Salmonella typhimurium

J Mol Biol, 1999 Jun 11, 289(3), 517 - 27
A newly identified, essential catalytic residue in a critical secondary structure element in the integrase family of site-specific recombinases is conserved in a similar element in eucaryotic type IB topoisomerases; Cao Y et al.; The integrase family of site-specific recombinases catalyzes conservative rearrangements between defined segments of DNA . A highly conserved tetrad (RHRY) of catalytic residues is essential for this process . This tetrad is dispersed in two motifs in the linear sequence, but is configured appropriately in the catalytic pocket to execute the strand cleavage and rejoining reactions . A third conserved motif has been identified in the Xer subgroup of the integrase family . Mutational analysis of 12 conserved residues in this motif in the XerD protein from Salmonella typhimurium led to the identification of an essential fifth catalytic residue (lysine 172) which is implicated in strand cleavage or exchange . This lysine residue occupies part of the turn of an antiparallel beta-hairpin which forms one side of the catalytic cleft in XerD, and is found at similar positions among evolutionarily diverse integrase family members . Related antiparallel beta-hairpins are present in eucaryotic type IB topoisomerase enzymes which also contain a critical lysine residue in the turn of the hairpin . In both the integrase family and eucaryotic type IB topoisomerases, the catalytic lysine residues are in close contact with the substrates and may play similar roles in influencing the reactivity of the phosphotyrosine intermediates formed during reactions catalyzed by both enzymes .

Biochemistry, 1999 Jun 1, 38(22), 7363 - 71
Mutagenesis of histidinol dehydrogenase reveals roles for conserved histidine residues; Teng H et al.; The dimeric zinc metalloenzyme L-histidinol dehydrogenase (HDH) catalyzes an unusual four-electron oxidation of the amino alcohol histidinol via the histidinaldehyde intermediate to the acid product histidine with the reduction of two molecules of NAD . An essential base, with pKa about 8, is involved in catalysis . Here we report site-directed mutagenesis studies to replace each of the five histidine residues (His-98, His-261, His-326, His-366, and His-418) in Salmonella typhimurium with either asparagine or glutamine . In all cases, the overexpressed enzymes were readily purified and behaved as dimers . Substitution of His-261 and His-326 by asparagine caused about 7000- and 500-fold decreases in kcat, respectively, with little change in KM values . Similar loss of activity was also reported for a H261N mutant Brassica HDH {Nagai, A., and Ohta, D . (1994) J . Biochem . 115, 22-25} . Kinetic isotope effects, pH profiles, substrate rescue, and stopped-flow experiments suggested that His-261 and His-326 are involved in proton transfers during catalysis . Sensitivity to metal ion chelator and decreased affinities for metal ions with substitutions at His-261 and His-418 suggested that these two residues are candidates for zinc ion ligands.

Biochemistry, 1999 Jun 1, 38(22), 7355 - 62
Mechanism of Salmonella typhimurium histidinol dehydrogenase: kinetic isotope effects and pH profiles; Grubmeyer C et al.; L-Histidinol dehydrogenase catalyzes the biosynthetic oxidation of L-histidinol to L-histidine with sequential reduction of two molecules of NAD . Previous isotope exchange results had suggested that the oxidation of histidinol to the intermediate histidinaldehyde occurred 2-3-fold more rapidly than overall catalysis . In this work, we present kinetic isotope effects (KIE) studies at pH 9.0 and at pH 6.7 with stereospecifically mono- and dideuterated histidinols . The data at pH 9.0 support minimal participation of the first hydride transfer and substantial participation of the second hydride transfer in the overall rate limitation . Stopped-flow experiments with protiated histidinol revealed a small burst of NADH production with stoichiometry of 0.12 per subunit, and 0.25 per subunit with dideuterated histidinol, indicating that the overall first half-reaction was not significantly faster than the second reaction sequence . Results from kcat and kcat/KM titrations with histidinol, NAD, and the alternative substrate imidazolyl propanediol demonstrated an essential base with pKa values between 7.7 and 8.4 . In KIE experiments performed at pH 6.7 or with a coenzyme analogue at pH 9 . 0, the first hydride transfer became more rate limiting . Kinetic simulations based on rate constants estimated from this work fit well with a mechanism that includes a relatively fast, and thermodynamically unfavorable, hydride transfer from histidinol and a slower, irreversible second hydride transfer from a histidinaldehyde derivative . Thus, although the chemistry of the first hydride transfer is fast, both partial reactions participate in the overall rate limitation.

Biochim Biophys Acta, 1999 May 18, 1431(2), 374 - 83
Interaction of FliI, a component of the flagellar export apparatus, with flagellin and hook protein; Silva-Herzog E et al.; FliI is a key component of the flagellar export apparatus in Salmonella typhimurium . It catalyzes the hydrolysis of ATP which is necessary for flagellar assembly . Affinity blotting experiments showed that purified flagellin and hook protein, two flagellar axial proteins, interact specifically with FliI . The interaction of either of the two proteins with FliI, increases the intrinsic ATPase activity . The presence of either flagellin or hook protein stimulates ATPase activity in a specific and reversible manner . A Vmax of 0.12 nmol Pi min-1 microgram-1 and a Km for MgATP of 0.35 mM was determined for the unstimulated FliI; the presence of flagellin increased the Vmax to 0.35 nmol Pi min-1 microgram-1 and the Km for MgATP to 1.1 mM . The stimulation induced by the axial proteins was fully reversible suggesting a direct link between the catalytic activity of FliI and the export process.

Arch Toxicol, 1999 Mar, 73(2), 71 - 9
Enzyme-mediated dichloromethane toxicity and mutagenicity of bacterial and mammalian dichloromethane-active glutathione S-transferases; Gisi D et al.; The kinetic properties of bacterial and rat liver glutathione S-transferases (GST) active with dichloromethane (DCM) were compared . The theta class glutathione S-transferase (rGSTTI-1) from rat liver had an affinity for dihalomethanes lower by three orders of magnitude (K(app) > 50 mM) than the bacterial DCM dehalogenase/GST from Methylophilus sp . DM11 . Unlike the bacterial DCM dehalogenase, the rat enzyme was unable to support growth of the dehalogenase minus Methylobacterium sp . DM4-2cr mutant with DCM . Moreover, the presence of DCM inhibited growth with methanol of the DM4-2cr transconjugant expressing the rat liver GSTT1-1 . In Salmonella typhimurium TA1535, expression of rat and bacterial DCM-active GST from a plasmid in the presence of DCM yielded up to 5.3 times more reversions to histidine prototrophy in the transconjugant expressing the rat enzyme . Under the same conditions, however, GST-mediated conversion of DCM to formaldehyde was lower in cell-free extracts of the transconjugant expressing the rat GSTT1 than in the corresponding strain expressing the bacterial DCM dehalogenase . This provided new evidence that formaldehyde was not the main toxicant associated with GST-mediated DCM conversion, and indicated that an intermediate in the transformation of DCM by GST, presumably S-chloromethylglutathione, was responsible for the observed effects . The marked differences in substrate affinity of rat and bacterial DCM-active GST, as well as in the toxicity and genotoxicity associated with expression of these enzymes in bacteria, suggest that bacterial DCM dehalogenases/GST have evolved to minimise the toxic effects associated with glutathione-mediated catalysis of DCM conversion.

J Toxicol Sci, 1999 May, 24(2), 87 - 94
Mutagenicity tests of 4-phenyl-1,3-dithia-2-thioxo-cyclopent-4-ene; Sadanobu S et al.; The mutagenicity of 4-phenyl-1,3-dithia-2-thioxo-cyclopent-4-ene (DT827B) was examined in reverse mutation tests using Salmonella typhimurium and Escherichia coli, in the chromosomal aberration test with Chinese hamster ovary (CHO) cells, and in the micronucleus test using mice bone-marrow . In reverse mutation assay on DT827B according to Ames' method, DT827B was not mutagenic to S . typhimurium or E . coli when tested in dimethylsulfoxide to the limit of its solubility where precipitation occurred . In chromosomal aberration assay using CHO cells, DT827B was not clastogenic to induce structural chromosomal aberration but capable of inducing polyploidy . In micronucleus test, DT827B did not show micronucleus-inducing potential at the maximum dose . In conclusion of the three mutagenicity studies, DT827B was considered to cause no mutagenicity under the conditions used in the present experiments except the increase in polyploidy, which probably is due to a toxic effect of the compound.

J Bacteriol, 1999 Jun, 181(11), 3610 - 2
Salmonella typhimurium IroN and FepA proteins mediate uptake of enterobactin but differ in their specificity for other siderophores; Rabsch W et al.; Salmonella typhimurium possesses two outer membrane receptor proteins, IroN and FepA, which have been implicated in the uptake of enterobactin . To determine whether both receptors have identical substrate specificities, fepA and iroN mutants and a double mutant were characterized . While both receptors transported enterobactin, the uptake of corynebactin and myxochelin C was selectively mediated by IroN and FepA, respectively.

J Bacteriol, 1999 Jun, 181(11), 3351 - 7
Mutant forms of Salmonella typhimurium sigma54 defective in transcription initiation but not promoter binding activity; Kelly MT et al.; Transcription initiation with sigma54-RNA polymerase holoenzyme (sigma54-holoenzyme) has absolute requirements for an activator protein and ATP hydrolysis . sigma54's binding to core RNA polymerase and promoter DNA has been well studied, but little is known about its role in the subsequent steps of transcription initiation . Following random mutagenesis, we isolated eight mutant forms of Salmonella typhimurium sigma54 that were deficient in transcription initiation but still directed sigma54-holoenzyme to the promoter to form a closed complex . Four of these mutant proteins had amino acid substitutions in region I, which had been shown previously to be required for sigma54-holoenzyme to respond to the activator . From the remaining mutants, we identified four residues in region III which when altered affect the function of sigma54 at some point after closed-complex formation . These results suggest that in addition to its role in core and DNA binding, region III participates in one or more steps of transcription initiation that follow closed-complex formation.

Braz J Med Biol Res, 1999 Feb, 32(2), 241 - 6
New vaccine strategies against enterotoxigenic Escherichia coli . II: Enhanced systemic and secreted antibody responses against the CFA/I fimbriae by priming with DNA and boosting with a live recombinant Salmonella vaccine; Lasaro MO et al.; The induction of systemic (IgG) and mucosal (IgA) antibody responses against the colonization factor I antigen (CFA/I) of enterotoxigenic Escherichia coli (ETEC) was evaluated in mice primed with an intramuscularly delivered CFA/I-encoding DNA vaccine followed by two oral immunizations with a live recombinant Salmonella typhimurium vaccine strain expressing the ETEC antigen . The booster effect induced by the oral immunization was detected two weeks and one year after the administration of the DNA vaccine . The DNA-primed/Salmonella-boosted vaccination regime showed a synergistic effect on the induced CFA/I-specific systemic and secreted antibody levels which could not be attained by either immunization strategy alone . These results suggest that the combined use of DNA vaccines and recombinant Salmonella vaccine strains can be a useful immunization strategy against enteric pathogens.

Appl Environ Microbiol, 1999 Jun, 65(6), 2396 - 401
Lethality of a heat- and phosphate-catalyzed glucose by-product to Escherichia coli O157:H7 and partial protection conferred by the rpoS regulon; Byrd JJ et al.; A by-product of glucose produced during sterilization (121 degrees C, 15 lb/in2, 15 min) at neutral pH and in the presence of phosphate (i.e., phosphate-buffered saline) was bactericidal to Escherichia coli O157:H7 (ATCC 43895) . Other six-carbon (fructose and galactose) and five-carbon (arabinose, ribose, and xylose) reducing sugars also produced a toxic by-product under the same conditions . Fructose and the five-carbon sugars yielded the most bactericidal activity . Glucose concentrations of 1% (wt/vol) resulted in a 99.9% decline in the CFU of stationary-phase cells per milliliter in 2 days at 25 degrees C . An rpoS mutant (pRR10::rpoS) of strain 43895 (FRIK 816-3) was significantly (P < 0.001) more sensitive to the glucose-phosphate by-product than the parent strain, as glucose concentrations from 0.05 to 0.25% resulted in a 2- to 3-log10 reduction in CFU per milliliter in 2 days at 25 degrees C . Likewise, log-phase cells of the wild-type strain, 43895, were significantly more sensitive (P < 0.001) to the glucose-phosphate by-product than were stationary-phase cells, which is consistent with the stability of rpoS and the regulation of rpoS-regulated genes . The bactericidal effect of the glucose-phosphate by-product was reduced when strains ATCC 43895 and FRIK 816-3 were incubated at a low temperature (4 degrees C) . Also, growth in glucose-free medium (i.e., nutrient broth) did not alleviate the sensitivity to the glucose-phosphate by-product and excludes the possibility of substrate-accelerated death as the cause of the bactericidal effect observed . The glucose-phosphate by-product was also bactericidal to Salmonella typhimurium, Shigella dysenteriae, and a Klebsiella sp . Attempts to identify the glucose-phosphate by-product were unsuccessful . These studies demonstrate the production of a glucose-phosphate by-product bactericidal to E . coli O157:H7 and the protective effects afforded by rpoS-regulated gene products . Additionally, the detection of sublethally injured bacteria may be compromised by the presence of this by-product in recovery media.

Electrophoresis, 1999 Apr-May, 20(4-5), 813 - 7
Regulation of virulence genes by environmental signals in Salmonella typhimurium; Deiwick J et al.; Sensing and responding to environmental signals is a crucial element of bacterial pathogenicity . For a successful progression of infection, virulence gene expression is coordinated in response to habitat-specific environmental signals from the host organism . We are interested in identifying environmental cues affecting the expression of genes within Salmonella Pathogenicity Island 2 (SPI2), a virulence locus important for systemic infections by S . typhimurium . We describe our approach starting with the identification of new virulence genes, and analysis of the regulation of these genes by environmental signals leading to the proteome analysis in order to define the SPI2 regulon.

Prev Vet Med, 1999 May 14, 40(1), 1 - 17
Generalised linear mixed models analysis of risk factors for contamination of Danish broiler flocks with Salmonella typhimurium; Chriel M et al.; We present a retrospective observational study of risk factors associated with the occurrence of Salmonella typhimurium (ST) in Danish broiler flocks . The study is based on recordings from 1994 in the ante-mortem database maintained by the Danish Poultry Council . The epidemiological units are the broiler flocks (about 4000 flocks) which are clustered within producers . Broiler flocks with ST-infected parent stocks show increased risk of salmonella infection, and also the hatchery affects the salmonella status significantly . Among the rearing factors, only the use of medicine as well as the time of rearing, and the sampling method are significant . Epidemiological control would seem most efficient on starting at the top levels of the production hierarchy from which a major part of the ST contamination is derived . A secondary purpose of the study is to evaluate different statistical approaches and software for the analysis of a moderately-sized data set of veterinary origin . We compare the results from five analyses of the generalised linear mixed model (GLMM) type . The first observation is that the results agree reasonably well and lead to similar conclusions . A closer look reveals certain patterns of bias and estimation accuracy that correspond well with theoretical findings and practical experience reported in the statistical literature.

Microb Pathog, 1999 Jun, 26(6), 299 - 305
Comparison of the abilities of Salmonella typhimurium rpoS, aroA and rpoS aroA strains to elicit humoral immune responses in BALB/c mice and to cause lethal infection in athymic BALB/c mice; Coynault C et al.; Salmonella typhimurium rpoS and rpoS aroA mutants are effective live vaccines in the murine model of salmonellosis (Coynault et al., Mol . Microbiol . 1996; 22: 149-60) . Here, we further investigate the characteristics of these vaccines . The systemic humoral response induced by S . typhimurium rpoS, aroA and rpoS aroA vaccine candidates against S . typhimurium LPS was studied by ELISA . In BALB/c mice, the rpoS aroA strain induced a systemic anti-LPS humoral response similar to that induced by the rpoS and aroA strains . The virulence of aroA and rpoS aroA vaccines in nude (nu/nu) BALB/c mice was also compared . Salmonella typhimurium aroA and rpoS aroA vaccines both produced slowly progressing lethal infections in athymic mice inoculated i.p . but the rpoS aroA strain was more attenuated than the aroA strain, as determined by time to death and bacterial counts in spleens . Finally, a rpoS mutant of Salmonella dublin conferred protection in mice against an oral challenge with a wild-type strain of S . dublin whereas a rpoS mutant of S . typhimurium did not . This suggests that the protection provided by the S . typhimurium rpoS vaccine is serotype-dependent .

JAMA, 1999 May 19, 281(19), 1811 - 6
Investigation of multidrug-resistant Salmonella serotype typhimurium DT104 infections linked to raw-milk cheese in Washington State; Villar RG et al.; CONTEXT: Multidrug-resistant Salmonella Typhimurium DT104 has recently emerged as a cause of human and animal illness in Europe and North America . In early 1997, health officials in Yakima County, Washington, noted a 5-fold increase in salmonellosis among the county's Hispanic population . OBJECTIVES: To characterize bacterial strains and identify risk factors for infection with Salmonella Typhimurium in Yakima County . DESIGN: Laboratory, case-control, and environmental investigations . SETTING AND PARTICIPANTS: Patients with culture-confirmed Salmonella Typhimurium infection living in Yakima County and age- and neighborhood-matched control subjects . MAIN OUTCOME MEASURES: Food vehicle implication based on case-control study and outbreak control . RESULTS: Between January 1 and May 5, 1997, 54 culture-confirmed cases of Salmonella Typhimurium were reported . The median age of patients was 4 years and 91% were Hispanic . Patients reported diarrhea (100%), abdominal cramps (93%), fever (93%), bloody stools (72%), and vomiting (53%); 5 patients (9%) were hospitalized . Twenty-two patients and 61 control subjects were enrolled in the case-control study . Seventeen case patients (77%) reported eating unpasteurized Mexican-style soft cheese in the 7 days before onset of illness compared with 17 control subjects (28%) (matched odds ratio, 32.3; 95% confidence interval, 3.0-874.6) . All case-patient isolates were phage definitive type 104 (DT104) (n = 10) or DT104b (n = 12), and 20 (91%) were resistant to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline . The cheese produced and eaten by 2 unrelated patients was made with raw milk traced to the same local farm . Milk samples from nearby dairies yielded Salmonella Typhimurium DT104 . The incidence of Salmonella Typhimurium infections in Yakima County returned to pre-1992 levels following interventions based on these findings . CONCLUSIONS: Multidrug-resistant Salmonella Typhimurium DT104 emerged as a cause of salmonellosis in Yakima County, and Mexican-style soft cheese made with unpasteurized milk is an important vehicle for Salmonella Typhimurium DT104 transmission . We postulate that recent increases in human salmonellosis reflect the emergence of Salmonella Typhimurium DT104 among dairy cows in the region . Continued efforts are needed to discourage consumption of raw milk products, promote healthier alternatives, and study the ecology of multidrug-resistant Salmonella Typhimurium.

JAMA, 1999 May 19, 281(19), 1805 - 10
Two outbreaks of multidrug-resistant Salmonella serotype typhimurium DT104 infections linked to raw-milk cheese in Northern California; Cody SH et al.; CONTEXT: Salmonella serotype Typhimurium definitive type 104 (DT104), with resistance to 5 drugs (ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline), has emerged as the most common multidrug-resistant Salmonella strain in the United States . However, illnesses resulting from this strain have not been associated definitively with a source in this country . OBJECTIVE: To determine the source of 2 outbreaks of Salmonella Typhimurium DT104 . DESIGN: Matched case-control study conducted between March 24 and April 5, 1997 (outbreak 1), enhanced surveillance for new cases dating from February 1, 1997 (outbreak 2), and environmental and laboratory investigations . SETTING AND PARTICIPANTS: The case-control study included residents of 2 adjacent counties in northern California infected with the outbreak strain of Salmonella Typhimurium var Copenhagen and age-matched controls . For enhanced surveillance, a case was defined as Salmonella Typhimurium infection in a person exposed to fresh Mexican-style cheese . MAIN OUTCOME MEASURES: Risk factors for infection and source of implicated food . RESULTS: Outbreak 1 peaked in February 1997; 31 patients were confirmed by culture as having Salmonella Typhimurium var Copenhagen infection, isolates of which showed indistinguishable pulsed-field gel electrophoresis (PFGE) patterns . The outbreak strain was phage type DT104 with the 5-drug resistance pattern . Sixteen cases and 25 controls were enrolled in the case-control study; 15 of 16 Salmonella Typhimurium var Copenhagen cases compared with 14 of 24 matched controls reported eating unpasteurized Mexican-style cheese, (matched odds ratio, 7.9; 95% confidence interval, 1.1-354.9) . Enhanced surveillance uncovered outbreak 2, which peaked in April 1997 and was caused by a non-Copenhagen variant of Salmonella Typhimurium . During outbreak 2, Salmonella Typhimurium was isolated from 79 persons who ate fresh Mexican-style cheese from street vendors and from cheese samples and raw milk . The PFGE pattern of the milk isolate matched 1 of the 3 patterns recovered from patients; all strains were phage type DT104b with the 5-drug resistance pattern . CONCLUSION: Raw-milk products pose a risk for multidrug-resistant Salmonella Typhimurium DT104 infections.

Regul Toxicol Pharmacol, 1999 Apr, 29(2 Pt 2), S36 - 42
Genotoxicity tests on D-tagatose; Kruger CL et al.; D-tagatose is a low-calorie sweetener that tastes like sucrose . Its genotoxic potential was examined in five standard assays: the Ames Salmonella typhimurium reverse mutation assay, the Escherichia coli/mammalian microsome assay, a chromosomal aberration assay in Chinese hamster ovary cells, a mouse lymphoma forward mutation assay, and an in vivo mouse micronucleus assay . D-tagatose was not found to increase the number of revertants per plate relative to vehicle controls in either the S . typhimurium tester strains or the WP2uvrA- tester strain with or without metabolic activation at doses up to 5000 microg/plate . No significant increase in Chinese hamster ovary cells with chromosomal aberrations was observed at concentrations up to 5000 microg/ml with or without metabolic activation . D-tagatose was not found to increase the mutant frequency in mouse lymphoma L5178Y cells with or without metabolic activation up to concentrations of 5000 microg/ml . D-tagatose caused no significant increase in micronuclei in bone marrow polychromatic erythrocytes at doses up to 5000 mg/kg . D-tagatose was not found to be genotoxic under the conditions of any of the assays described above .

Regul Toxicol Pharmacol, 1999 Apr, 29(2 Pt 1), 205 - 10
Genotoxicity evaluation of wood-derived and vegetable oil-derived stanol esters; Turnbull D et al.; Plant stanol esters from wood and vegetable oil sources were tested for genotoxicity in bacterial (Salmonella typhimurium) and mammalian cell (L5178Y) gene mutation assays and in a mammalian cell chromosome aberration assay (CHO cells) . The two stanol ester formulations were tested separately at doses up to the limit of solubility, with and without the addition of an Aroclor-induced rat liver microsome metabolic activation system (S9 mix) . All tests were performed in duplicate and gave negative results for both wood and vegetable oil stanol ester formulations . Thus, plant stanol esters are not genotoxic under the conditions of exposure tested .

J Food Prot, 1999 May, 62(5), 474 - 9
The effectiveness of triclosan-incorporated plastic against bacteria on beef surfaces; Cutter CN; Triclosan is a nonionic, broad-spectrum, antimicrobial agent that has been incorporated into a variety of personal hygiene products, including hand soaps, deodorants, shower gels, mouthwashes, and toothpastes . In this study, plastic containing 1,500 ppm of triclosan was evaluated in plate overlay assays and meat experiments as a means of reducing populations of bacteria . Plate overlay assays indicated that the triclosan-incorporated plastic (TIP) inhibited the following organisms: Brochothrix thermosphacta ATCC 11509, Salmonella Typhimurium ATCC 14028, Staphylococcus aureus ATCC 12598, Bacillus subtilis ATCC 6051, Shigella flexneri ATCC 12022, Escherichia coli ATCC 25922, and several strains of E . coli O157:H7 . In meat experiment 1, irradiated, lean beef surfaces inoculated with B . thermosphacta, Salmonella Typhimurium, E . coli O157:H7, or B . subtilis were covered with TIP, vacuum packaged, and stored for 24 h at 4 degrees C . Of the organisms tested, only populations of B . thermosphacta were slightly reduced . In meat experiment 2, prerigor beef surfaces were inoculated with E . coli O157: H7, Salmonella Typhimurium, or B . thermosphacta incubated at 4 degrees C for 24 h, wrapped in TIP or control plastic, vacuum packaged, and stored at 4 degrees C for up to 14 days . There was a slight reduction in the population of the organisms after initial application with TIP . However, bacterial populations following long-term, refrigerated (4 degrees C), vacuum-packaged storage up to 14 days were not statistically (P< or =0.05) or numerically different than controls . In meat experiment 3, even TIP-wrapped, vacuum-packaged beef samples that were temperature abused at 12 degrees C did not exhibit significant (P< or =0.05) or sustainable reductions after 14 days of 4 degrees C storage . Another study indicated that populations of E . coli O157:H7 or B . thermosphacta added directly to TIP were not affected after 2 h of refrigerated storage or that the antimicrobial activity could be extracted from the plastic . Additional experiments suggest that presence of fatty acids or adipose may diminish the antimicrobial activity of TIP on meat surfaces . This study demonstrates that while antimicrobial activity is detected against bacterial cultures in antimicrobial plate assays, plastic containing 1,500 ppm of triclosan does not effectively reduce bacterial populations on refrigerated, vacuum-packaged meat surfaces.

J Food Prot, 1999 May, 62(5), 431 - 7
Development of a rapid response biosensor for detection of Salmonella typhimurium; Seo KH et al.; An integrated optic interferometer for detecting foodborne pathogens was developed . The interferometer is a planar waveguide with two thin antibody-coated channels of immunochemically selective agents that interact with antigen molecules . One channel is coated with antibody to Salmonella as a sample, and the other is coated with human immunoglobulin G as a reference channel by using reductive amination . Salmonella was introduced onto the sensing channels through the flow cell on the channels . Phase shift (pi) generated by refractive index variation, as determined by interfering the perturbed sample channel with an unperturbed reference channel and observing the fringe shift, was used for detection . Salmonella Typhimurium (heat-treated or boiled) was detected by binding to antibody against Salmonella common structural antigen immobilized on a silane-derived sensor surface at concentrations in the range of 1x10(5) to 1x10(7) CFU/ml . Salmonella (1x10(7) CFU/ml) mixed with Escherichia coli (1x10(7) CFU/ml) were readily detected without any decrease in sensitivity by the direct assay . Application of a sandwich assay with a second antibody or a gold-conjugated antibody increased the detection limit to 1x10(5) CFU/ml within a 10-min reaction time . Various methods for the immobilization of the capture antibody to the biosensor channels were compared . The greatest binding response was observed in a direct reductive amination method with a long reaction period and increased the detection limit of direct binding of Salmonella antigen to 1x10(4) CFU/ml . The biosensor was able to detect Salmonella Typhimurium in chicken carcass wash fluid originally inoculated at a level of 20 CFU/ml after 12 h of nonselective enrichment . The planar optic biosensor shows promise as a fast, sensitive, reliable, and economical means of detecting food pathogens in the future.

FEMS Microbiol Lett, 1999 May 15, 174(2), 327 - 32
A new chloramphenicol and florfenicol resistance gene flanked by two integron structures in Salmonella typhimurium DT104; Arcangioli MA et al.; A new chloramphenicol resistance gene from Salmonella typhimurium DT104, designated floR, also conferring resistance to florfenicol, was characterized . Sequence analysis of the deduced FloR protein suggested that it belongs to the 12-TMS (transmembrane segments) multidrug efflux pumps family . The floR gene, and the downstream sequenced tetR and tetA tetracycline resistance genes, were surrounded by two class 1 integrons . The first one contained the resistance gene aadA2 and a deleted sulI resistance gene . The second one contained the beta-lactamase gene pse1 and a complete sulI gene . Thus, the floR gene is included in a multiresistance locus of at least 12.5 kb . Its particular organization and chromosomal location could be involved in the antibioresistance pattern stability of the DT104 Salmonella typhimurium strains.

Infect Immun, 1999 Jun, 67(6), 3121 - 7
Porcine epithelial beta-defensin 1 is expressed in the dorsal tongue at antimicrobial concentrations; Shi J et al.; Epithelial cells and phagocytes contain antimicrobial polypeptides that participate in innate host defense . A recently cloned porcine beta-defensin, PBD-1, was detected by Northern organ blots exclusively in the tongue epithelium . We generated recombinant PBD-1 peptide by using a baculovirus-insect cell expression system and obtained two forms (PBD-142 and PBD-138), which differed by N-terminal truncation . Only PBD-142 was found in scrapings of the surface of the dorsal tongue or the buccal mucosa . Immunohistochemical staining with antibody to PBD-142 revealed that PBD-1 was highly concentrated in an approximately 0.1-mm-thick layer in the cornified tips of the filiform (but not fungiform) papillae of the dorsal tongue and in the superficial squamous cell layers of the buccal mucosa . By scraping, extraction, and semiquantitative Western blotting, the concentration of PBD-1 in the dorsal tongue surface and the buccal mucosa was estimated at 20 to 100 micrograms/ml . PBD-1 had antibacterial activity against Escherichia coli, Salmonella typhimurium, Listeria monocytogenes, and Candida albicans in 10 mM sodium phosphate buffer (pH 7.4) . Added NaCl progressively inhibited the activity of PBD-1 against E . coli and C . albicans . In 10 mM sodium phosphate with 125 mM NaCl, the combinations of sublethal concentrations of PBD-1 and the porcine neutrophil peptide PG-3, PR-39, or PR-26 showed synergistic activity against E . coli or the multidrug-resistant S . typhimurium DT104 . At its physiologic concentration, PBD-1 has antimicrobial effects under both low- and high-salt conditions encountered in the oral cavity and may contribute to the antimicrobial barrier properties of the dorsal tongue and oral epithelium.

Infect Immun, 1999 Jun, 67(6), 2815 - 21
Evaluation of Salmonella typhimurium mutants in a model of experimental gastroenteritis; Everest P et al.; Salmonella typhimurium strains harboring independent, defined mutations in aroA, invA, ssrA, or msbB were assessed for their ability to induce fluid accumulation, tissue damage, and local inflammation in rabbit ileal loops . Three wild-type strains of S . typhimurium, TML, HWSH, and SL1344, and two mutant strains, S . typhimurium SL1344 ssrA and S . typhimurium SL1344 msbB, consistently induced fluid accumulation in the lumen of loops and inflammation of loop-associated tissues . In contrast, three different S . typhimurium aroA strains and an invA mutant of SL1344 did not induce significant fluid accumulation in the rabbit ileal loops . However, the S . typhimurium aroA strains did induce an inflammatory infiltrate and some local villus-associated damage, but the invA mutant did not . Histologically, wild-type S . typhimurium, S . typhimurium SL1344 ssrA, and S . typhimurium SL1344 msbB demonstrated more severe effects on villus architecture than S . typhimurium aroA strains, whereas S . typhimurium invA-infected loops showed no detectable damage . This suggests that villus damage most likely contributes to fluid accumulation within the loop.

Biotechniques, 1999 May, 26(5), 892 - 4, 896, 898 passim
Increasing DNA transfer efficiency by temporary inactivation of host restriction; Edwards RA et al.; E . coli and Salmonella typhimurium are widely used bacterial hosts for genetic manipulation of DNA from prokaryotes and eukaryotes . Introduction of foreign DNA by electroporation or transduction into E . coli and Salmonella is limited by host restriction of incoming DNA by the recipient cells . Here, we describe a simple method that temporarily inactivates host restriction, allowing high-frequency DNA transfer . This technique might be readily applied to a wide range of bacteria to increase DNA transfer between strains and species.

Environ Mol Mutagen, 1999, 33(3), 240 - 8
VITOTOX bacterial genotoxicity and toxicity test for the rapid screening of chemicals; Verschaeve L et al.; The VITOTOX test is a new bacterial genotoxicity test that was previously shown to be very rapid and sensitive . Initially only one Salmonella typhimurium strain (TA104 recN2-4) was used in the test . In this paper we introduce a second strain (TA104pr1) that can be used as an internal control to further enhance the reliability of the test . We demonstrate the usefulness of this pr1 strain in genotoxicity and toxicity testing . We also report on the results of a study where the VITOTOX test was performed on newly synthesized pharmaceutical compounds, or intermediate products in the synthesis of drug candidates . We demonstrate that the test gives identical results when performed independently in two different laboratories and that it correlates well with either the Ames test or SOS chromotest.

Environ Mol Mutagen, 1999, 33(3), 202 - 10
Combinations of chlorocatechols and heavy metals cause DNA degradation in vitro but must not result in increased mutation rates in vivo; Schweigert N et al.; Chlorocatechols introduced into the environment directly or as a result of degradation processes are highly toxic, particularly when combined with heavy metals . With in vitro DNA degradation assays, the high reactivity of chlorocatechols combined with heavy metals could be shown, whereby copper was shown to be more active than iron . Structure-activity analysis showed that the degradation potential of the chlorocatechols decreased with an increasing number of chloratoms . The addition of reactive oxygen species scavengers allowed the identification of hydrogen peroxide as an important agent leading to DNA damage in this reaction . The potential of other reactive compounds, however, can neither be determined nor excluded with this approach . Exposure of Escherichia coli and Salmonella typhimurium cultures to the same mixtures of chlorocatechols and copper surprisingly did not lead to an enhanced mutation rate . This phenomenon was explained by doing marker gene expression measurements and toxicity tests with E . coli mutants deficient in oxidative stress defense or DNA repair . In catechol-copper-exposed cultures an increased peroxide level could indeed be demonstrated, but the highly efficient defense and repair systems of E . coli avoid the phenotypical establishment of mutations . Increased mutation rates under chronic exposure, however, cannot be excluded.

Mutat Res, 1999 May 17, 441(2), 205 - 13
Substituent effect of a fluorine atom on the mutagenicity of nitroquinolines; Saeki K et al.; Some 16 nitroquinolines (NQs) and their fluorinated derivatives were tested for mutagenicity in Salmonella typhimurium TA100 without S9 mix to investigate the effect of fluorine-substitution on the mutagenicity . These NQs consist of 5-NQs, 5-nitroquinoline N-oxides (5-NQOs), N-methyl-5-nitroquinolinium methanesulfonates (N-Me-5-NQs) and 8-NQs, including three ortho-F-NQs, one meta-F-NQ, four para-F-NQs and four 3-F-NQs . For this purpose, eight F-NQs were newly synthesized . The data indicated that the ratio of the mutagenic activities (revertants/plate/nmol) of fluorinated NQs to those of the corresponding parent non-fluorinated compounds ranged from 0.6- to 119-fold . The fluorine atom located para to the nitro group markedly enhanced the mutagenicity (24-fold and more), while three ortho-fluorinated derivatives showed no significant increase in mutagenicity (enhancement ratio were 0.6, 0.8 and 1.7) . With respect to 8-NQs, its meta-fluorinated derivative also had an enhanced mutagenicity over the parent compound (53-fold) . In addition, although N-Me-5-NQ was less mutagenic than 5-NQ and 5-NQO, the mutagenicity of N-Me-5-NQ was most significantly enhanced by fluorine-substitution . These results suggest that introduction of a fluorine atom to the molecule in question may be a useful tool to modify their mutagenic potency and to better understand the mechanism of mutation .

Mol Biol Evol, 1999 Apr, 16(4), 512 - 24
CRITICA: coding region identification tool invoking comparative analysis; Badger JH et al.; Gene recognition is essential to understanding existing and future DNA sequence data . CRITICA (Coding Region Identification Tool Invoking Comparative Analysis) is a suite of programs for identifying likely protein-coding sequences in DNA by combining comparative analysis of DNA sequences with more common noncomparative methods . In the comparative component of the analysis, regions of DNA are aligned with related sequences from the DNA databases; if the translation of the aligned sequences has greater amino acid identity than expected for the observed percentage nucleotide identity, this is interpreted as evidence for coding . CRITICA also incorporates noncomparative information derived from the relative frequencies of hexanucleotides in coding frames versus other contexts (i.e., dicodon bias) . The dicodon usage information is derived by iterative analysis of the data, such that CRITICA is not dependent on the existence or accuracy of coding sequence annotations in the databases . This independence makes the method particularly well suited for the analysis of novel genomes . CRITICA was tested by analyzing the available Salmonella typhimurium DNA sequences . Its predictions were compared with the DNA sequence annotations and with the predictions of GenMark . CRITICA proved to be more accurate than GenMark, and moreover, many of its predictions that would seem to be errors instead reflect problems in the sequence databases . The source code of CRITICA is freely available by anonymous FTP (rdp.life.uiuc.edu in/pub/critica) and on the World Wide Web rdpwww.life.uiuc.edu).

Mikrobiol Z, 1999 Jan-Feb, 61(1), 32 - 45
{The antibacterial efficacy of preparations of interferon and its inducers}; Spivak NIa et al.; Experimental data have been presented which prove antibacterial efficiency of interferon preparations and inducers under infection processes evoked by Staphylococcus aureus, Salmonella typhimurium, Pseudomonas aeruginosa, Klebsiella pneumoniae . It is shown that the system of phagocytizing cells and natural cells-killers plays the basic protective role in the organism under the persistence of these microbes . The basic regularities of activating effect of interferon preparations and its inducers on the functional activity of the above mentioned cells have been found . The methods of treatment with interferon drugs based on experimental data have been developed for the first time . They were used to cure patients with pyo-septic diseases.

Aust N Z J Public Health, 1999 Apr, 23(2), 164 - 7
Consumer attitudes and behaviours--key risk factors in an outbreak of Salmonella typhimurium phage type 12 infection sourced to chicken nuggets; Kenny B et al.; OBJECTIVES: To identify the source and intervention methods for an outbreak of Salmonella Typhimurium phage type 12 in South Australia . METHOD: Ten cases of S . Typhimurium phage type (PT) 12 infection were notified in South Australia in a four-week period from 7 May 1998 . Nine cases and 27 controls were included in a case control study to test the hypothesis that illness was associated with the consumption of chicken nuggets . RESULTS: A significant association between illness and the consumption of one brand of chicken nuggets was determined, odds ratio undefined (95% CI undefined; p = undefined) . Nine of nine cases and one of 27 controls reported eating these chicken nuggets . S . Typhimurium PT 12 was isolated from an opened sample of this particular brand of nuggets which had been retrieved from the home of one case . CONCLUSIONS AND IMPLICATIONS: The implicated nuggets were essentially a raw product which had been 'flash fried' in contrast with other brands which were fully cooked . The investigation highlighted issues of inadequate labelling and consumer responses to labelling information which affect food safety . A media release to highlight to the consumer the need to cook frozen food properly and a voluntary recall of the 'flash fried' product was instigated as a result of these conclusions . Further action is needed to eliminate the potential hazard that consumers will perceive and handle 'flash fried' nuggets as if they are a cooked chicken product.

Aust Vet J, 1999 Apr, 77(4), 229 - 32
Concurrent Aelurostrongylus abstrusus infection and salmonellosis in a kitten; Barrs VR et al.; A 14-week-old kitten had a history of vomiting, diarrhoea and pyrexia, all of which resolved without treatment . Three weeks later the kitten developed a violent non-productive dry cough . Thoracic radiographs revealed pneumothorax and nodular alveolar disease . Aelurostrongylus abstrusus larvae and intracellular Gram-negative bacilli were seen in bronchial wash fluid and pleural exudate, and Salmonella Typhimurium was cultured from both fluids but not from faeces . Therapy included unilateral closed-tube thoracostomy, enrofloxacin and fenbendazole . Historical signs were compatible with gastrointestinal salmonellosis and secondary broncho-pneumonia . Seeding of the lungs with salmonellae may have occurred as a result of migration of A abstrusus from a gastro-intestinal tract residually infected or colonised by S Typhimurium . Alternatively, the development of lungworm infection in the cat may have activated quiescent S Typhimurium pulmonary granulomata from bacteraemia secondary to gastro-intestinal salmonellosis . Two years after diagnosis the cat was reportedly in good health.

EMBO J, 1999 May 17, 18(10), 2886 - 96
Mutations which alter the elbow region of tRNA2Gly reduce T4 gene 60 translational bypassing efficiency; Herr AJ et al.; Translating ribosomes bypass a 50 nucleotide coding gap in bacteriophage T4 gene 60 mRNA between codons 46 and 47 in order to synthesize the full-length protein . Bypassing of the coding gap requires peptidyl-tRNA2Gly detachment from a GGA codon (codon 46) followed by re-pairing at a matching GGA codon just before codon 47 . Using negative selection, based on the sacB gene from Bacillus subtilis, Escherichia coli mutants were isolated which reduce bypassing efficiency . All of the mutations are in the gene for tRNA2Gly . Most of the mutations disrupt the hydrogen bonding interactions between the D- and T-loops (G18*psi55 and G19*C56) which stabilize the elbow region in nearly all tRNAs . The lone mutation not in the elbow region destabilizes the anticodon stem at position 40 . Previously described Salmonella typhimurium mutants of tRNA2Gly, which reduce the stability of the T-loop, were also tested and found to decrease bypassing efficiency . Each tRNA2Gly mutant is functional in translation (tRNA2Gly is essential), but has a decoding efficiency 10- to 20-fold lower than wild-type . This suggests that rigidity of the elbow region and the anticodon stem is critical for both codon-anticodon stability and bypassing.

J Mol Biol, 1999 Apr 23, 288(1), 165 - 75
Protein-ligand interaction: grafting of the uridine-specific determinants from the CytR regulator of Salmonella typhimurium to Escherichia coli CytR; Thomsen LE et al.; Members of the LacI family of transcriptional repressors respond to the presence of small effector molecules . The binding of the ligands affect the proteins ability to repress transcription by stabilizing a conformation that, in most cases, is unfavorable for high-affinity DNA binding . The CytR anti-activator diverges from the other family members by relying on the cooperative DNA binding with the global regulator CRP . The inducers of CytR do not affect CytR-DNA binding per se, but alleviate repression by interrupting protein-protein interactions between the two regulators . Here, we have studied of the CytR-inducer interaction by exploring a discrepancy in the inducer response observed for the homologous CytR regulators of Escherichia coli and Salmonella typhimurium . CytR of S . typhimurium (CytRSt) appears to respond to the presence of both uridine and cytidine nucleosides, whereas E . coli CytR (CytREc) responds to cytidine only . We have used a combination of genetic and structural modeling studies to provide detailed information regarding the nature of this discrepancy . By analysis of hybrid CytR proteins followed by site-directed mutagenesis, we have successfully transferred the specificity determinants for uridine from CytRSt to CytREc, revealing that serine substitutions of only two residues (G131 and A152) in CytREc is required to make CytREc sensitive to uridine . In addition, by employing a genetic screen for induction of defective mutants, we have identified four amino acid residues in CytRSt that appear to be important for the response to uridine . The implications of these findings for the understanding of the ligand binding and induction of CytR are discussed in the context of the structural knowledge of CytR and homologous protein-ligand complexes .

J Math Biol, 1999 Apr, 38(4), 359 - 75
Model and analysis of chemotactic bacterial patterns in a liquid medium; Tyson R et al.; A variety of spatial patterns are formed chemotactically by the bacteria Escherichia coli and Salmonella typhimurium . We focus in this paper on patterns formed by E . coli and S . typhimurium in liquid medium experiments . The dynamics of the bacteria, nutrient and chemoattractant are modeled mathematically and give rise to a nonlinear partial differential equation system . We present a simple and intuitively revealing analysis of the patterns generated by our model . Patterns arise from disturbances to a spatially uniform solution state . A linear analysis gives rise to a second order ordinary differential equation for the amplitude of each mode present in the initial disturbance . An exact solution to this equation can be obtained, but a more intuitive understanding of the solutions can be obtained by considering the rate of growth of individual modes over small time intervals.

Intervirology, 1998, 41(6), 238 - 43
Antiviral efficacy of disinfectant solution MRI-1; Skinner GR et al.; Disinfectant MRI-1 was prepared by dissolution of non-ionic and ionic detergent in ethanol . The disinfectant inactivated extracellular and intracellular enveloped and non-enveloped viruses including herpes viruses, influenza A and human immunodeficiency disease virus in suspension or on surfaces by pre-exposure or post-exposure to the disinfectant; in addition, cells were disabled as potential hosts for viral infection using concentrations of MRI-1 which were 50-fold less than the operative concentration for disinfection . There was no evidence of in vitro mutagenicity using Salmonella typhimurium or sensitization or other adverse effect in a guinea pig model or in human subjects.

Mol Gen Genet, 1999 Apr, 261(3), 472 - 9
A coding segment of the virulence regulatory gene spvR enhances expression of spvR-lacZ and spvR-gfp translational fusions in Salmonella typhimurium; Robbe-Saule V et al.; The Salmonella gene spvR is regulated by RpoS, and encodes an autoregulatory DNA-binding protein of the LysR family, which is required for transcriptional activation of the virulence operon spvABCD . We found that the 12-bp sequence between codons 3 and 8 of spvR (12bp-box) increased the expression of spvR'-lacZ translational fusions by at least two orders of magnitude . The 12bp-box did not affect the level of expression of a transcriptional spvR'-lacZ fusion, as determined by measurement of beta-galactosidase activity and mRNA levels . This suggests that the 12bp-box does not play a major role at the transcriptional level . However, the amounts of both SpvR-LacZ hybrid protein and lacZ mRNA produced from a translational spvR'-lacZ fusion were significantly lower if the 12bp-box was removed . Thus, the 12bp-box directly or indirectly affects the amount of spvR'-lacZ mRNA produced from a translational fusion but not from a transcriptional fusion . The 12bp-box still appeared to be functional when the spvR'-lacZ mRNA was elongated at its 5' end . In that case, however, while the 12bp-box greatly reduced the level of downstream mRNA produced, it did not affect the level of upstream mRNA . The effect of the 12bp-box was not restricted to lacZ fusions because expression of a translational spvR'-gfp fusion was also decreased by removal of the 12bp-box . The sequence of the 12bp-box is complementary to a sequence near the decoding region of 16S rRNA thought to be involved in translation initiation . This box may thus function as a translational enhancer, in which case the effect of the 12bp-box on lacZ mRNA levels might result indirectly from the tight coupling of translation and mRNA degradation . However, a direct role of the 12bp-box in increasing mRNA stability or in reducing the incidence of premature termination of transcription cannot be excluded.

Int Immunol, 1999 Apr, 11(4), 481 - 9
Th1 dominance in the immune response to live Salmonella typhimurium requires bacterial invasiveness but not persistence; Pashine A et al.; Factors responsible for the predictable generation of Th1 or Th2 immune responses to microorganisms in vivo are not well characterized, although the ability of antigen presenting cells (APC) to provide co-stimulation, the kinetics of MHC-peptide ligand generation as well as the cytokine environment are all considered important factors for the differential Th1/Th2 priming of T cells . Our earlier findings of an IFN-gamma-dominant, Th1-type response to live Salmonella typhimurium (Stm) and a Th2-type response to killed Stm suggested that persistence of viable bacteria might be an important factor in the generation of IFN-gamma-dominant responses . Using genetically susceptible and resistant strains of mice to limit bacterial replication and persistence in vivo, we show that mice of the lty(r) genotype, capable of a 10-fold better clearance of Stm, mount an IFN-gamma-dominant immune response following immunization with live Stm similar to that in the lty(s) strain . Further, metabolically defective mutants of Stm, aroA and purA, when used in the live form, also elicit IFN-gamma-dominant immune responses similar to the wild-type Stm strain despite their inability to proliferate in vivo . While a laboratory strain of Escherichia coli, which is antigenically cross-reactive but non-invasive, elicits hardly any IFN-gamma in immune responses, an invasive strain of E . coil induces an IFN-gamma-dominant response . These data together indicate that, while entry of bacteria into macrophages is likely to be critical for the generation of IFN-gamma-dominant immune responses, their persistence is not.

Zhonghua Yu Fang Yi Xue Za Zhi, 1998 Mar, 32(2), 70 - 2
{Studies on mutagenicity of humic acid samples collected from arsenosis-affected areas in China}; Yu X et al.; OBJECTIVE: To explore whether there is mutagenicity of humic acid samples collected from arsenosis affected areas in China . METHODS: Ames' test was used for natural humic acid samples extracted from the coal containing high arsenic in Guizhou Province, and the well water in the areas with blackfoot disease in Taiwan and with arsenosis in Inner Mongolia of China . RESULTS: Coefficients of revertant mutation were 1.02-0.82 net revertant per microgram (rev/microgram) of humic acid extracted from the coal containing high arsenic in Guizhou without rat liver homogenate (-S9), and 1.53-1.12 rev/microgram of that with rat liver homogenate (+S9), with an obvious dose-response relationship . CONCLUSION: Humic acid extracted from the coal containing high arsenic produced in Guizhou could cause stronger direct mutation in Salmonella typhimurium TA98 (frameshift mutation) and that extracted from the well water in the areas with blackfoot disease in Taiwan could cause weaker direct mutation in Salmonella typhimurium (-S9), but that extracted from the well water with arsenosis in Inner Mongolia could not cause mutation . Mutagenicity of commercial humic acid, purchased from Aldrich Co . in the United States, arsenic and iron was also studied, and no mutagenicity was found for them independently, but arsenic combined with humic acid, at certain concentrations, could cause weak mutation in TA98 (-S9), with or without iron . No mutagenicity in TA100 (+/- S9, base pair substitution mutation) was found in all the samples mentioned above.

Zhonghua Yu Fang Yi Xue Za Zhi, 1998 Jul, 32(4), 214 - 8
{Plasmid profile typing of Salmonella typhimurium and its distribution in some provinces and cities of China}; Pan R et al.; OBJECTIVE: To study the relationship between the infection and prevalence of Salmonella typhimurium . METHODS: From 1980 to 1995, when Salmonella typhimurium infection was prevalent in north and south China, a total of 2,655 isolates of Salmonella typhimurium were collected from 19 provinces, municipalities, and autonomous regions and Hong Kong Special Administrative Region for plasmid profile analysis . The profiles consisting of some small plamids were recorded with capital letters A to J, and the profile which was free from small plasmid was recorded with capital letter O . Large plasmid in molecular weights of 140-20 Mdal showing in profiles was recorded with Arabic numerals 9 to 0 respectively . Having two or more than two large plasmids, the profile was recorded with two or more Arabic numerals . RESULTS: The 2,655 strains were divided into 11 plasmid profile groups and 168 plasmid profile types . Of 52 plasmid profile types frequently isolated, plasmid profile type O 4 was distributed extensively in 16 provinces, municipalities, and autonomous rehions and Hong Kong . Plasmid profile type O 5 was isolated in 5 provinces and Hong Kong, mostly in Jiangxi and Hubei . Plasmid profile type J 4 was isolated in 7 provinces, municipalities and autonomous regions, mostly in Jiangxi . Five plasmid profile types of Group A caused a big prevalence of nosocomial infection in Henan . Type A 6 was also isolated in Xinjiang and Anhui . Six plasmid profile types of Group E were prevalent in Xinjiang predominantely, three types caused nosocomial infection in Shanghai, type E 82 caused nosocomial infection and food poisoning in Liaoning . Type F 4 was isolated in 4 provinces, mostly in jiangxi and Fujian . Many plasmid profile types were prevalent local . Types B 5, BCEG 60, BE 6, B' 0 G 6, G 5 and EG 6 were prevalent in Jiangxi, Types IF'4, IF'H40 and F 9864 were prevalent in Sandong, Type IF'4 was also isolated in Jiangsu and Hubei, Type IF 40 in Anhui, Type BH 6 in Huibei, and Type EH 5 in Fujian . CONCLUSION: Plasmid profile analysis showed the same types in the prevance but different plasmid profiles in different provinces.

J Bacteriol, 1999 May, 181(10), 3096 - 104
A HilA-independent pathway to Salmonella typhimurium invasion gene transcription; Rakeman JL et al.; Salmonella typhimurium invasion of nonphagocytic cells requires the expression of a type III secretion system (TTSS) encoded within Salmonella pathogenicity island 1 (SPI1) . TTSS gene transcription is activated in response to environmental signals and requires transcriptional regulators encoded within (HilA) and outside (SirA) SPI1 . Two unique loci, sirB and sirC, which contribute to SPI1 gene transcription were defined . sirC is an SPI1-encoded transcription factor of the AraC family that contributes to the invasive phenotype . sirB is required for maximal expression of sirC and consists of two open reading frames located near kdsA, a gene involved in lipopolysaccharide biosynthesis . sirC expression, unlike expression of other SPI1 genes, does not require HilA . Overexpression of sirC or sirA restores expression of a subset of SPI1 genes, including invF and sspC, in the absence of HilA . These data define roles for SirC and SirA as part of a HilA-independent pathway to SPI1 gene expression . We postulate that HilA-independent activation of inv expression is important for efficient assembly and function of the SPI1 TTSS.

Biochim Biophys Acta, 1999 May 14, 1445(2), 196 - 206
Identification of a cis-acting regulatory sequence responsible for the repression of brnQ in Salmonella typhimurium; Ohnishi K et al.; brnQ is the gene encoding the LIV-II transport system for branched-chain amino acids in Salmonella typhimurium . The expression of the gene is transcriptionally repressed by an excess of glycyl-l-leucine added to the bacterial culture . To investigate the mechanism of regulation, we constructed brnQ-lacZ translational fusions with various deletions upstream from the promoter of brnQ, and examined the effects of the deletions on the regulation . We found a cis-acting region, 5'-GTGTTTTA-3', for the repression of brnQ expression, which was located 94 base pairs upstream from the transcription start site . Removal of the sequence resulted in derepression of brnQ . Two homologous sequences were found 45 base pairs downstream and 42 base pairs upstream from the sequence . We designated these sequences as O1, O2, and O3, in the order from the sequence proximal to the promoter to that distal to the promoter, respectively . The gleR1 mutation, which we reported previously to be a regulatory mutation enhancing transcription of brnQ, was a G-to-T transversion in the O1 sequence 50 base pairs upstream from the transcription start site . Insertion of five nucleotides between O1 and O2 resulted in derepression of brnQ . Further insertion of five nucleotides did not restore the original regulation of brnQ, indicating the importance of the proper spacing of these sequences . We also showed that the protein product of livS, the gene responsible for regulation of the LIV-I transport system, may bind to the O2 sequence . Furthermore, LivS was shown to be an allele of Lrp based on complementation experiments.

Parasite Immunol, 1999 Apr, 21(4), 219 - 24
Immunogenicity of the extracellular copper/zinc superoxide dismutase of the filarial parasite Acanthocheilonema viteae delivered by a two-phase vaccine strain of Salmonella typhimurium; Lattemann CT et al.; The recombinant extracellular copper/zinc superoxide dismutase of the filarial parasite Acanthocheilonema viteae (AVSOD2) was cloned in an expression vector under control of the bacteriophage T7 promoter and the resulting plasmid pLAT7 was introduced in tha aroA attenuated Salmonella typhimurium vaccine strain SL3261:pYZ84 . This vaccine strain carries a chromosomally integrated two phase expression system containing inducible T7 RNA polymerase . The recombinant AVSOD2 was efficiently expressed, constituting up to 5% of the total bacterial protein . Furthermore, the plasmid vector containing the AVSOD2 cDNA was shown to be stable over a long period of time in the vaccine strain without antibiotic selection in vitro and in vivo . Jirds which were immunised orally with the recombinant vaccine strain expressing the A . viteae EC-SOD produced a strong humoral immune response.

Mol Microbiol, 1999 May, 32(3), 595 - 606
Phage Mu transposition immunity reflects supercoil domain structure of the chromosome; Manna D et al.; Transposition immunity is the negative influence that the presence of one transposon sequence has on the probability of a second identical element inserting in the same site or in sites nearby . A transposition-defective Mu derivative (MudJr1) produced transposition immunity in both directions from one insertion point in the Salmonella typhimurium chromosome . To control for the sequence preference of Mu transposition proteins, Tn10 elements were introduced as targets at various distances from an immunity-conferring MudJr1 element . Mu transposition into a Tn10 target was not detectable when the distance of separation from MudJr1 was 5 kb, and transposition was unencumbered when the separation was 25 kb . Between 5 kb and 25 kb, immunity decayed gradually with distance . Immunity decayed more sharply in a gyrase mutant than in a wild-type strain . We propose that Mu transposition immunity senses the domain structure of bacterial chromosomes.

Science, 1999 May 7, 284(5416), 967 - 70
An essential role for DNA adenine methylation in bacterial virulence; Heithoff DM et al.; Salmonella typhimurium lacking DNA adenine methylase (Dam) were fully proficient in colonization of mucosal sites but showed severe defects in colonization of deeper tissue sites . These Dam- mutants were totally avirulent and were effective as live vaccines against murine typhoid fever . Dam regulated the expression of at least 20 genes known to be induced during infection; a subset of these genes are among those activated by the PhoP global virulence regulator . PhoP, in turn, affected Dam methylation at specific genomic sites, as evidenced by alterations in DNA methylation patterns . Dam inhibitors are likely to have broad antimicrobial action, and Dam- derivatives of these pathogens may serve as live attenuated vaccines.

Res Rep Health Eff Inst, 1999 Mar, (84), i - iv, 1-22; discussion 23-7
Evaluation of the potential health effects of the atmospheric reaction products of polycyclic aromatic hydrocarbons; Grosovsky AJ et al.; The genotoxic risks from exposure to polycyclic aromatic hydrocarbons (PAHs) have long been recognized . Less well understood are the potential genotoxic risks of the atmospheric reaction products of this class of compounds . In this investigation, we have utilized several human cell assays to evaluate the genotoxicity of naphthalene, phenanthrene, and their atmospheric reaction products 1-nitronaphthalene, 2-nitronaphthalene (2NN), 1-hydroxy-2NN, 2-hydroxy-1-nitronaphthalene, 1,4-naphthoquinone, and 2-nitrodibenzopyranone (2NDBP) . In addition, simulated atmospheric reaction products of naphthalene were generated in a 6,700 liter (L) Teflon environmental chamber, collected on a solid adsorbent, extracted, and fractionated by normal-phase high-performance liquid chromatography (HPLC) . Individual fractions were then analyzed using gas chromatography/mass spectrometry (GC/MS), and tested for genotoxic effects . Genotoxicity was primarily determined using the human B-lymphoblastoid cell line, MCL-5, which expresses several transfected P450 and epoxide hydrolase genes . Mutagenicity was evaluated at both the heterozygous thymidine kinase (tk) locus and the hemizygous hypoxanthine phosphoribosyl transferase (hprt) locus, permitting detection of both intragenic and chromosomal scale mutational events . Test compounds were also screened using the CREST modified micronucleus assay . The results indicate that 2NN and 2NDBP possess greater mutagenic potency than their parent compounds, and, interestingly, both compounds induced significant increases in mutation frequency at the tk but not the hprt locus . These findings suggest a mechanistic difference in human cell response to 2NN and 2NDBP as compared to bacteria, where both compounds were previously shown to induce point mutations in the Salmonella typhimurium reversion assay . The genotoxicity of 2NN and 2NDBP in human cells, together with their high concentrations in ambient air relative to nitro-PAHs directly emitted from combustion sources, emphasizes the need to consider atmospheric reaction products of PAHs in assessments of the genotoxicity of air pollutants . We also investigated whether transfected cytochrome P450 monooxygenase activities were required to activate 2NN and 2NDBP to genotoxic species, and whether a single enzyme could be sufficient for metabolic activation . Three directly related cell lines with multiple (MCL-5), single (AHH-1 1A1), or no (L3) transfected cytochrome P450 genes were used . AHH-1 is additionally distinguished by elevated mutagenic response at the tk locus, a heterozygous mutation in p53, and apoptosis capacity . The effect of these metabolic and genetic differences on genotoxicity of 2NN, 2NDBP, and beta-naphthylamine (beta NA) was also investigated . The results indicated that 2NN and 2NDBP were not activated to genotoxic species through nitroreduction pathways . Mutagenicity induced at the tk locus was dependent on oxidative metabolism, provided by transfected cytochrome P450 enzymes in MCL-5 and AHH-1 1A1 . Mutagenicity was not observed in the L3 cell line, which does not carry transfected cytochrome P450 activities . The negative response of beta NA in all cell lines indicates that, contrary to previous hypotheses, 2NN and beta NA are not activated by similar metabolic pathways in these human cell lines . Taken as a whole, these results suggest that the genotoxicity of nitro-PAHs in human cells requires oxidative metabolism.

Arch Pharm Res, 1994 Apr, 17(2), 71 - 5
Antimutagenic effect of plant flavonoids in the Salmonella assay system; Choi JS et al.; The antimutagenic effects of 27 kinds of plant flavonoids on the mutagenicity of aflatoxin B1(AFB1) and N-methyl-N'-nitro-N-nitrosoguanidine(MNNG) in Salmonella typhimurium TA100 were investigated . In the mixed applications of AFB1 (1 microgram/plate) with the flavonoids (300 micrograms/plate) in the presence of a mammalian metabolic activation system (S9 mix), chrysin, apigenin, luteolin and its glucoside, kaempferol, fisetin, morin, naringenin, hesperetin, persicogenin, (+)-catechin and (-)-epicatechin showed the antimutagenic effect against AFB1 with more than 70% inhibition rate . A little or no antimutagenicities except flavone against MNNG (0.5 microgram/plate) were observed . For the antimutagenicity of the flavonoids on AFB1, the flavonoid structure that contains the free 5-, 7-hydroxyl group seemed to be essential . However, saturation of the 2,3-double bond or elimination of the 4-keto group did not affect the activity.

Mol Microbiol, 1999 Apr, 32(2), 275 - 87
The genetic basis of tetrathionate respiration in Salmonella typhimurium; Hensel M et al.; A range of bacteria are able to use tetrathionate as a terminal respiratory electron acceptor . Here we report the identification and characterization of the ttrRSBCA locus required for tetrathionate respiration in Salmonella typhimurium LT2a . The ttr genes are located within Salmonella pathogenicity island 2 at centisome 30.5 . ttrA, ttrB and ttrC are the tetrathionate reductase structural genes . Sequence analysis suggests that TtrA contains a molybdopterin guanine dinucleotide cofactor and a {4Fe-4S} cluster, that TtrB binds four {4Fe-4S} clusters, and that TtrC is an integral membrane protein containing a quinol oxidation site . TtrA and TtrB are predicted to be anchored by TtrC to the periplasmic face of the cytoplasmic membrane implying a periplasmic site for tetrathionate reduction . It is inferred that the tetrathionate reductase, together with thiosulphate and polysulphide reductases, make up a previously unrecognized class of molybdopterin-dependent enzymes that carry out the reductive cleavage of sulphur-sulphur bonds . Cys-256 in TtrA is proposed to be the amino acid ligand to the molybdopterin cofactor . TtrS and TtrR are the sensor and response regulator components of a two-component regulatory system that is absolutely required for transcription of the ttrBCA operon . Expression of an active tetrathionate reduction system also requires the anoxia-responsive global transcriptional regulator Fnr . The ttrRSBCA gene cluster confers on Escherichia coli the ability to respire with tetrathionate as electron acceptor.

Mol Microbiol, 1999 Apr, 32(2), 243 - 52
Negative and positive regulation of the non-osmoregulated ompS1 porin gene in Salmonella typhi: a novel regulatory mechanism that involves OmpR; Oropeza R et al.; The Salmonella typhi ompS1 gene codes for an outer membrane protein of the OmpC/OmpF porin family . It is expressed at very low levels, relative to the major porins . However, deletion analysis of the 5' regulatory region showed that the gradual removal of nucleotides -310 to -88, upstream from the P1 major transcriptional start-point, resulted in a stepwise increase in expression, reaching levels 10-fold above those for the ompC major porin gene . Hence, this 222 bp segment contains cis-acting regulatory elements involved in negative control . Primer extension analysis revealed the presence of three promoters: P1 activity was OmpR dependent; P2 was expressed at a lower level in the absence of OmpR; and P3 had a minor constitutive activity . OmpR bound preferentially to box II, an 18 bp F1/C1 canonical site, the removal (-88 to -66) of which resulted in a decrease in expression thus supporting its role in positive control . Expression of ompS1 was not induced by a set of stress conditions, including a shift in osmolarity, nor was the IHF regulator involved in negative control . An ompS1 homologue was found in E . coli K-12, which contains a nonsense codon and a shift in the reading frame, whereas Salmonella typhimurium contains an open reading frame in this region . Thus, S . typhi ompS1 provides novel features in OmpR regulation.

Poult Sci, 1999 Apr, 78(4), 546 - 9
Effects of chicken-derived cecal microorganisms maintained in continuous culture on cecal colonization by Salmonella typhimurium in turkey poults; Hollister AG et al.; A characterized, chicken-derived, competitive exclusion culture of cecal bacteria was evaluated for effectiveness in the reduction of Salmonella typhimurium cecal colonization in growing turkey poults . The culture was administered by crop gavage on the day of hatch . All groups were challenged orally on Day 3 with 10(4) S . typhimurium . Compared with untreated controls, the percentage of poults that were Salmonella cecal-culture-positive at 10 d of age was significantly reduced (P < 0.05) in the poults provided culture . Additionally, the culture-treated poults had significantly (P < 0.05) fewer Salmonella per gram of cecal contents than the controls . The results indicated that treatment of turkey poults with the characterized chicken-derived culture effectively decreased Salmonella cecal colonization.

Chemosphere, 1999 Jun, 38(13), 3015 - 30
Frontier orbital energies, hydrophobicity and steric factors as physical QSAR descriptors of molecular mutagenicity . A review with a case study: MX compounds; Tuppurainen K; A review on QSARs (Quantitative Structure-Activity Relationships) in modelling molecular mutagenicity is given . The importance of hydrophobicity, frontier orbital (HOMO and LUMO) energies and steric factors as physical descriptors of mutagenicity is emphasized . In addition, some possible connections between QSAR models and the general electrophilic theory of genotoxic activity are discussed . As a detailed example, QSARs for the Ames Salmonella typhimurium TA100 mutagenicity of halogenated hydroxyfuranones including MX, one of the most potent bacterial mutagens ever identified, are discussed and a plausible mechanism for their mutagenic activity is proposed.

Mutagenesis, 1999 Mar, 14(2), 181 - 5
Microsomal metabolism of dictamnine: identification of metabolites and evaluation of their mutagenicity in Salmonella typhimurium; Klier B et al.; Incubation of the furoquinoline alkaloid dictamnine with microsomal fractions from phenobarbital-induced rat liver resulted in a metabolite mixture from which demethyl-dictamnine and dictamnic acid were identified by a GCMS technique . The identity of the two compounds was confirmed by synthesis . Other metabolites were characterized by their mass spectra only . The pattern of the metabolites suggested a possible pathway of metabolism in vitro . We assume that the metabolism of dictamnine is analogous to the metabolism of the related 8-methoxypsoralen and takes place via an unstable epoxide and subsequent oxidative opening of the furan ring . Direct evidence for the formation of an epoxide was, however, not obtained . The identified compounds as well as some putative metabolites were shown to be non-mutagenic in Salmonella typhimurium TA98 except for 8-hydroxydictamnine, which, however, was not detected in the metabolite mixture.

J Immunol, 1999 May 1, 162(9), 5398 - 406
T cell responses to Gram-negative intracellular bacterial pathogens: a role for CD8+ T cells in immunity to Salmonella infection and the involvement of MHC class Ib molecules; Lo WF et al.; Despite being a major group of intracellular pathogens, the role of class I-restricted T cells in the clearance of Gram-negative bacteria is not resolved . Using a murine typhoid model, a role for class I-restricted T cells in the immune response to the Gram-negative pathogen Salmonella typhimurium is revealed . Class I-deficient beta2-microglobulin-/- mice show increased susceptibility to infection with S . typhimurium . Following infection, CD8+ CTLs specific for Salmonella-infected targets can be readily detected . The Salmonella-specific CTLs recognize infected H-2-mismatched targets, suggesting the involvement of shared class Ib molecules . Studies using transfectants expressing defined class Ia and class Ib molecules indicate the involvement of the class Ib molecule, Qa-1 . Ab-blocking studies and the measurement of bacteria-specific CTL frequencies identified Qa-1 as a dominant restricting element . The Qa-1-restricted CTL recognition depends on TAP and proteasome functions . Surprisingly, Qa-1-restricted CTLs recognized cells infected with other closely related Gram-negative bacteria . Taken together, these observations indicate that Salmonella-specific CTLs recognize a cross-reactive epitope presented by Qa-1 molecules and, as such, may be novel targets for vaccine development.

Food Chem Toxicol, 1999 Feb-Mar, 37(2-3), 135 - 44
Chemical composition and toxicity of Taiwanese betel quid extract; Wang CK et al.; In this genotoxic study, the Ames Salmonella microsome test showed that an aqueous extract of betel quid did not induce mutagenicity in Salmonella typhimurium strains TA98 and TA100 . Mammalian cell studies (Chinese hamster ovary K1 cell; CHO-K1 cell) revealed that only higher concentrations (100 and 1000 microg/ml) of aqueous extract weekly increased the frequencies of sister-chromatid exchange (SCE) in the absence of S9 . Animal (male Sprague-Dawley rat) studies showed that low-dose feeding (0.53 g dry aqueous extract/kg diet) significantly increased the activities of glutathione (GSH) peroxidase and cytoplasmic glutathione S-transferase (cGST) of liver, high-dose feeding (26.5 g dry aqueous extract/kg diet) lowered the contents of GSH and total glutathione . The effect of an aqueous extract of betel quid on the oxidation of 2'-deoxyguanosine (2'-dG) to 8-hydroxy-2'-deoxyguanosine (8-OH-dG) evaluated that this aqueous extract may act as a pro-oxidant at lower dosage and may be dependent on the iron ions in the model system . However, the aqueous extract of betel quid showed antioxidant activity at higher doses by the ability of the scavenging effect of the hydroxyl radicals.

Food Chem Toxicol, 1999 Feb-Mar, 37(2-3), 95 - 103
Antimutagens in food plants eaten by Polynesians: micronutrients, phytochemicals and protection against bacterial mutagenicity of the heterocyclic amine 2-amino-3-methylimidazo{4,5-f}quinoline; Botting KJ et al.; We have previously suggested that differences in cancer incidence between Polynesians (including Maoris and people from several Pacific islands) and Europeans in New Zealand may at least partially relate to differences in the species of food plants (fruits, vegetables and cereals) preferentially eaten by these groups . Twenty-five food plants that are typically eaten in different amounts by these two population groups were selected for detailed study . Antimutagenic properties of three extracts from each of the selected plants were investigated using a preincubation mutagenicity assay with Salmonella typhimurium strain TA1538 against the mutagenicity of the heterocyclic amine 2-amino-3-methylimidazo{4,5-f}quinoline (IQ) . The data revealed strong antimutagenic properties in several of the food plants commonly eaten by Polynesians, especially rice, watercress, pawpaw, taro leaves, green banana and mango . Using the New Zealand food database, a number of nutrients and micronutrients with antimutagenic and anticarcinogenic potential were identified from the selected food plants . Some of these were tested for antimutagenic potential in parallel experiments to those done with the food plant extracts . Although some of these micronutrients are antimutagens against IQ, their concentrations in the food plants failed to explain the protection against mutagenicity found in the experiments with extracts of the food plants . Thus, other types of chemical, not identified in the database, must be leading to antimutagenesis . Possible active molecules include chlorophylls, carotenoids, flavonoids and coumarins, many of which are also known to be anticarcinogens . If human cancer data are to be interpreted in terms of cancer protection, these components need urgently to be quantified in food plants in the New Zealand diet, especially in those food plants eaten in large amounts by Polynesians.

FEMS Microbiol Lett, 1999 Apr 15, 173(2), 411 - 23
Sample sequencing of a Salmonella typhimurium LT2 lambda library: comparison to the Escherichia coli K12 genome; Wong RM et al.; As part of the ongoing sequencing of the complete Salmonella typhimurium LT2 genome, a partly ordered set of 416 lambda clones has been developed, representing over 90% of the genome . The average insert size is 17 kb . Sequences were obtained from both ends of each clone in this set . A total of over 600 kb of sequence has been deposited in the genome survey sequence section of GenBank . This resource of clones is available from the Salmonella Genome Stock Center . A preliminary comparison with the Escherichia coli K12 genome indicates that there are likely to be many hundred insertion deletion events, encompassing more than one gene, that distinguish these genomes . Fully 30% of the S . typhimurium sequences have no close homologs in the GenBank database.

Biosci Biotechnol Biochem, 1999 Mar, 63(3), 537 - 41
Antimutagenicity of sweetpotato (Ipomoea batatas) roots; Yoshimoto M et al.; Antimutagenicity of the water extracts prepared from the storage roots of four varieties of sweetpotato with different flesh colors was investigated using Salmonella typhimurium TA 98 . The extract from the whole roots of the purple-colored Ayamurasaki variety effectively decreased the reverse mutation induced not only by Trp-P-1, Trp-P-2, IQ, B{a}P, and 4-NQO but also by dimethyl sulfoxide extracts of grilled beef . Comparison of the inhibitory activity of the extracts from the normal Ayamurasaki and its anthocyanin-deficient mutant one suggested that the anthocyanin pigment in the flesh decreases the mutagenic activity of the mutagens as heterocyclic amines . Two anthocyanin pigments purified from purple-colored sweet-potato, 3-(6,6'-caffeylferulylsophoroside)-5-glucoside of cyanidin (YGM-3) and peonidin (YGM-6) effectively inhibited the reverse mutation induced by heterocyclic amines, Trp-P-1, Trp-P-2, and IQ in the presence of rat liver microsomal activation systems.

Anticancer Res, 1999 Jan-Feb, 19(1A), 569 - 72
Antimutagenicity of lignin in vitro; Ebringer L et al.; The inhibitory activity of lignin against nitrosoguanidine (MNNG)- and acridine orange (AO)- induced mutagenesis was examined using two microbial systems: green unicellular flagellate Euglena gracilis and Salmonella typhimurium TA100 and TA97 . To verify the hypothesis that the above mentioned mutagens may generate some oxidant species and subsequently free radicals, or they may interact with lignin, two physico-chemical measurements were performed . Lignin at a tested concentration (100 micrograms/ml) decreases Euglena-bleaching activity of MNNG by 67.7% and AO by 99.7% . Percentage of MNNG-induced revertants of S . typhimurium was also decreased substantially by lignin . We conclude that our results indicate the possible mechanisms behind the antimutagenic/anticarcinogenic effects of lignin: namely, scavening of reactive oxygen species produced by MNNG and binding of AO itself.

Infect Immun, 1999 May, 67(5), 2292 - 8
Characterization of the Escherichia coli AF/R1 pilus operon: novel genes necessary for transcriptional regulation and for pilus-mediated adherence; Cantey JR et al.; We isolated the genetic determinant of AF/R1 pilus production in attaching/effacing Escherichia coli RDEC-1 and identified seven genes required for pilus expression and function . DNA sequence analysis of the structural subunit gene afrA corrected an error in the published sequence and extended homology with the F18 pilus subunit of pig edema E . coli strains . AfrB and AfrC, encoded downstream from AfrA, were required for pilus expression . AfrB was related to the usher protein PefC of Salmonella typhimurium plasmid-encoded fimbriae, and AfrC was related to PefD, a chaperone protein . AfrD and AfrE, encoded downstream from AfrC, were not necessary for the expression of AF/R1 pili but were required for ileal adherence as assayed by ileal brush border aggregation . Thus, the adhesive subunit of the AF/R1 pilus is distinct from the structural subunit, as is the case for Pap pili and type 1 pili . AfrD was related to FedE of the F18 fimbrial operon of the E . coli strain that causes edema disease in pigs . AfrE was a novel protein . AfrR and AfrS are encoded upstream from AfrA, in the opposite orientation . AfrR is related to the AraC family of transcriptional regulators, and AfrR and AfrS interact to function in a novel mode of transcriptional activation of afrA . AF/R1 pili mediate the adherence to Peyer's patch M cells, ileal mucosa, and colonic mucosa in a rabbit model of diarrhea caused by enteropathogenic E . coli . Our observations will facilitate the further study of the phenomena of M-cell adherence.

Infect Immun, 1999 May, 67(5), 2225 - 32
Functional expression of Nramp1 in vitro in the murine macrophage line RAW264.7; Govoni G et al.; Mutations at the Nramp1 locus in vivo cause susceptibility to infection by unrelated intracellular microbes . Nramp1 encodes an integral membrane protein abundantly expressed in the endosomal-lysosomal compartment of macrophages and is recruited to the phagosomal membrane following phagocytosis . The mechanism by which Nramp1 affects the biochemical properties of the phagosome to control microbial replication is unknown . To devise an in vitro assay for Nramp1 function, we introduced a wild-type Nramp1(G169) cDNA into RAW 264.7 macrophages (which bear a homozygous mutant Nramp1(D169) allele and thus are permissive to replication of specific intracellular parasites) . Recombinant Nramp1 was expressed in a membranous compartment in RAW264.7 cells and was recruited to the membrane of Salmonella typhimurium and Yersinia enterocolitica containing phagosomes . Evaluation of the antibacterial activity of RAW264.7 transfectants showed that expression of the recombinant Nramp1 protein abrogated intracellular replication of S . typhimurium . Studies with a replication-defective S . typhimurium mutant suggest that this occurs through an enhanced bacteriostatic activity . The effect of Nramp1 expression was specific, since (i) it was not seen in RAW264.7 transfectants overexpressing the closely related Nramp2 protein, and (ii) control RAW264.7 cells, Nramp1, and Nramp2 transfectants could all efficiently kill a temperature-sensitive, replication-defective mutant of S . typhimurium . Finally, increased antibacterial activity of the Nramp1 RAW264.7 transfectants was linked to increased phagosomal acidification, a distinguishing feature of primary macrophages expressing a wild-type Nramp1 allele . Together, these results indicate that transfection of Nramp1 cDNAs in the RAW264.7 macrophage cell line can be used as a direct assay to study both Nramp1 function and mechanism of action as well as to identify structure-function relationships in this protein.

Virus Res, 1999 Mar, 60(1), 95 - 9
Specific interaction of fused H protein of bacteriophage phiX174 with receptor lipopolysaccharides; Suzuki R et al.; The DNA fragment encoding the spike H protein of bacteriophage phiX174 was amplified by polymerase chain reaction . The fragment was sub-cloned into pQE-30 to yield pQE-H . The histidine-tagged H protein (HisH) was obtained from the cell extract of Escherichia coli M15 (pREP4) harboring pQE-H and purified by nickel chelating and anion-exchange chromatographies . HisH was shown to bind dose-dependently to the lipopolysaccharides (LPSs) isolated from phiX174-sensitive strains, E . coli C or Salmonella typhimurium TV119 (Ra mutant) . In sharp contrast, HisH did not bind to the LPSs from insensitive strains, E . coli F583 (Rd2 mutant) or E . coli O111:B4 (smooth strain) . Since the same selectivity was observed in the plaque counting assay for in vitro inactivation of phiX174, the spike H protein was shown to recognize receptor LPS.

Kaohsiung J Med Sci, 1999 Mar, 15(3), 127 - 36
Epidemiological study of human salmonellosis during 1991-1996 in southern Taiwan; Chen YH et al.; Within a 6-year period from January 1991 to December 1996, 249 patients of salmonellosis admitted to Kaohsiung Medical College Hospital were enrolled for clinical and microbiological analysis . The number of patients increased by year from 1991 (14 patients) to 1996 (79 patients), especially in the case of nontyphoid salmonellosis . There were 57 different serotypes isolated during these period . Salmonella typhimurium was the most common clinical serotype of human origin in southern Taiwan, followed by S . choleraesuis, S . schwanzengrund, and S . derby . Fever (81.1%), diarrhea (68.9%), and anorexia (44.6%) were the most common manifestations of human salmonellosis . Relative bradycardia was a more important feature in S . typhi group (100%) than nontyphoid salmonellosis . Leukocytosis, especially lymphocytosis, was found especially in nontyphoid, but not in typhoid salmonellosis . Elevated liver function tests were found in the most severe patients, such as S . choleraesuis and S . typhi infections . Malignancy (8.8%), especially hematological malignancy (5.2%), gastrointestinal diseases (8.8%), and diabetes mellitus (6.4%) were the common underlying diseases . Case fatality rate of human salmonellosis was 8% (20/249), especially high in S . choleraesuis group . The severity of underlying diseases may be the major cause in S . choleraesuis group . There was no fatal case with typhoid fever . Very high resistance rate to commonly used antimicrobial agents in nontyphoid Salmonella was noted in southern Taiwan with overall rates of resistance to ampicillin, 67.9%, chloramphenicol, 66.7%, and TMP/SMZ, 42.2% . The emergence of ciprofloxacin-resistant and multiresistant strains was also a major therapeutic problem in this study.

Mutat Res, 1999 Apr 26, 441(1), 43 - 51
Exposure to mutagenic airborne particulate in a rubber manufacturing plant; Fracasso ME et al.; Epidemiological studies conducted in the 1980s revealed that people working in the rubber manufacturing industry had an increased risk of cancer . Even now, workers employed in rubber processing are still at risk despite the measures adopted to improve their working conditions . The aim of the study was to evaluate the presence of a genotoxic risk in a rubber industry and to verify whether or not it was possible to locate the most dangerous position among the different rubber-working processes . The mutagenic activity of airborne particulate was evaluated in samples collected in the mixing department of a rubber manufacturing plant . Ambient air samples were taken over 3-h period in two stable positions near the mixing (Banbury mixer) and calendering areas . Personal air samples were taken over 2-h period during a normal workday from five workers employed in different rubber processing operations (mixing, weighing, calendering, compounding and extruding) . The mutagenic activity of the air samples was determined by plate incorporation assay using Salmonella typhimurium strains (TA 98, TA 98NR, TA 100, YG 1021) with and without metabolic activation . Polycyclic aromatic hydrocarbon (PAH) concentrations were determined by high-performance liquid chromatography (HPLC); the presence of other presumable contaminants were carried out by gas chromatography-mass spectrometry (GC-MS) . The results showed substantial direct and indirect frameshift mutagenicity in both ambient and personal samples . No mutagenic activity was present in S . typhimurium TA 100, except in the personal sample from a worker employed on the Banbury mixer . HPLC analysis revealed very low concentrations of PAHs . GC-MS analysis showed the presence of compounds such as azulene derivative, 1,2-dihydro-2,2,4-trimethylquinoline, N-methyl N-phenylbenzenamine, diphenylamine, bis(2-ethylhexyl)phthalate and bis(methyl-propyl)phthalate . We conclude that the high levels of mutagenic activity in ambiental and personal samples indicate the presence of substances with high genotoxic potency; no substantial differences were seen among the several rubber processing operations . PAHs were not involved in indoor pollution . GC-MS analysis revealed the presence of compounds which may be produced by high temperatures to which the raw materials are subjected during rubber manufacturing processes . These substances are potential carcinogen though their mutagen properties have not been clearly determined .

Mutat Res, 1999 Apr 26, 441(1), 1 - 9
Antimutagenic effects of natural phenolic compounds in beans; de Mejia EG et al.; Polyphenols in fruits, vegetables (e.g., flavonols like quercetin) and tea (e.g., catechins such as epigallocatechin gallate) are good antioxidants with antimutagenic and anticarcinogenic properties . In the present study, the Salmonella typhimurium tester strain YG1024 was used in the plate-incorporation test to examine the antimutagenic effect of phenolic compounds, extracted from common beans (Phaseolus vulgaris), on 1-NP and B{a}P mutagenicity . Dose-response curves for 1-NP and B{a}P were obtained; the number of net revertants/plate at the peak mutagenic dosage were 880 for 1-NP and 490 for B{a}P . For the antimutagenicity studies doses of 0.1 microg/plate and 2 microg/plate for 1-NP and B{a}P, respectively, were chosen . We obtained a dose-response curve of ellagic acid (EA) against B{a}P and 1-NP mutagenicity . To test the bean extract, a dose of 300 microg/plate of EA was chosen as the antimutagenic control . The EA and bean extracts were not toxic to the bacteria at the concentrations tested . The inhibitory effects of the bean extracts and EA against B{a}P mutagenicity were dose-dependent . The percentages of inhibition produced against B{a}P (2 microg/plate) using 300 microg/plate of EA and for the extracts 500 microg equivalent catechin/plate were 82%, 83%, 81% and 83% for EA, water extract, water/methanol extract and methanol extract, respectively . However, for 1-NP mutagenicity, only the methanolic extract from beans showed an inhibitory effect . These results suggest that common beans, as other legumes, can function as health-promoting foods .

J Exp Med, 1999 May 3, 189(9), 1479 - 88
Requirement of p21-activated kinase (PAK) for Salmonella typhimurium-induced nuclear responses; Chen LM et al.; Salmonella typhimurium has sustained a long-standing association with its host and therefore has evolved sophisticated strategies to multiply and survive within this environment . Central to Salmonella pathogenesis is the function of a dedicated type III secretion system that delivers bacterial effector proteins into the host cell cytoplasm . These effectors stimulate nuclear responses and actin cytoskeleton reorganization leading to the production of proinflammatory cytokines and bacterial internalization . The stimulation of these responses requires the function of Cdc42, a member of the Rho family of small molecular weight GTPases, and SopE, a bacterial effector protein that stimulates guanine nucleotide exchange on Rho GTPases . However, nothing is known about the role of Cdc42 effector proteins in S . typhimurium-induced responses . We showed here that S . typhimurium infection of cultured epithelial cells results in the activation of p21-activated kinase (PAK), a serine/threonine kinase that is an effector of Cdc42-dependent responses . Transient expression of a kinase-defective PAK blocked both S . typhimurium- and SopE-induced c-Jun NH2-terminal kinase (JNK) activation but did not interfere with bacteria-induced actin cytoskeleton rearrangements . Similarly, expression of SH3-binding mutants of PAK did not block actin-mediated S . typhimurium entry into cultured cells . However, expression of an effector loop mutant of Cdc42Hs (Cdc42HsC40) unable to bind PAK and other CRIB (for Cdc42/Rac interacting binding)-containing target proteins resulted in abrogation of both S . typhimurium-induced nuclear and cytoskeletal responses . These results show that PAK kinase activity is required for bacteria-induced nuclear responses but it is not required for cytoskeletal rearrangements, indicating that S . typhimurium stimulates cellular responses through different Cdc42 downstream effector activities . In addition, these results demonstrate that the effector loop of Cdc42 implicated in the binding of PAK and other CRIB-containing target proteins is required for both responses.

Appl Environ Microbiol, 1999 May, 65(5), 1924 - 9
Combinations of intervention treatments resulting in 5-log10-unit reductions in numbers of Escherichia coli O157:H7 and Salmonella typhimurium DT104 organisms in apple cider; Uljas HE et al.; The U.S . Food and Drug Administration (FDA) recently mandated a warning statement on packaged fruit juices not treated to reduce target pathogen populations by 5 log10 units . This study describes combinations of intervention treatments that reduced concentrations of mixtures of Escherichia coli O157:H7 (strains ATCC 43895, C7927, and USDA-FSIS-380-94) or Salmonella typhimurium DT104 (DT104b, U302, and DT104) by 5 log10 units in apple cider with a pH of 3.3, 3.7, and 4.1 . Treatments used were short-term storage at 4, 25, or 35 degrees C and/or freeze-thawing (48 h at -20 degrees C; 4 h at 4 degrees C) of cider with or without added organic acids (0.1% lactic acid, sorbic acid {SA}, or propionic acid) . Treatments more severe than those for S . typhimurium DT104 were always required to destroy E . coli O157:H7 . In pH 3.3 apple cider, a 5-log10-unit reduction in E . coli O157:H7 cell numbers was achieved by freeze-thawing or 6-h 35 degrees C treatments . In pH 3.7 cider the 5-log10-unit reduction followed freeze-thawing combined with either 6 h at 4 degrees C, 2 h at 25 degrees C, or 1 h at 35 degrees C or 6 h at 35 degrees C alone . A 5-log10-unit reduction occurred in pH 4.1 cider after the following treatments: 6 h at 35 degrees C plus freeze-thawing, SA plus 12 h at 25 degrees C plus freeze-thawing, SA plus 6 h at 35 degrees C, and SA plus 4 h at 35 degrees C plus freeze-thawing . Yeast and mold counts did not increase significantly (P < 0.05) during the 6-h storage at 35 degrees C . Cider with no added organic acids treated with either 6 h at 35 degrees C, freeze-thawing or their combination was always preferred by consumers over pasteurized cider (P < 0.05) . The simple, inexpensive intervention treatments described in the present work could produce safe apple cider without pasteurization and would not require the FDA-mandated warning statement.

APMIS, 1999 Mar, 107(3), 318 - 24
The effect of hydrocarbon chain length, pH, and temperature on the binding and bactericidal effect of amphiphilic betaine esters on Salmonella typhimurium; Ahlstrom B et al.; Amphiphilic betaine esters are quaternary ammonium compounds (QAC) with rapid microbicidal action . They are often labeled 'soft antimicrobial agents', since the compounds hydrolyze spontaneously into betaine and fatty alcohols, thus not only losing their surface active properties and toxicity but also becoming amenable to metabolic use . The present results show that the bactericidal effects of 1-decyl (B10), 1-dodecyl (B12), and 1-tetradecyl (B14) betaine esters on Salmonella typhimurium 395 MS decreased with decreasing hydrocarbon chain lengths, decreased at pH below neutral, and were lower at 0 degrees C that at 30 degrees C . At least part of the decreased effect at pH 4.0 as compared to pH 6.0 can be explained by reduced binding . However, reduced binding cannot explain the decrease in the microbicidal effect at 0 degrees C since the binding of B 14 was the same at 0 degrees C and 30 degrees C although 10-30 times higher concentrations were required at 0 degrees C to achieve the same microbicidal effect as at 30 degrees C . Neither can differences in binding explain the great differences seen in microbicidal effect between QAC with different chain lengths . It is proposed that the membrane deformation resulting in killing of S . typhimurium is more efficiently achieved with QAC with longer hydrocarbon chains and that reduced fluidity of the outer membrane of the bacteria at lower temperatures antagonizes the bactericidal effect . Charge interaction seems to be more important for the binding and bactericidal effect for the QAC with shorter hydrocarbon chains . The different effects of pH, temperature, and hydrocarbon chain length on binding, bactericidal effect, and hydrolysis have to be taken into account when optimizing disinfection and the subsequent elimination of disinfectants.

Carcinogenesis, 1999 Apr, 20(4), 545 - 51
Heterocyclic aromatic amines induce DNA strand breaks and cell transformation; Pfau W et al.; Heterocyclic aromatic amines (HAAs), formed during the cooking of foods, are known to induce tumours in rodent bioassays and may thus contribute to human cancer risk . We tested six HAAs in a morphological transformation assay and in three in vitro genotoxicity assays . The morphological transforming abilities of HAAs were tested, in the presence of rat-liver S9, in the C3H/M2 fibroblast cell line . Concentration levels of 50 microM 2-amino-3,8-dimethylimidazo{4,5-f}quinoxaline (8-MeIQx), 100 microM 2-amino-3,4,8-trimethylimidazo-{4,5-f}quinoxaline (4,8-DiMeIQx), 50 microM 2-amino-3-methylimidazo{4,5-f}quinoline (IQ), 100 microM 2-amino-9H-pyrido{2,3-b}indole (AalphaC), 100 microM 2-amino-3-methyl-9H-pyrido{2,3-b}indole (MeAalphaC) and 15 microM 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine (PhIP) induced maximum transformation potencies of 5.5, 6.6, 6.3, 5.2, 7.3 and 9.2 transformed foci per 10(4) surviving cells, respectively . Bacterial mutagenic activity was determined in the presence of rat-liver S9 using the Salmonella typhimurium reverse-mutation assay employing strain YG1019 . Mutagenic potencies of 3800 revertants (revs)/ng with 8-MeIQx, 2900 revs/ng with 4,8-DiMeIQx, 3480 revs/ng with IQ, 1.6 revs/ng with AalphaC, 2.9 revs/ng with MeAalphaC and 5 revs/ng with PhIP were observed . Clastogenic activity in vitro was analysed by the micronucleus assay in metabolically competent MCL-5 cells . Dose-dependent induction of micronuclei was observed for all HAAs tested with 1-5.4% of cells containing micronuclei at 10 ng/ml . Micronucleus induction was in the order 4,8-DiMeIQx > 8-MeIQx > IQ > MeAalphaC > PhIP > AalphaC . DNA strand-breaking activity in MCL-5 cells was measured by the alkaline single cell-gel (comet) assay . The lowest effect doses for significant increases (P < or = 0.0007, Mann-Whitney test) in comet tail length (microm) were 45.5 microg/ml (200 microM) for PhIP, 90.9 microg/ml (410-510 microM) for 4,8-DiMeIQx, IQ, MeAalphaC and AalphaC, and 454.5 microg/ml (2130 microM) for 8-MeIQx . It is not yet clear which of these assays most accurately reflects the genotoxic potential to humans of compounds of this class of environmental carcinogens.

Emerg Infect Dis, 1999 Mar-Apr, 5(2), 216 - 23
Enteropathogenic E . coli, Salmonella, and Shigella: masters of host cell cytoskeletal exploitation; Goosney DL et al.; Bacterial pathogens have evolved numerous strategies to exploit their host's cellular processes so that they can survive and persist . Often, a bacterium must adhere very tightly to the cells and mediate its effects extracellularly, or it must find a way to invade the host's cells and survive intracellularly . In either case, the pathogen hijacks the host's cytoskeleton . The cytoskeleton provides a flexible framework for the cell and is involved in mediating numerous cellular functions, from cell shape and structure to programmed cell death . Altering the host cytoskeleton is crucial for mediating pathogen adherence, invasion, and intracellular locomotion . We highlight recent advances in the pathogenesis of enteropathogenic Escherichia coli, Salmonella Typhimurium, and Shigella flexneri . Each illustrates how bacterial pathogens can exert dramatic effects on the host cytoskeleton.

Biochemistry, 1999 Apr 27, 38(17), 5283 - 9
Selection and characterization of human cytochrome P450 1A2 mutants with altered catalytic properties; Parikh A et al.; Random mutagenesis is an approach that has the potential to provide useful information about cytochrome P450 (P450) enzymes but has not been extensively used to date . We applied our previously developed systems for generation of random libraries of human P450 1A2 with the putative substrate recognition sequences mutated (individual residues) and an Escherichia coli genotoxity assay involving reversion to lac prototrophy as a response to activation of the heterocyclic amine 2-amino-3,5-dimethylimidazo{4,5-f}quinoline (MeIQ) . A total of 27 mutants were screened from 6000 clones, a small portion of the library . The sequence changes were identified, and E . coli membranes containing each P450 (with NADPH-P450 reductase expressed using a bicistronic vector) were used to determine kcat and Km values for 7-ethoxyresorufin and phenacetin O-deethylation and the (in vitro) activation of MeIQ with another bacterial genotoxicity system (Salmonella typhimurium umu) . Within each assay, the values of kcat/Km varied by 2 orders of magnitude, and in some cases these parameters were 3-4-fold higher than for the native enzyme . The profiles of the mutants varied considerably for the three different reactions . Some of the mutants in the Asp-320 region may be compared with site-directed mutants of rat P450 1A2 already reported, with several differences noted . Other mutants have not been studied before in human P450 1A2 or homologues and are of interest; i.e., all Phe-226 mutants showed considerably reduced activity but Glu-225 mutants had increased catalytic activities . In principle, this approach may be applied to random mutagenesis of any enzyme that converts chemicals to mutagens and can be expressed in bacteria.

Shock, 1999 Apr, 11(4), 253 - 8
Lethality of endotoxin in mice genetically deficient in the respiratory burst oxidase, inducible nitric oxide synthase, or both; Nicholson SC et al.; Two classes of oxidants are thought to play a critical role in tissue damage in septic shock: reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) . Particular importance has been ascribed to peroxynitrite, a product arising from the reaction of nitric oxide with superoxide . A major source of ROI is the respiratory burst oxidase of neutrophils, eosinophils, monocytes, and macrophages . A major source of RNI is inducible nitric oxide synthase (iNOS), an enzyme expressed in leukocytes, hepatocytes, vascular smooth muscle cells, endothelium, and cardiac myocytes during inflammation . In previous studies using various mouse models of endotoxic shock, genetic deficiency of iNOS as a sole intervention did not consistently alter survival . Here, using Salmonella typhimurium endotoxic bacterial lipopolysaccharide (LPS) as a sole challenge, genetic deficiency of iNOS was associated with no protection or a reduction in survival, depending on the dose of LPS . Further, no protection from lethality was observed when LPS was injected into mice genetically deficient in the 91 kDa subunit of the respiratory burst oxidase (gp91phox) nor in mice genetically deficient in both gp91phox and iNOS (gp91phox-/-/NOS2-/- mice) . For the latter experiments, mice were challenged either with S . typhimurium LPS alone or with inactivated bacille Calmette-Guerin (BCG) followed by Escherichia coli LPS . Deficiency of gp91phox impaired the inflammatory response to inactivated Propionobacterium acnes, rendering survival studies following priming with P . acnes difficult to interpret . Thus, in two models of endotoxic shock, major reductions in the ability to form nitric oxide or superoxide, alone or in combination, failed to improve survival.

J Bacteriol, 1999 May, 181(9), 2938 - 41
Histidine operon deattenuation in dnaA mutants of Salmonella typhimurium correlates with a decrease in the gene dosage ratio between tRNA(His) and histidine biosynthetic loci; Blanc-Potard AB et al.; Expression of the histidine operon of Salmonella typhimurium is increased in dnaA(Ts) mutants at 37 degrees C . This effect requires an intact his attenuator and can be suppressed by increasing the gene copy number of the hisR locus, which encodes the tRNA(His) . We present data which suggest that the his deattenuation defect in dnaA(Ts) mutants results from the loss of a gene dosage gradient between the hisR locus, close to oriC, and the his operon, far from oriC . Some of the conclusions drawn here may apply to other operons as well.

FEBS Lett, 1999 Apr 1, 448(1), 131 - 4
A Cys-less variant of the bacterial ATP binding cassette protein MalK is functional in maltose transport and regulation; Hunke S et al.; The cysteine residues of the ABC protein MalK from Salmonella typhimurium maltose transport system (C40, C350, C360) were consecutively replaced by serines . Cys-less MalK was fully functional in maltose transport in vivo . Moreover, the activity of MalK as a repressor of other maltose-regulated genes was also retained . The absence of cysteine residues in the purified protein was verified by its failure to react with fluorescein-5-maleimide . In contrast to purified wild-type MalK, the ATPase activity of the C40S variant was insensitive to inhibition by N-ethylmaleimide.

Mol Microbiol, 1999 Apr, 32(1), 179 - 91
The regulation and role of the periplasmic copper, zinc superoxide dismutase of Escherichia coli; Gort AS et al.; The discovery of superoxide dismutase (CuZnSOD) within the periplasms of several Gram-negative pathogens suggested that this enzyme evolved to protect cells from exogenous sources of superoxide, such as the oxidative burst of phagocytes . However, its presence in some non-pathogenic bacteria implies that there may be a role for this SOD during normal growth conditions . We found that sodC, the gene that encodes the periplasmic SOD of Escherichia coli, is repressed anaerobically by Fnr and is among the many antioxidant genes that are induced in stationary phase by RpoS . Surprisingly, the entry of wild-type E . coli into stationary phase is accompanied by a several-hour-long period of acute sensitivity to hydrogen peroxide . Induction of the RpoS regulon helps to diminish that sensitivity . While mutants of E . coli and Salmonella typhimurium that lacked CuZnSOD were not detectably sensitive to exogenous superoxide, both were killed more rapidly than their parent strains by exogenous hydrogen peroxide in early stationary phase . This sensitivity required prior growth in air . Evidently, periplasmic superoxide is generated during stationary phase by endogenous metabolism and, if it is not scavenged by CuZnSOD, it causes an unknown lesion that augments or accelerates the damage done by peroxide . The molecular details await elucidation.

Biosci Biotechnol Biochem, 1999 Jan, 63(1), 216 - 9
Occurrence of stereoisomers of 1-(2'-pyrrolidinethione-3'-yl)- 1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid in fermented radish roots and their different mutagenic properties; Ozawa Y et al.; Stereoisomers of the tetrahydro-beta-carboline derivative, 1-(2-pyrrolidinethione)-3-yl)-1,2,3,4-tetrahydro-beta-carboline- 3-carboxylic acid (PTCC), were formed from L-tryptophan with 4-methylthio-3-butenyl isothiocyanate, and their mutagenic properties and contents in different types of the radish products were studied . The isomers were identified as (1S*, 3S*, 3R*)- and (1R*, 3S*, 3R*)-PTCCs; the former was found as the major compound but had no mutagenic activity, while the latter was mutagenic toward Salmonella typhimurium TA 98 in the presence of a rat microsomal fraction . Both (1S*, 3S*, 3R*)- and (1R*, 3S*, 3R*)-PTCC were detected in a ratio of about 4:1 in a product fermented for 8 months, but only a trace was apparent in products manufactured within a few weeks.

Lett Appl Microbiol, 1999 Apr, 28(4), 255 - 7
Induction of rpoS in Salmonella typhimurium by nutrient-poor and depleted media is slower than that achieved by a competitive microflora; Aldsworth TG et al.; The presence of a viable competitive microflora, at greater than 10(8) cfu ml-1, is known to advance the induction of RpoS-mediated gene expression in a sub-population of Salmonella typhimurium . As starvation is known to induce RpoS, one action of the competitive microflora could be to cause depletion of essential nutrients . The aim of the current experiments was to determine whether this was the case by examining RpoS induction in Salm . typhimurium in reduced nutrient media . RpoS-mediated gene expression in Salm . typhimurium was not advanced so significantly in 'conditioned' or diluted medium as it was in the presence of competitors, which indicates that nutrient depletion was not the responsible mechanism . The effect of a competitive microflora has implications for models of bacterial survival during food processing, as RpoS ultimately regulates both stress resistance and virulence.

Mol Microbiol, 1999 Mar, 31(6), 1759 - 73
Environmental regulation of Salmonella pathogenicity island 2 gene expression; Deiwick J et al.; The enteric pathogen Salmonella typhimurium co-ordinates the expression of virulence determinants in response to environmental cues from the host organism . S . typhimurium possesses Salmonella pathogenicity island 2 (SPI2), a large virulence locus encoding a type III secretion system for virulence determinants required for systemic infections and accumulation inside host cells . We have generated transcriptional fusions to SPI2 genes to analyse expression and used antibodies against recombinant SPI2 proteins to monitor levels of SPI2 proteins under various conditions . Here, we demonstrate that SPI2 gene expression is induced by Mg2+ deprivation and phosphate starvation . These conditions are likely to represent the environmental cues encountered by S . typhimurium inside the phagosome of infected host cells . The induction of SPI2 gene expression is modulated by the global regulatory system PhoPQ and is dependent on SsrAB, a two-component regulatory system encoded by SPI2.

Novartis Found Symp, 1999, 221, 55 - 69; discussion 70-4
Inducible acid tolerance mechanisms in enteric bacteria; Foster JW et al.; Enteric micro-organisms have developed several inducible mechanisms for surviving transient periods of extreme acid stress . Salmonella typhimurium possesses an acid tolerance response (ATR) induced in minimal medium by short exposures to mild acid stress . More than 50 acid shock proteins (ASPs) are induced during adaptation . Eight ASPs are regulated by the major iron regulatory protein, Fur, in an unusual iron-independent manner . The two-component regulator, PhoP, is an autoinduced ASP that controls the induction of three additional ASPs . The stress sigma factor sigma S is an ASP that regulates induction of eight ASPs . Acid induction of sigma S is due to its decreased proteolytic turnover via the ClpXP protease in conjunction with the two-component-type response regulator MviA (RssB in Escherichia coli) . Mutations in any of these three regulators leads to a defective ATR . Repair of pH stress-induced DNA damage appears to require the Ada protein (O6-methylguanine methyltransferase) since an ada mutant is both acid and alkaline sensitive . In contrast to S . typhimurium, E . coli and Shigella have acid resistance systems induced in complex media that include a glucose-repressed system protective at pH 2.5 without amino acid supplementation, a glutamate decarboxylase system that requires glutamate and an arginine decarboxylase system unique to E . coli.

Microbiology, 1999 Jan, 145 ( Pt 1), 221 - 9
Expression of disulphide-bridge-dependent conformational epitopes and immunogenicity of the carboxy-terminal 19 kDa domain of Plasmodium yoelii merozoite surface protein-1 in live attenuated Salmonella vaccine strains; Somner EA et al.; The 19 kDa carboxy-terminal domain of Plasmodium yoelii merozoite surface protein-1 (MSP1(19)) was expressed in Salmonella vaccine strains as a carboxy-terminal fusion to fragment C of tetanus toxin (TetC) . This study demonstrates that antibodies that recognize disulphide-dependent conformational epitopes in native MSP1 react with the TetC-MSP1(19) fusion protein expressed in Salmonella . The proper folding of MSP1(19) polypeptide is dependent on both the Salmonella host strain and the protein to which the MSP1(19) polypeptide is fused . Serum from mice immunized with Salmonella typhimurium C5aroD expressing TetC-MSP1(19) recognized native MSP1 as shown by immunofluorescence with P . yoelii-infected erythrocytes . Antibody levels to MSP1(19) were highest in out-bred mice immunized with S . typhimurium C5aroD carrying pTECH2-MSP1(19) and antibody was mostly directed against reduction-sensitive conformational epitopes . However, antibody levels were lower than in BALB/c mice immunized with a glutathione S-transferase (GST)-MSP1(19) fusion protein in Freund's adjuvant, and which were protected against P . yoelii challenge infection . In challenge experiments with P . yoelii the Salmonella-immunized mice were not protected, probably reflecting the magnitude of the antibody response . The results of this study have important implications in the design of live multivalent bacterial vaccines against eukaryotic pathogens.

Microbiology, 1999 Jan, 145 ( Pt 1), 15 - 31
The medium-/long-chain fatty acyl-CoA dehydrogenase (fadF) gene of Salmonella typhimurium is a phase 1 starvation-stress response (SSR) locus; Spector MP et al.; Salmonella enterica serovar Typhimurium (S . typhimurium) is an enteric pathogen that causes significant morbidity in humans and other mammals . During their life cycle, salmonellae must survive frequent exposures to a variety of environmental stresses, e.g . carbon-source (C) starvation . The starvation-stress response (SSR) of S . typhimurium encompasses the genetic and physiological realignments that occur when an essential nutrient becomes limiting for bacterial growth . The function of the SSR is to produce a cell capable of surviving long-term starvation . This paper reports that three C-starvation-inducible lac fusions from an S . typhimurium C-starvation-inducible lac fusion library are all within a gene identified as fadF, which encodes an acyl-CoA dehydrogenase (ACDH) specific for medium-/long-chain fatty acids . This identification is supported by several findings: (a) significant homology at the amino acid sequence level with the ACDH enzymes from other bacteria and eukaryotes, (b) undetectable beta-oxidation levels in fadF insertion mutants, (c) inability of fad insertion mutants to grow on oleate or decanoate as a sole C-source, and (d) inducibility of fadF::lac fusions by the long-chain fatty acid oleate . In addition, the results indicate that the C-starvation-induction of fadF is under negative control by the FadR global regulator and positive control by the cAMP:cAMP receptor protein complex and ppGpp . It is also shown that the fadF locus is important for C-starvation-survival in S . typhimurium . Furthermore, the results demonstrate that fadF is induced within cultured Madin-Darby canine kidney (MDCK) epithelial cells, suggesting that signals for its induction (C-starvation and/or long-chain fatty acids) may be present in the intracellular environment encountered by S . typhimurium . However, fadF insertion mutations did not have an overt effect on mouse virulence.

Bioorg Med Chem Lett, 1999 Mar 8, 9(5), 637 - 40
Enzymatic syntheses of 6-(4H-selenolo{3,2-b}pyrrolyl)-L-alanine, 4-(6H-selenolo{2,3-b}pyrrolyl)-L-alanine, and 6-(4H-furo{3,2-b}pyrrolyl-L-alanine; Welch M et al.; 6-(4H-Selenolo{3,2-b}pyrrolyl)-L-alanine 1, 4-(6H-selenolo{2,3-b}pyrrolyl)-L-alanine 2, and 6-(4H-furo{3,2-b}pyrrolyl)-L-alanine 3 have been synthesized via reactions of selenolo{3,2-b}pyrrole, selenolo{2,3-b}pyrrole, and furo{3,2-b}pyrrole, respectively, with L-serine . The reactions are catalyzed by Salmonella typhimurium tryptophan synthase.

J Pharm Pharmacol, 1999 Jan, 51(1), 105 - 9
Preventive effects of a mixed disulphide from dithiocarbamate and N-acetylcysteine on the genotoxicity of N-nitrosodiethylamine; Lee BH et al.; A mixed disulphide model compound, S-(N,N-diethyldithiocarbamoyl)-N-acetylcysteine (AC-DDTC), prepared from diethyldithiocarbamate and N-acetylcysteine, was investigated for protective effects against the genotoxicity of the environmental carcinogen N-nitrosodiethylamine (NDEA) . AC-DDTC was found to be a potent inhibitor of genotoxicity induced by NDEA . The mutagenicity of NDEA to Salmonella typhimurium was inhibited by 70% at 3.2 micromol AC-DDTC per plate and the effect was dose-dependent . In the chromosome aberration assay, NDEA elicited a significant increase in the number of aberrant cells . Pretreatment with AC-DDTC suppressed the chromosome-damaging effect of NDEA . The micronucleus-inducing capacity of NDEA was reduced by 32% by treatment with AC-DDTC at 1.5 mmol kg(-1) . These results suggest that AC-DDTC might have a role to play in reducing the risk of cancer induced by NDEA.

Vet Immunol Immunopathol, 1999 Feb 22, 67(3), 259 - 70
Interaction between Salmonella typhimurium and phagocytic cells in pigs . Phagocytosis, oxidative burst and killing in polymorphonuclear leukocytes and monocytes; Riber U et al.; Interactions between Salmonella typhimurium and peripheral blood leucocytes from healthy, Salmonella-free pigs were investigated in vitro . Both granulocytes and monocytes phagocytized FITC-labelled heat-killed Salmonella bacteria as shown by flow cytometry . Phagocytosis in whole blood and isolated leucocytes was measured as acquired fluorescence in the leukocytes and was both time and dose related . Living, serum-opsonized Salmonella bacteria induced a dose-dependent oxidative burst in PMNs and monocytes as measured by luminol-enhanced chemiluminescence (LC) . When opsonized in normal serum the Salmonella bacteria, in the range of 2-5 x 10(7) cfu, induced a LC response in monocytes comparable to the level of responses induced by phorbol myristate acetate (PMA) and opsonized zymosan, and the Salmonella-induced response was only marginally reduced by superoxide dismutase (SOD) . Intracellular killing of Salmonella by monocytes was assessed from plate colony counts of lysed monocytes and showed that Salmonella typhimurium was able to survive and proliferate in adherent monocytes in vitro despite a reduction in intracellular cfu during the first hour's incubation in cells from some pigs . Experiments with the exhaustion of oxidative burst in non-adherent monocytes were performed by prestimulation with PMA, heat-killed Salmonella or buffer . Prestimulation with PMA led to a strong reduction in oxidative burst induced by living opsonized Salmonella bacteria, whereas prestimulation with heat-killed bacteria gave rise to an enhanced response . In these experiments intracellular killing of the added living Salmonella gave variable results, in that monocytes from two out of three pigs showed no essential change in intracellular bactericidal activity, but with cells from one pig a less pronounced bactericidal activity was found after prestimulation with PMA.

Biotechnol Prog, 1999 Mar-Apr, 15(2), 245 - 9
Measurement of bacterial flagellar thrust by negative dielectrophoresis; Hughes MP et al.; The force produced by the flagella of the bacterium Salmonella typhimurium has been measured using negative dielectrophoretic methods . The bacteria are held in a force funnel, produced using a nonuniform electric field . When the motor force is balanced against an opposing negative dielectrophoretic force the bacteria become motionless . Numerical simulations have been used to estimate the electric field gradient in the electrodes . Together with experimental observations of bacterial motion the data gives a value of the force produced by the bacterial motor to be 0.37 pN.

J Infect Dis, 1999 May, 179(5), 1173 - 82
Multidrug-resistant human and animal Salmonella typhimurium isolates in France belong predominantly to a DT104 clone with the chromosome- and integron-encoded beta-lactamase PSE-1; Casin I et al.; Epidemiologic relationships were investigated in 187 ampicillin-resistant Salmonella typhimurium strains (86 human, 101 animal) from >2000 strains isolated in 1994 . Of 23 resistance patterns, the most frequent (ampicillin {Am}, chloramphenicol {Cm}, tetracycline {Tc}, streptomycin and spectinomycin {Sm}, and sulfonamides {Su}) was found in 69.5% of human and 64.8% of animal isolates . Four beta-lactamase genes were identified, blaTEM (24%), blaPSE-1 (78%), and blaSHV and oxa-2 (each <3%) . blaPSE-1 and the integrase gene, intI1, but not blaTEM, blaSHV or oxa-2, were chromosomeborne and found almost exclusively in the AmCmTcSmSu strains . In these, polymerase chain reaction mapping revealed two distinct integrons carrying blaPSE-1 or aadA2 . Lysotypes, plasmid profiles, and restriction fragment length polymorphisms (IS200) were determined for 50 representative isolates and for 3 DT104 strains from the United Kingdom (UK) . The phage type of the PSE-1-producing AmCmTcSmSu strains was 12 atypic, indistinguishable from that of the DT104 strains . The combined data indicate that the same multiresistant clone has spread through human and animal ecosystems in the UK and France.

FEMS Microbiol Lett, 1999 Mar 15, 172(2), 159 - 63
Survival of Salmonella enteritidis and Salmonella typhimurium in chicken manure at different levels of water activity; Himathongkham S et al.; Survival of Salmonella enteritidis and Salmonella typhimurium in chicken manure at different levels of water activity (aw) was determined . The aw was adjusted by means of saturated salts with defined equilibrium relative humidity and the manure samples were stored aerobically at 20 degrees C . At aw levels higher than 0.93, a moderate increase in colony-forming units over 8-9 h was found for both strains; at aw levels of 0.89-0.75, there was a thousand-fold reduction . Extended storage resulted in a million-fold reduction of Salmonella enteritidis in 8 days at an aw of 0.89 . At higher and lower levels of aw, the reduction was less extensive.

Antimicrob Agents Chemother, 1999 Apr, 43(4), 846 - 9
Molecular characterization of an antibiotic resistance gene cluster of Salmonella typhimurium DT104; Briggs CE et al.; Salmonella typhimurium phage type DT104 has become an important emerging pathogen . Isolates of this phage type often possess resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (ACSSuT resistance) . The mechanism by which DT104 has accumulated resistance genes is of interest, since these genes interfere with treatment of DT104 infections and might be horizontally transferred to other bacteria, even to unrelated organisms . Previously, several laboratories have shown that the antibiotic resistance genes of DT104 are chromosomally encoded and involve integrons . The antibiotic resistance genes conferring the ACSSuT-resistant phenotype have been cloned and sequenced . These genes are grouped within two district integrons and intervening plasmid-derived sequences . This sequence is potentially useful for detection of multiresistant DT104.

Chemosphere, 1999 Apr, 38(8), 1721 - 32
Multi-bioassay approach for assessing the potency of complex mixtures of polycyclic aromatic hydrocarbons; Mayura K et al.; The chick embryotoxicity screening test (CHEST) and the Salmonella/microsome bioassay were used to evaluate embryotoxic and mutagenic endpoints from crude coal tar (CT) and its fractionated polycyclic aromatic hydrocarbon (PAH) mixtures (designated as A, B, C, D and E) . In the CHEST assay, CT and PAH mixtures were injected into the egg yolk . A dose-dependent increase in embryo mortality was observed for all fractions . The E fraction resulted in 47% embryo mortality at a dose of 0.125 mg/kg and was more toxic than CT . At a dose of 1 mg/kg, 85-100% embryonic deaths occurred in fractions C and D and these two fractions were more potent than fractions A and B . The main visual toxic manifestations were liver lesions, discoloration of the liver, and edema . Both CT and fractionated PAH mixtures were also tested in the Salmonella/microsome plate incorporation assay with Salmonella typhimurium strain TA98 and were evaluated with and without metabolic activation at five dose levels . In the presence of S9, the CT and fractions C, D and E induced a dose-dependent positive response . Results from the Salmonella/microsome assay were in good agreement with findings from the CHEST assay suggesting that these two bioassays in combination may facilitate the rapid detection and ranking of complex PAH mixtures.

Int J Food Microbiol, 1999 Feb 18, 46(3), 263 - 9
Comparative survival of Salmonella typhimurium DT 104, Listeria monocytogenes, and Escherichia coli O157:H7 in preservative-free apple cider and simulated gastric fluid; Roering AM et al.; This study compared the survival of three-strain mixtures (ca . 10(7) CFU ml(-1) each) of Salmonella typhimurium DT104, Listeria monocytogenes, and Escherichia coli O157:H7 in pasteurized and unpasteurized preservative-free apple cider (pH 3.3-3.5) during storage at 4 and 10 degrees C for up to 21 days . S . typhimurium DT104 populations decreased by <4.5 log10 CFU ml(-1) during 14 days storage at 4 and 10 degrees C in pasteurized cider, and by > or =5.5 log10 CFU ml(-1) during 14 days in unpasteurized cider stored at these temperatures . However, after 7 days at 4 degrees C, the S . typhimurium DT104 populations had decreased by only about 2.5 log10 CFU ml(-1) in both pasteurized and unpasteurized cider . Listeria monocytogenes populations decreased below the plating detection limit (10 CFU ml(-1)) within 2 days under all conditions tested . Survival of E . coli O157:H7 was similar to that of S . typhimurium DT104 in pasteurized cider at both 4 and 10 degrees C over the 21-days storage period, but E . coli O157:H7 survived better (ca . 5.0 log10 CFU ml(-1) decrease) than S . typhimurium DT104 (> 7.0 log10 CFU ml(-1) decrease) after 14 days at 4 degrees C in unpasteurized cider . In related experiments, when incubated in simulated gastric fluid (pH 1.5) at 37 degrees C, S . typhimurium DT104 and L . monocytogenes were eliminated (5.5-6.0 log10 CFU ml(-1) decrease) within 5 and 30 min, respectively, whereas E . coli O157:H7 concentrations decreased only 1.60-2.80 log10 CFU ml(-1) within 2 h.

Microbiol Immunol, 1999, 43(1), 9 - 14
Intracellular Salmonella typhimurium induce lysis of human polymorphonuclear leukocytes which is not associated with the Salmonella virulence plasmid; Chiu CH et al.; The interaction between Salmonella typhimurium and human polymorphonuclear leukocytes (PMNs) was analyzed in vitro . Three S . typhimurium strains, the wild-type strain OU5043, its isogenic virulence plasmid-cured strain OU5048, and LT2, which represented the types that exhibited three mouse virulence levels, respectively, were used in this study . There was no correlation between the recovery of intracellular S . typhimurium from PMNs and the presence or absence of the virulence plasmid, or the strains' mouse virulence level . When the oxygen-dependent response of PMNs upon phagocytosis of S . typhimurium was examined by checking the intracellular reduction of nitroblue tetrazolium (NBT), the fraction of PMNs that reduced NBT on phagocytosis of the three strains was around 80%, whereas it was 58% with Escherichia coli, 95% with phorbol 12-myristate 13-acetate and 15% with a negative control . Thus there were no significant differences among the three Salmonella strains in terms of their ability to induce the oxidative response in PMNs . Microscopic analysis of Salmonella-infected PMNs indicated that the intracellular Salmonella induced lysis of PMNs . Both OU5043 and OU5048 exhibited a significant intracellular cytotoxic effect on PMNs after 24 hr of infection and this effect was not associated with the presence or absence of the virulence plasmid . On the other hand, lysis of PMNs was related to the intracellular survival of Salmnonella, as ofloxacin, an antibiotic, appeared to be able to protect human PMNs from Salmonella-induced cytotoxicity when this agent was added into the medium to inactivate the intracellular organism . The ability to induce lysis of PMNs by either wild-type or plasmid-cured strains of S . typhimurium may play a crucial role in the pathogenesis of non-typhoid Salmonella . The contribution of pSTV to human salmonellosis is likely to be limited . Furthermore, early institution of antibiotics with a high intracellular activity against Salmonella, such as fluoroquinolones, may be useful to prevent the dissemination of Salmonella infection.

RNA, 1999 Mar, 5(3), 395 - 408
Structural alterations of the tRNA(m1G37)methyltransferase from Salmonella typhimurium affect tRNA substrate specificity; Li JN et al.; In Salmonella typhimurium, the tRNA(m1G37)methyltransferase (the product of the trmD gene) catalyzes the formation of m1G37, which is present adjacent and 3' of the anticodon (position 37) in seven tRNA species, two of which are tRNA(Pro)CGG and tRN(Pro)GGG . These two tRNA species also exist as +1 frameshift suppressor sufA6 and sufB2, respectively, both having an extra G in the anticodon loop next to and 3' of m1G37 . The wild-type form of the tRNA(m1G37)methyltransferase efficiently methylates these mutant tRNAs . We have characterized one class of mutant forms of the tRNA(m1G37)methyltransferase that does not methylate the sufA6 tRNA and thereby induce extensive frameshifting resulting in a nonviable cell . Accordingly, pseudorevertants of strains containing such a mutated trmD allele in conjunction with the sufA6 allele had reduced frameshifting activity caused by either a 9-nt duplication in the sufA6tRNA or a deletion of its structural gene, or by an increased level of m1G37 in the sufA6tRNA . However, the sufB2 tRNA as well as the wild-type counterparts of these two tRNAs are efficiently methylated by this class of structural altered tRNA(m1G37)methyltransferase . Two other mutations (trmD3, trmD10) were found to reduce the methylation of all potential tRNA substrates and therefore primarily affect the catalytic activity of the enzyme . We conclude that all mutations except two (trmD3 and trmD10) do not primarily affect the catalytic activity, but rather the substrate specificity of the tRNA, because, unlike the wild-type form of the enzyme, they recognize and methylate the wild-type but not an altered form of a tRNA . Moreover, we show that the TrmD peptide is present in catalytic excess in the cell.

J Photochem Photobiol B, 1998 Dec, 47(2-3), 211 - 5
Mutagenicity and dark toxicity of the second-generation photosensitizer bacteriochlorin a; Schuitmaker JJ et al.; Bacteriochlorin a (BCA) is an effective second-generation photosensitizer both in vitro and in vivo . BCA has a high molecular absorption coefficient (32,000 M-1 cm-1) at 760 nm . At this wavelength tissue penetration of light is almost optimal and melanin absorption is relatively low . BCA is preferentially retained in a number of tumour model systems and is rapidly cleared from non-cancerous tissues, thus inducing no or minor skin photosensitivity . Mutagenicity of BCA has been tested using the Salmonella typhimurium strains TA97, TA98, TA100, TA102 and TA104 . In all tester strains used, BCA induces, in the dark, a minute increase in the number of revertants . No linear correlation between the number of revertants and the BCA dose is observed . Incubation of isolated rat hepatocytes with BCA, in the dark, does not result in increased cell death as measured by leakage of cytosolic lactate dehydrogenase . A convenient bioassay to test possible genotoxicity in vivo is the established Somatic Mutation and Recombination Test (SMART) on Drosophila melanogaster . Bacteriochlorin was tested for induction of loss of heterozygosity in the white/white+ eye mosaic assay, which predominantly measures homologous mitotic recombination in somatic cells of Drosophila after treatment of larval stages . BCA did not induce loss of heterozygosity above the level of the incorporated controls, with or without illumination . Based on these results, obtained in prokaryotic and eukaryotic cells and in vivo, we are inclined to conclude that the dark toxicity and mutagenic properties of BCA, as measured by the applied bioassays, are negligible.

Science, 1999 Mar 26, 283(5410), 2092 - 5
Role of the S . typhimurium actin-binding protein SipA in bacterial internalization; Zhou D et al.; Entry of the bacterium Salmonella typhimurium into host cells requires membrane ruffling and rearrangement of the actin cytoskeleton . Here, it is shown that the bacterial protein SipA plays a critical role in this process . SipA binds directly to actin, decreases its critical concentration, and inhibits depolymerization of actin filaments . These activities result in the spatial localization and more pronounced outward extension of the Salmonella-induced membrane ruffles, thereby facilitating bacterial uptake.

Acta Crystallogr D Biol Crystallogr, 1999 Jan, 55 ( Pt 1), 305 - 6 Epub 1999 Jan 01.
Crystallization and preliminary crystallographic studies of the anthranilate synthase partial complex from Salmonella typhimurium; Tolbert WD et al.; Anthranilate synthase catalyzes the first step in the biosynthesis of tryptophan from chorismate . The anthranilate synthase partial complex from Salmonella typhimurium has been crystallized in space group P21212 with unit-cell dimensions a = 116.7, b = 101.2 and c = 66.8 A.

Acta Crystallogr D Biol Crystallogr, 1999 Jan, 55 ( Pt 1), 297 - 8 Epub 1999 Jan 01.
Crystallization and preliminary crystallographic analysis of an NADH oxidase that functions in peroxide reduction in Thermus aquaticus YT-1; Mac Sweeney A et al.; NADH oxidase from Thermus aquaticus is a thermostable flavoenzyme that is similar in amino-acid sequence and other properties to the flavoenzyme component of the NADH peroxidase systems from Salmonella typhimurium and Amphibacillus xylanus . The enzyme has been isolated from T . aquaticus and crystallized using the hanging-drop method of vapour diffusion with sodium citrate as a precipitant at pH 8.5 . The crystals belong to the hexagonal space group P622 with unit-cell dimensions a = b = 89.9, c = 491.6 A, and diffract to 2.5 A resolution.

Acta Crystallogr D Biol Crystallogr, 1999 Jan, 55 ( Pt 1), 285 - 6 Epub 1999 Jan 01.
Crystallization and preliminary X-ray analysis of the bacterial ATP-binding-cassette (ABC) protein MalK; Schmees G et al.; The ATP-binding protein, MalK, of the bacterial ABC (ATP-binding-cassette) transport complex MalFGK2 provides the energy for the translocation of maltose and maltodextrins across the cytoplasmic membrane . The MalK protein from Salmonella typhimurium was overexpressed in Escherichia coli and crystallized by the hanging-drop method using (NH4)2SO4as a precipitant . The crystals belong to space group P6x22 (most probably x = 1 or 5) with cell dimensions a = 181.8 and c = 182.5 A, corresponding to three or four molecules per asymmetric unit . They diffract to a resolution of about 3 A on a synchrotron X-ray source and are suitable for structure determination.

Acta Crystallogr D Biol Crystallogr, 1999 Apr, 55 ( Pt 4), 865 - 8
Preliminary crystallographic studies on glutamine synthetase from Mycobacterium tuberculosis; Gill HS et al.; The etiologic agent of tuberculosis, Mycobacterium tuberculosis, has been shown to secrete the enzyme glutamine synthetase (TB-GS) which is apparently essential for infection . Four crystal forms of a recombinant TB-GS were grown . The one chosen for synchrotron X--ray data collection belongs to space group P212121 with unit-cell dimensions 208 x 258 x 274 A, yielding 2.4 A resolution data . A Matthews number of 2.89 A3 Da-1 is found, corresponding to 24 subunits of molecular mass 1300 kDa in the asymmetric unit . From earlier work, the structure of Salmonella typhimurium GS, which is 51% identical in sequence to TB-GS, is known to be dodecameric with 622 symmetry . Self-rotation calculations on the TB-GS X-ray data reveal only one set of sixfold and twofold axes of symmetry . A Patterson map calculated from the native X-ray data confirms that there are two dodecamers in the asymmetric unit, having both their sixfold and twofold axes parallel to one another.

Microb Pathog, 1999 Mar, 26(3), 121 - 30
Protection and immune responses induced by attenuated Salmonella typhimurium UK-1 strains; Zhang X et al.; We previously reported that Salmonella typhimurium SR-11 mutants with deletion mutations in the genes encoding adenylate cyclase (cya) and the cAMP receptor protein (crp) are avirulent and protective in mice . Salmonella typhimurium UK-1 is highly virulent for chicks (oral LD50 of 3x10(3) CFU) and mice (oral LD50 of 8.5x10(3) CFU) and is capable of lethal infections in pigs, calves and horses . We postulated that attenuated derivatives of this lethal strain would probably induce a higher level of protective immunity than achieved with attenuated derivatives of less virulent S . typhimurium strains such as SR11 . To test this hypothesis, we have constructed S . typhimurium UK-1 Deltacya-12Deltacrp-11 mutant strain chi3985 and its virulence plasmid cured derivative chi4095 to investigate their avirulence and immunogenicity in mice . We found that the mutants are avirulent and able to induce protective immune responses in BALB/c mice . These mutant strains retained wild-type ability to colonize the gut associated lymphoid tissue but reach and persist in spleen and liver at a significantly lower level than the wild-type parent strain . Mice survived oral infection with >1x10(9) CFU of chi3985 (the equivalent to 10(5) 50% lethal doses of wild-type S . typhimurium UK-1) and were fully protected against challenge with 10(5)times the LD50 of the wild-type parent . Immunized mice developed a high level of serum IgG titre to Salmonella LPS and delayed-type hypersensitivity (DTH) response to S . typhimurium outer membrane proteins . Compared to the virulence plasmid-containing strain chi3985, the virulence plasmid cured DeltacyaDeltacrp mutant strain chi4095 was more attenuated and less protective, as some mice immunized with chi4095 died when challenged with the wild-type UK-1 strain . This work demonstrates that S . typhimurium UK-1 Deltacrp Deltacya mutant strain may be a potential live vaccine to induce protective immunity against Salmonella infection or to deliver foreign antigens to the immune system .

Arch Biochem Biophys, 1999 Apr 1, 364(1), 75 - 82
Extracellular iron reductases: identification of a new class of enzymes by siderophore-producing microorganisms; Vartivarian SE et al.; This study identifies extracellular iron reductases in culture supernatant fluids of the siderophore-producing microorganisms Escherichia coli and Pseudomonas aeruginosa . These enzymes were constitutively produced and reduced and released iron from a variety of ferric chelators . Dialyzable cofactors, necessary for the transfer of electrons in the enzymatic reduction of iron, were identified . The reductases were sensitive to treatment with proteinase K and guanidine-HCl, were not associated with siderophore activity, and were apparently released from the cell as extracellular enzymes . The acquisition of 59Fe2+ by cell suspensions of E . coli and P . aeruginosa was saturable, suggesting that the ferrous iron generated by these reductases can be bound and transported . Salmonella typhimurium mutants feoB, tonB, entB, and entBfeoB, deficient in numerous known iron uptake pathways, were found to exhibit substantial extracellular iron-reducing activities over that of the parent . A hypothesis is proposed in which the extracellular iron reductases excreted by siderophore-producing microorganisms may be responsible for the mobilization of iron during conditions of iron repletion when siderophores are repressed and may also function in concert with siderophores during periods of iron starvation .

J Biol Chem, 1999 Mar 26, 274(13), 8757 - 63
Transcription initiation at the flagellin promoter by RNA polymerase carrying sigma28 from Salmonella typhimurium; Schaubach OL et al.; The sigma subunit of RNA polymerase is a critical factor in positive control of transcription initiation . Primary sigma factors are essential proteins required for vegetative growth, whereas alternative sigma factors mediate transcription in response to various stimuli . Late gene expression during flagellum biosynthesis in Salmonella typhimurium is dependent upon an alternative sigma factor, sigma28, the product of the fliA gene . We have characterized the intermediate complexes formed by sigma28 holoenzyme on the pathway to open complex formation . Interactions with the promoter for the flagellin gene fliC were analyzed using DNase I and KMnO4 footprinting over a range of temperatures . We propose a model in which closed complexes are established in the upstream region of the promoter, including the -35 element, but with little significant contact in the -10 element or downstream regions of the promoter . An isomerization event extends the DNA contacts into the -10 element and the start site, with loss of the most distal upstream contacts accompanied by DNA melting to form open complexes . Melting occurs efficiently even at 16 degrees C . Once open complexes have formed, they are unstable to heparin challenge even in the presence of nucleoside triphosphates, which have been observed to stabilize open complexes at rRNA promoters.

Infect Immun, 1999 Apr, 67(4), 2025 - 9
Clonal expansion of antigen-specific CD4 T cells following infection with Salmonella typhimurium is similar in susceptible (Itys) and resistant (Ityr) BALB/c mice; Chen ZM et al.; The results show that CD4 T cells specific for a recombinant antigen expressed in Salmonella typhimurium proliferate normally in mice that express the susceptible form of the Ity gene at early times after infection but do not retain the capacity to produce gamma interferon later in the infection.

Infect Immun, 1999 Apr, 67(4), 2005 - 9
Biological properties of structurally related alpha-helical cationic antimicrobial peptides; Scott MG et al.; A series of alpha-helical cationic antimicrobial peptide variants with small amino acid changes was designed . Alterations in the charge, hydrophobicity, or length of the variant peptides did not improve the antimicrobial activity, and there was no statistically significant correlation between any of these factors and the MIC for Pseudomonas aeruginosa, Escherichia coli, or Salmonella typhimurium . Individual peptides demonstrated synergy with conventional antibiotics against antibiotic-resistant strains of P . aeruginosa . The peptides varied considerably in the ability to bind E . coli O111:B4 lipopolysaccharide (LPS), and this correlated significantly with their antimicrobial activity and ability to block LPS-stimulated tumor necrosis factor and interleukin-6 production . In general, the peptides studied here demonstrated a broad range of activities, including antimicrobial, antiendotoxin, and enhancer activities.

Infect Immun, 1999 Apr, 67(4), 1974 - 81
Salmonella typhimurium encodes a putative iron transport system within the centisome 63 pathogenicity island; Zhou D et al.; Upon entry into the host, Salmonella enterica strains are presumed to encounter an iron-restricted environment . Consequently, these bacteria have evolved a variety of often-redundant high-affinity acquisition systems to obtain iron in this restricted environment . We have identified an iron transport system that is encoded within the centisome 63 pathogenicity island of Salmonella typhimurium . The nucleotide composition of this locus is significantly different from that of the rest of this pathogenicity island, suggesting a different ancestry and a mosaic structure for this region of the S . typhimurium chromosome . This locus, designated sit, consists of four open reading frames which encode polypeptides with extensive homology to the yfe ABC iron transport system of Yersinia pestis, as well as other ABC transporters . The sitA gene encodes a putative periplasmic binding protein, sitB encodes an ATP-binding protein, and sitC and sitD encode two putative permeases (integral membrane proteins) . This operon is capable of complementing the growth defect of the enterobactin-deficient Escherichia coli strain SAB11 in iron-restricted minimal medium . Transcription of the sit operon is repressed under iron-rich growth conditions in a fur-dependent manner . Introduction of a sitBCD deletion into wild-type S . typhimurium resulted in no apparent growth defect in either nutrient-rich or minimal medium and no measurable virulence phenotype . These results further support the existence of redundant iron uptake systems in S . enterica.

Infect Immun, 1999 Apr, 67(4), 1614 - 22
PhoP-PhoQ-regulated loci are required for enhanced bile resistance in Salmonella spp; van Velkinburgh JC et al.; As enteric pathogens, Salmonella spp . are resistant to the actions of bile . Salmonella typhimurium and Salmonella typhi strains were examined to better define the bile resistance phenotype . The MICs of bile for wild-type S . typhimurium and S . typhi were 18 and 12%, respectively, and pretreatment of log-phase S . typhimurium with 15% bile dramatically increased bile resistance . Mutant strains of S . typhimurium and S . typhi lacking the virulence regulator PhoP-PhoQ were killed at significantly lower bile concentrations than wild-type strains, while strains with constitutively active PhoP were able to survive prolonged incubation with bile at concentrations of >60% . PhoP-PhoQ was shown to mediate resistance specifically to the bile components deoxycholate and conjugated forms of chenodeoxycholate, and the protective effect was not generalized to other membrane-active agents . Growth of both S . typhimurium and S . typhi in bile and in deoxycholate resulted in the induction or repression of a number of proteins, many of which appeared identical to PhoP-PhoQ-activated or -repressed products . The PhoP-PhoQ regulon was not induced by bile, nor did any of the 21 PhoP-activated or -repressed genes tested play a role in bile resistance . However, of the PhoP-activated or -repressed genes tested, two (prgC and prgH) were transcriptionally repressed by bile in the medium independent of PhoP-PhoQ . These data suggest that salmonellae can sense and respond to bile to increase resistance and that this response likely includes proteins that are members of the PhoP regulon . These bile- and PhoP-PhoQ-regulated products may play an important role in the survival of Salmonella spp . in the intestine or gallbladder.

Infect Immun, 1999 Apr, 67(4), 1560 - 8
The alternative sigma factor, sigmaE, is critically important for the virulence of Salmonella typhimurium; Humphreys S et al.; In Escherichia coli, extracytoplasmic stress is partially controlled by the alternative sigma factor, RpoE (sigmaE) . In response to environmental stress or alteration in the protein content of the cell envelope, sigmaE upregulates the expression of a number of genes, including htrA . It has been shown that htrA is required for intramacrophage survival and virulence in Salmonella typhimurium . To investigate whether sigmaE-regulated genes other than htrA are involved in salmonella virulence, we inactivated the rpoE gene of S . typhimurium SL1344 by allelic exchange and compared the phenotype of the mutant (GVB311) in vitro and in vivo with its parent and an isogenic htrA mutant (BRD915) . Unlike E . coli, sigmaE is not required for the growth and survival of S . typhimurium at high temperatures . However, GVB311 did display a defect in its ability to utilize carbon sources other than glucose . GVB311 was more sensitive to hydrogen peroxide, superoxide, and antimicrobial peptides than SL1344 and BRD915 . Although able to invade both macrophage and epithelial cell lines normally, the rpoE mutant was defective in its ability to survive and proliferate in both cell lines . The effect of the rpoE mutation on the intracellular behavior of S . typhimurium was greater than that of the htrA mutation . Both GVB311 and BRD915 were highly attenuated in mice . Neither strain was able to kill mice via the oral route, and the 50% lethal dose (LD50) for both strains via the intravenous (i.v.) route was very high . The i.v . LD50s for SL1344, BRD915, and GVB311 were <10, 5.5 x 10(5), and 1.24 x 10(7) CFU, respectively . Growth in murine tissues after oral and i.v . inoculation was impaired for both the htrA and rpoE mutant, with the latter mutant being more severely affected . Neither mutant was able to translocate successfully from the Peyer's patches to other organs after oral infection or to proliferate in the liver and spleen after i.v . inoculation . However, the htrA mutant efficiently colonized the livers and spleens of mice infected i.v., but the rpoE mutant did not . Previous studies have shown that salmonella htrA mutants are excellent live vaccines . In contrast, oral immunization of mice with GVB311 was unable to protect any of the mice from oral challenge with SL1344 . Furthermore, i.v . immunization with a large dose ( approximately 10(6) CFU) of GVB311 protected less than half of the orally challenged mice . Thus, our results indicate that genes in the sigmaE regulon other than htrA play a critical role in the virulence and immunogenicity of S . typhimurium.

Berl Munch Tierarztl Wochenschr, 1997 Oct, 110(10), 391 - 6
{Significance of motility of Salmonella enteritidis and Salmonella typhimurium as a virulence factor and on the expression of the inhibition phenomenon in vitro and in vivo in SPF chickens}; Methner U et al.; Methods of immunoprophylaxis for poultry using live Salmonella vaccines are increasingly gaining in importance . Methods of a simple and reliable bacteriological as well as serological differentiation between vaccine and field strains will be of decisive importance for the acceptance and use of live Salmonella vaccines . The absence of motility in Salmonella strains may be a marker fulfilling these criteria . The studies described served to examine whether virulence and the ability to inhibit other Salmonella strains could be influenced by the absence of motility in Salmonella (S.) Enteritidis and (S.) Typhimurium . In a cell-culture model (IEC 6) under in vitro conditions, non-motile transposon mutants (TnphoA) of S . Enteritidis and S . Typhimurium exhibited a clearly reduced invasion potential in comparison with the respective motile parental strain . Under in vitro conditions (nutrient broth culture), the inhibitory potential of these non-motile mutants was also reduced compared to the motile original strains . In contrast, in vivo studies in a-few-days-old chickens revealed that there was no reduction of the virulence of non-motile mutants of S . Enteritidis and S . Typhimurium in comparison with the motile parental strain . In day-old chicks, the inhibitory potential of non-motile strains was significantly reduced and in some cases, had even become completely lost.

Zentralbl Hyg Umweltmed, 1999 Feb, 201(6), 487 - 512
{Genotoxicity of stack gas condensates of Bavarian waste incineration plants . II . Suitability of fast bacterial tests of emission monitoring}; Raabe F et al.; The genotoxicity of stack gas condensates of 21 waste incineration plants (located in Bavaria) was examined in the years 1990-1995 using two bacterial short time tests . The SOS chromotest was carried out with the tester strains Escherichia coli PQ37, PQ243 and PQ300 . In addition, for the purpose of comparison, the Ames-Test was performed for selected examples with the tester strains Salmonella typhimurium TA97, TA98, TA100 and TA1537 . In a pilot study, carried out in the years 1990 to 1991, the stack gas condensates from five plants were examined . They showed clear genotoxic and mutagenic effects . On the other hand, in subsequent tests we generally discovered only weak inductions for 9 of 18 crude and 24 of 78 clean gas condensate extracts, mostly after metabolic activation . Four plants were tested continuously in the years 1992 to 1995 . Three of them showed a clear reduction of the detectable genotoxic potential . The fourth one gave negligible SOS inducing emissions in the whole examining period . On the other hand, for 6 of the 21 tested plants we found chromotest positive results even at the last test point . Correlations between the SOS inducing potential of the stack gas condensates and the analytical parameters detected at the same time (6 summary parameters, 24 inorganic and 63 organic chemical parameters) were not evident . Only the two highest emissions of nitropyrenes were associated with SOS inductions . Organic substances which are not analytically detected or synergistic effects might be responsible for the SOS inducing potency of the other genotoxic stack gas condensates.

Proc R Soc Lond B Biol Sci, 1999 Feb 7, 266(1416), 299 - 304
A minimal mechanism for bacterial pattern formation; Tyson R et al.; Colonies of Escherichia coli or Salmonella typhimurium form geometrically complex patterns when exposed to, or feeding on, intermediates of the tricarboxylic acid (TCA) cycle . In response to the TCA cycle intermediate, the bacteria secrete aspartate, a potent chemo-attractant . As a result, the cells form high-density aggregates arranged in striking regular patterns . The simplest are temporary spots formed in a liquid medium by both E . coli and S . typhimurium . In semi-solid medium S . typhimurium forms concentric rings arising from a low-density bacterial lawn, which are either continuous or spotted, whereas E . coli forms complex patterns arising from a dense swarm ring, including interdigitated spots (also called sunflower spirals), radial spots, radial stripes and chevrons . We present a mathematical model that captures all three of the pattern-forming processes experimentally observed in both E . coli and S . typhimurium, using a minimum of assumptions.

Vaccine, 1999 Jan, 17(1), 31 - 9
Induction of specific cell-mediated immunity in mice by oral immunization with Salmonella expressing Onchocerca volvulus glutathione S-transferase; Catmull J et al.; Cellular and humoral immune responses of mice to Onchocerca volvulus glutathione S-transferase (OvGST) presented via in vivo expression in attenuated Salmonella typhimurium were examined and compared with the same antigen administered by subcutaneous injection with Freund's adjuvant . After infection with recombinant S . typhimurium, maximal numbers of bacteria were recovered from the mesenteric lymph nodes and spleens during the second week postinfection . By weeks 3-4, bacteria were absent from these tissues . Splenocytes from mice infected with S . typhimurium expressing OvGST showed significant and specific proliferative responses to OvGST, whereas the non-recombinant S . typhimurium controls and those which received the antigen by subcutaneous injection with Freund's adjuvant did not . Mice infected with recombinant S . typhimurium had elevated IFN-gamma levels over non-recombinant S . typhimurium and placebo controls . but IL-4 and IL-5 levels were low and did not differ significantly between these groups . Antibody responses to OvGST antigen expressed by a recombinant Salmonella vaccine or delivered in a purified form with Freund's adjuvant were moderate to high . These data suggest that Salmonella can be used as a vaccine delivery vector that induces specific cellular and humoral immune responses to Onchocerca volvulus antigens . This is the first report to describe the successful application of a filarial antigen in a live-vector delivery system as well as the first recombinant based filarial vaccine to elicit a cellular immune response similar to that described for putative immune endemics.

Vaccine, 1999 Jan, 17(1), 1 - 12
Immunogenicity of viral B-cell epitopes inserted into two surface loops of the Escherichia coli K12 LamB protein and expressed in an attenuated aroA strain of Salmonella typhimurium; Wang J et al.; We previously developed a general procedure which allows the genetic coupling of a chosen foreign linear epitope in different 'permissive' sites of a carrier protein . By using the outer membrane protein LamB of Escherichia coli K12 as a carrier, we were able to express a number of different foreign epitopes at the bacterial surface . In the present work, taking advantage of the recent determination of the crystal structure of LamB, we inserted two model B-cell epitopes i.e.--the C3 epitope from poliovirus (residues 93 to 103 of VP1) and the preS2 epitope from hepatitis B virus, (residues 132 to 145)--at the tip of the most distal and largest surface exposed region of LamB (after residues 386, into loop L9) . We also used two previously constructed LamB hybrids, corresponding to the insertion of the C3B or preSB epitope into permissive site 153 (lying in the middle of the fourth surface loop of LamB), to construct two LamB proteins corresponding to the simultaneous insertion of the two different epitopes (with one epitope per site) . The LamB hybrids were placed under the control of the anaerobically inducible pnirB promoter and expressed in a LamB-negative derivative of the aroA attenuated strain of S . typhimurium, SL3261 . In vitro, the recombinant proteins were expressed at a high level (up to 10% of whole cell proteins) and in vivo the recombinant plasmids were stably maintained . For both epitopes, genetic coupling at site 386 appeared to be more favorable for the induction of anti-epitope antibodies than coupling at site 153 . Moreover, the LamB hybrid corresponding to the simultaneous insertion of the preSB epitope at site 153 and of the C3B epitope at site 386 allowed the induction of both anti-poliovirus and anti-hepatitis B antibodies.

FEMS Microbiol Lett, 1999 Feb 15, 171(2), 203 - 7
Aerosol route enhances the contamination of intact eggs and muscle of experimentally infected laying hens by Salmonella typhimurium DT104; Leach SA et al.; Commercial laying hens were infected with Salmonella typhimurium DT104 strain 16 alternatively via the crop (10(7) cfu per bird) or by an aerosol delivered directly to the beaks using a Collison nebuliser and Henderson apparatus (2 x 10(2) or 2 x 10(4) cfu per bird) . Infection by both routes caused systemic infection and prolonged contamination of faeces . Contamination rates of eggs and muscle were much higher following the aerosol challenges despite the much lower doses given by this route . The frequency of Salmonella isolation from eggs rose from 1.7% following oral challenge to 14% and 25%, for each of the aerosol challenges respectively, and the frequency of isolation from muscle rose from 0% following the oral challenge to 27% following each of the aerosol challenges.

FEMS Microbiol Lett, 1999 Feb 15, 171(2), 179 - 82
Destruction of Salmonella typhimurium, Escherichia coli O157:H7 and Listeria monocytogenes in chicken manure by drying and/or gassing with ammonia; Himathongkham S et al.; Escherichia coli O157:H7 and Listeria monocytogenes were able to grow for a period of 2 days in fresh chicken manure at 20 degrees C with a resulting 1-2 log units increase in CFU; Salmonella typhimurium remained stable . Prolongation of the storage time to 6 days resulted in a 1-2 log decreases of S . typhimurium compared to the initial count and a 3-4 log decrease of E . coli O157:H7; the number of L . monocytogenes did not decrease below the initial . These changes were accompanied by an increase in pH and accumulation of ammonia in the manure . The destruction of the three microorganisms was greatly increased by drying the manure to a moisture content of 10% followed by exposure to ammonia gas in an amount of 1% of the manure wet weight; S . typhimurium and E . coli O157:H7 were reduced by 8 log units, L . monocytogenes by 4.

Microbiology, 1999 Feb, 145 ( Pt 2), 367 - 78
The Salmonella typhi melittin resistance gene pqaB affects intracellular growth in PMA-differentiated U937 cells, polymyxin B resistance and lipopolysaccharide; Baker SJ et al.; Salmonella typhi is the causative agent of typhoid fever in humans . A cell-culture based assay involving the human monocyte macrophage cell line U937 has been developed to examine S . typhi invasion and survival . An S . typhi PhoP- (null) mutant was shown to be restricted in net growth in phorbol myristate acetate (PMA) differentiated U937 (PMA-U937) cells, and an S . typhi PhoPc (constitutive) mutant showed a defect in invasion . Neither of the phoP/Q mutants were growth impaired in HeLa cells, however the PhoPc mutant was impaired in invasion . As opposed to what was found for S . typhi, Salmonella typhimurium wild-type, PhoP- and PhoPc mutants grew equally well in PMA-U937 cells, indicating that the PhoP(-)-mediated net growth restriction in the PMA-U937 cells was S . typhi specific . An S . typhi mutation, pqaB::MudJ, recently shown to be a PhoP-activated locus, was shown to have a net growth defect in PMA-U937 cells . Sequencing of the S . typhipqaB gene revealed it had 98% identity to the fifth gene in a S . typhimurium PmrA/B regulated operon necessary for 4-aminoarabinose lipid A modification and polymyxin B resistance . The pqaB locus was regulated by PmrA/B (whose activity is modulated by PhoP-PhoQ) and the pqaB transposon mutant was sensitive to polymyxin B . The lipopolysaccharides (LPS) of S . typhi and S . typhimurium wild-type, PhoP- and PhoPc mutants, were compared by SDS-PAGE and silver staining . Differences in the LPS profile between the two Salmonella species were observed, and shown to be affected differently by the PhoPc mutation . Additionally, the pqaB::MudJ mutation affected S . typhi LPS . The effects on LPS may have ramifications for the difference between S . typhi and S . typhimurium infection of hosts.

J Bacteriol, 1999 Mar, 181(6), 1934 - 8
Role of ArgR in activation of the ast operon, encoding enzymes of the arginine succinyltransferase pathway in Salmonella typhimurium; Lu CD et al.; The ast operon, encoding enzymes of the arginine succinyltransferase (AST) pathway, was cloned from Salmonella typhimurium, and the nucleotide sequence for the upstream flanking region was determined . The control region contains several regulatory consensus sequences, including binding sites for NtrC, cyclic AMP receptor protein (CRP), and ArgR . The results of DNase I footprintings and gel retardation experiments confirm binding of these regulatory proteins to the identified sites . Exogenous arginine induced AST under nitrogen-limiting conditions, and this induction was abolished in an argR derivative . AST was also induced under carbon starvation conditions; this induction required functional CRP as well as functional ArgR . The combined data are consistent with the hypothesis that binding of one or more ArgR molecules to a region between the upstream binding sites for NtrC and CRP and two putative promoters plays a pivotal role in modulating expression of the ast operon in response to nitrogen or carbon limitation.

J Bacteriol, 1999 Mar, 181(6), 1733 - 8
Physiological states of individual Salmonella typhimurium cells monitored by in situ reverse transcription-PCR; Holmstrom K et al.; The possibility of using levels of specific mRNAs in individual bacteria as indicators of single-cell physiology was investigated . Estimates of the numbers of groEL and tsf mRNAs per cell in Salmonella typhimurium cells in different physiological states were obtained by Northern analysis . The average number of groEL mRNAs per cell was estimated to be 22 in fast-growing cultures and 197 in heat-shocked cultures . The average number of tsf mRNAs per cell was estimated to be 37 in fast-growing cultures, 4 in slow-growing cultures, and 0 in nongrowing cultures . The potential of mRNA-targeted in situ reverse transcription (RT)-PCR to monitor quantitatively different levels of groEL and tsf mRNA in individual cells and thus monitor both specific gene induction and general growth activity was assessed . Neither groEL nor tsf mRNA was present in stationary-phase cells, but it was shown that stationary-phase cells contain other RNA species at high levels, which may provide a possibility for monitoring directly stationary-phase individual cells by the use of in situ RT-PCR . The outcome of the in situ RT-PCR analyses indicated that a population of fast-growing cells is heterogeneous with respect to groEL mRNA single-cell contents, suggesting a cell-cycle-controlled expression of groEL in S . typhimurium, whereas a fast-growing culture is homogeneous with respect to tsf mRNA single-cell contents, suggesting that the level of tsf mRNA is relatively constant during the cell cycle.

FEBS Lett, 1999 Feb 19, 445(1), 126 - 30
Domain organization of flagellar hook protein from Salmonella typhimurium; Uedaira H et al.; Hook forms a universal joint, which mediates the torque of the flagellar motor to the outer helical filaments . Domain organization of hook protein from Salmonella typhimurium was investigated by exploring thermal denaturation properties of its proteolytic fragments . The most stable part of hook protein involves residues 148 to 355 and consists of two domains, as revealed by deconvolution analysis of the calorimetric melting profiles . Residues 72-147 and 356-370 form another domain, while the terminal regions of the molecule, residues 1-71 and 371-403, avoid a compact tertiary structure in the monomeric state . These folding domains were assigned to the morphological domains of hook subunits known from EM image reconstructions, revealing the overall folding of hook protein in its filamentous state.

Vet Microbiol, 1999 Feb 23, 65(1), 21 - 36
Brucella melitensis 16M: characterisation of the galE gene and mouse immunisation studies with a galE deficient mutant; Petrovska L et al.; The galE gene of Streptomyces lividans was used to probe a cosmid library harbouring Brucella melitensis 16M DNA and the nucleotide sequence of a 2.5 kb ClaI fragment which hybridised was determined . An open reading frame encoding a predicted polypeptide with significant homology to UDP-galactose-4-epimerases of Brucella arbortus strain 2308 and other bacterial species was identified . DNA sequences flanking the B . melitensis galE gene shared no identity with other gal genes and, as for B . abortus, were located adjacent to a mazG homologue . A plasmid which encoded the B . melitensis galE open reading frame complemented a galE mutation in Salmonella typhimurium LB5010, as shown by the restoration of smooth lipopolysaccharide (LPS) biosynthesis, sensitivity to phage P22 infection and restoration of UDP-galactose-4-epimerase activity . The galE gene on the B . melitensis 16M chromosome was disrupted by insertional inactivation and these mutants lacked UDP-galactose-4-epimerase activity but no discernible differences in LPS structure between parent and the mutants were observed . One B . melitensis 16M galE mutant, Bm92, was assessed for virulence in CD-1 and BALB/c mice and displayed similar kinetics of invasion and persistence in tissues compared with the parent bacterial strain . CD-1 mice immunised with B . melitensis 16M galE were protected against B . melitensis 16M challenge.

Vaccine, 1999 Feb 26, 17(7-8), 923 - 32
Recombinant, attenuated Salmonella typhimurium stimulate lymphoproliferative responses to SIV capsid antigen in rhesus macaques; Steger KK et al.; Recombinant bacteria are useful vectors for delivering foreign antigens to mucosal surfaces and may elicit immune protection against sexually-transmitted pathogens . Recombinant, attenuated Salmonella typhimurium expressing the Simian Immunodeficiency Virus capsid protein (p27) were given to rhesus macaques by intragastric intubation . This route of immunization was compared with intramuscular injection of soluble p27 in adjuvant, and with immunization protocols that combined intragastric and intramuscular antigen exposures . Recombinant Salmonella stimulated p27-specific lymphoproliferative responses that were present transiently in peripheral blood, and were recalled easily by booster immunizations . Intramuscular p27 injection elicited strong serum antibody responses, but only low level capsid-specific proliferative responses . Recombinant Salmonella immunization elicited low levels of p27-specific antibodies in serum and did not suppress subsequent responses to parenteral immunization . Intragastric immunization of macaques with recombinant Salmonella typhimurium was safe and induced immune responses specific for the expressed, foreign antigen.

Eur J Immunol, 1999 Feb, 29(2), 693 - 9
Salmonella vaccine carrier strains: effective delivery system to trigger anti-tumor immunity by oral route; Medina E et al.; Recombinant Salmonella strains expressing heterologous antigens can be delivered by oral route triggering the elicitation of efficient antigen-specific humoral, T helper and cytotoxic responses . The potential of attenuated Salmonella spp . to trigger anti-tumor immunity was evaluated for the first time by using beta-galactosidase (beta-gal) as a model tumor-associated antigen (TAA) . Beta-gal was expressed in a Salmonella typhimurium aroA vaccine carrier strain either constitutively or under the control of a promoter activated upon infection . Oral immunization with both vaccine prototypes resulted in the elicitation of beta-gal-specific humoral and cell-mediated immunity . Although both strains were able to trigger antigen-specific CTL, responses were more efficient when the expression was controlled by the promoter activated upon infection . The anti-tumor efficacy of the stimulated response was validated by challenging vaccinated animals with an aggressive fibrosarcoma transfected with beta-gal, which operationally acts as a TAA . Both groups of vaccinated mice exhibited a significant reduction in tumor take and growth with respect to animals vaccinated with plasmidless carrier (p < 0.05) . However, the overall efficiency was better in the group in which beta-gal was controlled by the in vivo-activated promoter (85% versus 54%; p < 0.05).

Lett Appl Microbiol, 1999 Feb, 28(2), 113 - 7
Evaluation of a multiplex PCR assay for simultaneous identification of Salmonella sp., Salmonella enteritidis and Salmonella typhimurium from environmental swabs of poultry houses; Soumet C et al.; A Multiplex PCR-based assay (m-PCR) with three sets of primers was developed for the detection of all serotypes of Salmonella enterica and the identification of Salmonella Enteritidis and Salmonella Typhimurium . This method was evaluated against a bacteriological method for the analysis of environmental swabs of poultry houses . Samples were preenriched in phosphate-buffered peptone water for 24 h and subjected to three different protocols prior to PCR: (i) an immunomagnetic separation using Dynabeads anti-Salmonella (Dynal); (ii) a DNA extraction procedure using the Instagene matrix; (iii) an additional step of culture on an MSRV medium . With protocols 1 and 2, eight positive results were found by PCR and 20 with the bacteriological method . Protocol 3 combining MSRV and PCR gave similar results to those obtained from bacteriological methods and allowed Salmonella detection within 2 days.

J Bacteriol, 1999 Mar, 181(5), 1555 - 61
Peptidoglycan-hydrolyzing activity of the FlgJ protein, essential for flagellar rod formation in Salmonella typhimurium; Nambu T et al.; Because the rod structure of the flagellar basal body crosses the inner membrane, the periplasmic space, and the outer membrane, its formation must involve hydrolysis of the peptidoglycan layer . So far, more than 10 genes have been shown to be required for rod formation in Salmonella typhimurium . Some of them encode the component proteins of the rod structure, and most of the remaining genes are believed to encode proteins involved in the export process of the component proteins . Although FlgJ has also been known to be involved in rod formation, its exact role has not been understood . Recently, it was suggested that the C-terminal half of the FlgJ protein has homology to the active center of some muramidase enzymes from gram-positive bacteria . In this study, we showed that the purified FlgJ protein from S . typhimurium has a peptidoglycan-hydrolyzing activity and that this activity is localized in its C-terminal half . Through oligonucleotide-directed mutagenesis, we constructed flgJ mutants with amino acid substitutions in the putative active center of the muramidase . The resulting mutants produced FlgJ proteins with reduced enzymatic activity and showed poor motility . These results indicate that the muramidase activity of FlgJ is essential for flagellar formation . Immunoblotting analysis with the fractionated cell extracts revealed that FlgJ is exported to the periplasmic space, where the peptidoglycan layer is localized . On the basis of these results, we conclude that FlgJ is the flagellum-specific muramidase which hydrolyzes the peptidoglycan layer to assemble the rod structure in the periplasmic space.

J Bacteriol, 1999 Mar, 181(5), 1508 - 14
Environmental signals modulate ToxT-dependent virulence factor expression in Vibrio cholerae; Schuhmacher DA et al.; The regulatory protein ToxT directly activates the transcription of virulence factors in Vibrio cholerae, including cholera toxin (CT) and the toxin-coregulated pilus (TCP) . Specific environmental signals stimulate virulence factor expression by inducing the transcription of toxT . We demonstrate that transcriptional activation by the ToxT protein is also modulated by environmental signals . ToxT expressed from an inducible promoter activated high-level expression of CT and TCP in V . cholerae at 30 degrees C, but expression of CT and TCP was significantly decreased or abolished by the addition of 0.4% bile to the medium and/or an increase of the temperature to 37 degrees C . Also, expression of six ToxT-dependent TnphoA fusions was modulated by temperature and bile . Measurement of ToxT-dependent transcription of genes encoding CT and TCP by ctxAp- and tcpAp-luciferase fusions confirmed that negative regulation by 37 degrees C or bile occurs at the transcriptional level in V . cholerae . Interestingly, ToxT-dependent transcription of these same promoters in Salmonella typhimurium was relatively insensitive to regulation by temperature or bile . These data are consistent with ToxT transcriptional activity being modulated by environmental signals in V . cholerae and demonstrate an additional level of complexity governing the expression of virulence factors in this pathogen . We propose that negative regulation of ToxT-dependent transcription by environmental signals prevents the incorrect temporal and spatial expression of virulence factors during cholera pathogenesis.

Mol Microbiol, 1999 Feb, 31(3), 971 - 82
Salmonella SirA is a global regulator of genes mediating enteropathogenesis; Ahmer BM et al.; SirA of Salmonella typhimurium is known to regulate the hilA and prgH genes within Salmonella pathogenicity island 1 (SPI1) . To identify more members of the SirA regulon, we screened 10,000 random lacZY fusions (chromosomal MudJ insertions) for regulation by SirA and identified 10 positively regulated fusions . Three fusions were within the SPI1 genes hilA (an SPI1 transcriptional regulator), spaS (a component of the SPI1 type III export apparatus) and sipB (a substrate of the SPI1 export apparatus) . Two fusions were within the sopB gene (also known as sigD) . sopB is located within SPI5, but encodes a protein that is exported via the SPI1 export apparatus . In addition, five fusions were within genes of unknown function that are located in SPI4 . As spaS and sipB were likely to be hilA dependent, we tested all of the fusions (except hilA) for hilA dependence . Surprisingly, we found that all of the fusions require hilA for expression and that plasmid-encoded SirA cannot bypass this requirement . Therefore, SirA regulates hilA, the product of which regulates genes within SPI1, SPI4 and SPI5 . Both sirA and hilA mutants are dramatically attenuated in a bovine model of gastroenteritis, but have little or no effect in the mouse model of typhoid fever . This study establishes the SirA/HilA regulatory cascade as the primary regulon controlling enteropathogenic virulence functions in S . typhimurium . Because S . typhimurium causes gastroenteritis in both cattle and humans, we believe that this information may be directly applicable to the human disease.

Environ Mol Mutagen, 1999, 33(1), 28 - 41
Genotoxicity of iron compounds in Salmonella typhimurium and L5178Y mouse lymphoma cells; Dunkel VC et al.; The mutagenic activity of elemental and salt forms of iron (Fe), including compounds currently being used in dietary supplements and for food fortification, were evaluated for mutagenicity in Salmonella typhimurium and L5178Y mouse lymphoma cells . Except for the weak response obtained with ferrous fumarate, none of the compounds induced a mutagenic response in Salmonella . In the mouse lymphoma assay, responses were related to the Fe compound and/or reduction of ferric (Fe+3) to ferrous (Fe+2) . Responses with the elemental forms of Fe were divergent . Electrolytic Fe with a relatively larger particle size and irregular shape was negative . The smaller-sized carbonyl Fe, which after 4 hr attached to and was taken up by the cells, induced mutagenic responses both with and without S9 . With ferric chloride (FeCl3) and ferric phosphate (FePO4), there was an increase in mutant frequency only with S9 . With the Fe+2 compounds, ferrous sulfate (FeSO4) and ferrous fumarate (FeC4H2O4), positive responses were observed without S9 . The Fe chelate, sodium Fe(III)EDTA was positive in both the presence and absence of S9 . The lowest effective doses (LED) for induction of mutagenicity were identified for these compounds and an LED ratio calculated . The LED ratio ranges from 1 for FeSO4 to 30 for carbonyl Fe, which are similar to oral LD50 values obtained in animal studies.

Epidemiol Infect, 1998 Dec, 121(3), 569 - 77
Salmonella infections in Norway: descriptive epidemiology and a case-control study; Kapperud G et al.; The epidemiological progression of human salmonellosis in Norway is parallel to trends noted elsewhere in Europe . During the past two decades, the number of reported cases has increased steadily, with a special sharp rise in the early 1980s due to the emergence of Salmonella enteritidis, followed by a levelling off in recent years . However, in contrast to the situation in most other European countries, about 90% of the cases from whom a travel history is available, have acquired their infection abroad . The incidence of indigenous salmonella infections as well as the prevalence of the microorganism in the domestic food chain, are both comparatively low . In 1993-4, a national case-control study of sporadic indigenous salmonella infections was conducted to identify preventable risk factors and guide preventive efforts . Ninety-four case patients and 226 matched population controls were enrolled . The study failed to demonstrate any statistically significant association between salmonellosis and consumption of domestically produced red meat, poultry or eggs . The only factor which remained independently associated with an increased risk in conditional logistic regression analysis, was consumption of poultry purchased abroad during holiday visits to neighbouring countries . A separate analysis of Salmonella typhimurium infections incriminated food from catering establishments and foreign travel among household members, in addition to imported poultry.

J Food Prot, 1999 Feb, 62(2), 106 - 11
Response surface models for effects of temperature, pH, and previous growth pH on growth kinetics of Salmonella Typhimurium in brain heart infusion broth; Oscar TP; Response surface models were developed for effects of temperature (15 to 40 degrees C), pH (5.2 to 7.4), and previous growth pH (5.7 to 8.6) on lag time (lambda) and specific growth rate (mu) of Salmonella Typhimurium in brain heart infusion broth (BHIB) . Seventy-five growth curves for model development and 30 growth curves for model validation were fit to a two-phase linear growth model to obtain direct estimates of lambda and mu of Salmonella Typhimurium in BHIB . Response surface models for natural logarithm transformations of lambda and mu as a function of temperature, pH, and previous growth pH were obtained by regression analysis . Previous growth pH did not alter (P > 0.05) or interact with temperature or pH to alter subsequent growth kinetics of Salmonella Typhimurium . However, lambda and mu of Salmonella Typhimurium in BHIB were affected (P < 0.05) by linear and quadratic effects of temperature and pH . The models were validated against data not used in their development . Mean absolute relative error of predictions (model accuracy) was 7.8% for lambda and 6.6% for mu . Median relative error of predictions (model bias) was -1.8% for lambda and -2.8% for mu . Results of the current study indicated that the models developed accurately predicted growth kinetics of Salmonella Typhimurium in BHIB within the matrix of factors modeled and that the range of previous growth pH (5.7 to 8.6) investigated did not alter the subsequent growth kinetics of Salmonella Typhimurium in BHIB.

Mutat Res, 1999 Feb 2, 439(1), 69 - 75
Genotoxicity evaluation of tetramethylbenzenes; Janik-Spiechowicz E et al.; The three tetramethyl isomers of benzene (prenitene, 1,2,3,4-; izodurene, 1,2,3,5-; and durene, 1,2,4,5-tetramethylben- zene) were studied using in vitro mutagenicity and in vivo genotoxicity tests . Potency of mutate induction by these solvents was evaluated in Salmonella typhimurium cells with, and without S9-mix made from Aroclor 1254-induced rat liver S9 . The potency of induction of micronuclei (MN) and sister chromatid exchanges (SCEs) by solvents was evaluated in bone marrow of mice . Izodurene displayed mutagenic potency in strains TA97a, TA98 and TA100 only in the absence of the S9-mix . In MN tests, all three tetramethylbenzenes demonstrated no clastogenic activity on the bone marrow cells . All the tested solvents were active as genotoxic compounds in the SCE tests, demonstrating a dose-response relationships .

Mutat Res, 1999 Feb 2, 439(1), 63 - 7
Studies on the genotoxicity of endosulfan in bacterial systems; Chaudhuri K et al.; Endosulfan, an organochlorine pesticide, was subjected to the differential sensitivity assay in repair-deficient and repair-proficient strains of Escherichia coli K12, prophage lambda induction assay in WP2s (lambda) and mutation induction in E . coli K12 . The induction of umu gene expression with endosulfan was studied also in Salmonella typhimurium TA1535/pSK1002 cells . The differential sensitivity assay revealed that the recA 13 strain was the most sensitive . Endosulfan induced prophage lambda in E . coli and umu gene expression in S . typhimurium cells; however, the extent of the effects were low . Endosulfan also induced a dose-dependent increase in forward mutations in E . coli K12 cells from ampicillin sensitivity to ampicillin resistance . Our studies indicate the genotoxic potential of endosulfan and the role of the recA gene in the repair of endosulfan-induced DNA damage .

Mol Microbiol, 1999 Jan, 31(2), 585 - 95
Regulation of autoinducer production in Salmonella typhimurium; Surette MG et al.; Salmonella typhimurium strain LT2 secretes an organic signalling molecule that can be assayed by its ability to activate one of two specific quorum-sensing systems in Vibrio harveyi . Maximal activity is produced during mid- to late exponential phase when S . typhimurium is grown in the presence of glucose or other preferred carbohydrates . The signal is degraded by the onset of stationary phase or when the carbohydrate is depleted from the medium . Presumably, quorum sensing in S . typhimurium is operational during periods of rapid, nutrient-rich growth . Protein synthesis is required for degradation of the activity, suggesting that a complex regulatory circuitry controls signal production and detection in S . typhimurium . Increased signalling activity is observed if, after growth in the presence of glucose, S . typhimurium is transferred to a high-osmolarity (0.4 M NaCl) or to a low-pH (pH 5.0) environment . Degradation of the signal is induced by conditions of low osmolarity (0.1 M NaCl) . High osmolarity and low pH are two conditions encountered by S . typhimurium cells when they undergo the transition to a pathogenic existence inside a host organism, suggesting that quorum sensing may have a role in the regulation of virulence in S . typhimurium.

Mol Microbiol, 1999 Jan, 31(2), 499 - 510
The gene bglH present in the bgl operon of Escherichia coli, responsible for uptake and fermentation of beta-glucosides encodes for a carbohydrate-specific outer membrane porin; Andersen C et al.; The cryptic gene bglH from the Escherichia coli chromosome was cloned into a tacOP-driven expression vector . The resulting plasmid was transferred into the porin-deficient E . coli strain KS26 and the protein was expressed by addition of IPTG . The BglH protein was localized in the outer membrane . It was purified to homogeneity using standard methods . Reconstitution experiments with lipid bilayer membranes defined BglH as a channel-forming component, i.e . it is an outer membrane porin . The single-channel conductance of BglH (560 pS in 1 M KCl) was only one-third of that of the general diffusion porins of E . coli outer membrane . The presence of carbohydrates in the aqueous phase led to a dose-dependent block of ion transport through the channel, similar to that found for LamB (maltoporin) of E . coli and Salmonella typhimurium, which means that BglH is a porin specific for the uptake of carbohydrates . The binding constants of a variety of different carbohydrates were calculated from titration experiments of the BglH-induced membrane conductance . The tightest binding was observed with the aromatic beta-D-glucosides arbutin and salicin, and with gentibiose and cellobiose . Binding of maltooligosaccharides to BglH was in contrast to their binding to LamB in that it was much weaker, indicating that the binding site of BglH for carbohydrates is different from that of LamB (maltoporin) . The kinetics of cellopentaose binding to BglH was investigated using the carbohydrate-induced current noise and was compared with that of cellopentaose binding to LamB (maltoporin) and ScrY (sucroseporin).

Mol Microbiol, 1999 Jan, 31(2), 489 - 98
Molecular and functional analysis indicates a mosaic structure of Salmonella pathogenicity island 2; Hensel M et al.; Two large virulence loci encoding type III secretion systems are present on the chromosome of Salmonella typhimurium . Salmonella pathogenicity island 2 (SPI2) is important for the survival of S . typhimurium in host organs and forms an insertion of about 40 kb at the tRNA(Val) gene . However, several indications suggested that SPI2 was not the result of a single event of horizontal gene transfer . We characterized the portion of SPI2 towards the 30 cs boundary and performed mutational analysis to investigate the contribution of this region to S . enterica virulence . This analysis indicates that SPI2 may be composed of at least two different genetic elements . About 15 kb of the 40 kb of SPI2 contain genes without a significant contribution to systemic infections in the model of murine salmonellosis . Our study allowed us to define genes in SPI2 important for virulence further and indicated that this locus has a complex mosaic structure.

J Biol Chem, 1999 Feb 26, 274(9), 5797 - 809
Molecular basis for the enterocyte tropism exhibited by Salmonella typhimurium type 1 fimbriae; Thankavel K et al.; Salmonella typhimurium exhibits a distinct tropism for mouse enterocytes that is linked to their expression of type 1 fimbriae . The distinct binding traits of Salmonella type 1 fimbriae is also reflected in their binding to selected mannosylated proteins and in their ability to promote secondary bacterial aggregation on enterocyte surfaces . The determinant of binding in Salmonella type 1 fimbriae is a 35-kDa structurally distinct fimbrial subunit, FimHS, because inactivation of fimHS abolished binding activity in the resulting mutant without any apparent effect on fimbrial expression . Surprisingly, when expressed in the absence of other fimbrial components and as a translational fusion protein with MalE, FimHS failed to demonstrate any specific binding tropism and bound equally to all cells and mannosylated proteins tested . To determine if the binding specificity of Salmonella type 1 fimbriae was determined by the fimbrial shaft that is intimately associated with FimHS, we replaced the amino-terminal half of FimHS with the corresponding sequence from Escherichia coli FimH (FimHE) that contains the receptor binding domain of FimHE . The resulting hybrid fimbriae bearing FimHES on a Salmonella fimbrial shaft exhibited binding traits that resembled that of Salmonella rather than E . coli fimbriae . Apparently, the quaternary constraints imposed by the fimbrial shaft on the adhesin determine the distinct binding traits of S . typhimurium type 1 fimbriae.

Lupus, 1999, 8(1), 29 - 38
Modulation of autoimmune disease in the MRL-lpr/lpr mouse by IL-2 and TGF-beta1 gene therapy using attenuated Salmonella typhimurium as gene carrier; Huggins ML et al.; We have investigated the effects of interleukin-2 (IL-2) and transforming growth factor-beta (TGF-beta) gene therapy on the progress of autoimmune disease in MRL-lpr/lpr mice, a murine model of systemic lupus erythematosus (SLE) . These mice have uncontrolled proliferation of T cells, an impaired response to T cell mitogen and produce autoantibodies against nuclear antigens, including DNA . Immune complexes formed by these autoantibodies are believed to cause glomerulonephritis and vasculitis in lupus mice and human SLE . Since there is an imbalance of cytokine production in both SLE patients and lupus mice, we examined the effects of cytokine gene therapy on the progression of autoimmune disease in MRL-lpr/lpr mice . The mice were treated orally with a non-pathogenic strain of Salmonella typhimurium bearing the aroA-aroD- mutations and carrying the murine genes encoding IL-2 and TGF-beta . The bacteria synthesise and slowly release the cytokines in vivo . Our results show that, contrary to expectation, TGF-beta gene therapy produced no improvement in pathology and generally had opposite effects to those of IL-2 . IL-2 gene therapy restored the defective T cell proliferative response to mitogen and suppressed the autoantibody response, glomerulonephritis and growth of lymphoid tumours.

Infect Immun, 1999 Mar, 67(3), 1432 - 8
Effect of transforming growth factor beta on experimental Salmonella typhimurium infection in mice; Galdiero M et al.; We have investigated the effect of the in vivo administration of recombinant transforming growth factor beta (rTGF-beta) on the pathogenic mechanisms involved in Salmonella typhimurium experimental infection in mice . The protective response elicited by macrophages was induced by rTGF-beta1 by 2 days after experimental infection, as demonstrated by an increased NO production, while the humoral protective effect began with cytokine mRNA expression 2 days after the challenge and continued after 5 days with cytokine release and lymphocyte activation . We demonstrated that all mice who received rTGF-beta1 survived 7 days after infection . The number of bacteria recovered in the spleens and in the livers of rTGF-beta1-treated mice 2 and 5 days after infection was significantly smaller than that found in the same organs after phosphate-buffered saline (PBS) inoculation . Furthermore, 2 and 5 days after infection, splenic macrophages from rTGF-beta1-treated mice showed a greater NO production than did those from PBS-treated mice . The effect of rTGF-beta1 on S . typhimurium infection in mice was correlated with the expression of cell costimulatory CD28 molecules . Five days after S . typhimurium infection, the percentage of CD28(+)-expressing T cells in splenic lymphocytes from rTGF-beta1-treated mice increased with respect to that from control mice . Gamma interferon (IFN-gamma) mRNA was present in a greater amount in spleen cells from rTGF-beta1-treated mice after 2 days, although the intensity of the band decreased 5 days after the challenge . A similar pattern was obtained with the mRNAs for interleukin-1alpha (IL-1alpha), IL-6, TGF-beta, and inducible nitric oxide synthase, which showed greater expression in cells obtained from rTGF-beta1-treated and S . typhimurium-infected mice 2 days after challenge . The treatment with rTGF-beta1 induced an increase in IL-1alpha and IFN-gamma release in the supernatant of splenocyte cultures 5 days after the experimental infection with S . typhimurium . Moreover, we demonstrated that 5 days after infection, the IFN-gamma titer was significantly greater in the sera of rTGF-beta-treated mice than in those of PBS-treated mice . Also, hsp60 showed greater expression 2 days after the challenge in splenocytes from rTGF-beta1-treated mice . The role played by proinflammatory and immunoregulatory cytokines and by CD28 is discussed.

Infect Immun, 1999 Mar, 67(3), 1415 - 23
The virulence plasmid-encoded impCAB operon enhances survival and induced mutagenesis in Shigella flexneri after exposure to UV radiation; Runyen-Janecky LJ et al.; Upon exposure to UV radiation, Shigella flexneri SA100 displayed survival and mutation frequencies comparable to those of Escherichia coli AB1157, which contains a functional UmuDC error-prone DNA repair system . Survival of SA100 after UV irradiation was associated with the presence of the 220-kb virulence plasmid, pVP . This plasmid encodes homologues of ImpA and ImpB, which comprise an error-prone DNA repair system encoded on plasmid TP110 that was initially identified in Salmonella typhimurium, and ImpC, encoded upstream of ImpA and ImpB . Although the impB gene was present in representatives of all four species of Shigella, not all isolates tested contained the gene . Shigella isolates that lacked impB were more sensitive to UV radiation than isolates that contained impB . The nucleotide sequence of a 2.4-kb DNA fragment containing the imp operon from S . flexneri SA100 pVP was 96% identical to the imp operon from the plasmid TP110 . An SA100 derivative with a mutation in the impB gene had reduced survival following UV irradiation and less UV-induced mutagenesis relative to the parental strain . We also found that S . flexneri contained a chromosomally encoded umuDC operon; however, the umuDC promoter was not induced by exposure to UV radiation . This suggests that the imp operon but not the umuDC operon contributes to survival and induced mutagenesis in S . flexneri following exposure to UV radiation.

Infect Immun, 1999 Mar, 67(3), 1093 - 9
Pathogenicity island 2 mutants of Salmonella typhimurium are efficient carriers for heterologous antigens and enable modulation of immune responses; Medina E et al.; The potential use as vaccine delivery system of Salmonella typhimurium strains harboring defined mutations in the sseC (HH104) and sseD (MvP101) genes, which encode putative effector proteins of the type III secretion system of Salmonella pathogenicity island 2, was evaluated and compared with that of the well-characterized aroA mutant strain SL7207 by using beta-galactosidase (beta-Gal) as a model antigen . When orally administered to immune-competent or gamma interferon-deficient (IFN-gamma-/-) BALB/c mice, both mutants were found to be highly attenuated (50% lethal dose, >10(9) bacteria) . Both strains were also able to efficiently colonize and persist in Peyer's patches . Immunization with HH104 and MvP101 triggered beta-Gal-specific serum and mucosal antibody responses equivalent to or stronger than those observed in SL7207-immunized mice . Although immunoglobulin G2 (IgG2) serum antibodies were dominant in all groups, IgG1 was also significantly increased in mice vaccinated with MvP101 and SL7207 . Comparable beta-Gal-specific IgA and IgG antibodies were detected in intestinal lavages from mice immunized with the different strains . Antigen-specific CD4(+) T-helper cells were generated after vaccination with all vaccine prototypes; however, responses were significantly more efficient when HH104 and MvP101 were used (P < 0.05) . Significantly higher levels of IFN-gamma were produced by restimulated spleen cells from mice immunized with HH104 than from those vaccinated with the MvP101 or SL7207 derivatives (P </= 0.05) . Interestingly, the three strains induced major histocompatibility complex class I-restricted CD8(+) cytotoxic T cells against beta-Gal; however, cytotoxic T-lymphocyte responses were significantly stronger after immunization with HH104 (P < 0.05) . These novel S . typhimurium attenuated strains constitute promising delivery systems for vaccine antigens . The qualitative differences observed in the obtained responses with different carriers may be useful for those applications in which a targeted immunomodulation is required.

Infect Immun, 1999 Mar, 67(3), 1011 - 7
Salmonella typhimurium and lipopolysaccharide stimulate extracellularly regulated kinase activation in macrophages by a mechanism involving phosphatidylinositol 3-kinase and phospholipase D as novel intermediates; Procyk KJ et al.; Activation of the extracellularly regulated kinase (ERK) pathway is part of the early biochemical events that follow lipopolysaccharide (LPS) treatment of macrophages or their infection by virulent and attenuated Salmonella strains . Phagocytosis as well as the secretion of invasion-associated proteins is dispensable for ERK activation by the pathogen . Furthermore, the pathways used by Salmonella and LPS to stimulate ERK are identical, suggesting that kinase activation might be solely mediated by LPS . Both stimuli activate ERK by a mechanism involving herbimycin-dependent tyrosine kinase(s) and phosphatidylinositol 3-kinase . Phospholipase D activation and stimulation of protein kinase C appear to be intermediates in this novel pathway of MEK/ERK activation.

Mutat Res, 1999 Feb 19, 439(2), 267 - 76
Glycine betaine in beer as an antimutagenic substance against 2-chloro-4-methylthiobutanoic acid, the sanma-fish mutagen; Kimura S et al.; Beer can inhibit the mutagenicity of the sanma-fish mutagen, 2-chloro-4-methylthiobutanoic acid (CMBA) in Salmonella typhimurium TA100 and TA1535 . The antimutagenic component was isolated from beer and identified as glycine betaine, a compound known to be distributed widely in plants and animals including humans . Beer also contains components that interfere the antimutagenic action of glycine betaine . Glycine betaine seems to antagonize CMBA in a specific manner, since several other direct-acting mutagens tested were not subject to inhibition by glycine betaine . CMBA was stable in the presence of glycine betaine under neutral conditions . Since a treatment of Salmonella with glycine betaine before the bacteria was exposed to CMBA resulted in inhibition of the mutagenesis, the antimutagenic action of glycine betaine may be taking place inside the cells . These observations suggest that the mutagenic action of CMBA may be modified by the presence of both extracellular and intracellular glycine betaine .

Mutat Res, 1999 Feb 19, 439(2), 233 - 8
Glutathione activation of chloropicrin in the Salmonella mutagenicity test; Schneider M et al.; Chloropicrin (CCl3NO2) is a major soil fumigant for control of fungi, insects and nematodes and may by formed by chlorination of drinking water . It is also a strong lacrimator and induces sister chromatid exchanges in cultured human lymphocytes . Mutagenicity assays of CCl3NO2 in Salmonella typhimurium TA100 establish that it is toxic but not mutagenic at 500 nmol/plate but becomes mutagenic but not toxic on addition of S9 (previous work) or 1-2 molar equivalents of glutathione (GSH) (this study) . The preincubation assay is superior to the plate incorporation test giving 2-4-fold higher revertants/nmol . Using the preincubation assay with GSH at 5 mM (a biomimetic level) in the top agar gives linear dose-response relationships for CCl3NO2 and its dechlorination products CHCl2NO2 and CH2ClNO2 with 0.56, 0.56 and 1.8 revertants/nmol, respectively . The mutagenicity values for CHCl2NO2 and CH2ClNO2 are the same in the presence and the absence of GSH, which only improves the linearity at high levels by reducing toxicity to the bacteria . GSH activation of CCl3NO2 mutagenicity may be due to reductive dechlorination of the trichloro compound to the more active CHCl2NO2 and CH2ClNO2 . Alternatively, the mutagenicity may result from an intermediate GSH conjugate such as GSCCl2NO2 or GSCHClNO2 . In comparison, the mutagenicity of CH2Br2 and CH2I2 is affected little if any by addition of GSH and these dihalomethanes are much less active than the halonitromethane series . It therefore appears that CCl3NO2 is not mutagenic in the absence of activation and that the dechlorinated metabolites CHCl2NO2 and CH2ClNO2 are moderately potent bacterial mutagens, consistent with the possible genotoxicity of CCl3NO2 in mammals .

Mutat Res, 1999 Feb 19, 439(2), 149 - 57
Effects of oligofluorine substitution on the mutagenicity of quinoline: a study with twelve fluoroquinoline derivatives; Kato T et al.; A total of 12 variously fluorinated derivatives of quinoline (Q) were tested for their mutagenicity in Salmonella typhimurium TA100 in the presence of S9 mix to investigate the structure-mutagenicity relationship in oligofluorinated quinolines . Nine of them, 3,7-di-, 5,6-di-, 6,7-di-, 6,8-di-, 7,8-di-, 3,5,7-tri-, 5,6,8-tri-, 6,7, 8-tri-, and 5,6,7,8-tetrafluoroquinolines (FQs), were newly synthesized for this purpose . Those fluorinated at position 3 were all non-mutagenic . Mutagenicity was enhanced by fluorine-substitution at position 5 or 7, but not in 3-FQs (i.e., 3, 5-di-, 3,7-di-, and 3,5,7-triFQs) . Some of the 6-fluorinated derivatives showed less maximum induced-revertants with more mutagenic potencies in terms of induced-revertants per dose than quinoline . No marked change occurred by fluorine-substitution at position 8 . These results show that the effect of di- and trifluoro-substitution on mutagenicity is generally additive, while that of tetrafluorination approaches the deactivating effect of perfluorination . Our study suggests that 3-fluorine-substitution in the pyridine moiety may be a useful means of antimutagenic structural modification in pyridine-fused aromatic chemicals for medicinal and agricultural use .

Acta Microbiol Pol, 1998, 47(3), 275 - 81
The susceptibility of gram-negative rods and their adaptive forms resistant to colistine to the bactericidal action of sera; Doroszkiewicz W et al.; The susceptibility of Escherichia coli K1, Salmonella enteritidis, Salmonella typhimurium strains and their adaptative forms resistant to colistine (Colr forms) was compared with respect to their sensitivity to the bactericidal action of normal cord serum and normal bovine serum . It has been shown that the Colr forms are more susceptible to sera as compared to initial strains . The increase of sensitivity of the Colr forms is connected with structural changes within bacterial cell wall which is the target for complement as well as for colistine.

Proc Natl Acad Sci U S A, 1999 Feb 16, 96(4), 1639 - 44
Quorum sensing in Escherichia coli, Salmonella typhimurium, and Vibrio harveyi: a new family of genes responsible for autoinducer production; Surette MG et al.; In bacteria, the regulation of gene expression in response to changes in cell density is called quorum sensing . Quorum-sensing bacteria produce, release, and respond to hormone-like molecules (autoinducers) that accumulate in the external environment as the cell population grows . In the marine bacterium Vibrio harveyi two parallel quorum-sensing systems exist, and each is composed of a sensor-autoinducer pair . V . harveyi reporter strains capable of detecting only autoinducer 1 (AI-1) or autoinducer 2 (AI-2) have been constructed and used to show that many species of bacteria, including Escherichia coli MG1655, E . coli O157:H7, Salmonella typhimurium 14028, and S . typhimurium LT2 produce autoinducers similar or identical to the V . harveyi system 2 autoinducer AI-2 . However, the domesticated laboratory strain E . coli DH5alpha does not produce this signal molecule . Here we report the identification and analysis of the gene responsible for AI-2 production in V . harveyi, S . typhimurium, and E . coli . The genes, which we have named luxSV.h., luxSS.t., and luxSE.c . respectively, are highly homologous to one another but not to any other identified gene . E . coli DH5alpha can be complemented to AI-2 production by the introduction of the luxS gene from V . harveyi or E . coli O157:H7 . Analysis of the E . coli DH5alpha luxSE.c . gene shows that it contains a frameshift mutation resulting in premature truncation of the LuxSE.c . protein . Our results indicate that the luxS genes define a new family of autoinducer-production genes.

Biol Pharm Bull, 1999 Jan, 22(1), 107 - 10
An antimutagenic metabolite, streptovaricin C, isolated from Streptomyces sp; Ooka K et al.; Streptomyces sp . KM1-30 was isolated from soil as a producer of antimutagens by screening with a modified Ames test . The chemical structure of the antimutagenic metabolite was identified as streptovaricin C, which is known to inhibit DNA dependent RNA polymerase from E . coli and RNA dependent DNA polymerase from RNA tumor viruses, by MS and 1H-, 13C-NMR analyses . Addition of streptovaricin C to the cultures of UV treated Salmonella typhimurium TA100 or Trp-P-2-treated S . typhimurium TA98 decreased the frequency of mutation without a decrease in viable cell counts . The effect of streptovaricin C to the mutation induced by UV and Trp-P-2 was not desmutagenic, but antimutagenic.

Biol Pharm Bull, 1999 Jan, 22(1), 90 - 2
Synergistic effect of naphthoquinones on the mutagenicity of nitroarene; Ohmori K et al.; Nitro reduction is a critical step in the mutagenic activation of nitroarene . Nitroarene and quinone are known to be reduced by common enzymes, and thus, naphthoquinone (NQ) was studied for its effects on the mutagenicity of nitroarene in the Ames test using Salmonella typhimurium TA98 without S9 . The mutagenicity of 1,3-dinitropyrene in TA98 was found to increase 9- and 6-fold as much in the presence of 70 nmol/plate of 2-methyl-1,4-NQ and 2-hydroxy-1,4-NQ, respectively . Mutagenicity also became greater in 1,3,5-trinitronaphthalene, 1-nitropyrene and 3-nitrofluoranthene . Seventy nmol/plate of 2-methyl-1,4-NQ increased the mutagenicity of 1-nitropyrene by 10.5-fold as much.

Mol Microbiol, 1999 Jan, 31(1), 53 - 8
Novel ribosomal mutations affecting translational accuracy, antibiotic resistance and virulence of Salmonella typhimurium; Bjorkman J et al.; Many mutations in rpsL cause resistance to, or dependence on, streptomycin and are restrictive (hyperaccurate) in translation . Dependence on streptomycin and hyperaccuracy can each be reversed phenotypically by mutations in either rpsD or rpsE . Such compensatory mutations have been shown to have a ram phenotype (ribosomal ambiguity), increasing the level of translational errors . We have shown recently that restrictive rpsL alleles are also associated with a loss of virulence in Salmonella typhimurium . To test whether ram mutants could reverse this loss of virulence, we have isolated a set of rpsD alleles in Salmonella typhimurium . We found that the rpsD alleles restore the virulence of strains carrying restrictive rpsL alleles to a level close to that of the wild type . Unexpectedly, three out of seven mutant rpsD alleles tested have phenotypes typical of restrictive alleles of rpsL, being resistant to streptomycin and restrictive (hyperaccurate) in translation . These phenotypes have not been previously associated with the ribosomal protein S4 . Furthermore, all seven rpsD alleles (four ram and three restrictive) can phenotypically reverse the hyperaccuracy associated with restrictive alleles of rpsL . This is the first demonstration that such compensations do not require that the compensating rpsD allele has a ribosomal ambiguity (ram) phenotype.

Mol Microbiol, 1999 Jan, 31(1), 45 - 51
Type III secretion by Salmonella typhimurium does not require contact with a eukaryotic host; Daefler S; The type III secretion system encoded by pathogenicity island I in Salmonella typhimurium delivers proteins to the external milieu and into the eukaryotic host cell . The principal factor in induction of the secretion system was found to be a change in the pH of the culture medium from acidic to mildly alkaline . The synthesis of components of the secretion machinery and the production and secretion of substrates occur simultaneously and do not require contact with a eukaryotic host cell . This argues against the concept that type III secretion in S . typhimurium is a process in which the delivery of a presynthesized pool of substrates is triggered by contact with a eukaryotic host cell.

J Bacteriol, 1999 Feb, 181(4), 1364 - 8
The virulence plasmid of Salmonella typhimurium is self-transmissible; Ahmer BM et al.; Most isolates of Salmonella enterica serovar Typhimurium contain a 90-kb virulence plasmid . This plasmid is reported to be mobilizable but nonconjugative . However, we have determined that the virulence plasmid of strains LT2, 14028, and SR-11 is indeed self-transmissible . The plasmid of strain SL1344 is not . Optimal conjugation frequency requires filter matings on M9 minimal glucose plates with a recipient strain lacking the virulence plasmid . These conditions result in a frequency of 2.9 x 10(-4) transconjugants/donor . Matings on Luria-Bertani plates, liquid matings, or matings with a recipient strain carrying the virulence plasmid reduce the efficiency by up to 400-fold . Homologs of the F plasmid conjugation genes are physically located on the virulence plasmid and are required for the conjugative phenotype.

J Bacteriol, 1999 Feb, 181(4), 1343 - 7
Cloning and characterization of arylamine N-acetyltransferase genes from Mycobacterium smegmatis and Mycobacterium tuberculosis: increased expression results in isoniazid resistance; Payton M et al.; Arylamine N-acetyltransferases (NATs) are found in many eukaryotic organisms, including humans, and have previously been identified in the prokaryote Salmonella typhimurium . NATs from many sources acetylate the antitubercular drug isoniazid and so inactivate it . nat genes were cloned from Mycobacterium smegmatis and Mycobacterium tuberculosis, and expressed in Escherichia coli and M . smegmatis . The induced M . smegmatis NAT catalyzes the acetylation of isoniazid . A monospecific antiserum raised against pure NAT from S . typhimurium recognizes NAT from M . smegmatis and cross-reacts with recombinant NAT from M . tuberculosis . Overexpression of mycobacterial nat genes in E . coli results in predominantly insoluble recombinant protein; however, with M . smegmatis as the host using the vector pACE-1, NAT proteins from M . tuberculosis and M . smegmatis are soluble . M . smegmatis transformants induced to express the M . tuberculosis nat gene in culture demonstrated a threefold higher resistance to isoniazid . We propose that NAT in mycobacteria could have a role in acetylating, and hence inactivating, isoniazid.

J Bacteriol, 1999 Feb, 181(4), 1211 - 9
Conditional stability of the HemA protein (glutamyl-tRNA reductase) regulates heme biosynthesis in Salmonella typhimurium; Wang L et al.; In many bacteria, including the enteric species Salmonella typhimurium and Escherichia coli, heme is synthesized starting from glutamate by a pathway in which the first committed step is catalyzed by the hemA gene product, glutamyl-tRNA reductase (HemA) . We have demonstrated previously that when heme limitation is imposed on cultures of S . typhimurium, HemA enzyme activity is increased 10- to 25-fold . Western (immunoblot) analysis with monoclonal antibodies reactive with HemA revealed that heme limitation results in a corresponding increase in the abundance of the enzyme . Similar regulation was also observed for E . coli . The near absence of regulation of hemA-lac operon fusions suggested a posttranscriptional control . We report here the results of pulse-labeling and immunoprecipitation studies of this regulation . The principal mechanism that contributes to elevated HemA abundance is protein stabilization . The half-life of HemA protein is approximately 20 min in unrestricted cells but increases to >300 min in heme-limited cells . Similar regulation was observed for a HemA-LacZ hybrid protein containing almost all of the HemA protein (416 residues) . Sodium azide prevents HemA turnover in vivo, suggesting a role for energy-dependent proteolysis . This was confirmed by the finding that HemA turnover is completely blocked in a lon clpP double mutant of E . coli . Each single mutant shows only a small effect . The ClpA chaperone, but not ClpX, is required for ClpP-dependent HemA turnover . A hybrid HemA-LacZ protein containing just 18 amino acids from HemA is also stabilized in the lon clpP double mutant, but this shorter fusion protein is not correctly regulated by heme limitation . We suggest that the 18 N-terminal amino acids of HemA may constitute a degradation tag, whose function is conditional and modified by the remainder of the protein in a heme-dependent way . Several models are discussed to explain why the turnover of HemA is promoted by Lon-ClpAP proteolysis only when sufficient heme is available.

Br J Rheumatol, 1998 Dec, 37(12), 1330 - 3
Antibodies to Klebsiella pneumoniae lipopolysaccharide in patients with ankylosing spondylitis; Ahmadi K et al.; The role of microbial lipopolysaccharides (LPS) in the aetiopathogenesis of ankylosing spondylitis (AS) is a matter of continuing debate . In this study, class-specific IgG, IgA and IgM antibodies against Klebsiella pneumoniae, Escherichia coli, Salmonella typhimurium and Salmonella enteritidis LPS were measured by enzyme-linked immunosorbent assay (ELISA) in 100 AS patients, 50 rheumatoid arthritis (RA) patients and 50 healthy control subjects . The AS patients had significantly elevated levels of IgG and IgA antibodies against K . pneumoniae LPS (P < 0.001) and IgA antibodies against E . coli LPS (P < 0.05) compared to healthy controls . There were no significant elevations of antibody levels against S . typhimurium and S . enteritidis in the three study groups . In addition, there was a correlation between IgG and IgA anti-K . pneumoniae LPS antibody levels and the acute-phase reactant C-reactive protein (P < 0.001).

J Virol, 1999 Mar, 73(3), 1853 - 9
Acute effects of pathogenic simian-human immunodeficiency virus challenge on vaccine-induced cellular and humoral immune responses to Gag in rhesus macaques; Steger KK et al.; Simian-human immunodeficiency virus (SHIV) infection in macaques provides a convenient model for testing vaccine efficacy and for understanding viral pathogenesis in AIDS . We immunized macaques with recombinant, Salmonella typhimurium (expressing Gag) or soluble Gag in adjuvant to generate T-cell-dependent lymphoproliferative or serum antibody responses . Immunized animals were challenged by intrarectal inoculation with SHIV89.6PD . Virus infection was accompanied by rapid losses of lymphoproliferative responses to Gag or phytohemagglutinin . By 8 weeks, mitogen responses recovered to near normal levels but antigen-specific immunity remained at low or undetectable levels . Serum antibody levels were elevated initially by virus exposure but soon dropped well below levels achieved by immunization . Our studies show a rapid depletion of preexisting Gag-specific CD4(+) T cells that prevent or limit subsequent antiviral cellular and humoral immune responses during acute SHIV infection.

Vet Immunol Immunopathol, 1999 Jan 4, 67(1), 55 - 65
Effect of peroral anti-bacterial antiserum treatment on intestinal immune parameters of germ-free piglets intragastrically infected with virulent Salmonella typhimurium or enteropathogenic E . coli; Trebichavsky I et al.; Germ-free piglets were orally infected with either enteropathogenic E . coli 055 or a virulent strain of Salmonella typhimurium . Orally applied antiserum against E . coli protected infected animals in spite of the fact that the bacteria were consistently found in mesenteric lymph nodes and other organs . By contrast, the application of an antiserum against S . typhimurium was without any effect on the outcome of infection . The treatment with anti-bacterial antiserum prevented inflammation of ileal mucosa (TNF-alpha and heat shock protein 65 expression) only in piglets infected with E . coli . A decrease in the frequency of ileal MAC320+ cells was observed in all infected piglets treated with antiserum.

SAR QSAR Environ Res, 1998, 9(3-4), 187 - 215
Comparison of CoMFA models for Salmonella typhimurium TA98, TA100, TA98 + S9 and TA100 + S9 mutagenicity of nitroaromatics; Fan M et al.; Comparative Molecular Field Analysis (CoMFA) was applied to a comprehensive data set of heterogeneous nitroaromatics tested in Salmonella typhimurium TA98 and TA100 with and without S9 microsomal activation . The four CoMFA models developed agree with postulated mechanisms of mutagenicity, and explain over 70% of the corresponding mutagenic variance The standard deviation coefficient contours common in the four models included high electronic density regions equivalent to C4-C5 in the pyrene ring, and an electron deficient site equivalent to C6 . These areas are associated with high mutagenicity . Electron deficient areas may be related with the nitroreductive bioactivation of nitroaromatics . Electron rich sites may be involved with oxidative mechanisms analogous to the bioactivation pathway of polycyclic aromatic hydrocarbons . The contribution of steric factors to mutagenicity follows the order TA98 + S9 > TA98 > TA100 + S9 > TA100 . The models indicated that increasing bulk perpendicular to the aromatic plane would decrease mutagenicity, but increasing the aromatic ring system along a region corresponding to C6-C7 in 1-nitropyrene would increase mutagenicity.

FEMS Microbiol Lett, 1999 Jan 15, 170(2), 355 - 61
Iterative selection from a Salmonella typhimurium cosmid library can lead to the isolation of an atypically small plasmid; Lodge JM et al.; We have constructed a DNA library from a virulent Salmonella typhimurium strain, in an avirulent strain . The process of selecting the components of interest from the library involved iterative growth in liquid culture . This resulted, after four cycles, in the culture becoming homogeneous for a single plasmid, which was much smaller than the average size for the library . We have identified the larger precursor of this plasmid which has two regions of sufficient homology to allow recombination resulting in the formation of the small plasmid . S . typhimurium carrying the small plasmid have a smaller genetic burden than other members of the library and survive better in spent culture medium, facilitating selection on repeated subculture . Such rapid adventitious selection has important implications for isolation of clones of interest from genomic libraries.

FEMS Microbiol Lett, 1999 Jan 15, 170(2), 313 - 7
RecA-dependent viral burst in bacterial colonies during the entry into stationary phase; Ramirez E et al.; We have explored the nature of the sudden viral amplification observed during the ageing of P22-infected lysogenic colonies of Salmonella typhimurium {Ramirez, E, and Villaverde, A . (1997) Gene 202, 147-149} . By a comparative analysis of the wild-type P22 and a P22 integration mutant, it has been shown that the conditions promoting prophage induction occur in only a small portion of the bacterial population and briefly during the transition between the exponential growth and the stationary phase . The viral burst is RecA-dependent and cannot be reproduced in continuous culture by a mere decrease of the growth rate . This suggests that the limited viral propagation in colonies is probably linked to heterogeneous physiological conditions within colonial populations, distinct from those of the homogeneous liquid cultures.

Clin Oral Investig, 1998 Sep, 2(3), 125 - 9
Mutagenicity of the root canal sealer AHPlus in the Ames test; Schweikl H et al.; The mutagenic activity of the root canal sealing cement, AHPlus, was tested in a bacterial gene mutation assay (Ames test) . The material was mixed according to the manufacturer's instruction and tested immediately after mixing and after a setting time of 24 h at 37 degrees C in a humidified chamber . The set material was powdered and both the freshly mixed and the powdered material were eluted in dimethyl sulfoxide (DMSO) and physiological saline (0.1 g/2 ml) for 24 h at 37 degrees C . Aliquots of serially diluted eluates were then used in the standard plate incorporation assay . The Salmonella typhimurium tester strains TA98 and TA100 were employed to detect the induction of frameshift mutations and base pair substitutions both in the presence and in the absence of a metabolically active microsomal fraction from rat liver (S9 fraction) . No mutagenic and no toxic effects were found with physiological saline eluates of the freshly mixed material and of mixed material which was set for 24 h . However, DMSO eluates of the freshly mixed AHPlus were mutagenic in tester strain TA100 in a dose-related manner in the absence of S9 . A four- to fivefold increase of the mutation frequencies was induced by 2.5 mg AHPlus per plate compared with the number of spontaneous mutants . The mutagenic effect was completely abolished in the presence of a metabolically active S9 fraction . Also, no mutagenic effects were observed with DMSO eluates of AHPlus set for 24 h . However, the set material was more toxic towards bacteria than the freshly mixed sealer . This difference was indicated by a tenfold lower amount of material necessary to cause complete absence of the background lawn in both S . typhimurium tester strains . Therefore, we conclude that at least two different compounds of AHPlus are biologically active in DMSO eluates to cause mutagenic and toxic effects in S . typhimurium TA100 and TA98.

Infect Control Hosp Epidemiol, 1999 Jan, 20(1), 55 - 6
An outbreak of Salmonella typhimurium at a teaching hospital; McCall B et al.; An outbreak of Salmonella typhimurium infection in December 1996 affected 52 patients, relatives, and staff of a large teaching hospital in southeast Queensland . Assorted sandwiches were identified as the vehicle of transmission . This article describes the outbreak investigation and demonstrates the importance of food hygiene and timely public health interventions.

Toxicol Appl Pharmacol, 1999 Jan 15, 154(2), 126 - 34
Nitroarene reduction and generation of free radicals by cell-free extracts of wild-type, and nitroreductase-deficient and -enriched Salmonella typhimurium strains used in the umu gene induction assay; Metosh-Dickey CA et al.; Studies of the enzymatic properties of cell-free extracts prepared from overnight cultures of the normal, and nitroreductase-deficient and -enriched strains of Salmonella typhimurium, designed for use in the umu gene induction assay of Oda et al . (1992), were undertaken in an effort to clarify the nature of nitroreductase deficiency in relation to mutagenicity . The ability of these strains to promote oxygen consumption and free radical intermediates of representative nitroarene substrates was measured, respectively, by oxygen polarography and electron spin resonance (ESR) spectroscopy . The substrates 4-nitropyridine N-oxide (4NPO) and 4-nitroquinoline N-oxide (4NQO) stimulated the rate and extent of NADH-dependent oxygen consumption catalyzed by cell-free extracts prepared from wild-type, and nitroreductase-deficient and -enriched strains . The extent of oxygen consumption was greater than stoichiometric with respect to the amount of nitroaromatic substrate, which implied one-electron reduction of 4NQO by these bacterial extracts and subsequent redox cycling with oxygen . ESR spectroscopy confirmed the production of free radical metabolites of the nitroarene substrates, which were inferred by the oxygen consumption studies . At equal protein concentrations the cell-free extracts of each strain catalyzed univalent reduction of 4NPO yielding the 59 line signal characteristic of the 4NPO nitro anion radical . This ESR signal was potently inhibited by the flavoprotein inhibitors CuSO4 and PCMB, albeit a twofold or higher concentration of both inhibitors was required to inhibit the signal produced by extract from the nitroreductase-deficient strain than that produced by the other strains . The results indicate that the nitroreductase-deficient strain of Salmonella typhimurium developed for use in the umu gene induction assay is not deficient in either one-electron nitro group or quinone reductase activity .

J Mol Biol, 1999 Feb 5, 285(5), 2199 - 209
Intermolecular cleavage by UmuD-like enzymes: identification of residues required for cleavage and substrate specificity; McDonald JP et al.; The UmuD-like proteins are best characterized for their role in damage-induced SOS mutagenesis . An essential step in this process is the enzymatic self-processing of the UmuD-like proteins . This reaction is thought to occur either via an intramolecular or intermolecular self-cleavage mechanism . Here, we demonstrate that it can also occur via an heterologous intermolecular cleavage reaction . The Escherichia coli UmuD enzyme demonstrated the broadest substrate specificity, cleaving both E . coli and Salmonella typhimurium UmuD substrates in vivo . In comparison, the wild-type S . typhimurium UmuD (UmuDSt) and MucA enzymes catalyzed intermolecular self-cleavage, but did not facilitate heterologous cleavage . Heterologous cleavage by the UmuDSt enzyme was, however, observed with chimeric UmuD substrates that possess residues 30-55 of UmuDSt . We have further localized the residue predominantly responsible for UmuDSt-catalyzed heterologous cleavage to Ser50 in the substrate molecule . We hypothesize that changes at this residue affect the positioning of the cleavage site of a substrate molecule within the catalytic cleft of the UmuDSt enzyme by affecting the formation of a so-called UmuD "filament-dimer" . This hypothesis is further supported by the observation that mutations known to disrupt an E . coli UmuD' filament dimer also block intermolecular UmuDEc cleavage .

J Bacteriol, 1999 Feb, 181(3), 841 - 8
Biosynthesis of the pyrimidine moiety of thiamine independent of the PurF enzyme (Phosphoribosylpyrophosphate amidotransferase) in Salmonella typhimurium: incorporation of stable isotope-labeled glycine and formate; Enos-Berlage JL et al.; Genetic analyses have suggested that the pyrimidine moiety of thiamine can be synthesized independently of the first enzyme of de novo purine synthesis, phosphoribosylpyrophosphate amidotransferase (PurF), in Salmonella typhimurium . To obtain biochemical evidence for and to further define this proposed synthesis, stable isotope labeling experiments were performed with two compounds, {2-13C}glycine and {13C}formate . These compounds are normally incorporated into thiamine pyrophosphate (TPP) via steps in the purine pathway subsequent to PurF . Gas chromatography-mass spectrometry analyses indicated that both of these compounds were incorporated into the pyrimidine moiety of TPP in a purF mutant . This result clearly demonstrated that the pyrimidine moiety of thiamine was being synthesized in the absence of the PurF enzyme and strongly suggested that this synthesis utilized subsequent enzymes of the purine pathway . These results were consistent with an alternative route to TPP that bypassed only the first enzyme in the purine pathway . Experiments quantitating cellular thiamine monophosphate (TMP) and TPP levels suggested that the alternative route to TPP did not function at the same capacity as the characterized pathway and determined that levels of TMP and TPP in the wild-type strain were significantly altered by the presence of purines in the medium.

J Bacteriol, 1999 Feb, 181(3), 799 - 807
Coordinate intracellular expression of Salmonella genes induced during infection; Heithoff DM et al.; Salmonella typhimurium in vivo-induced (ivi) genes were grouped by their coordinate behavior in response to a wide variety of environmental and genetic signals, including pH, Mg2+, Fe2+, and PhoPQ . All of the seven ivi fusions that are induced by both low pH and low Mg2+ (e.g., iviVI-A) are activated by the PhoPQ regulatory system . Iron-responsive ivi fusions include those induced under iron limitation (e.g., entF) as well as one induced by iron excess but only in the absence of PhoP (pdu) . Intracellular expression studies showed that each of the pH- and Mg2+-responsive fusions is induced upon entry into and growth within three distinct mammalian cell lines: RAW 264.7 murine macrophages and two cultured human epithelial cell lines: HEp-2 and Henle-407 . Each ivi fusion has a characteristic level of induction consistent within all three cell types, suggesting that this class of coordinately expressed ivi genes responds to general intracellular signals that are present both in initial and in progressive stages of infection and may reflect their responses to similar vacuolar microenvironments in these cell types . Investigation of ivi expression patterns reveals not only the inherent versatility of pathogens to express a given gene(s) at various host sites but also the ability to modify their expression within the context of different animal hosts, tissues, cell types, or subcellular compartments.

J Food Prot, 1999 Jan, 62(1), 73 - 6
Eggshell membrane structure and penetration by Salmonella typhimurium; Berrang ME et al.; Eggshell membrane was removed from 10 broiler hatching eggs at approximately monthly intervals through the productive life of a commercial flock . A piece of membrane (2 by 2 cm) was used to cover an opening in an apparatus designed to test penetration by Salmonella Typhimurium . The membrane was placed between two chambers as the only means of liquid transfer . The chamber above the membrane was filled with a suspension of Salmonella Typhimurium cells . Sampling of the bottom chamber provided cultural evidence of the penetration by a marker strain of Salmonella Typhimurium . Samples were drawn at 15 min and at 1, 3, 5, 7, 12, 24, and 48 h . Following the cultural penetration experiment, the same pieces of membrane were removed and stained for microscopic examination . Membrane structure was examined with the use of a confocal laser scanning microscope . Each image consisted of a composite of eight 1-microm optical slices showing the fibers making up the outer surface of the membrane . These images were transferred to an image analysis software package that allowed the measurement of the interfiber area . No clear correlation could be made between the average size of all interfiber areas or the total interfiber area measured in the outer 8 microm of the outer membrane and the ability of Salmonella Typhimurium to penetrate the same piece of eggshell membrane.

J Food Prot, 1999 Jan, 62(1), 70 - 2
RpoS function in Salmonella Typhimurium LT2 monitored in a skim milk model food; Thompson JM et al.; In Salmonella Typhimurium, the stationary phase sigma factor RpoS regulates the expression of genes associated with adaptation, survival, and virulence . Expression of rpoS is known to be under environmental control and yet, to date, there have been no studies that assess the expression of this global regulator in real food systems . Skim milk represents a useful model food; using an spvRA::luxCDABE reporter construct, we have determined RpoS availability from an inoculum of Salmonella Typhimurium LT2 . We report that RpoS activity increases exponentially at the end of the logarithmic phase of growth, consistent with data from nutrient media.

FEMS Microbiol Lett, 1999 Jan 1, 170(1), 251 - 6
Transduction of multiple drug resistance of Salmonella enterica serovar typhimurium DT104; Schmieger H et al.; Epidemic strain Salmonella typhimurium DT104 is characterized by various multiresistance patterns . At least some of the resistance genes are organized as integrons . Resistance genes of DT104 isolates can be efficiently transduced by P22-like phage ES18 and by phage PDT17 which is released by all DT104 isolates so far analyzed . Cotransduction tests demonstrate that the resistance genes, although not organized in a unique integron, are tightly clustered on the Salmonella chromosome . The spread of resistance genes in this strain by generalized transduction is discussed.

Cent Eur J Public Health, 1998 Nov, 6(4), 307 - 13
Mutagenicity tests on the bacteria and the detection of genotoxicity of industrial complex mixtures containing PAHs; Malachova K; The study summarizes the results of an evaluation of mutagenicity of heterogeneous complex mixtures of substances, the main mutagenic component of which consists of polycyclic aromatic hydrocarbons . The testing was performed using bacterial assays of mutagenicity--the SOS chromotest and the S . typhimurium His-test (in modifications without and with metabolic activation in vitro) . It was found that samples of tested tar mixtures (crude tar, pitch, anthracene oils III and II, granulated pitch and some of its extraction portions) induced SOS repair functions and frameshift mutations in tests with metabolic activation . Some of samples as tar, pitch and anthracene oils III, granulated pitch and two its extraction portions--LRAC and LRBe--induced also frameshift mutations, and SOS repairs in tests without the metabolic activation . In one sample--LRBe--the ability to induce mutations in all variants of both tests, was also proved . The evaluation of mutagenicity of fly ashes showed that differences in the mutagenic activity of samples can be directly dependent on the extraction method chosen and on the type of extraction agent used . The study results demonstrate that the bacterial tests Salmonella typhimurium His- and the SOS chromotest are uninterchangeable and quite independent . Both tests can be used for orientative screening for genotoxicity in a wide range of various complex mixtures arising from industrial production and contaminating the environment.

J Nat Prod, 1999 Jan, 62(1), 102 - 6
Structure-antimutagenic activity relationship study of plicatin B; Menon SR et al.; A systematic structure-activity relationship study of plicatin B (1), an antimutagenic constituent of Psoralea juncea, was undertaken with a view toward elucidating its chemical mode of action and possibly optimizing its antimutagenic activity during the process . Compound 1 and its related analogues were examined for their antimutagenic activity against mutations induced by ethyl methanesulfonate, a direct acting mutagen and alkylating agent, in Salmonella typhimurium strain TA100, utilizing the modified Ames test protocol . The dihydro analogue 3 resulting from saturation of the conjugated alkene double bond of 1 was found to exhibit reduced cytotoxicity and enhanced efficacy relative to the parent compound . This result serves preliminarily to exclude a Michael acceptor role of the alpha,beta-unsaturated carbonyl moiety in connection with its antimutagenic activity.

Zentralbl Veterinarmed B, 1998 Dec, 45(10), 577 - 83
Salmonellosis in relation to chlamydiosis and pox and Salmonella infections in captive falcons in the United Arab Emirates; Wernery U et al.; During the spring of 1995, 1996 and 1997 following tests on six peregrine falcons (Falco peregrinus) and two gyr falcons (Falco rusticolus), Salmonella typhimurium was isolated from liver, spleen and small intestines . Four of the falcons (two peregrines and two gyrs) had also contracted Chlamydia infection, three peregrines a pox infection and one peregrine a Herpesvirus infection . It is believed that this dual infection was fatal for these birds . The disease was marked by anorexia, dehydration and green-coloured droppings . Necropsy of all falcons revealed discolouration of the liver and enlargement of liver and spleen . Miliary necrosis was detected in all livers . A total of 12 salmonella serovars, including S . typhimurium, were cultured from faeces of 48 falcons which showed no clinical signs.

Infect Immun, 1999 Feb, 67(2), 891 - 8
In vivo blockage of nitric oxide with aminoguanidine inhibits immunosuppression induced by an attenuated strain of Salmonella typhimurium, potentiates Salmonella infection, and inhibits macrophage and polymorphonuclear leukocyte influx into the spleen; MacFarlane AS et al.; Our laboratory has previously shown that after immunization with a strain of Salmonella typhimurium, SL3235, made avirulent by a blockage in the pathway of aromatic synthesis, murine splenocytes were profoundly suppressed in their capacity to mount an in vitro antibody plaque-forming cell (PFC) response to sheep erythrocytes . Evidence indicated that suppression was mediated by nitric oxide (NO), since the in vitro addition of NG-monomethyl-L-arginine blocked suppression . The present studies examined the effect of blocking NO production on Salmonella-induced immunosuppression by in vivo administration of aminoguanidine hemisulfate (AG) . AG was administered to C3HeB/FeJ mice in their drinking water (2.5% solution) for 7 days prior to intraperitoneal inoculation with SL3235 . AG treatment inhibited the increase in nitrate and nitrite levels in plasma and nitrite levels in the spleen seen in immunized mice . Importantly, AG treatment completely blocked suppression of the splenic PFC response and markedly attenuated the suppression of the response to concanavalin A in immunized mice, providing further evidence that Salmonella-induced immunosuppression is mediated by NO . AG treatment also alleviated the majority of the splenomegaly associated with SL3235 inoculation, which correlated with a blockage of influx of neutrophils and macrophages into spleens, as assessed by flow cytometry . AG treatment unexpectedly resulted in 90% mortality in mice injected with the highly attenuated vaccine strain of Salmonella, SL3235 . Increased mortality in AG-treated mice correlated with inability to clear organisms from the spleen by day 15 postinoculation and with persistent bacteremia, compared with control mice . Collectively, these in vivo results underscore the dual biological consequences of NO production following Salmonella infection, with NO being necessary for host defense, but also having the potentially adverse effect of immunosuppression . A unifying hypothesis to explain how these seemingly paradoxical effects could both result from NO production is presented.

Infect Immun, 1999 Feb, 67(2), 800 - 4
Bacterial invasion is not required for activation of NF-kappaB in enterocytes; Eaves-Pyles T et al.; Pathogenic enteric microorganisms induce the NF-kappaB-dependent expression of proinflammatory genes in intestinal epithelial cells . The purpose of the present study was to clarify the contribution of microbial invasion to the degradation of the regulatory protein Ikappa Balpha and the subsequent activation of NF-kappaB in cultured intestinal epithelial cells . Caco-2BBe cells were incubated with Salmonella dublin, Salmonella typhimurium, or a weakly invasive strain of E . coli . S . dublin and S . typhimurium (10(7) organisms/ml) induced equivalent concentration-dependent gel mobility shifts of an NF-kappaB consensus sequence that was preceded by Ikappa Balpha degradation . E . coli (10(7) organisms/ml) did not induce Ikappa Balpha degradation or NF-kappaB translocation . Pretreatment with cytochalasin D blocked invasion of all three strains but had no effect on Ikappa Balpha degradation or NF-kappaB activation . S . dublin and S . typhimurium adhered to Caco-2BBe cells 3- to 10-fold more than E . coli . NF-kappaB activation was prevented by physical separation of S . dublin from Caco-2BBe cells by a 0 . 4-micrometers-pore-size filter . Our results imply that bacterial adhesion, rather than invasion or release of a secreted factor, is sufficient to induce IkappaBalpha degradation and NF-kappaB activation in intestinal epithelial cells . Our data suggest that strategies to reduce enteric inflammation should be directed to the reduction of bacterial enterocyte adhesion.

Infect Immun, 1999 Feb, 67(2), 608 - 17
Orchestration of neutrophil movement by intestinal epithelial cells in response to Salmonella typhimurium can be uncoupled from bacterial internalization; Gewirtz AT et al.; Intestinal epithelial cells respond to Salmonella typhimurium by internalizing this pathogen and secreting, in a polarized manner, an array of chemokines which direct polymorphonuclear leukocyte (PMN) movement . Notably, interleukin-8 (IL-8) is secreted basolaterally and directs PMN through the lamina propria, whereas pathogen-elicited epithelial chemoattractant (PEEC) is secreted apically and directs PMN migration across the epithelial monolayer to the intestinal lumen . While most studies of S . typhimurium pathogenicity have focused on the mechanism by which this bacterium invades its host, the enteritis characteristically associated with salmonellosis appears to be more directly attributable to the PMN movement that occurs in response to this pathogen . Therefore, we sought to better understand the relationship between S . typhimurium invasion and epithelial promotion of PMN movement . First, we investigated whether S . typhimurium becoming intracellular was necessary or sufficient to induce epithelial promotion of PMN movement . Blocking S . typhimurium invasion by preventing, with cytochalasin D, the epithelial cytoskeletal rearrangements which mediate internalization did not reduce the epithelial promotion of PMN movement . Conversely, bacterial attainment of an intracellular position was not sufficient to induce model epithelia to direct PMN transmigration, since neither basolateral invasion by S . typhimurium nor apical internalization of an invasion-deficient mutant (achieved by inducing membrane ruffling with epidermal growth factor) induced this epithelial cell response . These results indicate that specific interactions between the apical surface of epithelial cells and S . typhimurium, rather than simply bacterial invasion, mediate the epithelial direction of PMN transmigration . To further investigate the means by which S . typhimurium induces epithelia to direct PMN movement, we investigated whether the same signaling pathways regulate secretion of IL-8 and PEEC . IL-8 secretion, but not PEEC secretion, was activated by phorbol myristate acetate and blocked by an inhibitor (mg-132) of the proteosome which mediates NF-kappabeta activation . Further, secretion of IL-8, but not PEEC, was activated by an entry-deficient (HilDelta) S . typhimurium mutant or by basolateral invasion of a wild-type strain . Together, these results indicate that distinct signaling pathways mediate S . typhimurium invasion, induction of IL-8 secretion, and induction of PEEC secretion in model intestinal epithelia.

Infect Immun, 1999 Feb, 67(2), 478 - 83
Interleukin 18 contributes to host resistance and gamma interferon production in mice infected with virulent Salmonella typhimurium; Mastroeni P et al.; Spleen and peritoneal macrophages obtained from innately resistant A/J mice released low levels of interleukin 18 (IL-18) upon infection with Salmonella typhimurium C5 RP4 . Incubating the cells with recombinant gamma interferon (rIFN-gamma) enhanced IL-18 production . A/J mice treated in vivo with anti-IL-18 antibodies showed impaired resistance to infection, with increased bacterial loads in the liver and spleen . Administration of rIL-18 could protect A/J mice from challenge with a lethal dose of virulent salmonellae, with a dramatic reduction in bacterial numbers in the tissues . rIL-18 administration did not ameliorate the disease in IFN-gamma-R-/- mice . IL-18 proved to be required for IFN-gamma production by mouse splenocytes from conventional, scid, and rag-1(-/-) mice; in vivo IL-18 neutralization caused a decrease in circulating IFN-gamma levels . Thus, IL-18 is a key factor in early host resistance to Salmonella and probably acts via IFN-gamma.

Arch Microbiol, 1999 Jan, 171(2), 122 - 6
Integration of minitransposons for expression of the Escherichia coli elt genes at a preferred site in Salmonella typhimurium identifies a novel putative fimbrial locus; Brocchi M et al.; An asd-complementing mini-Tn5 transposon was constructed for random insertion of the Escherichia coli LT enterotoxin genes (elt) into the genome of Deltaasd attenuated strains of Salmonella typhimurium . Transfer of the minitransposon to different S . typhimurium strains resulted in random integration only in strain chi4072, while in strain chi3987, which harbours the virulence plasmid, over 20% of the insertions occurred at the same site . Expression of elt was found to be highest in Salmonella isolates carrying the mini-Tn5 integrated at the preferred site, which was mapped to an uncharacterised region of the virulence plasmid . Sequence analysis of the integration site showed that it lies within an open reading frame with sequence similarity to E . coli leuO and contiguous to a novel fimbrial locus.

Chem Res Toxicol, 1999 Jan, 12(1), 46 - 52
Identification of an ethenoformyl adduct formed in the reaction of the potent bacterial mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone with guanosine; Munter T et al.; 3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) is a potent direct-acting bacterial mutagen and a rodent carcinogen occurring in chlorine-disinfected drinking water . In this study, we have reacted MX with guanosine, cytidine, thymidine, and calf thymus DNA in aqueous solutions . HPLC analyses of the reaction mixture of MX with guanosine showed that one main product peak was formed . In the reactions of MX with cytidine or thymidine, no product peaks representing base-modified nucleosides could be observed . The product from the MX guanosine reaction mixture was isolated by preparative chromatography on reversed phase C18 columns, and its structure was determined by UV absorbance, 1H and 13C NMR spectroscopy, and mass spectrometry . The product was identified as 3-(beta-D-ribofuranosyl)-7-formylimidazo{1,2-a}purin-9(4H)-one (epsilonfGuo), and the yield for the reaction carried out at pH 7.4 and 37 degrees C was about 0.3 mol % . The adduct could not be observed at the detection limit of five adducts per 10(7) bases in the hydrolysate of the calf thymus DNA reacted with MX . However, this failure does not rule out the possibility that lower amounts of the adduct might be involved in the observed mispairing (adenine incorporated opposite an adducted guanine base) caused by MX in the Salmonella typhimurium strain TA100.

Immunology, 1998 Dec, 95(4), 640 - 7
The full expression of the ity phenotype in ityr mice requires C3 activation by Salmonella lipopolysaccharide; Nishikawa F et al.; Our previous study has shown that the rapid and sufficient activation of complement by Salmonella lipopolysaccharide occurs in genetically resistant (Ityr) A/J mice . To assess whether the level of complement activation by a virulent strain of Salmonella typhimurium regulates the level of murine natural resistance, we compared levels of serum complement activation by S . typhimurium and kinetics of serum-opsonized S . typhimurium grown in macrophages using several strains of resistant (Ityr) and susceptible (Itys) mice . Itys macrophages killed intracellular S . typhimurium to the same extent as did Ityr macrophages when the pathogen was opsonized with Ityr serum . Opsonization of S . typhimurium with Itys serum reduced intracellular killing activity in Ityr macrophages to the same level as seen with Itys macrophages . Incubation of S . typhimurium with 25% Mg2+ EGTA (5 mm MgCl2-3 mm ethylene glycol-bis (beta-aminotheyl either)-N,N,N',N'-tetraacetic acid)-chelated Ityr serum resulted in higher levels of C3 deposition onto the surface of this bacteria, C3b generation and also C3 consumption, compared with that with Mg2+ EGTA-chelated Itys serum . Opsonization of S . typhimurium with A/J serum prior to infection increased early resistance in Itys mice . Infection with a virulent strain of S . typhimurium induced the expression of interleukin-10 (IL-10) mRNA at higher levels in C57BL/6 mice than in A/J mice . However, opsonization of S . typhimurium with A/J serum decreased bacterial growth in the spleen of C57BL/6 mice to the same level as observed for A/J mice in association with decreased expression levels of IL-10 mRNA . Moreover, administration of anti-C3 antibodies reduced the resistance of A/J mice in association with a decrease in serum levels of C3 . These results indicate that the high level of complement activation via the alternative pathway in Ityr serum by a virulent strain of S . typhimurium reduces the virulence of this pathogen, which may contribute to the full expression of Ity phenotype in Ityr mice.

Biochemistry, 1999 Jan 5, 38(1), 329 - 36
Identification of a site critical for kinase regulation on the central processing unit (CPU) helix of the aspartate receptor; Trammell MA et al.; Ligand binding to the homodimeric aspartate receptor of Escherichia coli and Salmonella typhimurium generates a transmembrane signal that regulates the activity of a cytoplasmic histidine kinase, thereby controlling cellular chemotaxis . This receptor also senses intracellular pH and ambient temperature and is covalently modified by an adaptation system . A specific helix in the cytoplasmic domain of the receptor, helix alpha6, has been previously implicated in the processing of these multiple input signals . While the solvent-exposed face of helix alpha6 possesses adaptive methylation sites known to play a role in kinase regulation, the functional significance of its buried face is less clear . This buried region lies at the subunit interface where helix alpha6 packs against its symmetric partner, helix alpha6' . To test the role of the helix alpha6-helix alpha6' interface in kinase regulation, the present study introduces a series of 13 side-chain substitutions at the Gly 278 position on the buried face of helix alpha6 . The substitutions are observed to dramatically alter receptor function in vivo and in vitro, yielding effects ranging from kinase superactivation (11 examples) to complete kinase inhibition (one example) . Moreover, four hydrophobic, branched side chains (Val, Ile, Phe, and Trp) lock the kinase in the superactivated state regardless of whether the receptor is occupied by ligand . The observation that most side-chain substitutions at position 278 yield kinase superactivation, combined with evidence that such facile superactivation is rare at other receptor positions, identifies the buried Gly 278 residue as a regulatory hotspot where helix packing is tightly coupled to kinase regulation . Together, helix alpha6 and its packing interactions function as a simple central processing unit (CPU) that senses multiple input signals, integrates these signals, and transmits the output to the signaling subdomain where the histidine kinase is bound . Analogous CPU elements may be found in other receptors and signaling proteins.

Biochemistry, 1999 Jan 5, 38(1), 284 - 95
Motional dynamics of the catalytic loop in OMP synthase; Wang GP et al.; In de novo pyrimidine biosynthesis, orotate phosphoribosyltransferase catalyzes the formation of orotidine 5'-monophosphate (OMP) from orotic acid and alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP) . The known three-dimensional structure of the dimeric enzyme from Salmonella typhimurium is similar to that of other Type I phosphoribosyltransferases (nucleotide synthases) with a solvent-exposed active site atop a Rossman-type nucleotide binding fold . The three-dimensional structure of an enzyme-inhibitor complex {Henriksen et al . (1996) Biochemistry 35, 3803-3809} indicates that one of the two identical solvent-exposed loops can descend to cover the active site of the adjacent subunit of the dimeric enzyme . Catalytically essential residues are known to reside on this loop . In the present work, sensitivity toward limited proteolysis by trypsin confirms that the loop is solvent-exposed . Protection by PRPP and, to a lesser extent, by OMP demonstrates the existence of a second, trypsin-inaccessible, loop position . Two-dimensional 1H-15N NMR relaxation experiments on {alpha-15N}histidine-labeled WT OPRTase yielded backbone 15N T1 and T2 relaxation times and 15N inverted question mark1H inverted question mark NOE for His-105 (a loop residue) that are characteristic of small peptides . These results document that the surface loop is highly flexible in the unliganded enzyme . Addition of a hydrolytically stable PRPP analogue to the enzyme resulted in a significant reduction of His-105 peak intensity, indicating a dramatic change in the dynamic properties of the loop backbone in the analogue-ligated enzyme . 1H NMR titrations on histidine C2 protons, coupled with 1H and 31P titrations monitoring the C1H and 5-phosphate PRPP resonances, allowed the quantitation of the rates of loop movement during product release, and relate protein motion to enzymatic catalysis . These results suggest that loop opening and PRPP release is a two-step process, whose overall rate is partially rate-limiting in the reverse pyrophosphorolysis reaction.

Adv Microb Physiol, 1998, 40, 233 - 79
The starvation-stress response (SSR) of Salmonella; Spector MP; Salmonella serovars are common etiologic agents of intestinal-based disease of animals and humans . As a result of their lifestyle, salmonellae occupy and survive in a wide range of niches where they can encounter an even broader range of environmental stresses . One of the most common stresses is starvation for an essential nutrient such as a carbon/energy (C)-source . The genetic and physiologic changes that the bacterium undergoes in response to starvation-stress are referred to as the starvation-stress response or SSR . The genetic loci whose expression increases in response to the starvation-stress compose the SSR stimulon . Several loci of the SSR stimulon have been identified in Salmonella typhimurium and grouped, based on putative or known functions or products, into transport systems, C-compound catabolic enzymes, known protective enzymes, respiratory enzyme systems, regulatory proteins, virulence loci and unclassified products . The majority of loci identified are under positive control by the rpoS-encoded sigma factor, sigma S . However, a few are under (indirect) negative control by sigma S, but only during starvation-induced stationary phase . Most of the loci identified are also under either positive or negative control by the cAMP:CRP complex . For many, additional regulatory proteins (e.g . FadR, OxyR, and RelA and others) play a role in their regulation as well . Furthermore, most of the SSR loci identified are induced during other stresses or environmental conditions . For example, some are induced during P- or N-starvation, in addition to C-starvation; some are induced by extremes in pH or osmolarity; and some are induced in the intracellular environment of epithelial cells, and/or macrophages, and/or medium designed to mimic the intracellular milieu of mammalian cells (ISM) . Several SSR loci are required for long-term starvation-survival (core SSR loci), e.g . narZ, dadA, stiC and rpoS . In addition, a few of the core SSR loci are also required for stress-specific-inducible and/or C-starvation-inducible resistance to H2O2 (e.g . stiC), thermal (e.g . stiC), and/or acid pH (e.g . narZ), challenge . Interestingly, C-starved cells are resistant to challenge with the antimicrobial peptide, polymyxin B . However, this resistance mechanism(s) is different from the resistance mechanisms for H2O2 and other environmental stresses . Furthermore, a link between the SSR and Salmonella virulence can be hypothesized since the two major regulators of the SSR, sigma s and cAMP:CRP, are required for full virulence of Salmonella . Moreover, the spv (Salmonella plasmid-associated virulence) genes, required for Salmonella to cause systemic disease, are C (and P- and N-)-starvation-inducible . However, a direct link between starvation-stress and virulence has not been established conclusively.

J Am Vet Med Assoc, 1999 Jan 1, 214(1), 67 - 70, 43-4
Outbreak of fatal salmonellosis in cats following use of a high-titer modified-live panleukopenia virus vaccine; Foley JE et al.; A 14-week-old kitten from a private cattery was examined because of an acute onset of recumbency and epistaxis 10 days after receiving a high-titer modified-live virus vaccine containing panleukopenia virus, calicivirus, and herpesvirus components . The kitten died the following day, and intestinal crypt necrosis; hepatic, splenic, and lymph node inflammation and necrosis; and pneumonia were seen at necropsy . Salmonella typhimurium was isolated from mesenteric lymph nodes and the spleen . The breeder reported that 4 other kittens had died in the previous month, each within 1 to 2 weeks after being vaccinated with the same modified-live virus vaccine . Carcasses of 3 kittens were available for examination, and Salmonella sp was isolated from mesenteric lymph nodes of all 3 . Villus crypt necrosis and secondary fibrosis were also found . Three of the remaining 12 kittens in the cattery were also found to be shedding Salmonella sp in their feces . Clinical and pathologic findings in these kittens were likely attributable to salmonellosis and panleukopenia, and suggest that mild immunosuppression induced by vaccination could have facilitated development of fatal salmonellosis in subclinical carrier kittens . However, we cannot prove that vaccination actually played any role.

Eur Spine J, 1998, 7(6), 509 - 11
Possible salmonella osteomyelitis of spine following laser disc decompression; Farrar MJ et al.; We report a case of chronic discitis and vertebral osteomyelitis that we believe was caused by Salmonella typhimurium following laser decompression of the L4/5 disc for symptomatic disc protrusion in a 50-year-old Asian man . The infection was successfully treated with intravenous ceftriaxone combined with oral ciprofloxacin . We believe this to be the only report of such a complication following this procedure, which is generally without infective complications.

J Bacteriol, 1999 Jan, 181(2), 689 - 94
Cyclic AMP receptor protein and TyrR are required for acid pH and anaerobic induction of hyaB and aniC in Salmonella typhimurium; Park KR et al.; Two acid-inducible genes, aniC and aciK, that require anaerobiosis and tyrosine for expression were identified as orf326a encoding a potential amino acid/polyamine antiporter and hyaB encoding hydrogenase I, respectively . Cyclic AMP (cAMP) receptor protein, cAMP, and TyrR, regulator of aromatic amino acid metabolism, were strong positive regulators of both genes.

J Bacteriol, 1999 Jan, 181(2), 648 - 55
NAD-dependent DNA-binding activity of the bifunctional NadR regulator of Salmonella typhimurium; Penfound T et al.; NadR is a 45-kDa bifunctional regulator protein . In vivo genetic studies indicate that NadR represses three genes involved in the biosynthesis of NAD . It also participates with an integral membrane protein (PnuC) in the import of nicotinamide mononucleotide, an NAD precursor . NadR was overexpressed and purified as a His-tagged fusion in order to study its DNA-binding properties . The protein bound to DNA fragments containing NAD box consensus sequences . NAD proved to be the relevant in vivo corepressor, but full NAD dependence of repressor activity required nucleotide triphosphates . DNA footprint analysis and gel shift assays suggest that NadR binds as a multimer to adjacent NAD boxes . The DNA-repressor complex would sequester a potential RNA polymerase binding site and thereby decrease expression of the nad regulon.

J Bacteriol, 1999 Jan, 181(2), 368 - 74
Molecular characterization of eutF mutants of Salmonella typhimurium LT2 identifies eutF lesions as partial-loss-of-function tonB alleles; Thomas MG et al.; The eutF locus of Salmonella typhimurium LT2 was identified as a locus necessary for the utilization of ethanolamine as a sole carbon source . Initial models suggested that EutF was involved in either ethanolamine transport or was a transcriptional regulator of an ethanolamine transporter . Phenotypic characterization of eutF mutants suggested EutF was somehow involved in 1,2-propanediol, propionate, and succinate utilization . Here we provide evidence that two alleles defining the eutF locus, Delta903 and eutF1115, are partial-loss-of-function tonB alleles . Both mutations were complemented by plasmids containing a wild-type allele of the Escherichia coli tonB gene . Immunoblot analysis using TonB monoclonal antibodies detected a TonB fusion protein in strains carrying eutF alleles . Molecular analysis of the Delta903 allele identified a deletion that resulted in the fusion of the 3' end of tonB with the 3' end of trpA . In-frame translation of the tonB-trpA fusion resulted in the final 9 amino acids of TonB being replaced by a 45-amino-acid addition . We isolated a derivative of a strain carrying allele Delta903 that regained the ability to grow on ethanolamine as a carbon and energy source . The molecular characterization of the mutation that corrected the Eut- phenotype caused by allele Delta903 showed that the new mutation was a deletion of two nucleotides at the tonB-trpA fusion site . This deletion resulted in a frameshift that replaced the 45-amino-acid addition with a 5-amino-acid addition . This change resulted in a TonB protein with sufficient activity to restore growth on ethanolamine and eut operon expression to nearly wild-type levels . It was concluded that the observed EutF phenotypes were due to the partial loss of TonB function, which is proposed to result in reduced cobalamin and ferric siderophore transport in an aerobic environment; thus, the eutF locus does not exist.

J Med Microbiol, 1998 Jun, 47(6), 483 - 7
The epidemiology of Salmonella typhimurium in cattle: plasmid profile analysis of definitive phage type (DT) 204c; Wray C et al.; During the period 1979-1991, Salmonella Typhimurium DT 204c was the cause of a major epidemic of salmonellosis in calves in the UK . Plasmid profile analysis of DT 204c isolates from England and Wales commenced in 1986 and isolates from all subsequent incidents were examined by this technique . Forty-three different plasmid profile types (PPTs) were detected, of which the commonest, designated type E, constituted 44.6-80.2% of the annual incidents during the study period . Some PPTs, e.g., F and P, were detected throughout most years of the study, whereas PPTs O and 6 persisted for short periods . Until 1984, most isolates were resistant to neomycin, but the subsequent predominant PP type E was sensitive to this antibacterial agent . It was concluded that during the epidemic there was an evolution of new genotypes, of which only some persisted; again, antibacterial resistance genes may be acquired or lost . The study demonstrated the value of PP typing for epidemiological studies.

J Med Microbiol, 1998 Feb, 47(2), 151 - 7
Secretory pathways in Salmonella Typhimurium-induced fluid accumulation in the porcine small intestine; Grondahl ML et al.; The involvement of 5-hydroxytryptamine (5-HT) and 5-HT3 receptors and prostaglandin E2 (PGE2) in Salmonella Typhimurium-induced fluid accumulation in the porcine small intestine was investigated . Salmonella Typhimurium (10(8) and 10(10) cfu) and cholera toxin (CT; 20 microg) were instilled for 8 and 11 h in ligated loops in the porcine jejunum and ileum . Fluid accumulation and concentrations of Na+, K+, Cl-, 5-HT and PGE2 in the fluid accumulated in the loops were measured . The fluid accumulation was also measured when Salmonella Typhimurium (10(10) cfu) and CT (20 microg) were instilled for 8 h in ligated loops in jejunum and ileum in pigs given subcutaneous injections of saline or the 5-HT3 receptor antagonist ondansetron (200 microg/kg) . Salmonella Typhimurium (10(10) cfu) and CT both induced fluid accumulation in jejunum and ileum after 8 and 11 h . Both treatments also induced an increase in luminal release of 5-HT and PGE2 . The accumulated fluid was iso-osmotic and hyperosmotic in CT- and Salmonella Typhimurium-treated loops, respectively . Ondansetron reduced the Typhimurium-induced fluid accumulation in both jejunum and ileum by c . 40%, while it failed to reduce the response to CT . These results demonstrate that 5-HT and PGE2 are released and 5-HT3 receptors activated in the secretory pathway of Typhimurium in the porcine small intestine.

FEMS Immunol Med Microbiol, 1998 Dec, 22(4), 341 - 9
DNA sequencing of the gene encoding Salmonella typhimurium-derived T-cell inhibitor (STI) and characterization of the gene product, cloned STI; Matsui K et al.; In a previous study, we found a novel protein which inhibited T-cell responsiveness to interleukin-2 (IL-2) in Salmonella typhimurium and called it S . typhimurium-derived T-cell inhibitor (STI) . In this study, we analyzed the DNA sequence of the gene encoding STI . The STI gene was cloned into a plasmid vector, pUC118, and expressed in Escherichia coli JM109 . Like native STI, the cloned STI inhibited IL-2-dependent CTLL-2 cell growth . Furthermore, this growth inhibition involved down-regulation of IL-2 receptor expression . These results indicate that the cloned STI expressed in E . coli was identical to native STI . Sequencing revealed that the STI gene contained an open reading frame of 2298 base pairs encoding a precursor form of 765 amino acid residues (molecular mass 83605) that is processed into a mature form of 745 amino acid residues with molecular mass 81 548 . Homology analysis revealed that its amino acid sequence was highly homologous with that of the beta-glucosidase of E . coli K-12 . We designated the gene encoding STI sti.

Mutat Res, 1999 Jan, 436(1), 69 - 97
Antimutagenic and anticarcinogenic activity of tea polyphenols; Kuroda Y et al.; Tea is the most popular beverage, consumed by over two thirds of the world's population . Tea is processed differently in different parts of the world to give green (20%), black (78%) or oolong tea (2%) . Green tea is consumed mostly in Japan and China . The antimutagenic and anticarcinogenic activities of green tea are extensively examined . The chemical components of green and black tea are polyphenols, which include EC, ECG, EGC, EGCG and TFs . This article reviews the epidemiological and experimental studies on the antimutagenicity and anticarcinogenicity of tea extracts and tea polyphenols . In Japan, an epidemiological study showed an inverse relationship between habitual green tea drinking and the standardized mortality rates for cancer . Some cohort studies on Chanoyu (Japanese tea ceremony) women teachers also showed that their mortality ratio including deaths caused by malignant neoplasms were surprisingly low . The antimutagenic activity against various mutagens of tea extracts and polyphenols including ECG and EGCG has been demonstrated in microbial systems (Salmonella typhimurium and Escherichia coli), mammalian cell systems and in vivo animal tests . The anticarcinogenic activity of tea phenols has been shown in experimental animals such as rats and mice, in transplantable tumors, carcinogen-induced tumors in digestive organs, mammary glands, hepatocarcinomas, lung cancers, skin tumors, leukemia, tumor promotion and metastasis . The mechanisms of antimutagenesis and anticarcinogenesis of tea polyphenols suggest that the inhibition of tumors may be due to both extracellular and intracellular mechanisms including the modulation of metabolism, blocking or suppression, modulation of DNA replication and repair effects, promotion, inhibition of invasion and metastasis, and induction of novel mechanisms .

J Mol Biol, 1999 Jan 15, 285(2), 443 - 8
Transcription-induced hypersupercoiling of plasmid DNA; Chen D et al.; Transcription can induce high levels of negative supercoiling into plasmid DNA under some circumstances . This is especially true when the plasmid carries a functional tetracycline-resistance gene tetA, and is borne in a topA strain of Escherichia coli or Salmonella typhimurium . An important mechanism in transcription-induced supercoiling is believed to be the twin supercoiled-domain effect resulting from hindered rotation of the transcriptional complex, and this is very much more efficient where there is coupled transcription, translation and membrane insertion of the gene product . However, we have noted that strong promoters inserted into tetA-carrying plasmids can greatly increase the fraction of hypersupercoiled DNA . We show here that this effect is clearly present when the inserted promoter transcribes a very short segment of DNA (down to transcript lengths of approximately 45 nt), and where there is no possibility of translation of the RNA transcript . We suggest that the repeated helical opening due to transcriptional initiation is a significant contributor to the induction of high levels of supercoiling .

J Mol Biol, 1998 Dec 18, 284(5), 1399 - 416
Mechanism of self-association and filament capping by flagellar HAP2; Vonderviszt F et al.; HAP2 forms a capping structure, which binds very tightly to the distal end of flagellar filaments and still allows insertion of flagellin subunits below the cap by an unknown mechanism . Terminal regions of HAP2 from Salmonella typhimurium were found to be quickly degraded by various proteases, indicating that HAP2 also possesses disordered terminal regions like other axial proteins of bacterial flagellum . Removal of these portions by trypsin results in a fragment of 40 kDa (HP40), which lacks 42 NH2-terminal and 51 COOH-terminal residues . HAP2 in solution readily associates into a decameric structure without any significant population of intermediate oligomeric forms . The HP40 fragments, however, do not form decamers, while they can assemble into pentamers, as revealed by chemical cross-linking and analytical ultracentrifugation . Decameric HAP2 also dissociates into pentamers and smaller oligomers upon a heat induced conformational transition around 36 degreesC . While the highly mobile terminal regions are immobilized in decameric HAP2 complexes, they are still largely disordered in the pentameric state . These results demonstrate that the intersubunit interactions within the pentamers are mainly through the HP40 portions, whereas the terminal regions are responsible for association of pentamers into decameric complexes . Several observations indicate that HAP2 performs its capping function as a pentamer . We suggest that binding of the pentameric HAP2 cap to the filament is mediated by the highly flexible terminal regions . Indeed, HP40 fragments are unable to cap the end of filaments, while removal of about 30 residues from both terminal regions of HAP2 results in a highly reduced capping ability . A model is presented to explain the molecular mechanism of capping, in which conformational entropy in the disordered terminal regions moderates the otherwise too tight HAP2-filament interactions to allow insertion of flagellin subunits below the cap .

J Food Prot, 1998 Dec, 61(12), 1649 - 56
Shelf life extension, safety, and quality of fresh pork loin treated with high hydrostatic pressure; Ananth V et al.; The optimal conditions of pressure, time, and processing temperature required to eliminate Listeria monocytogenes Scott A and Salmonella typhimurium ATCC 13311 in fresh pork loin and the effect of these optimal conditions on quality and shelf life were determined . Twenty-five grams of fresh pork loin were inoculated with either of the two organisms and were subjected to pressures between 414 and 827 MPa at either 2 or 25 degrees C for 30 min . The D414MPa(25 degrees C) was determined to be 2.17 min for L . monocytogenes and the D414 MPa (2 degrees C) was determined to be 1.48 min for S . typhimurium . Samples subjected to a 6D process were evaluated by sensory and objective tests as well as for shelf life . These samples were found to be different (P < 0.05) from controls when evaluated after cooking by a triangle test of difference, but only when the pressure was applied at 2 degrees C and not at 25 degrees C . The descriptive analysis test showed that cooked samples treated at 25 degrees C were not different (P > 0.05) from controls in flavor, juiciness, and firmness . Color, peak load, water-holding capacity, and moisture were not found to be different (P > 0.05) between samples treated at 25 degrees C and controls when both were cooked . However, in the raw state, differences were found in the values for color parameters L and b . The level of psychrotrophs was 5.7 log CFU/g for samples treated at 25 degrees C after 33 days of storage at 4 degrees C, as compared with 7.0 log CFU/g for controls . The color and peak load (texture) did not change over the storage period (P > 0.05) in any of the samples . All samples spoiled in 5 days when stored at 25 degrees C.

J Biol Chem, 1999 Jan 8, 274(2), 739 - 47
Both lobes of the soluble receptor of the periplasmic histidine permease, an ABC transporter (traffic ATPase), interact with the membrane-bound complex . Effect of different ligands and consequences for the mechanism of action; Liu CE et al.; The histidine permease of Salmonella typhimurium is an ABC transporter (traffic ATPase) . The liganded soluble receptor, the histidine-binding protein HisJ, interacts with the membrane-bound complex HisQMP2 and stimulates its ATPase activity, which results in histidine translocation . In this study, we utilized HisJ proteins with mutations in either of the two lobes and wild type HisJ liganded with different substrates to show that each lobe carries an interaction site and that both lobes are involved in inducing (stimulating) the ATPase activity . We suggest that the spatial relationship between the lobes is one of the factors recognized by the membrane-bound complex in dictating the efficiency of the induction signal and of translocation . Several of the key residues involved have been identified . In addition, using constitutive ATPase mutants, we show that the binding protein provides some additional essential function(s) in translocation that is independent of the stimulation of ATP hydrolysis, and one possible mechanism is proposed, which includes the notion that liganded HisJ has different optimal conformations for signaling and for translocation.

Nature, 1998 Dec 17, 396(6712), 703 - 7
Crystal structure of the ATP-binding subunit of an ABC transporter; Hung LW et al.; ABC transporters (also known as traffic ATPases) form a large family of proteins responsible for the translocation of a variety of compounds across membranes of both prokaryotes and eukaryotes . The recently completed Escherichia coli genome sequence revealed that the largest family of paralogous E . coli proteins is composed of ABC transporters . Many eukaryotic proteins of medical significance belong to this family, such as the cystic fibrosis transmembrane conductance regulator (CFTR), the P-glycoprotein (or multidrug-resistance protein) and the heterodimeric transporter associated with antigen processing (Tap1-Tap2) . Here we report the crystal structure at 1.5 A resolution of HisP, the ATP-binding subunit of the histidine permease, which is an ABC transporter from Salmonella typhimurium . We correlate the details of this structure with the biochemical, genetic and biophysical properties of the wild-type and several mutant HisP proteins . The structure provides a basis for understanding properties of ABC transporters and of defective CFTR proteins.

Poult Sci, 1998 Dec, 77(12), 1874 - 83
Evaluation of the efficacy of intraperitoneal immunization in reducing Salmonella typhimurium infection in chickens; Muir WI et al.; Conventional methods of parenteral immunization with killed bacterin vaccines have met with limited success in protecting the avian intestinal mucosa from pathogens such as Salmonella typhimurium . For mucosal vaccines to be successful they must be evaluated for their ability to stimulate local secretory immunoglobulin (SIgA) at the mucosal surface, which acts as the first line of defense against invading pathogens . Previously we have demonstrated the ability of i.p . immunization with nonreplicating antigen in an appropriate adjuvant to induce a primary immune response, which, after an oral booster immunization, stimulates enhanced intestinal IgA responses in chickens . In the experiments reported here we have applied this immunization protocol to vaccinate against S . typhimurium in chickens, and examined the protection provided against subsequent S . typhimurium challenge by placing vaccinated birds on seeded litter with cohabitant infected birds . Immunized+challenged birds displayed delayed onset of S . typhimurium infection, both at the mucosal surface and within the reticuloendothelial system . Elevated anti-S . typhimurium IgG and IgA titers were detected in serum after vaccination, which markedly increased after challenge, to levels higher than in control+challenged chickens . Anti-S . typhimurium IgA in bile and intestinal scrapings supernatant was also higher in the immunized+challenged birds than in the control+challenged birds 15 d after challenge . This study illustrates the potential for i.p . vaccination to induce a mucosal immune response to S . typhimurium in chickens, which, in the challenge model employed here, provided partial protection against intestinal challenge with the same pathogen and was reflected in deferred onset of bacterial infection and shedding.

Pathol Biol (Paris), 1998 Oct, 46(8), 587 - 90
{Growing incidence of nalidixic acid resistance and sensitivity to quinolones in Salmonella typhimurium strains isolated from man or animal}; Heurtin-Le Corre C et al.; To determine the prevalence of quinolone resistance in Salmonella typhimurium strains from humans or animals (cattle, poultry, swine), the S . typhimurium strains isolated at a teaching hospital and at the central veterinary laboratory of the same district between January 1, 1995, and December 31, 1996 were studied . Susceptibility to nalidixic acid was determined using the disk diffusion method . Strains with decreased susceptibility to nalidixic acid were subjected to minimal inhibitory concentration (MIC) determination for pefloxacin, ofloxacin, ciprofloxacin, norfloxacin, levofloxacin, and grepafloxacin . Decreased susceptibility to nalidixic acid was demonstrated for 41 of the 309 strains studied and increased from 8.5% in 1995 to 18.6% in 1996 . MIC90 values of fluoroquinolones for strains with decreased susceptibility to nalidixic acid were lower than 1 mg/L, which is the cutoff above which a strain is classified as susceptible, but were higher than for strains that were susceptible to nalidixic acid . These low levels of resistance may be the first step in selection of mutant strains with high levels of resistance to fluoroquinolones . This warrants continued monitoring of resistance of Salmonella to fluoroquinolones.

Arch Microbiol, 1998 Dec, 171(1), 66 - 8
Biosynthesis of cobalamin in Salmonella typhimurium: transformation of riboflavin into the 5,6-dimethylbenzimidazole moiety; Keck B et al.; In order to elucidate the biosynthesis of the base moiety of cobalamin in Salmonella typhimurium LT2, this organism was grown in the presence of {1'-14C}riboflavin . The vitamin B12 isolated was 14C-labeled . It was shown by chemical degradation that the 14C-label was exclusively localized in carbon atom 2 of the 5,6-dimethylbenzimidazole moiety . This demonstrated the precursor function of riboflavin in the biosynthesis of 5,6-dimethylbenzimidazole in S . typhimurium.

J Biol Chem, 1999 Jan 1, 274(1), 29 - 35
Interaction of the cyclic antimicrobial cationic peptide bactenecin with the outer and cytoplasmic membrane; Wu M et al.; Bactenecin, a 12-amino acid cationic antimicrobial peptide from bovine neutrophils, has two cysteine residues, which form one disulfide bond, making it a cyclic molecule . To study the importance of the disulfide bond, a linear derivative Bac2S was made and the reduced form (linear bactenecin) was also included in this study . Circular dichroism spectroscopy showed that bactenecin existed as a type I beta-turn structure regardless of its environment, while the reduced form and linear bactenecin adopted different conformations according to the lipophilicity of the environment . Bactenecin was more active against the Gram-negative wild type bacteria Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhimurium than its linear derivative and reduced form, while all three peptides were equally active against the outer membrane barrier-defective mutants of the first two bacteria . Only the two linear peptides showed activity against the Gram-positive bacteria Staphylococcus epidermidis and Enterococcus facaelis . Bactenecin interacted well with the outer membrane and its higher affinity for E . coli UB1005 lipopolysaccharide and improved ability to permeabilize the outer membrane seemed to account for its better antimicrobial activity against Gram-negative bacteria . The interaction of bactenecin with the cytoplasmic membrane was determined by its ability to dissipate the membrane potential by using the fluorescence probe 3, 3-dipropylthiacarbocyanine and an outer membrane barrier-defective mutant E . coli DC2 . It was shown that the linear derivative and reduced form were able to dissipate the membrane potential at much lower concentrations than bactenecin despite the similar minimal inhibitory concentrations of all three against this barrier-defective mutant.

Infect Immun, 1999 Jan, 67(1), 436 - 8
Glucose 6-phosphate dehydrogenase is required for Salmonella typhimurium virulence and resistance to reactive oxygen and nitrogen intermediates; Lundberg BE et al.; Salmonella typhimurium zwf mutants lacking glucose 6-phosphate dehydrogenase (G6PD) activity have increased susceptibility to reactive oxygen and nitrogen intermediates as well as attenuated virulence in mice . Abrogation of the phagocyte respiratory burst oxidase during experimental infection with zwf mutant Salmonella causes a prompt restoration of virulence, while inhibition of inducible nitric oxide synthase results in delayed lethality . These observations suggest that G6PD-dependent bacterial antioxidant defenses play an important pathogenic role during early salmonellosis and additionally may help to antagonize NO-dependent antimicrobial mechanisms later in the course of infection.

Infect Immun, 1999 Jan, 67(1), 319 - 26
The polysaccharide portion of lipopolysaccharide regulates antigen-specific T-cell activation via effects on macrophage-mediated antigen processing; Zirk NM et al.; The lipopolysaccharide (LPS) structure of Salmonella typhimurium has been correlated with the virulence of wild-type strain LT2 . Mutants of LT2 with truncated polysaccharide portions of LPS are less virulent than strains with a complete LPS structure . Polyclonal T cells and monoclonal T-cell hybridomas were more reactive to heat-killed rough mutants than to heat-killed smooth strains, as measured by interleukin-2 (IL-2) production . Using a large panel of strains with truncated LPS molecules, we found that T-cell reactivity decreased with certain lengths of polysaccharide . The decreased response was not due to differential phagocytic uptake, IL-12 production, or major histocompatibility complex class II surface expression by macrophages . Also, LT2 did not mediate any global suppression since addition of LT2 did not diminish the response of T cells specific for antigens unrelated to Salmonella . In an experiment in which processing times were varied, we found that antigens from rough strains were processed and presented more quickly than those associated with smooth strains . At longer processing times, epitopes from LT2 were presented well . We hypothesize that the slower antigen processing and presentation of wild-type Salmonella may be caused by masking of surface antigens by the longer polysaccharide portion of smooth LPS . This blocking of effective antigen presentation may contribute to the virulence of Salmonella.

Infect Immun, 1999 Jan, 67(1), 213 - 9
Influence of the Salmonella typhimurium pathogenicity island 2 type III secretion system on bacterial growth in the mouse; Shea JE et al.; We have investigated the in vivo growth kinetics of a Salmonella typhimurium strain (P11D10) carrying a mutation in ssaJ, a Salmonella pathogenicity island 2 (SPI2) gene encoding a component of a type III secretion system required for systemic growth in mice . Similar numbers of mutant and wild-type cells were recovered from the spleens and livers of BALB/c mice up to 8 h after inoculation by the intraperitoneal route . Thereafter, the numbers of wild-type cells continued to increase logarithmically in these organs, whereas those of P11D10 remained relatively static for several days before being cleared . Gentamicin protection experiments on spleen cell suspensions recovered from infected mice showed that viable intracellular wild-type bacteria accumulated over time but that intracellular P11D10 cells did not . Infection experiments were also performed with wild-type and P11D10 cells carrying the temperature-sensitive plasmid pHSG422 to distinguish between bacterial growth rates and killing in vivo . At 16 h postinoculation there were 10-fold more wild-type cells than mutant cells in the spleens of infected mice, but the numbers of cells of both strains carrying the nonreplicating plasmid were very similar, showing that there was little difference in the degree of killing sustained by the two strains and that the SPI2 secretion system must be required for bacterial replication, rather than survival, in vivo . The SPI2 mutant phenotype in mice is similar to that of strains carrying mutations in the Salmonella virulence plasmid spv genes . To determine if these two sets of genes interact together, a double mutant strain carrying SPI2 and spv mutations was constructed and compared with strains carrying single mutations in terms of virulence attenuation . These experiments failed to provide any evidence showing that the SPI2 and spv gene products interact together as part of the same virulence mechanism.

Infect Immun, 1999 Jan, 67(1), 120 - 5
Expression of the EspB protein of enteropathogenic Escherichia coli within HeLa cells affects stress fibers and cellular morphology; Taylor KA et al.; The EspB protein of enteropathogenic Escherichia coli (EPEC) is essential for the signaling events that lead to the accumulation of actin beneath intimately attached bacteria, a process that is known as the attaching and effacing effect . EspB is targeted to the host cell cytoplasm by a type III secretion apparatus . To determine the effect of intracellular EspB on the host cell cytoskeleton, we transfected HeLa cells with a plasmid containing the espB gene under the control of an inducible eukaryotic promoter . A HeLa cell clone that expressed espB mRNA and EspB protein after induction was selected for further study . The expression of EspB in these cells caused a dramatic change in cell morphology and a marked reduction in actin stress fibers . Cells expressing EspB were significantly impaired in their ability to support invasion by EPEC and Salmonella typhimurium . However, the expression of EspB within host cells could not compensate for the lack of EspB expression by an espB mutant strain of EPEC to restore attaching and effacing activity . These studies suggest that EspB is a cytoskeletal toxin that is translocated to the host cell cytoplasm, where it causes a redistribution of actin.

Structure, 1998 Dec 15, 6(12), 1529 - 39
Ligand size is a major determinant of specificity in periplasmic oxyanion-binding proteins: the 1.2 A resolution crystal structure of Azotobacter vinelandii ModA; Lawson DM et al.; Background: . Periplasmic receptors constitute a diverse class of binding proteins that differ widely in size, sequence and ligand specificity . Nevertheless, almost all of them display a common beta/alpha folding motif and have similar tertiary structures consisting of two globular domains . The ligand is bound at the bottom of a deep cleft, which lies at the interface between these two domains . The oxyanion-binding proteins are notable in that they can discriminate between very similar ligands . Results: . Azotobacter vinelandii is unusual in that it possesses two periplasmic molybdate-binding proteins . The crystal structure of one of these with bound ligand has been determined at 1.2 A resolution . It superficially resembles the structure of sulphate-binding protein (SBP) from Salmonella typhimurium and uses a similar constellation of hydrogen-bonding interactions to bind its ligand . However, the detailed interactions are distinct from those of SBP and the more closely related molybdate-binding protein of Escherichia coli . Conclusions: . Despite differences in the residues involved in binding, the volumes of the binding pockets in the A . vinelandii and E . coli molybdate-binding proteins are similar and are significantly larger than that of SBP . We conclude that the discrimination between molybdate and sulphate shown by these binding proteins is largely dependent upon small differences in the sizes of these two oxyanions.

Environ Health Perspect, 1998 Dec, 106 Suppl 6, 1373 - 6
Mixture design and multivariate analysis in mixture research; Eide I et al.; Mixture design has been used to identify possible interactions between mutagens in a mixture . In this paper the use of mixture design in multidimensional isobolographic studies is introduced . Mutagenicity of individual nitro-polycyclic aromatic hydrocarbons (PAH) was evaluated is an organic extract of diesel exhaust particles (DEPs) . The particles were extracted with dichloromethane (DCM) . After replacing DCM with dimethyl sulfoxide, the extract was spiked with three individual nitro-PAH: 1-nitropyrene, 2-nitrofluorene, and 1,8-dinitropyrene . The nitro-PAH were added separately and in various combinations to the extract to determine the effects of each variable and to identify possible interactions between the individual nitro-PAH and between the nitro-PAH and the extract . The composition of the mixtures was determined by mixture design (linear axial normal) with four variables (the DEP extract and the three nitro-PAH, giving 8 different mixtures plus a triplicate centerpoint, i.e., a total of 11 . The design supports a model with linear and interaction (product) terms . Two different approaches were used: traditional mixture design within a well-defined range on the linear part of the dose-response curves and an isobolographic mixture design with equipotent doses of each variable . The mixtures were tested for mutagenicity in the Ames assay using the TA98 strain of Salmonella typhimurium . The data were analyzed with projections to latent structures (PLS) . The three individual nitro-PAH and the DEP extract acted additively in the Ames test . The use of mixture design either within a well-defined range of the linear part on the dose-response curve or with equipotent doses saves experiments and reduces the possibility of false interaction terms in situations with dose additivity or response additivity.

Chem Res Toxicol, 1998 Dec, 11(12), 1501 - 7
Identification of 1,6- and 1,8-dinitropyrene isomers as major mutagens in organic extracts of soil from Osaka, Japan; Watanabe T et al.; The organic extracts of soil collected at parks in residential areas in Osaka and neighboring cities in the Kansai area, Japan, showed mutagenicity in Salmonella typhimurium strain TA98 in the presence or absence of a mammalian metabolic activation system (S9 mix) . The soil extracts from Ibaraki and two different sites in Osaka, i.e., Sumiyoshi-ku and Minato-ku, were mutagenic in strain TA100 as well as in strain TA98 . Direct-acting mutagenicity of soil extracts from Sumiyoshi-ku and Minato-ku toward strain TA98 were 66 or more times higher than that of the other cities . Both extracts exerted stronger mutagenicity in strains YG1021 and YG1024 than TA98 and TA100, and the potency was especially high in strain YG1024: Sumiyoshi-ku, 153 000 revertants/g of soil; and Minato-ku, 246 000 revertants/g of soil . Two mutagenic compounds (I and II) were isolated from the Soxhlet extract of soil from the park in Sumiyoshi-ku by repetitive separation using normal-phase and reversed-phase column chromatography . By comparing the mass and UV spectra and retention times for HPLC on two individual ODS columns of compounds I and II with those of authentic chemicals, we identified these two compounds as 1,6- and 1,8-dinitropyrene (DNPy) isomers . Amounts of DNPy isomers in soil from Sumiyoshi-ku and Minato-ku were 1.7-2.2 ng/g . Forty-three percent and 40% of the mutagenicity of soil from Sumiyoshi-ku and Minato-ku could be attributed to these DNPy isomers, respectively.

Mutat Res, 1999 Jan 2, 438(1), 71 - 8
Triethylene glycol dimethacrylate induces large deletions in the hprt gene of V79 cells; Schweikl H et al.; Acrylate esters are applied in industrial and consumer products often associated with polymers and resins . The difunctional methacrylate, triethylene glycol dimethacrylate (TEGDMA), is also frequently included in dental composite materials . Recently, mutagenicity testing of the compound revealed the induction of gene mutations at the hprt locus in V79 cell {H . Schweikl, G . Schmalz, K . Rackebrandt, The mutagenic activity of unpolymerized resin monomers in Salmonella typhimurium and V79 cells, Mutat . Res . 415 (1998) 119-130} . In the present study, TEGDMA caused a dose dependent increase of the number of micronuclei in V79 cells . Furthermore, the mutation spectra induced in exon sequences of the hprt gene in HPRT-deficient V79 cell clones were analyzed by the polymerase chain reaction (PCR) . No DNA sequence deletions were observed in spontaneously occurring HPRT-deficient cell clones at the molecular level after PCR analysis, indicating that all spontaneous mutations were caused by point mutations . However, TEGDMA treated V79 cell cultures exhibited different mutation spectra . Only one cell clone among a total of 25 contained all exon sequences of the hprt gene . Large DNA sequences were deleted in 24 cell clones . Partial gene deletions occurred in four clones from exon 5 through 9, and exon 1 was not amplified in one cell clone . Exon sequences of the hprt gene were totally deleted in 19 HPRT-deficient clones . The induction of mostly large deletions in the genome of mammalian cells, like the mutation spectra induced by TEGDMA in V79 cells here, is probably typical for crosslinking agents, including anticancer drugs . Identical types of mutations including chromosomal aberrations and the formation of micronuclei in vitro were observed for acrylates and methacrylates tested so far in various mutation assays . Therefore, we conclude by analogy that the induction of large DNA sequence deletions as shown here with the reactive dimethacrylate, triethylene glycol dimethacrylate, is probably common for acrylates and methacrylates .

Mutat Res, 1999 Jan 2, 438(1), 1 - 12
Cytochrome P450 1A1 in rat peripheral blood lymphocytes: inducibility in vivo and bioactivation of benzo{a}pyrene in the Salmonella typhimurium mutagenicity assay in vitro; Fung J et al.; The presence and inducibility of CYP1A1 in freshly isolated peripheral blood lymphocytes was examined in untreated rats and in rats pretreated with agents known to induce the enzyme in other tissues, as well as dexamethasone {CAS #50-02-2}, which is not commonly associated with CYP1A1 induction . CYP1A1 but not CYP1A2 was detected by Western blot analysis of lymphocytes from untreated rats and was induced in lymphocytes from rats treated with the known CYP1A inducers beta-naphthoflavone {CAS #6051-87-2} or 3-methylcholanthrene {CAS #56-49-5} (7.3-fold), cigarette smoke (2 . 8-fold), and pyridine {CAS #108-86-1} (2.6-fold) . CYP1A1 was also induced in lymphocytes from rats treated with the nonprototypic inducer dexamethasone (17.7-fold) or bromobenzene {CAS #108-86-1} (3 . 9-fold) . Lymphocyte homogenate from rats treated with the inducers also catalyzed NADPH-dependent bioactivation of benzo{a}pyrene {CAS #50-32-8} to mutagens . The benzo(a)pyrene mutagenicity was detected using Salmonella typhimurium TA100 in the Ames test, and correlated positively with lymphocyte CYP1A1 content . The data show that CYP1A1 is present in rat peripheral blood lymphocytes in vivo, and is inducible by prototypic, as well as nonprototypic, inducers of the enzyme .

Gene, 1998 Oct 9, 221(1), 151 - 7
Cloning and characterization of Planctomyces limnophilus rpoN: complementation of a Salmonella typhimurium rpoN mutant strain; Leary BA et al.; The rpoN gene, which encodes the alternative sigma factor sigma 54, was cloned from the budding, peptidoglycan-less bacterium Planctomyces limnophilus . P . limnophilus rpoN complemented the Ntr- phenotype of a Salmonella typhimurium rpoN mutant strain . The P . limnophilus rpoN gene encoded a predicted polypeptide that was 495 residues in length and shared a significant homology with other members of the sigma 54 family . The protein sequence displayed all of the characteristic motifs found in members of this family, including the C-terminal helix-turn-helix motif and the well-conserved RpoN box . A potential sigma 54-dependent activator was also identified in P . limnophilus . These findings extend the range of phylogenetic groups within the Domain Bacteria that are known to contain sigma 54.

Gene, 1998 Oct 9, 221(1), 25 - 34
A new membrane-bound OprI lipoprotein expression vector . High production of heterologous fusion proteins in gram (-) bacteria and the implications for oral vaccination; Cote-Sierra J et al.; We have previously described the development of cloning vectors for the production of OprI-based outer membrane fusion proteins in E . coli (Cornelis et al., 1996) and now describe the construction of a new vector, containing a lacI(q) gene, resulting in tight repression of the promotor and allowing its use in other Gram (-) bacteria . The new pVUB3 expression vector encodes a truncated but active LacI(q)(341) repressor which binds to the single operator in the vector . A high repression of the trc promotor was observed, resulting in a very low basal leakage of expression and very high production levels of OprI or derivatives after IPTG induction in E . coli . Bacterial viability was not affected under uninduced conditions, but the number of viable cell counts decreased after production of large amounts of the outer membrane-bound OprI lipoprotein and its derivatives, both in E . coli and Salmonella typhimurium . This highly repressible system allows us to extend the use of OprI vectors in other Gram (-) bacteria, resulting in the production of outer membrane-bound lipid-modified molecules, opening the possibility for its application in the design of potential live Salmonella-based subunit vaccines.

J Biol Chem, 1998 Dec 18, 273(51), 34028 - 32
Regulation of the Salmonella typhimurium flavohemoglobin gene . A new pathway for bacterial gene expression in response to nitric oxide; Crawford MJ et al.; Flavohemoglobins, a family of two-domain proteins with homology to vertebrate hemoglobins, are found in a variety of prokaryotic and eukaryotic microorganisms . Recent studies suggest a role for these proteins in nitrogen oxide metabolism . We now show that nitric oxide donors positively regulate a chromosomal flavohemoglobin (hmp)/lacZ operon fusion in Salmonella typhimurium . hmp gene expression in the presence of NO . is independent of the SoxS, OxyR, and FNR transcription factors and instead relies on inactivation of the iron-dependent Fur repressor . Other Fur-repressed promoters in S . typhimurium are also activated by an NO . donor . In contrast to the wild-type strain, an hmp- mutant requires markedly lower concentrations of NO to induce the hmp/lacZ fusion, whereas its response to iron chelation is equivalent to wild type . These data unveil a new pathway for NO-dependent gene expression in S . typhimurium.

J Bacteriol, 1998 Dec, 180(24), 6689 - 96
Selectivity of ferric enterobactin binding and cooperativity of transport in gram-negative bacteria; Thulasiraman P et al.; The ligand-gated outer membrane porin FepA serves Escherichia coli as the receptor for the siderophore ferric enterobactin . We characterized the ability of seven analogs of enterobactin to supply iron via FepA by quantitatively measuring the binding and transport of their 59Fe complexes . The experiments refuted the idea that chirality of the iron complex affects its recognition by FepA and demonstrated the necessity of an unsubstituted catecholate coordination center for binding to the outer membrane protein . Among the compounds we tested, only ferric enantioenterobactin, the synthetic, left-handed isomer of natural enterobactin, and ferric TRENCAM, which substitutes a tertiary amine for the macrocyclic lactone ring of ferric enterobactin but maintains an unsubstituted catecholate iron complex, were recognized by FepA (Kd approximately 20 nM) . Ferric complexes of other analogs (TRENCAM-3,2-HOPO; TREN-Me-3,2-HOPO; MeMEEtTAM; MeME-Me-3,2-HOPO; K3MECAMS; agrobactin A) with alterations to the chelating groups and different net charge on the iron center neither adsorbed to nor transported through FepA . We also compared the binding and uptake of ferric enterobactin by homologs of FepA from Bordetella bronchisepticus, Pseudomonas aeruginosa, and Salmonella typhimurium in the native organisms and as plasmid-mediated clones expressed in E . coli . All the transport proteins bound ferric enterobactin with high affinity (Kd </= 100 nM) and transported it at comparable rates (>/=50 pmol/min/10(9) cells) in their own particular membrane environments . However, the FepA and IroN proteins of S . typhimurium failed to efficiently function in E . coli . For E . coli, S . typhimurium, and P . aeruginosa, the rate of ferric enterobactin uptake was a sigmoidal function of its concentration, indicating a cooperative transport reaction involving multiple interacting binding sites on FepA.

J Bacteriol, 1998 Dec, 180(24), 6519 - 28
Complex metabolic phenotypes caused by a mutation in yjgF, encoding a member of the highly conserved YER057c/YjgF family of proteins; Enos-Berlage JL et al.; The oxidative pentose phosphate pathway is required for function of the alternative pyrimidine biosynthetic pathway, a pathway that allows thiamine synthesis in the absence of the PurF enzyme in Salmonella typhimurium . Mutants that no longer required function of the oxidative pentose phosphate pathway for thiamine synthesis were isolated . Further phenotypic analyses of these mutants demonstrated that they were also sensitive to the presence of serine in the medium, suggesting a partial defect in isoleucine biosynthesis . Genetic characterization showed that these pleiotropic phenotypes were caused by null mutations in yjgF, a previously uncharacterized open reading frame encoding a hypothetical 13.5-kDa protein . The YjgF protein belongs to a class of proteins of unknown function that exhibit striking conservation across a wide range of organisms, from bacteria to humans . This work represents the first detailed phenotypic characterization of yjgF mutants in any organism and provides important clues as to the function of this highly conserved class of proteins . Results also suggest a connection between function of the isoleucine biosynthetic pathway and the requirement for the pentose phosphate pathway in thiamine synthesis.

J Bacteriol, 1998 Dec, 180(24), 6511 - 8
Studies of regulation of expression of the propionate (prpBCDE) operon provide insights into how Salmonella typhimurium LT2 integrates its 1,2-propanediol and propionate catabolic pathways; Tsang AW et al.; Expression of the prpBCDE operon of Salmonella typhimurium LT2 required (i) the synthesis of propionyl-coenzyme A (CoA) by the PrpE protein or the acetyl-CoA-synthesizing systems of the cell and (ii) the synthesis of 2-methylcitrate from propionyl-CoA and oxaloacetate by the PrpC protein . We propose that either 2-methylcitrate or a derivative of it signals the presence of propionate in the environment . This as yet unidentified signal is thought to serve as a coregulator of the activity of PrpR, the member of the sigma-54 family of transcriptional activators needed for activation of prpBCDE transcription . The CobB protein was also required for expression of the prpBCDE operon, but its role is less well understood . Expression of the prpBCDE operon in cobB mutants was restored to wild-type levels upon induction of the propanediol utilization (pdu) operon by 1,2-propanediol . This effect did not require catabolism of 1,2-propanediol, suggesting that a Pdu protein, not a catabolite of 1,2-propanediol, was responsible for the observed effect . We explain the existence of these redundant functions in terms of metabolic pathway integration . In an environment with 1,2-propanediol as the sole carbon and energy source, expression of the prpBCDE operon is ensured by the Pdu protein that has CobB-like activity . Since synthesis of this Pdu protein depends on the availability of 1,2-propanediol, the cell solves the problem faced in an environment devoid of 1,2-propanediol where propionate is the sole carbon and energy source by having cobB located outside of the pdu operon and its expression independent of 1,2-propanediol . At present, it is unclear how the CobB and Pdu proteins affect prpBCDE expression.

Arch Toxicol, 1998 Oct, 72(10), 645 - 9
Mutagenicity testing of organic extracts of diesel exhaust particles after spiking with polycyclic aromatic hydrocarbons (PAH); Bostrom E et al.; In the present study, spiking was used as a strategy to evaluate the mutagenicity of individual compounds in a mixture . Mutagenicity of individual polycyclic aromatic hydrocarbons (PAH) was evaluated in an organic extract of diesel exhaust particles (DEP) . The particles were extracted with dichloromethane (DCM) . After replacing DCM with dimethylsulphoxide (DMSO), the extract was spiked with four individual PAH: benzo(a)pyrene, benzo(a)anthracene, pyrene and fluoroanthene . The PAH were added separately and in various combinations to the extract to determine the effects of each variable and to identify possible interactions between the individual PAH and between the PAH and the extract . The study was designed as a fractional factorial experiment with the five variables (the DEP extract and the four PAH), giving 16 (instead of 32) mixtures plus a triplicate centrepoint and background, i.e . a total of 20 . The fractionated factorial design used in the present work supports a model with linear and interaction terms . The mixtures were tested for mutagenicity in the Ames assay using four strains of Salmonella typhimurium in the presence of rat liver xenobiotic enzymes (S9-mix) . Projections to Latent Structures (PLS) was used to quantify the mutagenicity of each compound and possible interactions . The four individual PAH and the DEP extract acted additively in the Ames test with 10% S9-mix.

FEMS Microbiol Lett, 1998 Dec 1, 169(1), 147 - 53
High levels of transcription factor RpoS (sigma S) in mviA mutants negatively affect 1,2-propanediol-dependent transcription of the cob/pdu regulon of Salmonella typhimurium LT2; Rondon MR et al.; Expression of the cobalamin biosynthetic (cob) and 1,2-propanediol utilization (cob/pdu) regulon of Salmonella typhimurium LT2 is controlled at the transcriptional level by global and specific regulatory proteins . In this paper we show that mutations in the mviA gene negatively affect cob/pdu transcription in response to 1,2-propanediol in the environment . The effects of mviA mutations were consistent with its role in the regulation of RpoS levels in the cell . Null mutations in rpoS eliminated the negative effect of mviA mutations on cob/pdu transcription, and restored growth on succinate, propionate and 1,2-propanediol . In addition, mviA mutants were deficient in the utilization of succinate, propionate and 1,2-propanediol as carbon and energy sources.

Vet Res, 1998 Nov-Dec, 29(6), 547 - 56
High hydrostatic pressure inactivation of Salmonella typhimurium: effects of pressure, duration, pH and temperature studied by analysis of variance; Ritz M et al.; High hydrostatic pressure treatments are regarded as possible alternative methods for food preservation . One of the primary considerations for industrial applications is the ability of these methods to eradicate pathogenic microorganisms . This study subjected S . typhimurium suspensions, first in a phosphate buffer (pH 7.0) and then in a citrate phosphate buffer (pH 5.6), to high hydrostatic pressure treatments relative to the following variables: pressure (200-400 MPa), duration (3, 10 and 20 min), temperature (4, 20 and 40 degrees C) and the pH of the suspension medium (5.6 and 7.0) . An optimal design of 40 runs was obtained using the Fedorov algorithm, and responses were studied by analysis of variance in terms of cell survival on plate count agar . Efficiency was determined by Log10 comparisons of the numbers of live cells before and after treatment . A statistically significant relationship was found between the four variables considered (pressure, pH, duration and temperature), their interactions (duration x pressure, pH x temperature, pH x pressure) and the inactivation of S . typhimurium . R-squared statistical analysis indicated that the linear model used accounted for more than 98% of the variability in the inactivation of S . typhimurium.

Int J Food Microbiol, 1998 Oct 20, 44(1-2), 107 - 13
Adenylates and adenylate-energy charge in submerged and planktonic cultures of Salmonella enteritidis and Salmonella typhimurium; Walker SL et al.; Adenine nucleotide values and adenylate energy charge (AEC) were measured during the growth of Salmonella enteritidis and Salmonella typhimurium as submerged colonies in agarose gel and gelatin gel, and as planktonic cells in broth . Growth in all three systems showed similar trends with a ten-fold decrease in total adenylate pool during exponential growth, before attaining a fairly stable value throughout stationary phase . AEC values were generally low, (approximately 0.66), but did rise slightly during stationary phase . The large proportion of dead cells during early exponential phase may have contributed to the adenosine diphosphate and adenosine monophosphate pools, through cell lysis or excretion, and it is suggested that this was likely to account for the low values of AEC . In agarose and gelatin gelled cultures the percentage of adenosine triphosphate (ATP) in relation to the total adenylates showed random fluctuations . This was contrary to the broth culture where percentage ATP was highest after 12 h and the data formed a smooth curve . These data demonstrated that considerable physiological heterogeneity exists within a colony of bacteria growing in a gel matrix and by analogy a food material also, and that AEC is a poor indicator of cell viability in such systems.

Onderstepoort J Vet Res, 1998 Sep, 65(3), 213 - 20
The production of an auxotrophic marked, plasmid-cured Salmonella ser . Typhimurium as a live attenuated vaccine; Van Der Walt ML et al.; A number of amino acid requiring auxotrophic strains of Salmonella Typhimurium were produced by chemical mutagenesis . One of them, strain 81, was cured of the virulence plasmid and attenuated for mice . This strain had an auxotrophic requirement for serine, which could be used as a marker for the differentiation of the vaccine strain from other isolates in the field . The strain still contained the smooth form of the O-antigen, was resistant to Complement-mediated killing of serum and produced type 1 fimbriae . Of the six auxotrophic mutants only this mutant differed in its outer membrane protein profile from that of the parent strain in that an outer membrane protein of about 30 kDa was absent . With the use of the polymerase chain reaction, using total DNA of the cell as template, and with primers targeted to the virulence plasmid, it was shown that the virulence plasmid of Salmonella Typhimurium was completely cured from this strain . This strain also had a LD50-value of 4 log units lower for mice than the parent strain . The plasmid-cured strain gave a very high degree of protection to mice after systemic immunization, but not after oral vaccination . Compared to the parent, strain 81 also had a lower multiplication rate in the liver and spleen after intraperitoneal inoculation, characteristics that could be attributed to plasmid-loss, and it could also not be recovered from the spleen and liver of orally inoculated mice.

Biol Trace Elem Res, 1998 Summer, 64(1-3), 197 - 213
Transition metals as protease inhibitors; Duffy B et al.; An alternative approach to the development of clinically useful protease inhibitors was investigated . The approach utilized coordination chemistry of transition metal ions rather than substrate analogs to block active sites of these enzymes . In the case of serine proteases it was found that aqueous Ti(IV) is a potent inhibitor of the trypsin subclass, but not the chymotrypsin subclass . The direct binding of Ti(IV) to trypsin was made possible by the presence of a free carboxyl group at the bottom of the substrate binding pocket of the enzyme, and the five-coordinate geometry of TiO(SO4)(H2O) . Although initial binding of Ti(IV) was reversible, it was followed in time by irreversible inhibition . Direct binding of octahedral or tetrahedral metal ion complexes was prevented by the inability of the enzyme active sites to promote formation of a five-coordinate transition state of the metal ion required for reaction . These studies demonstrate the ability of direct metal ion binding as a way to enhance blocking of enzyme active sites as compared with that of traditional organic inhibitors . Application of these findings was investigated by measuring the affect Ti(IV) had on growth of Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa . Five-coordinate titanyl sulfate completely inhibited the growth of these organisms . This suggests that five-coordinate titanyl sulfate, which is easier and less expensive to manufacture than conventional antibiotics, may be useful in controlling endemic infections of E . coli and S . typhimurium.

Mutat Res, 1998 Dec 3, 420(1-3), 115 - 24
Pentachlorophenol-mediated mutagenic synergy with aromatic amines in Salmonella typhimurium; Gichner T et al.; Pentachlorophenol (PCP), a widely used pesticide, enhanced the mutagenic potency of plant- or mammalian-activated 2-aminofluorene (2AF) as well as the direct-acting mutagen 2-acetoxyacetylaminofluorene (2AAAF) when assayed with specific Salmonella typhimurium strains . With 2AF the mutagenic synergy was observed in strains YG1024, TA1538, and MP153 . With 2AAAF the PCP-mediated synergy was observed with these strains and with strain TA98/1,8-DNP6 . The synergy was dependent upon the presence of an activated N-acetoxy functional group and was only expressed at the hisD3052 allele and not at the hisG46 allele . Spectrophotometric analysis demonstrated that the rate of degradation of 2AAAF was reduced in the presence of PCP in phosphate buffer or with S . typhimurium cytosol and thus PCP may be affecting the stability of the N-acetoxy group of activated aromatic amines .

Mutat Res, 1998 Dec 3, 420(1-3), 109 - 14
Mutagenicity of cooked hamburger is reduced by addition of onion to ground beef; Kato T et al.; Addition of onion effectively reduced mutagenicity of cooked hamburger when tested on Salmonella typhimurium TA98 strain with metabolic activation . The components of onion that participated in the reduction of mutagenicity were sugars . Addition of starch or glucose to ground beef the amount equivalent to that in onion reduced the mutagenicity of cooked hamburger . Addition of onion may cause imbalance of the sugar content of ground beef that effectively produces mutagenicity . Mutagenicity of the heated model mixture of glucose/glycine/creatinine in diethyleneglycol-water was reduced by an excessive amount of glucose . Hence, Japanese cooking-style with addition of onion can reduce mutagenicity of hamburger .

Mutat Res, 1998 Dec 3, 420(1-3), 27 - 32
Mutagenicities of 2,4- and 2,6-dinitrotoluenes and their reduced products in Salmonella typhimurium nitroreductase- and O-acetyltransferase-overproducing Ames test strains; Sayama M et al.; Mutagenicities of 2,4- and 2,6-dinitrotoluene (2,4-and 2,6-DNT), and reduced metabolites formed by the incubation of 2,4- and 2,6-DNT with Salmonella typhimurium TA98, were tested using S . typhimurium YG strains possessing high level of nitroreductase (NR) and/or O-acetyltransferase (OAT) activities . All compounds tested showed greatest mutagenic activities toward strains YG1041 and YG1042, which possess high levels of NR and OAT activities . The relative mutagenic activities of 2,4-DNT and its related compounds toward YG1041 and YG1042 were aminonitrotoluenes<2,4-DNT<2,2'-dimethyl-5, 5'-dinitroazoxybenzene (2,2'-DM-5, 5'-DNAOB)4-hydroxylamino-2-nitrotoluene (4HA2NT)<<4, 4'-dimethyl-3,3'-dinitroazoxybenzene (4,4'-DM-3,3'-DNAOB), and aminonitrotoluenes (2A4AT, 4A2NT)<2,4-DNT<4HA2NT4,4'-dimethyl-3, 3'-dinitroazoxybenzene (4,4'-DM-3,3'-DNAOB)<2HA4NT, respectively . In addition, the relative mutagenic activities of 2,6-DNT and its related compounds toward YG1041 and YG1042 were 2, 6-DNT<2-hydroxylamino-6-nitrotoluene (2HA6NT)<2,2'-dimethyl-3, 3'-dinitroazoxybenzene (2,2'-DM-3,3'-DNAOB), and 2-amino-6-nitrotoluene (2A6NT)<2,6-DNT<2HA6NT, respectively . These results, together with previous findings, suggested that aminohydroxylamino dimethylazoxybenzenes or aminohydroxylamino dimethylazobenzenes produced either by the reduction of hydroxylaminonitrotoluenes or by the reduction of dimethyl dinitroazoxybenzenes are active metabolites responsible for the mutagenic activities of 2,4- and 2,6-DNT .

Appl Environ Microbiol, 1998 Dec, 64(12), 5033 - 8
Production of monoclonal antibodies specific for the i and 1,2 flagellar antigens of Salmonella typhimurium and characterization of their respective epitopes; de Vries N et al.; Salmonella typhimurium expresses two antigenically distinct flagellins, each containing a different H antigen (i and 1,2), the combination of which is highly specific for this serotype . In this study, overlapping recombinant flagellin fragments were constructed from the fliC (H:i) and fljB (H:1,2) flagellin genes, and the expression products were tested for binding to H antigen-specific monoclonal and polyclonal antibodies . A minimal area, 86 amino acids for H:i and 102 amino acids for H:1,2, located in the central variable domain of each flagellin was required for the binding of serotype-specific antibodies, providing further evidence for the presence of a discontinuous H epitope . Two peptides comprising these areas were shown to be highly suitable for application as antigens in an enzyme-linked immunosorbent assay detecting S . typhimurium-specific antibody.

Ophthalmic Res, 1999, 31(1), 42 - 6
PGE2 synthesis by corneal endothelial cells: effect of glucocorticoids and NSAIDs; Garcia-Cabanes C et al.; The aim of this work was to study the effect of various anti-inflammatory drugs on PGE2 synthesis in cultured bovine corneal endothelial cells (BCECs) stimulated with calcium ionophore A23187 or lipopolysaccharide (LPS) of Salmonella typhimurium . NSAIDs were more potent in inhibiting LPS-stimulated PGE2 synthesis . Diclofenac was more potent than indomethacin, although both drugs showed a 98% maximal inhibitory effect . Dexamethasone inhibited 80% of the A23187-stimulated PGE2 synthesis and only 53% of the LPS-stimulated PGE2 synthesis . Prednisolone did not show an inhibitory effect . The results demonstrate the inhibitory effect of NSAIDs and show differences between the activity of glucocorticoids on PGE2 synthesis in BCECs . Prednisolone could not inhibit PGE2 synthesis in these cells in our experimental conditions.

Res Microbiol, 1998 Oct, 149(9), 625 - 30
Salmonella typhimurium porin internalization by leukocytes; Galdiero M et al.; The location of Salmonella typhimurium porins has been found in human monocytes and lymphocytes by means of high resolution autoradiography . The results indicate that traces of porins are frequently visible on ultrathin sections of treated human lymphomonocytes and that they are located especially in the nuclear areas.

Infect Immun, 1998 Dec, 66(12), 5862 - 6
Salmonella typhimurium infection in mice induces nitric oxide-mediated immunosuppression through a natural killer cell-dependent pathway; Schwacha MG et al.; Splenocytes isolated from C57BL/6J female mice 3 to 7 days after inoculation with an attenuated strain of Salmonella typhimurium produced high levels of nitric oxide (39 to 77 microM) and gamma interferon (IFN-gamma) . Additionally, spleen cell cultures from Salmonella-inoculated mice were markedly suppressed in their ability to generate an in vitro plaque-forming cell (PFC) response to sheep erythrocytes . Depletion of natural killer (NK) cells from the immune splenocyte population markedly reduced nitric oxide production, prevented suppression of PFC responses, and completely abrogated IFN-gamma release . Treatment of NK cell-depleted immune cells with IFN-gamma restored nitric oxide production to levels comparable to those of intact immune cells and also restored the immunosuppression . These results suggest that NK cells regulate the induction of nitric oxide-mediated immunosuppression following infection with S . typhimurium through the production of IFN-gamma.

Infect Immun, 1998 Dec, 66(12), 5725 - 30
Identification and characterization of a phase-variable nonfimbrial Salmonella typhimurium gene that alters O-antigen production; Kwan LY et al.; Salmonella typhimurium 798, which was isolated from a pig, is known to phase vary from a nonadhesive to an adhesive phenotype . Cells of the adhesive phenotype adhere to porcine enterocytes, are more readily phagocytized by porcine neutrophils and macrophages, and once phagocytized can survive intracellularly, while cells of the nonadhesive phenotype die rapidly . The effect of phenotypic switching also can be visualized by changes in colony morphologies and the presence of between 10 and 15 proteins in the envelopes of cells in the adhesive phenotype . Mutants previously constructed with cells in the adhesive phenotype and the transposon TnphoA were screened to identify mutants lacking one or more of the unique proteins . One mutation was cloned and sequenced, and the mutation was shown to be in rfaL (O-antigen ligase) . Expression of O antigen was shown to be phase variable . The adhesive strain expressed an O antigen that was at least eightfold longer than that for the nonadhesive strain and by virtue of O-antigen production was resistant to porcine complement . The mutant survived intracellularly in phagocytic cells as well as its wild-type parent.

J Infect, 1998 Sep, 37(2), 136 - 9
A 3-year retrospective review of 132 patients with Salmonella enterocolitis admitted to a regional infectious diseases unit; McCarron B; Over a 3-year-period, 132 patients were admitted to the Infectious Diseases Unit, Ruchill Hospital, Glasgow, with non-typhoidal salmonellae enterocolitis . Salmonella enteritidis and typhimurium were isolated from 67% of admissions . Of all patients, 11% had recently travelled overseas, 12% were hypochlorhydric and 3.8% were bacteraemic . Salmonella typhimurium infection was associated with a younger age group, a more pronounced leucocytosis and an earlier and less marked summer peak than S . enteritidis.

J Biol Chem, 1998 Nov 27, 273(48), 31788 - 94
CobB, a new member of the SIR2 family of eucaryotic regulatory proteins, is required to compensate for the lack of nicotinate mononucleotide:5,6-dimethylbenzimidazole phosphoribosyltransferase activity in cobT mutants during cobalamin biosynthesis in Salmonella typhimurium LT2; Tsang AW et al.; The cobB gene of Salmonella typhimurium LT2 has been isolated and genetically and biochemically characterized . cobB was located by genetic means to the 27-centisome region of the chromosome . Genetic crosses established the gene order to be cobB pepT phoQ, and the direction of cobB transcription was shown to be clockwise . The nucleotide sequence of cobB (711 base pairs) predicted a protein of 237 amino acids length with a molecular mass of 26.3 kDa, a mass consistent with the experimentally determined one of approximately 28 kDa . The cobB gene was defined genetically by deletions (10), insertions (5), and point mutations (15) . The precise location of a Tn10d(Tc) element within cobB was established by sequencing . DNA sequence analysis of the region flanking cobB located it 81 base pairs 3' of the potABCD operon, with the potABCD operon and cobB being divergently transcribed . cobB was overexpressed to approximately 30% of the total soluble protein using a T7 overexpression system . In vitro activity assays showed that cell-free extracts enriched for CobB catalyzed the synthesis of the cobalamin biosynthetic intermediate N1-(5-phospho-alpha-D-ribosyl)-5, 6-dimethylbenzimidazole (also known as alpha-ribazole-5'-phosphate) from nicotinate mononucleotide and 5,6-dimethylbenzimidazole, the reaction known to be catalyzed by the CobT phosphoribosyltransferase enzyme (EC 2.4.2.21) (Trzebiatowski, J . R . and Escalante-Semerena, J . C . (1997) J . Biol . Chem . 272, 17662-17667) . Computer analysis of the primary amino acid sequence of the CobB protein identified the sequences GAGISAESGIRTFR and YTQNID which are diagnostic of members of the SIR2 family of eucaryotic transcriptional regulators . Possible roles of CobB as a regulator are discussed within the context of the catabolism of propionate, a pathway known to require cobB function (Tsang, A . W . and Escalante-Semerena, J . C . (1996) J . Bacteriol . 178, 7016-7019).

J Infect, 1998 Mar, 36(2), 175 - 7
The use of pulsed-field gel electrophoresis for subdivision of Salmonella typhimurium in an outbreak situation; Corbett-Feeney G et al.; An institutional outbreak of gastroenteritis provided an opportunity to investigate further the isolates of Salmonella typhimurium by pulsed-field gel electrophoresis (PFGE) . Three phage types were identified . Antibiograms identified two types and two distinct patterns were found on PFGE . If phage typing alone is used for epidemiological study of strains, it is possible that an association between strains may be missed.

Biochemistry, 1998 Nov 17, 37(46), 16369 - 77
The tyrosyl free radical of recombinant ribonucleotide reductase from Mycobacterium tuberculosis is located in a rigid hydrophobic pocket; Liu A et al.; The tyrosyl free radical in protein R2-2 of class Ib ribonucleotide reductase (RNR) fromMycobacterium tuberculosis is essential for the enzymatic activity and has an EPR spectrum remarkably similar to that of the tyrosyl radical YD* in PSII . The EPR relaxation properties of the radical suggest a very weak exchange coupling between the two redox centers, the radical and the diferric cluster . The tyrosyl radical gives almost identical EPR spectra in the temperature interval 10-293 K . We conclude that the tyrosyl radical sits in a rigid pocket . Two ring protons and one beta-methylene proton account for the major anisotropic hyperfine interactions . A high-frequency EPR spectrum of the radical showed a resolved gx = 2 . 0092, indicating that a hydrogen bond to the phenolic oxygen of the radical is absent . Theoretical modeling studies based on the structural data known for Salmonella typhimurium class Ib RNR protein R2F revealed a hydrophobic wall aligned with the radical harboring residue Y110 . The distance between the phenolic oxygen of the radical and the diferric cluster is longer in the two class Ib nrdF R2 proteins than in other characterized class Ia R2 proteins . The tyrosyl radical in protein R2-2 from M . tuberculosis was accessible to direct reduction by dithionite in the absence of a mediator . The radical could be partly regenerated when the system was exposed to O2 after the completion of anaerobic reduction . This indicates that the Fe3+ ions also had become reduced by dithionite.

Chem Res Toxicol, 1998 Nov, 11(11), 1258 - 64
Biodistribution of, antimutagenic efficacies in Salmonella typhimurium of, and inhibition of P450 activities by ellagic acid and one analogue; Castonguay A et al.; Ellagic acid (EA) is generated by hydrolysis of ellagitannins present in fruit berries and edible nuts and grapes . Large doses of EA prevent lung tumorigenesis induced by the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A/J mice . In this study, we document the efficacies of the EA structural analogue (3,4,7,8-tetrahydroxy-6H-benzo{b,d}pyran-6-one) (analogue 1) to inhibit specific P450 activities, pulmonary metabolism of NNK in A/J mice, and NNK-induced mutations in Salmonella typhimurium . Mouse lung microsomes metabolized benzyloxyresorufin, a marker of cytochrome P450 2B1 activity, more extensively than methoxyresorufin or ethoxyresorufin . The EA analogue was more effective than EA in inhibiting dealkylation of the three alkoxyresorufins, suggesting that it is a nonspecific inhibitor of P450s . Mouse lung microsomes hydroxylate testosterone in the 7alpha and 6beta positions, suggesting contributions of P450 2A1 and P450 3A2 isozymes, respectively . Inhibition of both pathways was more effective with the EA analogue than with EA . Mouse lung explants metabolized NNK by alpha-carbon hydroxylation (activation) and pyridine N-oxidation (deactivation) . Both pathways were inhibited when 100 microM EA was added to the culture medium . The EA analogue was a better inhibitor of the activation of NNK to electrophilic species than EA . Mouse lung microsomes activate NNK to intermediates mutagenic to S . typhimurium . Inhibition of NNK mutagenicity by EA or the EA analogue was 20 or 65%, respectively . The distribution of the EA analogue in lung and liver was determined following gavage with 1.7 mmol of the EA analogue . In the lung, a maximal level of EA analogue corresponding to 105 nmol was observed 30 min after administration of the analogue . The level in liver tissues was 4-fold lower than in the lung . Results of this study demonstrate that the EA analogue is more effective than EA in inhibiting the pulmonary activation of NNK and suggest that the EA analogue could be effective in preventing lung tumorigenesis.

J Mol Biol, 1998 Nov 27, 284(2), 521 - 30
Role of the outermost subdomain of Salmonella flagellin in the filament structure revealed by electron cryomicroscopy; Mimori-Kiyosue Y et al.; A mutant strain of Salmonella typhimurium, SJW46, has flagellar filaments supercoiled in the same form as the wild-type strain, SJW1103, and swims normally . However, its flagellar filaments are mechanically unstable and show anomalous behaviors of polymorphism . Flagellin from SJW46 has a large central deletion from Ala204 to Lys292 of SJW1103 flagellin, which has been thought to be located in the outer surface of the filament . Since the filament structure is determined by intersubunit interactions of the terminal regions in the densely packed core of the filament, no serious involvement of the deleted portion was expected in the filament stability and polymorphism . In order to locate the deleted portion and to understand the underlying mechanism of these anomalous characteristics, we carried out structure analysis of the L-type straight filament reconstituted from a mutant flagellin of SJW46 (SJW46S) and compared the structure with that of the SJW1660 filament, which is also the L-type but composed of flagellin with no deletion . The deleted portion was identified as the outermost subdomain, and the structure in the core region showed no appreciable differences . The structure revealed the previously identified folding of flagellin in further detail, and the significance of intersubunit interactions between outer domains, which are present in the SJW1660 filament but absent in the SJW46 filament . This suggests that these contacts have a significant contribution to the filament stability and polymorphic behavior, despite the fact that the contacting surface area occupies only a minor portion of the whole intersubunit interactions .

J Appl Microbiol, 1998 Oct, 85(4), 673 - 7
Use of polymerase chain reaction and restriction enzyme analysis to directly detect and identify Salmonella typhimurium in food; Cocolin L et al.; A primer set of oligonucleotides (Salm 3 and Salm 4) from the invA gene of Salmonellae has been evaluated for the specific detection of Salmonella spp . by the polymerase chain reaction (PCR) . This primer set amplified 33 Salmonella serovars but did not amplify 16 non-Salmonella bacteria . Moreover, after PCR amplification, it was possible to identify Salm . typhimurium by restriction enzyme analysis . The PCR-RE method developed could represent a helpful tool for detecting Salmonella spp., and for directly and rapidly identifying Salm . typhimurium in food.

J Bacteriol, 1998 Nov, 180(22), 5891 - 5
A combination of three mutations, dcd, pyrH, and cdd, establishes thymidine (Deoxyuridine) auxotrophy in thyA+ strains of Salmonella typhimurium; Krogan NJ et al.; The dum gene of Salmonella typhimurium was originally identified as a gene involved in dUMP synthesis (C . F . Beck et al., J . Bacteriol . 129:305-316, 1977) . In the genetic background used in their selection, the joint acquisition of a dcd (dCTP deaminase) and a dum mutation established a condition of thymidine (deoxyuridine) auxotrophy . In this study, we show that dum is identical to pyrH, the gene encoding UMP kinase . The level of UMP kinase activity in the dum mutant was found to be only 30% of that observed for the dum+ strain . Thymidine prototrophy was restored to the original dum dcd mutant (KP1361) either by transduction using a pyrH+ donor or by complementation with either of two pyrH+-carrying plasmids . Thymidine auxotrophy could be reconstructed in the dum+ derivative (KP1389) by the introduction of a mutant pyrH allele . To define the minimal mutational complement necessary to produce thymidine auxotrophy in thyA+ strains, a dcd::Km null mutation was constructed . In the wild-type background, dcd::Km alone or in combination with a pyrH (dum) mutation did not result in a thymidine requirement . A third mutation, cdd (cytidine-deoxycytidine deaminase), was required together with the dcd and pyrH mutations to impart thymidine auxotrophy.

Zentralbl Bakteriol, 1995 Oct, 282(4), 416 - 26
Immunogenicity and antigenic relationship of Salmonella enterotoxin with other enterotoxins; Rahman H et al.; The immunogenicity and antigenic relationship of Salmonella enterotoxin with other enterotoxins were studied . The purified enterotoxin of Salmonella typhimurium strains was immunogenic in rabbits and antiserum produced against it completely neutralized the enterotoxic activity of purified as well as crude enterotoxins . The Salmonella enterotoxin did not cross-react with cholera toxin, heat-labile enterotoxin of Escherichia coli or Shiga toxin . On the other hand, enterotoxins produced by different serotypes of Salmonella were antigenically related . Heat, pH and proteolytic enzymes had not only adversely affected the enterotoxigenicity but also the antigenicity of the Salmonella enterotoxin . However, formalin was found to completely destroy the enterotoxigenicity of enterotoxin without affecting the antigenicity . Formalin-treated enterotoxin may thus be tried as a vaccine to prevent the diarrhoeal syndrome induced by enterotoxigenic salmonellae.

Eur J Cell Biol, 1998 Sep, 77(1), 35 - 47
Isolation and characterization of Salmonella typhimurium and Yersinia pseudotuberculosis-containing phagosomes from infected mouse macrophages: Y . pseudotuberculosis traffics to terminal lysosomes where they are degraded; Mills SD et al.; The interaction of Salmonella and Yersinia with macrophages is critical to the pathogenesis of these organisms . After internalization into macrophages, these bacteria reside in membrane-enclosed vacuoles . In this report, we present an approach to isolate and characterize bacteria-containing vacuoles (BCVs) to study intracellular trafficking of pathogenic bacteria within the membrane system of host cells . Using the mouse monocyte-macrophage cell line J774A.1, we found that Salmonella typhimurium replicated intracellularly to approximately 5 times its original numbers over a 9 hour infection course, while Yersinia pseudotuberculosis and Escherichia coli did not replicate inside these cells . Analysis of isolated latex bead-containing vacuoles confirmed that they trafficked normally from endosomes to lysosomes within the endocytic pathway of J774A.1 cells . We isolated BCVs free of contaminating endosomes and lysosomes using sucrose step gradients, and used quantitative immunoblotting to characterize the contents of these vacuoles at different time points after internalization . We found that the isolated BCVs contained endosomal and lysosomal marker proteins including lamp-1, mannose 6-phosphate receptor (M 6-PR), cathepsin D and cathepsin L . Further, we report on differential processing of lysosomal hydrolases (such as cathepsin D and cathepsin L) associated with the isolated BCVs . Although there was some contamination of the S . typhimurium-containing vacuoles with endoplasmic reticulum (ER) marker protein calnexin, the Y . pseudotuberculosis-containing vacuoles were predominately free of ER contamination . The Y . pseudotuberculosis-containing vacuoles displayed properties of lysosomes, containing the M 6-PR-dependent lysosomal hydrolases cathepsin D and cathepsin L, which were shown to be processed to their mature forms incrementally over time . These results, coupled with intracellular growth and microscopic examination of infected cells over time, indicated that Y . pseudotuberculosis traffics to lysosomes where they are degraded . The described method for isolation and characterization of BCVs proved to be a valuable tool to characterize the vacuolar compartment occupied by Y . pseudotuberculosis, and has potential to be applied to other vacuole resident pathogens whose trafficking is thought to play a role in pathogenesis.

Carcinogenesis, 1998 Oct, 19(10), 1709 - 13
Rat, but not human, sulfotransferase activates a tamoxifen metabolite to produce DNA adducts and gene mutations in bacteria and mammalian cells in culture; Glatt H et al.; Tamoxifen increases the risk of human endometrial cancer and is a potent carcinogen in rat liver, in which it produces DNA adducts and cytogenetic damage . Nevertheless its prophylactic use against breast cancer in healthy women is under investigation in several large trials . To investigate whether rat hepatocarcinogenicity predicts human hepatocarcinogenicity we used genetically engineered bacterial and mammalian target cells to investigate how alpha-hydroxy-tamoxifen, a major phase I metabolite of tamoxifen, is further metabolised by rat and human phase II enzymes, sulfotransferases, to mutagenic and DNA-adduct-forming species . We expressed rat hydroxysteroid sulfotransferase a, a liver-specific enzyme, and corresponding human sulfotransferase in bacteria (Salmonella typhimurium) and in a mammalian cell line (Chinese hamster V79 cells) and tested alpha-hydroxytamoxifen for DNA adduct formation and mutagenicity in these systems, using unmodified cells as controls . In cells that expressed rat hydroxysteroid sulfotransferase, alpha-hydroxytamoxifen was mutagenic and formed the same pattern of DNA adducts as that found in the liver of tamoxifen-treated rats . Alpha-hydroxytamoxifen was not activated, or was at least 20 times less active in cells expressing human hydroxysteroid sulfotransferase . All the other six known human xenobiotic-metabolising sulfotransferases were also expressed in S . typhimurium . None activated alpha-hydroxytamoxifen to a mutagen . These results suggest that the risk of DNA adduct formation, and cancer, in the human liver is low and explain why tamoxifen is a powerful carcinogen to the rat liver, and why standard short-term tests fail to detect its mutagenicity.

Mutat Res, 1998 Nov 9, 419(1-3), 169 - 79
Antimutagenicity of hydrolyzable tannins from Terminalia chebula in Salmonella typhimurium; Kaur S et al.; A tannin fraction (TC-E) from the dried fruit pulp of Terminalia chebula was obtained by successfully extracting with 95% ethyl alcohol and ethyl acetate . TC-E was subjected to silica gel chromatography which yielded four fractions, viz., TC-EI, TC-EII, TC-EIII and TC-EIV . Thin layer chromatography (TLC) and 13C-NMR revealed that TC-EI was gallic acid (GA) derivative while the other fractions were tannin in nature . TC-E and its fractions were evaluated for their antimutagenic potential against two direct-acting mutagens, 4-nitro-o-phenylenediamine (NPD) and 4-nitroquinoline-N-oxide (4NQNO), and S9-dependent mutagen, 2-aminofluorene (2AF) in TA98 and TA100 strains of Salmonella typhimurium . The study revealed that the extract (TC-E) and its fractions were highly significant against S9-dependent mutagen, 2AF . The effect was found to be more or less corresponding with the nature of the fractions, as the monomeric TC-EI (a GA derivative) was least effective as compared to other fractions which were oligomeric, and the order of their effectiveness as per their IbD50 value being TC-EIV (8.9 micrograms)>TC-EIII (17.8 micrograms)>TC-EII (45 micrograms)>TC-EI (320 micrograms) in TA98; TC-EIV being 40 times more effective than TC-EI in inhibiting his+ revertants . A similar effect was noticed in TA100 too, where TC-EI was the least effective and TC-EII had the maximum effect . A similar result was noticed when the antimutagenicity of GA (a monomeric) was compared with tannic acid (TA, an oligomeric) . However, chebula tannins were found to be partly effective against NPD but not at all effective against 4NQNO .

Mutat Res, 1998 Nov 9, 419(1-3), 13 - 20
Detection of in vivo genotoxicity of haloalkanes and haloalkenes carcinogenic to rodents by the alkaline single cell gel electrophoresis (comet) assay in multiple mouse organs; Sasaki YF et al.; The micronucleus test is widely used to assess in vivo clastogenicity because of its convenience, but it is not appropriate for some carcinogenic chemical classes . Halogenated compounds, for example, are inconsistent micronucleus inducers . We assessed the genotoxicity of 7 haloalkanes and haloalkenes carcinogenic to rodents in 7 mouse organs-stomach, liver, kidney, bladder, lung, brain, and bone marrow-using the alkaline single cell gel electrophoresis (SCG) assay . The carcinogens we studied were 1, 2-dibromo-3-chloropropane (DBCP), 1,3-dichloropropene (mixture of cis and trans) (DCP), 1,2-dibromoethane (EDB), 1,2-dichloroethane (EDC), vinyl bromide, dichloromethane, and carbon tetrachloride; only DBCP induces micronuclei in mouse bone marrow . Except for carbon tetrachloride, halocompounds studied are mutagenic to Salmonella typhimurium . Mice were sacrificed 3 or 24 h after carcinogen administration . DCP and EDC induced DNA damage in all of the organs studied . Vinyl bromide yielded DNA damage in all of the organs except for bone marrow . DBCP induced DNA damage in the stomach, liver, kidney, lung, and bone marrow; EDB in the stomach, liver, kidney, bladder, and lung; and dichloromethane in the liver and lung . Since no deaths, morbidity, clinical signs, organ pathology, or microscopic signs of necrosis were observed, the DNA damage was not attributable to cytotoxicity . On the other hand, the positive response in the liver induced by carbon tetrachloride, which was accompanied by necrosis, was considered to be a false positive response . We suggest that the alkaline SCG assay can be used in multiple organs to detect in vivo genotoxicity that is not expressed in bone marrow cells in mice given non-necrogenic doses of halocompounds .

Vet Rec, 1998 Sep 26, 143(13), 351 - 4
Salmonella typhimurium DT104 infection in people and animals in Scotland: a collaborative epidemiological study 1993-96; Calvert N et al.; This paper describes a comparative analysis of human and farm animal salmonellosis in Scotland between 1993 and 1996, with particular reference to Salmonella typhimurium definitive type 104 (DT104) . Cattle were the main reservoir, accounting for 73.1 per cent of incidents involving all salmonellae and 69.5 per cent of those involving S typhimurium DT104 . The annual rates of incidence in people and cattle were recorded in each Health Board area . Dumfries and Galloway had the highest rate of incidence in cattle for all salmonellae (19.0 per 100,000) but people were affected uniformly across mainland Scotland . However, the rate of incidence of S typhimurium DT104 was highest in Dumfries and Galloway for both people (10.1 per 100,000) and cattle (13.0 per 100,000) . In Dumfries and Galloway, Wigtownshire had the highest rates for all salmonellae and for S typhimurium DT104 in both people and cattle . In Dumfries and Galloway, 37.8 per cent of the adult cases of S typhimurium DT104 in people were among those known to have had regular contact with animals, and children under six years of age accounted for 36.3 per cent of the human infections in this region.

Ocul Immunol Inflamm, 1998 Mar, 6(1), 51 - 6
Topical liposome-encapsulated FK506 for the treatment of endotoxin-induced uveitis; Whitcup SM et al.; PURPOSE: Liposome preparations of FK506 improve the penetration of topically administered drug into the aqueous humor . The purpose of the experiment was to compare topically administered highdose oil-dissolved FK506 (OD-FK506) and low-dose liposome-bound FK506 (LB-506) for the treatment of endotoxin-induced uveitis (EIU) . METHODS: Endotoxin-induced uveitis was produced in female Lewis rats with Salmonella typhimurium endotoxin . Four hours prior to endotoxin injection, one eye received 20 mul eyedrops every four hours containing either high-dose OD-FK506 at 3 mg/ml (N = 20), low-dose LB-FK506 at 0.16 mg/ml (N = 19), prednisolone acetate 1% (N = 20), or empty liposomes (N = 20) . Eyes were enucleated 24 hours after endotoxin injection and inflammatory cells were counted on histologic sections by two masked observers . RESULTS: The mean number of infiltrating inflammatory cells per section +/- S.E.M . was 127.8 +/- 20.1, 76.8 +/- 16.7, 75.0 +/- 19.1, and 3.6 +/- 0.4 for animals treated with empty liposomes, LB-FK506, OD-FK506, and prednisolone acetate, respectively . The difference in inflammation between the empty liposome controls and the LB-FK506- and OD-FK506-treated animals was statistically significant (p = 0.03 and p = 0.02, respectively) . The difference in inflammation between the high-dose OD-FK506- and low-dose LB-FK506-treated animals was not statistically significant (0 = 0.94) . CONCLUSION: In this study, low-dose LB-FK506 and high-dose (OD-FK506) were both effective in inhibiting EIU . Higher concentrations of LB-FK506 are being developed and should augment the therapeutic effect of topical FK506.

J Food Prot, 1998 Oct, 61(10), 1390 - 5
Susceptibility of antibiotic-resistant and antibiotic-sensitive foodborne pathogens to acid anionic sanitizers; Lopes JA; Acid anionic sanitizers for treatment of fruits and vegetables were prepared using ingredients generally recognized as safe by the U.S . Food and Drug Administration or anionic surfactants and organic acid food additives . They met the regulatory definition as sanitizers by showing bactericidal efficacy of 99.999% in 30 s against Staphylococcus aureus ATCC 6538 and Escherichia coli ATCC 11229 . These sanitizers showed a broad spectrum of microbicidal activity against both gram-positive and gram-negative bacteria . Antibiotic-sensitive and resistant strains of Listeria monocytogenes and Salmonella typhimurium were equally susceptible to these sanitizers . The acid anionic sanitizers showed microbicidal efficacy equal to that of hypochlorite against Aeromonas hydrophila, E . coli O157:H7, L . monocytogenes, Pseudomonas aeruginosa, S . typhimurium, and S . aureus . Unlike most other sanitizers, these agents do not covalently react with organic components of food; unlike cationic agents, they do not leave residues . The acid anionic sanitizers are prepared using stable, biodegradable, and nontoxic ingredients . Rapid microbicidal activity and the ease of storage, transportation, and use make these sanitizers an attractive alternative to hypochlorite for sanitizing fruits and vegetables.

J Food Prot, 1998 Oct, 61(10), 1378 - 80
Antibiotic resistance of Salmonella spp . and Listeria spp . isolates from traditionally made fresh sausages in Greece; Abrahim A et al.; Sixty-five samples of traditionally made fresh sausages obtained from retail shops and butcher shops in northern Greece were screened for the presence of Salmonella spp . and Listeria spp . Salmonella spp . were found in 20% of the samples tested (54% Salmonella typhimurium and 46% Salmonella enteritidis) . The prevalence of Listeria spp . in the samples was 26% (12% Listeria monocytogenes, 76% Listeria innocua, and 12% Listeria welshimeri) . Nine of 13 Salmonella isolates were found to be resistant to ampicillin and 4 of 13 showed intermediate sensitivity; 1 of 13 was found to be resistant to chloramphenicol and 1 of 13 to tetracycline . Two strains of Salmonella typhimurum were multiresistant (resistant to ampicillin, chloramphenicol, and norfloxacin) . All Listeria isolates were sensitive to the antibacterial agents tested that are commonly used for the treatment of human listeriosis.

Br J Ophthalmol, 1998 Jun, 82(6), 695 - 9
Interferon gamma immunoreactivity in iris nerve fibres during endotoxin induced uveitis in the rat; Yang P et al.; AIMS: Previous studies have implied that interferon gamma (IFN-gamma) is involved in the pathogenesis of endotoxin induced uveitis (EIU) in the rat . This study investigated the source of IFN-gamma in the iris during EIU . METHODS: Whole mounts of iris were isolated from Lewis rats before and at different times (from 4 hours to 14 days) after foot pad injection of 200 micrograms Salmonella typhimurium lipopolysaccharide (LPS) . Immunohistological analysis was performed using monoclonal antibodies (mAbs) specific to rat IFN-gamma (DB12 and DB13) . mAbs specific to monocytes, macrophages, and dendritic cells and MHC class II were used to asses the inflammatory response in the eye (ED-1, ED-2, and OX-6) . An antibody specific to neurofilaments (2H3) was used to stain nerve fibres in the normal iris . RESULTS: LPS administration induced acute intraocular inflammation, characterised by a massive infiltration of monocytes/macrophages and increased numbers of MHC class II positive cells in the iris . IFN-gamma immunoreactive cells were not detected in iris whole mounts of control rats . Strikingly, IFN-gamma immunoreactivity was found in fibres from 4 hours until 10 days after LPS injection, with the most intense staining at 48-72 hours . Other DB12 or DB13 positive cells were not detected in the iris . The pattern of DB12 and DB13 staining in the inflamed iris was similar to the 2H3 staining of neurons in the iris of control rats . CONCLUSION: These results show that systemic LPS administration induces IFN-gamma immunoreactivity in iris fibres and suggest that iris nerve fibres may be a source of IFN-gamma during EIU . The IFN-gamma immunoreactive material in the iris nerve fibres may be identical to neuronal IFN-gamma.

Antimicrob Agents Chemother, 1998 Nov, 42(11), 2950 - 5
Effects of Salmonella typhimurium infection and ofloxacin treatment on glucose and glutamine metabolism in Caco-2/TC-7 cells; Posho L et al.; The effects of both Salmonella typhimurium infection and 5 mM ofloxacin treatment on 2 mM glutamine and 5 mM glucose metabolism in the enterocyte-like Caco-2/TC-7 cell line were studied . These cells utilized glutamine (212.07 +/- 16.75 {mean +/- standard deviation} nmol per h per 10(6) viable cells) and, to a lesser extent, glucose (139.63 +/- 11.52 nmol per h per 10(6) viable cells) . Metabolism of these substrates in Caco-2/TC-7 cells resembled that in rat, pig, or human enterocytes . Infection by S . typhimurium C53-enhanced glucose and glutamine substrate utilization by 32 and 22%, respectively and enhanced glucose and glutamine substrate oxidation by eight- and twofold, respectively . These increases in glucose and glutamine metabolism (especially glucose metabolism) were due in part to the metabolism of intracellular bacteria and/or to the activation of cellular metabolism . Substrate metabolism (especially glucose metabolism) in C53-infected cells was partially reduced by treatment with ofloxacin . It was concluded that cellular fuel metabolism is stimulated at the earliest stage of infection (3 to 4 h) and that treatment with 5 mM ofloxacin does not completely restore substrate metabolism to the levels observed in uninfected cells, possibly because this treatment does not eradicate intracellular S . typhimurium completely.

J Biol Chem, 1998 Nov 6, 273(45), 29929 - 34
Suppression of leu-500 mutation in topA+ Salmonella typhimurium strains . The promoter relay at work; Fang M et al.; Suppression of leu-500 mutation in Salmonella typhimurium topA- strains has been one of the most fascinating examples for the DNA supercoiling effect on transcription initiation control . Previous studies have indicated possible involvement of transcription-driven DNA supercoiling in the activation of the leu-500 promoter in topA- strains . Our recent studies have shown that ilvIH transcription activity located 1.9 kilobase pairs upstream is the initial supercoiling signal for leu-500 activation via a promoter relay mechanism . In the present communication, we show that the ilvIH transcription activity-initiated promoter relay can result in leu-500 activation in topA+ strains . In addition, suppression of the chromosomal leu-500 mutation correlates with the transcription activities of ilvIH and leuO rather than the TopA level in the topA+ strain . It appears that the leu-500 suppression in a topA- strain is due to the constant ilvIH transcription activity.

Cell Immunol, 1998 Nov 1, 189(2), 116 - 24
Immune responsiveness of lymphotoxin-alpha-deficient mice: two reconstitution models; Davis IA et al.; The effects of lymphotoxin-alpha (LT-alpha) deficiency on mucosal immune status has not been defined . We utilized severe combined immunodeficiency (scid) mice as recipients of both mutant and wild-type whole splenocytes to determine whether lymphocytes from mutant mice had impaired homing ability . We also utilized irradiated mutant mice as recipients of wild-type whole splenocytes to determine whether lymphoid tissue anlages had, indeed, failed to develop as a consequence of LT-alpha deficiency . Subsequently, all mice were immunized orally with an attenuated strain of Salmonella typhimurium and mucosal IgA responses were monitored . The data presented here demonstrate that scid recipients generate mucosal responses equally well when reconstituted with mutant or wild-type lymphocytes . In contrast, reconstitution of mutant mice with wild-type cells failed to affect the efficiency of their mucosal immunity . The mutant phenotype, therefore, appears to involve neither impaired lymphocyte homing nor function in the generation of mucosal immunity . However, the mutant phenotype and immune responsiveness cannot be transformed merely by the provision of LT-alpha-expressing donor cell populations . The consequence of LT-alpha deficiency on mucosal immune responsiveness appears to be due to the lack of gut-associated lymphoid tissues, which may include the spleen, in mutant mice .

Blood, 1998 Nov 1, 92(9), 3172 - 6
Gene transfer in dendritic cells, induced by oral DNA vaccination with Salmonella typhimurium, results in protective immunity against a murine fibrosarcoma; Paglia P et al.; A live attenuated AroA- auxotrophic mutant of Salmonella typhimurium (SL7207) has been used as carrier for the pCMVbeta vector that contains the beta-galactosidase (beta-gal) gene under the control of the immediate early promoter of Cytomegalovirus (CMV) . We tested whether orally administered bacterial carrier could enter and deliver the transgene to antigen-presenting cells (APCs) through the natural enteric route of infection and whether beta-gal expression could generate a protective response against an aggressive murine fibrosarcoma transduced with the beta-gal gene (F1.A11) that behaves operationally as a tumor-associated antigen . After three courses, at 15-day intervals, mice developed both cell-mediated and systemic humoral responses to beta-gal . Mice vaccinated with the Salmonella harboring pCMVbeta, but not with plasmid-less carrier, showed resistance to a challenge with F1.A11 cells . These experiments suggest that Salmonella-based DNA immunization allows us to specifically target antigen expression in vivo to APCs . To prove that the transgene is actually expressed by APCs as a function of an eukaryotic promoter, the green fluorescent protein (GFP) was placed under the control of either the eukariotic CMV or a prokaryotic promoter . Using cytofluorometric analysis, GFP was detected only in splenocytes of mice receiving a Salmonella carrier harboring GFP under the CMV promoter . These results indicate that transgene expression occurs because of a Salmonella-mediated gene transfer to eukaryotic cells . Finally, approximately 19% of the splenocytes expressed GFP . Among them, F4/80(+) macrophages and CD11cbright dendritic cells (DCs) were scored as positive for GFP expression . Extensive work has been performed trying to optimize the way to transfect DCs, ex vivo, with genes coding for relevant antigens . We show here, for the first time, that DCs can be directly and specifically transduced in vivo such to induce DNA vaccination against tumors .

J Biol Chem, 1998 Oct 30, 273(44), 28663 - 9
The CorA Mg2+ transport protein of Salmonella typhimurium . Mutagenesis of conserved residues in the third membrane domain identifies a Mg2+ pore; Smith RL et al.; The CorA transport system is the major Mg2+ influx pathway for bacteria and the Archaea . CorA contains three C-terminal transmembrane segments . No conserved charged residues are apparent within the membrane, suggesting that Mg2+ influx does not involve electrostatic interactions . We have mutated conserved residues within the third transmembrane segment to identify sites involved in transport . Mutation of conserved aromatic residues at either end of the membrane segment to alternative aromatic amino acids did not affect total cation uptake or cation affinity . Mutation to alanine greatly diminished uptake with little change in cation affinity implying that the conserved aromatic residues play a structural role in stabilizing this membrane segment of CorA at the interface between the bilayer and the aqueous environment . In contrast, mutation of Tyr292, Met299, and Tyr307 greatly altered the transport properties of CorA . Y292F, Y292S, Y292C, or Y292I mutations essentially abolished transport, without effect on expression or membrane insertion . M299C and M299A mutants exhibited a decrease in cation affinity for Mg2+, Co2+, or Ni2+ of 10-50-fold without a significant change in uptake capacity . Mutations at Tyr307 had no significant effect on cation uptake capacity; however, the affinity of Y307F and Y307A mutations for Mg2+ and Co2+ was decreased 3-10-fold, while affinity for Ni2+ was unchanged compared with the wild type CorA . In contrast, the affinity of the Y307S mutant for all three cations was decreased 2-5-fold . Projection of the third transmembrane segment as an alpha-helix suggests that Tyr292, Met299, and Tyr307 all reside on the same face of the alpha-helix . We interpret the transport data to suggest that a hydroxyl group is important at Tyr307, and that these three residues interact with Mg2+ during transport, forming part of the cation pore or channel within CorA.

J Antimicrob Chemother, 1998 Sep, 42(3), 341 - 7
Escherichia coli O157 interactions with human intestinal Caco-2 cells and the influence of fosfomycin; Izumikawa K et al.; It is not clear how Escherichia coli O157 invades human enteric epithelium and causes the haemolytic uraemic syndrome (HUS), and nor has the most appropriate treatment of E . coli O157 infection been established . Verotoxins, leucocytes and proinflammatory cytokines, such as tumour necrosis factor-alpha (TNF-alpha), interleukin (IL)-6 and IL-8, are considered essential for the development of HUS . We used the Caco-2 cell monolayer system, well-known as an in-vitro model of human intestinal infection, to determine how E . coli O157 interacts with intestinal epithelial cells and also studied the influence of fosfomycin on the virulence of the bacteria . Results showed that the E . coli O157 used in this study did not penetrate the Caco-2 cell monolayer system, unlike Salmonella typhimurium SL1344, and verotoxin 1 (VT 1), but not VT 2, translocated across the system . In an in-vitro conventional assay, fosfomycin increased the amount of verotoxins but it did not influence penetration of bacteria and translocation of verotoxins in the Caco-2 cell monolayer system . The production of both IL-8 (a potent neutrophil activator) and TNF-alpha in the human monocytic THP-1 cell line was reduced by fosfomycin-treated basolateral medium in this system . These results indicate that fosfomycin may be a potent drug for preventing HUS caused by E . coli O157 infection.

Mol Microbiol, 1998 Oct, 30(1), 91 - 105
In vivo analysis of the interactions of the LysR-like regulator SpvR with the operator sequences of the spvA and spvR virulence genes of Salmonella typhimurium; Sheehan BJ et al.; The interaction of the Salmonella typhimurium virulence gene regulator, SpvR, with its operator sites upstream of the spvA and spvR genes was analysed in vivo by dimethyl sulphate (DMS) footprinting and site-directed mutagenesis . DMS methylation protection assays showed that, in vivo, SpvR forms direct protein-DNA contacts with nucleotides clustered in two regions (+1 to -27 and -51 to -71) of the spvA regulatory region . These regions were subjected to site-directed mutagenesis and the effects on SpvR binding and gene activation assessed . Mutations that prevented occupancy of the promoter distal site (-51 to -71) in vivo also prevented occupancy of the promoter proximal site (+1 to -27), whereas mutations in the proximal site affected binding only at the proximal site and not the distal site . SpvR binding at the promoter proximal site was an essential prerequisite for transcription activation . These findings demonstrated a hierarchy of SpvR binding in which the promoter distal site is dominant to the proximal . The spvR gene was found to possess an operator site that resembled closely the distal SpvR binding site of the spvA operator . Nonetheless, SpvR interaction with the spvR operator was difficult to detect in vivo . When the nucleotide sequence of the spvR operator was altered at two nucleotides so that it corresponded more precisely to that of the distal site of the spvA operator, strong SpvR-DNA interactions were detected, with nucleotides in the region -31 to -67 being protected from DMS methylation in vivo . However, despite the improved interaction with the transcriptional activator, the altered regulatory region was poorer at promoting spvR gene transcription than the wild type . We describe a two-step model for activation of the spvA promoter and discuss the possibility that a specific cofactor in addition to sigma factor RpoS is required for SpvR action at this promoter in vivo.

Mol Microbiol, 1998 Oct, 30(1), 47 - 56
Salmonella InvG forms a ring-like multimer that requires the InvH lipoprotein for outer membrane localization; Crago AM et al.; Salmonella species translocate virulence effector proteins from the bacterial cytoplasm into mammalian host cells by means of a type III secretion apparatus, encoded by the pathogenicity island-1 (SPI-1) . Little is known about the assembly and structure of this secretion apparatus, but the InvG protein is essential and could be an outer membrane secretion channel for the effector proteins . We observed that in recombinant Escherichia coli, the yield of InvG was enhanced by co-expression of InvH, and showed that mutation of invH decreased the level of InvG in wild-type Salmonella typhimurium . In E . coli, InvG alone was able to form an SDS-resistant multimer, but InvG localization to the outer membrane was dependent upon InvH, a lipoprotein itself located in the outer membrane, and no other SPI-1 specific protein . InvG targeted to the outer membrane by InvH became accessible to extracellular protease . InvG and InvH did not, however, appear to form a stable complex . Electron microscopy of InvG membrane protein purified from E . coli revealed that it forms an oligomeric ring-like structure with inner and outer diameters, 7 nm and 15 nm respectively.

Mol Microbiol, 1998 Oct, 30(1), 1 - 6
Temperature sensing in bacterial gene regulation--what it all boils down to; Hurme R et al.; Many bacterial gene regulatory circuits are controlled by temperature . Temperature-mediated regulation occurs at the level of transcription and translation . Supercoiling, changes in mRNA conformation and protein conformation are all implicated in thermosensing . Bacterial virulence functions are often temperature regulated and thus many an example of thermoregulation comes from pathogenic organisms . H-NS is at the crossroads of regulation in many such systems . mRNA melting has also been shown to act as a thermosensing mechanism in various contexts . Proteins can also act as temperature sensors as exemplified by the gene regulator TlpA in Salmonella typhimurium.

Infect Immun, 1998 Nov, 66(11), 5470 - 6
Oral immunization with a Salmonella typhimurium vaccine vector expressing recombinant enterotoxigenic Escherichia coli K99 fimbriae elicits elevated antibody titers for protective immunity; Ascon MA et al.; Bovine enterotoxigenic Escherichia coli (ETEC) continues to cause mortality in piglets and newborn calves . In an effort to develop a safe and effective vaccine for the prevention of F5(+) ETEC infections, a balanced lethal asd+ plasmid carrying the complete K99 operon was constructed and designated pMAK99-asd+ . Introduction of this plasmid into an attenuated Salmonella typhimurium Deltaaro Deltaasd strain, H683, resulted in strain AP112, which stably expresses E . coli K99 fimbriae . A single oral immunization of BALB/c and CD-1 mice with strain AP112 elicited significant mucosal immunoglobulin A (IgA) titers that remained elevated for >11 weeks . IgA and IgG responses in serum specific for K99 fimbriae were also induced, with a prominent IgG1, as well as IgG2a and IgG2b, titer . To assess the derivation of these antibodies, a K99 isotype-specific B-cell ELISPOT analysis was conducted by using mononuclear cells from the lamina propria of the small intestines (LP), Peyer's patches (PP), and spleens of vaccinated and control BALB/c mice . This analysis revealed elevated numbers of K99 fimbria-specific IgA-producing cells in the LP, PP, and spleen, whereas elevated K99 fimbria-specific IgG-producing cells were detected only in the PP and spleen . These antibodies were important for protective immunity . One-day-old neonates from dams orally immunized with AP112 were provided passive protection against oral challenge with wild-type ETEC, in contrast to challenged neonates from unvaccinated dams or from dams vaccinated with a control Salmonella vector . These results confirm that oral Salmonella vaccine vectors effectively deliver K99 fimbriae to mucosal inductive sites for sustained elevation of IgA and IgG antibodies and for eliciting protective immunity.

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