genes: evidence for a role in protein secretion; Pegues DA et al.; Previously, the PhoP-repressed locus prgH was identified as important for signalling epithelial cells to endocytose Salmonella typhimurium . Characterization of prgH revealed that it is an operon of four genes encoding polypeptides of 392 (prgH), 80 (prgI), 101 (prgJ) and 252 amino acid residues (prgK) . Synthesis of the 2.6 kb prgHIJK transcript was repressed in bacteria that activate PhoP/PhoQ, indicating that PhoP/PhoQ regulates prgHIJK by transcriptional repression . The prgI, prgJ and prgK predicted gene products were similar to Shigella flexneri and Yersinia enterocolitica proteins required for secretion of Ipa and Yop virulence factors . Analysis of the culture supernatants from wild-type S . typhimurium demonstrated that at least 25 polypeptides larger than 14 kDa could be detected . In contrast, prgH1::TnphoA, phoP-constitutive and hil-deletion mutants had significant defects in their supernatant protein profiles . The invasion and supernatant protein profile defects of the prgH1::TnphoA mutant were both complemented by a 5.1 kb plasmid that included prgHIJK . These results suggest that PhoP/PhoQ regulates extracellular transport of proteins by transcriptional repression of secretion determinants and that secreted proteins may be involved in signalling epithelial cells to endocytose bacteria. Mol Microbiol, 1995 Jul, 17(1), 155 - 67 The stationary-phase sigma factor sigma S (RpoS) is required for a sustained acid tolerance response in virulent Salmonella typhimurium; Lee IS et al.; The acid tolerance response (ATR) of log-phase Salmonella typhimurium is induced by acid exposures below pH 4.5 and will protect cells against more extreme acid . Two systems are evident: a transiently induced system dependent on the iron regulator Fur that provides a moderate degree of acid tolerance and a more effective sustained ATR that requires the alternate sigma factor sigma S encoded by rpoS . Differences between the acid responses of virulent S . typhimurium and the attenuated laboratory strain LT2 were attributed to disparate levels of RpoS caused by different translational starts . The sustained ATR includes seven newly identified acid shock proteins (ASPs) that are dependent upon sigma S for their synthesis . It is predicted that one or more of these ASPs is essential for the sustained system . The sustained ATR also provided cross-protection to a variety of other environmental stresses (heat, H2O2 and osmolarity); however, adaptation to the other stresses did not provide significant acid tolerance . Therefore, in addition to starvation, acid shock serves as an important signal for inducing general stress resistance . Consistent with this model, sigma S proved to be induced by acid shock . Our results also revealed a connection between the transient and sustained ATR systems . Mutations in the regulator atbR are known to cause the overproduction of ten proteins, of which one or more can suppress the acid tolerance defect of an rpoS mutant . One member of the AtbR regulon, designated atrB, was found to be co-regulated by sigma S and AtbR . Both regulators had a negative effect on atrB expression . The results suggest AtrB serves as a link between the sustained and transient ATR systems . When sigma S concentrations are low, a compensatory increase in AtrB is required to engage the transiently induced, RpoS-independent system of acid tolerance . Results also suggest different acid-sensitive targets occur in log-phase versus stationary-phase cells. Mol Gen Genet, 1995 Jun 25, 247(6), 680 - 92 RNA polymerase (rpoB) mutants selected for increased resistance to gyrase inhibitors in Salmonella typhimurium; Blanc-Potard AB et al.; Some rifampicin-resistance (RifR) mutations make bacteria slightly resistant to the gyrase inhibitors novobiocin (Nov) and nalidixic acid (Nal) . This suggested that it might be possible to isolate rpoB mutants using either drug for positive selection . In an initial test, we confirmed the presence of Rif-resistant isolates among clones selected for Nov resistance . These mutants are also more resistant to Nal . In a subsequent experiment, we found that mutants selected for low-level resistance to Nal include isolates harboring mutations genetically linked to the rpoB locus; of two such mutants studied, one is temperature-sensitive for growth . These two mutants, which are only marginally affected in their response to Nov, are normally sensitive to Rif and thus might be representative of a new class of rpoB alleles . The Rif-resistant and Rif-sensitive rpoB alleles that increase resistance to gyrase inhibitors have one property in common: they all suppress, to varying degrees, the defect in his operon regulation (transcriptional deattenuation) caused by a gyrase defect or inhibition by novobiocin . To further analyse the transcription-supercoiling relationships in these mutants, we examined the ability of RNA polymerase to recruit gyrase activity during transcription . This was done by two independent approaches: (i) observing transcription-induced accumulation of hyper-negatively supercoiled plasmid DNA in a topA mutant background and (ii) measuring transcription-induced plasmid DNA cleavage in the presence of oxolinic acid . Results indicate that the rpoB alleles described in this study diminish the recruitment of gyrase activity by the transcription process . This property correlates with a decrease in the rate of transcription initiation.(ABSTRACT TRUNCATED AT 250 WORDS) J Mol Biol, 1995 Jun 16, 249(4), 700 - 13 Constitutive forms of the enhancer-binding protein NtrC: evidence that essential oligomerization determinants lie in the central activation domain; Flashner Y et al.; Nitrogen regulatory protein C (NtrC) is a bacterial enhancer-binding protein that activates transcription by the sigma 54-holoenzyme . To activate transcription, NtrC must hydrolyze ATP, a reaction that depends upon its being phosphorylated and forming an appropriate oligomer . In this paper we characterize "constitutive" mutant forms of the NtrC protein from Salmonella typhimurium; unlike wild-type NtrC, these forms are able to hydrolyze ATP and activate transcription in vitro without being phosphorylated . The amino acids altered in NtrCconstitutive proteins are located in both the N-terminal regulatory domain and the central domain, which is directly responsible for transcriptional activation . The residues that are altered are not conserved among activators of the sigma 54-holoenzyme, and are not identical even among NtrC proteins from members of different subgroups of the proteobacteria (purple bacteria) . NtrCconstitutive proteins are phosphorylated normally; phosphorylation increases their ability to hydrolyze ATP and activate transcription . Moreover, the oligomerization of these proteins that occurs when they bind to an enhancer also increases the ATPase activity of both unmodified and phosphorylated forms . Removal of the N-terminal regulatory domain from two NtrCconstitutive proteins with amino acid substitutions in the central domain (NtrCS160F and NtrCV2881) leaves them active, indicating that essential oligomerization determinants lie outside the regulatory domain . This conclusion is confirmed by the observation that the ATPase activity of delta N-NtrCS160F is greatly stimulated when it binds to an enhancer, and by the ability of this protein to activate transcription synergistically with a form of NtrC incapable of DNA-binding . Together with previous results indicating that oligomerization determinants do not lie in the C-terminal DNA-binding domain of NtrC; these results provide evidence that they lie in the central domain. J Mol Biol, 1995 Jun 9, 249(3), 529 - 34 Cold denaturation of CheY; DeKoster GT et al.; The thermal stability of the bacterial chemotaxis protein CheY from Salmonella typhimurium has been examined by thermal denaturation at pH 7.0 in the presence of guanidine-HCl and urea . For both denaturants, thermal denaturation monitored by circular dichroism spectropolarimetry consists of transitions both above and below 25 degrees C, which is strong evidence for a heat capacity change that is > or = 1500 cal/(mol K) upon unfolding . While many data for chemical and thermal denaturation are consistent with data for CheY from Escherichia coli, the observation of cold denaturation for S . typhimurium CheY is inconsistent with the small heat capacity change, 600 to 850 cal/(mol K), reported for denaturation of the E . coli protein. J Natl Cancer Inst, 1995 Jun 7, 87(11), 836 - 41 Mutagens from heated Chinese and U.S . cooking oils; Shields PG et al.; BACKGROUND: The lung cancer incidence in Chinese women is among the highest in the world, but tobacco smoking accounts for only a minority of the cancers . Epidemiologic investigations of lung cancer among Chinese women have implicated exposure to indoor air pollution from wok cooking, where the volatile emissions from unrefined cooking oils are mutagenic . PURPOSE: This study was conducted to identify and quantify the potentially mutagenic substances emitted from a variety of cooking oils heated to the temperatures typically used in wok cooking . METHODS: Several cooking oils and fatty acids were heated in a wok to boiling, at temperatures (for the cooking oils) that ranged from 240 degrees C to 280 degrees C (typical cooking temperatures in Shanghai, China) . The oils tested were unrefined Chinese rapeseed, refined U.S . rapeseed (known as canola), Chinese soybean, and Chinese peanut in addition to linolenic, linoleic, and erucic fatty acids . Condensates of the emissions were collected and tested in the Salmonella mutation assay (using Salmonella typhimurium tester strains TA98 and TA104) . Volatile decomposition products also were subjected to gas chromatography and mass spectroscopy . Aldehydes were detected using high-performance liquid chromatography and UV spectroscopy . RESULTS: 1,3-Butadiene, benzene, acrolein, formaldehyde, and other related compounds were qualitatively and quantitatively detected, with emissions tending to be highest for unrefined Chinese rapeseed oil and lowest for peanut oil . The emission of 1,3-butadiene and benzene was approximately 22-fold and 12-fold higher, respectively, from heated unrefined Chinese rapeseed oil than from heated peanut oil . Lowering the cooking temperatures or adding an antioxidant, such as butylated hydroxyanisole, before cooking decreased the amount of these volatile emissions . Among the individual fatty acids tested, heated linolenic acid produced the greatest quantities of 1,3-butadiene, benzene, and acrolein . Separately, the mutagenicity of individual volatile emission condensates was correlated with linolenic acid content (r = .83; P = .0004) . Condensates from heated linolenic acid, but not linoleic or erucic acid, were highly mutagenic . CONCLUSIONS: These studies, combined with experimental and epidemiologic findings, suggest that high-temperature wok cooking with unrefined Chinese rapeseed oil may increase lung cancer risk . This study indicates methods that may reduce that risk . IMPLICATIONS: The common use of wok cooking in China might be an important but controllable risk factor in the etiology of lung cancer . In the United States, where cooking oils are usually refined for purity, additional studies should be conducted to further quantify the potential risks of such methods of cooking. Med Oncol, 1995 Jun, 12(2), 103 - 8 Immunotherapy of a plasmacytoma with attenuated salmonella; Eisenstein TK et al.; An attenuated strain of Salmonella typhimurium, SL3235, developed as a prototypic typhoid vaccine, is shown to retard growth of a murine plasmacytoma, TEPC-183, and to prolong survival of tumor-bearing mice . Live salmonella, but not acetone-killed organisms, had antitumor activity . The immunotherapeutic effect was demonstrable when the tumor was injected intralesionally or intraperitoneally . Increased survival, longer mean time to death, and retardation of tumor growth were found when the salmonella were given intralesionally as late as the sixth day post-tumor injection . Timing of salmonella inoculation, as well as the salmonella dose, had an effect on treatment efficacy . Injection of salmonella intraperitoneally exerted a strong antitumor effect when given as late as the third day post-tumor inoculation . The highest dose (2 x 10(6)) of salmonella was less effective than doses 10- or 100-fold lower . TEPC-183 plasmacytoma is rapidly growing and highly immunosuppressive, so the ability of the salmonella to exert therapeutic activity against it is a measure of the potency of the vaccine . These observations are of interest, as they show that a genetically engineered, avirulent strain of Salmonella has immunotherapeutic properties similar to those of BCG and other biological response modifiers, and might have clinical potential as an antitumor agent. Food Chem Toxicol, 1995 Jun, 33(6), 491 - 500 Evaluation of the genotoxicity potential and chronic inhalation toxicity of 1,1-dichloro-1-fluoroethane (HCFC-141b); Millischer RJ et al.; A battery of in vitro and in vivo tests were conducted on HCFC-141b as a vapour . Bacterial gene mutation assays with Escherichia coli and Salmonella typhimurium were negative in all tester strains . In vitro chromosomal aberration assays were positive on CHO cells but negative on human lymphocytes . Moreover, HCFC-141b was negative in vivo in a mouse micronucleus inhalation assay . On the basis of these data and previously reported genotoxicity testing, HCFC-141b is considered non-genotoxic . Groups of 80 male and 80 female Sprague-Dawley rats were exposed, by inhalation (6 hr/day, 5 days/wk) to vapours of HCFC-141b for 104 wk at target concentrations of 0 (control), 1500, 5000 and 20,000 ppm (increased from 15,000 ppm after 17 wk of exposure) . No exposure-related effects of toxicological significance were noted with respect to survival, clinical signs, ophthalmoscopy, haematology, clinical chemistry, urinalysis or organ weight analysis . Reduced food intake and body weight gain were noted in both sexes of the 15,000 ppm group during the first 16 wk; thereafter, body weight gains in all groups were similar although the intergroup differences in body weight remained evident . Reduced food intake persisted in both sexes through wk 52 and in females during the second year of exposure . Treatment-related effects on macroscopic pathology were confined to increased incidences of testicular masses and altered appearance . Microscopic pathology examinations confirmed the testes as the target organ with findings of increased incidences of benign interstitial cell tumours and hyperplasia at 5000 and 20,000 ppm . The no-observable-adverse-effect level (NOAEL) was 1500 ppm . The testicular changes at high exposure levels were considered to be due to a change of the senile hormonal imbalance in geriatric rats and of little significance for the assessment of human health effects. Invest Ophthalmol Vis Sci, 1995 Jun, 36(7), 1344 - 51 Systemic immunization with Hsp60 alters the development of chlamydial ocular disease; Rank RG et al.; PURPOSE: To determine whether immunization with recombinant Hsp60 would exacerbate ocular pathology on challenge with viable chlamydial elementary bodies . METHODS: Guinea pigs were immunized either subcutaneously with recombinant Hsp60 or both subcutaneously with recombinant Hsp60 and ocularly with attenuated Salmonella typhimurium expressing the guinea pig inclusion conjunctivitis (GPIC) Hsp60 antigen . All animals were challenged in the conjunctiva with the agent of GPIC, and the degree of gross ocular pathology was determined . Immunoglobulin G (IgG) and immunoglobulin A (IgA) antibody titers to Hsp60 were measured in ocular secretions as a measure of the degree of immunization . RESULTS: In primary and challenge GPIC infection, the degree of gross ocular pathology was lower in the immunized group . The presence of high IgA and IgG antibody titers to Hsp60 in tears suggested that the response may have been modified by the presence of blocking antibodies that either may have removed the antigen quickly or prevented interaction with sensitized T cells . In contrast to subcutaneous immunization, the combined immunization regimen, consisting of subcutaneous recombinant Hsp60 followed by ocular inoculation of the attenuated Salmonella, resulted in no difference in gross pathology after reinfection of guinea pigs with GPIC . CONCLUSIONS: These data indicated that the immunization with Hsp60 did not produce exacerbated disease on challenge with viable organisms; however, the data suggested that the route of administration, form of antigen, or both may be critical in the disease process. Mutat Res, 1995 Jun, 329(1), 1 - 9 Specific disruption of samAB genes in a 60-megadalton cryptic plasmid of Salmonella typhimurium; Nohmi T et al.; The expression of umuDC operon is required for UV and most chemical mutagenesis in Escherichia coli . The closely related species Salmonella typhimurium has two sets of umuDC-like operons, i.e., umuDCST in the chromosome and samAB in a 60-megadalton cryptic plasmid . In this study, we specifically disrupted the samAB genes to investigate their exact roles in UV mutagenesis in S . typhimurium . The specific gene disruption was carried out by the preligation method . Deletion of samAB did not lower the UV mutability of S . typhimurium TA2659 but rather increased the UV mutability about twofold . The samAB-umuDCST double deletion mutant as well as the umuDCST deletion mutant was UV nonmutable . These results suggest that the samAB genes do not considerably contribute to the UV mutability of S . typhimurium and raise the question of why such quiet umuDC-like genes are present in the cryptic plasmid of S . typhimurium. J Bacteriol, 1995 Jun, 177(12), 3485 - 95 Regulated underexpression of the FliM protein of Escherichia coli and evidence for a location in the flagellar motor distinct from the MotA/MotB torque generators; Tang H et al.; The FliM protein of Escherichia coli is essential for the assembly and function of flagella . Here, we report the effects of controlled low-level expression of FliM in a fliM null strain . Disruption of the fliM gene abolishes flagellation . Underexpression of FliM causes cells to produce comparatively few flagella, and most flagella built are defective, producing subnormal average torque and fluctuating rapidly in speed . The results imply that in a normal flagellar motor, multiple molecules of FliM are present and can function independently to some degree . The speed fluctuations indicate that stable operation requires most, possibly all, of the normal complement of FliM . Thus, the FliM subunits are not as fully independent as the motility proteins MotA and MotB characterized in earlier work, suggesting that FliM occupies a location in the motor distinct from the MotA/MotB torque generators . Several mutations in fliM previously reported to cause flagellar paralysis in Salmonella typhimurium (H . Sockett, S . Yamaguchi, M . Kihara, V.M . Irikura, and R . M . Macnab, J . Bacteriol . 174:793-806, 1992) were made and characterized in E . coli . These mutations did not cause flagellar paralysis in E . coli; their phenotypes were more complex and suggest that FliM is not directly involved in torque generation. J Bacteriol, 1995 Jun, 177(11), 3355 - 7 I-CeuI reveals conservation of the genome of independent strains of Salmonella typhimurium; Liu SL et al.; The enzyme I-CeuI, encoded by a class I mobile intron inserted in the gene for 23S rRNA in Chlamydomonas eugamatos, cleaves a specific 19-bp sequence in this gene . This sequence is present only in the seven genes for rRNA in Salmonella typhimurium and Escherichia coli . Partial digestion with I-CeuI of DNA from 17 wild-type strains of S . typhimurium indicates that the chromosome of these strains is strongly conserved, for the digestion products closely resemble those of strain LT2 . The lengths and order of chromosomal segments are conserved in 15 of the strains; 2 show some rearrangements . XbaI digestion indicated heterogeneity without revealing the genomic structure . Because of conservation of I-CeuI sites in genes for rRNA and conservation of the number and locations of these genes, I-CeuI provides an excellent tool for the rapid examination of the chromosomes of related species of bacteria; differences in the fingerprints indicate the occurrence of chromosomal rearrangements such as insertions or inversions. J Bacteriol, 1995 Jun, 177(11), 3326 - 31 Cloning and characterization of the Escherichia coli hemN gene encoding the oxygen-independent coproporphyrinogen III oxidase; Troup B et al.; Coproporphyrinogen III oxidase, an enzyme involved in heme biosynthesis, catalyzes the oxidative decarboxylation of coproporphyrinogen III to form protoporphyrinogen IX . Genetic and biochemical studies suggested the presence of two different coproporphyrinogen III oxidases, one for aerobic (HemF) and one for anaerobic (HemN) conditions . Here we report the cloning of the hemN gene encoding the oxygen-independent coproporphyrinogen III oxidase from Escherichia coli by complementation of a Salmonella typhimurium hemF hemN double mutant . An open reading frame of 1,371 bp encoding a protein of 457 amino acids with a calculated molecular mass of 52.8 kDa was identified . Sequence comparisons revealed 92% amino acid sequence identity to the recently cloned S . typhimurium hemN gene and 35% identity to the Rhodobacter sphaeroides gene . The hemN gene was mapped to 87.3 min of the E . coli chromosome and found identical to open reading frame o459 previously discovered during the genome sequencing project . Complementation of S . typhimurium hemF hemN double mutants with the E . coli hemN gene was detected under aerobic and anaerobic conditions, indicating an aerobic function for HemN . The previously cloned E . coli hemF gene encoding the oxygen-dependent enzyme complemented exclusively under aerobic conditions . Primer extension experiments revealed a strong transcription initiation site 102 bp upstream of the translational start site . DNA sequences with homology to a sigma 70-dependent promoter were detected . Expression of the hemN gene in response to changing environmental conditions was evaluated by using lacZ reporter gene fusions . Under anaerobic conditions, hemN expression was threefold greater than under aerobic growth conditions . Removal of iron from the growth medium resulted in an approximately fourfold decrease of aerobic hemN expression . Subsequent addition of iron restored normal expression. J Bacteriol, 1995 Jun, 177(11), 3259 - 68 Functional equivalence of Escherichia coli sigma E and Pseudomonas aeruginosa AlgU: E . coli rpoE restores mucoidy and reduces sensitivity to reactive oxygen intermediates in algU mutants of P . aeruginosa; Yu H et al.; Mucoid colony morphology is the result of the overproduction of the exopolysaccharide alginate and is considered to be a major pathogenic determinant expressed by Pseudomonas aeruginosa during chronic respiratory infections in cystic fibrosis . Conversion to mucoidy can be caused by mutations in the second or third gene of the stress-responsive system algU mucA mucB . AlgU is 66% identical to the alternative sigma factor RpoE (sigma E) from Escherichia coli and Salmonella typhimurium and directs transcription of several critical alginate biosynthetic and regulatory genes . AlgU is also required for the full resistance of P . aeruginosa to reactive oxygen intermediates and heat killing . In this work, we report that E . coli sigma E can complement phenotypic defects of algU inactivation in P . aeruginosa: (i) the rpoE gene from E . coli complemented an algU null mutant of P . aeruginosa to mucoidy; (ii) the presence of the E . coli rpoE gene in P . aeruginosa induced alginate production in the standard genetic nonmucoid strain PAO1; (iii) the plasmid-borne E . coli rpoE gene induced transcription of algD, a critical algU-dependent alginate biosynthetic gene; and (iv) when present in algU::Tcr mutants, E . coli rpoE partially restored resistance to paraquat, a redox cycling compound that increases intracellular levels of superoxide radicals . A new gene, mclA, encoding a polypeptide with an apparent molecular mass of 27.7 kDa was identified immediately downstream of rpoE in E . coli . The predicted product of this gene is 28% identical (72% similar) to MucA, a negative regulator of AlgU activity in P . aeruginosa . The results reported in this study demonstrate that RpoE and AlgU are functionally interchangeable in P . aeruginosa and suggest that elements showing sequence similarity to those known to regulate AlgU activity in P . aeruginosa are also present in other bacteria. J Bacteriol, 1995 Jun, 177(11), 3080 - 6 The superinfection exclusion gene (sieA) of bacteriophage P22: identification and overexpression of the gene and localization of the gene product; Hofer B et al.; Previous work has shown that the sieA gene of Salmonella bacteriophage P22 is located between the genes mnt and 16 . We cloned DNA fragments of the region into multicopy vectors and tested the transformants for mediating superinfection exclusion . Subcloning, phenotypical tests, and DNA sequencing resulted in the identification of the sieA gene . There are two possible initiation codons within one open reading frame of 492 or 480 bp . The deduced amino acid sequence leads to a hypothetical polypeptide with a calculated molecular mass of 18.8 or 18.3 kDa, respectively . According to three hydrophobic regions, all of which are long enough to span the membrane, the product of sieA should be a protein of the inner membrane of a P22-lysogenic cell of Salmonella typhimurium . The SieA protein was moderately overproduced from an expression vector in cultures of Escherichia coli and could be recovered from the membrane fraction. Infect Immun, 1995 Jun, 63(6), 2302 - 9 Transepithelial signaling to neutrophils by salmonellae: a novel virulence mechanism for gastroenteritis; McCormick BA et al.; Salmonella serotypes which elicit human enteritis cannot be distinguished from those that do not on the basis of their in vitro interactions with eukar