Salmonella typhimurium

Biochim Biophys Acta, 1996 Feb 7, 1305(1-2), 71 - 8
Opposing effects of nitroxide free radicals in Escherichia coli mutants deficient in DNA repair; Wang G et al.; Nitroxide free radicals have been previously shown to function as superoxide dismutase (SOD) mimics and to protect bacterial and mammalian cells against oxidative damage, particularly from superoxide and hydrogen peroxide . Although nitroxides are generally considered to be non-toxic nor mutagenic, there is no agreement regarding their potential adverse effect . Some toxic effects were observed upon using high concentration of six-membered ring derivatives . Conflicting evidence has also been reported regarding the mutagenic activity of nitroxides toward Salmonella typhimurium . It was also demonstrated that nitroxides exert two opposing effects on exonuclease III deficient cells of Escherichia coli upon exposure to naphthoquinones . The attempts to use nitroxides as contrast agents in nuclear magnetic resonance imaging (MRI) and as a new class of anti-oxidants underscore the need to examine their potential adverse effects . Since nitroxides protected xthA cells from DNA scission caused by H2O2, it was anticipated that they would provide even greater protection for recA DNA repair-deficient cells of E . coli, which are more sensitive to H2O2-induced oxidative stress . The results of the present study showed that: (a) nitroxides exert bactericidal and bacteriostatic effects on recA but not on xthA or wild-type E . coli K12 cells; (b) nitroxides and H2O2 act synergistically on recA cells, both under aerobic and hypoxic conditions; (c) the nitroxide-induced toxicity in recA cells and the synergistic effect with H2O2 were not accompanied by a decrease in the cellular level of reduced glutathione; (d) TEMPAMINE protected against DNA scission induced by H2O2 and 1,10-ortho-phenanthroline chelate of Cu(II) in xthA cells, but potentiated DNA double-strand breakage in recA cells.

Mol Gen Genet, 1996 Feb 5, 250(2), 197 - 206
Molecular analysis of the scrA and scrB genes from Klebsiella pneumoniae and plasmid pUR400, which encode the sucrose transport protein Enzyme II Scr of the phosphotransferase system and a sucrose-6-phosphate invertase; Titgemeyer F et al.; The Klebsiella pneumoniae genes scrA and scrB are indispensable for sucrose (Scr) utilisation . Gene scrA codes for an Enzyme IIScr (IIScr) transport protein of the phosphoenolpyruvate-dependent carbohydrate: phosphotransferase system (PTS), while scrB encodes a sucrose 6-phosphate specific invertase . A 3.7 kbscr AB DNA fragment has been cloned from K . pneumoniae and expressed in Escherichia coli . Its nucleotide sequence was determined and the coding regions for scrA (1371 bp) and scrB (1401 bp) were identified by genetic complementation, enzyme activity test and radiolabelling of the gene products . In addition, the nucleotide sequence of the scrB gene from conjugative plasmid pUR400 isolated from Salmonella typhimurium was also determined and errors in the previously published sequence of the scrA gene of pUR400 were corrected . Extensive similarity was found between the sequences of ScrA and other Enzymes II, as well as between the two invertases and other sucrose hydrolysing enzymes . Based on the analysis of seven IIScr proteins, a hypothetical model of the secondary structure of IIScr is proposed.

J Biol Chem, 1996 Feb 2, 271(5), 2448 - 54
A novel human serum lectin with collagen- and fibrinogen-like domains that functions as an opsonin; Matsushita M et al.; Collectins are C-type animal lectins with both collagenous and carbohydrate recognition domains and are involved in the first line host defense against pathogens . We report here a novel Ca(2+)-dependent and GlcNAc-binding lectin consisting of subunits of 35 kDa (P35) with a collagen-like sequence . When P35 is isolated from human serum, it forms a homopolymer by means of intermolecular disulfide bonding, as is the case with collectins . P35 cDNA was cloned from a human liver cDNA library, and the deduced amino acid sequence of 313 residues revealed that the mature form of P35 consists mainly of collagen- and fibrinogen-like domains . The latter contained two potential Ca(2+)-binding sites that may be involved in carbohydrate binding . The overall sequence of P35 was highly homologous to porcine ficolins alpha and beta . Northern blots of various human tissues showed that the major product of the 1.3-kilobase-long P35 transcript is expressed in liver . P35 enhanced phagocytosis of Salmonella typhimurium by neutrophils, suggesting an opsonic effect via the collagen region . P35 was found to bind to GlcNAc-conjugated bovine serum albumin, a neoglycoprotein, as well as to neoglycolipids containing complex-type oligosaccharides derived from glycoproteins, suggesting that P35 recognizes GlcNAc residues such as those found in microbial glycoconjugates and complex-type oligosaccharides . Therefore, P35 represents a new type of GlcNAc-binding lectin with structural and functional similarities to collectins involved in innate immunity.

Biosci Biotechnol Biochem, 1996 Feb, 60(2), 328 - 9
Antimutagenic activity of caffeic acid and related compounds; Yamada J et al.; Effects of caffeic acid and chlorogenic acid on mutagenicity were studied using the Salmonella typhimurium system . These compounds had inhibitory effects on the mutagenicity of Trp-P-1 and Glu-P-2 . Caffeic acid completely eliminated the mutagenicity induced by activated Glu-P-2 . Some compounds analogous to caffeic acid, such as cinnamic acid, coumaric acid, and ferulic acid, also significantly decreased the mutagenicity of Glu-P-2.

Vet Microbiol, 1996 Feb, 48(3-4), 293 - 303
Time course of the serological response to Yersinia enterocolitica O:3 in experimentally infected pigs; Nielsen B et al.; A total of 25 pigs inoculated with Yersinia enterocolitica serovar O:3 and 25 un-inoculated controls were followed weekly by sampling blood and faeces for 70 days post infection (p.i.) . All inoculated pigs were faeces culture positive from day 5 to 21 p.i., whereafter shedding of bacteria declined to < 10% of the pigs at day 49 p.i . and to 0% at day 68 p.i . All control pigs remained Y . enterocolitica O:3 culture negative . When examined in an indirect ELISA using purified LPS from Y . enterocolitica 0:3, sera from all inoculated pigs showed significantly higher optical densities (OD) as compared to the control group . All inoculated pigs had seroconverted at day 19 p.i . and remained seropositive until slaughter at day 70 p.i . The maximum mean anti-LPS response was observed at day 33 p.i . with a positive/negative ratio of 780 . No cross-reactions were observed with sera from 21 pigs, infected with Salmonella typhimurium . At necropsy at day 70 p.i., Y . enterocolitica O:3 was isolated from the tonsils of 20 inoculated pigs, whereas the rest of the gastrointestinal tract and associated lymph nodes were culture negative . The remaining inoculated pigs and all control pigs were culture negative at necropsy at day 70 p.i . The ELISA seems to be a promising alternative to bacteriological culture for detection of Y . enterocolitica O:3 infection in pig herds.

Vet Microbiol, 1996 Feb, 48(3-4), 257 - 68
Enzyme-linked immunosorbent assay and immunoblot analysis for detection of antibodies to Borrelia burgdorferi in dogs . The impact of serum absorption with homologous and heterologous bacteriae; Wittenbrink MM et al.; Sera from 665 apparently healthy dogs were examined for antibodies to the Lyme disease spirochete Borrelia (B.) burgdorferi by using an ELISA with a whole cell sonicate of B . burgdorferi sensu stricto reference strain B31 (ATCC 35210) as antigen . To discover false positive reactions due to the unsatisfactory specificity of conventional enzyme-linked immunosorbent assays for B . burgdorferi, sera were absorbed in parallel with both B . burgdorferi and a heterologous sorbent consisting of whole cells of Escherichia coli, Salmonella typhimurium, and eight serovars of Leptospira interrogans . The difference between optical densities obtained in the ELISA after serum absorption with the heterologous sorbent and the B . burgdorferi sorbent was defined as a new value, "ODdiff", for ELISA reactivity specific for B . burgdorferi . ELISA results were confirmed by immunoblot studies . By testing unabsorbed sera, 48 of 665 serum samples (7.2%) were considered ELISA positive . 37 of these 48 sera (77.1%) were apparently false positive: here a similar reduction of ELISA reactivity was obtained after absorption with B . burgdorferi antigen and with the heterologous sorbent (ODdiff approximately equal to 0) . None of these 37 sera gave immunoblot patterns characteristic for canine B . burgdorferi infection.

Microbiology, 1996 Feb, 142 ( Pt 2), 217 - 30
Catabolite repression and inducer control in Gram-positive bacteria; Saier MH Jr et al.; Results currently available clearly indicate that the metabolite-activated protein kinase-mediated phosphorylation of Ser-46 in HPr plays a key role in catabolite repression and the control of inducer levels in low-GC Gram-positive bacteria . This protein kinase is not found in enteric bacteria such as E . coli and Salmonella typhimurium where an entirely different PTS-mediated regulatory mechanism is responsible for catabolite repression and inducer concentration control . In Table 2 these two mechanistically dissimilar but functionally related processes are compared (Saier et al., 1995b) . In Gram-negative enteric bacteria, an external sugar is sensed by the sugar-recognition constituent of an Enzyme II complex of the PTS (IIC), and a dephosphorylating signal is transmitted via the Enzyme IIB/HPr proteins to the central regulatory protein, IIAGlc . Targets regulated include (1) permeases specific for lactose, maltose, melibiose and raffinose, (2) catabolic enzymes such as glycerol kinase that generate cytoplasmic inducers, and (3) the cAMP biosynthetic enzyme, adenylate cyclase that mediates catabolite repression (Saier, 1989, 1993) . In low-GC Gram-positive bacteria, cytoplasmic phosphorylated sugar metabolites are sensed by the HPr kinase which is allostericlaly activated . HPr becomes phosphorylated on Ser-46, and this phosphorylated derivative regulates the activities of its target proteins . These targets include (1) the PTS, (2) non-PTS permeases (both of which are inhibited) and (3) a cytoplasmic sugar-P phosphatase which is activated to reduce cytoplasmic inducer levels . Other important targets of HPr(ser-P) action are (4) the CcpA protein and probably (5) the CepB transcription factor . These two proteins together are believed to determine the intensity of catabolite repression . Their relative importance depends on physiological conditions . Both proteins may respond to the cytoplasmic concentration of HPr(ser-P) and appropriate metabolites . CepA possibly binds sugar metabolites such as FBP as well as HPr(ser-P) . Because HPr(his-P, ser-P) does not bind to CepA, the regulatory cascade is also sensitive to the external PTS sugar concentration . Mutational analyses (unpublished results) suggest that CepA may bind to a site that includes His-15 . Interestingly, both the CepA protein in the Gram-positive bacterium, B . subtilis, and glycerol kinase in the Gram-negative bacterium, E . coli, sense both a PTS protein and a cytoplasmic metabolic intermediate . The same may be true of target permeases and enzymes in both types of organisms, but this possibility has not yet been tested . The parallels between the Gram-negative and Gram-positive bacterial regulatory systems are superficial at the mechanistic level but fundamental at the functional level . Thus, the PTS participates in regulation in both cases, and phosphorylation of its protein constituents plays key roles . However, the stimuli sensed, the transmission mechanisms, the central PTS regulatory proteins that effect allosteric regulation, and some of the target proteins are completely different . It seems clear that these two transmission mechanisms evolved independently . They provide a prime example of functional convergence.

Mol Microbiol, 1996 Feb, 19(3), 505 - 9
Multicopy single-stranded DNA of Escherichia coli enhances mutation and recombination frequencies by titrating MutS protein; Maas WK et al.; Multicopy single-stranded DNA (msDNA) molecules consist of single-stranded DNA covalently linked to RNA . In Escherichia coli, such molecules are encoded by genetic elements called retrons . The DNA moieties of msDNAs have characteristic stem-loop structures, and most of these structures contain mismatched base pairs . Previously, we showed that retrons encoding msDNAs with mismatched base pairs are mutagenic when present in multicopy plasmids . In this study we show that such msDNAs, in a similar manner to genetic defects in mismatch repair, increase the frequency of interspecies recombination in matings between Salmonella typhimurium and E . coli . To demonstrate interference with mismatch repair by msDNA, we show that the addition of a plasmid containing the gene for MutS protein suppresses the mutagenic and recombinogenic effects of msDNAs . We also show that in mutS mutants, msDNA does not increase the frequency of either mutations or interspecies recombination . We conclude from these findings that the mutagenic and recombinogenic effects of msDNAs are due to titrating out MutS protein.

Mol Microbiol, 1996 Feb, 19(4), 791 - 801
A Salmonella typhimurium htrA live vaccine expressing multiple copies of a peptide comprising amino acids 8-23 of herpes simplex virus glycoprotein D as a genetic fusion to tetanus toxin fragment C protects mice from herpes simplex virus infection; Chabalgoity JA et al.; Multiple tandem copies of an immunogenic epitope comprising amino acids 8-23 of glycoprotein D of herpes simplex virus (HSV) were expressed as C-terminal fusions to tetanus toxin fragment C (TetC) in different Salmonella typhimurium live vaccine strains . Expression of the longer fusions was best in strains harbouring a lesion in htrA, a stress protein gene . SL3261, an aroA strain, did not effectively express the longer fusions . Mice immunised with an S . typhimurium C5 htrA mutant expressing fusions with two or four copies of the peptide made an antibody response to both the peptide and TetC, whereas constructs expressing one copy of the peptide only elicited antibody to TetC . A non-immunogenic octameric fusion underwent rearrangements in vivo resulting in a predominantly monomeric fusion . In contrast, the S . typhimurium SL3261 aroA vaccine expressing the TetC-tetrameric fusion did not elicit antibody to the peptide . Sera from mice immunised with a single dose of the dimer and tetramer fusions in the htrA strain neutralised HSV in vitro, and the mice were protected from HSV infection as measured by a reduction in virus load in the ear pinna . We have previously shown that mice vaccinated with salmonella expressing TetC are protected against tetanus toxin and virulent salmonella challenge . These results suggest that it may be possible to develop a multivalent vaccine against salmonellosis, tetanus and HSV.

Mol Microbiol, 1996 Feb, 19(4), 777 - 90
Promoter identification and expression analysis of Salmonella typhimurium and Escherichia coli nrdEF operons encoding one of two class I ribonucleotide reductases present in both bacteria; Jordan A et al.; Salmonella typhimurium and Escherichia coli cells have two different class I ribonucleotide reductases encoded by the nrdEF and nrdAB operons . Despite the presence of one additional ribonucleotide reductase, the nrdAB-encoded enzyme is essential to the aerobic growth of the cell because nrdAB-defective mutants of both species are not viable in the presence of oxygen . Several factors controlling nrdAB gene transcription have been analysed intensively . Nothing is known about the expression of the nrdEF genes . To study this subject, and after cloning of E . coli nrdEF genes and sequencing of their 5' ends, the promoter of this operon has been identified by primer extension in both bacterial species . The +1 position was 691 bp and 692 bp upstream of the translational start points of the nrdE genes of S . typhimurium and E . coli, respectively . Downstream of the +1 position, and before the nrdE gene, two open reading frames (ORFs) of 81 and 136 amino acid residues are present in both bacteria . The synthesis of a polypeptide with a molecular mass of 9 kDa, corresponding to the first of these two ORFs, was observed by using the T7 RNA polymerase expression system . Comparison of the amino acid predicted sequence of this ORF reveals a significant similarity with glutaredoxin proteins . Competitive, reverse-transcription polymerase chain reaction experiments indicate that transcription from the nrdEF promoter normally takes place in wild-type cells . nrdEF transcription is increased by hydroxyurea, which inhibits class I ribonucleotide reductase activity, in both RecA+ and RecA- cells . nrdA(ts) mutants show a higher level of nrdEF transcription than wild-type cells at either the permissive or the restrictive temperature . nrdEF expression was unaffected by changes in DNA supercoiling whether caused by the introduction of either topA::Tn10 and hns::Tn10 mutations or by the inhibition of DNA gyrase with the antibiotic novobiocin . In contrast to the nrdAB genes, the nrdEF operon is not essential to the cells because nrdEF-defective mutants are viable under both aerobic and anaerobic conditions.

FEMS Immunol Med Microbiol, 1996 Feb, 13(2), 155 - 60
Purification of a product from Salmonella typhimurium with the ability to inhibit mitogen-induced proliferation of murine splenic T-lymphocytes; Matsui K; We attempted to purify a substance that inhibits mitogen-induced proliferation of murine splenic T-lymphocytes from Salmonella typhimurium . The soluble fraction of a suspension of bacteria disrupted by sonication was chromatographed serially on Mono Q HR, Superdex 200 HR and HiLoad Superdex 75 p.g . columns . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the purified active substance migrated as a single band corresponding to a molecular mass of 87 kDa . We designated the purified substance S . typhimurium-derived inhibitor of T-cell proliferation (STI), which, at 0.2 microgram/ml and above, inhibited proliferation and augmented CD25 expression of phytohemagglutinin-stimulated murine splenic lymphocytes . These findings suggested that the immunosuppression induced by Salmonella infection may be attributable to STI.

Genetika, 1996 Feb, 32(2), 233 - 9
{Genotoxic and mutagenic activity of antineoplastic anthracyclines and their aglycones: study in two test-systems}; Vasil'eva SV et al.; Mutagenic (Ames tests) and genotoxic (SOS chromotest) activities of highly-efficient natural anthracycline monosaccharides possessing antitumor activity-daunorubicin (also known as daunomycin or rubomycin), doxorubicin (adriamycin), and carminomycin-were studied . At the same time, the hypothesis was tested that intercalation of the antibiotic moiety into the helix of cell DNA, which was mediated by the saccharide amino group, played a crucial role in genotoxicity of these anthracyclines . The hydrolysis products of these antibiotics (the corresponding aglycones) and aclacynomycin A (an anthracycline trisaccharide), as well as aclavinone (its derivative aglycone), were studied . All these compounds lacked the saccharide amino group necessary for intercalation . It was found that all anthracycline monosaccharides studied had a strong mutagenic effect on strain TA98 and a moderate effect on strain TA100 of Salmonella typhimurium . Aclacynomycin A was found to have no mutagenic effect on any strain . Lack of the glycoside amino group did not necessarily result in loss of mutagenic activity in the derivative aglycones of anthracycline monosaccharides: they exhibited moderate mutagenic activity in strain TA98 and low but significant activity in strain TA100 . The S9 microsomal fraction did not alter the mutagenic activity of either anthracycline monosaccharides or their aglycones; however, it dramatically increased the mutagenic activity of aclavinone: correspondence between positive responses in Ames tests and the SOS chromotest was found . Apparently, the mutagenic activity of the substances studied in bacterial cells was mediated by inducing the SOS-repair process . If the compound contained the amino glycoside moiety, functional and structural precursors of the SOS response were formed via intercalation of the reagents into the DNA duplex; if the substance did not contain this moiety, the precursors were formed via ionic interaction.

J Antimicrob Chemother, 1996 Feb, 37(2), 351 - 6
Mutations of the gyrA gene of clinical isolates of Salmonella typhimurium and three other Salmonella species leading to decreased susceptibilities to 4-quinolone drugs; Brown JC et al.; The region of the gyrA gene encoding nucleotides 72 to 557 in ten Salmonella typhimurium clinical isolates received from Vellore, India and six British clinical isolates of a number of Salmonella species has been sequenced . Those exhibiting a decreased susceptibility to 4-quinolone drugs (MICs of ciprofloxacin ranging from 0.128 mg/L to 1.0 mg/L) have been shown to possess a number of different mutations in this region of the gene (Asp-87 to Gly, Asp-87 to Asn and Ser-83 to Phe) . The mutations Asp-87 to Gly and Ser-83 to Phe/Asp-87 to Asn have yet to be reported occurring in S . typhimurium.

J Antimicrob Chemother, 1996 Feb, 37(2), 253 - 63
The effect of antibiotics on bacteria under hyperbaric conditions; Hind J et al.; The sensitivity of selected bacteria to a range of antibiotics was tested under hyperbaric conditions used in saturation diving . The effect of hyperbaric helium and oxygen (heliox) on antibiotic stability and on induction of beta-lactamase was also determined . Increased resistance to penicillin (up to 23%) was shown by Staphylococcus aureus and to gentamicin (up to 46%) and rifampicin (up to 18%) by Escherichia coli and Salmonella typhimurium at 36 and 71 bar pressure . Exposure to 71 bar heliox did not affect antibiotic activity but increased the production of beta-lactamase in inducible S . aureus and Bacillus subtilis and production of beta-galactosidase in inducible E . coli . Increased resistance to antibiotics in saturation diving conditions can be attributed in some cases to the influence of hyperbaric pressure on induction mechanisms in bacteria . The experimental system devised for this work is suitable for more detailed examination of the influence of hyperbaric stress on antibiotic resistance and of its effect on induction mechanisms in general.

Yeast, 1996 Feb, 12(2), 149 - 67
Analysis of a 36.2 kb DNA sequence including the right telomere of chromosome VI from Saccharomyces cerevisiae; Eki T et al.; The nucleotide sequence of a 36.2-kb distal region containing the right telomere of chromosome VI was determined . Both strands of DNA cloned into cosmid clone 9965 and plasmid clone pEL174P2 were sequenced with an average redundancy of 7.9 per base pair, by both dye primer and dye terminator cycle sequencing methods . The G+C content of the sequence was found to be 37.9% . Eighteen open reading frames (ORFs) longer than 100 amino acids were detected . Four of these ORFs (9965orfR017, 9965orfF016, 9965orfR009 and 9965orfF003) were found to encode previously identified genes (YMR31, PRE4, NIN1 and HXK1, respectively) . Six ORFs (9965orfR013, 9965orfF018, 9965orfF006, 9965orfR014, 9965orfF013 and 9965orfR020) were found to be homologous to hypothetical 121.4-kDa protein in the BCK 5' region, Bacillus subtilis DnaJ protein, hypothetical Trp-Asp repeats containing protein in DBP3-MRPL27, putative mitochondrial carrier YBR291C protein, Salmonella typhimurium nicotinate-nucleotide pyrophosphorylase, and Escherichia coli cystathionine beta-lyase, respectively . The putative proteins encoded by 9965orfF018, 9965orfR014 and 9965orfR020 were found to be, respectively, a new member of the family of DnaJ-like proteins, the mitochondrial carrier protein and cystathionine lyase.

Mutat Res, 1996 Feb, 367(2), 83 - 92
Inhibition of mutagenicity of N-nitrosamines by tobacco smoke and its constituents; Lee CK et al.; Tobacco smoke is a complex chemical mixture including pyridine alkaloids and N-nitrosamines, with the concentration of the former several orders of magnitude higher that that of the N-nitrosamines . The major biologically important N-nitrosamines present in tobacco smoke are N-nitrosodimethylamine (NDMA), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N(1)-nitrosonornicotine (NNN) . These nitrosamines require metabolic activations by cytochrome P-450s for the expression of mutagenicity . Although nicotine, the major pyridine alkaloid in tobacco, has been shown to inhibit the metabolic activation of NNK, its effect on the mutagenicity of NNK and other N-nitrosamines has not been reported, In the present study, the ability of three pyridine alkaloids (nicotine, cotinine, nornicotine) and aqueous cigarette smoke condensate extract (ACE) to inhibit the mutagenicity of tobacco-related N-nitrosamines was tested on Salmonella typhimurium strain TA1535 in the presence of a metabolic activation system (S9) . All three of the pyridine alkaloids tested, as well as ACE, inhibited the mutagenicity of NDMA and NNK, but not NNN, in a concentration-dependent manner . The induction of SCEs in mammalian cells (CHO) by NNK in the presence of metabolic activation was also significantly reduced by nicotine and cotinine . None of the observed reductions in mutagenicity could be explained by cytotoxicity . These results demonstrate that tobacco smoke contains chemicals, pyridine alkaloids and other unidentified constituent(s), which inhibit the mutagenicity of N-nitrosamines.

Indian J Exp Biol, 1996 Feb, 34(2), 98 - 102
Antimutagenic effects of polyphenols isolated from Terminalia bellerica myroblan in Salmonella typhimurium; Padam SK et al.; Antimutagenic effect of 2 polyphenolic fractions isolated from T . bellerica in TA98 and TA100 strains of S . typhimurium against 2AF, NPD and 4NQNO has been characterized . Both the fractions were significantly effective against S9-dependent 2AF; less effective against NPD and almost not effective against 4NQNO in TA100 strain . Using 13C-NMR spectral analysis, the TB-3 fraction, which was significantly more effective against 2AF compared to TB-4, was found to be a mixture of 3 tannins while TB-4 was non-tannin fraction . Interaction of polyphenols with S9 proteins may be the probable cause of inhibitory effect of these polyphenols, though the possibility of other mechanisms cannot be ruled out.

DNA Cell Biol, 1996 Feb, 15(2), 113 - 23
Structural organization, sequence, and expression of the chicken NRAMP1 gene encoding the natural resistance-associated macrophage protein 1; Hu J et al.; One of the most common causes of food poisoning in humans is salmonellosis, which is frequently caused by ingestion with Salmonella-contaminated poultry products . Several lines of evidence suggest that genetic factors control resistance and susceptibility of chickens to infection with Salmonellae . In the mouse, innate resistance to infection with intracellular pathogens such as Salmonella typhimurium, several species of Mycobacteria, and Leishmania donovani is controlled by the mouse chromosome 1 Nramp1Bcg gene . To investigate the role of NRAMP1 in the differential resistance and susceptibility of chickens to infections with S . typhimurium, we have cloned and characterized cDNA clones corresponding to the chicken NRAMP1 gene . Nucleotide and predicted amino acid sequence analyses indicate that the chicken NRAMP1 polypeptide encodes a 555-amino-acid residue membrane protein with 12 putative transmembrane domains, two N-linked glycosylation sites, and an evolutionary conserved consensus transport motif . The peptide sequence identity among chicken, mouse, and human NRAMP1 is 68% . The chicken NRAMP1 gene contains 15 exons and spans 5 kb of genomic DNA . One major and two minor transcription initiation sites were detected using primer extension . Nucleotide sequencing of the promoter region revealed the presence of a classical TATAA element and consensus sequences for binding the myeloid specific PU.1 factor and several lipopolysaccharide (LPS) (NF-IL6 and NF-kappa B) and interferon-gamma (IFN-gamma)-inducible response elements . Similar regulatory elements are found in the promoters of mouse and human NRAMP1 . Northern blot analyses revealed NRAMP1 expression in reticuloendothelial organs (spleen and liver), lung, and thymus . As demonstrated in mice and humans, the macrophage is also a major site of NRAMP1 mRNA expression in chickens . However, the high levels of expression detected in chicken thymus contrast with the absence of expression of the mammalian Nramp1 gene in this tissue.

Carcinogenesis, 1996 Feb, 17(2), 297 - 302
A new Salmonella typhimurium NM5004 strain expressing rat glutathione S-transferase 5-5: use in detection of genotoxicity of dihaloalkanes using an SOS/umu test system; Oda Y et al.; The Escherichia coli mu operon was subcloned into a pKK233-2 vector containing rat glutathione S-transferase (GST) 5-5 cDNA and the plasmid thus obtained was introduced into Salmonella typhimurium TA1535 . The newly developed strain S.typhimurium NM5004, was found to have 52-fold greater GST activity than the original umu strain S.typhimurium TA1535/pSK1002 . We compared sensitivities of these two tester strains, NM5004 and TA1535/pSK1002, for induction of umuC gene expression with several dihaloalkanes which are activated or inactivated by GST 5-5 activity . The induction of umuC gene expression by these chemicals was monitored by measuring the cellular beta-galactosidase activity produced by umuC"lacZ fusion gene in these two tester strains . Ethylene dibromide, 1-bromo-2-chloroethane, 1,2-dichloroethane, and methylene dichloride induced umuC gene expression more strongly in the NM5004 strain than the original strain . 4-Nitroquinoline 1-oxide and N-methyl-N'-nitro-N-nitrosoguanidine were found to induce umuC gene expression to similar extents in both strains . In the case of 1-nitropyrene and 2-nitrofluorene, however, NM5004 strain showed weaker umuC gene expression responses than the original TA1535/pSK1002 strain . 1,2-Epoxy-3-(4'-nitrophenoxy)propane, a known substrate for GST 5-5, was found to inhibit umuC induction caused by 1-bromo-2-chloroethane . These results indicate that this new tester NM5004 strain expressing a mammalian GST theta class enzyme may be useful for studies of environmental chemicals proposed to be activated or inactivated by GST activity.

Carcinogenesis, 1996 Feb, 17(2), 277 - 82
CYP2E1-mediated mechanism of anti-genotoxicity of the broccoli constituent sulforaphane; Barcelo S et al.; The broccoli constituent sulforaphane (1-isothiocyanate-4-methylsulfinylbutane) has previously been shown to protect rats against 9,10-dimethyl-1,2-benz{a}anthracene tumorigenesis, thought to be due, at least in part, to induction of phase II detoxification . We investigated the ability of sulforaphane to also inhibit the phase I enzyme cytochrome P450 isoenzyme 2E1 (CYP2E1), which is responsible for activation of several carcinogens, including dialkylnitrosamines . Using the p-nitrophenol hydroxylation assay in microsomes from livers of acetone-treated Sprague-Dawley rats, sulforaphane was shown to be a potent competitive inhibitor of CYP2E1 with a Ki of 37.0 +/- 4.5 microM . In view of this result, we studied the capacity of sulforaphane to inhibit the genotoxicity of N-nitrosodimethylamine (NDMA) . Sulforaphane at concentrations of > 0.8 microM inhibited the mutagenicity of NDMA (4.4 mg/plate) in Salmonella typhimurium strain TA100 after pre-incubation for 45 min with cytosol extract from livers of Balb/c mice pre-treated with acetone . Unscheduled DNA synthesis induced by NDMA (33.5 microM) in mouse hepatocytes was inhibited in a dose-dependent manner by sulforaphane at 0.064-20 microM . Sulforaphane was unable to inhibit mutagenicity of sodium azide (5 micrograms/plate), a direct acting mutagen, in the Salmonella assay . It was not itself genotoxic in hepatocytes, as measured by unscheduled DNA synthesis, or mutagenic in the strain of Salmonella employed and cytotoxic only at high concentrations (> or = 0.5 mM) . These findings suggest that inhibition of CYP2E1 by sulforaphane may offer chemoprotection against carcinogenic substrates of this enzyme.

Mutat Res, 1996 Feb 1, 349(2), 201 - 8
Efficiency of MucAB and Escherichia coli UmuDC proteins in quinolone and UV mutagenesis in Salmonella typhimurium: effect of MucA and UmuD processing; Clerch B et al.; The role of MucAB and Escherichia coli UmuDC proteins in mutagenesis by 4-quinolone (4-Q) compared to that in UV mutagenesis has been studied in hisG428 Salmonella typhimurium strains . A low-copy plasmid carrying mucAB genes, but not umuDC, promotes reversion of the hisG428 mutation by the 4-Q ciprofloxacin . In contrast, a umuDC plasmid mediates the reversion of hisG428 by UV, although less efficiently than a mucAB one . In addition, a unique copy of mucAB genes is enough to promote UV mutagenesis, whereas, several copies of them are required to detect ciprofloxacin mutagenesis . Therefore, the mutagenic repair of quinolone damage by MucAB proteins is not a very efficient process . The presence of an umuD'C plasmid but not a mucA'B one, slightly increases the reversion of the hisG428 mutation by ciprofloxacin and this finding is further discussed . In contrast, MucA'B are still more active than UmuD'C proteins in UV mutagenesis . These results suggest that the enhanced processing of MucA compared to UmuD would not explain all functional differences between MucAB and UmuDC proteins in the error-prone DNA repair.

Arch Microbiol, 1996 Feb, 165(2), 126 - 31
Purification and characterization of fumarase from the syntrophic propionate-oxidizing bacterium strain MPOB; Van Kuijk BL et al.; Fumarase from the syntrophic propionate-oxidizing bacterium strain MPOB was purified 130-fold under anoxic conditions . The native enzyme had an apparent molecular mass of 114 kDa and was composed of two subunits of 60 kDa . The enzyme exhibited maximum activity at pH 8.5 and approximately 54 degrees C . The Km values for fumarate and L-malate were 0.25 mM and 2.38 mM, respectively . Fumarase was inactivated by oxygen, but the activity could be restored by addition of Fe2+ and β-mercaptoethanol under anoxic conditions . EPR spectroscopy of the purified enzyme revealed the presence of a {3Fe-4S} cluster . Under reducing conditions, only a trace amount of a {4Fe-4S} cluster was detected . Addition of fumarate resulted in a significant increase of this {4Fe-4S} signal . The N-terminal amino acid sequence showed similarity to the sequences of fumarase A and B of Escherichia coli (56%) and fumarase A of Salmonella typhimurium (63%).

Appl Environ Microbiol, 1996 Feb, 62(2), 587 - 92
Immunomagnetic-electrochemiluminescent detection of Escherichia coli O157 and Salmonella typhimurium in foods and environmental water samples; Yu H et al.; Hemorrhagic Escherichia coli O157:H7 strains and other virulent enteric pathogens can pose a serious health threat in tainted meats, poultry, and even drinking water . Traditional culture-based methods for assay of enteric pathogens in foods and water sources are relatively slow, and results can be ambiguous . Immunomagnetic separation (IMS) and detection methods have been investigated and appear promising for rapid bacterial assay of foods and environmental samples . In this work, a commercial sensor which combines IMS with electrochemiluminescence (ECL) detection is evaluated for detection of E . coli O157 and Salmonella typhimurium in foods and fomites . Results indicate that detection limits are in the range of 100 to 1,000 bacteria per ml in pristine buffer for E . coli O157 and S . typhimurium, respectively, or 1,000 to 2,000 bacteria per ml in food samples (depending on the sample) and that total processing and assay time is rapid (< 1 h) even in food samples . An immunologic "hook" or high-antigen-concentration prozone effect was observed above 10(4) and 10(5) bacteria per ml for E . coli O157 and S . typhimurium, respectively . IMS was accomplished in milk, juices, serum, supernatant fluids from ground beef, finely minced chicken, and fish suspensions as well as several freshwater sources and followed by ECL assay . Some samples, especially fish, gave unexpectedly high background ECL . Conversely, low ECL intensity was observed in nonfat and 2% fat milk samples, which appeared to be related to binding or entrapment of the antibody-coated magnetic beads by particulates in the milk, as revealed by microscopy . Results of this evaluation suggest the feasibility of immunomagnetic-ECL methodology for rapid, sensitive, and facile preliminary screening of various foods and fomites for the presence of virulent enteric pathogens.

J Bacteriol, 1996 Feb, 178(4), 1187 - 96
Metabolic effects of inhibitors of two enzymes of the branched-chain amino acid pathway in Salmonella typhimurium; Epelbaum S et al.; The metabolic effects of inhibitors of two enzymes in the pathway for biosynthesis of branched-chain amino acids were examined in Salmonella typhimurium mutant strain TV105, expressing a single isozyme of acetohydroxy acid synthase (AHAS), AHAS isozyme II . One inhibitor was the sulfonylurea herbicide sulfometuron methyl (SMM), which inhibits this isozyme and AHAS of other organisms, and the other was N-isopropyl oxalylhydroxamate (IpOHA), which inhibits ketol-acid reductoisomerase (KARI) . The effects of the inhibitors on growth, levels of several enzymes of the pathway, and levels of intermediates of the pathway were measured . The intracellular concentration of the AHAS substrate 2-ketobutyrate increased on addition of SMM, but a lack of correlation between increased ketobutyrate and growth inhibition suggests that the former is not the immediate cause of the latter . The levels of the keto acid precursor of valine, but not of the precursor of isoleucine, were drastically decreased by SMM, and valine, but not isoleucine, partially overcame SMM inhibition . This apparent stronger effect of SMM on the flux into the valine arm, as opposed to the isoleucine arm, of the branched-chain amino acid pathway is explained by the kinetics of the AHAS reaction, as well as by the different roles of pyruvate, ketobutyrate, and the valine precursor in metabolism . The organization of the pathway thus potentiates the inhibitory effect of SMM . IpOHA has strong initial effects at lower concentrations than does SMM and leads to increases both in the acetohydroxy acid substrates of KARI and, surprisingly, in ketobutyrate . Valine completely protected strain TV105 from IpOHA at the MIC . A number of explanations for this effect can be ruled out, so that some unknown arrangement of the enzymes involved must be suggested . IpOHA led to initial cessation of growth, with partial recovery after a time whose duration increased with the inhibitor concentration . The recovery is apparently due to induction of new KARI synthesis, as well as disappearance of IpOHA from the medium.

J Bacteriol, 1996 Feb, 178(4), 1113 - 9
Effect of the surface composition of motile Escherichia coli and motile Salmonella species on the direction of galvanotaxis; Shi W et al.; We have reported that motile Escherichia coli K-12 placed in an electric field swims toward the anode but that motile Salmonella typhimurium strains swim toward the cathode, a phenomenon called galvanotaxis (J . Adler and W . Shi, Cold Spring Harbor Symp . Quant . Biol . 53:23-25, 1988) . In the present study, we isolated mutants with an altered direction of galvanotaxis . By further analyses of these mutants and by examination of E . coli and Salmonella strains with altered cell surface structure, we have now established a correlation between the direction of galvanotaxis and the surface structure of the cell: motile rough bacteria (that is, those without O polysaccharide; for example, E . coli K-12 and S . typhimurium mutants of classes galE and rfa) swam toward the anode, whereas motile smooth bacteria (that is, those with O polysaccharide; for example, wild-type S . typhimurium LT2) swam toward the cathode . However, smooth bacteria with acidic polysaccharide capsules (K1 in E . coli and Vi in Salmonella typhi) swam toward the anode . Measurements of passive electrophoretic mobility of strains representative of each set were made . We propose that the different directions of galvanotaxis of rough (or capsulate) bacteria and of smooth bacteria are explicable if the negative electrophoretic mobility of flagellar filaments is less than that of rough bodies but greater than that of smooth bodies.

J Bacteriol, 1996 Feb, 178(3), 899 - 901
Negative regulation by fliD, fliS, and fliT of the export of the flagellum-specific anti-sigma factor, FlgM, in Salmonella typhimurium; Yokoseki T et al.; The fliD operon of Salmonella typhimurium consists of three flagellar genes, fliD, fliS, and fliT, and is transcribed in this order . It has been shown that an fliD::Tn10 mutation causes an excess export of the flagellum-specific anti-sigma factor, FlgM, resulting in an overexpression of flagellar class 3 operons . In this study, using gene-disruption mutants in the individual genes in the fliD operon, we showed that mutations in any one of the genes in the operon enhanced both FlgM export and the expression of flagellar regulon . This indicates that all three genes in the operon are involved in the negative regulation of FlgM export.

J Bacteriol, 1996 Feb, 178(3), 638 - 46
Transcription of the glutamyl-tRNA reductase (hemA) gene in Salmonella typhimurium and Escherichia coli: role of the hemA P1 promoter and the arcA gene product; Choi P et al.; In Salmonella typhimurium and Escherichia coli, the hemA gene encodes the enzyme glutamyl-tRNA reductase, which catalyzes the first committed step in the heme biosynthetic pathway . It has recently been reported that a lac operon fusion to the hemA promoter of E . coli is induced 20-fold after starvation for heme . Induction was dependent on the transcriptional regulator ArcA, with a second transcriptional regulator, FNR, playing a negative role specifically under anaerobic conditions (S . Darie and R . P . Gunsalus, J . Bacteriol . 176:5270-5276, 1994) . We have investigated the generality of this effect by examining the response to heme starvation of a number of lac operon fusions to the hemA promoters of both E . coli and S . typhimurium . We confirmed that such fusions are induced during starvation of a hemA auxotroph, but the level of induction observed was maximally sixfold and for S . typhimurium fusions it was only two- to fourfold . Sequences required for high-level expression of hemA lie within 129 bp upstream of the major (P1) promoter transcriptional start site . Mutants defective in the P1 promoter had greatly reduced hemA-lac expression both in the presence and in the absence of ALA . Mutations in arcA had no effect on hemA-lac expression in E . coli during normal growth, although the increase in expression during starvation for ALA was half that seen in an arcA+ strain . Overexpression of the arcA gene had no effect on hemA-lac expression . Primer extension analysis showed that RNA 5' ends mapping to the hemA P1 and P2 promoters were not expressed at significantly higher levels in induced cultures . These results differ from those previously reported.

Infect Immun, 1996 Feb, 64(2), 548 - 56
Variation of Brucella abortus 2308 infection in BALB/c mice induced by prior vaccination with salt-extractable periplasmic proteins from Brucella abortus 19; Pugh GW Jr et al.; The study compared the immune and protective responses induced in BALB/c mice vaccinated with six salt-extractable periplasmic protein fractions (Brucella cell surface proteins {BCSP}) of Brucella abortus 19 and later challenge exposed with B . abortus 2308 . BCSP70 was precipitated with ammonium sulfate at 70% saturation, and BCSP100 was precipitated with ammonium sulfate at 100% saturation by use of supernatant fluid of BCSP70 that had been precipitated with 70% ammonium sulfate . Four subfractions were separated from BCSP100 by anion-exchange high-performance liquid chromatography (HPLC) . Monophosphoryl lipid A (MPL) from Salmonella typhimurium Re mutant strain was used as a potential immune response modifier in some vaccines . Reduced or increased numbers of CFU and increased spleen size in the principal groups of mice relative to that of the nonvaccinated control group were considered protectiveness or virulence (survival) criteria . Results indicated that vaccines prepared from BCSP70 and BCSP100 were moderately protective and immunogenic . The subfractions designated BCSP100-A through BCSP100-D purified by anion-exchange HPLC were not protective when MPL was not used as an immune response modifier . However, two subfractions were associated with significant (P < 0.05) increases in CFU per spleen and splenomegaly in vaccinated mice compared with those in nonvaccinated challenge-exposed mice . MPL enhanced protection or was neutral when used with BCSP70, BCSP100, BCSP100-C, and BCSP100-D . Serologic results of an enzyme-linked immunosorbent assay indicated that MPL modulated the immunoglobulin G responses induced by BCSP70, BCSP100, and subfraction BCSP100-B vaccines only . The overall results suggest that certain proteinaceous periplasmic fractions might serve as virulence or survival factors in B . abortus infections.

J Biotechnol, 1996 Jan 26, 44(1-3), 161 - 70
Bacterial ghosts: non-living candidate vaccines; Szostak MP et al.; Expression of cloned PhiX174 gene E in bacteria results in lysis of bacteria . It is unique among phage lysis systems as it introduces a transmembrane tunnel structure through the cell envelope complex of Gram-negative bacteria . The resulting bacterial ghosts have intact envelope structures devoid of cytoplasmic contents . E-mediated lysis has been achieved in a variety of Gram-negative bacteria including Escherichia coli, Salmonella typhimurium, Vibrio cholerae, Klebsiella pneumoniae, and Actinobacillus pleuropneumoniae . Such ghosts, derived from human or animal pathogens, have been proposed as non-living candidate vaccines and represent an alternative to heat or chemically inactivated bacteria . In 'recombinant ghosts', foreign proteins (e.g., viral proteins) are inserted into the inner membrane via specific N-, or C-, or N- and C-terminal anchor sequences prior to lysis . Relevant advantages of (recombinant) bacterial ghosts as immunogens include: (i) inactivation procedures that denature relevant immunogenic determinants are not employed in the production of ghosts used as vaccines or as carriers of relevant antigens; (ii) the recombinant proteins are inserted into a highly immune stimulatory environment; (iii) there is no size limitation of the foreign protein moieties: multiple antigenic determinants can be presented simultaneously; (iv) bacterial ghosts can be produced inexpensively in large quantities; (v) (recombinant) ghosts are stable for long periods of time and do not require the cold chain storage system . Intraperitoneal, subcutaneous or intramuscular applications of recombinant ghosts in experimental animals induced specific humoral and cellular immune responses against bacterial and viral components . Initial aerosol vaccinations of swine with ghosts from Actinobacillus pleuropneumoniae showed that protective immunity can be established by this route of application and that the well-preserved surface structures of ghosts obtained by E-mediated lysis are able to target the mucosal immune system.

J Biotechnol, 1996 Jan 26, 44(1-3), 91 - 6
Hybrid hepatitis B virus core antigen as a vaccine carrier moiety: I . presentation of foreign epitopes; Schodel F et al.; Hepatitis B virus (HBV) core antigen (HBcAg) is a highly immunogenic subviral particle . Here, we review recent progress in the use of HBcAg as a carrier moiety for heterologous epitopes . To define surface exposed and immunogenic insertion sites for foreign epitopes in HBcAg, peptidic epitopes representing binding sites for virus neutralizing antibodies on the HBV surface antigens were inserted at different positions within HBcAg using genetic engineering in an Escherichia coli expression system (Schodel et al . (1992) J . Virol . 66, 106-114) . While fusion to the N-terminus required a linker to become surface accessible, both fusion to the N-terminus and to the C-terminus was compatible with particle assembly and preserved the native antigenicity and immunogenicity of HBcAg . Fusion to an immunodominant internal site of HBcAg reduced the HBcAg immunogenicity and antigenicity and most drastically enhanced the immunogenicity of the inserted foreign epitope . This internal site of HBcAg was used to express circumsporozoite antigen (CS) repeat epitopes of two rodent malaria parasites and of Plasmodium falciparum (Schodel et al . (1994b) J . Exp . Med . 180, 1037-1046 and Schodel et al . (1995a) 95th ASM General Meeting, Washington DC, Abstr . E61) . When purified from recombinant Salmonella typhimurium, the hybrid HBcAg-CS proteins were particulate and displayed CS antigenicity as well as reduced HBc antigenicity, as compared to native HBcAg . Immunization of several mouse strains with HBcAg-CS hybrid particles resulted in high titered serum anti-CS antibodies representing all murine IgG isotypes . Immunization of mice with HBcAg or HBcAg-CS particles formulated on alum, complete Freunds or incomplete Freunds adjuvant resulted in equivalent anti-CS and anti-HBc serum antibody titres . The possible influence of carrier-specific immunosuppression was examined and pre-existing immunity to HBcAg did not significantly alter the immunogenicity of hybrid HBcAg particles suggesting that they would be useful carrier moieties for repeated immunizations against multiple haptens or in immune subjects after HBV infection . Examination of T cell recognition of HBcAg-CS particles revealed that HBcAg-specific T cells were universally primed and CS-specific T cells were primed if the insert contained a CS-specific T cell recognition site . This indicates that the internal amino acid position in HBcAg is permissive for the inclusion of heterologous functional T helper as well as B cell epitopes . BALB/c mice immunized with HBcAg-CS1 were protected against P . berghei challenge to 90% and 100%, respectively, in two independent experiments.

J Mol Biol, 1996 Jan 26, 255(3), 458 - 75
Geometry of the flagellar motor in the cytoplasmic membrane of Salmonella typhimurium as determined by stereo-photogrammetry of quick-freeze deep-etch replica images; Katayama E et al.; The precise geometry of the flagellar basal structure anchored in the cytoplasmic membrane was determined by digital stereo-photogrammetry of the images captured by quick-freeze deep-etch replica electron microscopy . In order to examine the structure on the periplasmic side of the membrane, we analyzed the MS ring complexes of Salmonella typhimurium overproduced in the cytoplasmic membrane of Escherichia coli . The rod, the S ring, and the shoulder of the M ring were exposed to the periplasm . On the cytoplasmic side of the membrane, small bumps corresponding to the cytoplasmic rod were discernible . We also examined the intact inner surface of the cells of polyhook mutant which was prepared by a new protocol and found the bell-shaped structure extending from the membrane towards the cytoplasm . It was identified as the C ring, since it was located at the base of the polyhook . Various dimensions of the MS ring complex and the C ring projecting from the membrane were determined by digital stereo-photogrammetry, and a three-dimensional model of the total basal structure is presented.

Proc Natl Acad Sci U S A, 1996 Jan 23, 93(2), 906 - 7
Muller's ratchet decreases fitness of a DNA-based microbe; Andersson DI et al.; Muller proposed that an asexual organism will inevitably accumulate deleterious mutations, resulting in an increase of the mutational load and an inexorable, ratchet-like, loss of the least mutated class {Muller, H.J . (1964) Mutat . Res . 1, 2-9} . The operation of Muller's ratchet on real populations has been experimentally demonstrated only in RNA viruses . However, these cases are exceptional in that the mutation rates of the RNA viruses are extremely high . We have examined whether Muller's ratchet operates in Salmonella typhimurium, a DNA-based organism with a more typical genomic mutation rate . Cells were grown asexually under conditions expected to result in high genetic drift, and the increase in mutational load was determined . S . typhimurium accumulated mutations under these conditions such that after 1700 generations, 1% of the 444 lineages tested had suffered an obvious loss of fitness, as determined by decreased growth rate . These results suggest that in the absence of sex and with high genetic drift, genetic mechanisms, such as back or compensatory mutations, cannot compensate for the accumulation of deleterious mutations . In addition, we measured the appearance of auxotrophs, which allowed us to calculate an average spontaneous mutation rate of approximately 0.3-1.5 x 10(-9) mutations per base pair per generation . This rate is measured for the largest genetic target studied so far, a collection of about 200 genes.

Cancer Lett, 1996 Jan 19, 99(1), 15 - 21
All-trans beta-carotene enhances mitogenic responses and ornithine decarboxylase activity of BALB/c 3T3 fibroblast cells induced by tumor promoter and fetal bovine serum but suppresses mutagen-dependent umu C gene expression in Salmonella typhimurium (TA 1535/pSK 1002); Okai Y et al.; Although previous epidemiological studies have indicated that beta-carotene is an important agent for the chemical prevention against carcinogenesis, a recent prospective study has strikingly suggested that supplementation with beta-carotene significantly increased the incidence of some types of cancer (The alpha-Tocopherol and beta-Carotene Cancer Prevention Study Group, New Engl . J . Med., 330 (1994) 1031-1035) . To analyze the discrepancy of this problem, the authors analyze the effects of beta-carotene on biochemical and biological events associated with carcinogenesis by in vitro experiments . (1) All-trans beta-carotene enhanced the proliferation and DNA synthesis of BALB/c 3T3 cells induced by a tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and fetal bovine serum, although beta-carotene itself did not show mitogenic activity . (2) All-trans beta-carotene caused a remarkable stimulation for the early induction of ornithine decarboxylase (ODC) activity after the stimulation of TPA and fetal bovine serum . (3) All-trans beta-carotene exhibited significant antimutagenic activity which suppresses umu C gene expression in Salmonella typhimurium (TA 1535/pSK 1002) induced by a typical mutagen, 2-aminoanthracene (2-AA) . These experimental results suggest that all-trans beta-carotene might cause beneficial and harmful effects on different phases of carcinogenesis.

Mutat Res, 1996 Jan 17, 349(1), 137 - 44
Mutagenicity of nitrophenanthrene derivatives for Salmonella typhimurium: effects of nitroreductase and acetyltransferase; Sera N et al.; To determine the mutagenicity of nitrophenanthrenes, three mononitrophenanthrenes (NPhs), 11 dinitrophenanthrenes (diNPhs) and eight trinitrophenanthrenes (tiNPhs) were synthesized, and their mutagenicity was investigated by using Salmonella typhimurium his- strains TA98, TA100, and TA98NR, nitroreductase-deficient, and TA98/1,8-DNP6, O-acetyltransferase-deficient mutants, and strains YG1021 and YG1026, nitroreductase-overproducing mutants of TA98 and TA100, respectively, and strains YG1024 and YG1029, O-acetyltransferase-overproducing mutants of TA98 and TA100, respectively . 1-, 3- and 9-NPhis induced 329, 620 and 438 revertants per nmol in strain TA100, respectively, and 4,839, 11,309 and 16728 revertants per nmol, respectively, in strain YG1029 . Mutagenicity of 1,6-, 2,6-, 2,9-, 2,10-, 3,5-, 3,6- and 3,10-diNPh was elevated in strains YG1021, YG1024, YG1026 and YG1029 . Among these derivatives, 1,6-, 2,6-, 3,6- and 3,10-diNPhs were more mutagenic in strains YG1024 and YG1029 than YG1021 and YG1026, and they showed a structure-activity relationship between mutagenicity and NO2-substitution . Nitro derivatives substituted at the 3 and 6 positions of their chemical structure strongly mutated both strains YG1024 and YG1029, whereas those substituted at the 9 and 10 positions showed weak mutagenicity . In addition, nitro substituents at positions 4 and 5 were perpendicular while those on positions 2,3,6 and 7 were nearly coplanar to the aromatic ring . Furthermore, 2,6,9-, 3,6,9- and 1,6,9-trinitrophenanthrenes (triNPhs) were mutagenic for strain TA100, and their mutagenicity was more enhanced in YG1024 and YG1029 than in YG1021 and YG1026 . Of the eight triNPhs all except 1,5,10-triNP were mutagenic in TA98 and TA100, and their mutagenicity was more enhanced in YG1024 and YG1029 than in YG1021 and YG1026 . These results suggest that these compounds are mutagens that are activated by O-acetyltransferase esterification following nitroreductase . The nitrated derivatives substituted at the 2(7) and 3(6) positions of the phenanthrene ring were highly mutagenic . The relationship between chemical structure and the mutagenicity of NPh derivatives is discussed.

Mutat Res, 1996 Jan 16, 359(1), 17 - 24
DNA strand break by 2,5-dimethyl-4-hydroxy-3(2H)-furanone, a fragrant compound in various foodstuffs; Hiramoto K et al.; 2,5-Dimethyl-4-hydroxy-3(2 H)-furanone (DMHF), produced by Maillard reaction of sugar/amino acid and found in various foodstuffs, showed mutagenicity to Salmonella typhimurium TA100 strain with and without S9 mix, and induced micronucleated mouse peripheral reticulocytes . DNA strand breaking activity of the compound at pH 7.4 increased with the increasing dose of the compound and with the increasing incubation time . The breaking activity was inhibited in the presence of superoxide dismutase, catalase, hydroxyl radical scavengers, spin trapping agents, thiol compounds and metal chelators, and also by removal of dissolved oxygen from the incubation mixture . Addition of Fe(III) ion to the incubation mixture enhanced the breaking activity . Incubation of DMHF with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) gave electron spin resonance signals characteristic to DMPO-OH adduct, indicating generation of hydroxyl radical . It was found that DMHF generated hydroxyl radical with an aid of a trace amount of metal ions, and induced DNA strand breaking . Mutagenicity and induction of micronucleated reticulocytes by DMHF may be caused as a result of DNA modification via hydroxyl radical.

FEMS Microbiol Lett, 1996 Jan 15, 135(2-3), 281 - 5
The Salmonella typhimurium flgM gene, which encodes a negative regulator of flagella synthesis and is involved in virulence, is present and functional in other Salmonella species; Schmitt CK et al.; FlgM inhibits the flagella-specific sigma factor FliA and is involved in the mouse-virulence of Salmonella typhimurium . In recent experiments, we observed that: (i) a flgM gene that could function to negatively regulate flagella synthesis was present in a variety of salmonellae; and (ii) the flgM gene derived from Salmonella species that are not normally virulent in mice could complement the S . typhimurium flgM mutant for virulence . Our results suggest that a functional flgM has been retained in most, and perhaps all, Salmonella species, regardless of the motility or virulence phenotype of the strain.

FEMS Microbiol Lett, 1996 Jan 15, 135(2-3), 161 - 7
Salmonella typhimurium acrB-like gene: identification and role in resistance to biliary salts and detergents and in murine infection; Lacroix FJ et al.; Salmonella serotype typhimurium transpositional mutants altered in resistance to biliary salts and detergents were isolated previously . We have characterized further the LX1054 mutant strain, the most sensitive of them . The chromosomal DNA segment flanking transposon insertion was cloned and sequenced . The highest level of identity was found for the acrB (formerly acrE) gene of Escherichia coli, a gene encoding a drug efflux pump of the Acr family . LX1054 exhibited a reduced capacity to colonize the intestinal tract . After passages in mice, the mutant strain lost the sensitive phenotype . In vitro, a resumption of growth appeared after 17 h of culture in medium with cholate or other tested biological or chemical detergents . Then, the acquired resistant phenotype seemed stable . The data suggested a role of S . typhimurium acrB-like gene in resistance to biliary salts and detergents and in mice intestinal colonization . However, the local and transient sensitivity observed in vivo, and the in vitro adaptations suggest that several detergent-resistance mechanisms operate in S . typhimurium.

J Biol Chem, 1996 Jan 12, 271(2), 1232 - 6
Mutants with defective phosphatase activity show no phosphorylation-dependent oligomerization of CheZ . The phosphatase of bacterial chemotaxis; Blat Y et al.; CheZ is the phosphatase of CheY, the response regulator in bacterial chemotaxis . The mechanism by which the activity of CheZ is regulated is not known . We used cheZ mutants of Salmonella typhimurium, which had been isolated by Sockett et al . (Sockett, H., Yamaguchi, S., Kihara, M., Irikura, V . M., and Macnab, R . M . (1992) J . Bacteriol . 174, 793-806), for cloning the mutant cheZ genes, overexpressing and purifying their products . We then measured the phosphatase activity, binding to CheY and to phosphorylated CheY (CheY approximately P), and CheY approximately P dependent oligomerization of the mutant CheZ proteins . While all the mutant proteins were defective in their phosphatase activity, they bound to CheY and CheY approximately P as well as wild-type CheZ . However, unlike wild-type CheZ, all the four mutant proteins failed to oligomerize upon interaction with CheY approximately P . On the basis of these and earlier results it is suggested that (i) oligomerization is required for the phosphatase activity of CheZ, (ii) the region defined by residues 141-145 plays an important role in mediating CheZ oligomerization and CheY approximately P dephosphorylation but is not necessary for the binding to CheY approximately P, (iii) the oligomerization and hence the phosphatase activity are regulated by the level of CheY approximately P, and (iv) this regulation plays a role in the adaptation to chemotactic stimuli.

Cell, 1996 Jan 12, 84(1), 165 - 74
Mg2+ as an extracellular signal: environmental regulation of Salmonella virulence; Garcia Vescovi E et al.; Ions are not traditionally thought to act as first messengers in signal transduction cascades . However, while searching for genes regulated by the PhoP/PhoQ virulence regulatory system of Salmonella typhimurium, we recovered two loci whose expression is controlled by the concentration of Mg2+ . To determine whether Mg2+ is the signal modulating the whole PhoP/PhoQ system, we evaluated the gene expression pattern of six PhoP-activated genes . Growth in physiological concentrations of divalent cations repressed transcription of PhoP-activated genes and rendered wild-type Salmonella phenotypically PhoP- . Mg2+ changed the conformation of the periplasmic domain of PhoQ, identifying this protein as a Mg2+ sensor . A mutation in the sensing domain of PhoQ altered the set point for Mg2+ and rendered Salmonella avirulent.

Cell Immunol, 1996 Jan 10, 167(1), 8 - 17
Treatment of Candida albicans mannan-specific downregulatory cell populations with divergent concentrations of monophosphoryl lipid A and intact lipopolysaccharide in vitro abrogates their effect on delayed hypersensitivity; Domer JE et al.; We have shown previously that splenocytes from mice injected with Candida albicans mannan (MAN) suppress MAN-specific delayed hypersensitivity (DH) when transferred to immunized recipients and that treatment of donor mice with monophosphoryl lipid A (MLA) derived from Salmonella typhimurium or Salmonella minnesota shortly before transfer abrogated the downregulatory activity . We now show that treatment of splenocytes in vitro at 4 degrees C with 5 ng/ml MLA or 0.05 ng/ml S . typhimurium lipopolysaccharide (LPS) for 30 min before transfer also abrogated downregulatory activity . Higher or lower doses of MLA, 5 micrograms or 5 pg, appeared to increase the suppressor activity slightly (5 micrograms) or had no effect (5 pg) . LPS induced similar effects but the concentrations of LPS required to show the effects were 100-fold less than those of MLA . The effect of MLA appeared to be on cell(s) in the transfer population involved in MAN-specific DH, in that spleen cells from normal mice treated with MLA prior to transfer had no effect on DH . Finally, the population of MLA-responsive cells mediating downregulation could not be concentrated on MLA-coated plates, suggesting that the MAN-specific downregulatory cell(s) either did not bind to MLA or did not bind to MLA with sufficient avidity to remain attached during the washing procedures . The feasibility of abrogating suppression by treatment of lymphoid cells in vitro will allow a more detailed analysis of the mechanism of abrogation.

Biochemistry, 1996 Jan 9, 35(1), 65 - 75
Flavin-dependent alkyl hydroperoxide reductase from Salmonella typhimurium . 2 . Cystine disulfides involved in catalysis of peroxide reduction; Poole LB; The two-component alkyl hydroperoxide reductase enzyme system from Salmonella typhimurium catalyzes the pyridine nucleotide-dependent reduction of alkyl hydroperoxide and hydrogen peroxide substrates . This system is composed of a flavoenzyme, AhpF, which is related to the disulfide-reducing enzyme thioredoxin reductase, and a smaller protein, AhpC, which lacks a chromophoric cofactor . We have demonstrated that NADH-linked reduction of AhpF under anaerobic conditions converts two cystine disulfide centers to their dithiol forms . The AhpC cystine disulfide center, shown to exist as an intersubunit disulfide bond, is stoichiometrically reducible by NADH in the presence of a catalytic amount of AhpF and can be reoxidized by ethyl hydroperoxide . Disulfide bridges within oxidized AhpF form between Cys129 and Cys132 and between Cys345 and Cys348; the two C-terminal half-cystine residues, Cys476 and Cys489, exist as free thiol groups in oxidized AhpF and play no role in catalysis . Removal of the N-terminal 202-amino acid segment containing the Cys129-Cys132 disulfide center obliterates the ability of AhpF to transfer electrons to 5,5'-dthiobis(2-nitrobenzoic acid) (DTNB) and AhpC . NADH added anaerobically to AhpF causes spectral changes consistent with preferential reduction of both disulfides relative to flavin reduction; the reduction potentials of the disulfide centers are thus appropriately poised for electron transfer from NADH and flavin to disulfide-containing substrates (AhpC or DTNB), and ultimately to peroxides from AhpC . Blue, neutral flavin semiquinone is also generated in high yields during reductive titrations (91% yield during dithionite titrations), although the relatively slow formation of this species indicates its catalytic incompetence . A long wavelength absorbance band beyond 900 nm attributable to an FADH2-->NAD+ charge transfer interaction is generated during NADH, but not dithionite, titrations and may be indicative of a species directly involved in the catalytic cycle . A catalytic mechanism including the transient formation of cysteine sulfenic acid within AhpC is proposed.

Biochemistry, 1996 Jan 9, 35(1), 56 - 64
Flavin-dependent alkyl hydroperoxide reductase from Salmonella typhimurium . 1 . Purification and enzymatic activities of overexpressed AhpF and AhpC proteins; Poole LB et al.; The two components, AhpF and AhpC, of the Salmonella typhimurium alkyl hydroperoxide reductase enzyme system have been overexpressed and purified from Escherichia coli for investigations of their catalytic properties . Recombinant proteins were isolated in high yield (25-33 mg per liter of bacterial culture) and were shown to impart a high degree of protection against killing by cumene hydroperoxide to the host E . coli cells . We have developed quantitative enzymatic assays for AhpF alone and for the combined AhpF/AhpC system which have allowed us to address such issues as substrate specificity and inhibition by thiol reagents for each protein . All assays gave identical results whether overexpressed S . typhimurium proteins from E . coli or proteins isolated directly from S . typhimurium were used . Anaerobic hydroperoxide reductase assays have demonstrated that cumene hydroperoxide, ethyl hydroperoxide, and hydrogen peroxide can all be reduced by the combined enzyme system . AhpF possesses multiple pyridine nucleotide-dependent activities {5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) reductase, oxidase, transhydrogenase, and, in the presence of AhpC, peroxide reductase activities} . Although AhpF can use either NADH or NADPH as the electron donor for these activities, NADH is the preferred reductant (Km,app of AhpF for NADH was more than 2 orders of magnitude lower than that for NADPH when analyzed using DTNB reductase assays) . Thiol-modifying reagents react readily with each reduced protein, leading to complete loss of hydroperoxide and DTNB reductase activities . In contrast, thiol modification of reduced AhpF does not affect transhydrogenase or oxidase activities . These data provide the first direct evidence for a catalytic mechanism for peroxide reduction involving redox-active disulfides within each protein.

Biochemistry, 1996 Jan 9, 35(1), 14 - 21
Transition state structure of Salmonella typhimurium orotate phosphoribosyltransferase; Tao W et al.; Orotate phosphoribosyltransferase (OPRTase) catalyzes the magnesium-dependent conversion of alpha-D-phosphoribosylpyrophosphate (PRPP) and orotate to orotidine 5'-monophosphate (OMP) and pyrophosphate . We have determined kinetic isotope effects on the reaction of OMP with pyrophosphate and with the pyrophosphate analog phosphonoacetic acid . In the latter case, full expression of the kinetic isotope effects allowed us to calculate the structure of the transition state for the pyrophosphorylytic reaction . The transition state resembles a classical oxocarbonium ion . Using the recently reported three-dimensional structures of the OPRTase-OMP (Scapin et al., 1994) and the OPRTase-PRPP complexes (Scapin et al., 1995a), we have modeled the calculated transition state structure into the active site of OPRTase . We propose a detailed chemical mechanism which is consistent with these results.

Proc Natl Acad Sci U S A, 1996 Jan 9, 93(1), 279 - 83
The lpf fimbrial operon mediates adhesion of Salmonella typhimurium to murine Peyer's patches; Baumler AJ et al.; We investigated the role of the Salmonella typhimurium fimbrial operon formed by the genes lpfABCDE in infection of mice . A mutant in lpfC, the gene encoding the fimbrial outer membrane usher, had an approximately 5-fold increased 50% lethal dose when administered orally to mice . When mice were infected with a mixture of the lpfC mutant and isogenic wild-type S . typhimurium, the lpfC mutant was recovered in lower numbers from Peyer's patches, mesenteric lymph nodes, liver, and spleen . In an organ culture model using murine intestinal loops, lpfC mutants were shown to be associated in lower numbers than wild-type bacteria with Peyer's patches but not with villous intestine . The defect of the lpfC mutant in adhesion to Peyer's patches could be complemented by introducing lpfABCDE on a cosmid . Similarly, heterologous expression of the Salmonella lpf operon in Escherichia coli resulted in an increased adhesion to histological thin sections of Peyer's patch lymph follicles . Electron microscopic analysis of histological sections taken from Peyer's patches after intragastric infection of mice showed that, in contrast to the S . typhimurium wild type, the isogenic lpfC mutant did not destroy M cells of the follicle-associated epithelium . These data show that the Salmonella lpf operon is involved in adhesion to murine Peyer's patches.

Chem Biol Interact, 1996 Jan 5, 99(1-3), 55 - 72
Cytotoxicity and mutagenicity of polycyclic aromatic hydrocarbon ortho-quinones produced by dihydrodiol dehydrogenase; Flowers-Geary L et al.; Eight polycyclic aromatic hydrocarbon (PAH) ortho-quinones that can be generated by dihydrodiol dehydrogenase (DD) were examined for their cytotoxicity in H-4-II-e (rat hepatoma) cells and for their mutagenicity in the Ames test . Seven of the PAH otrtho-quinones were potent cytotoxins yielding IC50 values for cell survival in the range 1-30 microns . PAH ortho-quinones were grouped into three classes based on their cytotoxicity profiles: group I contained ortho-quinones (e.g., naphthalene-1,2-dione and 7,12-dimethylbenz{alpha}anthracene-3,4-dione) which reduced cell viability and cell survival; group II contained ortho-quinones (e.g., benz{alpha}anthracene-3,4-dione and 5-methylchrysene-1,2-dione which reduced cell survival but had no effect on cell viability; and group III contained ortho-quinones (e.g., benzo{alpha}pyrene-7,8-dione) which had a pronounced effect on cell viability but minimal effects on cell survival . Using hepatoma cell suspensions and rat liver subcellular fractions, it was found that ortho-quinones underwent preferential enzymatic one-electron redox-cycling and produced superoxide anion radical (O2-.) and/or ortho-semiquinone anion or alternant radicals . ortho-Quinones that reduced cell viability produced O2- . and caused the most total free radical formation, while those that reduced cell survival produced ortho-semiquinone anion or alternant radicals only . PAH ortho-quinones were also tested as direct-acting mutagens in Salmonella typhimurium tester strains TA97a, TA98, TA100, TA102 and TA104 . They were found to be more mutagenic than the test mutagens used for each tester strain, and were predominantly frameshift mutagens . The presence of an activating system (Aroclor-induced rat liver S9 plus NADPH) did not increase the mutagenicity of ortho-quinones in tester strains that are sensitive to oxidative mutagens (TA102 and TA104) . These data suggest that PAH ortho-quinones produced by DD are cytotoxic and mutagenic by different mechanisms . The mechanism of cytotoxicity involves the formation of reactive oxygen species and/or ortho-semiquinone anion or alternant radicals . The mechanism of mutagenicity is independent of free radical formation and is related to the ability of PAH orthooffinones to intercalate and covalently modify DNA.

Chem Biol Interact, 1996 Jan 5, 99(1-3), 193 - 204
Cell-cycle specific cytotoxicity mediated by rearranged ent-kaurene diterpenoids isolated from Parinari curatellifolia; Lee IS et al.; Two structurally novel cytotoxic ent-kaurene diterpenoids, 13-methoxy-15-oxozoapatlin and 13-hydroxy-15-oxozoapatlin, were isolated from the root bark of Parinari curatellifolia, together with the known compound, 15-oxozoapatlin, on the basis of bioactivity-guided chromatographic fractionation and found to demonstrate broad-spectrum cytotoxic activity against a panel of cultured human cancer cell lines . The structures of these compounds were determined by analysis of their spectroscopic data . The presence of an alpha, beta-unsaturated carbonyl group in 13-methoxy-15-oxozoapatlin suggested that the cytotoxic potential of this compound could be mediated through reaction with cellular nucleophiles by means of a Michael-type addition . The compound 13-methoxy-15-oxozoapatlin reacted with the nucleophiles L-cysteine and beta-mercaptoethanol . The adduct with beta-mercaptoethanol was isolated, structurally characterized and found to be approximately 5-fold less cytotoxic than 13-methoxy-15-oxozoapatlin itself . The compound 13-methoxy-15-oxozoapatlin did not interact with DNA nor guanosine, and it was not mutagenic for Salmonella typhimurium strain TM677 . The effects of 13-methoxy-15-oxozoapatlin on the growth of human cancer cells were analyzed utilizing cultured ZR-75-1 breast cancer cells . Biosynthesis of DNA, RNA and protein was reduced in treated cells, and accumulation at the G2/M phase of the cell cycle was observed . The compound 13-methoxy-15-oxozoapatlin did not mediate antimitotic activity with dibutyryl cAMP-treated cultured astrocytoma cells, suggesting that the cell cycle effect is G2 specific . No antitumor activity was observed when athymic mice carrying KB cells were treated with 13-methoxy-15-oxozoapatlin . These data indicate that the cytotoxic activity of 13-methoxy-15-oxozoapatlin is mediated in part by covalent reaction with a cellular component (such as sulfhydryl-containing protein) by means of a Michael-type addition, and this results in the blockage of cell-cycle progression.

Biochem Biophys Res Commun, 1996 Jan 5, 218(1), 356 - 9
A novel expression system for Salmonella typhimurium allowing high production levels, product secretion and efficient recovery; Liljeqvist S et al.; A novel expression system for heterologous production in Salmonella typhimurium, taking advantage of the promoter, signal sequence and two IgG-binding domains (ZZ) from staphylococcal protein A, has been investigated . The production of two different fusion proteins, ZZ-M3 and ZZ-M5, was characterized in terms of production levels, product localization (periplasma or culture medium) and product quality after affinity purification . High expression levels and efficient product secretion were obtained, making the system attractive for vaccine development . The potential use of S . typhimurium as host for heterologous production in biotechnology is discussed.

Microbios, 1996, 88(357), 199 - 204
Ultrastructural localization of acid phosphatase in some bacteria, after treatment with Lubrol W1; Cherepova N et al.; The ultracytochemical localization of acid phosphatase from some bacteria (Listeria monocytogenes, Salmonella typhimurium, Pseudomonas pseudomallei and Pseudomonas aeruginosa) was dependent on the changes in the lipoprotein content of the membranes as a result of the action of the Lubrol W1.

Biochimie, 1996, 78(11-12), 1025 - 34
Structure and regulation of the Salmonella typhimurium rnc-era-recO operon; Anderson PE et al.; The Escherichia coli rnc-era-recO operon encodes ribonuclease III (RNase III; a dsRNA endonuclease involved in rRNA and mRNA processing and decay), Era (an essential G-protein of unknown functions and RecO (involved in the RecF homologous recombination pathway) . Expression of the rnc and era genes is negatively autoregulated: RNase III cleaves the rncO 'operator' in the untranslated leader, destabilizing the operon mRNA . As part of a larger effort to understand RNase III and Era structure and function, we characterized rnc operon structure, function and regulation in the closely related bacterium Salmonella typhimurium . Construction of a S typhimurium strain conditionally defective for RNase III and Era expression showed that Era is essential for cell growth . This mutant strain also enabled selection of recombinant clones containing the intact S typhimurium rnc-era-recO operon, whose nucleotide sequence, predicted protein sequence, and predicted rncO RNA secondary structure were all highly conserved with those of E coli . Furthermore, genetic and biochemical analysis revealed that S typhimurium rnc gene expression is negatively autoregulated by a mechanism very similar or identical to that in E coli, and that the cleavage specificities of RNase IIIs.t . and RNase IIIE.c . are indistinguishable with regard to rncO cleavage and S typhimurium 23S rRNA fragmentation in vivo.

Folia Microbiol (Praha), 1996, 41(1), 48 - 52
Euglena gracilis as a supplementary test organism for detecting biologically active compounds; Macor M et al.; The mutagenic activity of more than 120 antimicrobial agents and protective components was investigated . Only Kathon showed a consistent increase in revertant counts in the Ames test on Salmonella typhimurium . The hereditary bleaching test on Euglena gracilis used for detecting extranuclear mutations, showed positive results for Kathon, triethanolamine and diamine silver tetraborate.

J Environ Pathol Toxicol Oncol, 1996, 15(1), 21 - 8
Biochemical and genetic interactions of two commercial pesticides with the monooxygenase system and chlorophyllin; Della Croce C et al.; Two commercial preparations of atrazine and zineb were tested on a diploid D7 strain of the yeast Saccharomyces cerevisiae using cells from logarithmic growth phase (with a high level of cytochrome P-450) and from stationary growth phase . The compounds induced marked increases of both gene conversion and point mutation frequencies in the logarithmic phase cells, while in the stationary phase no genotoxic effect was observed . The results obtained employing TA98 and TA100 strains of Salmonella typhimurium (Ames test) confirmed that neither atrazine nor zineb were mutagenic . The interaction between zineb and chlorophyllin, a known antimutagen in several biological systems, has been evaluated in yeast cells from logarithmic growth phase . The results showed that chlorophyllin seems to have a protective role against the genotoxic effects of zineb . The in vivo effects on cytochrome P-450 content (Cyt . P-450) and on monooxygenase activities were examined in hepatic microsomes of induced animals (rat, pig, and rabbit) 24 hrs after acute treatment . The results obtained with atrazine showed that it caused different effects in the three animal species . Preliminary data with zineb indicated that it can act both as an inducer or as an inhibitor of the monooxygenase system, depending on the dose used.

Microbios, 1996, 87(351), 89 - 96
Effect of bisquaternary ammonium salts on Salmonella typhimurium; Majtan V et al.; The effect of 4,7-dioxo-3,8-dioxadekan-1, 1{bis(alkyl-dimethyldiammonium dibromides)} on growth, synthesis of macromolecules, respiration and induction of prophage in Salmonella typhimurium cells was studied . Only those compounds with alkyl chains of C10-C12 (octyl, decyl) in their molecules showed antimicrobial efficacy and inhibition incorporation of {14C}-adenine and {14C}-leucine as precursors of macromolecular biosynthesis . The highest inhibition of endogenous respiration was evoked by compounds with a long alkyl chain in the molecule (tetradecyl, hexadecyl) . Endogenous respiration of the cells was more sensitive to a compound with hexadecyl than the respiration of various intermediates of the Krebs cycle . The sub-MICs of compounds with octyl induced a prophage of a lysogenic S . typhimurium strain.

Int Arch Occup Environ Health, 1996, 69(1), 33 - 8
Cyto- and genotoxic effects of coordination complexes of platinum, palladium and rhodium in vitro; Bunger J et al.; The growing industrial use of platinum group elements as catalysts, especially in automobile exhaust detoxification (trimetal catalytic converters), is causing increasing occupational and environmental pollution . The cytotoxic and mutagenic properties of industrially used coordination complexes of platinum, palladium and rhodium were investigated using the neutral red cytotoxicity assay on two established cell lines and the Salmonella typhimurium/microsome test system (Ames test) . Cytotoxic effects of the platinum complexes, measured as ED50, occurred at test concentrations of 0.2 mM . The analogous palladium salts tested were 3 times less toxic with ED50 being 0.6 mM, while the rhodium salts proved to be 30 times less toxic (ED50 = 6 mM) . Levels of toxicity of the different complexes of a particular metal did not differ significantly from each other, which indicates that the metal itself is responsible for the toxic effects . In the Ames test, the spontaneous mutation rates increased by factors of 3 to 20 when the four tester strains were exposed to the platinum complexes . The analogous rhodium compounds proved to be considerably less mutagenic, and palladium demonstrated no mutagenic potential . As all of the four tester strains contain different mutations, the mutagenic potential of platinum and rhodium complexes appears to be based on a variety of mechanisms that damage DNA . From these in vitro experiments, it can be concluded that water-soluble complex salts of rhodium are less toxic and have a smaller mutagenic potential than the analogous platinum complexes . For palladium there is no evidence of any mutagenic property . From this point of view, the development of a catalytic converter containing predominantly palladium may be a possible means of minimizing potential health risks from this exhaust detoxification technique.

Acta Microbiol Pol, 1996, 45(2), 169 - 80
The mechanism of bactericidal action of normal human serum against Salmonella rods; Mokracka-Latajka G et al.; Forty Salmonella strains sensitive to the bactericidal action of serum were investigated . All these strains were susceptible to complement activated by the classical pathway though in part (60%) of these strains the presence of lysozyme was necessary for killing . S . typhimurium rods were susceptible to only one mechanism of the action of bactericidal factors . On the contrary, S . enteritidis strains were sensitive to three various mechanisms of bactericidal action of serum . Next eight forms of Salmonella typhimurium LT2 differing with respect to the structure of LPS were studied . Original strain was a smooth, form S, and remaining strains were various rough (R) forms such as Ra, Rb1, Rb2, Rc, Rd1, Rd2, Re . The S form was susceptible only to the complete human serum what means that all bactericidal factors of serum were needed for killing . Ra form was susceptible to two independent bactericidal mechanisms: complement (C) activated by the alternative pathway in the presence of lysozyme and C activated simultaneously by both bactericidal C pathways without the participation of lysozyme (al, ac) . The next form, Rb1, besides the mechanism mentioned above was also susceptible to C activated by the classical pathway in the presence of lysozyme (al, ac, cl) . Other forms (Rb2, Rc, Rd1, Rd2, Re) were susceptible to three mechanisms (al, ac, cl) as well as to C activated by the classical pathway without lysozyme (c) . The mechanism (c) was weakly efficient against forms Rb2 and Rc and somewhat more efficient against Rd1 and Rd2 and the most efficient against Re.

Acta Pol Pharm, 1996 Jan-Feb, 53(1), 13 - 7
Characteristics of mutagenesis by bleomycin and adriamycin in salmonella typhimurium: action of superoxide dismutase; Ejchart A et al.; The role of reactive oxygen species in adriamycin and bleomycin-induced mutagenicity was investigated in Salmonella typhimurium TA98 and TA102 respectively . Activity of superoxide dismutase (SOD) was inhibited by preincubation of bacteria with diethyldithiocarbamate (DEDTC) . Results of Ames test may suggest the involvement of active oxygen species in bleomycin induced mutagenesis and an absence of their participation in adriamycin induced mutagenesis.

Clin Rheumatol, 1996 Jan, 15(1), 83 - 5
Acute anterior uveitis in juvenile Reiter's syndrome; Fischel JD et al.; We describe the case of an acute anterior uveitis (AAU) and reactive arthritis following a gastrointestinal infection of Salmonella typhimurium in a 9-year-old girl . Reiter's syndrome mainly affects men and is unusual in children especially in young females.

Clin Rheumatol, 1996 Jan, 15(1), 72 - 4
Recurrent salmonella sepsis with different species in a systemic lupus erythematosus patient; Green L et al.; Infections are a common cause of morbidity and mortality in systemic lupus erythematosus (SLE) patients . The primary disease process and complications of drug management may contribute to this increased susceptibility . A high incidence of salmonella infections have been reported in SLE patients . We report an unusual case of a SLE patient who developed recurrent salmonella sepsis . The first episode with salmonella typhimurium was followed a few months later by an episode of salmonella enteritides sepsis.

Chem Res Toxicol, 1996 Jan-Feb, 9(1), 58 - 66
Gastric carcinogenesis: 2-chloro-4-methylthiobutanoic acid, a novel mutagen in salted, pickled Sanma hiraki fish, or similarly treated methionine; Chen W et al.; The customary salting and pickling of fish in high risk gastric cancer regions were modeled to explore the relevant causative chemicals . The fish Sanma hiraki was treated with sodium chloride and sodium nitrite at pH 3 . Previously, it had been found that an extract of the treated fish was mutagenic in Salmonella typhimurium TA 1535 without S9 and also that it induced glandular stomach cancer upon gavage to rats . We now demonstrate that the mutagenicity was enhanced by preincubation of the raw meat for several days before salt-nitrite treatment . HPLC techniques showed that three mutagens were present in the fish extract . One of the mutagens was found to be stable over the pH range of 1.0-9.0 . This mutagen was purified by silica gel solid phase extraction, followed by a series of reverse phase HPLC steps, and was characterized by low and high resolution MS, NMR, and FT-IR . While N-nitroso compounds were generally believed to be associated with gastric carcinogenesis, it was unexpectedly found that the mutagen has the novel structure 2-chloro-4-methylthiobutanoic acid (CMBA) . Based on the structure, it seemed likely that methionine might be the precursor, and this was, indeed, proven . Both salt and nitrite are essential factors for forming this mutagen . The yield of CMBA was linear for chloride concentrations from 0 to 800 mM NaCl . Of 20 amino acids reacted with nitrite and chloride at pH 3, only methionine generated a mutagen for S . typhimurium TA 1535 . Tryptophan gave a product mutagenic in S . typhimurium TA 100 and TA 98, but not TA 1535, and in the case of tyrosine, the mutagen was active only for TA 100 . These results suggest an important role for salt in gastric carcinogenesis and provide new approaches for exploring the formation of mutagens/carcinogens for specific target organs.

Chem Res Toxicol, 1996 Jan-Feb, 9(1), 333 - 40
Activation and inactivation of carcinogenic dihaloalkanes and other compounds by glutathione S-transferase 5-5 in Salmonella typhimurium tester strain NM5004; Shimada T et al.; A newly developed tester Salmonella typhimurium NM5004 strain was constructed by introducing a plasmid containing both rat GSH S-transferase (GST) 5-5 cDNA and the umuC"lacZ operon into the host strain Salmonella typhimurium TA1535 and used to examine whether or not GST modified the genotoxic activities of several dihaloalkanes and other compounds . Twenty-nine chemicals that were suggested to be conjugated by GST were compared with regard to their abilities to induce umu gene expression and cause cytotoxicity responses in both the NM5004 strain and the original tester strain (S . typhimurium TA1535/pSK1002, which is devoid of GST activity toward 1,2-epoxy-3-(4'-nitrophenoxy)propane) . Ten chemicals--1,2-dibromoethane,N-(2,3-epoxypropyl)phthalimide, 1,3-dichloroacetone, CH2I2, 1,2-epoxy-3-phenoxypropane, 2,3-epoxypropyl p-methoxyphenyl ether, 1-bromo-2-chloroethane, 1-bromo-2,3-dichloropropane, CH2BrCl, and CH2Br2--were found to enhance induction of umu gene expression in the NM5004 strain as compared with the TA1535/pSK1002 strain . 1,2-Epoxy-3-(4'-nitrophenoxy)propane and 2,3-dibromo-1-chloropropane were inactivated by GST 5-5 in the NM5004 tester strain, although these chemicals were cytotoxic in both tester strains . Roles of GST 5-5 were also examined for the inactivation of reactive metabolites of several procarcinogens that were formed through oxidation by liver microsomes of polychlorinated biphenyl-treated rats . The results suggest that reactive metabolites (possibly epoxides) of aflatoxin B1, sterigmatocystin, 1,2-dihydro-1,2-dihydroxy-6-aminochrysene, and (+)- and (-)-enantiomers of 7,8-dihydroxy-7,8-dihydrobenzo{a}pyrene could be trapped as inactivated GSH conjugates in the NM5004 strain . High-performance liquid chromatographic analysis suggested that exo-aflatoxin B1-8,9-oxide--GSH conjugate was formed during the oxidation of aflatoxin B1 by rat and human liver microsomes in the presence of GSH and several GST enzymes including purified rat theta class GST Yrs-Yrs and rat liver GST (a mixture of alpha and mu class enzymes) . Thus, the present results support the view that the theta class rat GST 5-5 enzyme participates in the activation and inactivation of potential environmental carcinogenic chemicals . This newly developed NM5004 tester strain is of use in the elucidation of roles of GST 5-5 in transformations.

Microbiol Immunol, 1996, 40(9), 681 - 4
Purified protein from Salmonella typhimurium inhibits the interleukin-2 response of murine splenic T-lymphocytes activated with anti-CD3 antibody; Matsui K; Previously, we demonstrated that the immunosuppression induced by a purified preparation of Salmonella typhimurium-derived inhibitor of T-cell proliferation (STI) can be observed in terms of suppression of the proliferation of murine spleen cells stimulated with a mitogenic lectin . In the present study, I observed that STI inhibited the interleukin-2 (IL-2) response of purified murine splenic T lymphocytes stimulated with anti-CD3 antibody . The flow cytometric analysis of IL-2 receptor (IL-2R) expression on T cells showed that STI specifically suppressed the expression of IL-2R beta and IL-2R gamma . Furthermore, when the IL-2-dependent T-cell line CTLL-2 was incubated with STI, the growth of CTLL-2 cells was significantly inhibited . These results suggest that the target cells for STI are T cells themselves, and that the suppression of T-cell proliferation induced by STI might involve a defect in the IL-2 receptor (IL-2R) function of T cells.

Teratog Carcinog Mutagen, 1996, 16(2), 125 - 38
An investigation of some Turkish herbal medicines in Salmonella typhimurium and in the COMET assay in human lymphocytes; Basaran AA et al.; Medicinal plants play a major role in the life of Turkish people and of late medicinal plant usage has increased in many countries . Green plants in general contain mutagenic and carcinogenic substances, but there is little information about the biological activities of herbal medicine . In the present study, therefore, various Turkish medicinal herbs were investigated for their genotoxic potential in the Salmonella typhimurium microsomal activation assay and the alkaline single cell gel electrophoresis (COMET) assay . Extracts from these medicinal herbs and some fractions of these extracts were examined . The species investigated were Arctium minus, Ecballium elatterium, Momordica charantia, Plantago major, Urtica dioica, Viscum album, Salvia triloba, Euphorbia rigida, Stachys lavandulifolia, Acteoside, Abies nordmannia . They are used for various immune disorders and are applied either topically or taken orally as a herbal tea . Of the 19 samples of the extracts and fractions investigated, none produced a positive response in strains TA98 and TA100 with or without metabolic activation, but all produced an increase above negative control values in the COMET assay . Some extracts were investigated further and produced dose-related increases . In the case of Urtica and Euphorbia species, where two fractions from these plants were examined, one fraction produced a greater response than the other . It is suggested that the lesser response of the fractions might be due to less DNA strand-breaking agents in the fractions or they may have antigenotoxic properties . The breaks that are detected in the COMET assay could be alkali-labile AP-sites and intermediates in base- or nucleotide-excision repair and are difficult to interpret in terms of hazard for man . Further studies with additional genotoxicity assays would be required to make such a prediction.

Environ Mol Mutagen, 1996, 28(2), 127 - 32
Mutagenicity of cosmetic products containing Kathon; Connor TH et al.; A variety of shampoos, conditioners, skin-care lotions, and other cosmetic products contain the biocide Kathon CG, which is a mixture of two heterocyclic isothiazolinones: methylisothiazolinone and methylchloroisothiazolinone . This mixture and the related biocide, Kathon 886, have been shown to be potent sensitizers and bacterial mutagens . Five cosmetic products that list the components of Kathon on their labels and two that do not were screened for mutagenicity with Salmonella typhimurium TA100 without S-9 . Five of these products and Kathon 886 were further evaluated in TA100 without and with S-9 . Kathon 886, a cosmetic product that contained Kathon, and thin layer chromatography-separated components of Kathon 886 were identified by GC/MS analysis . Three of the five products that listed Kathon were direct acting mutagens with TA100 . The remaining two products were considerably more toxic than the other products and could not be evaluated for mutagenicity . The addition of S-9 reduced toxicity but did not eliminate mutagenicity . The mutagenic evaluation of Kathon 886 resulted in a dose response similar to that seen with some cosmetic products but at a 1,000-fold lower concentration, and activity was also reduced by the addition of S-9 mix . S-9 reduced activity both with and without cofactors present . Thin layer chromatography separation of the components and subsequent identification by GC/MS indicated that methylisothiazolinone was nonmutagenic while methylchloroisothiazolinone was mutagenic . Additionally, a dichlorinated compound was identified which was also mutagenic . In light of these findings and the reported skin sensitization by Kathon CG in various cosmetics, we recommend that additional testing be done to assure the safety of products containing Kathon CG.

Clin Rheumatol, 1996 Jan, 15 Suppl 1, 79 - 85
Immunization of HLA-B27 transgenic and non transgenic mice with Salmonella typhimurium results predominantly in the generation of proliferative T cell responses; Ringrose JH et al.; Reactive arthritis (ReA) due to Gram-negative intestinal bacteria or Chlamydia, is associated by an unknown mechanism with HLA-B27 . Like other MHC class I molecules, HLA-B27 presents antigenic peptides derived from intracellular proteins to CD8+ cytotoxic T cells (CTL) . In humans however, CTL specific for ReA associated bacteria have been reported in a limited number of studies . This may be caused by an inefficient in vivo induction of CTL against such micro-organisms . In the present study we addressed the question whether and to what extend mice transgenic for HLA-B27 are able to generate CTL against Salmonella typhimurium after immunization . To this end both HLA-B27 transgenic and non transgenic mice were immunized i.p., i.v . or orally, receiving a secondary challenge four weeks later . One day after infection with Salmonella, bacteria could be cultured from spleen and liver . There was no significant difference in the number of bacteria cultured from these organs between both groups of mice . Spleen cells from all immunized mice proliferated specifically in the presence of heat killed Salmonella but not in the presence of heat killed Yersinia . No proliferation of spleen cells from naive mice was observed in the presence of heat killed Salmonella, excluding the possibility that Salmonella antigens were mitogenic . Only in one out of 6 mice immunized i.v . with Salmonella Salmonella specific CTL could be generated . In order to rule out the possibility that in HLA-B27 transgenic mice the HLA-B27 molecule is not used as a restriction element by murine T cells, CTL were raised against the male minor histocompatibility (mH) antigen H-Y . Both murine class I as well as HLA-B27 restricted CTL could be generated . In conclusion this study demonstrates that MHC class I restricted CTL specific for the Gram-negative bacterium Salmonella typhimurium are difficult to generate in contrast to proliferative responses which can be easily demonstrated . This may comparable in humans where in the majority of studies bacteria specific T cells isolated from ReA patients appear to be CD4+ and class II restricted.

Mol Microbiol, 1996 Jan, 19(1), 139 - 44
The specificity of fumarate as a switching factor of the bacterial flagellar motor; Barak R et al.; Fumarate restores to flagella of cytoplasm-free, Che Y-containing envelopes of Escherichia coli and Salmonella typhimurium the ability to switch from one direction of rotation to another . To examine the specificity of this effect, we studied flagellar rotation of envelopes which contained, instead of fumarate, one of its analogues . Malate, maleate and succinate promoted switching, but to a lesser extent than fumarate . These observations were made both with wild-type envelopes and with envelopes of a mutant which lacks the enzymes succinate dehydrogenase and fumarase, indicating that the switching-promoting activity of the analogues was not caused by their conversion to fumarate . Aspartate and lactate did not promote switching . Using strains defective in specific enzymes of the tricarboxylic acid cycle and lacking the cytoplasmic chemotaxis proteins as well as some of the chemotaxis receptors, we demonstrated that, in intact bacteria, unlike the situation in envelopes, fumarate promoted clockwise rotation via its metabolites acetyl phosphate and acetyladenylate, but did not promote switching (presumably because of the presence of cytoplasmic fumarate) . All of the results are consistent with the notion that fumarate acts as a switching factor, presumably by lowering the activation energy of switching . Thus fumarate and some of its metabolites may serve as a connection point between the bacterial metabolic state and chemotactic behaviour.

Mol Microbiol, 1996 Jan, 19(1), 1 - 5
Flagellar assembly in Salmonella typhimurium; Aizawa SI; The bacterial flagellum is a motility apparatus in which a long helical filament--the propeller--is driven by a rotary motor embedded in the cell surface . Out of more than 40 genes required for construction of a fully functional flagellum in Salmonella typhimurium, only 18 gene products have been identified in the mature structure . Some other flagellar proteins play logistical roles during construction, which involves the selective export of flagellar components through a central hole in the flagellum . The whole structure is constructed from base to tip by linear assembly; that is, by adding new components on the growing end, resulting in the distal growth of each substructure . Components of the substructures do not necessarily self-assemble, but often demand the help of other proteins . Recent progress in the understanding of flagellar assembly, which has been most extensively studied in S . typhimurium, is reviewed.

Vaccine, 1996 Jan, 14(1), 19 - 24
Evaluation of a phoP/phoQ-deleted, aroA-deleted live oral Salmonella typhi vaccine strain in human volunteers; Hohmann EL et al.; Improved live oral typhoid fever vaccines may be engineered by deletion of Salmonella-specific virulence genes in Salmonella typhi . Ty445, an aroA-deleted S . typhi Ty2 strain also deleted for the phoP/phoQ Salmonella typhimurium virulence regulatory locus, was tested in human volunteers . Volunteers received escalating single doses of the vaccine; subsequently 14 individuals received two doses of 10(10) c.f.u . without significant side-effects . Control vaccinees received four doses of the live oral typhoid vaccine Ty21a . Of controls, 5/8 seroconverted as measured by increases in serum IgG directed against S . typhi O antigen or whole bacterial antigens in ELISAs . Only 2/14 volunteers receiving the experimental vaccine Ty445 seroconverted . Although a delta aroA delta phoP/phoQ S . typhi strain is overattenuated for use as a typhoid fever vaccine, our data demonstrate that the deletion of the phoP/phoQ locus in S . typhi significantly attenuates this human pathogen.

Arzneimittelforschung, 1996 Jan, 46(1), 64 - 7
Effects of amphiphilic compounds on metabolic and biological activities of Salmonella typhimurium; Majtanova L et al.; The effect of two quaternary ammonium salts, (1-methyldodecyl)trimethylammonium bromide (ATDBr) and tetramethylammonium bromide (TMABr), as well as that of two amine oxides, (1-methyldodecyl)dimethylamine oxide (ATDNO) and trimethylamine oxid (TMANO), on both metabolic and biological activities of a S . typhimurium strain isolated from nosocomial infection were studied . The compounds with long alkyl chain in the molecule (ATDBr, ATDNO) affected the growth, synthesis of biomacromolecules (DNA, RNA, proteins) and respiration . The compounds lacking the alkyl chain (TMABr, TMANO) were ineffective with exception of the endogenous respiration that they inhibited in a less degree in the highest concentrations tested . The highest inhibition of respiration was evoked by ATDBr and ATDNO in the presence of pyruvate as an exogenous carbon substrate . The sub-MICs of quaternary ammonium salts induced a prophage of the lysogenic S . typhimurium strain, amine oxides were ineffective . The tested compounds decreased at one-fourth of the MICs the permeability reaction . The results showed that both physiologic and metabolic processes were inhibited in dependence on the length of the alkyl chain, however, amine oxides were less effective in biological activities.

FEMS Immunol Med Microbiol, 1996 Jan, 13(1), 19 - 28
Differentiated U937 cells and human monocytes exhibit a differential production of extracellular oxygen species: O2.- excretion versus H2O2 diffusion; Chateau MT et al.; The nature and the localization of the oxidative response triggered by different stimuli in either differentiated U937 cells and peripheral blood-derived human monocytes was investigated using luminometric and cytofluorometric techniques . Differentiated U937 cells essentially produced extracellular superoxide anion (O2.-), whatever the stimulus used . Monocytes, however, responded to Salmonella typhimurium, phorbol esters, and opsonized zymosan by an intracellular, an extracellular, and both an intra- and extracellular production of oxygen species, respectively . Furthermore, H2O2 but not O2.- was detected in the extracellular oxidative response of monocytes . Using differentiated U937 cells, luminol was found to be as efficient as lucigenin in the detection of extracellular O2.-, providing sufficient concentrations of extracellular horseradish peroxidase were present . However, both azide and histidine inhibited the lucigenin-enhanced chemiluminescence, suggesting an initial and transient production of singlet oxygen by differentiated U937 cells . Taken together these results strongly suggest that, when stimulated, differentiated U937 cells directly excrete O2.- in the extracellular medium while, within monocytes, O2.- is rapidly dismutated in H2O2 which can eventually diffuse outside the cell . Such differences in the oxidative response between the two cell types could be explained by the lack of total closure of the phagosome, only observed in differentiated U937 cells.

Rev Argent Microbiol, 1996 Jan-Mar, 28(1), 1 - 8
{Genotoxicity resulting from a complex mixture of environmental origin using 2 bacterial systems}; Lopez L et al.; The genotoxicity of environmental complex mixtures was tested with Ames mutagenicity assay and with Bacillus subtilis rec assay for estimation of primary DNA damage . Soil samples obtained from an area contaminated with petrochemical wastes were sequentially extracted with ethyl ether and methanol . After evaporation to dryness the extracts were resuspended in dimethylsulfoxide for their use in biological tests . No mutagenic response was detected with the Ames test . Ether extract from parcel 1 showed an unusually high toxicity for Salmonella typhimurium TA100 and induced DNA damage in Bacillus subtilis with a clear relationship dose-genotoxic response . The analysis of the results obtained in both assays and the nature of the samples indicate that the genotoxicity detected in the complex mixture is compatible with the one caused by DNA intercalating compounds.

Genetics, 1996 Jan, 142(1), 11 - 24
Evolution of coenzyme B12 synthesis among enteric bacteria: evidence for loss and reacquisition of a multigene complex; Lawrence JG et al.; We have examined the distribution of cobalamin (coenzyme B12) synthetic ability and cobalamin-dependent metabolism among enteric bacteria . Most species of enteric bacteria tested synthesize cobalamin under both aerobic and anaerobic conditions and ferment glycerol in a cobalamin-dependent fashion . The group of species including Escherichia coli and Salmonella typhimurium cannot ferment glycerol . E . coli strains cannot synthesize cobalamin de novo, and Salmonella spp . synthesize cobalamin only under anaerobic conditions . In addition, the cobalamin synthetic genes of Salmonella spp . (cob) show a regulatory pattern different from that of other enteric taxa tested . We propose that the cobalamin synthetic genes, as well as genes providing cobalamin-dependent diol dehydratase, were lost by a common ancestor of E . coli and Salmonella spp . and were reintroduced as a single fragment into the Salmonella lineage from an exogenous source . Consistent with this hypothesis, the S . typhimurium cob genes do not hybridize with the genomes of other enteric species . The Salmonella cob operon may represent a class of genes characterized by periodic loss and reacquisition by host genomes . This process may be an important aspect of bacterial population genetics and evolution.

Rocz Panstw Zakl Hig, 1996, 47(1), 77 - 85
{Evaluation of mutagenic activity in water disinfected with chlorine}; Bilyk A et al.; City of Wroclaw is supplied with water from Olawa . The main contaminations of water are high concentration of organic compounds and bacteria count . Raw and drinking water show some mutagenic and carcinogenic properties in Ames tests . To improve the quality of drinking water now technology bored on infiltrated water composed of, coagulation, filtration and disinfection was tested . The goal of investigation and was to examine mutagenic and carcinogenic properties of raw and treated water . Potential carcinogenic activity of volatile disinfection-by-products was estimated by direct analysis of THMs, while for nonvolatile halogenated organic substances Ames test was used . Carcinogenic risk based on THMs concentration could be estimate as 10(-5) for chlorine and 10(-6) for chlorine dioxide . Ozonation and post chlorination did not lowered the risk . Positives results of Ames test obtained for raw water no 2 with Salmonella typhimurium TA100, and for chlorinated treated water with Salmonella typhimurium TA98 . The treatment of water with chlorine transforms same compounds into carcinogenic chlorinated derivatives and does not eliminate its harmful properties . Our results suggest that not all methods of treatment remove harmful to the health components from the water . Consequently in the case of the presence of such compounds in surface water it is necessary to employ appropriate methods and procedures the used Ames test allows rapid determination of the presence of carcinogenic compound in water . In Poland determination of the presence of potential carcinogens in water destined for the supply of urban areas is not obligatory and standard analyses of chemical composition do not give such information . It seems that the mentioned test could be considered for the control of the quality of raw and treated water as an indispensable measure contributing to reducing the health hazard for the population.

Jpn J Ophthalmol, 1996, 40(1), 12 - 7
Early expression of intercellular adhesion molecule-1 in the corneal endothelium stimulated by endotoxin: an immuno-scanning electron microscopical study; Yamaguchi K et al.; Three-dimensional localization of the intercellular adhesion molecule-1 (ICAM-1) in the corneal endothelium stimulated by Salmonella typhimurium endotoxin was investigated using immuno-scanning electron microscopy (SEM) . Samonella typhimurium endotoxin, 100 micrograms, was injected in Lewis rats weighing 200 grams . The animals, including the controls, were sacrificed and both eyes enucleated at 0, 3, 12 and 24 hours after injection (n = 3 each time) . After resection, the corneas were immersed in hypothermic University of Wisconsin solution with monoclonal mouse-anti-rat ICAM-1 IgG and then goat-anti-mouse IgG coupled to 15 nm gold particles . Then the corneas were prepared conventionally for scanning electron microscopy . Histotopographical examination with immuno-SEM revealed that ICAM-1 antigen increased on the corneal endothelium by 3 hours postinjection . The particles were arranged along the cytoplasmic processes, especially at the summits . The number of particles was 3.3 +/- 0.8/microns2 in the control, 3.6 +/- 0.8/microns2 at 0 hour, 14.4 +/- 0.9/microns2 at 3 hours, 25.4 +/- 1.4/microns2 at 12 hours, and 22.7 +/- 2.6/microns2 at 24 hours postinjection . ANOVA indicated that the time-course was an important factor (P < 0.01) . Our results showed that ICAM-1 could be augmented in the corneal endothelium by endotoxin . The interrelationship between ICAM-1 expression and cytoplasmic processes seems to be important for the neutrophil-binding mechanism.

Mikrobiologiia, 1996 Jan-Feb, 65(1), 84 - 8
{Dependence of the intensity of 2,4,6-trinitrotoluene-induced mutagenesis on the physiological state of tester strains of Salmonella typhimurium}; Il'inskaia ON et al.; The dependence of the mutagenic potential of 2,4,6-trinitrotoluene (TNT) on the growth phase of the Salmonella typhimurium tester strains TA100 and TA98 is shown . The mutagenic potential proved to be maximal for log-phase cultures, being higher with strain TA100 than TA98 . The amount of TNT absorbed by bacteria in the first minutes of contact varied, depending on culture age, and was the largest at the beginning of the exponential growth phase . A direct relation between the number of microbial cells and the amount of TNT absorbed from the medium was shown . The analysis of infrared (IR) spectra of cells after a 3-min contact with TNT, demonstrated that, at physiological temperatures, 2.4% of the original amount of TNT was present in cells of the tester strain in an unaltered form, while, at a lowered temperature, this value was 10.3% . Thus, the retardation of physiological processes in the cell changes the intensity of mutagen absorption and transformation . It is emphasized that an accurate assessment of mutagenicity should take into account the physiological state of tester bacteria.

Adv Exp Med Biol, 1996, 397, 15 - 21
Hybrid hepatitis B virus core antigen as a vaccine carrier moiety . II . Expression in avirulent Salmonella spp . for mucosal immunization; Schodel F et al.; Hepatitis B virus (HBV) core antigen (HBcAg) is a highly immunogenic subviral particle . We and others have defined insertion sites for heterologous epitopes and successfully used hybrid particles to generate B and T cell immunity (reviewed in: Schodel et al . 1994a, 1995) . Here we shall review recent progress in constructing avirulent Salmonella spp . expressing hybrid HBcAg particles carrying different epitopes . Hybrid HBcAg particles carrying virus neutralizing epitopes of the hepatitis B virus pre-S region or repeat epitopes of plasmodial circumsporozoite antigens were previously described (Schodel et al . 1992, 1994b) . Salmonella spp . can be attenuated by defined genetic means so that they become avirulent, yet preserve invasiveness after oral uptake . Hybrid HBcAg-pre-S particles were expressed in Salmonella typhimurium and S . typhi vaccine strains . A single oral immunization of mice with such live recombinant S . typhimurium strains elicited a high titered serum anti-pre-S1 IgG response . Similarly, circumsporozoite repeat epitopes of three different malaria parasites were expressed as HBcAg-CS hybrids in recombinant S . spp . and were found to be highly immunogenic after oral immunization . To analyze mucosal immune responses, BALB/c mice were immunized with recombinant phoPc S . typhimurium expressing HBcAg by various mucosal routes (Hopkins et al., 1995) . All routes of immunization resulted in high titered serum and local antibodies against HBcAg and S . typhimurium LPS . However, nasal immunization was most efficient in generating pulmonary IgA and rectal immunization in eliciting rectal IgA, suggesting some compartmentalization of the mucosal immune response.

Avian Dis, 1996 Jan-Mar, 40(1), 28 - 36
The effect of bacterial surface structures on the pathogenesis of Salmonella typhimurium infection in chickens; Lee MD et al.; Mutants of Salmonella typhimurium strain SR11 containing Tn10 insertions in genes encoding for the motility and fimbriation phenotypes were evaluated for adhesion, invasion, and virulence in avian models . Mutations abolishing mannose-sensitive or mannose-resistant hemagglutination did not influence adherence or invasion in epithelial cells in vitro . A double hemagglutinin-deficient mutant, lacking both mannose-sensitive and mannose-resistant hemagglutinins, was diminished in ability to adhere to chick kidney epithelial cells in vitro, but invasion in vitro was not significantly affected . Compared with the wild-type parent, mutations that decreased motility reduced invasion levels in vitro and increased the peroral LD50 in one-day-old chicks . A mutant deficient in both motility and mannose-sensitive hemagglutination was greatly reduced in its ability to invade epithelial cells in vitro and persist in the liver and spleen of orally challenged chicks . Results of this study indicate that loss of motility in S . typhimurium attenuates peroral virulence in chicks.

Gene, 1996, 173(1 Spec No), 47 - 52
Applications for green fluorescent protein (GFP) in the study of host-pathogen interactions; Valdivia RH et al.; The green fluorescent protein (GFP) from Aequorea victoria is a novel fluorescent marker that has potential use in the study of bacterial pathogenicity . To explore some of the potential applications of GFP to the study of host-parasite interactions, we constructed two GFP expression vectors suitable for different facultative intracellular bacterial pathogens . The first expression vector was tested in the enteric pathogens, Salmonella typhimurium and Yersinia pseudotuberculosis, and the second vector tested in Mycobacterium marinum (Mm) . Both expression vectors were found to be stable and to direct high levels of GFP synthesis . Standard epifluorescence microscopy was used to detect all three bacterial pathogenic species during the early and late stages of infection of live mammalian cells . Mm expressing gfp was also visualized in infected animal tissues . gfp expression did not adversely affect bacterial survival, nor did it compromise entry into mammalian cells or their survival within macrophages . In addition, all three gfp-expressing bacterial pathogens could be detected and sorted in a flow cytometer, either alone or in association with epithelial cells or macrophages . Therefore, GFP not only provides a convenient tool to image pathogenic bacteria, but allows the quantitative measurement of bacterial association with mammalian cells.

Life Sci, 1996, 59(3), 263 - 71
Lack of an EMF-induced genotoxic effect in the Ames assay; Morandi MA et al.; A few epidemiological studies have linked exposure to electromagnetic fields (EMF) and the incidence of cancer . Since many carcinogens are mutagens in the Ames assay, the purpose of this study was to determine if exposure of four tester strains of Salmonella typhimurium (TA97a, TA98, TA100, and TA102) to EMF would increase their rate of mutation . Parallel plate electrodes and Helmholtz coils were used to create uniform field properties (300 V/in., 0.3 mT) . Separate and combined alternating electric and magnetic fields effects were studied at a combined field frequency of 60, 600, and 6000 Hz at room temperature . These fields did not elevate the temperature of the culture plates above room temperature, Petri dishes containing each tester strain in top agar were exposed to an electric field (E), magnetic field (M), combined electric and magnetic field (EM), or no additional field above ambient conditions in the lab (control) . Four plates containing each strain were exposed in each condition: two plates had the appropriate positive-control mutagen for each strain included in the top agar and two plates did not . Plates were exposed to either E, M, EM, or control conditions at room temperature for 48 hr . and then incubated an additional 24 hr . at 37 deg . C . The plates containing mutagen in the top agar showed an increased number of colonies consistent with mutagenesis . However, the rate of mutation in the S . typhimurium strains TA97a, TA98, TA100, and TA102 in either the presence or absence of mutagen was not affected by 48 hr . exposure at room temperature to E, M, or EM fields at 60, 600, or 6000 Hz.

Microb Pathog, 1996 Jan, 20(1), 11 - 30
Construction of recombinant aroA salmonellae stably producing the Shigella dysenteriae serotype 1 O-antigen and structural characterization of the Salmonella/Shigella hybrid LPS; Falt IC et al.; The TN501 mercury resistant transposon containing the rfp and rfb loci encoding biosynthesis of the O-antigen of Shigella dysenteriae serotype 1 lipopolysaccharide (LPS) was constructed and introduced into aroA mutants of Salmonella typhimurium and Salmonella dublin . In five recombinant strains, both homologous LPS and hybrid LPS, consisting of Salmonella lipid A-core and Shigella O-antigen, were produced . All derivatives but one (SL3235) stably inherited the new trait . Immunofluorescence microscopy, using mixtures of differentially-labelled antibodies specific for either the Salmonella or the Shigella O-antigen, demonstrated that individual bacteria produced both types of LPS . Qualitative and quantitative analysis of polysaccharides obtained by mild hydrolysis of purified LPS was carried out by methylation analysis and NMR spectroscopy, and revealed that the ratio of Salmonella to Shigella O-antigen repeating units in the high molecular weight fraction of isolated polysaccharides varied from 1.3: 1 to 8.4:1 as based on the relative proportions of 1,4,5-tri-O-acetyl-2,3-di-O-methyl-L- rhamnitol (Salmonella repeating unit) and 1,3,5-tri-O-acetyl-2,4-di-O-methyl-L-rhamnitol (Shigella repeating unit) . The attachment site of the Shigella O-antigen to the Salmonella core was investigated by construction of a mutant rfp-rfb gene cluster encoding the synthesis of only one repeat unit of the Shigella dysenteriae type 1 O-antigen, and its introduction into a rough Salmonella strain . This hybrid organism produced a polysaccharide with the following structure, {formula: see text} demonstrating that the Shigella dysenteriae type 1 O-antigen is linked at position O-4 of the subterminal D-glucose unit in the Salmonella core.

Mutagenesis, 1996 Jan, 11(1), 37 - 41
Relationship between antimutagenic activity and major components of various teas; Yen GC et al.; The objectives of this study were to determine the major components in tea leaves and tea extracts and to study the relationship between chemical content and antimutagenic activity of various tea extracts . The amount of catechins in various tea extracts was in the order: green tea (26.7%) > oolong tea (23.2%) > pouchong tea (15.8%) > black tea (4.3%) . The amounts of caffeine and phenolic compounds in oolong tea extracts were 8.3 and 32.4%, respectively; these amounts were greater than those in the other three tea extracts . The ascorbic acid in green tea extracts was a little higher than in oolong and pouchong tea extracts . The amount of catechins in tea leaves also showed the order: nonfermented (green tea) > semifermented (pouchong tea and oolong tea) > fermented tea (black tea) . The amounts of caffeine and phenolic compounds in oolong tea leaves are also higher than in other tea leaves . Besides water soluble components, tea leaves also contain several lipid soluble chemicals such as beta-carotene and tocopherols . The tea extracts, especially oolong and pouchong teas, markedly inhibited the mutagenicity of 2-amino-3-methylimidazo (4,5-f)quinoline (IQ), 3-amino-1,4-dimethyl-5H-pyrido-(4,3-b)indole (Trp-P-1), 2-amino-6-methyl-dipyrido(1,2-a:3',2'-d) imidazole (Glu-P-1), benzo{a}pyrene (B{a}P) and aflatoxin B1 (AFB1) . The inhibitory effect of tea extracts against the mutagenicity of IQ and Glu-P-1 in Salmonella typhimurium TA100 showed a significant (P < 0.05) correlation to the contents of catechins and ascorbic acid . The antimutagenic activity of tea extracts to Trp-P-1 in TA98 or TA100 was well correlated (P < 0.05) to the caffeine contents . No significant (P > 0.05) correlation was found between the antimutagenicity of tea extracts to B{a}P and AFB1 in TA100 and the content of major components in tea extracts.

Mutagenesis, 1996 Jan, 11(1), 27 - 31
Bacterial mutagenic evaluation of Luxabendazole, a new broad spectrum anthelmintic, with the Salmonella typhimurium His- and the Escherichia coli Tryp- reversion tests; Ortiz AI et al.; Luxabendazole is a new benzimidazole carbamate chemotherapeutic agent, which has proved to be effective against adult and immature stages of the major gastrointestinal nematodes, trematodes and cestodes . The mutagenic properties of Luxabendazole were investigated in the in vitro Ames Salmonella and E . coli tests . The product was tested at concentrations of 0.5, 5, 50, 500, 1250 and 2500 micrograms/plate in the TA1535, TA1538, TA98 and TA100 strains of Salmonella typhimurium, and 0.5, 5, 50 and 500 micrograms/plate in the WP2, WP2uvrA- and its pKM 101-containing derivative CM891 (WP2 uvrA- pKM101) strains of Escherichia coli, with and without S9 microsomal activation (post-mitochondrial liver fraction from Wistar rats pretreated with Aroclor(R)) . Positive and negative controls were included in each experiment . From the present study it can be concluded that Luxabendazole, over a dose range of 0.5-2500 micrograms/plate, is unlikely to present a mutagenic hazard, as demonstrated by the Ames test.

J Antimicrob Chemother, 1996 Jan, 37(1), 85 - 91
Increasing ciprofloxacin resistance in salmonellas in England and Wales 1991-1994; Frost JA et al.; Ciprofloxacin is now widely used as the drug of choice for those severe salmonella infections where antibiotic therapy is indicated . Between 1991 and 1994 ciprofloxacin resistance in salmonellas isolated from humans in England and Wales increased from 0.3% to 2.1% . Among the most prevalent serotypes the highest incidence was seen in Salmonella hadar where ciprofloxacin resistance has increased from 2.0% in 1991 to 39.6% in 1994 . The incidence of ciprofloxacin resistance remains uncommon in other serotypes and in 1994 5.1% of Salmonella virchow and Salmonella newport were resistant compared with 1.4% of Salmonella typhimurium and 0.4% of Salmonella enteritidis . There has been a number of examples of development of resistance to quinolone drugs during treatment of human infections . Ciprofloxacin resistance also occurs in salmonellas isolated from food animals and human food . This increasing incidence of ciprofloxacin resistance reflects the more widespread use of fluoroquinolone drugs in both humans and food animals.

J Antimicrob Chemother, 1996 Jan, 37(1), 105 - 15
Intracellular distribution of ampicillin in murine macrophages infected with Salmonella typhimurium and treated with (3H)ampicillin-loaded nanoparticles; Balland O et al.; The intracellular distribution of (3H)ampicillin-loaded polyisohexylcyanoacrylate nanoparticles was studied in murine macrophages (peritoneal cells and the J774 cell line) infected by Salmonella typhimurium C5, using ultrastructural autoradiography . Ampicillin penetration and retention into the cells obviously increased by means of nanoparticles . After short-term (2-4 h) treatment with the nanoparticle formulation, numerous intracellular bacteria were seen to be in the process of destruction . The tritium labelling was located in the cell cytoplasm and inside vacuoles in which bacteria undergoing degradation were often present . After long-term (12 h) treatment, numerous spherical bodies (d: 100 nm to 500 nm) and larger forms (2 microns) were seen in the vacuoles . Radioactivity was mainly found to be localized on the spherical bodies, indicating marked damaging action of the ampicillin on the bacterial walls . The targeting of ampicillin therefore allowed its penetration into the macrophages and vacuoles infected with S . typhimurium.

Eur J Clin Microbiol Infect Dis, 1996 Jan, 15(1), 85 - 8
Changes in susceptibility of Salmonella enteritidis, Salmonella typhimurium, and Salmonella virchow to six antimicrobial agents in a Spanish hospital, 1980-1994; Ramos JM et al.; To determine changes in the susceptibility patterns of Salmonella enteritidis, Salmonella typhimurium, and Salmonella virchow over time, resistance to ampicillin, chloramphenicol, tetracycline, gentamicin, trimethoprim/sulfamethoxazole, and nalidixic acid was studied by the disk diffusion method in 1,024, 191, and 61 clinical isolates of these organisms, respectively . All isolates were recovered from 1980 to 1994 at a hospital in Madrid, Spain . Salmonella enteritidis isolates were less resistant (10.9%) than Salmonella typhimurium (43.5%) and Salmonella virchow (36.1%; p < 0.001) . The incidence of resistance of Salmonella enteritidis to ampicillin increased from 2.7% during the period 1980-1982 to 15.6% during 1992-1994 (p < 0.001) . The resistance of Salmonella typhimurium to ampicillin, chloramphenicol, and tetracycline increased from 15.2%, 7.6%, and 21.2% respectively in 1980-1982 to 73.3%, 46.7%, and 73.3% in 1992-1994 (p < 0.001) . These marked increases in antimicrobial resistance suggest the need for public health interventions, several of which are discussed.

Environ Mol Mutagen, 1996, 27(3), 202 - 10
Unexpected genetic toxicity to rodents of the N',N'-dimethyl analogues of MNU and ENU; Tinwell H et al.; Lijinsky and his colleagues have reported that the N',N'-dimethyl analogues of ENU and MNU {N',N'-dimethyl-N-ethyl-N-nitrosourea (DMENU) and trimethylnitrosourea (TMNU), respectively} are carcinogenic to rats despite their extreme hydrolytic stability which would reduce or preclude generation of alkylating species analogous to those formed upon hydrolysis of ENU and MNU . Lijinsky and his colleagues were unable to rationalize those activities of DMENU and TMNU despite extensive experimentation . We therefore decided to study this problem further . Whichever mode is accepted for the generation of electrophilic/mutagenic/carcinogenic reactive species from ENU and MNU, blocking of the free-NH2 group with methyl groups (-NMe2) should ablate or abolish activity . Consistent with this DMENU and TMNU gave negative results in the NBP alkylation test while the parent compounds gave an instantaneous deep blue coloration . Studies of the rate of hydrolysis of these four compounds revealed ENU and MNU to have half-lives of 8 min, while the alkylated analogues (DMENU and TMNU) had half-lives of 25 and 41 days, respectively . Hydrolysis of ENU and MNU, to yield the alkylating species, proceeds either via proton abstraction from the -NH2 group or by attack by water on the carbon of the carbonyl group . Methylation will inhibit both of these pathways, the first absolutely (no -NH2 protons) and the second partially, via steric inhibition . The slow hydrolysis observed for DMENU and TMNU suggests that the latter route of hydrolysis is applicable . Studies with strain TA1535 of Salmonella typhimurium (without S9 mix) confirmed the potent mutagenic activity for ENU and MNU (approximately 300-fold increase in revertants at 2,000 micrograms/plate and approximately 180-fold increase in revertants at 150 micrograms/plate respectively) . In contrast, the methylated analogues showed only weak mutagenic activity (approximately 3-fold) at approximately 100-fold higher dose-levels . Addition of S9 mix did not affect the mutagenicity of DMENU or TMNU . To this point, hypothesis and data coincide . ENU and MNU are potent micronucleus-inducing agents to the mouse bone marrow, and given the above data, it was expected that DMENU and TMNU would show weak or no activity in that assay . In fact, the methylated analogues were as effective as ENU and MNU as clastogens to the mouse bone marrow . Four possible reasons for this conflict of theory and data are explored . The speculative explanation we favour for these effects is that the net alkylation of bone marrow DNA is the same for all four chemicals . With ENU and MNU, most of the alkylating activity is dissipated by rapid hydrolysis . Thus, only a small fraction of the administered dose survives to alkylate the bone marrow . Due to the enhanced stability of the methyl analogues most of the delivered dose will reach the bone marrow . However, because of their lower intrinsic reactivity, only a small fraction of the target dose will alkylate the bone marrow DNA during the time window of the experiment . If these opposing influences happen to balance out, the essentially identical bone marrow genetic toxicity for the four chemicals could be explained.

Food Chem Toxicol, 1996 Jan, 34(1), 33 - 42
Effect of eugenol on the genotoxicity of established mutagens in the liver; Rompelberg CJ et al.; The influence of in vivo treatment with eugenol on established mutagens was studied to determine whether eugenol has antigenotoxic potential . The effects of eugenol in rats was investigated in the unscheduled DNA synthesis (UDS) assay with established mutagens and the Salmonella typhimurium mutagenicity assay . In addition, the effect of in vivo treatment with eugenol on benzo{a}pyrene (B{a}P)-induced genotoxicity in human hepatoma cell line Hep G2 was investigated in the single-cell gel electrophoresis assay . The mutagenicity of B{a}P in the S . typhimurium mutagenicity assay was lower in liver S-9 fractions from control rats . Incubation of liver S-9 fractions from eugenol-treated rats with dimethylbenzanthracene (DMBA) had no antimutagenic effect . Eugenol did not modify UDS activity in hepatocytes isolated from rats pretreated with eugenol orally after exposure of these cells in vitro to DMBA and aflatoxin B1 . Four different treatment schemes of combinations of B{a}P and eugenol were examined in Hep G2 cells: pre-treatment with eugenol; simultaneous treatment with eugenol and B{a}P; a combination of these (pretreatment/simultaneous treatment); and post-treatment with eugenol . An increase in the genotoxicity of B{a}P was found in Hep G2 cells . No effect of eugenol on the genotoxicity of B{a}P was found with the pre- and post-treatments . It is concluded that the effect of eugenol on genotoxicity induced by established mutagens is not univocal; in vivo treatment of rats with eugenol resulted in a reduction of the mutagenicity of B{a}P in the S . typhimurium mutagenicity assay, while in the UDS assay no effect of eugenol was found . In vitro treatment of cultured cells with eugenol resulted in an increase in genotoxicity of B{a}P . These findings indicate that there is only limited support for the antigenotoxic potential of eugenol in vivo.

Environ Mol Mutagen, 1996, 27(2), 110 - 5
Analysis of the ciprofloxacin-induced mutations in Salmonella typhimurium; Clerch B et al.; The mutagenic events induced by ciprofloxacin, a potent antimicrobial agent, have been characterized . For this, a battery of His mutants of Salmonella typhimurium (hisG428, his G46, His C9070, and his G1775 targets) that detects the six possible transitions and transversions {Levin and Ames (1986): Environ Mutagen 8:9-28} and two additional His strains (hisC3076 and his D3052 targets) carrying frameshift mutations have been used . Our results indicate that GC-TA transversions are the major base-pair substitution induced by ciprofloxacin and that GC-At transitions are also produced, but to a lesser degree . However, we cannot discard the fact that At-Ta transversions are also induced . In addition, the data indicate that the mutational specificity of ciprofloxacin depends on the location of the target . Intragenic base-pair substitutions are the most frequent mutations at the hisG428 target when it is on the chromosome, whereas 3 or 6 base-pair deletions are the major mutagenic events when this target is on the plasmid pAQ1 . We have shown that ciprofloxacin also induces deletions/insertions at the hisC3076 and hisD3052 frameshift targets . Therefore, this inhibitor of DNA gyrase promotes a wide pattern of mutations including different kinds of base-pair substitutions, 3 or 6 base-pair deletions, and insertions/deletions resulting in frameshifts . All of these mutagenic events require the MucAb proteins involved in the error-prone repair, with the exception of base-pair insertions/deletions at the hisD3052 target, which are independent of the presence of plasmid pKM101.

Mutat Res, 1996 Jan, 367(1), 43 - 9
Mutagenicity of 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid treated with nitrite in the presence of alcohols; Higashimoto M et al.; The mutagenicity of a product produced from 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid (MTCCA), which is a component in soy sauce, after treatment with 50 mM nitrite at pH 3, 37 degrees C, for 60 min in the presence of 7.5% ethanol was much higher than that in the absence of ethanol during the nitrite treatment . The enhancement of the mutagenicity of nitrite-treated MTCCA by ethanol required simultaneous treatment of MTCCA with nitrite and ethanol . The mutagenicity of MTCCA treated with nitrite in the presence and absence of ethanol was detected in the same fractions on HPLC and was highest for Salmonella typhimurium strain YG1029 possessing elevated O-acetyltransferase activity among the several Salmonella test strains, suggesting that the same mutagen belonging to aromatic compounds was produced both in the presence and absence of ethanol . Methanol, n-propanol and isopropanol as well as ethanol were also observed to have an augmenting effect . However, the sugars glucose and sucrose had no effect . When MTCCA was treated with nitrite in the presence of commercial alcoholic beverages equivalent to 1.25-10% ethanol, Japanese 'sake' and 'shochu' were demonstrated to have a highly augmenting effect and beer, wine, whisky and brandy to have a mildly augmenting effect.

Appl Environ Microbiol, 1996 Jan, 62(1), 221 - 9
Isolation, DNA sequence analysis, and mutagenesis of a proline dehydrogenase gene (putA) from Bradyrhizobium japonicum; Straub PF et al.; We report here the cloning and sequencing of the gene for proline dehydrogenase (putA) of Bradyrhizobium japonicum . An open reading frame coding for 1,016 amino acids was identified . The B . japonicum gene codes for a bifunctional protein with proline dehydrogenase and pyrroline-5-carboxylate (P5C) dehydrogenase activities, as it does in Escherichia coli and Salmonella typhimurium . Comparison of the sequences of these proteins with other proline and P5C dehydrogenase sequences identified proline dehydrogenase and P5C dehydrogenase catalytic domains . Within the proline dehydrogenation domain, several areas of high identity were observed between B . japonicum, E . coli, S . typhimurium, Saccharomyces cerevisiae put1, and Drosophila melanogaster slgA . Within the P5C dehydrogenase domain, several areas of high identity were observed between B . japonicum, E . coli, S . typhimurium, Bacillus subtilis ipa76d, and S . cerevisiae put2 . A consensus catalytic site for semialdehyde dehydrogenase was observed in the P5C dehydrogenase domain . This suggests that the substrate for this domain may be the open-chain gamma-glutamylsemialdehyde, not its cyclized form, P5C . Unlike the gene isolated from E . coli, S . typhimurium, and K . pneumoniae, the B . japonicum putA gene does not appear to be part of an operon with the proline porter gene (putP) . Additionally, the B . japonicum gene lacks the putative C-terminal regulatory domain present in the E . coli and S . typhimurium genes . The gene was disrupted by insertion of antibiotic resistance gene cassettes, which were then recombined into the bacterial chromosome . Symbiotically active mutant strains that were devoid of putA activity were isolated . With this proline dehydrogenase clone, we will test the hypothesis that putA in symbiotic nitrogen-fixing B . japonicum bacteroids is transcriptionally regulated by drought and other stresses.

Appl Environ Microbiol, 1996 Jan, 62(1), 1 - 5
Production and characterization of monoclonal antibodies against the O-5 antigen of Salmonella typhimurium lipopolysaccharide; Jaradat ZW et al.; Three murine monoclonal antibodies (MAbs) were produced by fusion of P3X63-Ag8.653 myeloma cells and splenocytes of a mouse immunized with heat-attenuated (20 min, 80 degrees C) Salmonella typhimurium cells . MAbs 5A5 and 5B2 were of the immunoglobulin M (IgM) class, while MAb 4A8 was IgG2a . All possessed the kappa light chains . The MAbs were specific to the lipopolysaccharide (LPS) O-5 antigen of Salmonella B serogroup, as determined by electrophoresis followed by immunoblotting . All MAbs recognized the same epitope, as determined by an additive enzyme-linked immunosorbent assay (ELISA), although IgM MAbs exhibited higher avidity than the IgG MAb . ELISA analyses revealed that all three MAbs reacted with S . typhimurium (LPS O:1, 4, 5, and 12) while failing to recognize S . typhimurium var . copenhagen (LPS O:1, 4, and 12) . The MAbs reacted equally with live and heat-attenuated Salmonella B serovars containing LPS O-5 antigen . The ability of the MAbs to detect live bacterial cells was further confirmed by transmission electron microscopy . Treatment of bacteria with cholic acid and extremely low pH did not affect antibody binding to S . typhimurium . However, when S . typhimurium cells were exposed to alkaline conditions prior to reaction with all three MAbs, no binding was observed . The use of MAbs to discriminate between S . typhimurium and S . typhimurium var . copenhagen in meat samples was investigated.

Carcinogenesis, 1996 Jan, 17(1), 163 - 6
Human glutathione S-transferase T1-1 enhances mutagenicity of 1,2-dibromoethane, dibromomethane and 1,2,3,4-diepoxybutane in Salmonella typhimurium; Thier R et al.; The rat theta class glutathione S-transferase (GST) 5-5 has been shown to affect the mutagenicity of halogenated alkanes and epoxides . In Salmonella typhimurium TA1535 expressing the rat GST5-5 the number of revertants was increased compared to the control strain by CH2Br2, ethylene dibromide (EDB) and 1,2,3,4-diepoxybutane (BDE); in contrast, mutagenicity of 1,2-epoxy-3-(4'-nitro-phenoxy)propane (EPNP) was reduced . S.typhimurium TA1535 cells were transformed with an expression plasmid carrying the cDNA of the human theta ortholog GST1-1 either in sense or antisense orientation, the latter being the control . These transformed bacteria were utilized for mutagenicity assays . Mutagenicity of EDB, BDE, CH2Br2, epibromohydrin and 1,3-dichloroacetone was higher in the S.typhimurium TA1535 expressing GSTT1-1 than in the control strain . The expression of active enzyme did not affect the mutagenicity of 1,2-epoxy-3-butene or propylene oxide . GSTT1-1 expression reduced the mutagenicity of EPNP . Glutathione S-transferase 5-5 and GSTT1-1 modulate genotoxicity of several industrially important chemicals in the same way . Polymorphism of the GSTT1 locus in humans may therefore cause differences in cancer susceptibility between the two phenotypes.

Carcinogenesis, 1996 Jan, 17(1), 115 - 9
The role of acetylation in the mutagenicity of the antitumor agent, batracylin; Stevens GJ et al.; The role of acetylation in the genotoxicity of the heterocyclic amine, batracylin, was evaluated in Salmonella typhimurium strains expressing various levels of N- and O-acetyltransferase activity . A significant correlation was observed between batracylin-induced mutagenicity and bacterial N-acetyltransferase activity . Strains with the greatest capacity for N-acetylating batracylin (YG 1012 and YG 1024) were the most sensitive to the mutagenic effects of the drug . The number of revertants/nmol batracylin and the formation of acetylbatracylin were approximately 50-fold greater in YG 1024 compared to TA 98 which expresses endogenous levels of N-acetyltransferase . A similar response was observed with strains YG 1012 and TA 1538 . Strains (TA 98/1,8-DNP6 or TA 1538/1,8-DNP6) which lack the ability to N-acetylate batracylin were the least sensitive to the mutagenic effects of the drug . At 1 microgram/plate of batracylin, the number of revertants in TA 1538 and TA 98 was 4-fold higher than that observed in TA 1538/1,8-DNP6 and TA 98/1,8-DNP6 . To determine if batracylin was a substrate for human N-acetyltransferases, assays were performed in bacteria expressing NAT1 or NAT2 . Both strains were capable of N-acetylating batracylin . The strain expressing NAT2 (DJ 460) formed a significantly greater amount of acetylbatracylin, as well as batracylin-induced revertants, compared to the strain expressing NAT1 (DJ 400) . These results demonstrate that the mutagenicity of batracylin is directly related to N-acetyltransferase activity . Data obtained in bacteria expressing either human NAT1 and NAT2 show that batracylin is capable of being bioactivated by both human enzymes . In addition, the higher enzyme activity and mutagenicity in bacteria expressing NAT2 suggests that batracylin is a substrate of this enzyme in humans.

Infect Immun, 1996 Jan, 64(1), 61 - 8
The pef fimbrial operon of Salmonella typhimurium mediates adhesion to murine small intestine and is necessary for fluid accumulation in the infant mouse; Baumler AJ et al.; We investigated the role of the pef operon, containing the genes for plasmid-encoded (PE) fimbriae of Salmonella typhimurium, in adhesion to the murine small intestine . In an organ culture model, a mutant of S . typhimurium carrying a tetracycline resistance cassette inserted in pefC was found to be associated in lower numbers with murine small intestine than the wild-type . Similarly, heterologous expression of PE fimbriae in Escherichia coli increased the bacterial numbers recovered from the intestine in the organ culture model . Adhesion to villous intestine mediated by PE fimbriae was further demonstrated by binding of an E . coli strain expressing PE fimbriae to thin sections of mouse small intestine . The contribution of pef-mediated adhesion on fluid accumulation was investigated in infant mice . Intragastric injection of S . typhimurium 14028 and SR-11 caused fluid accumulation in infant mice . In contrast, pefC mutants of S . typhimurium 14028 and SR-11 were negative in the infant mouse assay . Introduction of a plasmid containing pefBACD and orf5, the first five genes of the pef operon, into the pefC mutant complemented for fluid accumulation in the infant mouse assay . However, heterologous expression of PE fimbriae in E . coli did not result in fluid accumulation in the infant mouse, suggesting that factors other than fimbriae are involved in causing fluid accumulation.

Infect Immun, 1996 Jan, 64(1), 319 - 25
Intracellular fate of Mycobacterium avium: use of dual-label spectrofluorometry to investigate the influence of bacterial viability and opsonization on phagosomal pH and phagosome-lysosome interaction; Oh YK et al.; Mycobacterium avium is a facultative intracellular pathogen that can survive and replicate within macrophages . We tested the hypotheses that survival mechanisms may include alteration of phagosomal pH or inhibition of phagosome-lysosome fusion . M . avium was surface labeled with N-hydroxysuccinimidyl esters of carboxyfluorescein (CF) and rhodamine (Rho) to enable measurement of the pH of individual M . avium-containing phagosomes and the interactions of bacterium-containing phagosomes with labeled secondary lysosomes . CF fluorescence is pH sensitive, whereas Rho is pH insensitive; pH can be calculated from their fluorescence ratios . Surface labeling of M . avium did not affect viability in broth cultures or within J774, a murine macrophage-like cell line . By fluorescence spectroscopy, live M . avium was exposed to an environmental pH of approximately 5.7 at 6 h after phagocytosis, whereas similarly labeled Salmonella typhimurium, zymosan A, or heat-killed M . avium encountered an environmental pH of < 5.0 . Video fluorescence and laser scanning confocal microscopy gave consistent pH results and demonstrated the heterogeneity of intracellular fate early in infection . pH became more homogeneous 6 h after infection . M . avium cells were coated with immunoglobulin G (IgG) or opsonized to investigate whether phagocytosis by the corresponding receptors would alter intracellular fate . Opsonized, unopsonized, and IgG-coated M . avium cells entered compartments of similar pH . Finally, the spatial distribution of intracellular bacteria and secondary lysosomes was compared . Only 18% of live fluorescent M . avium cells colocalized with fluorescent lysosomes, while 98% of heat-killed bacteria colocalized . Thus, both inhibition of phagosome-lysosome fusion and alteration of phagosomal pH may contribute to the intracellular survival of M . avium.

Infect Immun, 1996 Jan, 64(1), 189 - 96
Effect of interleukin 12 neutralization on host resistance and gamma interferon production in mouse typhoid; Mastroeni P et al.; Innately resistant (Ityr) A/J mice infected with the virulent Salmonella typhimurium C5 strain suppress the early exponential bacterial growth in the reticuloendothelial system toward the end of the first week of infection, with spleen and liver bacterial counts reaching a plateau phase . In vivo administration of neutralizing anti-interleukin-12 (IL-12) antibodies did not affect early bacterial growth in the tissues (days 1 to 3) but impaired the establishment of the plateau, with higher spleen and liver counts by day 7 of the infection in anti-IL-12 treated mice than in untreated controls . Gamma interferon (IFN-gamma) was detectable in the sera and spleen homogenates of both control and anti-IL-12-treated mice on days 3 and 7 of the infection . Noticeably, IFN-gamma levels were significantly lower in anti-IL-12 treated mice than in control animals . Splenocytes from uninfected A/J mice released IFN-gamma in response to concanavalin A (ConA) or to S . typhimurium C5 . In vitro IL-12 neutralization dramatically impaired the IFN-gamma response to S . typhimurium but not to ConA . Splenocytes harvested from infected anti-IL-12 treated mice on day 7 of the infection produced significantly lower amounts of IFN-gamma upon in vitro stimulation with ConA and with a Salmonella protein-rich extract than did cells from similarly infected untreated control animals . Spleen cells from infected mice showed lower proliferative (mitogenic) responses to ConA and to a Salmonella soluble extract than did cells from uninfected mice . In vivo anti-IL-12 treatment significantly restored the ability of splenocytes from infected mice to proliferate in response to the antigens and ConA . In vivo neutralization of IL-12n in innately susceptible BALB/c mice ((ItyS)) immunized with a live attenuated aromatic-dependent Salmonella vaccine reduced host resistance to virulent oral challenge with S . typhimurium C5 . Thus, in primary Salmonella infections, IL-12 mediates the suppression of growth of virulent salmonellae in the reticuloendothelial system, positively modulates IFN-gamma production, and is involved in the immunosuppression which accompanies the acute stages of the disease . IL-12 also contributes to host resistance to virulent organisms in secondary infections.

Infect Immun, 1996 Jan, 64(1), 135 - 45
Adherence of Salmonella typhimurium to Caco-2 cells: identification of a glycoconjugate receptor; Giannasca KT et al.; The mechanism by which Salmonella species adhere to the epithelium of the intestine is not well understood . To identify components on intestinal epithelial cells that may be involved in the initial adherence of Salmonella typhimurium, we correlated patterns of adherence to well-differentiated Caco-2BBe cell monolayers with expression of brush border membrane components and lectin binding sites . This cloned cell line shows heterogeneous expression of sucrase-isomaltase and most lectin receptors . S . typhimurium adhered to a subpopulation of living or formaldehyde-fixed cells with a high multiplicity (up to 150 bacteria per cell) . Bacterial binding to selected cells was not correlated with expression of the brush border hydrolases dipeptidyl-peptidase IV and sucrase-isomaltase or with binding of 10 of the 12 lectins tested . However, binding was correlated with the presence of binding sites for peanut agglutinin (PNA) {specific for Gal beta (1-3) GalNAc} and soybean agglutinin (specific for terminal GalNAc) . Preincubation of live and fixed Caco-2BBe monolayers with PNA inhibited bacterial binding, while preincubation with soybean agglutinin did not . Electron microscopic analysis demonstrated that the initial adherence of S . typhimurium to Caco-2 cells in vitro involved peripheral components of the glycocalyx on apical microvilli . These results suggest that a Gal beta (1-3)GalNAc epitope recognized by PNA and located in the glycocalyx is involved in the early recognition events between S . typhimurium and Caco-2 cells and that differences in glycosylation patterns among individual epithelial cells may be a determinant in cell-selective adherence of S . typhimurium.

J Bacteriol, 1996 Jan, 178(2), 542 - 5
Salmonella typhimurium fimbrial phase variation and FimA expression; Clegg S et al.; Bacteria in a nonfimbriate phase because of continuous aeration of liquid cultures produce FimA in amounts similar to those produced by fimbriate bacteria . However, relatively low FimA production was observed in nonfimbriate-phase cultures obtained by growth on solid media or by anaerobic incubation . Regardless of the fimbrial phase of Salmonella typhimurium, the fimA promoter region was always oriented in the direction that might allow fimA transcription.

J Bacteriol, 1996 Jan, 178(2), 327 - 31
D-histidine utilization in Salmonella typhimurium is controlled by the leucine-responsive regulatory protein (Lrp); Hecht K et al.; A new class of D-histidine-utilizing mutants which carry mutations in the gene encoding the leucine-responsive regulatory protein (Lrp) has been identified in Salmonella typhimurium . The lrp mutations arise as suppressors of mutations in the genes encoding the histidine permease which drastically decrease the level of histidine transport activity . However, the suppressor effect is not exerted by elevating the level of the permease . Rather, the properties of the suppressor mutants are consistent with the notion that the parent permease mutants transport D-histidine at a low level and that in the suppressor mutants D-histidine is utilized effectively through elevated levels of racemization . The enzymatic activity of D-alanine dehydrogenase (Dad) is shown to be elevated in the suppressor mutants and is a possible pathway of D-histidine utilization . The suppressor mutations are located in the helix-turn-helix region of Lrp.

J Bacteriol, 1996 Jan, 178(1), 293 - 7
Serratia marcescens contains a heterodimeric HU protein like Escherichia coli and Salmonella typhimurium; Oberto J et al.; Homologs of the dimeric HU protein of Escherichia coli can be found in every prokaryotic organism that has been analyzed . In this work, we demonstrate that Serratia marcescens synthesizes two distinct HU subunits, like E . coli and Salmonella typhimurium, suggesting that the heterodimeric HU protein could be a common feature of enteric bacteria . A phylogenetic analysis of the HU-type proteins (HU and IHF) is presented, and a scheme for the origin of the hup genes and the onset of HU heterodimericity is suggested.

J Bacteriol, 1996 Jan, 178(1), 258 - 65
FliG and FliM distribution in the Salmonella typhimurium cell and flagellar basal bodies; Zhao R et al.; Salmonella typhimurium FliG and FliM are two of three proteins known to be necessary for flagellar morphogenesis as well as energization and switching of flagellar rotation . We have determined FliG and FliM levels in cellular fractions and in extended flagellar basal bodies, using antibodies raised against the purified proteins . Both proteins were found predominantly in the detergent-solubilized particulate fraction containing flagellar structures . Basal flagellar fragments could be separated from partially constructed basal bodies by gel filtration chromatography . FliG and FliM were present in an approximately equimolar ration in all gel-filtered fractions . FliG and FliM copy numbers, estimated relative to that of the hook protein from the early fractions containing long, basal, flagellar fragments, were (means +/- standard errors) 41 +/- 10 and 37 +/- 13 per flagellum, respectively . Extended structures were present in the earliest identifiable basal bodies . Immunoelectron microscopy and immunoblot gel analysis suggested that the FliG and, to a less certain degree, the FliM contents of these structures were the same as those for the complete basal bodies . These facts are consistent with the postulate that FliG and FliM affect flagellar morphogenesis as part of the extended basal structure, formation of which is necessary for assembly of more-distal components of the flagellum . The determined stoichiometries will provide important constraints to modelling energization and switching of flagellar rotation.

Gene, 1995 Dec 29, 167(1-2), 75 - 9
Two different operons for the same function: comparison of the Salmonella typhimurium nrdAB and nrdEF genes; Jordan A et al.; By using a P22 phage-mediated cloning system, the nrdAB genes of Salmonella typhimurium (St), encoding a ribonucleotide reductase (RR) of class I, have been isolated . The coding regions of the St nrdAB operon show a very high identity with those of the homologous operon of Escherichia coli (Ec) . Nevertheless, there are significant differences in their promoter regions since, although the promoters of both operons present two DnaA boxes, these boxes are located downstream from the transcription start point in St, being upstream in Ec . Moreover, the deduced amino-acid sequences of the St nrdAB showed a very limited overall identity (28%) with the products of St nrdEF, which encode a second class-I RR . Expression of St nrdAB and nrdEF is inducible by hydroxyurea, an inhibitor of RR activity . Alignment of the promoter regions of the nrdAB and nrdEF operons of both St and Ec reveals the presence of a consensus sequence . St is the first organism from which two different RR belonging to the same biochemical class are known.

Structure, 1995 Dec 15, 3(12), 1395 - 406
The crystal structures of the oligopeptide-binding protein OppA complexed with tripeptide and tetrapeptide ligands; Tame JR et al.; BACKGROUND: The periplasmic oligopeptide-binding protein OppA has a remarkably broad substrate specificity, binding peptides of two or five amino-acid residues with high affinity, but little regard to sequence . It is therefore an ideal system for studying how different chemical groups can be accommodated in a protein interior . The ability of the protein to bind peptides of different lengths has been studied by co-crystallising it with different ligands . RESULTS: Crystals of OppA from Salmonella typhimurium complexed with the peptides Lys-Lys-Lys (KKK) and Lys-Lys-Lys-Ala (KKKA) have been grown in the presence of uranyl ions which form important crystal contacts . These structures have been refined to 1.4 A and 2.1 A, respectively . The ligands are completely enclosed, their side chains pointing into large hydrated cavities and making few strong interactions with the protein . CONCLUSIONS: Tight peptide binding by OppA arises from strong hydrogen bonding and electrostatic interactions between the protein and the main chain of the ligand . Different basic side chains on the protein form salt bridges with the C terminus of peptide ligands of different lengths.

FEMS Microbiol Lett, 1995 Dec 15, 134(2-3), 239 - 44
Effect of cloned Salmonella typhimurium enterotoxin on rabbit intestinal motility; Reeves-Darby VG et al.; We analysed the small intestine myoelectric responses of anesthetized New Zealand albino rabbits to Escherichia coli lysates containing an entertoxin cloned from Salmonella typhimurium . Migrating action potential complex, which consisted of rapid bursts of actions potentials and secretion of fluid, was observed only in ileal loops, injected with the enterotoxin-containing lysate . Migrating action potential complex produced by Stn usually propagated aborally, which was typical of cholera toxin, but orad or bidirectional propagation occurred from a single point of origin when activity was intense . Cell lysates from an E . coli clone containing vectors alone, as well as proximal control segments injected with phosphate-buffered saline, gave neither a change in motility nor fluid secretion . These results show that Stn caused dramatic changes in intestinal motility and substantial fluid production.

J Biol Chem, 1995 Dec 15, 270(50), 29936 - 44
Kinetic characterization of channel impaired mutants of tryptophan synthase; Anderson KS et al.; Tryptophan synthase, an alpha 2 beta 2 tetrameric complex, is a classic example of an enzyme that is thought to "channel" a metabolic intermediate (indole) from the active site of the alpha subunit to the active site of the beta subunit . The solution of the three-dimensional structure of the enzyme from Salmonella typhimurium provided physical evidence for a 25-A hydrophobic tunnel which connects the alpha and beta active sites (Hyde, C . C., Ahmed, S . A., Padlan, E . A., Miles, E . W., and Davies, D . R . (1988) J . Biol . Chem . 263, 17857-17871) . Using rapid reaction kinetics, we have previously established that indole is indeed channeled and have identified three essential kinetic features which govern efficient channeling . In the current study we have probed the necessity of these features by using site-directed mutagenesis to alter these requirements . We now report the kinetic characterization of two mutants which contain substitutions to block or restrict the tunnel (beta C170F and beta C170W) . Preliminary kinetic and structural evidence of a restricted tunnel in the beta C170W has been provided (Schlichting, I., Yang, X . W., Miles, E . W., Kim, A . Y., and Anderson, K . S . (1994) J . Biol . Chem . 269, 26591-26593) . The rapid kinetic analysis of these mutant proteins shows that these mutations interfere with efficient channeling of the indole metabolite such that indole can be observed in single enzyme turnover of the physiologically relevant alpha beta reaction . In addition, the beta C170W mutant appears to be impaired in alpha beta intersubunit communication.

Mol Gen Genet, 1995 Dec 10, 249(4), 417 - 24
Molecular dissection of the flagellum-specific anti-sigma factor, FlgM, of Salmonella typhimurium; Iyoda S et al.; In the flagellar regulon of Salmonella typhimurium, the flagellar operons are divided into three classes, 1, 2 and 3, with respect to transcriptional hierarchy . Class 3 operons are controlled positively by FliA, a flagellum-specific sigma factor, and negatively by FlgM, an anti-sigma factor which binds to FliA and inhibits its activity . The sequential expression of flagellar operons is coupled to the assembly process of flagellar structures . This coupling is achieved by the fact that FlgM is exported out of the cell through the flagellar structures that are formed by the functions of the class 1 and 2 genes . Therefore, FlgM has a dual function: it can bind to FliA and is capable of being exported through the flagellar structure . In this study, using a set of deletion mutants of flgM in high-expression plasmids, we demonstrated that polypeptides containing the C-terminal portion of FlgM could inhibit the FliA-dependent transcription of the class 3 genes . Loss of amino acids near the N-terminus eliminated the export of the protein, while loss of C-terminal amino acids did not affect this function . These results indicate that the domain essential for export lies in the N-terminal region and that for FliA-binding in the C-terminal region.

J Toxicol Sci, 1995 Dec, 20 Suppl 2, 325 - 34
{Mutagenicity studies of montirelin hydrate (NS-3)}; Iwakura K et al.; Montirelin hydrate (NS-3), a new drug for the treatment of disturbance of consciousness, was examined for mutagenicity in the reverse mutation test, the chromosome aberration test in vitro, and the micronucleus test in mice . The reverse mutation test was performed at dose range of 156.25-5,000 micrograms/plate using Salmonella typhimurium strains, TA1535, TA100, TA1537, and TA98, and Escherichia coli WP2uvrA . The drug did not increase revertant colonies significantly in any of the test strains with or without metabolic activation system (S-9mix) . The chromosome aberration test was carried out at dose range of 300-4,800 micrograms/ml using cultured Chinese hamster lung cells (CHL/IU) . No significant increases of the frequencies of cells with chromosomal aberrations were observed with or without metabolic activations . The micronucleus test was conducted in the bone marrow cells of Slc:ddY male mice . Mice were given the drug by a single intraperitoneal administration at doses of 0, 250, 500, 1,000, and 2,000 mg/kg . There were no significant increases in the frequencies of micronucleated polychromatic erythrocytes at any dose levels . These results show that montirelin hydrate has no mutagenic activity in vitro or in vivo.

Microb Pathog, 1995 Dec, 19(6), 421 - 7
Effect of deoxymannojirimycin and Brefeldin A on Yersinia pseudotuberculosis invasin--eukaryotic cell interaction; Frankel G et al.; The cell surface receptors of the Yersinia pseudotuberculosis invasin protein are the alpha 3-6 beta 1 integrins . Invasin and the extracellular matrix protein fibronectin bind to the same or closely located sites on alpha 5 beta 1 integrin, although invasin bind with a much greater affinity . Invasin-integrin interaction promotes bacterial penetration into eukaryotic cells . Binding of fibronectin to its integrin receptor seems to be dependent on terminal oligosaccharides processing . In this paper, we have examined the effect of 1-deoxymannojirimycin (dMNJ), an inhibitor of Golgi alpha-mannosidases involved in processing of N-glycan precursors, and of Brefeldin A (BFA), a natural product of fungi which has profound effects on the structure and function of the Golgi apparatus, on invasin-cell interaction . We found that unlike fibronectin, the interaction of invasin with cells was resistance to dMNJ . However, preincubating cells with BFA caused a dose dependent inhibition of invasin-mediated cell entry, while cell invasion by Salmonella typhimurium was not affected.

Fundam Appl Toxicol, 1995 Dec, 28(2), 223 - 31
Toxicological evaluation of 1,1,1,2,2-pentafluoroethane (HFC-125); Kawano T et al.; Acute, subacute, and subchronic inhalation toxicity studies, developmental toxicity studies, a cardiac sensitization evaluation, and mutagenicity assays were conducted with pentafluoroethane (HFC-125) . In the acute study, rats were exposed to a single concentration of 800,000 ppm for 4 hr . Ataxic gait and abnormal respiration were observed during exposure but not after exposure . There was no mortality or other signs of toxicity . Repeated exposures of rats to 50,000 ppm, 6 hr/day, 5 days/week for either 4 or 13 weeks elicited no effects on body weight, food consumption, clinical signs, hematology, biochemistry, urinalysis, organ weight, or tissue morphology . Positive evidence of cardiac sensitization in response to an intravenous epinephrine challenge in dogs was seen at 100,000 ppm and above, but not at 75,000 ppm . HFC-125 was not mutagenic in Salmonella typhimurium and Escherichia coli strains at concentrations of 20 to 100% (v/v) with and without activation . No evidence of clastogenic activity was observed in cultured Chinese hamster ovary (CHO) cells or human lymphocytes at < or = 70% HFC-125 when treatments were conducted for 3-4 hr with activation or for 24 and 48 hr (human lymphocytes only) without activation . However, a statistically significant increase in chromosomally aberrant cells was observed in CHO cells at 60% HFC-125 when treatment without activation was extended to 48 hr . The biological significance of this effect is questionable since signs of severe toxicity were also present . In vivo, no micronuclei were induced in mouse bone marrow at concentrations as high as 600,000 ppm HFC-125 for a 6-hr exposure . In addition, HFC-125 did not induce embryotoxic or teratogenic effects in either the rat or the rabbit at exposure concentrations as high as 50,000 ppm.

Biotechniques, 1995 Dec, 19(6), 956 - 65
Single-step purifications of His6-MutH, His6-MutL and His6-MutS repair proteins of escherichia coli K-12; Feng G et al.; The MutS, MutH and MutL proteins mediate methyl-directed-mismatch (MDM) repair in Escherichia coli and Salmonella typhimurium . These proteins have been developed into powerful tools for screening genomes for polymorphisms and detecting and localizing mutations . In an ongoing study of the regulation of MDM repair, we developed one-step schemes to purify the E . coli MutS, MutH and MutL proteins fused to a polyhistidine (His6) affinity tag . The E . coli K-12 mutS+, mutH+ and mutL+ genes were cloned from the Clarke-Carbon plasmid or Kohara-phage library into expression vector pET-15b, which allows fusion to the His6 affinity tag . Each of the resulting recombinant plasmids complemented the corresponding mutHLS mutation in the Cupples-Miller CC106 mutator tester strain, indicating that the His6-MutHLS fusion proteins were individually functional in vivo . The His6-MutHLS proteins were separately purified by variations of batch binding to Ni(2+)-chelation affinity resin . The yield of purified His6-MutHLS proteins from these procedures was 0.4-0.6 mg from 40 mL of induced culture . The binding properties of one-step-purified His6-MutS protein were characterized further . His6-MutS exhibited the same mismatch-binding activity and specificity as native MutS in side-by-side bandshift assays . These constructs and purification methods should be useful to laboratories wishing to apply or develop MutS-mismatch mapping and to other applications or studies of the E . coli MutHLS repair proteins.

FEMS Immunol Med Microbiol, 1995 Dec, 12(3-4), 251 - 8
Salmonella typhimurium and Salmonella enteritidis induce gut growth and increase the polyamine content of the rat small intestine in vivo; Naughton PJ et al.; The effects of infection by Salmonella enteritidis and S . typhimurium on the small and large intestines, liver, spleen and mesenteric nodules of rats were studied in vivo . Both Salmonella serotypes persisted and proliferated in the gastrointestinal tract and invaded sub-epithelial tissues, mainly the ileum, leading to the systemic distribution of these pathogens . Coincidental with infection, the rate of crypt cell proliferation increased resulting in substantial growth of the small intestine . The extent of this and the accompanying accumulation of polyamines was particularly dramatic in the ileum where there was also some disruption of the villus epithelium . It is possible that these effects of the infection on the metabolism and morphology of the small bowel, which strongly resembled the changes induced by some plant lectins, may facilitate the colonisation and invasion of the gut by Salmonellae.

FEMS Immunol Med Microbiol, 1995 Dec, 12(3-4), 175 - 86
Surface display compared to periplasmic expression of a malarial antigen in Salmonella typhimurium and its implications for immunogenicity; Haddad D et al.; Two different expression systems were investigated for the production of an 80 amino acid polypeptide, M3, from the C-terminus of the Plasmodium falciparum blood stage antigen Pf155/RESA in an attenuated Salmonella typhimurium vaccine strain . Upon expression, the malarial polypeptide was targeted either to the periplasm as a soluble fusion protein containing two IgG-binding domains (ZZ) from the staphylococcal protein A or, to the bacterial surface as an insert within a chimeric outer membrane protein A (OmpA) derived from Escherichia coli and Shigella dysenteriae . Both the ZZM3 and the OmpAM3 proteins were stably expressed in the periplasm or on the surface of Salmonella, respectively . The ZZ expression system yielded 10-100 times more malarial immunogen than did the OmpA system . Live recombinant Salmonella expressing ZZM3 or OmpAM3 were used to immunize mice intraperitoneally . Both the ZZM3 and OmpAM3 genes persisted for up to three weeks in bacteria isolated from different lymphoid organs . Bacteria expressing ZZM3 induced antibodies to M3, ZZ and to the Pf155/RESA antigen whereas, bacteria producing OmpAM3 induced similar levels of antibodies reactive with M3 but not with Pf155/RESA . Both recombinants induced a memory response of antibodies reactive with both M3 and Pf155/RESA . The high levels of M3 produced by the ZZ expression system make it suitable for the expression of heterologous antigens in Salmonella . Nevertheless, in spite of the quantitative difference in M3 expression, the ZZ and OmpA constructs elicited comparable immune responses to M3.

Vaccine, 1995 Dec, 13(17), 1697 - 705
Construction and immunogenicity of Salmonella typhimurium vaccine vectors that express HIV-1 gp120; Fouts TR et al.; Since the human immunodeficiency virus (HIV-1) is transmitted either parenterally or sexually, both mucosal and systemic immune responses may be required to provide protective immunity . Attenuated Salmonella vectors expressing heterologous antigen can stimulate responses in both compartments . To evaluate the utility of Salmonella vectors as an HIV-1 vector vaccine, a gene expression cassette encoding recombinant HIV-1 gp120 (rgp120) was integrated into the hisOGD locus of Salmonella typhimurium aroA strain, SL3261 (SL3261::120) . To test if increased antigen expression potentiates immunogenicity, strains were constructed that express rgp120 from a multicopy asd-stabilized plasmid (SL7207 pYA:120) . Immunoblot analysis demonstrated that SL7207 pYA:120 expressed approximately 50-fold more rgp120 than SL3261::120 . Oral immunization of BALB/c mice with these strains did not stimulate an env-specific CTL response or a significant rise in antigp120 antibody titer as compared to controls . However, splenic T cells from SL7207 pYA::120 immunized mice proliferated upon restimulation with gp120 in vitro while splenocytes from SL3261::120 immunized mice did not, gp120 restimulated splenic T cells from SL7207 pYA:120 immune mice also produced IFN-gamma but no IL-5 . Two conclusions can be drawn from these results . First, high level expression of rgp120 in Salmonella vectors is necessary to stimulate a gp120-specific immune response in mice . Second, Salmonella::rgp120 stimulates a gp120-specific Th1 response in mice . This is the first report to describe the construction of a Salmonella::rgp120 vector vaccine that is immunogenic in mice.

Indian J Biochem Biophys, 1995 Dec, 32(6), 368 - 71
Exclusion of temperature bacteriophage P22 by virulent bacteriophage MB78 of Salmonella typhimurium: competition for association with the replication complex; Khan SA et al.; MB78, a virulent bacteriophage of S . typhimurium does not allow other bacteriophages like P22 and 9NA to multiply in its presence . The exclusion of P22 by MB78 is found to be due to competition for common binding site(s) in the host cell membrane . As a result, P22 DNA fails to replicate in presence of MB78 DNA . Further, the sedimentation profile of P22 DNA in cells infected simultaneously with P22 and MB78 suggested fragmentation of P22 DNA . This may also contribute to the exclusion phenomenon.

Jpn J Cancer Res, 1995 Dec, 86(12), 1131 - 5
Effects of nine active ingredients in Chinese herbal medicine sho-saiko-to on 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide mutagenicity; Ohtsuka M et al.; The antimutagenic effects of nine active compounds in the Chinese herbal medicine "sho-saiko-to" on mutagenesis induced by a direct-acting mutagen, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2) were investigated in Salmonella typhimurium, strain TA100 . The active compounds examined were classified into two major groups, saponins and flavonoids, the former comprising glycyrrhizin, saikosaponins a, c, and d, and ginsenosides Rb1 and Rg1, and the latter, baicalin, baicalein and wogonin . Saikosaponin a and ginsenoside Rb1 were found to reduce the mutagenicity of AF-2 significantly when applied post-AF-2-treatment in the Salmonella mutagenicity assay . Ginsenoside Rb1 also decreased the mutagenic activity of AF-2 in a simultaneous treatment protocol . The results indicate that saikosaponin a and ginsenoside Rb1 may enhance DNA repair, and ginsenoside Rb1 may also have the ability to inactivate the mutagenic activity of AF-2 directly . On the other hand, saikosaponin d and baicalin showed a slight enhancing effect . None of the compounds, except baicalein, showed any toxic effect on the test strain . These findings may be useful for the development of chemopreventive agents.

Genetics, 1995 Dec, 141(4), 1245 - 52
Recombination between chromosomal IS200 elements supports frequent duplication formation in Salmonella typhimurium; Haack KR et al.; Spontaneous tandem chromosomal duplications are common in populations of Escherichia coli and Salmonella typhimurium . They range in frequency for a given locus from 10(-2) to 10(-4) and probably form by RecA-dependent unequal sister strand exchanges between repetitive sequences in direct order . Certain duplications have been observed previously to confer a growth advantage under specific selective conditions . Tandem chromosomal duplications are unstable and are lost at high frequencies, representing a readily reversible source of genomic variation . Six copies of a small mobile genetic element IS200 are evenly distributed around the chromosome of S . typhimurium strain LT2 . A survey of 120 independent chromosomal duplications (20 for each of six loci) revealed that recombination between IS200 elements accounted for the majority of the duplications isolated for three of the loci tested . Duplications of the his operon were almost exclusively due to recombination between repeated IS200 elements . These data add further support to the idea that mobile genetic elements provide sequence repeats that play an important role in recombinational chromosome rearrangements, which may contribute to adaptation of bacteria to stressful conditions.

Am J Vet Res, 1995 Dec, 56(12), 1549 - 54
Enzyme-linked immunosorbent assay for screening of milk samples for Salmonella typhimurium in dairy herds; Hoorfar J et al.; We investigated the ability of an antibody-specific, O antigen-based ELISA to document Salmonella typhimurium herd infections by screening of milk samples . Three cattle populations, 20 herds with no history of salmonellosis, 8 herds with history of S typhimurium episodes within the previous 7 months, and 220 herds of unknown disease status, were tested . A herd was considered ELISA positive if at least 5% of the cows had OD values > 0.3 . Among the 20 herds without history of salmonellosis, only 2 herds were ELISA positive, whereas all 8 herds with a known history of salmonellosis were ELISA positive herd specificity, 0.9 and herd sensitivity, 1.0) . A significant correlation (P < 0.001) was found between the OD values of serum and milk samples from cows in the herds with a history of salmonellosis . It was concluded that ELISA testing of individual milk samples can be used for surveillance of herds for S typhimurium infections, but further modifications are needed to test bulk tank milk samples.

Microbiology, 1995 Dec, 141 ( Pt 12), 3241 - 5
Determination of a 17,484 bp nucleotide sequence around the 39 degrees region of the Bacillus subtilis chromosome and similarity analysis of the products of putative ORFs; Akagawa E et al.; We have determined a 17,484 bp nucleotide sequence around the 39 degrees region, located about 480 kb downstream from the zero position of the Bacillus subtilis chromosome physical map . Among the 17 putative ORFs identified, orf1 and orf2 seem to correspond to mtlA and mtlB, encoding mannitol-specific phosphotransferase enzyme II and mannitol-1-phosphate dehydrogenase, respectively . orf4 seems to be another signal peptidase I gene (sipS) of B . subtilis . The putative products of six ORFs were similar to known proteins in data banks, namely a hypothetical 29.7 kDa protein of Escherichia coli (Orf7), a lactam utilization protein (Orf8), the urea amidolyase of yeast (Orf12), the IcIR regulatory protein for aceAB of Salmonella typhimurium (Orf13), penicillin-binding protein 2 (Orf16) and aryl-alcohol dehydrogenase (Orf17) . The amino acid sequence of Orf3 showed 34% identity with that of LeuC of B . subtilis, though they seem to be functionally different . The remaining seven ORFs did not show similarity to any known proteins.

Immunology, 1995 Dec, 86(4), 612 - 9
Interleukin-1 and interleukin-6 gene expression in human monocytes stimulated with Salmonella typhimurium porins; Galdiero M et al.; The aim of this study was to verify whether Salmonella typhimurium porins can affect the expression of interleukin-1 (IL-1) and interleukin-6 (IL-6) genes . Human monocytes were treated with porins, and total RNAs were analysed by Northern blotting to evaluate the expression of IL-1 alpha, IL-1 beta and IL-6 in both treated and untreated cell cultures . Porins induced a significant increase in IL-1 and IL-6 transcripts . This increase was related to the dose of porins, and it peaked 5 hr after treatment . The same results were obtained when polymyxin B was added to the porin preparation to eliminate eventual traces of lipopolysaccharide (LPS) associated with porins . The porins-mediated increase in interleukin transcripts did not require de novo protein synthesis, and it was because of the enhanced half-life of IL-1 and IL-6 mRNAs, rather an increased rate of gene transcription . These data suggest that porins may affect inflammatory and immunological responses by enhancing the expression of cytokine genes.

Br J Nutr, 1995 Dec, 74(6), 787 - 95
High doses of dietary arginine during repletion impair weight gain and increase infectious mortality in protein-malnourished mice; Peck MD et al.; There is considerable evidence for the beneficial effects of dietary arginine, a conditionally-essential amino acid that enhances anabolism and T-cell function . However, the safety and efficacy of higher doses of arginine supplementation following infection have not been investigated completely . These issues were explored therefore, in a murine model of malnutrition and infection . Severe protein malnutrition was induced by feeding mice for 6 weeks on an isoenergetic diet containing only 10 g protein/kg . Mice were then allowed to consume diets with normal amounts of protein (200 g/kg) with 50 g/kg provided as amino acid mixtures of glycine and arginine in which the arginine content ranged from 0 to 50 g/kg . During the repletion period a significant weight gain was noted in the groups fed on diets with either 10 or 20 g arginine/kg, but not in the group fed on the diet with 50 g arginine/kg, compared with the diet with 0 g arginine/kg . Mortality rates after infection with Salmonella typhimurium were not decreased by the addition of 10 or 20 g arginine/kg to the diet, and were in fact worsened by supplementation with 50 g arginine/kg . The results of the present study showed that not only are the beneficial effects of arginine supplementation after infection lost when high doses are administered, but also that these high doses become toxic . Mice fed on higher doses showed significant impairment of weight gain and an increase in mortality rates.

Mutat Res, 1995 Dec, 345(3-4), 137 - 46
A miniaturized Ames mutagenicity assay employing bioluminescent strains of Salmonella typhimurium; Cote C et al.; Increased awareness of the role of environmental factors in carcinogenesis has led to an emphasis on preventing or minimizing exposure to genotoxicants . This is presently promoting the development of simple, rapid, cost-effective mutagenicity screening assays . We have developed a test system based on the well-known Salmonella mutagenicity assay . The lux genes, which permit cells to emit light through bioluminescence, were introduced into Salmonella typhimurium strain TA98 . These bacteria were exposed for 48 h to chemicals or complex mixtures in 48-well microplates containing an appropriate liquid medium . Cells were subsequently centrifuged and resuspended in buffer . The final postexposure revertant biomass was then estimated using a microluminometer . Replication trials confirmed methodological reproducibility . Clear dose-response relationships were obtained with the direct frameshift mutagens 4NQO and 2NF . Mutagenicity threshold effect concentrations found for these compounds were comparable to those reported in the literature . Industrial effluents and environmental extracts (effluents, suspended solids) were also tested and results compared well with those of the SOS Chromotest . While further validation of this new adaptation of the Ames test will be required, it appears at this time that it could be well suited for routine screening of xenobiotics and environmental samples.

Microbiol Rev, 1995 Dec, 59(4), 623 - 45
Control of rRNA transcription in Escherichia coli; Condon C et al.; The control of rRNA synthesis in response to both extra- and intracellular signals has been a subject of interest to microbial physiologists for nearly four decades, beginning with the observations that Salmonella typhimurium cells grown on rich medium are larger and contain more RNA than those grown on poor medium . This was followed shortly by the discovery of the stringent response in Escherichia coli, which has continued to be the organism of choice for the study of rRNA synthesis . In this review, we summarize four general areas of E . coli rRNA transcription control: stringent control, growth rate regulation, upstream activation, and anti-termination . We also cite similar mechanisms in other bacteria and eukaryotes . The separation of growth rate-dependent control of rRNA synthesis from stringent control continues to be a subject of controversy . One model holds that the nucleotide ppGpp is the key effector for both mechanisms, while another school holds that it is unlikely that ppGpp or any other single effector is solely responsible for growth rate-dependent control . Recent studies on activation of rRNA synthesis by cis-acting upstream sequences has led to the discovery of a new class of promoters that make contact with RNA polymerase at a third position, called the UP element, in addition to the well-known -10 and -35 regions . Lastly, clues as to the role of antitermination in rRNA operons have begun to appear . Transcription complexes modified at the antiterminator site appear to elongate faster and are resistant to the inhibitory effects of ppGpp during the stringent response.

Gene, 1995 Dec 1, 166(1), 57 - 64
Identification and sequences of the Treponema pallidum fliM', fliY, fliP, fliQ, fliR and flhB' genes; Hardham JM et al.; Information regarding the biology and virulence attributes of Treponema pallidum (Tp) is limited due to the lack of genetic exchange mechanisms and the inability to continuously cultivate this spirochete . We have utilized TnphoA mutagenesis of a Tp genomic DNA library in Escherichia coli (Ec) to identify genes encoding exported proteins, a subset of which are likely to be important in treponemal pathogenesis . We report here the identification and nucleotide (nt) sequence of a 5-kb treponemal DNA insert that contains seven open reading frames (ORFs) . The proteins encoded by six of these ORFs have homology with members of a newly described protein family involved in the biogenesis/assembly of flagella and the control of flagellar rotation in Ec, Salmonella typhimurium (St) and Bacillus subtilis (Bs) . Certain members of this family are also involved in the export of virulence factors in Yersinia (Yr) spp., St and Shigella flexneri (Sf) . We have named these six ORFs fliM', fliY, fliP, fliQ, fliR and flhB' . The operon containing these ORFs has been designated as the fla operon . We hypothesize that the protein products of these genes are involved in the biogenesis/assembly of flagella and the control of flagellar rotation in Tp.

Mutat Res, 1995 Dec, 335(3), 309 - 16
Cryopreserved and hypothermically stored rat liver parenchymal cells as metabolizing system in the Salmonella mutagenicity assay; Diener B et al.; Freshly isolated and preserved rat liver parenchymal cells were used as metabolizing system in the Salmonella mutagenicity assay . The liver cells were isolated with EDTA perfusion without the addition of collagenase and had a viability of 96% as judged by trypan blue exclusion . When freshly isolated liver parenchymal were cryopreserved with a computer controlled freezing protocol and stored at -196 degrees C they had a mean viability of 89% after thawing . Furthermore, freshly isolated cells were stored at 0 degree C in University of Wisconsin organ transplantation solution . After 1 day of hypothermic storage they had a viability of 95% . Four different indirect mutagens, 2-aminoanthracene, benzo{a}pyrene, 7,12-dimetylbenz{a}anthracene and cyclophosphamide, were used with the liver cells as metabolizing system in the preincubation assay with Salmonella typhimurium TA100 . After cryopreservation, liver parenchymal cells were able to activate all tested indirect mutagens to ultimate mutagens . However, the induction of revertants was lower with three of the four tested compounds . Only 2-aminoanthracene was activated to the same extent by freshly isolated and cryopreserved liver cells . 7-Hydroxymethyl-12-methylbenz{a}anthracene, which is activated to its ultimate mutagen by sulfotransferase, also induced a reduced mutagenic effect with cryopreserved liver cells in comparison to freshly isolated liver parenchymal cells . This indicates that phase I and phase II enzyme activities are effected by cryopreservation . However, identical mutation frequencies were obtained when freshly isolated liver parenchymal cells or 1 day hypothermically preserved liver parenchymal cells were used in the cell-mediated Salmonella mutagenicity test . The use of hypothermic short-time storage of liver parenchymal cells could help to make the liver cell-mediated genotoxicity test simpler and thereby more attractive.

Mutat Res, 1995 Dec, 335(3), 219 - 27
Mutagenic activation of aromatic amines by a human hepatoma cell (Hep G2) supernatant tested by means of Salmonella typhimurium strains with different acetyltransferase activities; Duverger-van Bogaert M et al.; The study was carried out to characterize hepatoma cells (Hep G2) as activation system relevant to man and to investigate which are the tester strains most suitable for the mutagenic assay of aromatic amines . A supernatant prepared from the human hepatoma cell line Hep G2 was used to activate benzidine, 2-aminofluorene (2-AF) and 2-acetylaminofluorene (2-AAF) in the Salmonella typhimurium reversion assay . Activation by Hep G2 supernatant was studied with increasing concentrations of the three compounds, in tester strains TA98, YG1024, DJ400 and DJ460 . Benz{alpha}anthracene (BA) pretreatment of cells increases the mutagenicity of benzidine in strains YG1024, DJ460 and DJ400 . Activation of 2-AAF and 2-AF was observed in strains YG1024, DJ400 and, at the highest tested dose, in DJ460 . These results were compared with those obtained with S9 from control and Aroclor 1254 (Aro)-pretreated rat liver . With strain TA98 comparable responses were obtained except for 2-AF which was better activated using rat liver S9 . The use of strain YG1024 greatly increases the sensitivity of the response . Strain DJ460 makes it possible to detect activation of 2-AF and 2-AAF by Aro-induced rat liver . These results indicate that Hep G2 supernatant is a useful metabolic activation system of human origin that can be used to replace rat liver S9 . An appropriate choice of the Salmonella strain not only can increase the sensitivity of the response, but may also help to overcome certain metabolic shortcomings of the Hep G2 cell line and rat liver S9.

Mutat Res, 1995 Dec, 335(3), 207 - 11
Mutagenicity of tetranitroazoxytoluenes: a preliminary screening in Salmonella typhimurium strains TA100 and TA100NR; Spanggord RJ et al.; Tetranitroazoxytoluenes are polynitroaromatic compounds that can be produced during the microbial reduction of the explosive, 2,4,6-trinitrotoluene (TNT) . The three major tetranitroazoxytoluenes were synthesized and tested in Salmonella typhimurium strains TA100 and TA100NR . All compounds were mutagenic in TA100 but not in TA100NR, indicating the need for nitroreductase activity to induce mutagenicity . The most active chemical was 4,4',6,6'-tetranitro-2,2'-azoxytoluene (2735 rev/mumol) followed by 2',4,6,6'-tetranitro-2',4-azoxytoluene (929 rev/mumol) and 2,2'-6,6'-tetranitro-4,4'-azoxytoluene (320 rev/mumol) . These chemicals were more active than the aminodinitrotoluenes (298 rev/mumol for 2-amino-4,6-dinitrotoluene and 115 rev/mumol for 4-amino-2,6-dinitrotoluene) and only 4,4',6,6'-tetranitro-2,2'-azoxytoluene was more active than the parent compound, TNT (1022 rev/mumol).

J Cell Biol, 1995 Dec, 131(6 Pt 1), 1599 - 608
Surface attachment of Salmonella typhimurium to intestinal epithelia imprints the subepithelial matrix with gradients chemotactic for neutrophils; McCormick BA et al.; During intestinal disease induced by Salmonella typhimurium transepithelial migration of neutrophils (PMN) rapidly follows attachment of the bacteria to the epithelial apical membrane . Among the events stimulated by these interactions is the release of chemotaxins that guide PMN through the subepithelial matrix and subsequently through the epithelium itself (McCormick, B.A., S.P . Colgan, C . Delp-Archer, S.I . Miller, and J.L . Madara . 1993 . J . Cell Biol . 123:895-907) . Given the substantial volume flow that normally characterizes matrix compartments underlying transporting epithelia, it is unclear how such transmatrix signaling is sustained . Here we show that when underlying matrices are isolated from biophysically confluent polarized monolayers of the human intestinal epithelial cell line T84, they fail to support substantial transmatrix migration of PMN unless an exogenous chemotactic gradient is imposed . In contrast, such matrices isolated from confluent monolayers apically colonized with S . typhimurium support spontaneous transmatrix migration of PMN . Such chemotactic imprinting of underlying matrices is resistant to volume wash and is paralleled by secretion of the known matrix-binding chemokine IL-8 . Chemotactic imprinting of the matrix underlying S . typhimurium-colonized monolayers is dependent on epithelial protein synthesis, is directional implying the existence of a gradient, and is neutralized by antibodies either to IL-8 or to the IL-8 receptor on PMN . An avirulent S . typhimurium strain, PhoPc, which attaches to epithelial cells as efficiently as wild-type S . typhimurium, fails to induce basolateral secretion of IL-8 and likewise fails to imprint matrices . Together, these observations show that the epithelial surface can respond to the presence of a luminal pathogen and subsequently imprint the subepithelial matrix with retained IL-8 gradients sufficient to resist washout effects of the volume flow that normally traverses this compartment . Such data further support the notion that the primary role for basolateral secretion of IL-8 by the intestinal and likely other epithelia is recruitment of PMN through the matrix to the subepithelial space, rather than directing the final movement of PMN across the epithelium.

J Bacteriol, 1995 Dec, 177(24), 7119 - 24
DNA polymerase I function is required for the utilization of ethanolamine, 1,2-propanediol, and propionate by Salmonella typhimurium LT2; Rondon MR et al.; Evidence documenting the requirement for a functional DNA polymerase I when Salmonella typhimurium LT2 uses ethanolamine (EA), 1,2-propanediol (1,2-PDL), or propionate (PRP) as the sole carbon and energy source is presented . Providing rat polymerase beta in trans demonstrated that the growth phenotypes observed were due exclusively to the lack of DNA polymerase I functions . The location of the mutation (a MudI1734 insertion) that rendered cells unable to grow on EA, 1,2-PDL, or PRP was determined by DNA sequencing to be within the polA gene . polA mutants of this bacterium may be unable to repair the damage caused by reactive aldehydes generated during the catabolism of EA, 1,2-PDL, or PRP . Consistent with this hypothesis, the inhibitory effects of acetaldehyde and propionaldehyde on the growth of this polA mutant were demonstrated . A derivative of the polA mutant unable to synthesize glutathione (GSH) was markedly more sensitive to acetaldehyde and propionaldehyde than was the polA mutant proficient in GSH synthesis . This finding was in agreement with the recently proposed role of GSH as a mechanism for quenching reactive aldehydes generated during the catabolism of these compounds (M . R . Rondon, R . Kazmierczack, and J . C . Escalante-Semerena, J . Bacteriol . 177:5434-5439, 1995).

J Bacteriol, 1995 Dec, 177(24), 7078 - 85
Identification of two targets of the type III protein secretion system encoded by the inv and spa loci of Salmonella typhimurium that have homology to the Shigella IpaD and IpaA proteins; Kaniga K et al.; An important virulence factor of Salmonella spp . is their ability to gain access to host cells . A type III secretion system encoded in the inv and spa loci of these organisms is essential for this phenotype . We have identified two proteins, SipA and SipD, whose secretion from the bacterial cells is dependent on this system . The genes encoding these proteins are located at centisome 63 on the S . typhimurium chromosome, immediately downstream of the previously identified sipB and sipC genes (K . Kaniga, S . Tucker, D . Trollinger, and J . E . Galan, J . Bacteriol . 177:3965-3971, 1995) . Nucleotide sequence analysis of the genes encoding these proteins indicated that SipA and SipD have significant sequence similarity to the Shigella IpaA and IpaD proteins . A nonpolar null mutation in sipD rendered S . typhimurium severely deficient for entry into cultured epithelial cells . In addition, this mutant strain exhibited increased secretion of a selected group of proteins whose export is controlled by the inv- and spa-encoded translocon . In contrast, a nonpolar mutation in sipA did not result in an invasion defect or in a significant decreased in virulence in a mouse model of infection . In addition, we have found an open reading frame immediately downstream of SipA that encodes a predicted protein with significant similarity to a family of acyl carrier proteins.

J Bacteriol, 1995 Dec, 177(23), 6874 - 80
Characterization of a chimeric proU operon in a subtilin-producing mutant of Bacillus subtilis 168; Lin Y et al.; The ability to respond to osmotic stress by osmoregulation is common to virtually all living cells . Gram-negative bacteria such as Escherichia coli and Salmonella typhimurium can achieve osmotolerance by import of osmoprotectants such as proline and glycine betaine by an import system encoded in an operon called proU with genes for proteins ProV, ProW, and ProX . In this report, we describe the discovery of a proU-type locus in the gram-positive bacterium Bacillus subtilis . It contains four open reading frames (ProV, ProW, ProX, and ProZ) with homology to the gram-negative ProU proteins, with the B . subtilis ProV, ProW, and ProX proteins having sequence homologies of 35, 29, and 17%, respectively, to the E . coli proteins . The B . subtilis ProZ protein is similar to the ProW protein but is smaller and, accordingly, may fulfill a novel role in osmoprotection . The B . subtilis proU locus was discovered while exploring the chromosomal sequence upstream from the spa operon in B . subtilis LH45, which is a subtilin-producing mutant of B . subtilis 168 . B . subtilis LH45 had been previously constructed by transformation of strain 168 with linear DNA from B . subtilis ATCC 6633 (W . Liu and J . N . Hansen, J . Bacteriol . 173:7387-7390, 1991) . Hybridization experiments showed that LH45 resulted from recombination in a region of homology in the proV gene, so that the proU locus in LH45 is a chimera between strains 168 and 6633 . Despite being a chimera, this proU locus was fully functional in its ability to confer osmotolerance when glycine betaine was available in the medium . Conversely, a mutant (LH45 deltaproU) in which most of the proU locus had been deleted grew poorly at high osmolarity in the presence of glycine betaine . We conclude that the proU-like locus in B . subtilis LH45 is a gram-positive counterpart of the proU locus in gram-negative bacteria and probably evolved prior to the evolutionary split of prokaryotes into gram-positive and gram-negative forms.

J Bacteriol, 1995 Dec, 177(23), 6861 - 5
Construction and characterization of a fimZ mutant of Salmonella typhimurium; Yeh KS et al.; The Salmonella typhimurium fimA gene is controlled by several ancillary fim genes . One of these genes, fimZ, appears to be involved in increasing the expression of fimA . A fimZ mutant of S . typhimurium was constructed by allelic exchange, and this mutant was found to be nonfimbriate . The fimZ mutant demonstrated decreased levels of fimA expression compared with the parental strain when both were grown under conditions favoring fimbrial expression . An examination of the predicted amino acid sequence, deduced from the nucleotide sequence of fimZ, indicated that the FimZ polypeptide possessed a DNA binding motif . Bacterial lysates, derived from strains transformed with recombinant plasmids possessing a fimZ gene, demonstrated DNA binding activity with a fragment containing the fimA promoter . Lysates without a FimZ polypeptide did not exhibit any binding activity . These data are consistent with FimZ being a transcriptional activator of fimA, and FimZ acts by binding to the promoter region.

J Bacteriol, 1995 Dec, 177(23), 6711 - 7
Isolation of NAD cycle mutants defective in nicotinamide mononucleotide deamidase in Salmonella typhimurium; Cheng W et al.; The NAD or pyridine nucleotide cycle is the sequence of reactions involved in the breakdown of NAD to nicotinamide mononucleotide (NMN) and regeneration of NAD . This cycle is fivefold more active during aerobic growth of Salmonella typhimurium and under this condition breaks down half of the NAD pool every 90 min . DNA ligase is known to convert NAD to NMN but is only a minor contributor to the NAD cycle during aerobic growth . The dominant aerobic route of NMN formation is otherwise uncharacterized . Accumulated NMN generated by either of these routes is potentially dangerous in that it can inhibit the essential enzyme DNA ligase . The reactions which recycle NMN to NAD may serve to minimize the inhibition of ligase and other enzymes by accumulated NMN . The predominant recycling reaction in S . typhimurium appears to be NMN deamidase, which converts NMN directly to the biosynthetic intermediate nicotinic acid mononucleotide . Mutants defective in this recycling step were isolated and characterized . By starting with a ligase-deficient (lig mutant) parent strain that requires deamidase to assimilate exogenous NMN, two classes of mutants that are unable to grow on minimal NMN media were isolated . One class (pncC) maps at 83.7 min and shows only 2% of the wild-type levels of NMN deamidase . Under aerobic conditions, a lig+ allele allows a pncC mutant to grow on NMN and restores some deamidase activity . This growth ability and enzyme activity are not found in lig+ strains grown without oxygen . This suggests that the existence of a second NMN deamidase (pncL) dependent on ligase and stimulated during aerobic growth . The second class of mutants (pncD) gains a requirement for isoleucine plus valine with growth in the presence of exogenous NMN . We propose that pncD mutations reduce the activity of an ilv biosynthetic enzyme that is naturally sensitive to inhibition by NMN.

Infect Immun, 1995 Dec, 63(12), 4933 - 8
Immune responses to novel Escherichia coli and Salmonella typhimurium vectors that express colonization factor antigen I (CFA/I) of enterotoxigenic E . coli in the absence of the CFA/I positive regulator cfaR; Wu S et al.; An asd-stabilized plasmid carrying enterotoxigenic Escherichia coli cfaABCE genes was constructed and called pJGX15C-asd+ . Expression of colonization factor antigen I (CFA/I) by this plasmid occurs independently of the cfaABCE positive regulator cfaR in attenuated Salmonella delta aro delta asd strain H683 and nonpathogenic laboratory E . coli asd strain chi 6212 . Oral immunization of mice with nonpathogenic E . coli chi 6212 (pJGX15C-asd+) does not elicit significant serum or mucosal responses against CFA/I . In contrast, oral immunization with a single dose of attenuated S . typhimurium H683(pJGX15C-asd+) elicits a 10(5)-fold increase in CFA/I-specific serum immunoglobulin G and significant elevation of CFA/I-specific immunoglobulin A-secreting B cells in the lamina propria, mesenteric lymph nodes, and spleen . Thus, only the Salmonella-CFA/I construct effectively delivered CFA/I to the inductive sites of the gut-associated and systemic lymphoid tissues.

Infect Immun, 1995 Dec, 63(12), 4600 - 5
Antigenic determinants of the OmpC porin from Salmonella typhimurium; Singh SP et al.; The antigenic determinants of Salmonella typhimurium OmpC were investigated by the analysis of cyanogen bromide (CNBr)-generated porin peptides with antiporin monoclonal antibodies (MAbs) . We identified six bands (f1 to f6) with estimated molecular masses of 35.5, 31.0, 25.0, 22.5, 13.8, and 10.0 kDa, respectively . In addition, two small fragments (f7 and f8; 3.0 to 6.0 kDa) were detected only infrequently . The OmpC monomer or its CNBr-generated peptides were electrophoretically transferred to a polyvinylidene difluoride membrane and then subjected to amino acid composition analysis and N-terminal sequencing . A comparison of the amino acid composition data with known compositions of Escherichia coli and Salmonella typhi OmpC showed some differences; however, the amino acid sequences of 71 residues identified in S . typhimurium showed 88 and 98% identity with OmpC from E . coli and S . typhi, respectively . The screening of CNBr peptides with the 12 anti-(S . typhimurium) OmpC MAbs by Western blot (immunoblot), in conjunction with the prediction of the OmpC folding pattern based on the known three-dimensional structure of E . coli OmpF, showed that four MAbs reacted with surface-exposed epitopes on loops L2, L8, and L4 to L7, four MAbs reacted with a region in the eyelet structure on loop L3, and four MAbs reacted with the buried epitopes on transmembrane beta strands . The MAbs reacting with surface-exposed loops showed no cross-reaction with E . coli OmpC, whose sequence has diverged extensively from that of S . typhi and (probably) S . typhimurium OmpC only in regions of the externally exposed loops . In contrast, MAbs reacting with transmembrane beta strands, whose sequence is strongly conserved, showed strong cross-reaction with E . coli OmpC . These results show that comparison with the E . coli OmpF structure predicts the folding pattern of S . typhimurium OmpC rather accurately and that evolutionary divergence in sequences is confined to the external loops . The possible roles of these surface-exposed and buried epitopes as potentially useful antigenic regions for diagnostic assays and vaccine development are discussed.

Arch Biochem Biophys, 1995 Dec 1, 324(1), 71 - 7
Resolution of pyridoxal 5'-phosphate from O-acetylserine sulfhydrylase from Salmonella typhimurium and reconstitution of apoenzyme with cofactor and cofactor analogues as a probe of the cofactor binding site; Schnackerz KD et al.; A procedure has been developed to prepare the apoenzyme of O-acetylserine sulfhydrylase (apoOASS) by first converting the native enzyme to the alpha-aminoacrylate intermediate and dialyzing against 5 M guanidinium chloride . Aposulfhydrylase is stable for at least a month in buffers containing phosphate or phosphate analogues . Reconstitution of aposulfhydrylase with pyridoxal 5'-phosphate (PLP), 2'-methyl PLP (2'-MePLP), and pyridoxal 5'-deoxymethylenephosphonate (PDMP) results in enzymatically competent proteins . Pyridoxal in the absence and presence of phosphate and pyridoxal 5'-phosphate monomethyl ester are unable to form a Schiff base with apoOASS . The reconstitution of apoOASS with PLP is highly cooperative judged by the initial rate of activity regained and shows no evidence of saturation with PLP . The reconstituted enzymes have been studied using 31P NMR spectroscopy . The 31P NMR of the aposulhydrylase reconstituted with PLP exhibits a chemical shift of 5.2 ppm, identical to that of native enzyme . The latter has been interpreted in terms of a strong ionic interaction between enzyme and the 5'-phosphate of PLP (P . F . Cook, S . Hara, S . Nalabolu, and K . D . Schnackerz, 1992, Biochemistry 31, 2298-2303) . Reconstitution with 2'-MePLP gives a lower chemical shift of 4.95 ppm, suggesting a weaker ionic interaction at the 5'-phosphate when compared to native enzyme . The PDMP-reconstituted enzyme gives a chemical shift of 23.7 ppm, consistent with the monoanionic form of the bound phosphonate . All of the chemical shifts are pH independent . The apoenzyme has also been reconstituted with pyridoxal 5'-sulfate . Although the resulting enzyme is not active in the overall reaction, it forms the external Schiff base . The PDMP- and 2'-MePLP-reconstituted enzymes have also been studied in the presence of amino acid reactants and analogues, and results are discussed in terms of the mechanism of OASS.

J Theor Biol, 1995 Nov 21, 177(2), 171 - 9
The inhibition of maltose transport by the unliganded form of the maltose-binding protein of Escherichia coli: experimental findings and mathematical treatment; Merino G et al.; Binding protein-dependent transport systems in Gram-negative enteric bacteria are multicomponent systems in which a soluble periplasmic binding protein of high substrate binding affinity establishes the major substrate recognition site . Usually, there are two integral membrane proteins which are thought to interact with the substrate loaded form of the binding protein to allow transport of substrate to occur . Transport is against the concentration gradient and needs energization by an ATP hydrolizing polypeptide . Overall transport is considered mainly unidirectional due to the high energy of ATP hydrolysis coupled to transport . In the study reported here, maltose transport in membrane vesicles in the presence of varying concentrations of unliganded maltose-binding protein but with constant amounts of maltose was measured . The conditions were chosen such that the concentration of maltose was always smaller than that of the binding protein and the initial concentration of the liganded binding protein was essentially constant . It was found that the initial rate of transport went through a maximum with increasing amounts of binding protein and declined thereafter . This finding strongly supports the conclusion that both the liganded and the unliganded forms of the binding protein interact with the membrane components of the transport system . The mathematical treatment of the experimental data allowed the ratio of the affinities for the membrane components of the substrate loaded and unloaded binding protein to be estimated . Published data on the binding protein-dependent transport of histidine in membrane vesicles of Salmonella typhimurium were also used . The data allowed the ratio of the binding affinity of the membrane components to the substrate-loaded and free binding protein to be determined . In addition, the KM of transport to the KD of binding protein was approximated.

Life Sci, 1995 Nov 17, 57(26), 2413 - 23
Effects of benzodiazepines on immunodeficiency and resistance in mice; Galdiero F et al.; Our results indicate that benzodiazepine (Bz) treatment time, greater than 2-3 months, induce a decrease of both specific and nonspecific responses . Mice treated for different times with diazepam or chlordemethyldiazepam showed decreased survival to experimental Salmonella typhimurium infections after three months of treatment . Adherence, expressed as the polymorphonuclear cells (PMN) capacity to attach to nylon wool, was impaired after 7 days of treatment . Longer treatments further increase this impairment . PMN from mice treated with Bz for 90 days also demonstrate on impaired chemotaxis and phagocytosis for Saccharomyces cerevisiae . Monocytes from mice treated for 7 days secreted more IL-1 alpha then controls; the antibody titer in mice given to prolonged treatment progressively diminished compared to controls . Con A or LPS stimulated lymphocytes showed an increase of H3-thymidine incorporation from mice treated for a short time and conversely a decreased incorporation when taken from mice that underwent longer treatments . Benzodiazepines were therefore found to affect PMN chemotaxis and phagocitosis, general immunity and survival of mice to infections.

J Mol Biol, 1995 Nov 17, 254(1), 96 - 105
Strange bedfellows: interactions between acidic side-chains in proteins; Flocco MM et al.; The oxygen atoms of two acidic side-chains are frequently found within hydrogen-bonding distance of each other in proteins . Two distinct types of cases are common . In metal-binding sites, the oxygen atoms are brought near (average closest approach 3.0 A) by their common role as metal ligands . In a different location, either buried or on the protein surface, the two acidic groups can share a proton . The corresponding O-O distances in the latter case are shorter (usually 2.7 or less), and the geometry is typical of hydrogen-bonding interactions . The glucose/galactose-binding protein of Salmonella typhimurium provides an example of a well-ordered Asp-Glu pair on the surface of a protein with a very short O-O distance, at a pH of 7.0 . Other instances have been found at pH values as high as 8.0, suggesting substantial alteration of the pKa involved . These observations have implications for the study of enzymes that use pairs of acidic residues in binding and catalysts.

EMBO J, 1995 Nov 15, 14(22), 5690 - 700
DNA twist, flexibility and transcription of the osmoregulated proU promoter of Salmonella typhimurium; Jordi BJ et al.; Transcription from many bacterial promoters is sensitive to the level of DNA supercoiling . We have investigated the mechanism by which environmentally induced changes in DNA supercoiling might regulate transcription . For the proU promoter of Salmonella typhimurium, osmotically induced changes in DNA topology appear to play a primary regulatory role . Changes in DNA supercoiling (linking number; delta Lk) are partitioned into changes in the winding of the strands of the double helix about themselves (twist; delta Tw) and/or elastic deformations or flexibility of the DNA helix (writhe; delta Wr) . Mutations of the proU promoter were isolated in vivo, or generated in vitro, which altered the spacing between the -10 and -35 motifs . Studies on these mutant promoters, both in vivo and in vitro, exclude models in which changes in DNA twist play a regulatory role . Instead, our data suggest that increased DNA flexibility, reflecting the osmotically induced increase in negative supercoiling of DNA, is required for promoter activation.

Biochem J, 1995 Nov 15, 312 ( Pt 1), 281 - 6
Catalysis by the large subunit of the second beta-galactosidase of Escherichia coli in the absence of the small subunit; Calugaru SV et al.; Plasmids containing the ebgAo and ebgAa genes of Escherichia coli under the control of the lac repressor and promoter have been constructed and inserted into Salmonella typhimurium CH3 . This system expresses the large subunit of the ebgo and ebga beta-galactosidase in high yield (20-60% of total protein) . The large subunits have been purified to homogeneity . As isolated they are tetramers of significant catalytic activity; the N-terminal amino acid residue is Met, but it is not formylated . The kcat . values for a series of aryl galactosides were 6-200-fold reduced from the corresponding values for the holoenzymes . kcat/Km Values for glycosides of acidic aglycones, though, were unchanged, whilst kcat./Km values for galactosides of less acidic aglycones showed a modest (up to 10-fold) decrease . The kcat . values for glycosides of acidic aglycones hydrolysed by ebgo and ebga large subunits were essentially invariant with aglycone pK, suggesting that hydrolysis of the galactosyl-enzyme intermediate had become rate-determining for these substrates . Rate-determining hydrolysis of the glycosyl-enzyme intermediate was confirmed by pre-steady-state measurements and nucleophilic competition with methanol . Absence of the small subunit was thus estimated to cause a 200-fold decrease in degalactosylation rate for ebgo and a 20-fold one for ebga . beta 1g(V/K) values of -0.57 +/- 0.08 for ebgo and -0.54 +/- 0.08 for ebga isolated subunits were significantly more negative than for holoenzymes . It is suggested that the small subunit is associated with the optimal positioning of the electrophilic Mg2+ ions in these enzymes . Use of PCR in the construction of the plasmid also inadvertently led to the production of psi ebgo large subunit in which there was a PCR-introduced Leu9-->His change . Values of kcat . for aryl galactosides, calculated on the assumption that the psi ebgo large subunit, like the ebgo and ebga large subunits, was 100% active as isolated, were about an order of magnitude lower than for true ebgo large subunit, whilst Km values were similar . The very significant kinetic effect of this inadvertant site-undirected mutagenesis indicates that quite large kinetic effects of amino-acid replacements in enzymes may have no obvious mechanistic significance.

Biochem Biophys Res Commun, 1995 Nov 13, 216(2), 589 - 94
Vanadate and bafilomycin A1 are potent inhibitors of the ATPase activity of the reconstituted bacterial ATP-binding cassette transporter for maltose (MalFGK2); Hunke S et al.; Vanadate and Bafilomycin A1 were shown to inhibit the ATPase activity of the reconstituted binding protein-dependent ATP-binding Cassette (ABC) transporter for maltose (MalFGK2) of Salmonella typhimurium in the micromolar range . This is in sharp contrast to the recent finding that the isolated ATPase subunit MalK was insensitive to both compounds . Our data provide the first experimental evidence for the view that functional coupling of the ATPase domain of an ABC transporter to the membrane-integral domains is crucial for conferring sensitivity to vanadate and bafilomycin A1 . Possible consequences for the mode of action of ABC transport proteins are discussed.

J Mol Biol, 1995 Nov 3, 253(4), 547 - 58
Radial mass analysis of the flagellar filament of Salmonella: implications for the subunit folding; Yamashita I et al.; X-ray fiber diffraction patterns of the R-type straight flagellar filament of Salmonella typhimurium SJW1655 strain showed layer-lines with an axial spacing of 1/437 A-1, which could be resolved only due to very small disorientation angles (< 2 degrees) of the filaments in oriented sol specimens . Although the equatorial layer-line was situated between the relatively strong first layer-lines right above and below it, these small disorientation angles and a new method of two-dimensional angular deconvolution allowed us to determine the equatorial layer-line intensities quite accurately . The equatorial data were phased by using the amplitude difference between the native flagellar filament and its heavy atom derivatives . One of the heavy-atom derivatives was prepared by introducing a cysteine residue by site-directed mutagenesis and applying a mercury compound . From the equatorial structure factors, the radial density distribution of the filament was calculated at 11 A resolution . A prominent feature was two pairs of high density peaks at radii of around 25 and 45 A and a deep density trough between them, which corresponds to the concentric double tubular structure in the core region that has been found in the density map recently deduced by helical image reconstruction from electron micrographs of frozen hydrated filaments . The molecular masses were estimated for four radial segments that correspond to the morphological domains identified in the map of helical image reconstruction . Then the domains were assigned to sequence positions by correlating the estimated masses with those of proteolytic fragments of flagellin . The assignment is consistent with the distributions of secondary structures and in particular alpha-helical coiled-coils that were predicted from the sequence . It also helps to understand how the polymerization behaviour is affected by truncation of the disordered terminal regions of flagellin and why mutations in a specific region are responsible for changes in the polymorphic shape of the filament.

Mol Microbiol, 1995 Nov, 18(4), 715 - 27
hilA is a novel ompR/toxR family member that activates the expression of Salmonella typhimurium invasion genes; Bajaj V et al.; During infection of its hosts, Salmonella enters intestinal epithelial cells . Many Salmonella typhimurium genes required for bacterial entry into host cells are encoded on a 40 kb 'pathogenicity island' . We report here the identification of hilA, a gene within the 'island' that appears to encode an activator of invasion gene expression . By using a set of lacZY transcriptional fusions to S . typhimurium invasion genes, we found that hilA activates the expression of invasion genes located on the 'pathogenicity island' . hilA is required for efficient entry into HEp-2 cells in vitro . The predicted amino acid sequence of hilA shares significant homology with the DNA-binding domains of the OmpR-ToxR family of transcriptional activators . However, unlike OmpR and ToxR, HilA contains neither a phosphoryl acceptor nor a membrane-spanning domain, and, therefore, its activity may be modulated by a novel mechanism . Many environmental conditions modulate the ability of Salmonella to enter non-phagocytic mammalian cells . It has been proposed that induction of Salmonella invasion proteins in response to a combination of environmental cues ensures that bacterial entry is limited to specific sites and times during infection . Our results are consistent with the hypothesis that hilA plays a key role in the regulation of Salmonella invasion during infection.

Mol Microbiol, 1995 Nov, 18(3), 479 - 90
Salmonella typhimurium secreted invasion determinants are homologous to Shigella Ipa proteins; Hueck CJ et al.; Salmonella typhimurium secreted proteins (Ssp) were previously implicated in epithelial cell invasion . Here we describe four genes (SspB, sspC, sspD, and sspA), located between spaT and prgH, which encode proteins of 63, 42, 36, and 87 kDa, respectively . These Ssp are homologous to Shigella flexneri secreted proteins IpaB, IpaC, IpaD and IpaA . A non-invasive mutant with a transposon insertion in sspC lacks Ssp of 87, 42 and 36 kDa . Complementation and analyses show that sspC and sspD encode the 42 and the 36 kDa Ssp, while the 87 kDa Ssp is encoded by sspA . sspC and sspD, but not sspA, are required for invasion . Amino-terminal sequencing shows that SspC and SspA are secreted without amino-terminal processing . We further demonstrate that Ssp secretion requires proteins encoded by prgHIJK, homologous to the Shigella Ipa secretion system, since SspA is abundantly secreted by wild-type bacteria but is completely retained within the cellular fraction of a prgHIJK mutant . A precipitate containing abundant SspC and three other major Ssp of 63, 59 and 22 kDa was isolated from culture supernatants of wild-type bacteria . These data indicate that major secreted invasion determinants of S . typhimurium are structurally and functionally homolgous to S . flexneri Ipa proteins.

J Toxicol Sci, 1995 Nov, 20(5), 619 - 27
Mutagenicity studies with palm fruit carotene; Masuda M et al.; The mutagenicity of palm fruit carotene was examined using the reverse mutation test with bacteria, the chromosomal aberration test with mammalian cells and the micronucleus test in mice . The carotene induced neither reverse mutation in Salmonella typhimurium TA98, TA1537, TA100, TA1535 and in Escherichia coli WP2uvrA, nor structural and numerical (polyploidy) chromosomal aberrations in the Chinese hamster fibroblast cell line (CHL) . In addition, no increase in micronucleated polychromatic erythrocytes was elicited in the micronucleus test in CD-1(ICR) male mice . It is concluded that palm fruit carotene had no mutagenic activity in these in vitro and in vivo tests.

Exp Eye Res, 1995 Nov, 61(5), 629 - 32
Endotoxin induced uveitis in the mouse: susceptibility and genetic control; Li Q et al.; Endotoxin induced uveitis in the mouse provides a useful animal model for acute anterior uveitis in humans . We have investigated the susceptibility of endotoxin-induced uveitis among various mouse strains, and have examined the relationship between genetic background and the resultant inflammatory response to endotoxin . We studied ten strains with differing major histocompatibility-2 genes, lipopolysaccharide response gene, and strains with mast cell depletion and its sham control . Anterior uveitis was induced by injecting 300 micrograms of Salmonella typhimurium endotoxin into one hind footpad . Mice were then killed 8, 12, 16, 20, 24, 48 and 72 hr after endotoxin injection, and vertical sections of the eyes through the pupil-optic nerve axis were evaluated for ocular inflammation . C3H/HeN mice developed severe uveitis . In contrast, C3H/HeJ mice (lipopolysaccharide response gene-) did not develop uveitis even though it has the same genetic background and shares the same major histocompatibility-2 haplotype with C3H/HeN mice (lipopolysaccharide response gene+) . The strain that was mast-cell deficient (W/Wv) developed minimal uveitis; however, W/+ mice, with mast cells, developed more inflammation at 48 and 72 hr after endotoxin injection . C3H.SW and FVB/N mice also developed severe uveitis, and BALB/C, CBA/J, and B10.A developed mild uveitis . In conclusion, there is a wide variation in the magnitude and susceptibility to endotoxin among mouse strains . Multiple factors appear to influence this variability, including non-histocompatibility-2 genetic background, the lipopolysaccharide response gene, and the presence of mast cells.

J Endod, 1995 Nov, 21(11), 537 - 42
Investigation of mutagenicity of mineral trioxide aggregate and other commonly used root-end filling materials; Kettering JD et al.; Little information is available regarding the mutagenicity of root-end filling materials . To study mutagenicity of Intermediate Restorative Material (IRM), Super-EBA, and a potential root-end filling material, mineral trioxide aggregate (MTA), strains of Salmonella typhimurium LT-2 (TA 98, R-factor strain and TA 1535, non-R-factor strains) were used in a standard Ames mutagenicity assay . Positive controls (S9 protein and benzo-(a)-pyrene and N-methyl-N'-nitro-N-nitrosoguanidine) operated properly . No increase in revertant bacteria colony counts occurred with any of the test materials . Based on these results, it seems that IRM, Super-EBA, and MTA are not mutagenic as measured by the Ames Test.

Mutagenesis, 1995 Nov, 10(6), 497 - 504
Metabolic activation of the genotoxic environmental contaminants 2- and 3-nitrofluoranthene in variants of Salmonella typhimurium TA98; Ball LM et al.; The mutagenic environmental pollutants 2-nitrofluoranthene (2-NFA) and 3-nitrofluoranthene (3-NFA), labelled with 3H and 14C respectively, were incubated with Salmonella typhimurium strain TA98, its nitroreductase-deficient variant TA98NR and its O-acetytransferase-deficient variant TA98/1,8-DNP6, to investigate the activity of these metabolic pathways under conditions approximating those of the Ames assay, hence their contribution to mutagenic potency . 2-Aminofluoranthene (2-AFA) was the major metabolite of 2-NFA (4 microM) in all three TA98 variants, isolated by reverse-phase HPLC and identified by UV-vis and NMR spectroscopy and mass spectrometry . 2-AFA was formed more slowly in TA98NR (65 pmol/h/ml resting phase bacterial broth, 1 to 2 x 10(9) bacteria/ml) than in TA98 (295 pmol/h/ml) or TA98/1,8-DNP6 (82 pmol/h/ml) . 2-Acetamidofluoranthene (2-AAFA) was also identified in incubations with TA98 (80 pmol/h/ml), TA98NR (21 pmol/h/ml), and TA98/1,8-DNP6 (8 pmol/h/ml) . 3-Aminofluoranthene (3-AFA, confirmed by UV-vis and NMR spectroscopy and mass spectrometry) was formed by all three variants from 3-NFA (4 microM): TA98, 1.76 nmol/h/ml; TA98NR, 0.55 nmol/h/ml; TA98/1,8-DNP6, 2.93 nmol/h/ml . 3-Acetamidofluoranthene (3-AAFA) was not detected in any of the variants . 3-AFA and 3-AAFA were less mutagenic than 3-NFA, and required S9 for activation . Mutagenicity of 3-NFA relative to initial nitroreduction rate was similar in TA98 and in TA98NR, but almost 10-fold lower in TA98/1,8-DNP6; hence O-acetylation considerably enhances the mutagenicity of reduction products of 3-NFA . Mutagenicity of 2-NFA relative to initial nitroreduction rate was similar in TA98 and in TA98/1,8-DNP6; the bacterial genotoxicity of 2-NFA is therefore largely independent of O-acetyltransferase activity . Ratios of mutagenicity to nitroreduction rate were similar in TA98 for 2-NFA and 3-NFA; differences in the potency of these isomers arise primarily from their respective suitabilities as substrates for nitroreductase enzymes.

Res Microbiol, 1995 Nov-Dec, 146(9), 751 - 9
Characterization of non-virulence plasmids with homology to the virulence plasmid of Salmonella dublin; Aabo S et al.; Six wild-type (wt) strains of Salmonella typhimurium, one wt strain of S . heidelberg and 12 wt strains of Escherichia coli were isolated based on both hybridization to a 6-kb HindIII fragment of the non-virulence coding part of the S . dublin serovar-specific virulence plasmid and the absence of hybridization to the virulence genes (spv genes) of the same plasmid . Such hybridization was shown to be caused by resident plasmids in all strains and to involve the same region of 30 to 37 kb of consecutive HindIII fragments on the S . dublin virulence plasmid, suggesting a common origin of this plasmid DNA . Nine of the plasmids were selected for detailed characterization and were shown not to be of the same plasmid species . They varied in size between 44 and 88 kb, they showed incompatibility with the plasmid K-MP10, or belonged to incompatibility group X, and with the exception of five plasmids from E . coli, they showed different HindIII restriction profile patterns.

J Clin Microbiol, 1995 Nov, 33(11), 2888 - 93
Combined PCR-oligonucleotide ligation assay for rapid detection of Salmonella serovars; Stone GG et al.; We have developed a rapid and sensitive assay for the detection of Salmonella serovars in veterinary clinical specimens . This method utilizes a short cultivation period followed by PCR . For detection of the amplified product, an enzyme-linked immunosorbent assay (ELISA)-based oligonucleotide ligation assay (OLA) was used . In this study, the PCR-OLA technique was compared with conventional culture and membrane hybridization for the detection of Salmonella bacteria . In evaluating the PCR-OLA with Salmonella serovars and non-Salmonella strains of bacteria, A490 readings for 51 Salmonella strains, representing 28 serovars, were significantly higher (P < 0.05) than those for 25 non-Salmonella bacteria . With serial 10-fold dilutions of Salmonella CFU or with known concentrations of purified chromosomal DNA from Salmonella typhimurium ATCC 29946, the PCR-OLA was able to detect > or = 20 CFU per assay or > or = 80 fg of chromosomal DNA (corresponding to 160 molecules of DNA) . Of 102 suspect clinical specimens screened, 15 were positive for Salmonella bacteria by both culture and the PCR-OLA procedure (100% sensitivity), and 3 samples were positive only by PCR-OLA (96.6% specificity), indicating a positive predictive value of 83.3% and a negative predictive value of 100% . In all experiments, the PCR-OLA was as sensitive as membrane hybridization . These results indicate that a limited enrichment cultivation and PCR-OLA could be used as a presumptive screening test for the detection of Salmonella serovars from any sample that currently requires extensive cultivation and that this assay would be adaptable to automation.

FEMS Microbiol Lett, 1995 Nov 1, 133(1-2), 113 - 8
Streptococcus pneumoniae type 3 encodes a protein highly similar to the human glutamate decarboxylase (GAD65); Garcia E et al.; A 2.5-kb ScaI fragment of the type 3 pneumococcal strain 406 DNA containing a 1425-nucleotide open reading frame (gadA) and encoding a 475-amino acid protein (M(r) 54,427) was characterised . The gene gadA was expressed in Salmonella typhimurium . Pulsed-field gel electrophoresis and Southern blotting analysis of DNAs prepared from several pneumococcal serotypes showed that only those clinical isolates belonging to serotype 3 harbour the gadA gene . Sequence comparison of GadA with proteins included in the data banks revealed the highest similarity with human glutamate decarboxylase (GAD65) (59% similarity, 28% identity) . Auto-antibodies to GAD65 have been associated with the onset of insulin-dependent diabetes mellitus . Interestingly, several epitopes of GAD65 that have been identified as immunodominant are particularly well conserved in the pneumococcal GadA.

Protein Sci, 1995 Nov, 4(11), 2327 - 34
Crystal structure of the dipeptide binding protein from Escherichia coli involved in active transport and chemotaxis; Dunten P et al.; The Escherichia coli periplasmic dipeptide binding protein functions in both peptide transport and taxis toward peptides . The structure of the dipeptide binding protein in complex with Gly-Leu (glycyl-L-leucine) has been determined at 3.2 A resolution . The binding site for dipeptides is designed to recognize the ligand's backbone while providing space to accommodate a variety of side chains . Some repositioning of protein side chains lining the binding site must occur when the dipeptide's second residue is larger than leucine . The protein's fold is very similar to that of the Salmonella typhimurium oligopeptide binding protein, and a comparison of the structures reveals the structural basis for the dipeptide binding protein's preference for shorter peptides.

Biosci Biotechnol Biochem, 1995 Nov, 59(11), 2151 - 2
Assessment of the mutagenicity of extracts of TMV-coat-protein-gene induced transgenic tomato by the umu-test; Shinmoto H et al.; We examined the mutagenicity of extracts (juice and ethanol extract) from a transgenic tomato that was established by transfection of a gene encoding the coat protein of tobacco mosaic virus (TMV) to the F1 hybrid between Lycopersicon esculentum LA1000 and L . peruvianum PI128650, by the umu-test with Salmonella typhimurium TA1535/pSK1002 as the test organism . The extracts showed no detectable mutagenicity . The extracts from the above-mentioned F1 hybrids and wild tomatoes and cultivars (L . peruvianum PI128650, L . peruvianum PI126944, L . pimpinellifolium LS1524, L . pimpinellifolium LA722, L . hirsutum LS503, Mini-carol, Sun-cherry, Momotaro, Odoriko, Kagome77, and Ponderosa) also showed no detectable mutagenicity.

Microbiology, 1995 Nov, 141 ( Pt 11), 2945 - 53
Aerotaxis in Halobacterium salinarium is methylation-dependent; Lindbeck JC et al.; The behavioural response to a gradient of oxygen (aerotaxis) has been characterized in the archaeon, Halobacterium salinarium . When the gas surrounding a drop of H . salinarium strain S9-P culture was changed abruptly from 10% (v/v) O2 to 100% N2, the bacteria transiently increased the frequency of reversing before they adapted and resumed random swimming . When the gas was returned to 10% O2 the bacteria responded by swimming smoothly for approximately 45 s . Aerotaxis was strongest when respiration in H . salinarium was highest and when bacteriorhodopsin and halorhodopsin were not contributing to the proton motive force . Starvation for methionine of the auxotrophic H . salinarium essentially abolished the step-down aerotactic response . Methanol production from demethylation of methyl-accepting chemotaxis proteins was transiently increased in H . salinarium S9-P by a step down or step up in oxygen concentration, as observed in methylation-dependent chemotaxis in H . salinarium . The taxis-negative and methyltransferase-deficient mutant, H . salinarium strain Pho72 did not exhibit changes in methanol release in response to aerotaxis or chemotaxis stimuli . This is the first report of an aerotactic response that is dependent on methylation of methyl-accepting chemotaxis proteins . Aerotaxis in Escherichia coli and Salmonella typhimurium is independent of transducer methylation.

Appl Environ Microbiol, 1995 Nov, 61(11), 3781 - 7
Bioactivation of cysteine conjugates of 1-nitropyrene oxides by cysteine conjugate beta-lyase purified from Peptostreptococcus magnus; Kataoka K et al.; To determine the role of cysteine conjugate beta-lyase (beta-lyase) in the metabolism of mutagenic nitropolycyclic aromatic hydrocarbons, we determined the effect of beta-lyase on the mutagenicities and DNA binding of cysteine conjugates of 4,5-epoxy-4,5-dihydro-1-nitropyrene (1-NP 4,5-oxide) and 9,10-epoxy-9,10-dihydro-1-nitropyrene (1-NP 9,10-oxide), which are detoxified metabolites of the mutagenic compound 1-nitropyrene . We purified beta-lyase from Peptostreptococcus magnus GAI0663, since P . magnus is one of the constituents of the intestinal microflora and exhibits high levels of degrading activity with cysteine conjugates of 1-nitropyrene oxides (1-NP oxide-Cys) . The activity of purified beta-lyase was optimal at pH 7.5 to 8.0, was completely inhibited by aminooxyacetic acid and hydroxylamine, and was eliminated by heating the enzyme at 55 degrees C for 5 min . The molecular weight of beta-lyase was 150,000, as determined by fast protein liquid chromatography . S-Arylcysteine conjugates were good substrates for this enzyme . As determined by the Salmonella mutagenicity test, 5 ng of beta-lyase protein increased the mutagenicity of the cysteine conjugate of 1-NP 9,10-oxide (10 nmol per plate) 4.5-fold in Salmonella typhimurium TA98 and 4.1-fold in strain TA100 . However, beta-lyase had little effect on the cysteine conjugate of 1-NP 4,5-oxide (10 nmol per plate) . Both conjugates exhibited only low levels of mutagenicity with nitroreductase-deficient strain TA98NR . In vitro binding of 1-NP oxide-Cys to calf thymus DNA was increased by adding purified beta-lyase or xanthine oxidase.(ABSTRACT TRUNCATED AT 250 WORDS)

Toxicol Lett, 1995 Nov, 81(1), 33 - 8
Absorption of 3-chloro-4-(dichloromethyl)-5-hydroxy-2-{5H} furanone (MX) through rat small intestine in vitro; Clark NW et al.; The intestinal absorption of 3-chloro-4-(dichloromethyl)-5-hydroxy-2-{5H} furanone (MX), a highly mutagenic furanone found in chlorinated waters, was studied using an in vitro everted rat gut sac system, using reverse mutation in Salmonella typhimurium to detect mutagens transported from the mucosal to the serosal compartments . Absorption was measurable, but limited, with significant increase in bacterial revertants (serosal compartment) noted at a dose of 50 micrograms/ml MX (mucosal compartment, p < 0.05) . Gut sac incubation with MX and glutathione (GSH, 1.0 mM) resulted in no detectable absorption of mutagens . Preincubation with diethylmaleate to deplete mucosal GSH resulted in increased absorption of MX-derived mutagens compared to controls (a significant induction of revertant colonies was noted at a dose of 25 micrograms/ml MX p < 0.05) . Gut sac incubation with chlorinated fulvic acids resulted in no detectable absorption of mutagens . In vitro studies to assess the possibility of beta-lyase activation of the postulated MX-GSH conjugate showed no mutagenic activation.

Mutat Res, 1995 Nov, 348(3), 137 - 45
Mutagenicity of nitrobenzyl derivatives: potential bioreductive anticancer agents; Juneja TR et al.; Ortho-, meta- and para-nitrobenzyl bromides, alcohols, ethers and esters were synthesised and tested for their mutagenicity toward Salmonella typhimurium TA100, TA100NR (nitroreductase deficient) and TA98 in absence of S9 mix and in TA100 with S9 mix . Compounds of the ortho- and meta-series were non mutagenic with and without S9 mix . Except for the alcohol and ether, the compounds of the para-series were mutagenic in TA100 with activity sequence propionate > butyrate > benzoate > acetate > bromide and this specific activity was reduced considerably by S9 mix . The Ames Salmonella test system does not seem to be an appropriate model to evaluate mutagenicity of o-nitrobenzyls . However, further work is in progress to test all the compounds for mutagenicity in mammalian system.

J Bacteriol, 1995 Nov, 177(22), 6593 - 600
1-Methylguanosine deficiency of tRNA influences cognate codon interaction and metabolism in Salmonella typhimurium; Li JN et al.; 1-Methylguanosine (m1G) is present next to the 3' end of the anticodon (position 37) in tRNA(1,2,3,Leu), tRNA(1,2,3,Pro), and tRNA(3Arg) . A mutant of Salmonella typhimurium lacks m1G in these seven tRNAs when grown at or above 37 degrees C, as a result of a mutation (trmD3) in the structural gene (trmD) for the tRNA(m1G37)methyltransferase . The m1G deficiency induced 24 and 26% reductions in the growth rate and polypeptide chain elongation rate, respectively, in morpholinepropanesulfonic acid (MOPS)-glucose minimal medium at 37 degrees C . The expression of the leuABCD operon is controlled by the rate with which tRNA(2Leu) and tRNA(3Leu) read four leucine codons in the leu-leader mRNA . Lack of m1G in these tRNAs did not influence the expression of this operon, suggesting that m1G did not influence the efficiency of tRNA(2,3Leu) . Since the average step time of the m1G-deficient tRNAs was increased 3.3-fold, the results suggest that the impact of m1G in decoding cognate codons may be tRNA dependent . The trmD3 mutation rendered the cell more resistant or sensitive to several amino acid analogs . 3-Nitro-L-tyrosine (NT), to which the trmD3 mutant is sensitive, was shown to be transported by the tryptophan-specific permease, and mutations in this gene (mtr) render the cell resistant to NT . Since the trmD3 mutation did not affect the activity of the permease, some internal metabolic step(s), but not the uptake of the analog per se, is affected . We suggest that the trmD3-mediated NT sensitivity is by an abnormal translation of some mRNA(s) whose product(s) is involved in the metabolic reactions affected by the analog . Our results also suggest that tRNA modification may be a regulatory device for gene expression.

J Bacteriol, 1995 Nov, 177(22), 6585 - 92
The chromosome of Salmonella paratyphi A is inverted by recombination between rrnH and rrnG; Liu SL et al.; Salmonella paratyphi A, a human-adapted bacterial pathogen, causes paratyphoid enteric fever . We established the genome map of strain ATCC 9150 by the use of four endonucleases, XbaI, I-CeuI, AvrII (= BlnI), and SpeI, which generated 27, 7, 19, and 38 fragments, respectively; the sum of the fragments in each case indicates a genome size of ca . 4,600 kb . With phage P22, we transduced Tn10 insertions in known genes from Salmonella typhimurium LT2 to S . paratyphi A ATCC 9150 and located these insertions on the S . paratyphi A chromosome through the XbaI and AvrII sites in Tn10 and through the increased size of the SpeI fragment bearing a Tn10 . Compared with the maps of other Salmonella species, the S . paratyphi A genomic map showed two major differences: (i) an insertion of about 100 kb of DNA between rrnH/G and proB and (ii) an inversion of half the genome between rrnH and rrnG, postulated to be due to homologous recombination between the rrn genes . We propose that during the evolution of S . paratyphi A, the first rearrangement event was the 100-kb insertion, which disrupted the chromosomal balance between oriC and the termination of replication, forcing the rrnH/G inversion to restore the balance . The insertion and the inversion are both present in all 10 independent wild-type S . paratyphi A strains tested.

J Bacteriol, 1995 Nov, 177(22), 6432 - 9
Expression of the putA gene encoding proline dehydrogenase from Rhodobacter capsulatus is independent of NtrC regulation but requires an Lrp-like activator protein; Keuntje B et al.; Four Rhodobacter capsulatus mutants unable to grow with proline as the sole nitrogen source were isolated by random Tn5 mutagenesis . The Tn5 insertions were mapped within two adjacent chromosomal EcoRI fragments . DNA sequence analysis of this region revealed three open reading frames designated selD, putR, and putA . The putA gene codes for a protein of 1,127 amino acid residues which is homologous to PutA of Salmonella typhimurium and Escherichia coli . The central part of R . capsulatus PutA showed homology to proline dehydrogenase of Saccharomyces cerevisiae (Put1) and Drosophila melanogaster (SlgA) . The C-terminal part of PutA exhibited homology to Put2 (pyrroline-5-carboxylate dehydrogenase) of S . cerevisiae and to aldehyde dehydrogenases from different organisms . Therefore, it seems likely that in R . capsulatus, as in enteric bacteria, both enzymatic steps for proline degradation are catalyzed by a single polypeptide (PutA) . The deduced amino acid sequence of PutR (154 amino acid residues) showed homology to the small regulatory proteins Lrp, BkdR, and AsnC . The putR gene, which is divergently transcribed from putA, is essential for proline utilization and codes for an activator of putA expression . The expression of putA was induced by proline and was not affected by ammonia or other amino acids . In addition, putA expression was autoregulated by PutA itself . Mutations in glnB, nifR1 (ntrC), and NifR4 (ntrA encoding sigma 54) had no influence on put gene expression . The open reading frame located downstream of R . capsulatus putR exhibited strong homology to the E . coli selD gene, which is involved in selenium metabolism . R . capsulatus selD mutants exhibited a Put+ phenotype, demonstrating that selD is required neither for viability nor for proline utilization.

J Bacteriol, 1995 Nov, 177(22), 6371 - 80
The cobalamin (coenzyme B12) biosynthetic genes of Escherichia coli; Lawrence JG et al.; The enteric bacterium Escherichia coli synthesizes cobalamin (coenzyme B12) only when provided with the complex intermediate cobinamide . Three cobalamin biosynthetic genes have been cloned from Escherichia coli K-12, and their nucleotide sequences have been determined . The three genes form an operon (cob) under the control of several promoters and are induced by cobinamide, a precursor of cobalamin . The cob operon of E . coli comprises the cobU gene, encoding the bifunctional cobinamide kinase-guanylyltransferase; the cobS gene, encoding cobalamin synthetase; and the cobT gene, encoding dimethylbenzimidazole phosphoribosyltransferase . The physiological roles of these sequences were verified by the isolation of Tn10 insertion mutations in the cobS and cobT genes . All genes were named after their Salmonella typhimurium homologs and are located at the corresponding positions on the E . coli genetic map . Although the nucleotide sequences of the Salmonella cob genes and the E . coli cob genes are homologous, they are too divergent to have been derived from an operon present in their most recent common ancestor . On the basis of comparisons of G+C content, codon usage bias, dinucleotide frequencies, and patterns of synonymous and nonsynonymous substitutions, we conclude that the cob operon was introduced into the Salmonella genome from an exogenous source . The cob operon of E . coli may be related to cobalamin synthetic genes now found among non-Salmonella enteric bacteria.

Infect Immun, 1995 Nov, 63(11), 4456 - 62
Spacious phagosome formation within mouse macrophages correlates with Salmonella serotype pathogenicity and host susceptibility; Alpuche-Aranda CM et al.; Light microscopic studies indicated a correlation between the virulence for mice of different Salmonella serotypes and the ability to form or maintain spacious phagosomes (SP) within mouse macrophages . Although Salmonella typhimurium induced membrane ruffling, macropinocytosis, and SP formation in macrophages from BALB/c mice, serotypes which are nonpathogenic for mice produced markedly fewer SP . SP formation correlated with both serotype survival within mouse macrophages and reported lethality for mice . Time-lapse video microscopy demonstrated that the human pathogen S . typhi induced generalized macropinocytosis and SP formation in human monocyte-derived macrophages, indicating a similar morphology for the initial phases of this host-pathogen interaction . In contrast to bone marrow-derived macrophages from BALB/c mice, macrophages from S . typhimurium-resistant outbred (CD-1) and inbred (CBA/HN) mice did not initiate generalized macropinocytosis after bacterial infection and formed markedly fewer SP . These deficiencies were not due to the Ihy resistance genotype of these mice, as macrophages from mice that were congenic except for the Ihy locus demonstrated equal SP formation in response to S . typhimurium . The observation that S . typhimurium-resistant CD-1 and CBA/HN mice are deficient in the ability to form and/or maintain SP indicates that a variable host component is important for SP formation and suggests that the ability to induce or form SP affects susceptibility to S . typhimurium . When serotypes nonpathogenic for mice were used to infect BALB/c macrophages, or when CD-1 or CBA/HN mouse macrophages were infected by S . typhimurium, some of the SP that formed shrank within seconds . This rapid shrinkage suggests that SP maintenance is also important for S . typhimurium survival within macrophages . These studies indicate that both host and bacterial factors contribute to SP formation and maintenance, which correlate with Salmonella intracellular survival and the ability to cause lethal enteric (typhoid) fever.

Infect Immun, 1995 Nov, 63(11), 4402 - 8
Granulation in livers of mice infected with Salmonella typhimurium is caused by superoxide released from host phagocytes; Umezawa K et al.; The pathophysiological roles of superoxide (O2.-) at the site of infection of facultative intracellular bacteria were examined in this study . To evaluate the actual in vivo generation of the superoxide, an ex vivo chemiluminescence assay was newly developed . When ICR mice were infected with a sublethal dose (8 x 10(4) CFU) of Salmonella typhimurium, the number of bacteria in the liver reached its peak at 5 days after infection (10(5.05) CFU/g of liver) and decreased thereafter . At 21 days after infection, the bacteria became undetectable . On the other hand, phorbol myristate 13-acetate-stimulated O2.- generation reached a maximum at 7 days after infection (mean photon count, 1,249 cps versus 28.8 cps before infection; n = 4) and decreased thereafter to a level similar to that before infection at 21 days after infection (28.8 cps) . Histological examinations revealed that the total area of the lesions reached a peak at 7 days after infection (7.2 x 10(4) microns 2/10 visual fields) . In the early phase, a microabscess with infiltration of polymorphonuclear cells was noted, and then, in the late stage, the lesion was replaced by granulation with mononuclear cell infiltration . When microscopic lesions were measured histologically, a significant correlation between the area of the lesions and phorbol myristate 13-acetate-stimulated O2.- generation was observed, which suggested that superoxide was responsible for the generation of the lesions . Modified superoxide dismutase, i.e., alpha-4-({6-(N-maleimido)hexanoyloxymethyl} cumyl)half-butyl-esterified poly(stylrene-co-malelic acid)-conjugated superoxide dismutase (SM-SOD), was then applied . When SM-SOD was administered to suppress the O2.- generation in vivo, the number of bacteria increased (10(6.1) CFU) . However, the lesion formation was inhibited (total lesion area, 0.3 x 10(4) microns 2) . These results suggest that the establishment of the microabscess and granuloma formation after S . typhimurium infection is not due to the bacteria per se but rather to the O2.- from the host's phagocytes . Two aspects of the O2.-, i.e., the bactericidal role and the tissue-injurious effect, were clearly demonstrated in this study . Therefore, the information obtained from these results is useful in designing treatment strategy for similar kinds of infection.

Infect Immun, 1995 Nov, 63(11), 4329 - 35
Host restriction phenotypes of Salmonella typhi and Salmonella gallinarum; Pascopella L et al.; Salmonella typhi and Salmonella gallinarum phenotypes correlated with mouse host restriction have been identified by using in vitro and in vivo systems . S . typhi is capable of entering the murine intestinal epithelium via M cells, as is Salmonella typhimurium, which causes systemic infection in the mouse . But, unlike S . typhimurium, S . typhi does not destroy the epithelium and is cleared from the Peyer's patches soon after M-cell entry . S . gallinarum appears to be incapable of entering the murine Peyer's patch epithelium . Our in vitro evidence suggests that S . gallinarum is taken up in murine phagocytic cells by a mechanism different from that of S . typhimurium . S . typhimurium is taken up at a higher frequency and is maintained at higher viable counts throughout a 24-h time course in a murine macrophage-like cell line than are S . gallinarum and S . typhi.

Mutat Res, 1995 Nov, 332(1-2), 63 - 71
An empirical and theoretical study on mechanisms of mutagenic activity of hydrazine compounds; Poso A et al.; Hydrazine compounds are important industrial and laboratory chemicals . Many of them are carcinogenic in animal tests . Although the carcinogenicity is well established, the results of mutagenicity tests performed on alkylhydrazines vary greatly in different studies . In an attempt to clarify the situation we have applied Salmonella typhimurium TA102 tests to hydrazine and its mono- and dimethyl derivatives . These compounds were also tested by an Escherichia coli DNA repair-assay . The results of the repair tests indicate that unsymmetrically alkylated hydrazines can cause DNA-lesions which are lethal in repair-deficient strains . Finally QSAR (Quantitative Structure Activity Relationships) was used to develop a model to describe the genotoxic mechanism of hydrazine compounds, taking advantage of the results of previous mutagenicity studies . Energy of the lowest unoccupied molecular orbital together with octanol-water partition coefficient explains nearly completely the mutagenic activity of alkylated hydrazine compounds included in the analysis . The mutagenic activity of unsubstituted hydrazine is apparently based on different mechanisms.

J Biol Chem, 1995 Oct 27, 270(43), 25645 - 50
Amphibacillus xylanus NADH oxidase and Salmonella typhimurium alkyl-hydroperoxide reductase flavoprotein components show extremely high scavenging activity for both alkyl hydroperoxide and hydrogen peroxide in the presence of S . typhimurium alkyl-hydroperoxide reductase 22-kDa protein component; Niimura Y et al.; The flavoprotein NADH oxidase from Amphibacillus xylanus consumes oxygen to produce hydrogen peroxide . The amino acid sequence of this flavoprotein shows 51.2% identity to the F-52a component, denoted AhpF, of the alkyl-hydroperoxide reductase from Salmonella typhimurium . AhpF also catalyzes NADH-dependent hydrogen peroxide formation under aerobic conditions, albeit at a somewhat slower rate than the Amphibacillus protein . In the presence of the 22-kDa colorless component (AhpC) of the Salmonella alkyl-hydroperoxide reductase, both proteins catalyze the 4-electron reduction of oxygen to water . Both flavoproteins are active as AhpC reductases and mediate electron transfer, resulting in the NADH-dependent reduction of hydrogen peroxide and cumene hydroperoxide . Both enzymes' Km values for hydrogen peroxide, cumene hydroperoxide, and NADH are so low that they could not be determined accurately . Vmax values for hydrogen peroxide or cumene hydroperoxide reduction are > 10,000 min(-1) at 25 degrees C . These values are almost the same as the reduction rate of the flavoprotein component by NADH . The involvement in catalysis of a redox-active disulfide of the A . xylanus flavoprotein was shown by construction of three mutant enzymes, C337S, C340S, and C337S/C40SC337S/C340S . Very little activity for hydrogen peroxide or cumene hydroperoxide was found with the single mutants (C337S and C340S), and none with the double mutant (C337S/C340S) . Analysis of the DNA sequence upstream of the Amphibacillus flavoprotein structural gene indicated the presence of a partial open reading frame homologous to the Salmonella ahpC structural gene (64.3% identical at the amino acid sequence level), suggesting that the NADH oxidase protein of A . xylanus is also part of a functional alkyl-hydroperoxide reductase system within these catalase-lacking bacteria.

Biochem Pharmacol, 1995 Oct 26, 50(9), 1407 - 12
Induction of cytochrome P450-dependent monooxygenase in serum-free cultured Hep G2 cells; Nakama A et al.; We examined the induction of cytochrome P450-dependent mixed-function monooxygenase (MFO) in the human hepatoma cell line Hep G2 by means of several factors . The MFO activities induced in the cells cultured in medium containing five commercial sera varied significantly, and the activity in the cells cultured in the absence of serum was about twice as high as that in cells supplemented with serum . The activity of ethoxycoumarin O-deethylase was highest 12 hr after adding 3-methylcholanthrene, and it was induced by several polycyclic aromatic hydrocarbons such as benzo(a)anthracene and benzo(a)pyrene, which are usually found in urban air as environmental contaminants . Furthermore, an extract from the total suspended particles collected using a high volume air sampler, which was mutagenic in the Ames assay using Salmonella typhimurium TA98, induced the same enzyme activities in Hep G2 cells . These findings suggest that serum-free culture allows the stable and highly sensitive measurement of induced MFO activity, and that studies of MFO induction by environmental samples using human hepatoma Hep G2 cells should provide helpful information regarding the risk associated with environmental contaminants.

Mol Cell Biochem, 1995 Oct 18, 151(2), 141 - 8
Binding between lipopolysaccharide and cecropin A; De Lucca AJ et al.; Cecropin A (CA), a bioactive peptide, produced significant lethality to Pantoea agglomerans (PA) at low concentrations . Significant mortality occurred immediately after addition of CA . Separate preincubations of lipopolysaccharides (LPS) from the following bacteria: PA, Serratia marcescens, Escherichia coli (EC), and Salmonella typhimurium with CA were performed prior to the bioassay . CA was also preincubated with diphosphoryl lipid A (DPL-A) from EC and S . minnesota (SM), trilinolein, palmitic, lauric and myristic acids (fatty acids contained in the lipid A of PA-LPS) and bovine brain gangliosides . Spectral analysis to determine the interaction between glycosphingolipids (sphingomyelin, bovine brain gangliosides, and galactocerebrosides) and CA were performed . Results showed that all types of LPS and DPL-A as well as gangliosides studied blocked CA lethality to PA . The level of inhibition of CA antibacterial properties was dependent on LPS and DPL-A concentration . The individual fatty acids and trilinolein did not affect CA lethality to PA . Spectral studies showed complexation between CA and PA-LPS, both types of DPL-A, and the glycosphingolipids . Biological and chemical analyses confirm that CA binds to the diphosphoryl lipid A moiety of LPS.

Gene, 1995 Oct 16, 164(1), 95 - 100
Sequence of a novel virulence-mediating gene, virC, from Vibrio anguillarum; Milton DL et al.; Previously, the double-transposon (Tn) mutant VAN20 of Vibrio anguillarum (Va) 775.17B was isolated . This mutant lacked a major surface antigen (MSA) suggested to be a lipopolysaccharide (LPS) and showed a 10(5)-fold increase in the 50% lethal dose (LD50) when fish were infected intraperitoneally . In this study, the two Tn insertion sites within the chromosome were identified, a plasmid insertion mutation was made at each locus in a more virulent strain of Va, NB10, and the virulence was analyzed . One mutant displayed a 10(4)-fold increase in LD50, whereas the second mutant showed the wild-type (wt) phenotype . However, both mutants still expressed the MSA, suggesting that there may be more than two Tn insertions in VAN20 or that a double mutation is required to prevent production of the MSA . The DNA locus for the virulent phenotype was cloned and sequenced . A potential transcriptional unit consisting of three putative open reading frames (ORFs) was identified . The Tn was located in the second ORF, virC (virulence) . The first ORF (34.8 kDa) showed 30% homology to the Escherichia coli and Salmonella typhimurium cysG (cysteine) genes . The virC gene (51.4 kDa) and the third ORF (24 kDa) showed no homology to other proteins in GenBank . Plasmid insertion mutants were made within each of these ORFs and the virulence was assayed . Only the virC mutant showed a loss in virulence, indicating that virC is a novel gene that is essential for the virulence of Va.

Gene, 1995 Oct 16, 164(1), 85 - 7
Revision of the amino-acid sequence of 3-isopropylmalate dehydrogenase from Salmonella typhimurium by means of X-ray crystallography; Kryger G et al.; The amino acid (aa) sequence of the leuB gene product of Salmonella typhimurium, 3-isopropylmalate dehydrogenase (IPMDH), has been revised using electron density maps from X-ray structure determination . The nucleotide (nt) sequence of both strands of leuB has been redetermined to confirm the crystallographic findings . It does not agree with the previously reported S . typhimurium leuB nucleotide sequence {Andreadis and Rosenthal, Biochim . Biophys . Acta 1129 (1992) 228-230}.

Gene, 1995 Oct 16, 164(1), 81 - 4
An alternative sigma factor controls transcription of flagellar class-III operons in Escherichia coli: gene sequence, overproduction, purification and characterization; Liu X et al.; Based on the studies of the FliA protein in Bacillus subtilis (Bs) and Salmonella typhimurium (St), the Escherichia coli (Ec) fliA gene has been proposed to encode a flagellar-specific sigma factor, sigma 28 . In this study, the complete nucleotide (nt) sequence of Ec fliA was determined . The fliA coding region consists of 717 nt starting with a GTG start codon and ending with a TAA stop codon . The gene product is predicted to be 239 amino acids (26,435 Da) . Sequence comparison between Ec FliA and the sigma 28 of St revealed 93.7% identity . Gene fliA was amplified by the polymerase chain reaction, subcloned into expression vector pT7-7, and overexpressed . The overproduced 28-kDa FliA protein, recognized by the St anti-sigma 28 antibody, was purified to homogeneity . The purified protein was able to initiate transcription from the tar promoter in the presence of RNP core enzyme . We conclude that FliA functions as an alternative sigma factor sigma 28 which is specific for flagellar operons in Ec.

Vet Rec, 1995 Oct 7, 137(15), 374 - 9
Evaluation of an O-antigen ELISA for screening cattle herds for Salmonella typhimurium; Hoorfar J et al.; A total of 2585 serum samples from 62 dairy herds located in four different regions of Denmark were tested in an O-antigen (0:1,4,5,12)-based ELISA for the detection of antibodies against Salmonella typhimurium . Ten closed herds from an island with no reported occurrence of salmonellosis for several years, and 12 herds from a salmonella enzootic area which had had clinical outbreaks of S typhimurium were used to define a herd ELISA cut-off value . When herds with at least 5 per cent of the serum samples having an optical density of > 0.5 were considered ELISA-positive, all 10 herds from the salmonellosis-free island were ELISA-negative, and all but one of the 12 S typhimurium-infected herds were ELISA-positive, which resulted in a herd test sensitivity of 0.92 and herd test specificity of 1.0 . Eleven of the 12 S typhimurium-infected herds were negative in a blocking ELISA based on a monoclonal antibody to the 0:9 antigen of the serogroup D salmonellas, indicating the possibility of rapid serogroup-specific screening of herds by means of these two tests . Ten other randomly selected herds with clinical outbreaks of S dublin were all, to a large extent, positive in the 0:1,4,5,12-ELISA, whereas a S dublin (0:1,9,12)-ELISA described previously appeared to be more serogroup D-specific . Thus, the 0:1,4,5,12-ELISA appears to be useful for detecting herd infections with S typhimurium, and positive reactions may be further discriminated by the serogroup D-specific ELISA.

J Biol Chem, 1995 Oct 6, 270(40), 23560 - 9
Purification and characterization of the bifunctional CobU enzyme of Salmonella typhimurium LT2 . Evidence for a CobU-GMP intermediate; O'Toole GA et al.; The CobU protein of Salmonella typhimurium was overexpressed and purified to approximately 94% homogeneity . N-terminal sequencing of purified CobU confirmed the first 22 amino acids . In vitro assays showed that CobU has kinase and guanylyltransferase activities which catalyze the synthesis of adenosyl-cobinamide-GDP from adenosyl-cobinamide, via an adenosyl-cobinamide-phosphate intermediate . We present evidence that the transfer of the guanylyl moiety of GTP to adenosyl-cobinamide-phosphate proceeds via an phosphoramidate-linked, enzyme-guanylyl intermediate . In the presence of oxygen, kinase and guanylyltransferase activities of CobU were lost . Treatment of inactive CobU with dithiothreitol restored approximately 20% of the kinase and guanylyltransferase activities, indicating the involvement of sulfhydryl groups in enzyme activity . The sulfhydryl modifying agents 5,5'-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide abolished both CobU activities . Native CobU protein was a dimer (approximately 40 kDa) that functioned optimally at pH 8.8-9.0 and 37 degrees C . Substrates and kinetic parameters for both activities were determined . The preferred corrinoid substrate for this enzyme was adenosyl-cobinamide . In vitro experiments are consistent with previous genetic studies which had suggested that adenosyl-cobinamide was the preferred substrate of CobU, and that CobU functioned more efficiently in the absence of oxygen.

Microb Drug Resist, 1995 Fall, 1(3), 211 - 8
Identification of DNA gyrase A mutations in ciprofloxacin-resistant isolates of Salmonella typhimurium from men and cattle in Germany; Heisig P et al.; Six multiply resistant isolates of Salmonella typhimurium var . copenhagen with high-level resistance to fluoroquinolones (e.g., MIC of ciprofloxacin: 32 micrograms/ml) were isolated from human patients (n = 3) and from cattle (n = 3) . The isolates were examined by complementation tests using a set of broad-host-range plasmids, which carry either the gyrA+ or the gyrB+ genes or a combination of both from Escherichia coli K-12 . The results indicated a combination of gyrA and gyrB mutations in all isolates . Subsequent direct sequencing of PCR-generated internal DNA fragments of gyrA revealed an identical double mutation in all six isolates (Ser-83-->Ala and Asp-87-->Asn) . In addition, the results of phenotypic (i.e., phagetype, biotype, serotype) and genotypic characterization {i.e., ribotyping and polymerase chain reaction fingerprinting (PCR-fingerprinting)} were identical for all six isolates and were distinguishable from a quinolone-susceptible strain of the same serovar and an unrelated isolate of S . typhimurium . These data indicate the clonal identity of the fluoroquinolone-resistant strains of S . typhimurium isolated from men and cattle in Germany.

Clin Infect Dis, 1995 Oct, 21 Suppl 2, S146 - 51
Biology and clinical significance of virulence plasmids in Salmonella serovars; Guiney DG et al.; Non-typhoid Salmonella strains containing virulence plasmids are highly associated with bacteria and disseminated infection in humans . These plasmids are found in Salmonella serovars adapted to domestic animals, such as Salmonella dublin and Salmonella choleraesuis, as well as in the widely distributed pathogens Salmonella typhimurium and Salmonella enteritidis . Although virulence plasmids differ between serovars, all contain a highly conserved 8-kb region containing the spv locus that encodes the spvR regulatory gene and four structural spvABCD genes . Studies in mice suggest that the spv genes enhance the ability of Salmonella strains to grow within cells of the reticuloendothelial system . The spv genes are not expressed during exponential growth in vitro but are rapidly induced following entry of Salmonella strains into mammalian cells, including macrophages . Transcription of the spv genes is controlled by the stationary-phase sigma factor RpoS, and mutations in RpoS abolish virulence . These studies suggest that the ability of Salmonella strains to respond to starvation stress in the host tissues is an essential component of virulence.

Microb Pathog, 1995 Oct, 19(4), 193 - 201
Induction of the phoE promoter upon invasion of Salmonella typhimurium into eukaryotic cells; Janssen R et al.; Live attenuated Salmonella typhimurium strains expressing foreign antigens can be used for vaccination purposes . Due to deleterious effects of constitutive, high-level expression of the heterologous antigens, there is often strong selection pressure against plasmids encoding these antigens, resulting in rapid segregation in vivo . In vivo-inducible promoters may be a good alternative for constitutive promoters . The outer membrane protein PhoE of Escherichia coli is being used as a carrier for foreign antigenic determinants . Here we studied whether its expression from a plasmid is induced in S . typhimurium upon invasion of eukaryotic cells . This appeared to be the case . Furthermore, a S . typhimurium phoE mutant was constructed and the effects of the mutation on invasion, intracellular survival and virulence were studied . Survival in HEp-2 cells or in the macrophage-like cell line J744 was not, or only slightly, affected . Furthermore, the mutant appeared to be as virulent for mice as the wild-type strain.

Avian Dis, 1995 Oct-Dec, 39(4), 867 - 72
Invasion of Caco-2 cells by Salmonella typhimurium (Copenhagen) isolates from healthy and sick chickens; Kottom TJ et al.; In a previous study, Salmonella isolates of sick birds were distinguished from those of apparently healthy birds by their high degree of invasion of tissue culture cells . In this study, a single pair of Salmonella isolates was examined to determine the source of this observed difference in invasion . When isolates were allowed to invade Caco-2 cells for 8 hours, the isolate from the sick bird (S) appeared to invade in greater numbers than did the isolate from the healthy bird (H) . However, when invasion was distinguished from intracellular growth/survival, it was found that H invaded in greater numbers than S, but once inside the cell, H declined in number, and S increased . Inhibition of RNA, protein, and DNA syntheses lessened the degree to which both invaded . The presence of mannose inhibited invasion by S but did not appear to inhibit invasion by H . Trypsin treatment of monolayers affected invasion of S and H, whereas neuraminidase treatment did not . There was no significant difference noted between S and H in ability to adhere to fixed monolayers . Therefore, the two isolates tested differ in their mechanisms of entry into Caco-2 cells, the efficiency with which they invade, and their ability to survive within Caco-2 cells.

Immunol Cell Biol, 1995 Oct, 73(5), 452 - 6
Prolactin and insulin regulate the release of IL-1-alpha and IFN-gamma from murine splenocytes activated with porins or LPS of Salmonella typhimurium; Vitiello M et al.; Murine splenocytes treated with prolactin (PRL) or insulin were stimulated in vitro with porins or lipopolysaccharide (LPS) of Salmonella typhimurium . It was seen that PRL inhibits the release of IFN-gamma from splenocytes treated with porins by about 20% while having no effect on the release of IL-1-alpha . Splenocytes porin-stimulated splenocytes exhibited a remarkable increase in IL-1-alpha release (100%) and a diminished release of IFN-gamma (about 50%) in the presence of insulin . The splenocytes stimulated with LPS had a reduced release of IL-1-alpha (75%) and IFN-gamma (about 50%) when insulin was added . The data suggest that classical endocrine system participates in a bioregulatory feedback loop that may prevent unwanted toxicity from cytokine excess . However, some bacterial products sometimes enormously unbalance this regulatory network.

Res Microbiol, 1995 Oct, 146(8), 659 - 70
Salmonella typhimurium TnphoA mutants with increased sensitivity to biological and chemical detergents; Lacroix FJ et al.; Salmonella typhimurium is a ubiquitous pathogenic bacterium able to sustain the environmental conditions of the gastrointestinal tract, including biliary salts . To understand the mechanisms involved in bile salt resistance and, more generally, detergent resistance, we investigated S . typhimurium mutants produced with the random mutagenic TnphoA transposon . A total of 3,000 transpositional mutants were isolated . Three strains among the 1,432 first mutants lost the ability to grow in the presence of biological and chemical detergents . They were prototrophic and exhibited normal lipopolysaccharide and outer membrane protein profiles after SDS-PAGE . They did not show sensitivity to dyes but showed very different sensitivities to antibiotics . For each mutant strain, Southern blotting analysis revealed a unique TnphoA insertion at different chromosomal locations . These observations were confirmed by transduction experiments.

Indian J Physiol Pharmacol, 1995 Oct, 39(4), 347 - 53
Mutagenicity and anti-mutagenicity of selected spices; Soudamini KK et al.; Using Salmonella typhimurium strains TA 100 and TA 1535, the mutagenicity and anti-mutagenicity of extracts of several spices were checked . Spices like pepper, pippali, ginger and mustard increased the number of revertants indicating their mutagenic potential . Garlic extract on the other hand was found to inhibit the mutagenicity produced by direct acting mutagens such as N-methyl N'-nitro-N-nitrosoguanidine and sodium azide . Asafoetida and turmetic extract were found to inhibit microsomal activation dependent mutagenicity of 2-acetamidofluorene . Similar results were also obtained using curcumin and eugenol which are phenolics present in turmeric and clove respectively . These results indicated that some of the spices may ameliorate the effect of environmental mutagens especially present in the food.

J Vet Diagn Invest, 1995 Oct, 7(4), 481 - 7
Enzyme-linked immunosorbent assay for Salmonella serology using lipopolysaccharide antigen; Smith BP et al.; Enzyme-linked immunosorbent assay (ELISA) using Salmonella lipopolysaccharide (LPS) to measure specific IgG titers in cattle has proven useful . Serology can be used to assess vaccine responses and infection rates, to detect carriers, and to aid in epidemiologic studies . The objective of this study was to assess cross-reactions using sera from cattle vaccinated with different Salmonella serogroups . ELISA plates using lipopolysaccharide from serogroup B, C1, C3, D1 or E1 as the plate antigens were tested . LPS was extracted from Salmonella typhimurium (Serogroup B; somatic antigens 01, 4, 12), S . montevideo (C1; 06, 7), S . kentucky (C3; 08, 20), S . dublin (D1; 01, 9, 12) and S . anatum (E1; 03, 10) using the Westphal method . Fifteen cows were found to be seronegative for all 5 of these serogroup antigens . Each cow was then vaccinated 3 times at 2-week intervals with a killed Salmonella bacterin . The 15 different serotypes used for vaccination were chosen to represent a wide array of Salmonella serogroups with a wide array of somatic "O" antigens expressed, including somatic antigens 1, 2, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13, 14, 18, 19, 20, 22, 23, 25, and 27 . With each antigen tested, the highest ELISA titers were seen with sera from cattle vaccinated with homologous O antigens, indicating that reactions were highly O antigen-specific . Some cross reactions between subgroups sharing one O factor antigen were found; these titers were lower than those found with homologous serogroups sharing 2 or more antigens . Only serum from the cow vaccinated from S . anatum (group E; antigens 03, 10) cross-reacted at a low titer with group C1 (O somatic antigens 6, 7) and D1 (O somatic antigens 1, 9, 12) plate antigens, with which no somatic antigens were shared . We conclude from these results that Salmonella serology using LPS antigens is highly O antigen-specific and predictable.

Int J Food Microbiol, 1995 Oct, 27(2-3), 117 - 28
Combined effect of gamma radiation and heating on the destruction of Listeria monocytogenes and Salmonella typhimurium in cook-chill roast beef and gravy; Grant IR et al.; The effect of heating alone (60, 65 or 70 degrees C), heating after irradiation (0.8 kGy) and heating after irradiation and storage for 14 days at 2-3 degrees C on the destruction of Listeria monocytogenes and Salmonella typhimurium in artifically inoculated minced cook-chill roast beef and gravy was investigated . Inoculated minced roast beef samples (5 g) were heated in Stomacher bags completely immersed in a water bath at each of the test temperatures . Survivors were enumerated and D and z values were determined for each of the pathogens . Observed thermal D values for two strains of L . monocytogenes at 60, 65 and 70 degrees C in the absence of pre-irradiation were 90.0-97.5 s, 34.0-53.0 s and 22.4-28.0 s, respectively, whereas thermal D values after pre-irradiation were 44.0-46.4 s, 15.3-16.8 s and 5.5-7.8 s at 60, 65 and 70 degrees C, respectively . This reduction in D values provides evidence for radiation-induced heat-sensitisation in L . monocytogenes . There was some evidence of heat-sensitisation of S . typhimurium at 60 degrees C, but not at either 65 or 70 degrees C . The z value also decreased as a consequence of pre-irradiation to a dose of 0.8 kGy (11.0-12.7 degrees C) . The radiation-induced heat-sensitivity in L . monocytogenes was found to persist for up to 2 weeks storage at 2-3 degrees C prior to heating . As cook-chill products are intended to be reheated prior to consumption the results of the present study suggest that any L . monocytogenes present in a cook-chill product would be more easily killed during reheating if it were to be treated with a low dose of gamma radiation during manufacture.

Zentralbl Veterinarmed B, 1995 Oct, 42(8), 493 - 502
Copro-antibody in calves from dams vaccinated against Salmonella typhimurium; Staak C et al.; Calves received colostrum either with (positive colostrum) or without (negative colostrum) anti-Salmonella typhimurium antibodies . Regarding the detectability of copro-antibodies, the following observations have been made . In calves that had been given positive colostrum on day 4 post natum (p.n.), copro-antibodies were detectable over 7 days, while in those that had received positive colostrum on day 1 p.n . copro-antibodies were detectable over 45 days . After supplying highly positive colostrum, copro-antibodies were found over a period of 8 weeks, and after supplying weakly positive colostrum, they were found over a period of 5 weeks . It is concluded that serum antibodies are transferred to the intestine for maximum local protection, and that there exists a preference for the intestinal system regarding the distribution of salmonella antibodies . Challenge infection on day 5 p.n . of calves that had received highly positive colostrum resulted in a copro-antibody gap that reached the limit of detectability in two calves that were excreting salmonellas . Challenge infection of calves that had received negative colostrum provoked a local IgM and IgA antibody response.

Pharmacol Toxicol, 1995 Oct, 77(4), 288 - 92
Cassia senna inhibits mutagenic activities of benzo{a}-pyrene, aflatoxin B1, shamma and methyl methanesulfonate; al-Dakan AA et al.; Ethanol extract of Senokot tablets (Cassia senna concentrate used as vegetable laxative), was found to be non-mutagenic while it inhibited the mutagenicity of benzo{a}pyrene, shamma, aflatoxin B1 and methyl methanesulfonate in the Ames histidine reversion assay using the Salmonella typhimurium tester strain TA98 . While the Senokot extract completely inhibited the mutagenicity of promutagens (i.e . metabolic activation dependent) like benzo{a}pyrene and shamma, it reduced the mutagenic activity of the direct acting mutagen methyl methanesulfonate by only 58% . The mutagen aflatoxin B1 showed a 25-fold increase in the number of histidine revertants per plate at low concentrations (1.0-4.0 micrograms/plate) in the presence of metabolic activation system while at high concentrations (10.0-30.0 micrograms/plate) it proved to be weakly mutagenic (with a 5-fold increase in the number of histidine revertants/plate) without metabolic activation . The Senokot extract completely inhibited the mutagenic effect of low concentrations of aflatoxin B1 in the presence of metabolic activation but not that resulting from higher concentrations without metabolic activation . The results obtained with benzo{a}pyrene, shamma and aflatoxin B1 indicated that the antimutagenic effects of Senokot extract could be largely due to an interaction with the metabolic process involved in the activation of procarcinogens . However, the results obtained with methyl methanesulfonate suggested that factors in Senokot may also interact with direct mutagens to produce some antimutagenic effects . An ethanol extract of crude senna leaves found to be weakly mutagenic also inhibited (though less than Senokot) the mutagenic effect of benzo{a}pyrene suggesting that the antimutagenic principle is present in the complex plant material itself.

Med Microbiol Immunol (Berl), 1995 Oct, 184(3), 123 - 7
Interactions of Yersinia enterocolitica with polarized human intestinal Caco-2 cells; Curfs JH et al.; The in vitro interactions of Yersinia enterocolitica, Salmonella typhimurium and Escherichia coli with polarized human colonic carcinoma (Caco-2) cells are described . Invasion of a confluent Caco-2 cell monolayer by Yersinia and Salmonella took place within 4 h after contact, which was in marked contrast to E . coli which did not invade Caco-2 cells . Cytoplasmic extrusions developed on the apical membrane and indicated the site of entrance of bacteria into the Caco-2 cells . Intracellular Yersinia and Salmonella were surrounded by a vacuolar membrane . Single as well as multiple bacteria were enclosed within a single vacuole . At 6 h after contact some of the intracellular yersiniae were found free in the cytoplasm . Furthermore, morphological signs of degeneration of Caco-2 cells such as vacuolization and autophagy were observed . Caco-2 cells infected with Salmonella also showed degenerative changes but the salmonellae resided within membrane-bound vacuoles in contrast to Yersinia . These observations are in contrast to those described for the invasion of other cells lines (not derived from intestinal epithelium) by Yersinia and may reflect more closely the interactions between Yersinia and the intestinal epithelium during gastrointestinal infection.

Genetika, 1995 Oct, 31(10), 1380 - 5
{Starvation-induced mutagenesis in Salmonella typhimurium}; Gizatullin FSh et al.; Spontaneous reversion to prototrophy of two his alleles of Salmonella typhimurium--hisG428 (ochre) and hisC527 (amber)--was studied . Strains containing hisG428 allele in the chromosome and within the multicopy plasmid pAQ1 were used . When the hisG428 allele was chromosomally located, no sharp fluctuations in the number of His+ revertant were observed in fluctuation experiments and the distribution of revertants obeyed the Poisson law . The pattern of revertants distribution in strains carrying plasmid pAQ1 was intermediate between the Poisson distribution and jackpot distribution described by Luria and Delbruck . Conversely, sharp fluctuations were observed in the distribution of His+ revertants of the hisC527 allele obtained from independent cultures . The data presented here suggest that His+ revertants, including ochre suppressors, appear under the influence of histidine starvation, when the hisG428 allele is chromosomally located . At the same time, reversion to prototrophy at the hisC527 allele does not strictly depend on the presence or absence of this amino acid in the medium . Plasmid location and novobiocin-resistance mutation promoted the identifying of preexisting mutants among His+ revertants of the hisG428 allele . The data obtained also demonstrate that the presence of the mutator plasmid pKM101 preferentially increases the frequency of adaptive His+ reversions.

Voen Med Zh, 1995 Oct, (10), 49 - 52, 80
{Experience in eliminating hospital salmonellosis in a large general treatment institution}; Akimkin VG; The author made a report on the results of epidemic examination of salmonellosis infection in a large medical institution . Peculiarities of the Salmonella typhimurium agent are described, as well as mechanism of transmission and most inflicted groups of patients . The factors which provided for proliferation of salmonellosis are depicted, taking into account the characteristic features of infectious process inside medical establishment.

J Trop Pediatr, 1995 Oct, 41(5), 301 - 3
Viral diarrhoea in a rural coastal region of Karnataka India; Shetty M et al.; A total of 106 children below 5 years of age admitted to the Kasturba Medical College Hospital Manipal Karnataka (South India) were investigated over a period of 6 months to determine the aetiological role of viruses in acute diarrhoea . Viral aetiological agents isolated were Rotaviruses in 12 (11 per cent) cases, Adenoviruses in 3 (3 per cent) cases, coronavirus and astroviruses in two (2 per cent) cases each . Non-viral isolates were Cryptosporidium and Salmonella typhimurium in two cases each, and Entamoeba histolytica and Shigella flexneri in one case each.

J Bacteriol, 1995 Oct, 177(20), 5784 - 9
Heterogeneity of genome sizes among natural isolates of Escherichia coli; Bergthorsson U et al.; Comparisons of the genetic maps of Escherichia coli K-12 and Salmonella typhimurium LT2 suggest that the size and organization of bacterial chromosomes are highly conserved . Employing pulsed-field gel electrophoresis, we have estimated the extent of variation in genome size among 14 natural isolates of E . coli . The BlnI and NotI restriction fragment patterns were highly variable among isolates, and genome sizes ranged from 4,660 to 5,300 kb, which is several hundred kilobases larger than the variation detected between enteric species . Genome size differences increase with the evolutionary genetic distance between lineages of E . coli, and there are differences in genome size among the major subgroups of E . coli . In general, the genomes of natural isolates are larger than those of laboratory strains, largely because of the fact that laboratory strains were derived from the subgroup of E . coli with the smallest genomes.

J Bacteriol, 1995 Oct, 177(20), 5778 - 83
Anaerobic protoporphyrin biosynthesis does not require incorporation of methyl groups from methionine; Bollivar DW et al.; It was recently reported (H . Akutsu, J.-S . Park, and S . Sano, J . Am . Chem . Soc . 115:12185-12186, 1993) that in the strict anaerobe Desulfovibrio vulgaris methyl groups from exogenous L-methionine are incorporated specifically into the 1 and 3 positions (Fischer numbering system) on the heme groups of cytochrome c3 . It was suggested that under anaerobic conditions, protoporphyrin IX biosynthesis proceeds via a novel pathway that does not involve coproporphyrinogen III as a precursor but instead may use precorrin-2 (1,3-dimethyluroporphyrinogen III), a siroheme and vitamin B12 precursor which is known to be derived from uroporphyrinogen III via methyl transfer from S-adenosyl-L-methionine . We have critically tested this hypothesis by examining the production of protoporphyrin IX-based tetrapyrroles in the presence of exogenous {14C}methyl-L-methionine under anaerobic conditions in a strict anaerobe (Chlorobium vibrioforme) and a facultative anaerobe (Rhodobacter capsulatus) . In both organisms, 14C was incorporated into the bacteriochlorophyll precursor, Mg-protoporphyrin IX monomethyl ester . However, most of the label was lost upon base hydrolysis of this compound to yield Mg-protoporphyrin IX . These results indicate that although the administered {14C}methyl-L-methionine was taken up, converted into S-adenosyl-L-methionine, and used for methyl transfer reactions, including methylation of the 6-propionate of Mg-protoporphyrin IX, methyl groups were not transferred to the porphyrin nucleus of Mg-protoporphyrin IX . In other experiments, a cysG strain of Salmonella typhimurium, which cannot synthesize precorrin-2 because the gene encoding the enzyme that catalyzes methylation of uroporphyrinogen III at positions 1 and 3 is disrupted, was capable of heme-dependent anaerobic nitrate respiration and growth on the nonfermentable substrate glycerol, indicating that anaerobic biosynthesis of protoporphyrin IX-based hemes does not require the ability to methylate uroporphyrinogen III . Together, these results indicate that incorporation of L-methionine-deprived methyl groups into porphyrins or their precursors is not generally necessary for the anaerobic biosynthesis of protoporphyrin IX-based tetrapyrroles.

J Appl Bacteriol, 1995 Oct, 79(4), 353 - 9
The effect of sodium chloride concentration and pH on the growth of Salmonella typhimurium colonies on solid medium; McKay AL et al.; The growth of Salmonella typhimurium colonies on a model food system (agar solidified culture medium) was followed . Colony radius, determined using computer image analysis (IA) techniques, and viable cell number per colony were measured as indices of colony growth, and the effect of {NaCl} (0.5-3.5% (w/v)) and pH (7.0-5.0) on colony growth at 30 degrees C was observed; colonies were point inoculated from serial dilutions . Colony growth (between 13 and 26 h after inoculation) was linear when expressed in terms of radius, and exponential when expressed in terms of viable cell number per colony . Overall, both increasing the {NaCl} and decreasing the pH had little effect on colony growth, other than to delay the onset of linear radial growth . Initial specific growth rate (mu) ranged from 0.73 to 0.87 h-1 . Thin films of agar medium on microscope slides allowed the growth of microcolonies to be observed after just 4 h incubation . A greater understanding of the growth kinetics of bacterial colonies, and the effects of environment on such data, may enable better control of foodborne bacterial pathogens, and consequently an improvement in food product safety.

Food Chem Toxicol, 1995 Oct, 33(10), 803 - 14
DNA strand breaks induced through active oxygen radicals by fragrant component 4-hydroxy-2-hydroxymethyl-5-methyl-3(2H)-furanone in Maillard reaction of hexose/amino acid; Hiramoto K et al.; The Maillard reaction of glucose/amino acid produces components that induce strand breakage of supercoiled DNA . This study was designed to elucidate the structure of the active components and the mechanisms of their DNA strand breakage . When an aqueous mixture of glucose/glycine heated under reflux for 4 hr was extracted with ethyl acetate, the extract showed mutagenicity to Salmonella typhimurium TA100 and strand-breaking activity for supercoiled DNA . One of the components with DNA strand-breaking activity was isolated by repeated HPLC using a reverse phase column . The component was identified as 4-hydroxy-2-hydroxymethyl-5-methyl-3(2H)-furanone (HHMF), a fragrant component in the Maillard reaction mixture . HHMF was similarly produced in the reaction of glucose/alanine and fructose/glycine . DNA strand-breaking activity of the component at pH 7.4 increased with increasing dose of the component and with increasing incubation time . The strand-breaking activity of the component was greater at pH 7.4 than at pH 4.4 and 9.4; it was inhibited in the presence of superoxide dismutase, catalase, hydroxyl radical scavengers, spin-trapping agents, thiol compounds and metal chelators, and also by removal of dissolved oxygen from the reaction mixture . The strand-breaking activity was enhanced in the presence of ionic iron . Incubation of HHMF with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) gave electron spin resonance signals characteristics of the DMPO-OH adduct, indicating generation of the hydroxyl radical . HHMF generated superoxide, hydrogen peroxide and then hydroxyl radical with the aid of a trace amount of metal ions, which effectively cleaved the DNA strand.

FEMS Microbiol Lett, 1995 Oct 1, 132(1-2), 73 - 8
Fts insertional mutant of Salmonella typhimurium; Cerquetti MC et al.; A temperature-sensitive filamentation (fts) Salmonella typhimurium mutant was isolated after transposon mutagenesis with mini-Tn 10dTc . The mutant was unable to form colonies after 20 h incubation at 37 degrees C on LB agar . Colonies appeared, however, after longer incubation at the restrictive temperature . Filamentation affected only part of the bacterial population . Rapid mapping using Mu dP22 hybrid phages revealed that the mutation, ftsD220, lies within minutes 68.5 and 73.6 on the genetic map . Further analysis revealed that the ftsD220 mapped at min 73 and that it is linked to cysG (6%) and to aroB (39%) . Complementation tests suggested that the ftsD220 mutation is not homologous to a Escherichia coli ftsH mutation.

FEMS Microbiol Lett, 1995 Oct 1, 132(1-2), 57 - 60
A novel gyrB mutation in a fluoroquinolone-resistant clinical isolate of Salmonella typhimurium; Gensberg K et al.; In order to study the role of gyrB in antibiotic resistance in post-ciprofloxacin therapy fluoroquinolone-resistant clinical isolates of Salmonella typhimurium, plasmid pBP548, which contains the Escherichia coli gyrB gene, was used in complementation studies . In a heterodiploid strain, the wild-type (quinolone sensitive) allele is dominant over the resistant allele therefore, eleven clinical isolates were complemented with gyrB encoded on pBP548 . Only one transformant, L18pBP548, exhibited increased susceptibility to the quinolones nalidixic acid, ciprofloxacin and sparfloxacin . The amino acid sequence of the gyrase B protein from a wild-type and the pre-therapy S . typhimurium (deduced from the nucleotide sequence) was identical to that of E . coli from codons 436 to 470; however, a point mutation was identified in codon 463 of gyrB of the quinolone-resistant post-therapy isolate L18, giving rise to an amino acid substitution of serine to tyrosine.

Carcinogenesis, 1995 Oct, 16(10), 2467 - 71
Chemoprotective effects of capsaicin and diallyl sulfide against mutagenesis or tumorigenesis by vinyl carbamate and N-nitrosodimethylamine; Surh YJ et al.; Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide) is a major pungent and irritating ingredient of hot chilli peppers, which are frequently consumed as spices . This dietary phytochemical has been found to interact with microsomal xenobiotic metabolizing enzymes in rodents . Capsaicin and its saturated analog dihydrocapsaicin (trans-8-methyl-N-vanillyl-6-nonanamide) have been proposed to inactivate cytochrome P-450 HE1 by irreversibly binding to the active sites of the enzyme . Besides cytochrome P-450 HE1, other isoforms of the P-450 superfamily were also reported to be inhibited by capsaicin . The inhibition by capsaicin of microsomal monooxygenases involved in carcinogen activation implies its chemopreventive potential . As part of a program to investigate chemoprotective properties of capsaicin we initially determined the effect of capsaicin on vinyl carbamate (VC)- and N-nitrosodimethylamine (NDMA)-induced mutagenesis in Salmonella typhimurium TA100 . Capsaicin (0.42 mM) attenuated the bacterial mutagenicity of VC and NDMA by 50% and 42% respectively . Diallyl sulfide, a thioether found in garlic with selective P-450 HE1 inhibitory activity, also lessened the mutagenicity of the above carcinogens in a concentration-dependent manner . The suppression of VC- and NDMA-induced mutagenesis by capsaicin and diallyl sulfide correlated with their inhibition of P-450 IIE1-mediated p-nitrophenol hydroxylation and NDMA N-demethylation . Pretreatment of female ICR mice with a topical dose of capsaicin lowered the average number of VC-induced skin tumors by 62% at 22 weeks after promotion . A similar degree of protection was attained with oral administration of diallyl sulfide before carcinogen treatment . The results of this study suggest that capsaicin and diallyl sulfide suppress VC- and NDMA-induced mutagenesis or tumorigenesis in part through inhibition of the cytochrome P-450 IIE1 isoform responsible for activation of these carcinogens.

Carcinogenesis, 1995 Oct, 16(10), 2315 - 20
Genotoxicity of zearalenone, an estrogenic mycotoxin: DNA adduct formation in female mouse tissues; Pfohl-Leszkowicz A et al.; Zearalenone is a non-steroidal estrogenic mycotoxin produced by several species of Fusarium which colonize maize, barley, oats, wheat and sorghum and have been implicated in numerous mycotoxicoses in farm animals, especially pigs . In a NTP mouse study a dose-related incidence of hepatocellular adenomas was seen in female mice . A limited number of genotoxicity assays have been conducted with zearalenone . Zearalenone was found to be negative in the Salmonella typhimurium assay . It was also negative in a eukaryotic cell mutation assay with Saccharomyces cerevisae . However, zearalenone and its estrogenic metabolites showed a positive DNA damaging effect in recombination tests with Bacillus subtilis . In this study DNA adducts in female mice and rat treated i.p or orally with zearalenone were determined using a 32P-postlabeling method . Several DNA adducts (12-15) were found in the kidney and liver of female mice treated with a single dose of zearalenone (2 mg/kg i.p . or orally) . The total DNA adduct levels reached 114 +/- 37 and 1393 +/- 324 adducts/10(9) nucleotides respectively in kidney and liver after i.p . treatment and 548 +/- 50 adducts/10(9) nucleotides in liver after oral treatment . In mice ovary DNA adducts appeared only after repeated doses (1 mg/kg body wt on days 1, 5, 7, 9 and 10) . The total DNA adducts after 10 days in this organ were 17 +/- 5 adducts/10(9) nucleotides . Some adducts were common to all organs . Others were specific to an organ . In contrast, no DNA adducts could be detected in rat organs after i.p . treatment . These results confirm the genotoxicity of zearalenone and its ability to induce hepatocellular adenomas, rather than tumours of genital organs, in mice.

J Submicrosc Cytol Pathol, 1995 Oct, 27(4), 445 - 9
Morphological changes induced in HEp-2 cells by Salmonella typhimurium porins; De Martino L et al.; The effects of different doses of Salmonella typhimurium porins were investigated on human laryngeal epithelial (HEp-2) cells by fluorescence and transmission electron microscopy . The changes observed in microfilament organization suggested an involvement of cytoskeleton in the cell response . Furthermore, morphologic phenomena characterized by ultrastructural membrane modifications also involved the nuclear membrane.

Microbiology, 1995 Oct, 141 ( Pt 10), 2749 - 56
Role of LPS length in clearance rate of bacteria from the bloodstream in mice; Ohno A et al.; Strains of Pseudomonas aeruginosa isolated from patients with cystic fibrosis (CF) never spread systemically . This may be due to serum sensitivity since these strains are very sensitive to complement-mediated bactericidal activity . A serum-resistant mutant, P . aeruginosa TUM3 HSR, was obtained from serum-sensitive strain TUM3 from a CF patient in order to clarify the mechanism of failure of systemic spread . LPS profiles on silver-stained gels and immunological analysis revealed that a long O-polysaccharide side chain was overproduced on the LPS molecules of TUM3 HSR as compared with the LPS of TUM3 . The clearance rate from the bloodstream in mice was compared in the two strains . The number of TUM3 bacteria in 1 ml of blood, 10 min after injection into the tail vein, significantly decreased from 1.7 x 10(8) to 3.7 x 10(5) c.f.u . ml-1 . In contrast, TUM3 HSR was not eliminated during the same period (decrease from 1.9 x 10(8) to 3.4 x 10(7) c.f.u . ml-1) . Interestingly, these isogenic strains were not killed by 40% murine serum, probably reflecting immaturity of the complement-mediated killing system in mice . These results pointed to a correlation between LPS structure and blood clearance rate in mice . This was confirmed by examining blood clearance kinetics using the smooth-LPS strain Salmonella typhimurium LT2 and LPS-deficient mutants derived from it . S . typhimurium LT2 resisted blood clearance while the LPS-deficient mutants were cleared rapidly . None of the S . typhimurium strains were killed by murine serum . The number of P . aeruginosa TUM3 and S . typhimurium LPS-deficient mutants trapped in the liver following injection into the peripheral circulation was greater than that of their counterparts.(ABSTRACT TRUNCATED AT 250 WORDS)

Radiat Res, 1995 Oct, 144(1), 36 - 42
Radiation-induced cell lethality of Salmonella typhimurium ATCC 14028: cooperative effect of hydroxyl radical and oxygen; Kim AY et al.; The lethality of gamma-radiation doses of 0.2 to 1.0 kGy for Salmonella typhimurium ATCC 14028 was measured in the presence of air, N2 and N2O and with the hydroxyl radical scavengers formate and polyethylene glycol (PEG), M(r) 8,000 . Saturation of cell suspensions with either N2O or N2/N2O (1:1, v/v) gas was expected to double the number of hydroxyl radicals (OH.) and to produce an equivalent increase in lethality, but this did not occur . Adding 10% (v/v) O2 to either N2 or N2O gas produced approximately the same gamma-irradiation lethality for S . typhimurium as did air . Addition of hydroxyl radical scavengers, 40 mM formate and 1.5% (w/v) PEG, significantly reduced the lethality of gamma radiation for S . typhimurium in the presence of air but not in the presence of N2 or N2O gases . Membrane-permeable formate provided slightly better protection than nonpermeable PEG . Cells of S . typhimurium grown under anaerobic conditions were more sensitive to radiation, and were less protected by hydroxyl radical scavengers, especially formate, than when cells grown under aerobic conditions were irradiated in the presence of oxygen . Hydroxyl radical scavengers provided no further protection during irradiation in the absence of oxygen . These results indicated that the increased radiation sensitivity of cells grown under anaerobic conditions may be related to superoxide radicals which could increase intercellular damage during irradiation in the presence of oxygen . However, endogenous superoxide dismutase and catalase activities did not protect cells from the radiation-induced lethality of S . typhimurium . Cytoplasmic extracts protected bacterial DNA in vitro in either the presence or absence of oxygen, and no radiation-induced lipid peroxidation of the cellular components was identified by measuring the levels of 2-thiobarbituric acid . These results suggest that most radiation-induced cell lethality was related to the cooperative effects of extracellular OH . and O2 on the cell surface as the radiation dose increased.

J Mol Evol, 1995 Oct, 41(4), 449 - 56
Synonymous substitution-rate constants in Escherichia coli and Salmonella typhimurium and their relationship to gene expression and selection pressure; Berg OG et al.; Based on the differences in synonymous codon use between E . coli and S . typhimurium, the synonymous substitution rates can be estimated . In contrast to previous studies on the substitution rates in these two organisms, we use a kinetic model that explicitly takes the selection bias into account . The selection pressure on synonymous codons for a particular amino acid can be calculated from the observed codon bias . This offers a unique opportunity to study systematically the relationship between substitution-rate constants and selection pressure . The results indicate that the codon bias in these organisms is determined by a mutation-selection balance rather than by stabilizing selection . A best fit to the data implies that the mutation rate constant increases about threefold in genes at low expression levels relative to those that are highly expressed.

J Med Microbiol, 1995 Oct, 43(4), 294 - 9
Resistance to extended-spectrum cephalosporins, caused by PER-1 beta-lactamase, in Salmonella typhimurium from Istanbul, Turkey; Vahaboglu H et al.; Two Salmonella typhimurium isolates were studied, one as a representative from a series of neonatal meningitis cases treated at an Istanbul teaching hospital, the other from a gastro-enteritis case seen at a different Istanbul hospital . Both isolates were resistant to extended-spectrum cephalosporins, as well as penicillins, aminoglycosides and chloramphenicol . Cephalosporin resistance depended on production of PER-1 beta-lactamase, which is an extended-spectrum class A enzyme that is only distantly related to TEM and SHV enzymes, and which was previously known only from Pseudomonas aeruginosa isolates . The PER-1 gene was carried by an 81-MDa plasmid, which also determined resistance to aminoglycosides and chloramphenicol . Although it was not self-transmissible to Escherichia coli, this element did transfer if mobilised with plasmid pUZ8 . The two S . typhimurium isolates gave indistinguishable DNA restriction patterns and, in addition to their 81-MDa plasmid, also contained 52- and 2.8-MDa plasmids, the last of these encoded TEM-1 enzyme . The two isolates were identical in serotype, antibiogram and plasmid-profile but nevertheless differed in phage type, and, therefore, represented distinct strains . The emergence of cefotaxime and ceftriaxone resistance in salmonellae is disturbing, since these agents are preferred therapy for neonatal meningitis caused by members of the genus.

J Bacteriol, 1995 Oct, 177(19), 5670 - 9
Multiple promoters and induction by heat shock of the gene encoding the alternative sigma factor AlgU (sigma E) which controls mucoidy in cystic fibrosis isolates of Pseudomonas aeruginosa; Schurr MJ et al.; Overproduction of the exopolysaccharide alginate causes mucoid colony morphology in Pseudomonas aeruginosa and is considered a major virulence determinant expressed by this organism during chronic respiratory infections in cystic fibrosis . One of the principal regulatory elements governing conversion to mucoidy in P . aeruginosa is AlgU, an alternative sigma factor which is 66% identical to and functionally interchangeable with sigma E from Escherichia coli and Salmonella typhimurium . sigma E has been implicated in the expression of systems enhancing bacterial resistance to environmental stress . In this study, we report that the gene encoding AlgU is transcribed in wild-type nonmucoid P . aeruginosa from multiple promoters (P1 through P5) that fall into three categories: (i) the P1 and P3 promoters, which display strong similarity to the -35 and -10 canonical sequences of sigma E promoters and were found to be absolutely dependent on AlgU; (ii) the P2 promoter, which was less active in algU mutants, but transcription of which was not completely abrogated in algU::Tcr cells; and (iii) the transcripts corresponding to P4 and P5, which were not affected by inactivation of algU . Introduction of E . coli rpoE (encoding sigma E) or algU into P . aeruginosa algU::Tcr strains restored P1 and P3 transcription and brought the P2 signal back to the wild-type level . The AlgU-dependent promoters P1 and P3 were inducible by heat shock in wild-type nonmucoid P . aeruginosa PAO1 . At the protein level, induction of AlgU synthesis under conditions of extreme heat shock was detected by metabolic labeling of newly synthesized proteins, two-dimensional gel analysis, and reaction with polyclonal antibodies raised against an AlgU peptide . Another AlgU-dependent promoter, the proximal promoter of algR, was also found to be induced by heat shock . Under conditions of high osmolarity, growth at elevated temperature induced alginate synthesis in the wild-type nonmucoid P . aeruginosa PAO1 . Cumulatively, these results suggest that algU itself is subject to complex regulation and is inducible by extreme heat shock, that the alginate system is a subset of the stress-responsive elements controlled by AlgU, and that AlgU and, by extension, its homologs in other organisms (e.g., sigma E in S . typhimurium) may play a role in bacterial virulence and adjustments to adverse growth conditions.

J Bacteriol, 1995 Oct, 177(19), 5547 - 53
Comparative characterization of release factor RF-3 genes of Escherichia coli, Salmonella typhimurium, and Dichelobacter nodosus; Kawazu Y et al.; The termination of protein synthesis in bacteria requires two codon-specific release factors, RF-1 and RF-2 . A gene for a third factor, RF-3, that stimulates the RF-1 and RF-2 activities has been isolated from the gram-negative bacteria Escherichia coli and Dichelobacter nodosus . In this work, we isolated the RF-3 gene from Salmonella typhimurium and compared the three encoded RF-3 proteins by immunoblotting and intergeneric complementation and suppression . A murine polyclonal antibody against E . coli RF-3 reacted with both S . typhimurium and D . nodosus RF-3 proteins . The heterologous RF-3 genes complemented a null RF-3 mutation of E . coli regardless of having different sequence identities at the protein level . Additionally, multicopy expression of either of these RF-3 genes suppressed temperature-sensitive RF-2 mutations of E . coli and S . typhimurium by restoring adequate peptide chain release . These findings strongly suggest that the RF-3 proteins of these gram-negative bacteria share common structural and functional domains necessary for RF-3 activity and support the notion that RF-3 interacts functionally and/or physically with RF-2 during translation termination.

J Bacteriol, 1995 Oct, 177(19), 5434 - 9
Glutathione is required for maximal transcription of the cobalamin biosynthetic and 1,2-propanediol utilization (cob/pdu) regulon and for the catabolism of ethanolamine, 1,2-propanediol, and propionate in Salmonella typhimurium LT2; Rondon MR et al.; Transcription of the cob/pdu regulon of Salmonella typhimurium is activated by the PocR regulatory protein in response to 1,2-propanediol (1,2-PDL) in the environment . Nutritional analysis and DNA sequencing confirmed that a strain defective in expression of the cob/pdu regulon in response to 1,2-PDL lacked a functional gshA gene . gshA encodes gamma-glutamylcysteine synthetase (L-glutamate:L-cysteine gamma-ligase {ADP forming}; EC 6.3.2.2), the enzyme that catalyzes the first step in the synthesis of glutathione (GSH) . The DNA sequence of gshA was partially determined, and the location of gshA in the chromosome was established by two-factor crosses . P22 cotransduction of gshA with nearby markers showed 21% linkage to srl and 1% linkage to hyd; srl was 9% cotransducible with hyd . In light of these data, the gene order gshA srl hyd is suggested . The level of reduced thiols in the gshA strain was 87% lower than the levels measured in the wild-type strain in both aerobically and anaerobically grown cells . 1,2-PDL-dependent transcription of cob/pdu was studied by using M . Casadaban's Mu-lacZ fusions . In aerobically grown cells, transcription of a cbi-lacZ fusion (the cbi genes are the subset of cob genes that encode functions needed for the synthesis of the corrin ring) was 4-fold lower and transcription of a pdu-lacZ fusion was 10-fold lower in a gshA mutant than in the wild-type strain . Expression of the cob/pdu regulon in response to 1,2-PDL was restored when GSH was included in the medium . In anaerobically grown cells, cbi-lacZ transcription was only 0.4-fold lower than in the gshA+ strain; pdu-lacZ transcription was reduced only by 0.34-fold, despite the lower thiol levels in the mutant . cobA-lacZ transcription was used as negative control of gene whose transcription is not controlled by the PocR/1,2-PDL system; under both conditions, cobA transcription remained unaffected . The gshA mutant strain was unable to utilize 1,2-PDL, ethanolamine, or propionate as a carbon and energy source . The defect in ethanolamine utilization appears to be at the level of ethanolamine ammonia-lyase activity, not at the transcriptional level . Possible roles for GSH in ethanolamine, 1,2-PDL, and propionate catabolism are proposed and discussed.

J Bacteriol, 1995 Oct, 177(19), 5401 - 10
Five promoters integrate control of the cob/pdu regulon in Salmonella typhimurium; Chen P et al.; Propanediol is degraded by a B12-dependent pathway in Salmonella typhimurium . The enzymes for this pathway are encoded in a small region (minute 41) that includes the pdu operon (controlling B12-dependent degradation of propanediol) and the divergent cob operon (controlling synthesis of cobalamin, B12) . Expression of both operons is induced by propanediol and globally controlled by the ArcA and Crp systems . The region between the two operons encodes two proteins, PduF, a transporter of propanediol, and PocR, which mediates the induction of the regulon by propanediol . Insertion mutations between the pdu and cob operons have been characterized, and their exact positions have been correlated with mutant phenotypes . The region includes five promoters, four of which are controlled by the PocR protein and induced by propanediol . The cob and pdu operons each have one regulated promoter; the pduF gene is expressed from two regulated promoters (P1 and P2) . The P1 and P2 transcripts extend beyond pduF to include the pocR gene; thus the PocR protein autoregulates its expression from these promoters . The fifth promoter, PPoc, is adjacent to the pocR gene and associated with a Crp binding site . We suggest that all global control of the regulon is exerted by regulating the level of PocR protein at the P1, P2, and PPoc promoters . A putative binding site for the PocR protein has been identified by computer analysis . Eight close matches to this proposed site were found in regions near the four promoters known to be regulated by PocR protein: PPdu, P1, P2, and PCob . A three-state model is proposed in which the regulon uses all five of its promoters to control expression.

Infect Immun, 1995 Oct, 63(10), 4170 - 3
Influence of the Bcg locus on macrophage response to the dimorphic fungus Candida albicans; Puliti M et al.; The Bcg/Ity/Lsh gene (candidate Nramp) controls natural resistance to several parasites, such as Mycobacterium bovis, Leishmania donovani, and Salmonella typhimurium . Using two macrophage (M phi) cell lines (B10R and B10S) derived from mouse strains congenic at Bcg, we found that M phi s from resistant mice (B10R M phi s) act more effectively against the two morphogenetic forms of the dimorphic fungus Candida albicans compared with M phi s from susceptible mice (B10S M phi s) . Moreover, when assessed for tumor necrosis factor secretion in response to the hyphal form of C . albicans, B10R M phi s are significantly more effective at expressing this secretory function than are B10S M phi s, closely resembling the trend of response to lipopolysaccharide . Overall, these results provide insight into the influence of the Bcg locus on the M phi response to C . albicans.

Infect Immun, 1995 Oct, 63(10), 4024 - 8
Contact with cultured epithelial cells stimulates secretion of Salmonella typhimurium invasion protein InvJ; Zierler MK et al.; Contact of Salmonella typhimurium with cultured epithelial cells results in the assembly of surface appendages termed invasomes which are presumably required for the internalization of these organisms into host cells . The assembly of these structures requires the function of a dedicated protein secretion system encoded in the inv locus . We show in this report that contact of wild-type S . typhimurium with cultured Henle-407 cells stimulated the secretion of InvJ, a recently identified target of the inv-encoded type III protein secretion system . Stimulation of InvJ secretion also occurred upon bacterial contact with bovine calf serum-coated culture dishes but did not occur upon S . typhimurium contact with glutaraldehyde-fixed Henle-407 cells . The stimulation of InvJ secretion did not require de novo protein synthesis . Invasion-defective invC and invG mutants of S . typhimurium failed to secrete InvJ upon contact with live Henle-407 cells . In contrast, contact-dependent secretion of InvJ in S . typhimurium invE mutants occurred at levels equivalent to those of the wild type . These results indicate that the presence of Henle-407 cells and/or serum is capable of activating the type III secretion system encoded in the inv locus, further supporting the notion that Salmonella entry into cultured cells is the result of a biochemical cross-talk between the bacteria and the host cells.

Mutat Res, 1995 Oct, 344(3-4), 135 - 40
A new anthracycline with potent antileukemic activity exhibits reduced mutagenicity; Andrivon W et al.; The mutagenicity of a new anthracycline (moflomycin) with potent antileukemic activity was studied by the Ames test in four strains of Salmonella typhimurium (TA97a, TA98, TA100 and TA102), and compared to the mutagenicity of doxorubicin, widely used as antineoplastic agent . Unlike doxorubicin, moflomycin displayed no mutagenic activity in strains TA98 and TA100 . Low mutagenicity was only observed in TA102 strain and was not enhanced after metabolic activation . This result indicates that moflomycin induce mutagenicity by reverting base-pair substitution . The structural changes in the sugar moiety may be involved in the reduced mutagenicity of moflomycin . The low mutagenicity of moflomycin shown in this study enhances the potential advantage of this new derivative which displays a high antileukemic activity.

Immunology, 1995 Oct, 86(2), 206 - 11
Conversion of Salmonella typhimurium to L-forms contributes to the maintenance of acquired immunity against murine typhoid; Kita E et al.; Conversion of Salmonella typhimurium to L-forms, both in vitro and in vivo, resulted in the expression of proteins cross-reacting to the mycobacterial 65,000 MW heat-shock protein (hsp) . Immunization of C3H/HeJ mice with a protective dose of stable L-form S . typhimurium induced gamma delta T cells in the liver, in accordance with the multiplication of L-form Salmonella in Kupffer cells . The number of gamma delta T cells decreased after the intracellular growth of L-form Salmonella plateaued . Persistance of the L-forms in Kupffer cells, however, allowed hepatic gamma delta T cells to increase within 48 hr of infection with virulent S . typhimurium . Thus, the intrahepatic colonization of L-form Salmonella seems to keep gamma delta T cells on standby, but the emergence of these T cells does not correlate with the expression of L-form hsp . In addition, Kupffer cells colonized by L-forms constitutively synthesized mRNA for interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-alpha) . These results suggest that conversion of S . typhimurium to L-forms in phagocytic cells builds up and maintains acquired resistance, conferred by live-cell vaccines of S . typhimurium, against murine typhoid.

Mutat Res, 1995 Oct, 348(2), 67 - 73
Inhibitory effect of curcumin on SOS functions induced by UV irradiation; Oda Y; The antigenotoxic effects of curcumin, including the inhibition of SOS induction and mutagenesis by UV light, were investigated in Salmonella typhimurium TA1535/pSK1002 and Escherichia coli K-12 strains . Induction of the SOS gene (umuC) expression was assayed by measuring accumulated beta-galactosidase activity . We found that curcumin blocked umuC induction promoted by UV irradiation in a dose-dependent manner . Also, with another SOS response, Weigle reactivation, we observed that curcumin effectively inhibited phage reactivation by UV irradiation . Furthermore, we tested the effect of curcumin on UV mutagenesis . We showed that mutagenesis induced by UV irradiation was suppressed by the addition of curcumin . Together these results indicate that curcumin acts as an inhibitor of SOS functions including UV mutagenesis.

Biochemistry, 1995 Sep 26, 34(38), 12311 - 22
Acid-base chemical mechanism of O-acetylserine sulfhydrylases-A and -B from pH studies; Tai CH et al.; The pH dependence of kinetic parameters using natural and alternative reactants was determined in order to obtain information on the chemical mechanisms of the A and B isozymes of O-acetylserine sulfhydrylase (OASS) from Salmonella typhimurium . A general mechanism is proposed for OASS in which OAS binds with its alpha-amine unprotonated to carry out a nucleophilic attack on C4' of the protonated Schiff base and with the acetyl carbonyl hydrogen-bonded to a protonated enzyme group (or a water molecule), which aids in the beta-elimination of acetate . The enzyme lysine that was in Schiff base linkage with the active site pyridoxal 5'-phosphate deprotonates the alpha-carbon in the beta-elimination reaction, and a proton is likely released with the acetate product . Sulfide likely binds as HS- to undergo nucleophilic attack on the alpha-aminoacrylate intermediate, followed by protonation of the alpha-carbon by the enzyme lysine . In OASS-A, HS- is hydrogen-bonded to the enzyme group that assists in the beta-elimination of acetate, but this is not the case for OASS-B . The pH independent equilibrium constant for the first half-reaction of OASS-A is 1.6 x 10(-3), while the second half-reaction is practically irreversible.

J Chromatogr A, 1995 Sep 22, 711(2), 277 - 88
Lipopolysaccharide determination by reversed-phase high-performance liquid chromatography after fluorescence labeling; Parlesak A et al.; A sensitive method for the quantitative determination of lipid A, the covalently bound hydrophobic component of lipopolysaccharides (endotoxins), has been developed . The determination of lipid A was based on the quantitative measurement of beta-hydroxymyristic acid and beta-hydroxylauric acid by reversed-phase HPLC . beta-Hydroxy acids were liberated from ester and amide linkages in endotoxins by acid catalyzed methanolysis . The resulting methyl esters were derivatized with 9-anthracene-carboxyl chloride, 9-fluorene-carboxyl chloride and 4-(1-pyrenyl)butyric acid chloride and quantified with a fluorescence detector . The effectiveness of the three derivatizing agents was compared . As internal standards-beta-hydroxytridecanoic acid {beta-OH(13:0)} and beta-hydroxypentadecanoic acid {beta-OH(15:0)} ethyl esters were used . The limits of detection of beta-hydroxymyristic acid were 0.5 pg for the 9-anthroyl and 2 pg for the fluorenoyl and 4-(1-pyrenyl)butyroyl ester per sample (signal-to-noise ratio of 3) . The detection limits of lipopolysaccharide from a smooth strain (Escherichia coli 0111:B4) was 20 pg, while that from two rough strains (E . coli Nissle 1917 and Salmonella typhimurium SL 1181) was 5 pg per sample after methanolysis and derivatization with 9-anthroyl chloride.

Gene, 1995 Sep 22, 163(1), 1 - 11
Engineering proteins without primary sequence tryptophan residues: mutant trp repressors with aliphatic substitutions for tryptophan side chains; Chapman D et al.; Combinatorial mismatch-primer mutagenesis was used to make simultaneous changes of codons for residues Trp19 and Trp99 of the Escherichia coli trp aporepressor (TrpR protein) to codons for other residues . Among 21 different single- and double-mutant repressors obtained from this round of mutagenesis, proteins with Trp-->Leu and Trp-->Met changes at one or both positions were found to be nearly as active as the wild type (wt) . Genes encoding repressors with each of the eight possible combinations of single- and double-mutant changes of Trp19 and Trp99 to Leu and Met were constructed by recombination in vitro . Whereas three of these eight mutant repressors are unstable in E . coli, all are made at similar steady-state levels in Salmonella typhimurium . Three of the eight mutant holorepressors are lethal when overproduced in S . typhimurium, because they confer an induced auxotrophy . Two different activity assays in vivo show that one of the four double-mutant repressors (Trp19-->Leu; Trp99-->Met) is similar to wt TrpR in its interactions with both Trp and DNA . These results show that more general approaches to engineering active proteins with fewer Trp residues may give rise to functional mutants without aromatic substitutions, and that aliphatic changes should be considered in cases where engineered changes of Trp to Phe or Tyr do not work.

Zhonghua Yi Xue Za Zhi (Taipei), 1995 Sep, 56(3), 199 - 204
Salmonella pericarditis and empyema: a case report; Yang CH et al.; A 54-year-old female diabetic patient who had been under regular oral hypoglycemic agent control for 2 years was admitted because of having dyspnea and orthopnea for several days . Massive pericardial effusion and a moderate amount of bilateral pleural effusion were noted at the time of admission . Pericardiocentesis and thoracocentesis were performed; both cultures grew Salmonella typhimurium . Antibiotic therapy was started immediately, but pleural effusion persisted . After repeated thoracocentesis and pleural effusion drainage, the patient was discharged 51 days later with complete recovery . Pericarditis and empyema are rare complications of Salmonella infections . Coexistence is even more rare, and only three cases have been reported in the English literature . Therefore, this case is presented with a review of the previous literature followed by discussion.

Bioessays, 1995 Sep, 17(9), 747 - 9
Recombination, mutation and the origin of species; Cox EC; A major barrier to recombination between bacterial species lies in the mismatch repair system, a complex of proteins that has evolved to proof-read freshly replicated DNA . It now appears that a second system, involving an inducible DNA recombination, repair and mutagenesis pathway, also regulates interspecies recombination, but in a positive way, being required for recombination between Escherichia coli and Salmonella typhimurium . Thus the rate at which newly emerging species of bacteria diverge can be seen as a balance between a permissive state associated with inducible repair and recombination, and the proof-reading of intermediates in the recombination pathway by the mismatch correction system.

Vet Med (Praha), 1995 Sep, 40(9), 273 - 8
Mutagenicity of stable dust and drinking water on swine and cattle farms; Raszyk J et al.; Single pilot examinations of mutagenicity of stable dust and drinking water were made on three swine farms (D., M., T.) and one cattle farm (N.) in the district of Hodonin in summer 1994 . The mutagenicity was examined by the Ames test using the indicator strains Salmonella typhimurium TA 98 and TA 100 with (+S9) or without (-S9) metabolic activation . At the same time the contents of selected pesticides (PES) and polychlorinated biphenyls (PCB) in stable dust and drinking water and that of polycyclic aromatic hydrocarbons in stable dust were determined . Increased mutagenicity was demonstrated in drinking water (strain TA 98 with metabolic activation; index Rt/Rk 3.6-7.7) and stable dust (strain TA 100 with metabolic activation; index Rt/Rk 2.2) collected on the swine farm M . High contents of PAH (8.246 mg/kg) and PCB (0.263 mg/kg) were also found in the dust samples collected on this farm . Only drinking water showed mutagenic activity (strain TA 98 without metabolic activation; index Rt/Rk 2.6) on the swine farm D . On both the farms, the number of revertants was dose-dependent . Increased content of PAH (2.553 mg/kg) was also demonstrated on the dust samples collected on the farm D . No significant increase (twofold or higher when compared with negative controls) of mutagenic activity of stable dust or drinking water was demonstrable on the swine farm T . and the cattle farm N . Substances responsible for the mutagenicity of drinking water on the farms D . and M . have not yet been identified . Anyway, the increase of mutagenicity of stable dust and drinking water should be taken as a warning that mutagens that can jeopardise animal and human health have penetrated into the stable environment.

Mol Microbiol, 1995 Sep, 17(5), 981 - 8
Participation of the purine repressor in control of the carbamoylphosphate synthetase operon in Salmonella typhimurium; Lu CD et al.; Previous work has shown that the carAB operon of Salmonella typhimurium is transcribed from tandem promoters, P1 and P2, that are negatively controlled by pyrimidines and arginine, respectively . The results reported here show that purines also negatively control expression of carAB and that this effect is absent in a purR::Tn10 derivative . Primer-extension experiments established that the purine effect is exerted at P1, thus redefining this promoter as sensitive to both purines and pyrimidines . The results of gel-retardation experiments as well as DNase I and premethylation footprintings indicate that the purine repressor interacts with a PUR box 85 bp upstream of P1 . Modification of this PUR box by site-directed mutagenesis abolishes the repression by purines in a carA::lacZ fusion, confirming that this box functions in vivo in purine control of carAB expression.

FEMS Immunol Med Microbiol, 1995 Sep, 12(1), 51 - 3
A cell-free Salmonella typhimurium extract induces inhibition of tyrosine phosphorylation in murine splenic T-lymphocytes; Matsui K et al.; In a previous study, we observed that suppression of T-cell proliferation induced by Salmonella infection is associated with inhibition of tyrosine phosphorylation in T-cells, and that a cell-free Salmonella typhimurium LT2 extract (LT2 extract) also suppressed mitogen-induced T-cell proliferation . In the present study, therefore, we attempted to clarify whether the T-cell suppression induced by LT2 extract involved inhibition of tyrosine phosphorylation in T-cells . Western blotting using anti-phosphotyrosine antibodies showed that the mitogen-induced tyrosine phosphorylation of 120-, 106-, 94-, 76-, 68-, 57- and 36-kDa proteins in murine splenic T-cells was inhibited by treatment with LT2 extract . These results suggest that the suppression of T-cell proliferation induced by LT2 extract is also associated with inhibition of tyrosine phosphorylation in T-cells.

Vaccine, 1995 Sep, 13(13), 1220 - 5
Role of lipopolysaccharide and a major outer membrane protein from Francisella tularensis in the induction of immunity against tularemia; Fulop M et al.; A crude outer membrane preparation from Francisella tularensis Live Vaccine Strain (LVS) was used to immunize mice . Immunized mice were completely protected from a F . tularensis challenge . We evaluated the role of two major outer membrane antigens in the induction of protective immunity, namely lipopolysaccharide and an outer membrane protein FopA . We presented FopA to the immune system using an aromatic amino acid-dependent Salmonella typhimurium as a vector . Although mice mounted an immune response to cloned FopA no significant protection was induced . However, LPS immunized mice were completely protected . We conclude that LPS is a major protective antigen whereas FopA has a limited or no role in the induction of protective immunity.

J Diarrhoeal Dis Res, 1995 Sep, 13(3), 166 - 71
Enterotoxin production and plasmid profiles in Salmonella typhimurium isolated from man; Mohan VP et al.; One hundred twenty-six isolates of Salmonella typhimurium from various clinical sources were tested for enterotoxin production and characterization of plasmid profile . Cell-free culture supernates and polymyxin B extracts of all the strains were assayed by rabbit ileal loop and skin permeability tests . Enterotoxic activity was detected in culture supernates of 32 strains . Twenty-one strains by both rabbit ileal loop and skin permeability tests, nine strains by skin permeability test, and two strains by rabbit ileal loop test were positive . Live culture of three enterotoxic strains, positive in culture supernates produced ileal secretion . Polymyxin B extracts from 6 hours and 18 hours broth cultures of all the strains were devoid of enterotoxicity . Ileal mucosa exposed to culture supernate of enterotoxigenic strains showed swollen and blunted villi with submucosal oedema while those exposed to polymyxin B extracts showed shortening of villi and sloughing of epithelial lining . Plasmid profiles of enterotoxigenic strains were heterogenous and grouped into 20 different profiles . No correlation could be established between plasmid profile, R-pattern, and enterotoxin production.

Rev Esp Salud Publica, 1995 Sep-Oct, 69(5), 393 - 408
{The evaluation of the mutagenic activity of public drinking water by the Ames test}; Albaladejo Vicente R et al.; BACKGROUND: The sources of potential mutagens in our environment are many, but the most important of these is water for public consumption . This is a result of the chlorinating process which is the main reason for the appearance of these mutagens . With this in mind, the aim of our study was to check a possible mutagenic activity, using the Ames test, in organic concentrates taken from water for public consumption in Madrid . METHODS: Several bacterial strains were used, namely Salmonella histidine dependent TA1535, TA1538, TA98 and TA100, taken originally from Salmonella typhimurium LT2 . Each test was performed twice, with or without the introduction of the mammalian-microsome activation (S9 mix), as per the indications in Ames . The plate incorporation assay was used to test the mutagenicity . All samples of the water in question were processed and treated so as to create concentrates of organic chlorinated compounds . RESULTS: The highest levels of mutagenicity appeared in the TA1535 strain and in the tests where the microsome fraction was not used (IM = 1.94) . CONCLUSIONS: With regard to mutagenic evaluation in organic concentrates taken from water for public consumption, no positive activity was found in any of the tester strains.

Biomed Environ Sci, 1995 Sep, 8(3), 240 - 5
Study on the mutagenicity of diesel exhaust particles; Song J et al.; The mutagenicity of diesel exhaust particles (DEP) was studied by using Salmonella typhimurium strains TA98 and TA100 in vitro and mice micronucleus in vivo test . DEP from six kinds of medium and heavy-duty diesel vehicles, which were made in China and imported, were tested . The vehicles were operated under free accelerating condition . The results showed that the DEP contained mutagenic activity . An increase in the number of the Salmonella TA98 was observed in the presence and especially in the absence of S9 mix . Positive results were also obtained from mice micronucleus assay . The frequency of mice bone marrow micronucleated polychromatic erythrocytes (M PCE) was increased and it showed a definite dose-response relationship . Comparing the different types of the vehicles, we found that the mutagenicity of DEP from domestic made vehicles was stronger than that from the imported ones.

Plasmid, 1995 Sep, 34(2), 144 - 7
Construction of a cloning vector from a naturally occurring plasmid of Salmonella typhimurium; Roy RK et al.; A naturally occurring plasmid isolated from a drug-resistant strain of Salmonella typhimurium (993) has been used to construct a plasmid vector for cloning in a wild strain of Salmonella . The strain (993) contains at least two plasmids . The smaller plasmid (9 kb) contains an ampicillin-resistant marker, while the larger one (25 kb) is cryptic . Physical mapping of the 9-kb plasmid and construction of a 3.5-kb derivative have been carried out . This plasmid has been used for cloning in a restriction+modification+strain of S . typhimurium using a conventional calcium chloride method . It exhibited better efficiency of transformation than other commonly used plasmids such as pBR322 or its derivatives and transformants were found to be stable in the absence of antibiotic selection . The vector is compatible with pBR322 and can be used to study the expression of cloned genes in minicells.

Comp Immunol Microbiol Infect Dis, 1995 Sep, 18(4), 283 - 90
Plasmid profile and phage type of Salmonella typhimurium strains encountered in different regions of India; Mohan VP et al.; A total of 190 Salmonella typhimurium strains encountered in different parts of India were characterized on the basis of plasmid profile, phage type and antimicrobial resistance pattern . Recent trends in the epidemiology of R-plasmids were also studied . The majority of S . typhimurium strains (90.5%) were untypable by phage typing . Only 18 strains (9.5%) were phage typable . The phage untypable strains isolated from northern (57) central (65), and southern (50) regions of India could be subgrouped into 24, 12 and 16 different plasmid profiles respectively . Heterogeneity was the prominent feature although most of the plasmid profiles were related among strains isolated from particular place . A great diversity among small plasmids (2.7-8.3 kb) made subgrouping of majority strains (71%) with R-pattern ApCmKmSmSuTcTp possible . Conjugation studies and plasmid profile analysis of transconjugants revealed all the strains to harbour non conjugative non-auto transmissible plasmids with the exception of 7.2 and 2.7 kb plasmids which were not mobilizable.

Mutagenesis, 1995 Sep, 10(5), 433 - 8
A new mutagenicity assay method for frameshift mutagens based on deleting or inserting a guanosine nucleotide in the beta-lactamase gene; Hour TC et al.; The conventional method of site-directed mutagenesis was used to develop two Salmonella strains, JK-1 and JK-2, for detecting frameshift mutagens . The JK-1 strain was derived from Salmonella typhimurium TA1537 strain transformed by a mutant construct . A guanosine nucleotide was inserted between nucleotide residues 312 and 313 of the beta-lactamase gene . The JK-2 strain was obtained by the same procedure, but a guanosine nucleotide in position 315 of the beta-lactamase gene was deleted . The strains were tested with ten frameshift mutagens and the revertants were selected by ampicillin resistance . Representative mutagens including 2-nitrofluorene (2-NF), 2-acetylaminofluorene (AAF), 9-aminoacridine (9-AA), 2,7-diaminofluorene (2,7-DAF) and 2-methoxy-6-chloro-9-(3-(ethyl-2-chloro-ethyl)-aminopropylamino)acridine (ICR-170) were more potent in the JK-1 strain than the JK-2 strain, and the number of revertant colonies were dose related . Under the same conditions, the ampicillin test was more sensitive than the Ames test . Other types of compounds such as 2-methoxy-6-chloro-9-(2-chloroethylaminopropylamino)acridine (ICR-191), benzo{a}pyrene (BP), 4-nitroquinoline N-oxide (4-NQNO), hycanthone and aflatoxin B1 (AFB1) were not as mutagenic to these new strains . The method is quite promising for studying certain specific frameshift mutagens, but more chemical mutagens should be tested to validate its applicability and reproducibility in general use.

Mutagenesis, 1995 Sep, 10(5), 425 - 31
A correlation of Salmonella mutagenicity with DNA adducts induced by the cooked-food mutagen 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine; Malfatti MA et al.; The correlation of bacterial mutagenicity with DNA adducts from the heterocyclic amine cooked-food mutagen 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine (PhIP) was investigated in Salmonella typhimurium strains TA98 (uvrB deficient) and TA1978 (uvrB proficient) . Bacterial cells were exposed to PhIP using a modification of the Ames/Salmonella microsuspension assay . Half of the cells, generated from a 90 min pre-incubation and washing, were plated for revertant formation while the remaining half was subjected to DNA adduct analysis via 32P-postlabeling . In TA98, DNA adducts were detected at an RAL (relative adduct labeling) of 10 x 10(-7) and 21 x 10(-7) at PhIP concentrations of 5.5 and 17 microM, respectively . This corresponded to 28.8 and 20.9 adducts/revertant, respectively . These values were based on the assumption that only four repeating GC bases within a 75 DNA base region is the gene target site for PhIP induced mutations . In TA1978, no revertants above background were detected at any concentration of PhIP tested . DNA adducts, however, were detected at 11 x 10(-7) and 21 x 10(-7) adducts per nucleotide at 223 and 1116 microM PhIP, respectively . The lack of detectable revertants, but the presence of DNA adducts, suggests pre-mutational lesions did occur during the 90 min pre-incubation . Presumably, when the S9 activating system and PhIP were removed (via washing with phosphate buffered saline) prior to plating, the cells containing an intact uvrB repair system repaired the lesions during the incubation time on the plates . In conclusion, the induction of revertants by adducts appears quite efficient, as approximately 25 adducts are required for one mutational event in the excision repair deficient bacteria.

Mikrobiologiia, 1995 Sep-Oct, 64(5), 651 - 6
{Mechanism of reactivating Escherichia coli inactivated by ultraviolet light with cell extracts of propionic acid bacteria}; Vorob'eva LI et al.; Two mechanisms of reactivation of UV-inactivated Escherichia coli cells--photoreactivation (PhR) and reactivation by the dialysates of propionic acid bacteria--are shown to be different but not completely additive . PhR displays an insignificant negative effect on the reactivation by active substances (peptides) of the dialysate, whereas reactivation by dialysate inhibits PhR . The maximal reactivation can be attained under complete PhR followed by the protective action of dialysate . The dialysate protects UV-irradiated E . coli cells with PolA, UvrA, and RecA mutations and Salmonella typhimurium TA 100 (UvrB) cells and also exerts an antimutagenic effect on S . typhimurium TA 100 . Protection by dialysate is suggested to be due to restoration of the cell division mechanism damaged by UV irradiation.

Int J Food Microbiol, 1995 Sep, 27(1), 45 - 60
The effect of transient temperatures on the growth of Salmonella typhimurium LT2 in gelatin gel; Brocklehurst TF et al.; The growth of colonies of Salmonella typhimurium derived from single immobilised cells was studied while subjected to constant and sinusoidally-varying temperatures . The bacteria grew in microbiological culture media adjusted to different pH and sodium chloride (NaCl) concentration and solidified with gelatin that was contained within a cassette formed between sheets of PVC film that allowed gaseous exchange . At pH 7.0 and 0.5% (w/v) NaCl and either 12 degrees C or 20 degrees C, S . typhimurium grew at a rate similar to that in liquid medium . The decrease in growth rate at 20 degrees C at a lower pH or higher NaCl concentration was greater in the case of immobilised cells than for cells in liquid medium . The change in the numbers of viable bacteria was measured with time under sinusoidally-varying temperatures between 4 and 22 degrees C and between 12 and 22 degrees C of period in the range 12 to 480 min . The experimental growth curves were compared with predictions based on isothermal growth in liquid medium . The discrepancies between experiment and prediction were greater for gels stressed by NaCl or pH than for gels at pH 7.0 and containing 0.5% (w/v) NaCl, consistent with the isothermal observations.

Food Addit Contam, 1995 Sep-Oct, 12(5), 715 - 23
Quantitative structure-activity relationships and COMPACT analysis of a series of food mutagens; Lewis DF et al.; Quantitative structure-activity relationships between chemical structure and Ames mutagenicity for a group of 24 food mutagens, including 17 cooked-food heterocyclic amines, have been determined . For the TA98 strain of Salmonella typhimurium (frameshift mutagens) the best correlation of mutagenicity is with molecular diameter (R = 0.91), while for the TA100 strain (base-pair mutations) the best correlation is with delta E, the energy difference between the lowest unoccupied and highest occupied molecular orbitals . High mutagenicity is related to high values of molecular diameter, hence to planarity and to high values of the COMPACT ratio ({area/depth2}/delta E) . High mutagenicity is also related to low values of delta E . Consequently, highly mutagenic and potentially carcinogenic food chemicals can readily be identified as substrates of cytochrome P4501 (CYP1) and may therefore be detected by the COMPACT procedure . Highly mutagenic compounds also exhibit high values of dipole moment.

Mutat Res, 1995 Sep, 331(1), 161 - 70
Use of monoclonal antibodies to cytochrome P450s to indicate the critical dealkylation and the P450s involved in methyl-n-amylnitrosamine mutagenicity in the presence of induced rat liver microsomes; Mirvish SS et al.; The mutagenicity for Salmonella typhimurium TA 1535 of the carcinogen methyl-n-amylnitrosamine (MNAN) was examined in the presence of rat liver microsomes from uninduced and induced rats . The number of mutations followed the order phenobarbital- and Aroclor-induced > 3-methylcholanthrene- and isoniazid-induced > uninduced microsomes . The MNAN metabolite 4-hydroxy-MNAN was not mutagenic . Using each type of induced liver microsomes, we examined the effect on MNAN mutagenicity of four monoclonal antibodies (MAbs) that inhibit cytochrome P450s . The MAbs inhibited MNAN mutagenicity in seven MAb-microsome combinations by up to 49% . Taken together, these results indicated that CYP (P450) 2B1/2B2 was responsible for one half and CYP 2C11 for one quarter of MNAN mutagenicity with phenobarbital-induced microsomes, CYP 1A1/1A2 accounted for about 40% of the mutagenicity with 3-methylcholanthrene-induced microsomes, CYP 2B1/2B2 accounted for half and CYP 1A1/1A2 and 2C11 for smaller proportions of the mutagenicity with Aroclor-induced microsomes, and CYP 1A1/1A2 accounted for about 30% of the mutagenicity with isoniazid-induced microsomes . With isoniazid-induced microsomes, MAb 2-66-3 to CYP 2B1/2B1 caused an unexpected 219% increase and MAb 1-68-11 caused a moderate increase in MNAN mutagenicity . The test MAbs also inhibited the microsome-catalyzed demethylation and depentylation of MNAN by up to 83%, confirming previous results . Four comparisons between individual mutagenic and metabolic results supported the view that depentylation of MNAN was more critical for its mutagenicity than was demethylation, e.g., with 3-methylcholanthrene- and Aroclor-induced microsomes, MAb 1-7-1 to CYP 1A1/1A2 inhibited mutagenesis and depentylation, but did not affect demethylation.

Mutat Res, 1995 Sep, 331(1), 127 - 32
Metabolic activation of m-phenylenediamine to products mutagenic in Salmonella typhimurium by medium isolated from tobacco suspension cell cultures; Gichner T et al.; Both tobacco cells in suspension and the medium recovered from the suspension cultures (TX1MX) activated the aromatic amine m-phenylenediamine (m-PDA) into a product that was mutagenic in Salmonella typhimurium TA98 and YG1024 . Medium recovered from stationary-phase tobacco cell cultures exhibited the highest level of m-PDA activation . No cytochrome P-450 was detected in the activating medium . A high molecular weight matrix having the highest m-PDA activating capacity and associated with a substantial fraction of the total peroxidase activity was isolated by Centricon-100 ultrafiltration of TX1MX . The data suggest that the peroxidases present in the recovered cell culture medium or in the high molecular weight matrix are responsible for the plant activation of m-PDA.

J Bacteriol, 1995 Sep, 177(17), 5099 - 107
Genomic cleavage map of Salmonella typhi Ty2; Liu SL et al.; The genomic cleavage map of Salmonella typhi Ty2, 4,780 kb in size, was determined through digestion of the genomic DNA with endonucleases and separation of the fragments by pulsed-field gel electrophoresis . The chromosome has 33, 26, 7, and 35 sites for the enzymes XbaI, BlnI, I-CeuI, and SpeI, respectively . The fragments were arranged around the chromosome through excision of fragments from the gel, redigestion with a second enzyme, and labelling with 32P, and reelectrophoresis and named in alphabetical order . Tn10 transposons inserted in 82 different genes of Salmonella typhimurium were transduced by phage P22 into S . typhi, and the location of Tn10, and thus of the gene, was mapped through the XbaI and BlnI sites of Tn10 . All seven I-CeuI sites (in rrl genes for 23S rRNA) were conserved, and the gene order within the I-CeuI fragments resembles that of S . typhimurium LT2, but the order of I-CeuI fragments is rearranged from ABCDEFG in S . typhimurium LT2 to AGCEFDB in S . typhi . In addition, there is a 500-kb inversion which covers the terminus region . Comparisons of lengths of segments between genes showed that S . typhi has segments which differ in size from those in S . typhimurium . The viaB locus, for synthesis of the Vi antigen of S . typhi, was shown to be within a 118-kb loop (a segment of DNA with no homolog in most other Salmonella species) between mel and poxA on the chromosome.

J Bacteriol, 1995 Sep, 177(17), 5040 - 7
Characterization of the Salmonella typhimurium pagC/pagD chromosomal region; Gunn JS et al.; The PhoP/PhoQ two-component system regulates Salmonella typhimurium genes that are essential to bacterial virulence and survival within macrophages . The best characterized of these PhoP-activated genes (pag) is pagC, which encodes a 188-amino-acid envelope protein (W . S . Pulkkinen and S . I . Miller, J . Bacteriol . 173:86-93, 1991) . We here report the identification of four genes (pagD, envE, msgA, and envF) located 5' to pagC . Each gene is transcribed from its own promoter, two of which (msgA and pagD) were defined by primer extension analysis . Three of these genes (pagD, envE, and envF) are predicted to encode envelope proteins . The pagD gene is transcribed in a direction opposite from that of and adjacent to pagC and is positively regulated by PhoP/PhoQ . Transposon insertions within pagD and msgA attenuate bacterial virulence and survival within macrophages; however, deletion of pagD has no effect on virulence . The product of the envF gene is predicted to be a lipoprotein on the basis of the presence of a consensus lipid attachment site . The low G + C content of these genes and the homology of msgA to Shigella plasmid DNA suggest that this region may have been acquired by horizontal transmission.

J Bacteriol, 1995 Sep, 177(17), 4841 - 50
Suppression of the pleiotropic effects of HisH and HisF overproduction identifies four novel loci on the Salmonella typhimurium chromosome: osmH, sfiW, sfiX, and sfiY; Flores A et al.; Insertion mutations that suppress some or all the pleiotropic effects of HisH and HisF overproduction were obtained by using transposons Tn10dTet and Tn10dCam . All suppressor mutations proved to be recessive, indicating that their effects were caused by loss of function; thus, the suppressors identify genes that are necessary to trigger the pleiotropic response when HisH and HisF are overproduced . Genetic mapping of the suppressor mutations identifies four novel loci on the Salmonella typhimurium genetic map . Mutations in osmH (min 49) behave as general suppressors that abolish all manifestations of the pleiotropic response . Mutations in sfiY (min 83) suppress cell division inhibition and thermosensitivity but not osmosensitivity . Mutations that suppress only cell division inhibition define another locus, sfiX (min 44) . A fourth novel locus, sfiW (min 19), is also involved in cell division inhibition . The phenotype of sfiW mutations is in turn pleiotropic: they suppress cell division inhibition, make S . typhimurium unable to grow in minimal media, and cause slow growth and abnormal colony and cell shape . The inability of sfiW mutants to grow in minimal medium cannot be relieved by any known nutritional requirement or by the use of carbon sources other than glucose . The hierarchy of suppressor phenotypes and the existence of epistatic effects among suppressor mutations suggest a pathway-like model for the Hisc pleiotropic response.

Invest Ophthalmol Vis Sci, 1995 Sep, 36(10), 1960 - 7
In vivo study of leukocyte-endothelium interaction in endotoxin-induced uveitis; Baatz H et al.; PURPOSE . To analyze leukocyte-endothelium interaction in iris venules of living rats and to quantify changes of leukocyte dynamics in endotoxin-induced uveitis (EIU) . METHODS . Lewis rats received an intraperitoneal injection of 100 micrograms of lipopolysaccharide (LPS; Salmonella typhimurium) . Using intravital fluorescence microscopy, the iris vessels were examined, 2, 4, 6, 10, 14, 24, and 72 hours after LPS injection . A setup for intravital fluorescence microscopy of iris venules in the rat is described . Images are recorded with a video camera and stored on S-VHS videotape for off-line analysis . For contrast enhancement, erythrocytes and plasma were stained with fluorescein isothiocyanate (FITC) and FITC-hydroxyethylstarch, respectively . Rhodamine 6G was used for intravital staining of leukocytes . Resolution and magnification (x850) of the system facilitates observation of individual cells in the bloodstream in real time . Leukocytes were either flowing in the center stream, rolling along the endothelium, or firmly adherent . Image analysis provided data on microvascular leukocyte flux and leukocyte velocity . RESULTS . The percentage of leukocytes rolling on postcapillary venular endothelium increased significantly (P < 0.05) 4 hours after endotoxin administration, as did the number of firmly adherent cells . Leukocyte-endothelium interaction reached its maximum 6 to 10 hours before an increase of inflammatory cells in the aqueous humor . The response to endotoxin was reversible, subsiding to near-normal values after 72 hours . CONCLUSIONS . Intravital fluorescence microscopy provides data on microvascular parameters, including the number of rolling and sticking leukocytes on vascular endothelium . Inflammation of the anterior uvea was characterized with regard to leukocyte recruitment from blood to the vessel wall.

J Exp Med, 1995 Sep 1, 182(3), 655 - 66
The Ity/Lsh/Bcg locus: natural resistance to infection with intracellular parasites is abrogated by disruption of the Nramp1 gene; Vidal S et al.; In mice, natural resistance or susceptibility to infection with intracellular parasites is determined by a locus or group of loci on chromosome 1, designated Bcg, Lsh, and Ity, which controls early microbial replication in reticuloendothelial organs . We have identified by positional cloning a candidate gene for Bcg, Nramp1, which codes for a novel macrophage-specific membrane transport protein . We have created a mouse mutant bearing a null allele at Nramp1, and we have analyzed the effect of such a mutation on natural resistance to infection . Targeted disruption of Nramp1 has pleiotropic effects on natural resistance to infection with intracellular parasites, as it eliminated resistance to Mycobacterium bovis, Leishmania donovani, and lethal Salmonella typhimurium infection, establishing that Nramp1, Bcg, Lsh, and Ity are the same locus . Comparing the profiles of parasite replication in control and Nramp1-/- mice indicated that the Nramp1Asp169 allele of BcgS inbred strains is a null allele, pointing to a critical role of this residue in the mechanism of action of the protein . Despite their inability to control parasite growth in the early nonimmune phase of the infection, Nramp1-/- mutants can overcome the infection in the late immune phase, suggesting that Nramp1 plays a key role only in the early part of the macrophage-parasite interaction and may function by a cytocidal or cytostatic mechanism distinct from those expressed by activated macrophages.

Infect Immun, 1995 Sep, 63(9), 3279 - 86
A recombinant Salmonella typhimurium vaccine induces local immunity by four different routes of immunization; Hopkins S et al.; Immunization of mice with an attenuated Salmonella typhimurium strain (Phopc) carrying a plasmid encoding a hybrid form of the hepatitis B virus core antigen (HBc) induced specific antibody responses against the bacterial lipopolysaccharide (LPS) and HBc . Different mucosal routes of immunization, i.e., oral, nasal, rectal, and vaginal, were compared for their ability to induce a systemic as well as a mucosal response at sites proximal or distant to the site of immunization . Anti-LPS and anti-HBc immunoglobulin A (IgA) antibodies were measured in saliva, in feces, and in genital, bronchial, and intestinal secretions . Specific antibodies in serum and secretions were observed after immunization via all routes; however, the response to LPS was independent of that against HBc . In serum, saliva, and genital and bronchial secretions, high amounts of anti-HBc IgA were obtained by the nasal route of immunization . Vaginal immunization resulted in two different responses in mice: high and low . We observed a correlation between the level of specific immune response and the estrous status of these mice at the time of immunization . Rectal immunization induced high amounts of IgA against HBc and LPS in colonorectal secretions and feces but not at distant sites . These data suggest that S . typhimurium is able to invade different mucosal tissues and induce long-lasting local IgA responses against itself and a carried antigen after a single immunization.

Appl Environ Microbiol, 1995 Sep, 61(9), 3463 - 5
Use of bioluminescence to model the thermal inactivation of Salmonella typhimurium in the presence of a competitive microflora; Duffy G et al.; The survival of Salmonella typhimurium was investigated by bioluminescence and standard plating techniques in pure cultures and in the presence of competitors after the cultures were heated to 55 degrees C for increasing lengths of time . Decimal reduction (D) values increased from 0.43 to 2.09 min in the presence of 10(8) CFU of competitors ml-1, indicating a significant protective effect.

J Pharmacol Exp Ther, 1995 Sep, 274(3), 1099 - 104
In vitro formation, disposition and toxicity of N-acetoxy-sulfamethoxazole, a potential mediator of sulfamethoxazole toxicity; Nakamura H et al.; Variation in the formation and disposition of the hydroxylamine of (SMX-HA) is thought to play an important role in the pathogenesis of sulfamethoxazole (SMX)-induced idiosyncratic adverse drug reactions . We hypothesized that, in analogy to carcinogenic arylamines, SMX-HA might be further converted to an electrophilic N-acetoxy metabolite which could play a role in mediating SMX toxicity . Accordingly, we chemically synthesized N-acetoxy-SMX, and examined the characteristics of its formation, metabolism, cytotoxicity and mutagenicity in human and bacterial test systems . The human arylamine N-acetyl-transferases, (NAT)1 and NAT2, were capable of converting SMX-HA to N-acetoxy-SMX . NAT1 and NAT2 possessed similar affinities for SMX-HA (apparent Km values of 650 and 520 microM, respectively), but the apparent maximal velocity of the NAT1-mediated acetylation was higher than that of NAT2 . (1332 vs . 37 nmol/min/U of immunoreactive NAT protein) . Human peripheral blood mononuclear cells 12,000 x g supernatant fractions converted N-acetoxy-SMX mainly back to SMX-HA, and also to a lesser extent to SMX, at clinically relevant concentrations . Similar pathways were observed in human hepatic cytosolic fractions . In a cytotoxicity assay, N-acetoxy-SMX was significantly more toxic to human peripheral blood mononuclear cells than SMX-HA (16.6 vs . 11.5% dead cells at a concentration of 300 microM) . N-acetoxy-SMX was weakly mutagenic to the Salmonella typhimurium TA100 strain in the Ames test . These data suggest that the N-acetoxy metabolites of sulfonamides could potentially play a role in mediating sulfonamide idiosyncratic adverse drug reactions.

FEMS Microbiol Lett, 1995 Sep 1, 131(2), 167 - 72
Silent genes in bacteria: the previously designated 'cryptic' ilvHI locus of 'Salmonella typhimurium LT2' is active in natural isolates; Burns DM et al.; Gene ilvG in Escherichia coli K-12 and ilvI in 'Salmonella typhimurium LT2' (S . enterica serotype Typhimurium, strain LT2) are inactive due to frameshift or nonsense mutations, respectively . These inactive genes have been suggested to be part of 'cryptic' genetic systems which are defined as being of long-term regulatory and evolutionary significance . We have shown that the nonsense mutation in ilvI is present only in derivatives of the laboratory strain 'S . typhimurium LT2' . All natural isolates of Salmonella examined have an arginine codon at the corresponding location of their ilvI sequences . Further, two randomly selected natural isolates of serotype Typhimurium are shown to each have an active ALS III isozyme . Our findings strongly suggest that the only Salmonella strains which lack a functional ilvHI locus are LT2 isolates . We suggest that the mutations leading to inactivation of both ilvI in 'S . typhimurium LT2' and ilvG in E . coli K-12 are more likely to have been acquired during laboratory storage and/or cultivation, rather than representing cryptic systems of gene regulation.

EMBO J, 1995 Sep 1, 14(17), 4187 - 95
Functional conservation of the secretion and translocation machinery for virulence proteins of yersiniae, salmonellae and shigellae; Rosqvist R et al.; Virulent bacteria of the genera Yersinia, Shigella and Salmonella secrete a number of virulence determinants, Yops, Ipas and Sips respectively, by a type III secretion pathway . The IpaB protein of Shigella flexneri was expressed in Yersinia pseudotuberculosis and found to be secreted under the same conditions required for Yop secretion . Likewise, YopE was secreted by the wild-type strain LT2 of Salmonella typhimurium, but YopE was not secreted by the isogenic invA mutant . Secretion of both IpaB and YopE required their respective chaperones, IpgC and YerA . In addition, yopE-containing S . typhimurium expressed a YopE-mediated cytotoxicity on cultured HeLa cells . YopE was detected in the cytosol of the infected HeLa cells and the amount of translocated YopE correlated with the degree of cytotoxicity . Both translocation and cytotoxicity were prevented by the addition of gentamicin . Treatment of HeLa cells with cytochalasin D prior to infection prevented internalization of bacteria, but translocation of YopE was still observed . These results favour the hypothesis that YopE is translocated through the plasma membrane by surface-located bacteria . We propose that virulent Salmonella and Shigella deliver virulence effector molecules into the target cell through the utilization of a functionally conserved secretion/translocation machinery similar to that shown for Yersinia.

Carcinogenesis, 1995 Sep, 16(9), 2167 - 70
Procarcinogen activation by cytochrome P450 3A4 and 3A5 expressed in Escherichia coli and by human liver microsomes; Yamazaki H et al.; Recent studies indicate that cytochrome P450 (P450) 3A4 plays important roles in the activation of procarcinogens such as aflatoxin B1 and sterigmatocystin, as well as in the oxidation of a number of structurally diverse chemicals and endogenous compounds . Since P450 3A5 has been reported to be present at significant levels in liver microsomes in approximately 25% of human adults, we examined and compared the role of P450 3A4 and 3A5 in procarcinogen activation in humans . Immunoblot experiments with liver microsomes from 60 human samples suggested that 4/30 Japanese and 4/30 Caucasians contained considerable levels of P450 3A5, although P450 3A4 could be determined at relatively high levels in all of the human samples examined . Good correlation was observed between P450 3A4, but not P450 3A5, levels versus activation of aflatoxin B1 and stergmatocystin in these human samples . Comparisons of the activation of procarcinogens in reconstituted monooxygenase systems containing modified P450 3A4 and 3A5 enzymes expressed in Escherichia coli were carried out in Salmonella typhimurium TA1535/pSK1002 or NM2009 tester strain for genotoxicity assay, and it was found that P450 3A4 had similar activities to or higher rates than P450 3A5 for the 24 procarcinogens tested.

Arzneimittelforschung, 1995 Sep, 45(9), 1021 - 3
Effect of quaternary ammonium salts on energy-yielding and energy-requiring processes in Salmonella typhimurium; Majtan V et al.; The effect of 2-(dodecanoylamino) ethylalkyldimethyl-ammonium bromides on energy-yielding (respiration) and energy-requiring (biosynthesis of nucleic acids and proteins) processes in Salmonella typhimurium cells was studied . The quaternary ammonium salts represented a homologous series (n = 7) . The efficacy of the compounds increased with the prolongation of the alkyl carbon chain up to octyl (C8), where the maximum was noted and then the efficacy decreased . This phenomenon known from the antimicrobial efficacy of these substances (cut-off effect) was shown also by influencing the rate of {14C}adenine and {14C}leucine incorporation and the inhibition of oxygen consumption (endogenous respiration) . The tested compounds influencing these processes interfere with the energy metabolism of Salmonella typhimurium cells.

Biochemistry, 1995 Aug 29, 34(34), 10764 - 70
Structure and function of Salmonella typhimurium orotate phosphoribosyltransferase: protein complementation reveals shared active sites; Ozturk DH et al.; A solvent-exposed loop, comprising residues 98-119 of S . typhimurium orotate phosphoribosyltransferase (OPRTase), is at the subunit interface of the dimeric enzyme, and its amino acid side chains potentially contact active sites on either subunit . A portion of the loop (103-107) appears to be mobile on the basis of the X-ray structures of enzyme.OMP {Scapin, G., Grubmeyer, C., & Sacchettini, J . C . (1994) Biochemistry 33, 1287-1294} and enzyme.PRPP.orotate complexes {Scapin, G., Ozturk, D . H., Grubmeyer, C., & Sacchettini, J . C . (1995) Biochemistry 34, 10744-10754} . Lys-103, which is essential for activity {Ozturk, D . H., Dorfman, R . H . Scapin, G., Sacchettini, J . C., & Grubmeyer, C . (1995) Biochemistry 34, 10755-10763}, may thus be functional in the active site formed by the adjacent subunit . Asp-125 is an essential residue that is in the middle of the active site . Equimolar mixtures of the nearly inactive K103A and D125N mutant ORPTase subunits produced approximately 21-23% of the enzymatic activity of the wild-type OPRTase . Heterodimer formation in the complemented mixtures was evidenced by various physical methods . Thus, the active site of OPRTase requires Asp-125 from one subunit and Lys-103 from the adjacent subunit . As predicted from the three-dimensional structure, increased activity resulting from complementation was also observed with mixtures of the K103A mutant and the poorly active K73A and K73Q mutants but not with mixtures of D125N and either K73A or K73Q mutants . Neither K103A nor D125N mutants exhibited negative complementation with the wild-type enzyme . A K103A/D125N double mutant enzyme was also constructed and was able to inactivate wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1995 Aug 29, 34(34), 10755 - 63
Locations and functional roles of conserved lysine residues in Salmonella typhimurium orotate phosphoribosyltransferase; Ozturk DH et al.; Salmonella typhimurium orotate phosphoribosyltransferase (OPRTase) catalyzes the formation of orotidine 5'-monophosphate (OMP) from orotate and alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP) . There are five highly conserved lysine residues (Lys-19, -26, -73, -100, and -103) in S . typhimurium OPRTase . Here, we report the results of mutagenesis and substrate analog studies to investigate the functional roles of these lysines . Together with information from X-ray crystallography {Scapin, G., Grubmeyer, C., & Sacchettini, J . C . (1994) Biochemistry 33, 1287-1294; Scapin, G., Ozturk, D . H., Grubmeyer, C., & Sacchettini, J . C . (1995) Biochemistry 34, 10744-10754}, sequence comparisons, and chemical modification {Grubmeyer, C., Segura, E., & Dorfman, R . (1993) J . Biol . Chem . 268, 20299-20304}, this work permits the assignment of functions of the five conserved lysines . Lys-19 is external to the active site, and its mutation to glutamine had little effect on enzyme activity . Lys-26 forms a hydrogen bond to OMP at the 3'-hydroxyl group, and its mutation produced 3-10-fold decreases in kcat . Lys-73 extends into the active site, and a conformational change allows it to interact with either the 5'-phosphate of OMP or the 2-hydroxyl and alpha-phosphoryl oxygen of PRPP in their respective substrate complexes . Mutation of Lys-73 produced a 50-100-fold decrease in kcat and an 8-12-fold increase in the KM value for PRPP . Mutation of Lys-100 produced a 5-fold decrease in kcat and a 3-fold increase in the KM for PRPP, consistent with its location within the active site, near the pyrophosphate moiety of PRPP.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1995 Aug 29, 34(34), 10744 - 54
The crystal structure of the orotate phosphoribosyltransferase complexed with orotate and alpha-D-5-phosphoribosyl-1-pyrophosphate; Scapin G et al.; The three-dimensional structure of Salmonella typhimurium orotate phosphoribosyltransferase (OPRTase) in complex with the ribose 5-phosphate donor alpha-D-5--phosphoribosyl-1-pyrophosphate (PRPP) and the nitrogenous base orotic acid has been solved and refined with X-ray diffraction data extending to 2.3 A resolution to a crystallographic R-factor of 18.7% . The complex was generated by carrying out catalysis in the crystal . Comparison of this structure with the previously reported structure of the orotidine 5'-monophosphate (OMP) complex {Scapin, G., Grubmeyer, C., and Sacchettini, J . C . (1994) Biochemistry 33, 1287-1294} revealed that the enzyme backbone undergoes only small movements . The most significant differences occur near the active site, at Ala71-Gly74, with the largest difference involving the side chains of Lys73, Val127-Ala133, the 5'-phosphate binding loop, and a long, solvent-exposed loop at the dimer interface . The position of the ribose moiety is, on the other hand, very different in the OMP and PRPP.orotate complexes, with its anomeric carbon moving approximately 7 A across the binding cavity . In the PRPP.orotate complex the highly conserved acidic side chain of Asp124 interacts with the ribose of PRPP, whereas there are no interactions of this aspartate with the substrate in the OMP complex.

J Mol Biol, 1995 Aug 25, 251(4), 520 - 32
Structural organization and assembly of flagellar hook protein from Salmonella typhimurium; Vonderviszt F et al.; The terminal regions of monomeric hook protein from Salmonella typhimurium are known to be highly mobile and exposed to the solvent . Although hook protein exhibits an unusual far-UV circular dichroism spectrum, resembling that of random coil structures, our calorimetric experiments clearly demonstrate that the molecule has a compact ordered core . The compact part probably consists of three domains as suggested by deconvolution analysis of the calorimetric melting profiles . Secondary structure prediction, together with the analysis of far-UV circular dichroism spectra, has shown that the domains of monomeric hook protein contain beta-sheeted structures without significant alpha-helical content . The polymerization of hook protein is accompanied by the stabilization of its disordered terminal regions into a predominantly alpha-helical domain . Evaluation of circular dichroism data suggests that about 45 terminal residues are involved in helical segments . Coiled-coil prediction indicates that whereas the whole carboxy-terminal helical region of hook protein has a strong bundle-forming potential, there is only a single short amino-terminal segment exhibiting weak coiled-coil forming tendencies . The formation of alpha-helical bundles is commonly believed to be a key event during the polymerization of the axial structure of bacterial flagella . To clarify the role of helical bundle formation in hook assembly, proteolytic fragments of hook protein with truncations of various lengths in their carboxy-terminal disordered regions were generated, and their polymerization behavior was investigated . We found that even fragments completely lacking the main helix-forming carboxy-terminal regions can polymerize into filaments in vitro under appropriately high salt concentrations . Our results suggest that, although helical bundle formation may occur during self-assembly, governing precise subunit packing and playing an important role in the stabilization of hook filaments, it is not the principal interaction mainly responsible for the development of their filamentous structure.

Biochem Biophys Res Commun, 1995 Aug 24, 213(3), 1010 - 6
Possible function of SP-22, a substrate of mitochondrial ATP-dependent protease, as a radical scavenger; Watabe S et al.; SP-22 was found to be a substrate protein of a mitochondrial ATP-dependent protease in bovine adrenal cortex . Its amino acid sequence was homologous to that of some prokaryotic and eukaryotic proteins such as thioredoxin peroxidase (formerly called thiol-specific antioxidant) in yeast and mammalian brains and the C22 component of alkyl hydroperoxide reductase in Salmonella typhimurium . In the present study, we found SP-22 to have the ability to scavenge reactive oxygen species, thus protecting radical-sensitive proteins such as tryptophan hydroxylase, glutamine synthetase and hemoglobin from oxidation . The protecting activity was enhanced by the addition of horse serum . The "serum factor(s)" seemed to be protein(s), since the physiological roles of SP-22 in adrenocortical mitochondria are discussed.

J Mol Biol, 1995 Aug 18, 251(3), 400 - 12
Structural effects of mutations in Salmonella typhimurium flagellar switch complex; Zhao R et al.; Mutations in Salmonella typhimurium fliG, fliM and fliN give rise either to non-flagellate, non-motile or non-chemotactic mutant bacteria . The FliG, FliM and FliN proteins form part of recently characterized extended flagellar basal structures, and have been postulated to form a mutually interacting structural complex . We have examined basal body preparations from non-motile or non-chemotactic fliG, fliM and fliN mutant strains by electron microscopy and immunoblot gel analysis . Most flagellar preparations isolated from the non-motile mutants lacked FliM, but contained FliG . The basal bodies lacked the belled morphology characteristic of the wild-type structures, but had protrusions which could be labelled with anti-FliG . Non-motile mutant preparations severely depleted of FliG but containing FliM were also obtained . These preparations contained extended, belled flagellar structures that were labelled with anti-FliM . Thus, FliM is part of the shell of the extended structures responsible for the belled morphology, while FliG may be part of the inner substructure . The extended basal structures from a FliG temperature-sensitive mutant strain rapidly lost FliM, as well as FliG, upon a shift to a non-permissive temperature, implying interaction between the FliG- and FliM-containing substructures . In dramatic contrast to non-motile mutants, extended basal structures isolated from non-chemotactic mutants were indistinguishable from wild-type structures . This difference may reflect the energetics of the different protein-protein interactions operative during torque generation and the switching of rotation sense.

Proc Natl Acad Sci U S A, 1995 Aug 15, 92(17), 7710 - 3
Selenophosphate synthetase: detection in extracts of rat tissues by immunoblot assay and partial purification of the enzyme from the archaean Methanococcus vannielii; Kim IY et al.; In Escherichia coli and Salmonella typhimurium it has been shown that selenophosphate serves as the selenium donor for the conversion of seryl-tRNA to selenocysteyl-tRNA and for the synthesis of 2-selenouridine, a modified nucleoside present in tRNAs . Although selenocysteyl-tRNA also is formed in eukaryotes and is used for the specific insertion of selenocysteine into proteins, the precise mechanism of its biosynthesis from seryl-tRNA in these systems is not known . Because selenophosphate is extremely oxygen labile and difficult to identify in biological systems, we used an immunological approach to detect the possible presence of selenophosphate synthetase in mammalian tissues . With antibodies elicited to E . coli selenophosphate synthetase the enzyme was detected in extracts of rat brain, liver, kidney, and lung by immunoblotting . Especially high levels were detected in Methanococcus vannielii, a member of the domain Archaea, and the enzyme was partially purified from this source . It seems likely that the use of selenophosphate as a selenium donor is widespread in biological systems.

Genes Dev, 1995 Aug 15, 9(16), 2034 - 41
Protein phosphorylation on serine, threonine, and tyrosine residues modulates membrane-protein interactions and transcriptional regulation in Salmonella typhimurium; Ostrovsky PC et al.; There exists a plethora of tyrosine kinases that play essential roles in regulation of eukaryotic proteins . Several dual specificity kinases that phosphorylate proteins on threonine, serine, and tyrosine residues also play critical roles in eukaryotic phosphorylation cascades . In contrast, very few prokaryotic proteins have been shown to be phosphorylated on tyrosine residues, and the functions of the rare examples remain obscure . Furthermore, no dual specificity kinases have been described in prokaryotes . Our results indicate that PutA protein from the bacterium Salmonella typhimurium autophosphorylates on several threonine, serine, and tyrosine residues . PutA protein both represses the proline utilization (put) operon and degrades proline to glutamate . These two opposing functions are regulated by the availability of proline and the membrane sites needed for the proline dehydrogenase activity of PutA protein . In addition, these functions are modulated by phosphorylation of PutA protein . The rate of dephosphorylation of PutA protein is determined by the availability of proline and membranes . Dephosphorylated PutA protein has a higher DNA binding affinity than the phosphorylated protein and thus may prevent toxic overexpression of PutA protein in the absence of available membrane sites.

Mol Microbiol, 1995 Aug, 17(4), 675 - 86
A third periplasmic transport system for L-arginine in Escherichia coli: molecular characterization of the artPIQMJ genes, arginine binding and transport; Wissenbach U et al.; A new binding-protein-dependent transport system of Escherichia coli specific for L-arginine was characterized by genetic and biochemical means . The system is encoded by five adjacent genes, artPIQMJ (art standing for arginine transport), which are organized in two transcriptional units (artPIQM and artJ) . The artl and artJ gene products (Artl and ArtJ) are periplasmic binding proteins with sequence similarity to binding proteins for polar (basic) amino acids . The artQ, artM and artP products are similar to the transmembraneous proteins and the ATPase of binding-protein-dependent carriers . The mature Artl and J proteins were localized in the periplasm and lacked signal peptides of 19 amino acid residues . Artl and ArtJ were isolated from overproducing strains . ArtJ specifically binds L-arginine with high affinity and overproduction of ArtJ stimulated L-arginine uptake by the bacteria . The substrate for Artl is not known, and isolated Artl did not bind common amino acids, various basic uncommon amino acids or amines . It is concluded that the artPIQM artJ genes encode a third arginine-uptake system in addition to the known argT hisJQMP system of Salmonella typhimurium and E . coli and the arginine (-ornithine) carrier (aps) of E . coli.

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