Arch Toxicol, 1999 Nov, 73(8-9), 489 - 92 Differential substrate behaviours of ethylene oxide and propylene oxide towards human glutathione transferase theta hGSTT1-1; Thier R et al.; The transformation of ethylene oxide (EO), propylene oxide (PO) and 1-butylene oxide (1-BuO) by human glutathione transferase theta (hGSTT1-1) was studied comparatively using 'conjugator' (GSTT1 + individuals) erythrocyte lysates . The relative sequence of velocity of enzymic transformation was PO > EO >> 1-BuO . The faster transformation of PO compared to EO was corroborated in studies with human and rat GSTT1-1 (hGSTT1-1 and rGSTT1-1, respectively) expressed by Salmonella typhimurium TA1535 . This sequence of reactivities of homologous epoxides towards GSTT1-1 contrasts to the sequence observed in homologous alkyl halides (methyl bromide, MBr; ethyl bromide, EtBr; n-propyl bromide, PrBr) where the relative sequence MeBr >> EtBr > PrBr is observed . The higher reactivity towards GSTT1-1 of propylene oxide compared to ethylene oxide is consistent with a higher chemical reactivity . This is corroborated by experimental data of acid-catalysed hydrolysis of a number of aliphatic epoxides, including ethylene oxide and propylene oxide and consistent with semi-empirical molecular orbital modelings. Mutat Res, 2000 Jan 24, 464(2), 161 - 7 The 2-phenylbenzotriazole-type water pollutant PBTA-2 has cytochalasin B-mimetic activity; Matsuoka A et al.; The 2-phenylbenzotriazole (PBTA)-type water pollutant, 2-{2-(acetylamino)-4-{N-(2-cyanoethyl)ethylamino}-5-methoxyphenyl}-5- amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-2), has been recently identified in samples from the Nishitakase River in Kyoto, Japan, and shows potent mutagenic activities in Salmonella typhimurium in the presence of a microsomal metabolizing system (S9 mix) . In the present study, we conducted the in vitro micronucleus (MN) test on PBTA-2 in the absence and presence of S9 mix in two Chinese hamster cell lines, CHL and V79-MZ . In the MN test, PBTA-2 was weakly positive in CHL cells and strongly positive in V79-MZ cells . Because the positive results were accompanied by a statistically significant increase in the number of polynuclear (PN) and/or mitotic (M) cells, we examined treated cells in metaphase to see if numerical chromosome aberrations were being induced . We found that PBTA-2 induces polyploidy in both CHL and V79-MZ cells . A detailed analysis of MN preparations showed that in CHL cells, PBTA-2 predominantly induces equal-sized binucleated cells . Rhodamine phalloidin staining revealed that PBTA-2 causes actin filament abnormalities in both cell lines similar to those caused by cytochalasin B . Cytochalasin B induced PN cells predominantly and dose dependently, and almost all the cells were equal-sized and binucleate . The results suggest that PBTA-2 has cytochalasin B-mimetic activity, although agents affecting actin filaments, such as cytochalasins, phallotoxins and chloropeptide, have been derived only from molds so far . This study also suggests that our MN test protocol may be used to identify chemicals that have cytochalasin B-mimetic activity as well as those that induce numerical aberrations. Cell Immunol, 1999 Dec 15, 198(2), 139 - 42 B1 B cell numbers and antibodies against phosphorylcholine and LPS are increased in IL-6 gene knockout mice; Bao S et al.; Peritoneal cavity cells were isolated from IL6-gene knockout (IL6(-/-)) and wild-type mice and stained for expression of IgM, CD5, and CD23 . B1 cell (IgM(+)/CD23(-), CD5(+)/IgM(+)) numbers were increased twofold in IL6(-/-) mice compared to normals while IgM(+)/CD23(+) (B2) cell numbers were reduced threefold . Intestinal antibody levels were also determined for both total immunoglobulin and phosphorylcholine (PC)-specific and LPS-specific antibody following oral challenge with attenuated Salmonella typhimurium . Total immunoglobulin levels (IgM, IgG, and IgA) were reduced 60-80% in intestinal secretions of IL6(-/-) mice compared to wild-type controls; however, PC-specific antibody was significantly higher in IL6(-/-) mice . Anti-LPS antibodies were also three- to sevenfold higher in IL6(-/-) mice compared to controls following Salmonella challenge . These data suggest that in IL6(-/-) mice the development of mucosal B2 cells is impaired but that intestinal B1 cells responding to microbial antigens such as PC and LPS develop normally and are fully functional . Arch Microbiol, 2000 Jan, 173(1), 76 - 7 Salmonella typhimurium forms adenylcobamide and 2-methyladenylcobamide, but no detectable cobalamin during strictly anaerobic growth; Keck B et al.; Under microaerophilic conditions Salmonella typhimurium LT2 synthesizes cobalamin, during which 5,6-dimethylbenzimidazole is formed from riboflavin . We report here that in an anoxic environment S . typhimurium did not form cobalamin, but rather adenylcobamide, 2-methyladenylcobamide, and cobyric acid . This indicated that S . typhimurium, like other microorganisms that synthesize 5,6-dimethylbenzimidazole from riboflavin, requires oxygen for the formation of the cobalamin base. Structure Fold Des, 1999 Dec 15, 7(12), 1547 - 56 The structure of the signal receiver domain of the Arabidopsis thaliana ethylene receptor ETR1; Muller-Dieckmann HJ et al.; BACKGROUND: In Arabidopsis thaliana, ethylene perception and signal transduction into the cell are carried out by a family of membrane-bound receptors, one of which is ethylene resistant 1 (ETR1) . The large cytoplasmic domain of the receptor showed significant sequence homology to the proteins of a common bacterial regulatory pathway, the two-component system . This system consists of a transmitter histidine kinase and a response regulator (or signal receiver) . We present the crystal structures of the first plant receiver domain ETRRD (residues 604-738) of ETR1 in two conformations . RESULTS: The monomeric form of ETRRD resembles the known structure of the bacterial receiver domain . ETRRD forms a homodimer in solution and in the crystal, an interaction that has not been described previously . Dimerization is mediated by the C terminus, which forms an extended beta sheet with the dimer-related beta-strand core . Furthermore, the loop immediately following the active site adopts an exceptional conformation . CONCLUSIONS: The three-dimensional structure of ETRRD shows the expected conformational conservation to prokaryotic receiver proteins, such as CheY and CheB, both of which are part of the chemotaxis signaling pathway . ETRRD provides the first detailed example of a dimerized receiver domain . Given that the dimer interface of ETRRD coincides with the phosphorylation-dependent interfaces of CheY and CheB, we suggest that the monomerization of ETRRD is phosphorylation-dependent too . In the Mg(2+)-free form of ETRRD, the gamma-loop conformation does not allow a comparable interaction as observed in the active-site architectures of Mg(2+)-bound CheY from Escherichia coli and Salmonella typhimurium. Microb Drug Resist, 1999 Winter, 5(4), 279 - 87 Characterization of integrons in Escherichia coli of the normal intestinal flora of swine; Sunde M et al.; Multiresistant Escherichia coli isolates of the normal intestinal flora of healthy fattening pigs were examined for the presence of integron class 1 by XL (extra long) PCR . The class 1 integron was detected in 17 isolates originating from 14 healthy animals on seven different farms . One isolate contained two class 1 integrons . The inserted gene cassettes were characterized by DNA sequencing and PCR . The ant(3")-Ia gene responsible for resistance to streptomycin/spectinomycin was inserted in all integrons detected . Fifteen isolates contained this gene cassette as the only inserted cassette . Three isolates contained integrons with two gene cassettes . Two isolates contained integrons with the trimethoprim resistance gene dfr1 and one isolate contained the oxa1 beta-lactamase gene upstream to the ant(3")-Ia gene . Detection of these three different resistance gene cassettes in bacteria from swine shows that cassettes occurring in integrons in human clinical isolates also appear in bacteria of the normal intestinal flora of healthy swine . Two integron-harboring strains were obtained from each of three different animals . These strains were probably not clonal derivatives of each other, suggesting the existence of different multiresistance clones within the intestinal normal flora of one specific animal . The oxa1 nucleotide sequence found in E . coli from swine differ by seven nucleotides from the oxa1 nucleotide sequence of the gene from the R-plasmid RGN238 . The fact that these two sequences are not identical might indicate that the two genes have evolved separately in different surroundings from the common ancestor . Transmissible plasmids of approximately 200 kb containing integron class I were detected in eight of the isolates when conjugation experiments were performed with E . coli DH5 as recipient strain . The transfer frequency ranged from 4x10(-4) to 6x10(-2) transconjugants per recipient cell . This study shows that the enteric commensals of domestic animals may be considered as a reservoir of integron-containing transmissible plasmids and gene cassettes that might be transferable to the pathogens of swine and to important zoonotic bacteria associated with the enteric flora of swine such as Salmonella typhimurium DT104. Indian J Exp Biol, 1999 Mar, 37(3), 283 - 9 Immunomodulation of macrophages by radio-detoxified lipopolysaccharide of Salmonella typhimurium; Naidu MD et al.; Lipopolysaccharide (LPS) and ratio-detoxified LPS (Rd-LPS) from Salmonella typhimurium were analysed for their ability to stimulate murine peritoneal exudate cells (PEC) and macrophages . Rd-LPS induced much more inflammatory response as compared to LPS . PEC numbers/mouse obtained were significantly higher (3-fold) in response to Rd-LPS than LPS . The haemorrhage was induced in mice by LPS but not by Rd-LPS . Activation of macrophages in vivo by Rd-LPS was significantly higher as compared to LPS . This was evident from the increase levels of their lysosomal enzymes and cytokines . Rd-LPS induced 10-fold increase in acid phosphatase contents of macrophages as compared to controls while only 7-fold increase was obtained with LPS . Arylsulfatase and beta-glucuronidase increased by about 2-fold by Rd-LPS and LPS . Macrophages incubated with Rd-LPS in vitro showed 16-fold and 20-fold increase in the cell associated levels of arylsulfatase and beta-glucuronidase respectively as compared to unstimulated cells . On the other hand, only 6-fold increase was observed in response to LPS in the levels of both the enzymes . TNF-{symbol: see text} and IL-1 secreted by macrophages increased considerably in response to Rd-LPS as compared to those released by LPS . Rd-LPS, thus seems to be a better immunomodulator than untreated LPS. J Immunol, 2000 Feb 1, 164(3), 1625 - 33 Linear PADRE T helper epitope and carbohydrate B cell epitope conjugates induce specific high titer IgG antibody responses; Alexander J et al.; Linear carbohydrate-peptide constructs based on the 13 amino acid nonnatural pan DR epitope (PADRE) and carbohydrate B cell epitopes are demonstrated to be potent immunogens . These data support our belief that PADRE should be considered as an alternative to more complex carriers for use in prophylaxis and therapeutic vaccines . Two model carbohydrate-PADRE glycoconjugates were used to demonstrate that PADRE could effectively provide T cell help for carbohydrate-specific Ab responses . Conjugates of PADRE covalently linked to the human milk oligosaccharide, lacto-N-fucopentose II or a dodecasaccharide derived from Salmonella typhimurium O-Ag induced high titer IgG Ab responses in mice, which were comparable to glycoconjugates employing human serum albumin (HSA) as the carrier protein . Different adjuvants, in combination with PADRE conjugates, allowed for the modulation of the isotype profile with alum supporting an IgG1 profile; QS-21 an IgG2a, 2b profile, while an alum/QS-21 mixture generated a balanced IgG1/IgG2b isotype profile . As defined by binding to synthetic glycoconjugates, dodecasaccharide-specific Abs exhibited fine specificity similar to protective polyclonal Ab responses previously reported for dodecasaccharide-protein conjugates . The same Abs bound to intact S . typhimurium cells, suggesting that biologically relevant specificities were produced . The affinity of the dodecasaccharide-specific Abs was further shown to be comparable to that of a well-characterized, high affinity monoclonal anti-carbohydrate Ab recognizing the same epitope. J Immunol, 2000 Feb 1, 164(3), 1470 - 7 The neuronal apoptosis inhibitory protein (Naip) is expressed in macrophages and is modulated after phagocytosis and during intracellular infection with Legionella pneumophila; Diez E et al.; Legionella pneumophila is an intracellular pathogen that causes Legionnaires' disease in humans . Inbred mouse strains are uniformly resistant to L . pneumophila infection with the notable exception of A/J, where the chromosome 13 locus Lgn1 renders A/J macrophages permissive to L . pneumophila replication . The mouse Lgn1 region is syntenic with the spinal muscular atrophy (SMA) locus on human chromosome 5 and includes several copies of the neuronal apoptosis inhibitory protein (Naip) gene . We have analyzed a possible link among Lgn1, Naip, and macrophage function . RNA expression studies show that Naip (mostly copy 2) mRNA transcripts are expressed in macrophage-rich tissues, such as spleen, lung, and liver and are abundant in primary macrophages . Immunoblotting and immunoprecipitation analyses identify Naip protein expression in mouse macrophages and in macrophage cell lines RAW 264.7 and J774A . Interestingly, macrophages from permissive A/J mice express significantly less Naip protein than their nonpermissive C57BL/6J counterpart . Naip protein expression is increased after phagocytic events . Naip protein levels during infection with either virulent or avirulent strains of L . pneumophila increase during the first 6 h postinfection and remain elevated during the 48-h observation period . This enhanced expression is also observed in macrophages infected with Salmonella typhimurium . Likewise, an increase in Naip protein levels in macrophages is observed 24 h after phagocytosis of Latex beads . The cosegregation of Lgn1 and Naip together with the detected Naip protein expression in host macrophages as well as its modulation after phagocytic events and during intracellular infection make it an attractive candidate for the Lgn1 locus. IUBMB Life, 1999 Nov, 48(5), 493 - 7 Molecular cloning, sequencing, and expression of two late proteins of bacteriophage MB78; Kolla V et al.; Bacteriophage MB78, a virulent phage of Salmonella typhimurium, does not allow other phages, such as P22 and 9NA, to grow in its presence . A detailed physical map of this phage has been constructed in our laboratory . In an ongoing effort to understand the genetics of this interesting phage, various genes were characterized . Here, we report cloning, sequencing, and expression of two late proteins, coded in a SalI-HindIII fragment (SH9), by using the minicell expression system . Further, we performed a kinetic study of phage proteins by infection the host LT2 cells and compared the proteins produced, with proteins obtained by the minicell expression system . Both sets of proteins run exactly parallel and migrated as 14- and 15-kDa proteins on a polyacrylamide gel . The synthesis of these two proteins started 15 min after infection with MB78 and was prominent after 45 min . One of the proteins exhibited 57% homology to the structural protein of mycobacteriophage L5. J Toxicol Sci, 1999 Nov, 24 Suppl 1, 89 - 94 {Mutagenicity study of gadobenate dimeglumine formulation (E7155) (1)--Reverse mutation assays in S . typhimurium and E . coli tester strains}; Hakura A et al.; The ability of gadobenate dimeglumine formulation (E7155) to cause gene mutations was assessed in five strains of Salmonella typhimurium (TA100, TA1535, TA98, TA1538, and TA1537) and a strain of Escherichia coli (CM891; WP2, uvrA-, pKM101) using the Ames test (agar plate assay) . The results suggest that E7155 is non-mutagenic towards these bacterial tester strains. Mutat Res, 2000 Jan 3, 464(1), 97 - 104 Semi-quantitative evaluation of genotoxic activity of chemical substances and evidence for a biological threshold of genotoxic activity; Sofuni T et al.; In Japan, the Chemical Substances Control Law requires evaluation of the genotoxic potential of chemical substances semi-quantitatively by application of a ranking system . During the past 10 years under the law, 1049 new chemical substances were evaluated by a reverse mutation assay in bacteria (RMA) and a chromosome aberration test in cultured mammalian cells (CAT) . Of them, 130 (12.4%) were positive in the RMA and 402 (38.3%) were positive in the CAT . Eighty (7.6%) were positive in both tests . Fifty (4.8%) were positive only in the RMA, 322 (30.7%) were positive only in the CAT, and 452 (43.1%) were positive in either the RMA or the CAT . Thus, the tests complement each other in detecting genotoxic substances in vitro . To explore the "threshold" concept, we compared the genotoxic responses of Salmonella typhimurium tester strains with and without DNA repair capacity . Recently constructed strains of TA1535 lacking O(6)-methylguanine DNA methyltransferase genes (ogt(ST) or ada(ST) and ogt(ST)) showed dose-related increases in the number of revertants induced by N-ethyl-N'-nitro-N-nitrosoguanidine, methyl methanesulfonate, dimethylnitrosamine, and ethylnitrosourea, while in the same dose ranges the parental strain TA1535 did not . This finding suggests that there is a threshold at which all DNA damage induced by low dose levels of genotoxic chemicals are repaired . That biological threshold seems to exist for both DNA and non-DNA targeting chemicals. Mol Cell Biochem, 1999 Nov, 201(1-2), 169 - 81 The effect of immunization with porins on gut pathophysiological response in rats infected with Salmonella typhimurium; Mittal A et al.; Attachment of Salmonella typhimurium to epithelial surfaces elicit significant alterations in different cell signalling events which lead to the development of disease . The present investigation was conducted to evaluate the effect of immunization of rats with porins, on gut physiologic markers following challenge with S . typhimurium . Male albino Wistar rats were immunized with purified porins and challenged by intragastric infection with S . typhimurium . Electrolyte transport, levels of different second messengers and inflammatory mediators were studied . A net absorption of transepithelial fluxes of Na+ and Cl- in immunized-challenged group and secretion in infected group was found . Ca2+ and 3-O-methyl-D-glucose fluxes did not show any change . Significant increase in the levels of {Ca2+}i, cAMP, membrane form of protein kinase C, prostaglandins, NADPH oxidase, Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, total oxygen free radicals, reactive nitrogen intermediates, citrulline and lipid peroxidation was found in the infected group . However, in the immunized-challenged group, the values of all the parameters were found to be almost the same as that of control as well as immunized groups . Na+, K+-ATPase and calmodulin levels were unaltered in all the groups of animals . The results of this study thus suggest that immunization of rats with purified Salmonella porins followed by subsequent challenge with the organism might be helpful for the prevention of multiple physiologic derangements in isolated ileal cells. Toxicol Sci, 1999 Dec, 52(2), 226 - 31 DNA-damaging effects of genotoxins in mixture: nonadditive effects of aflatoxin B1 and N-acetylaminofluorene on their mutagenicity in Salmonella typhimurium; Said B et al.; Most animal genotoxicity studies have used exposures to single chemicals; humans, however, are potentially exposed to mixtures of genotoxins . Cancer and developmental toxicity risks associated with genotoxins in mixture are generally estimated by assuming additivity of the components . Two or more genotoxins acting sequentially or simultaneously may present a greater or lesser hazard than that predicted by simple addition of their potencies . Previously, we studied the effect of one genotoxin on the binding of a second genotoxin to DNA in an in vitro system and demonstrated that consecutive binding of the two toxins was not additive . In the present study, the effect of one genotoxin on the mutagenicity of another was evaluated for two well-known genotoxins using the Salmonella assay . Pretreatment of frameshift strains TA98 and TA1538 with AFB1-8,9-epoxide (17.3 ng/plate) enhanced the mutagenicity induced by subsequent exposure to N-acetoxy-acetylaminofluorene (N-AcO-AAF) approximately 2-3 times above theoretical values for additivity . Pretreatment of base-substitution strain TA100 with N-AcO-AAF (0.1 microg/plate) inhibited the mutagenicity following subsequent exposure to AFB1-8,9-epoxide by 3 times below the theoretical additive value . Concentration-response relationships for these enhancing or inhibitory effects were demonstrated using increasing concentrations of the first genotoxin during pretreatment . These results demonstrate effects, other than additive, of sequential exposures to two genotoxins on the induction of mutations in a bacterial system. Mol Gen Genet, 1999 Dec, 262(4-5), 721 - 9 Identification and localization of differences between Escherichia coli and Salmonella typhimurium genomes by suppressive subtractive hybridization; Bogush ML et al.; The availability of bacterial genome sequences raises an important new problem - how can one move from completely sequenced microorganisms as a reference to the hundreds and thousands of other strains or isolates of the same or related species that will not be sequenced in the near future? An efficient way to approach this task is the comparison of genomes by subtractive hybridization . Recently we developed a sensitive and reproducible subtraction procedure for comparison of bacterial genomes, based on the method of suppression subtractive hybridization (SSH) . In this work we demonstrate the applicability of subtractive hybridization to the comparison of the related but markedly divergent bacterial species Escherichia coli and Salmonella typhimurium . Clone libraries representing sequence differences were obtained and, in the case of completely sequenced E . coli genome, the differences were directly placed in the genome map . About 60% of the differential clones identified by SSH were present in one of the genomes under comparison and absent from the other . Additional differences in most cases represent sequences that have diverged considerably in the course of evolution . Such an approach to comparative bacterial genomics can be applied both to studies of interspecies evolution - to elucidate the "strategies" that enable different genomes to fit their ecological niches - and to development of diagnostic probes for the rapid identification of pathogenic bacterial species. J Med Microbiol, 2000 Jan, 49(1), 103 - 10 Evolution of chloramphenicol resistance, with emergence of cross-resistance to florfenicol, in bovine Salmonella Typhimurium strains implicates definitive phage type (DT) 104; Arcangioli MA et al.; The prevalence of resistance to florfenicol, a phenicol drug newly introduced in veterinary therapy, was determined in 86 chloramphenicol-resistant Salmonella Typhimurium isolates from cattle collected during 1985-1995 . All were highly resistant to chloramphenicol (MICs > or = 128 mg/L) and 38 were simultaneously resistant to florfenicol (MICs >16 mg/L) and to beta-lactam agents, spectinomycin, streptomycin, sulphonamides and tetracyclines . The isolates susceptible to florfenicol harboured the chloramphenicol acetyl transferase gene, cat of type I . All the florfenicol-resistant isolates harboured the floR resistance gene and the characteristic multiple resistance genetic locus, previously characterised in a S . Typhimurium DT104 strain and identified by a multiplex PCR . Plasmid profiles and ribotype patterns were determined for all the isolates . The florfenicol-resistant isolates were grouped into the same ribotyping pattern and presented similar plasmid profiles, whereas the florfenicol-susceptible isolates showed a wider genetic diversity that is usual for S . Typhimurium . Thus, the florfenicol-resistant isolates could represent a clonal cluster, closely related to, if not of DT104 phage type, which appeared in 1989 and is now predominant within chloramphenicol-resistant S . Typhimurium . The multiplex PCR provided a useful tool to survey further evolution of multiresistant S . Typhimurium strains. J Immunol, 2000 Jan 15, 164(2), 986 - 93 Characterization of CD4+ T cell responses during natural infection with Salmonella typhimurium; McSorley SJ et al.; CD4+ T cells are important for resistance to infection with Salmonella typhimurium . However, the Ag specificity of this T cell response is unknown . Here, we demonstrate that a significant fraction of Salmonella-specific CD4+ T cells respond to the flagellar filament protein, FliC, and that this Ag has the capacity to protect naive mice from lethal Salmonella infection . To characterize this Ag-specific response further, we generated FliC-specific CD4+ T cell clones from mice that had resolved infection with an attenuated strain of Salmonella . These clones were found to respond to an epitope from a constant region of FliC, enabling them to cross-react with flagellar proteins expressed by a number of distinct Salmonella serovars. Microb Pathog, 2000 Jan, 28(1), 37 - 44 Identification of diminished tissue culture invasiveness among multiple antibiotic resistant Salmonella typhimurium DT104; Carlson SA et al.; Salmonella infections continue to cause gastrointestinal and systemic disease throughout the world . Salmonella typhimurium further poses a major health concern due to its apparent enhanced ability to acquire multiple antibiotic resistance genes . Currently it is unclear if multiresistant S . typhimurium are more or less pathogenic than non-resistant counterparts . Using an in vitro invasion assay, we evaluated the relative pathogenicity of over 400 multiresistant S . typhimurium isolates . Our studies failed to identify any <<<<hyperinvasive>>>> isolates . However, we identified 12 isolates exhibiting invasive phenotypes that were constrained relative to controls . These <<<<hypoinvasive>>>> strains were found in a variety of phagetypes all possessing at least a hexaresistant profile . Further studies revealed that the alterations in invasion were not due to changes in adherence . Limited studies exploring in vivo virulence revealed a mildly decreased ability to cause murine lethality for the hypoinvasive strain examined . These results indicate that the ability to cause disease is not increased but is rather mildly attenuated for certain isolates of multiresistant S . typhimurium . FEMS Microbiol Lett, 2000 Jan 15, 182(2), 355 - 60 Detection of multidrug-resistant Salmonella typhimurium DT104 by multiplex polymerase chain reaction; Khan AA et al.; Salmonella typhimurium definitive type 104 (DT104) is a virulent pathogen for humans and animals with many strains having multiple drug resistance characteristics . The organism typically carries resistance to ampicillin, chloramphenicol, florfenicol, streptomycin, sulfonamides, and tetracycline (ACSSuT-resistant) . A multiplex PCR method was developed to simultaneously amplify four genes, florfenicol (flo(st)), virulence (spvC), invasion (invA), and integron (int) from S . typhimurium DT104 (ACSSuT-type) . Twenty-two ACSSuT-resistant DT104 isolates in our collection gave 100% positive reactions to this PCR assay by amplifying 584-, 392-, 321- and 265-bp PCR products, using primers specific to the respective target genes . One Salmonella strain DT23, ACSSuT-resistant, phage type 711 failed to amplify the 584-bp fragment, indicating that this method is specific for DT104-type ACSSuT-resistant S . typhimurium strains . One clinical and one bovine ASSuT-resistant strains that were sensitive to chloramphenicol and florfenicol did not yield a 584-bp fragment, indicating the absence of the flo(st) gene . This method will be useful for rapid identification of ACSSuT-type DT104 strains from clinical, food and environmental samples. FEMS Microbiol Lett, 2000 Jan 15, 182(2), 219 - 23 Vectors to achieve selective expression of vaccine antigens within eukaryotic cells using Salmonella spp . as carrier strains; Basso H et al.; A set of expression vectors was constructed which allows the expression of recombinant antigens under the control of Salmonella typhi promoters that are selectively activated after infection of eukaryotic cells . The pUC18Not derivatives contain a promoter downstream of the early transcriptional terminator from phage T7 and followed by a multiple cloning site, whereas the pBluescript II S/K derivatives contain the ribosomal RNA T(1) transcriptional terminator and also the strong translation signals of the Escherichia coli atpE gene . The expression cassettes are flanked by NotI or PacI sites to simplify their subcloning where required . The resulting vectors were validated using the S1 subunit of pertussis toxin as a model antigen and Salmonella typhimurium aroA SL7207 as a carrier . The S1 subunit was efficiently expressed by recombinant Salmonella within Henle 407 cells. Tohoku J Exp Med, 1999 Sep, 189(1), 1 - 9 Modification of mutagenic activities of pro-mutagens by glyco-ursodeoxycholic acid in the Ames assay; Shibuya N et al.; Mutagenicity, co-mutagenicity and anti-mutagenicity of glycoursodeoxycholic acid (GUDCA) were examined by the Ames assay using Salmonella typhimurium strain TA98 with S9 . As pro-mutagens, 2-aminoanthracene (2AA), Benzo{a}pyrene (BaP), 3-amino-1-dimethyl-5H-pyrido{4, 3-b}indole (Trp-P-2), 2-amino-3-methylimidazo{4, 5-f}quinoline (IQ) and 2-amino-3, 4-dimethylimidazo{4, 5-f}quinoline (MeIQ) were used . In addition to these pro-mutagens, blue-chitin extracts of human gallbladder bile (BCE) collected from the cholecystectomized patients with cholelithiasis were used in order to investigate the role of GUDCA on mutagen(s) actually existing in human bile . It was found that GUDCA did not show mutagenicity in this test system . Concerning the modification of mutagenic activities of pro-mutagens, GUDCA showed the different directions . GUDCA acted as co-mutagen, since it enhanced the mutagenic activities of 2AA and BaP . But, acted as anti-mutagen, since it suppressed the activities of Trp-P-2, IQ and MeIQ, all of which were classified as heterocyclic amines . GUDCA also suppressed the mutagen(s) in human bile . Because of the use of blue-chitin absorbed method for testing bile mutagenicity, the chemicals involved were considered to be heterocyclic amines and other polycyclic compounds . In these we suspect the bile mutagens are heterocyclic amines . Further examination should be directed towards the investigation into the mechanism of anti-mutagenic effects of GUDCA on mutagen(s) actually existing in human bile. J Clin Invest, 2000 Jan, 105(1), 79 - 92 Salmonella typhimurium induces epithelial IL-8 expression via Ca(2+)-mediated activation of the NF-kappaB pathway; Gewirtz AT et al.; Interactions between the enteric pathogen Salmonella typhimurium and the luminal surface of the intestine provoke an acute inflammatory response, mediated in part by epithelial cell secretion of the chemokine IL-8 and other proinflammatory molecules . This study investigated the mechanism by which this pathogen induces IL-8 secretion in physiologically polarized model intestinal epithelia . IL-8 secretion induced by both the prototypical proinflammatory cytokine TNF-alpha and S . typhimurium was NF-kappaB dependent . However, NF-kappaB activation and IL-8 secretion induced by S . typhimurium, but not by TNF-alpha, was preceded by and required an increase in intracellular {Ca(2+)} . Additionally, agonists that increased intracellular {Ca(2+)} by receptor-dependent (carbachol) or independent (thapsigargin, ionomycin) means also induced IL-8 secretion . Furthermore, the ability of S . typhimurium mutants to induce IkappaB-alpha degradation, NF-kappaB translocation, and IL-8 transcription and secretion correlated precisely with their ability to induce an intracellular {Ca(2+)} increase in model intestinal epithelia, but not with their ability to invade these cells . Finally, S . typhimurium, but not TNF-alpha, induced a Ca(2+)-dependent phosphorylation of IkappaB-alpha . These results indicate that S . typhimurium-induced activation of NF-kappaB-dependent epithelial inflammatory responses proceeds by a Ca(2+)-mediated activation of an IkappaB-alpha kinase . These observations raise the possibility that pharmacologic intervention of the acute inflammatory response can be selectively matched to the specific class of initiating event. Vaccine, 2000 Feb 14, 18(15), 1543 - 54 Murine immune responses to mucosally delivered Salmonella expressing Lassa fever virus nucleoprotein; Djavani M et al.; Arenaviruses are emerging pathogens known to infect via the mucosa, however no formal attempts to make mucosal vaccines have been undertaken . Here we describe a recombinant aroA attenuated Salmonella typhimurium that expresses the nucleoprotein (NP) gene of Lassa fever virus (LAS) . The complete NP gene was cloned downstream of the bacterial groEL promotor and integrated into the aroA locus of S . typhimurium . Lassa NP protein was detected in whole cell extracts from the recombinant Salmonella by immunoblot analysis with serum from Lassa-infected people . Mice were inoculated by intragastric intubation with 5 x 10(9) S . typhimurium and boosted with the same recombinant Salmonella 21 days after the primary inoculation . Both local mucosal IgA and serum immunoglobulins against Lassa NP were observed . Splenic cytotoxic T-lymphocyte responses to LAS NP were detected after the boost and they cross-reacted with target cells infected with the related arenavirus, lymphocytic choriomeningitis virus . Recombinant Salmonella elicits humoral and cell mediated immune responses against Lassa fever virus in mice and should be considered as a potential vaccine strategy in man. Vaccine, 2000 Jan 31, 18(14), 1298 - 306 Use of the stationary phase inducible promoters, spv and dps, to drive heterologous antigen expression in Salmonella vaccine strains; Marshall DG et al.; We have investigated the ability of the growth phase regulated promoters dps and spv, to drive expression of heterologous antigens in Salmonella vaccine strains . Reporter plasmids were constructed which directed beta-galactosidase expression from dps (pDpslacZ) or spv (pSpvlacZ) and these were introduced independently into the Salmonella typhimurium vaccine strain SL3261 (aroA(-)) . beta-galactosidase expression was induced 20-fold and 100-fold when broth cultures of SL3261 (pDpslacZ) or SL3261 (pSpvlacZ) respectively, entered the stationary phase of growth . Within macrophages, beta-galactosidase expression was induced 3.5-fold with SL3261 (pDpslacZ) and 7-fold with SL3261 (pSpvlacZ) . The spv and dps promoters were used to drive independent expression of the C fragment domain of tetanus toxin (TetC) from plasmids harboured in S . typhimurium SL3261 . Levels of anti-TetC antibodies were significantly higher in the sera of BALB/c mice perorally inoculated with SL3261 (pSpvtetC) or SL3261 (pDpstetC) compared to unvaccinated controls . This suggests that these promoter systems may be used to drive foreign antigen expression in live oral Salmonella vaccines. Life Sci, 1999, 65(24), 2591 - 602 In vitro and in vivo effects of naringin on cytochrome P450-dependent monooxygenase in mouse liver; Ueng YF et al.; In vitro and in vivo effects of naringin on microsomal monooxygenase were studied to evaluate the drug interaction of this flavonoid . In vitro addition of naringin up to 500 microM had no effects on benzo(a)pyrene hydroxylase (AHH) activity of mouse liver microsomes . In contrast, the aglycone naringenin at 300 to 500 microM decreased AHH activity by 50% to 60% . Analysis of Lineweaver-Burk and Dixon plots indicated that naringenin competitively inhibited AHH activity with an estimated Ki of 39 microM . Naringenin at 100 microM also reduced metabolic activation of benzo(a)pyrene to genotoxic products as monitored by umuC gene expression response in Salmonella typhimurium TA1535/pSK1002 . In the presence of equimolar naringenin and benzo(a)pyrene, umuC gene expression presented as beta-galactosidase activity was reduced to a level similar to the control value . Administration of a liquid diet containing 10 mg/ml naringin for 7 days caused 38% and 49% decreases of AHH and 7-methoxyresorufin O-demethylase activities, respectively . In contrast, the administration had no effects on cytochrome P450 (P450)-catalyzed oxidations of 7-ethoxyresorufin, 7-ethoxycoumarin, N-nitrosodimethylamine, nifedipine, erythromycin and testosterone . Microsomal P450 and cytochrome b5 contents and NADPH-P450 reductase activity were not affected . Immunoblot analysis using MAb 1-7-1, which immunoreacted with both P450 1A1 and 1A2, revealed that the level of P450 1A2 protein was decreased by 38% . These results demonstrate that naringenin is a potent inhibitor of AHH activity in vitro and naringin reduces the P450 1A2 protein level in vivo . These effects may indicate a chemopreventive role of naringin against protoxicants activated by P450 1A2. Mutat Res, 1999 Oct 29, 446(1), 95 - 102 Genotoxicity of ochratoxin A by Salmonella mutagenicity test after bioactivation by mouse kidney microsomes; Obrecht-Pflumio S et al.; Ochratoxin A (OTA) was, up to now, believed to be non-mutagenic in the classical Salmonella typhimurium reverse mutation test . This was confirmed using rat liver microsomal fractions with the strains, TA1535, TA1538 and TA98, and up to 1210 micrograms/plate, utilizing an Ames microtest . However, using mice kidney microsomal fractions as metabolic activators, reverse mutations were obtained with the three strains used, in the presence of either NADP or arachidonic acid as cofactors . The mutagenicity was higher with arachidonic acid than with NADP using the TA1535 strain . This lends support to the results concerning the DNA or dGMP adducts obtained in vitro which were also higher in the presence of arachidonic acid, and indicate that several metabolic pathways of OTA can lead to genotoxic compounds . In addition, both base pair substitutions and frameshift mutations can be caused by OTA after metabolic activation. Mutat Res, 1999 Oct 29, 446(1), 83 - 94 Genotoxicity of fractionated organic material in airborne particles from São Paulo, Brazil; De Martinis BS et al.; Particulate matter less than 10 microns aerodynamic diameter (PM10) is associated with adverse health effects including increased respiratory problems and mortality . PM10 is also associated with increases in cancer in some urban areas . Identification of toxic compounds in PM10 is a step toward estimating exposure to these compounds and evaluating their public health risk . However, the toxic compounds on PM10 are part of a highly complex mixture of compounds that makes chemical characterization difficult . Before this study, there has been little investigation of genotoxic compounds in particulate matter from Latin American cities . Here, both bioassay (mutagenicity) and chemical analyses were conducted with organic solvent extracts of PM10 collected from Sao Paulo, a major Brazilian city . Sequential extraction in dichloromethane (DCM) followed by acetone (ACE) yielded 20.3% and 10.2% of the total mass, respectively . Non-polar and moderately polar organic material solubilized in DCM . ACE extracted more polar organic species and some inorganic ions . Both extracts were fractionated separately using cyanopropyl-bonded silica chromatography with organic solvents of increasing polarity . The mass distribution among the fractions was measured . The mutagenic activity of the fractions was assayed using the microsuspension procedure with the Salmonella typhimurium tester strain TA98, with and without addition of metabolic enzymes (S9) . The DCM extract had about four times higher mutagenic activity than the ACE extract . In general, addition of S9 resulted in an increase in mutagenicity of DCM fractions, but a decrease for the ACE extract . Most of the activity was concentrated in fractions in the mid-range of polarity within both the DCM and ACE extracts . The fractions were analyzed by gas chromatography with mass selective detection (GC/MS) without derivatization . The most mutagenic fractions in the DCM extract contained ketones, aldehydes, and quinolines . The most mutagenic ACE fraction had ketones, carboxylic acids, and aldehydes. Mutat Res, 1999 Oct 29, 446(1), 67 - 81 Genotoxicity tests with 6-acetyl-1,1,2,4,4,7-hexamethyltetraline and 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-gamma-2-benzopyr an; Api AM et al.; 6-Acetyl-1,1,2,4,4,7-hexamethyltetraline (AHTN) and 1,3,4,6,7,8-hexahydro-4,6,6,7,8,8-hexamethylcyclopenta-gamma-2-ben zopyran (HHCB), synthetic fragrance ingredients, were evaluated for potential genotoxicity in a battery of short-term tests . Salmonella typhimurium/Escherichia coli plate incorporation and liquid preincubation assays were conducted on AHTN using tester strains TA97, TA98, TA100, TA102, TA1535, TA1537 and WP2 uvrA +/- S9 activation at doses from 8 to 5000 micrograms/plate . The plate incorporation mutagenicity assay was conducted on HHCB using tester strains TA98, TA100, TA1535, TA1537, TA1538 and WP2 uvrA +/- S9 activation at doses from 10 to 5000 micrograms/plate . An in vitro cytogenetics assay in Chinese hamster ovary (CHO) cells was conducted with AHTN and HHCB at three concentrations each with +/- S9 activation . In the non-activated study, the exposure/harvest periods were 4/20-, 20/20- and 44/44-h . In the S9 activated study, the exposure/harvest periods were 4/20- and 4/44-h . In vitro unscheduled DNA synthesis (UDS) assays were conducted in primary rat hepatocytes at concentrations between 0.15 and 50 micrograms/ml for AHTN and HHCB . In vivo mouse micronucleus assays were conducted with high doses of 1600 mg AHTN/kg and of 1500 mg HHCB/kg in corn oil . No positive responses were observed in any of the tests with HHCB . With AHTN, no positive responses were observed except for cells with structural aberrations in the in vitro cytogenetics assay in CHO cells with S9 activation at the treatment/harvest time of 4/20 h . In initial studies with AHTN, the high dose of 7.8 micrograms/ml showed 0.5% aberrant cells, with the mitotic index at 41% relative to vehicle control and cell growth inhibition in the range of 25-50% . Thus the genotoxicity findings with AHTN were limited to this one positive response; all other genotoxicity tests with AHTN were considered as negative . In particular, the negative finding in the in vivo assay supports AHTN as not likely to be mutagenic in mammalian systems . These considerations, along with other negative published data, lead to the conclusion that both AHTN and HHCB do not have significant potential to act as genotoxic carcinogens. Mutat Res, 1999 Oct 29, 446(1), 35 - 48 Cytotoxic and genotoxic effect of inhibitor of vulcanisation N-cyclohexylthiophthalimide in a battery of in vitro assays; Slamenova D et al.; Mutagenicity of N-cyclohexylthiophthalimide (Duslin P) was tested first by the Ames test in the bacteria, Salmonella typhimurium . The negative results of the Ames test suggested that this compound does not induce mutations in the genome of S . typhimurium under the conditions used . To estimate the cytotoxicity of Duslin P to human cells, we measured cellular DNA and protein as well as cell proliferation, i.e., the mitotic index of treated and control cells . The genotoxic effects were assayed by two biochemical methods developed for detection of single-strand breaks of DNA in mammalian cells, i.e., by the alkaline single cell gel electrophoresis (comet assay) and by the DNA unwinding method, respectively . The DNA unwinding method showed that this compound did not induce DNA damage at concentrations < 7 micrograms/ml . Alkaline single cell gel electrophoresis revealed approximately double the level of DNA damage (in comparison to untreated control DNA) at a concentration of 2 micrograms/ml, which reduced proliferation to approximately 30%, and triple the level of DNA damage at higher concentrations (6 and 7 micrograms/ml), which inhibited completely both DNA synthesis and proteosynthesis . Cells with moderately damaged DNA were more common than cells with heavily damaged DNA . Parallel experiments with the strong mutagen and carcinogen MNNG showed that MNNG induced in cells a high level of DNA damage at concentrations which did not reduce the mitotic index or proteosynthesis, while DNA synthesis inhibited only partially . After treatment with MNNG, cells with heavily damaged DNA were more common than cells with moderately damaged DNA . Duslin P-treated VH10 cells were also tested cytogenetically, confirming that Duslin P induced neither chromosomal aberrations nor aneuploidy . We conclude that Duslin P has no mutagenic effect on bacteria, does not induce chromosomal aberrations and CREST positive or CREST negative micronuclei in human cells and induces only a small increase of DNA damage in human cells which is consistent with DNA fragmentation due to cell death. Microbes Infect, 1999 Jul, 1(8), 615 - 9 Genomic clues for defining bacterial pathogenicity; Salama NR et al.; Genomic sequences are becoming available from both pathogenic and nonpathogenic bacteria . Here we analyze an increasing body of information available on the molecular mechanisms Salmonella typhimurium uses to cause disease, in order to divine clues for identifying sequences that play a role in pathogenesis in other bacterial pathogens. Microbes Infect, 1999 Jul, 1(8), 581 - 7 Defective priming of the phagocyte oxidative burst in a child with recurrent intracellular infections; Elbim C et al.; Human phagocytes (polymorphonuclear neutrophils and monocytes) play a critical role in host defense against invading microorganisms . Recent studies reported that circulating phagocytes undergo a final maturation process, in particular in terms of oxidative burst, during extravasation and migration to local sites of inflammation . This process is known as priming . We report here on a nine-year-old boy with successive disseminated infections due to intracellular microorganisms (Mycobacterium bovis, BCG, and Salmonella typhimurium) . No T- or B-cell quantitative or qualitative defects were found . Polymorphonuclear neutrophil (PMN) migration and NADPH oxidase in PMNs and monocytes stimulated with various agents at optimal concentrations were normal, ruling out a leukocyte adhesion deficiency syndrome, a Chediak Higashi syndrome, and a chronic granulomatous disease . Nevertheless, the patient's PMNs and monocytes showed defective priming capacity, as measured by H(2)O(2) production after pretreatment with LPS (5 microg/mL for 30 min), TNFalpha (100 units/mL for 30 min), or IL-8 (50 ng/mL for 30 min) in response to bacterial N-formyl peptides (fMLP 10(-6) M for 5 min) . In these conditions, H(2)O(2) production of PMNs and monocytes from the patient did not exceed that of the samples treated with fMLP or LPS alone, while the controls strongly produced H(2)O(2) . Moreover, monocytes from the patient showed an impaired capacity to kill S . typhimurium in vitro . Such an impairment could be related at least in part to the priming deficiency of phagocyte oxidative burst . This case suggests, for the first time, that in vivo priming processes are critical in host defence against intracellular pathogens. Avian Dis, 1999 Oct-Dec, 43(4), 788 - 91 Multiple drug-resistant Salmonella typhimurium DT104 and DT104b isolated in bobwhite quail (Colinus virginianus); Helm JD et al.; Multiple drug-resistant Salmonella typhimurium, definitive type (DT) 104 and DT104b, were isolated in three separate hunting preserve bobwhite quail (Colinus virginianus) outbreaks . The cases involved 4-day-old and 3-wk-old quail with increased mortality of 5%-8.6%, respectively . Postmortem lesions included emaciation, distended abdomens, and dark colon contents, which were gaseous and fluid in consistency . Salmonella typhimurium isolated from the intestines and/or livers was resistant to ampicillin, chloramphenicol, sulfonamides, and tetracycline . The isolate involving the 3-wk-old quail was phage typed as S . typhimurium DT104 . The isolates involving the two cases of 4-day-old quail were phage typed as S . typhimurium DT104b. J Agric Food Chem, 1999 Dec, 47(12), 5239 - 44 Antimutagenic activity of polymethoxyflavonoids from Citrus aurantium; Miyazawa M et al.; The methanol extract from Citrus aurantium showed a suppressive effect on umu gene expression of SOS response in Salmonella typhimurium TA1535/pSK1002 against the mutagen 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (furylfuramide) . The methanol extract from C . aurantium was successively re-extracted with hexane, dichloromethane, butanol, and water . A dichloromethane fraction showed a suppressive effect . The suppressive compounds in the dichloromethane fraction were isolated by SiO(2) column chromatography and identified as tetra-O-methylscutellarein (1), sinensetin (2), and nobiletin (3) by EI-MS and (1)H- and (13)C NMR spectroscopy . These compounds suppressed the furylfuramide-induced SOS response in the umu test . Gene expression was suppressed 67%, 45%, and 25% at a concentration of 0.6 micromol/mL, respectively . The ID(50) value (50% inhibition dose) of compound 1 was 0 . 19 micromol/mL . These compounds were assayed with other mutagens, 3-amino-1,4-dimethyl-5H-pyrido{4,3-b}indole (Trp-P-1), which requires liver metabolizing enzymes, activated Trp-P-1, and UV irradiation . These compounds showed of all mutagen-induced SOS response in the umu test . In addition, compounds 1-3 exhibited antimutagenic activity in the S . typhimurium TA100 Ames test. J Food Prot, 1999 Dec, 62(12), 1470 - 4 Response surface models for effects of temperature and previous growth sodium chloride on growth kinetics of Salmonella typhimurium on cooked chicken breast; Oscar TP; Response surface models were developed and validated for effects of temperature (10 to 40 degrees C) and previous growth NaCl (0.5 to 4.5%) on lag time (lambda) and specific growth rate (mu) of Salmonella Typhimurium on cooked chicken breast . Growth curves for model development (n = 55) and model validation (n = 16) were fit to a two-phase linear growth model to obtain lambda and mu of Salmonella Typhimurium on cooked chicken breast . Response surface models for natural logarithm transformations of lambda and mu as a function of temperature and previous growth NaCl were obtained by regression analysis . Both lambda and mu of Salmonella Typhimurium were affected (P < 0.0001) by temperature but not by previous growth NaCl . Models were validated against data not used in their development . Mean absolute relative error of predictions (model accuracy) was 26.6% for lambda and 15.4% for mu . Median relative error of predictions (model bias) was 0.9% for lambda and 5.2% for mu . Results indicated that the models developed provided reliable predictions of lambda and mu of Salmonella Typhimurium on cooked chicken breast within the matrix of conditions modeled . In addition, results indicated that previous growth NaCl (0.5 to 4.5%) was not a major factor affecting subsequent growth kinetics of Salmonella Typhimurium on cooked chicken breast . Thus, inclusion of previous growth NaCl in predictive models may not significantly improve our ability to predict growth of Salmonella spp . on food subjected to temperature abuse. Infect Immun, 2000 Jan, 68(1), 221 - 6 Development of a DeltaglnA balanced lethal plasmid system for expression of heterologous antigens by attenuated vaccine vector strains of Vibrio cholerae; Ryan ET et al.; We have previously shown that more prominent immune responses are induced to antigens expressed from multicopy plasmids in live attenuated vaccine vector strains of Vibrio cholerae than to antigens expressed from single-copy genes on the V . cholerae chromosome . Here, we report the construction of a DeltaglnA derivative of V . cholerae vaccine strain Peru2 . This mutant strain, Peru2DeltaglnA, is unable to grow on medium that does not contain glutamine; this growth deficiency is complemented by pKEK71-NotI, a plasmid containing a complete copy of the Salmonella typhimurium glnA gene, or by pTIC5, a derivative of pKEK71-NotI containing a 1 . 8-kbp fragment that directs expression of CtxB with a 12-amino-acid epitope of the serine-rich Entamoeba histolytica protein fused to the amino terminus . Strain Peru2DeltaglnA(pTIC5) produced 10-fold more SREHP-12-CtxB in supernatants than did ETR3, a Peru2-derivative strain containing the same fragment inserted on the chromosome . To assess immune responses to antigens expressed by this balanced lethal system in vivo, we inoculated germfree mice on days 0, 14, 28, and 42 with Peru2DeltaglnA, Peru2DeltaglnA(pKEK71-NotI), Peru2(pTIC5), Peru2DeltaglnA(pTIC5), or ETR3 . All V . cholerae strains were recoverable from stool for 8 to 12 days after primary inoculation, including Peru2DeltaglnA; strains containing plasmids continued to harbor pKEK71-NotI or pTIC5 for 8 to 10 days after primary inoculation . Animals were sacrificed on day 56, and serum, stool and biliary samples were analyzed for immune responses . Vibriocidal antibody responses, reflective of in vivo colonization, were equivalent in all groups of animals . However, specific anti-CtxB immune responses in serum (P </= 0.05) and bile (P </= 0 . 001) were significantly higher in animals that received Peru2DeltaglnA(pTIC5) than in those that received ETR3, confirming the advantage of higher-level antigen expression in vivo . The development of this balanced lethal system thus permits construction and maintenance of vaccine and vector strains of V . cholerae that express high levels of immunogenic antigens from plasmid vectors without the need for antibiotic selection pressure. J Biol Chem, 1999 Dec 24, 274(52), 36973 - 9 The CorA Mg(2+) transport protein of Salmonella typhimurium . Mutagenesis of conserved residues in the second membrane domain; Szegedy MA et al.; Salmonella typhimurium CorA is the archetypal member of the largest family of Mg(2+) transporters of the Bacteria and Archaea . It contains three transmembrane segments . There are no conserved charged residues within these segments indicating electrostatic interactions are not used in Mg(2+) transport through CorA . Previous mutagenesis studies of CorA revealed a single face of the third transmembrane segment that is important for Mg(2+) transport . In this study, we mutated hydroxyl-bearing and other conserved residues in the second transmembrane segment to identify residues involved in transport . Residues Ser(260), Thr(270), and Ser(274) appear to be important for transport and are oriented such that they would also line a face of an alpha-helix . In addition, the sequence (276)YGMNF(280), found in virtually all CorA homologues, is critical for CorA function because even conservative mutations are not tolerated at these residues . Finally, mutations of residues in the second transmembrane segment, unlike those in the third transmembrane segment, revealed cooperative behavior for the influx of Mg(2+) . We conclude that the second transmembrane segment forms a major part of the Mg(2+) pore with the third transmembrane segment of CorA. Toxicol Lett, 1999 Nov 22, 110(3), 203 - 7 Studies on the mutagenic activity of hydralazine and dihydralazine in Salmonella typhimurium strains differing in expression of antioxidant genes; Chlopkiewicz B; The mutagenic activity of two antihypertensive drugs, hydralazine and dihydralazine was investigated in oxyR- proficient (TA104) and -deficient (TA4125) Salmonella typhimurium strains showing different ability to induce proteins involved in protection of the cells against oxidative damage . The results of the Ames test demonstrated that dihydralazine, in contrast to hydralazine, was mutagenic for oxyR- strain at concentrations that were nonmutagenic for oxyR+ strain . The scavenger of superoxide anion, superoxide dismutase decreased in both strains the number of revertants induced by dihydralazine but not by hydralazine . The results may suggest that active oxygen species generated by dihydralazine contribute to its mutagenicity. J Agric Food Chem, 1999 May, 47(5), 2163 - 7 Moscatilin from Dendrobium nobile, a naturally occurring bibenzyl compound with potential antimutagenic activity; Miyazawa M et al.; A bibenzyl compound that possesses antimutagenic activity was isolated from the storage stem of Dendrobium nobile . The isolated compound suppressed the expression of the umu gene following the induction of SOS response in Salmonella typhimurium TA1535/pSK1002 that have been treated with various mutagens . The suppressive compound was mainly localized in the n-hexane extract fraction of the processed D . nobile . This n-hexane fraction was further fractionated by silica gel column chromatography, which resulted in the purification and subsequent identification of the suppressive compound . EI-MS and (1)H and (13)C NMR spectroscopy were then used to delineate the structure of the compound that confers the observed antimutagenic activity . Comparison of the obtained spectrum with that found in the literature indicated that moscatilin is the secondary suppressive compound . When using 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (furylfuramide) as the mutagen, moscatilin suppressed 85% of the umu gene expression compared to the controls at <0.73 micromol/mL, with an ID(50) value of 0.41 micromol/mL . Additionally, moscatilin was tested for its ability to suppress the mutagenic activity of other well-known mutagens such as 4-nitroquinoline-1-oxide (4NQO), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), UV irradiation, 3-amino-1,4-dimethyl-5H-pyrido{4,3b}indole (Trp-P-1), benzo{a}pyrene (B{a}P), and aflatoxin B(1) (AFB(1)) . With all of the aforementioned chemicals or treatments, moscatilin showed a dramatic reduction in their mutagenic potential . Interestingly, moscatilin almost completely suppressed (97%) the AFB(1)-induced SOS response at concentrations <0.73 micromol/mL, with an ID(50) of 0.08 micromol/mL . Finally, the antimutagenic activities of moscatilin against furylfuramide and Trp-P-1 were assayed by the Ames test using the S . typhimurium TA100 strain . The results those experiments indicated that moscatilin demonstrated a dramatic suppression of the mutagenicity of only Trp-P-1 but not furylfuramide. Microbes Infect, 1999 Feb, 1(2), 113 - 21 Introduction of protein or DNA delivered via recombinant Salmonella typhimurium into the major histocompatibility complex class I presentation pathway of macrophages; Catic A et al.; Recombinant (r) Salmonella typhimurium aroA strains which display the hen egg ovalbumin OVA(257-264) peptide SIINFEKL in secreted form were constructed . In addition, attenuated rS . typhimurium pcDNA-OVA constructs harbouring a eukaryotic expression plasmid encoding complete OVA were used to introduce the immunodominant OVA(257-264) epitope into the major histocompatibility complex (MHC) class I presentation pathway . Both modes of antigen delivery (DNA and protein) by Salmonella vaccine carriers stimulated OVA(257-264)-specific CD8 T-cell hybridomas . An in vitro infection system was established that allowed both rSalmonella carrier devices to facilitate MHC class I delivery of OVA(257-264) by coexpression of listeriolysin (Hly) or by coinfection with rS . typhimurium Hlys (Hess J., Gentschev I., Miko D., Welzel M., Ladel C., Goebel W., Kaufmann S.H.E., Proc . Natl . Acad . Sci . USA 93 (1996) 1458-1463) . Coexpression of Hly and coinfection with rS . typhimurium Hlys slightly improved MHC class I processing of OVA . Our data provide further evidence for the feasibility of attenuated, Hly-expressing rS . typhimurium carriers secreting heterologous antigens or harbouring heterologous DNA as effective vaccines for stimulating CD8 T cells in addition to CD4 T cells. Mol Microbiol, 1999 Oct, 34(1), 112 - 23 Effects of DksA and ClpP protease on sigma S production and virulence in Salmonella typhimurium; Webb C et al.; Salmonella typhimurium responds to a variety of environmental stresses by accumulating the alternative sigma factor sigmaS . The repertoire of sigmaS -dependent genes that are subsequently expressed confers tolerance to a variety of potentially lethal conditions including low pH and stationary phase . The mechanism(s) responsible for triggering sigmaS accumulation are of considerable interest, because they help to ensure survival of the organism during encounters with suboptimal environments . Two genes associated with regulating sigmaS levels in S . typhimurium have been identified . The first is clpP, encoding the protease known to be responsible for degrading sigmaS in Escherichia coli . The second is dksA, encoding a protein of unknown function not previously associated with regulating sigmaS levels . As predicted, clpP mutants accumulated large amounts of sigmaS even in log phase . However, dksA mutants failed to accumulate sigmaS in stationary phase and exhibited lower accumulation during acid shock in log phase . DksA appears to be required for the optimal translation of rpoS based upon dksA mutant effects on rpoS transcriptional and translational lacZ fusions . The region of rpoS mRNA between codons 8 and 73 is required to see the effects of dksA mutations . This distinguishes the role of DksA from that of HF-I (hfq ) in rpoS translation, as the HF-I target area occurs well upstream of the rpoS start codon . DksA appears to be involved in the expression of several genes in addition to rpoS based on two-dimensional SDS-PAGE analysis of whole-cell proteins . As a result of their effects on gene expression, mutations in clpP and dksA decreased the virulence of S . typhimurium in mice, consistent with a role for sigmaS in pathogenesis. J Biol Chem, 1999 Dec 17, 274(51), 36439 - 45 Structure and function of the tryptophan synthase alpha(2)beta(2) complex . Roles of beta subunit histidine 86; Ro HS et al.; To probe the structural and functional roles of active-site residues in the tryptophan synthase alpha(2)beta(2) complex from Salmonella typhimurium, we have determined the effects of mutation of His(86) in the beta subunit . His(86) is located adjacent to beta subunit Lys(87), which forms an internal aldimine with the pyridoxal phosphate and catalyzes the abstraction of the alpha-proton of L-serine . The replacement of His(86) by leucine (H86L) weakened pyridoxal phosphate binding approximately 20-fold and abolished the circular dichroism signals of the bound coenzyme and of a reaction intermediate . Correlation of these results with previous crystal structures indicates that beta-His(86) plays a structural role in binding pyridoxal phosphate and in stabilizing the correct orientation of pyridoxal phosphate in the active site of the beta subunit . The H86L mutation also altered the pH profiles of absorbance and fluorescence signals and shifted the pH optimum for the synthesis of L-tryptophan from pH 7.5 to 8.8 . We propose that the interaction of His(86) with the phosphate of pyridoxal phosphate and with Lys(87) lowers the pK(a) of Lys(87) in the wild-type alpha(2)beta(2) complex and thereby facilitates catalysis by Lys(87) in the physiological pH range. Mol Microbiol, 1999 Oct, 34(1), 181 - 94 Analysis of Shigella flexneri wzz (Rol) function by mutagenesis and cross-linking: wzz is able to oligomerize; Daniels C et al.; The modal length or degree of polymerization (dp) of the Shigella flexneri O-antigen is determined in an unknown manner by the Wzz/Rol protein . The Wzz protein is anchored into the cytoplasmic membrane by two transmembrane domains (TM1 amino acids 32-52; TM2 amino acids 295-315) with the central loop of the protein located in the periplasm . Plasmids were constructed encoding hybrid Wzz proteins consisting of regions of S . flexneri Wzz (WzzSF) and Salmonella typhimurium Wzz (WzzST) . These imparted O-antigen modal chain lengths that implied that the carboxy-terminal region of Wzz was involved in chain length determination . Site-directed mutagenesis was undertaken to investigate the functional significance of highly conserved residues in amino-/carboxy-terminal domains of WzzSF . Some of the WzzSF variants resulted in O-antigen modal chain lengths much shorter than those of wild-type WzzSF, whereas other mutants inactivated WzzSF function entirely and a third class had a longer O-antigen chain length distribution . The data indicate that amino acids throughout the length of the WzzSF protein are important in determination of O-antigen modal chain length . In vivo cross-linking experiments were performed to investigate the interactions between Wzz proteins . The experiments indicated that the WzzSF protein is able to form dimers and oligomers of at least six WzzSF proteins . A carboxy-terminal-truncated WzzSF protein having the amino terminal 194 amino acids was able to oligomerize, indicating that the amino-terminal region is sufficient for the Wzz-Wzz interaction observed . Shortened WzzSF proteins having internal deletions in the amino-terminal region were also able to oligomerize, suggesting that residues 59-194 are not essential for oligomerization . Cross-linking of WzzSF proteins with mutationally altered residues showed that loss of WzzSF function may be correlated to a reduced/altered ability to form oligomers, and that mutational alteration of glycine residues in the TM2 segment affects WzzSF-WzzSF dimer mobility in SDS polyacrylamide gels . These results provide the first evidence of protein-protein interactions for proteins involved in O-antigen polysaccharide biosynthesis. Mol Microbiol, 1999 Oct, 34(1), 134 - 45 A role for the leucine-responsive regulatory protein and integration host factor in the regulation of the Salmonella plasmid virulence (spv ) locus in Salmonella typhimurium; Marshall DG et al.; The Salmonella plasmid virulence (spv ) genes of Salmonella typhimurium are activated at the level of transcription as the bacteria enter stationary phase in vitro or in response to signals received during intracellular growth . Activation requires the LysR-like transcription factor SpvR and the alternative sigma factor RpoS . In this report, we show by biochemical and genetic analyses that two chromosomally encoded DNA-binding proteins contribute to the control of spv expression . These are the integration host factor (IHF), which binds to DNA sequences upstream of the spvR regulatory gene, and the leucine-responsive regulatory protein (Lrp), which binds to sequences upstream of the spvABCD operon . Under all conditions tested, inactivation of IHF expression reduces the level of spvR transcription by twofold . It also alters the response of the spv regulon to loss of DNA gyrase activity, consistent with a role for IHF in organizing DNA structure in the vicinity of the spvR promoter . Lrp represses spvA gene expression by up to fivefold and Lrp-mediated repression is antagonized by leucine . The Lrp binding site upstream of the spvA gene overlaps one of the binding sites for the positive regulator SpvR, suggesting a mechanism by which Lrp repression is exerted . This is a first demonstration of a role for Lrp in controlling genes that are also subject to intracellular regulation . These data show that the spv virulence genes belong simultaneously to several regulons in the cell, raising the possibility that spv expression can be fine-tuned in response to multiple environmental inputs. Microb Pathog, 1999 Dec, 27(6), 369 - 76 The Legionella pneumophila prp locus; required during infection of macrophages and amoebae; Stone BJ et al.; Transposon mutagenesis was performed using mTn 10phoA to identify Legionella pneumophila genes that are expressed under certain in vitro conditions, and are required for intracellular replication . Of the 1653 PhoA fusions examined, 19 PhoA(+)fusion mutants were isolated and screened for differential expression of fusion proteins after growth at 30 or 37 degrees C, in the presence of low iron, or increased magnesium concentrations . The mutants were examined for their cytopathogenicity and intracellular replication within U937 macrophage-like cells and the protozoan Hartmannella vermiformis . One of the mutants generated, BS10, was defective in its multiplication within U937 macrophage-like cells and H . vermiformis . The defect in BS10 was complemented with a cosmid clone containing the wild type locus . The open reading frame interrupted by the insertion was homologous to prpD of Salmonella typhimurium and mmgE of Bacillus subtilis . Genes Genet Syst, 1999 Jun, 74(3), 105 - 11 Structure and transcriptional control of the flagellar master operon of Salmonella typhimurium; Yanagihara S et al.; The flhD and flhC genes constitute the flagellar master operon whose products are required for expression of all the remaining flagellar operons in Salmonella typhimurium . Here we report the molecular structure and in vivo and in vitro expression of the flhD operon . Nucleotide sequence analysis revealed that the upstream region of this operon contains the consensus sequence for the cAMP-CRP binding site . Primer extension analysis demonstrated six possible transcription start sites for this operon . They include CRP-dependent and CRP-repressible transcription start sites . The CRP-dependent transcription start site is located 203 bp upstream of the initiation codon of the flhD gene and preceded by the consensus sequences of the -10 and -35 regions of the sigma 70-dependent promoter . The putative cAMP-CRP binding site is located centered 70 bp upstream of this start site . The CRP-repressible transcription start site is located within this putative cAMP-CRP binding site . These two start sites were confirmed by in vitro transcription experiments using sigma 70-RNA polymerase with or without cAMP-CRP. Microbiology, 1999 Nov, 145 ( Pt 11), 3035 - 45 The rpoS-dependent starvation-stress response locus stiA encodes a nitrate reductase (narZYWV) required for carbon-starvation-inducible thermotolerance and acid tolerance in Salmonella typhimurium; Spector MP et al.; The starvation-stress response (SSR) of Salmonella typhimurium includes gene products necessary for starvation avoidance, starvation survival and virulence for this bacterium . Numerous genetic loci induced during carbon-source starvation and required for the long-term-starvation survival of this bacterium have been identified . The SSR not only protects the cell against the adverse effects of long-term starvation but also provides cross-resistance to other environmental stresses, e.g . thermal challenge (55 degrees C) or acid-pH challenge (pH 2.8) . One carbon-starvation-inducible lac fusion, designated stiA was previously reported to be a sigma(S)-dependent SSR locus that is phosphate-starvation, nitrogen-starvation and H2O2 inducible, positively regulated by (p)ppGpp in a relA-dependent manner, and negatively regulated by cAMP:cAMP receptor protein complex and OxyR . We have discovered through sequence analysis and subsequent biochemical analysis that the stiA::lac fusion, and a similarly regulated lac fusion designated sti-99, lie at separate sites within the first gene (narZ) of an operon encoding a cryptic nitrate reductase (narZYWV) of unknown physiological function . In this study, it was demonstrated that narZ was negatively regulated by the global regulator Fnr during anaerobiosis . Interestingly, narZ(YWV) was required for carbon-starvation-inducible thermotolerance and acid tolerance . In addition, narZ expression was induced approximately 20-fold intracellularly in Madin-Darby canine kidney epithelial cells and 16-fold in intracellular salts medium, which is believed to mimic the intracellular milieu . Also, a narZ1 knock-out mutation increased the LD50 approximately 10-fold for S . typhimurium SL1344 delivered orally in the mouse virulence model . Thus, the previously believed cryptic and constitutive narZYWV operon is in fact highly regulated by a complex network of environmental-stress signals and global regulatory functions, indicating a central role in the physiology of starved and stressed cells. Folia Microbiol (Praha), 1999, 44(2), 177 - 80 The influence of the growth phase of enteric bacteria on electrotransformation with plasmid DNA; Szostkova M et al.; Salmonella typhimurium LB5000 and Escherichia coli JM109 were transformed by electroporation . In accordance with the chemical transformation methods, the growth phase of these electrocompetent bacteria had a strong impact on transformation efficiency . Survival of bacteria after the high-voltage electrical pulse was also influenced by the growth phase . Both bacterial species were most successfully electrotransformed when microbial cells were harvested at the late lag phase . The second optimum for transformation reached E . coli cells in the mid-exponential and S . typhimurium cells in the late exponential phase . Transformation efficiencies ranged from 3.4 x 10(4) to 2.7 x 10(5) transformants per microgram DNA in the case of S . typhimurium and from 2.8 x 10(2) to 8.8 x 10(5) transformants per microgram DNA in the case of E . coli . Survival of cells after the electrical pulse in late lag and late exponential phases was about 20% higher than during other phases of growth . Preparing electrocompetent cells from later phases of their growth is more useful for practice, because it provides more biomass with good yield of transformants. Biochemistry, 1999 Dec 7, 38(49), 16125 - 35 The three-dimensional structures of nicotinate mononucleotide:5,6- dimethylbenzimidazole phosphoribosyltransferase (CobT) from Salmonella typhimurium complexed with 5,6-dimethybenzimidazole and its reaction products determined to 1.9 A resolution; Cheong CG et al.; Nicotinate mononucleotide:5,6-dimethylbenzimidazole phosphoribosyltransferase (CobT) from Salmonella typhimurium plays a central role in the synthesis of alpha-ribazole, which is a key component of the lower ligand of cobalamin . Two X-ray structures of CobT are reported here at 1.9 A resolution . First, a complex of CobT with 5,6-dimethylbenzimidazole, and second, a complex of CobT with its reaction products, nicotinate and alpha-ribazole-5'-phosphate . CobT was cocrystallized with 5,6-dimethylbenzimidazole (DMB) in the space group P2(1)2(1)2 with unit cell dimensions of a = 72.1 A, b = 90.2 A, and c = 47.5 A and one protomer per asymmetric unit . Subsequently, the crystals containing DMB were soaked in nicotinate mononucleotide whereupon the physiological reaction occurred in the crystal lattice to yield nicotinate and alpha-ribazole-5'-phosphate . These studies show that CobT is a dimer where each subunit consists of two domains . The large domain is dominated by a parallel six-stranded beta-sheet with connecting alpha-helices that exhibit the topology of a Rossmann fold . The small domain is made from components of the N- and C-terminal sections of the polypeptide chain and contains a three-helix bundle . The fold of CobT is unrelated to the type I and II phosphoribosylpyrophosphate dependent transferases and does not appear to be related to any other protein whose structure is known . The enzyme active site is located in a large cavity formed by the loops at the C-terminal ends of the beta-strands and the small domain of the neighboring subunit . DMB binds in a hydrophobic pocket created in part by the neighboring small domain . This is consistent with the broad specificity of this enzyme for aromatic substrates {Trzebiatowski, J . R., Escalante-Semerena (1997) J . Biol . Chem . 272, 17662-17667} . The binding site for DMB suggests that Glu317 is the catalytic base required for the reaction . The remainder of the cavity binds the nicotinate and ribose-5'-phosphate moieties, which are nestled within the loops at the ends of the beta-strands . Interestingly, the orientation of the substrate and products are opposite from that expected for a Rossmann fold. J Immunol, 1999 Dec 15, 163(12), 6769 - 76 Critical role of CD28 in protective immunity against Salmonella typhimurium; Mittrucker HW et al.; Efficient T cell activation requires both TCR signals and costimulatory signals . CD28 is one of the molecules that provide costimulatory signals for T cells . We used mice deficient in CD28 expression (CD28-/- mice) to analyze the role of CD28 in the immune response against the intracellular bacterium Salmonella typhimurium, the causative agent of murine typhoid fever . CD28-/- mice were highly susceptible to infection with wild-type S . typhimurium and even failed to control infection with attenuated aroA- S . typhimurium . More detailed analysis revealed that CD28-/- animals did not mount a T-dependent Ab response and were highly impaired in the production of IFN-gamma . Thus, CD28 cosignaling is crucial for immunity against S . typhimurium . To our knowledge, this is the first report describing an essential role for CD28 in protective immunity against an intracellular microbial pathogen. Microbios, 1999, 100(396), 109 - 16 Effect of the raw extract of Arthrinium strains (Hyphomycetes, Dematiaceae) on the growth of pathogenic bacteria in poultry feed; Aissaoui H et al.; Poultry feed contains a significant reservoir of bacteria and is a possible source of Salmonella typhimurium, Staphylococcus aureus and Escherichia coli, which can potentially infect farm animals and humans . The objective of this study was to determine whether the extract obtained from the culture of some Arthrinium species was able to inhibit the growth of these bacteria . The results obtained showed that the raw extracts of Arthrinium aureum, Arthrinium serenensis and Arthrinium phaeospermum inhibited the growth of Escherichia coli in poultry feed . In some cases the percentage inhibition of the growth of Escherichia coli was > 80% . With the raw extract of Arthrinium in poultry feed, the rate of growth of S . typhimurium fell to between 50% and 80% . The raw extract of A . serenensis had the lowest inhibitory activity . S . aureus counts were not affected by any Arthrinium raw extracts. EMBO J, 1999 Dec 1, 18(23), 6800 - 8 Cysteine-scanning mutagenesis provides no evidence for the extracellular accessibility of the nucleotide-binding domains of the multidrug resistance transporter P-glycoprotein; Blott EJ et al.; Multidrug resistance of cancer cells is, at least in part, conferred by overexpression of P-glycoprotein (P-gp), a member of the ATP-binding cassette (ABC) superfamily of active transporters . P-gp actively extrudes chemotherapeutic drugs from cells, thus reducing their efficacy . As a typical ABC transporter, P-gp has four domains: two transmembrane domains, which form a pathway through the membrane through which substrates are transported, and two hydrophilic nucleotide-binding domains (NBDs), located on the cytoplasmic side of the membrane, which couple the energy of ATP hydrolysis to substrate translocation . It has been proposed that the NBDs of ABC transporters, including the histidine permease of Salmonella typhimurium and the cystic fibrosis transmembrane conductance regulator, are accessible from the extracellular surface of the cell, spanning the membrane directly or potentially contributing to the transmembrane pore . Such organization would have significant implications for the transport mechanism . We determined to establish whether the NBDs of P-gp are exposed extracellularly and which amino acids are accessible, using cysteine-scanning mutagenesis and limited proteolysis . In contrast to other transporters, the data provided no evidence that the P-gp NBDs are exposed to the cell surface . The implications for the structure and mechanism of P-gp and other ABC transporters are discussed. Mol Cell Biol Res Commun, 1999 Jul, 2(1), 42 - 6 Direct visualization of gram-negative bacterial lipopolysaccharide self-assembly; Aurell CA et al.; Bacterial lipopolysaccharides (LPS) are outer cell wall components of gram-negative bacteria that may cause septic shock in mammals . The exact morphology of LPS when interacting with macromolecular complexes of the septic shock pathway in blood is still uncertain . Here, the geometry and morphology of hydrated bacterial LPS, dispersed in solution, at and below its the critical aggregate concentration, were directly examined by tapping mode atomic force microscopy (TMAFM) and dynamic light scattering . High-resolution phase-shift TMAFM images of hydrated LPS of Salmonella minnesota Re595, Salmonella typhimurium, and Escherichia coli 0111:B4 adsorbed on mica surfaces unveiled nanosized lipidic particles with a species-specific organization . The complex hydrodynamic geometry exhibited by LPS in dilute suspensions may have consequences for the interpretation of LPS biological activity in the host immune response. J Bacteriol, 1999 Dec, 181(23), 7285 - 90 A periplasmic location is essential for the role of the ApbE lipoprotein in thiamine synthesis in Salmonella typhimurium; Beck BJ et al.; ApbE is a lipoprotein in Salmonella typhimurium, and mutants unable to make this protein have a reduced ability to make thiamine (vitamin B(1)) and require it as a supplement for optimal growth in minimal glucose medium . Polyclonal antibodies specific to ApbE were used to determine that wild-type ApbE is located exclusively in the inner membrane . The periplasmic, monotopic topology of ApbE was determined by using computer-based hydrophobicity plots, LacZ and PhoA gene fusions, and proteinase protection experiments . This extracellular location of ApbE is required for its function, since a cytoplasmic form (ApbE(cyto)) did not allow an apbE mutant to grow in the absence of thiamine . A periplasmic form of ApbE (ApbE(peri)) lacking the lipoprotein modification allowed an apbE mutant to grow in the absence of thiamine, indicating that soluble ApbE could function in thiamine synthesis and that lipoation and membrane association were not required . Alteration of the amino acid implicated in membrane sorting for other lipoproteins did not result in a relocalization of ApbE to the outer membrane, suggesting that additional sorting determinants exist for ApbE. J Bacteriol, 1999 Dec, 181(23), 7274 - 84 General nitrogen regulation of nitrate assimilation regulatory gene nasR expression in Klebsiella oxytoca M5al; Wu SQ et al.; Klebsiella oxytoca can assimilate nitrate and nitrite by using enzymes encoded by the nasFEDCBA operon . Expression of the nasF operon is controlled by general nitrogen regulation (Ntr) via the NtrC transcription activator and by pathway-specific nitrate and nitrite induction via the NasR transcription antiterminator . This paper reports our analysis of nasR gene expression . We constructed strains bearing single-copy Phi(nasR-lacZ) operon fusions within the chromosomal rhaBAD-rhaSR locus . The expression of DeltarhaBS::{Phi(nasR-lacZ)} operon fusions was induced about 10-fold during nitrogen-limited growth . Induction was reduced in both ntrC and rpoN null mutants, indicating that Ntr control of nasR gene expression requires the NtrC and sigma(N) (sigma(54)) proteins . Sequence inspection of the nasR control region reveals an apparent sigma(N)-dependent promoter but no apparent NtrC protein binding sites . Analysis of site-specific mutations coupled with primer extension analysis authenticated the sigma(N)-dependent nasR promoter . Fusion constructs with only about 70 nucleotides (nt) upstream of the transcription initiation site exhibited patterns of beta-galactosidase expression indistinguishable from Phi(nasR-lacZ) constructs with about 470 nt upstream . Expression was independent of the Nac protein, implying that NtrC is a direct activator of nasR transcription . Together, these results indicate that nasR gene expression does not require specific upstream NtrC-binding sequences, as previously noted for argT gene expression in Salmonella typhimurium (G . Schmitz, K . Nikaido, and G . F.-L . Ames, Mol . Gen . Genet . 215:107-117, 1988). J Bacteriol, 1999 Dec, 181(23), 7256 - 65 Identification of the miaB gene, involved in methylthiolation of isopentenylated A37 derivatives in the tRNA of Salmonella typhimurium and Escherichia coli; Esberg B et al.; The tRNA of the miaB2508::Tn10dCm mutant of Salmonella typhimurium is deficient in the methylthio group of the modified nucleoside N(6)-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms(2)io(6)A37) . By sequencing, we found that the Tn10dCm of this strain had been inserted into the f474 (yleA) open reading frame, which is located close to the nag locus in both S . typhimurium and Escherichia coli . By complementation of the miaB2508::Tn10dCm mutation with a minimal subcloned f474 fragment, we showed that f474 could be identified as the miaB gene, which is transcribed in the counterclockwise direction on the bacterial chromosome . Transcriptional studies revealed two promoters upstream of miaB in E . coli and S . typhimurium . A Rho-independent terminator was identified downstream of the miaB gene, at which the majority (96%) of the miaB transcripts terminate in E . coli, showing that the miaB gene is part of a monocistronic operon . A highly conserved motif with three cysteine residues was present in MiaB . This motif resembles iron-binding sites in other proteins . Only a weak similarity to an AdoMet-binding site was found, favoring the idea that the MiaB protein is involved in the thiolation step and not in the methylating reaction of ms(2)i(o)(6)A37 formation. Gene, 1999 Sep 30, 238(1), 231 - 9 Lack of hotspot targets: a constraint for IS30 transposition in Salmonella; Casadesus J et al.; IS30 is an insertion element common in E . coli strains but rare or absent in Salmonella . Transfer of the IS30-flanked transposon Tn2700 to Salmonella typhimurium was assayed using standard delivery procedures of bacterial genetics (conjugation and transduction) . Tn2700 'hops' were rare and required transposase overproduction, suggesting the existence of host constraints for IS30 activity . Sequencing of three Tn2700 insertions in the genome of S . typhimurium revealed that the transposon had been inserted into sites with a low homology to the IS30 consensus target, suggesting that inefficient Tn2700 transposition to the Salmonella genome might be caused by a lack of hotspot targets . This view was confirmed by the introduction of an IS30 'hot target sequence', whose sole presence permitted Tn2700 transposition without transposase overproduction . Detection of IS30-induced DNA rearrangements in S . typhimurium provided further evidence that the element undergoes similar activities in E . coli and S . typhimurium . Thus, hotspot absence may be the main (if not the only) limitation for IS30 activity in the latter species . If these observations faithfully reproduce the scenario of natural populations, establishment of IS30 in the Salmonella genome may have been prevented by a lack of DNA sequences closely related to the unusually long (24 bp) IS30 consensus target. Mutagenesis, 1999 Nov, 14(6), 587 - 94 Novel nitrated derivatives of 5,8-diazabenzo{c}phenanthrene and 9,14-diazadibenz{a,e}acephenanthrylene: new classes of potent mutagenic compounds; Upton M et al.; We report the synthesis of 4-nitro-5,8-diazabenzo{c}phenanthrene (4-NDBP) and 11-nitro-9,14-diazadibenz{a,e}acephenanthrylene (11-NDDA) and the remarkable mutagenic activity of the latter . These two compounds and their non-nitrated parents, 5, 8-diazabenzo{c}phenanthrene (DBP) and 9,14-diazadibenz{a, e}acephenanthrylene (DDA), were screened in Ames plate incorporation assays against Escherichia coli WP2uvrA and Salmonella typhimurium TA98 both in the presence and absence of S9 liver fraction from Aroclor 1254-induced rats . None of the four compounds were cytotoxic up to the limits of their solubility and none showed mutagenic activity in E.coli WP2uvrA, which suggested that any such activity they may have had was not mediated via a base substitution mechanism . DBP and DDA also displayed a lack of activity in TA98 up to their precipitating doses (560 and 33.5 microg/plate, respectively) . The two nitrated compounds, however, were genotoxic . 4-NDBP was active at a dose of 500 ng/plate, in the absence of S9, producing 80.0 +/- 28.0 prototrophic organisms (equivalent to 44 revertants/nmol) and at 0.5 ng/plate, in the presence of S9, giving 147 +/- 6.6 revertants (equivalent to 81 000/nmol) and allowed the description of this tetracycle as a potent mutagen . Much more striking was the activity of 11-NDDA: in the absence of S9 a dose of 8.0 ng produced 2000 revertants/nmol and, remarkably, in the presence of S9 80 pg produced the equivalent of 643 000 revertants/nmol . This makes the hexacyclic 11-NDDA the most potent mutagen to date, in the Ames procedures described here. Mutagenesis, 1999 Nov, 14(6), 547 - 56 Comet assay application in environmental monitoring: DNA damage in human leukocytes and plant cells in comparison with bacterial and yeast tests; Poli P et al.; Urban airborne particulate is a complex mixture of air pollutants, many of which have not been identified . However, short-term mutagenesis tests together with chemicophysical parameter analysis are able to better assess air quality and genotoxic load . The findings of continuous monitoring (January 1991-August 1998) of urban air genotoxicity of a Po Valley town (Italy) on Salmonella typhimurium and Saccharomyces cerevisiae are reported . During this period, various measures (catalytic devices, unleaded fuels, annual vehicle overhaul, etc.) to improve air-dispersed pollutant control were enforced . However, a continuous presence of genotoxic compounds is shown and more qualitative than quantitative changes are evident . We also demonstrate the ability of the Comet assay to detect DNA-damaging agents in airborne particulate samples . We applied the test to human leukocytes and, with major improvements, to plant cells (Allium cepa roots and epigean tissues of Impatiens balsamina) . The first findings on human leukocytes confirm the sensitivity of this assay, its peculiarity and its applicability in assessing genotoxicity in environmental samples . The capability of plants to show the response of multicellular organisms to environmental pollutants largely counterbalances a probable lowering in sensitivity . Moreover, application of the Comet test to epigean tissues could be useful in estimating the bioavailability of and genotoxic damage by air pollutants, including volatile compounds (ozone, benzene, nitrogen oxides, etc.) to higher plants. Infect Immun, 1999 Dec, 67(12), 6249 - 56 Expression of recombinant enterotoxigenic Escherichia coli colonization factor antigen I by Salmonella typhimurium elicits a biphasic T helper cell response; Pascual DW et al.; Protective immunity to enterotoxigenic Escherichia coli (ETEC) is antibody (Ab) dependent; however, oral immunization with purified ETEC fimbriae fails to elicit protective immunity as a consequence of antigenic alteration by the gastrointestinal (GI) tract . Unless unaltered ETEC fimbriae can reach the inductive lymphoid tissues of the GI tract, immunity to ETEC cannot be induced . To produce immunity, live vectors, such as Salmonella typhimurium, can effectively target passenger antigens to the inductive lymphoid tissues of the GI tract . By convention, oral immunizations with Salmonella vectors induce CD4(+) T helper (Th) cell responses by gamma interferon (IFN-gamma)-dominated pathways both to the vector and passenger antigen, resulting in serum immunoglobulin G2a (IgG2a) and modest mucosal IgA Ab responses . In the present study, mice orally immunized with a Salmonella vector engineered to stably express ETEC colonization factor antigen I (CFA/I) showed initially elevated serum IgG1 and mucosal IgA anti-CFA/I Ab responses . As expected, mice orally immunized with an E . coli-CFA/I construct elicited poor anti-CFA/I Ab responses . In fact, the addition of cholera toxin during oral E . coli-CFA/I immunization failed to greatly enhance mucosal IgA Ab responses . Seven days after immunization with the Salmonella-CFA/I construct, cytokine-specific ELISPOT showed induction of predominant Th2-type responses in both mucosal and systemic immune compartments supporting the early IgG1 and IgA anti-CFA/I Abs . By 4 weeks, the Th cell response became Th1 cell dominant from the earlier Th2-type responses, as evidenced by increased mucosal and systemic IFN-gamma-producing T cells and a concomitant elevation of serum IgG2a Ab responses . This biphasic response offers an alternative strategy for directing Salmonella vector-induced host immunity along a Th2 cell-dependent pathway, allowing for early promotion of mucosal and systemic Abs. Infect Immun, 1999 Dec, 67(12), 6242 - 8 Role of endogenous interleukin-18 in resolving wild-type and attenuated Salmonella typhimurium infections; Dybing JK et al.; The stimulation of gamma interferon (IFN-gamma) has been shown to be essential in resolving infections by intracellular pathogens . As such, several different cytokines including, interleukin-12 (IL-12) and IL-18, can induce IFN-gamma . To resolve Salmonella infections, the stimulation of IL-12 and IFN-gamma are important for mediating its clearance . In this present study, the relevance of IL-18 in protection against oral challenge with Salmonella typhimurium was investigated to determine the role of this IFN-gamma-promoting cytokine . Rabbit anti-murine IL-18 antisera was generated and administered prior to the oral challenge of BALB/c and IL-12p40-deficient knockout (IL-12KO) mice with a wild-type S . typhimurium strain . The median survival time was reduced by 2 days for the anti-IL-18-treated BALB/c mice, while no significant reduction in survival rate for the anti-IL-18-treated IL-12KO mice was observed compared to vehicle-treated mice . To investigate the contribution of IL-18 to resolving Salmonella infections, an attenuated aro-negative mutant (H647) was orally administered to BALB/c mice . This Salmonella infection induced both IL-12 and IFN-gamma in both the Peyer's patches and the spleens . In vehicle-treated mice, Peyer's patch IL-12 peaked by 24 h, while IL-18 levels peaked at 3 days, suggesting sequential support by these cytokines for IFN-gamma . Anti-IL-18 treatment exerted its greatest effect upon the mucosal compartment, limiting early IFN-gamma production . However, anti-IL-18 treatment had little effect upon splenic IFN-gamma levels until late in the response . Infection of IL-12KO mice with H647 strain induced IFN-gamma, but it was not supported by IL-18, although IL-18 levels were reduced by this treatment . These results suggest that IL-18 does contribute to the clearance of S . typhimurium and that endogenously induced IL-18 could not substitute for IL-12. Free Radic Biol Med, 1999 Nov, 27(9-10), 1008 - 18 Implication of reactive oxygen species in the antibacterial activity against Salmonella typhimurium of hepatocyte cell lines; Lajarin F et al.; We recently described the antibacterial activity of a murine hepatocyte cell line stimulated with interferon-gamma (IFN-gamma), interleukin-1 (IL-1), and lipopolysaccharide (LPS) against intracellular Salmonella organisms . Here we show for the first time the existence of basal antibacterial activity in cultured hepatocyte cell lines . Thus treatment of resting and stimulated hepatocytes with catalase or superoxide dismutase increased bacterial number recovered per monolayer, which suggests that the mechanism involved with antibacterial activity of hepatocytes is mediated by reactive oxygen species (ROS) . Also, the capacity of these cell lines to generate intracellular peroxides under resting and stimulated conditions was investigated . This revealed that IL-1 and LPS did not induce any increase in the amount of intracellular peroxides by themselves, but they primed IFN-gamma for maximal induction of peroxides . The intracellular amount of peroxides was highly increased on stimulation with IFN-gamma, IL-1, and LPS, and it was strongly inhibited by catalase . This explains that the mechanism whereby this enzyme inhibits antibacterial activity takes place by decreasing the intracellular pool of peroxides . In turn, experiments performed in the presence of several inhibitors of metabolic pathways involved in ROS generation suggested that cyclo-oxygenase are a source of these species in hepatocyte cell lines . These results attribute a prominent role to the generation of peroxides as effector molecules of antibacterial activity in hepatocyte cell lines . Thus these cells displayed a moderate basal level, which increased on stimulation with proinflammatory cytokines such as IFN-gamma, IL-1, and bacterial products such as LPS . Finally, it has been also shown for the first time that IFN-gamma stimulation induces production of peroxides in human and murine hepatocyte cell lines. Curr Microbiol, 2000 Jan, 40(1), 72 - 7 Induction of the cysteine regulon of Salmonella typhimurium in LB medium affects the response of cysB mutants to mecillinam; Anton DN; Rapid growth of Salmonella typhimurium LT2 in rich LB-broth caused the induction of the cysteine regulon when the culture reached an optical density at 650 nm of 1.5 . Addition of 0.05 mM L-cystine to LB-broth abolished induction, while 0.025 mM L-cystine delayed it for a doubling time (30 min) . Induction did not occur when lack of oxygen or a temperature shift to 19 degrees C slowed down growth in LB-broth . Uninduced cultures reached growth yield similar to that of induced cultures after overnight incubation . Growth on solid LB-medium also brought about induction, but to a lower level than in liquid medium . Leaky cysB mutants, which are sensitive to the beta-lactam antibiotic mecillinam, displayed partial induction, whereas mecillinam-resistant cysB and cysE mutants showed no induction . It is suggested that induction develops in these mutants when constitutive transport systems fail to satisfy the high uptake of cysteine demanded by fast-growing cultures . The behavior of cysB mutants agrees with the hypothesis that, under some conditions, mecillinam action would be dependent on expression of the cysteine regulon. Curr Microbiol, 2000 Jan, 40(1), 67 - 71 Cloning and characterization of the gene encoding O-acetylserine lyase from Streptococcus suis; Osaki M et al.; We have cloned and sequenced a gene encoding O-acetylserine lyase from Streptococcus suis . The gene encodes a protein of 309 amino acids with a calculated molecular mass of 32,038 Da . The deduced amino acid sequence showed more extensive similarities to the CysK proteins than to the CysM proteins of other bacteria . The cloned gene was inserted into a pTrcHisB histidine hexamer expression vector . A 38-kDa fusion protein was expressed in a cysMK auxotrophic mutant of Salmonella typhimurium and complemented the auxotrophic properties of the mutant . Furthermore, the transformants could grow in minimal defined media supplemented with not only sulfide but also thiosulfate as a sole sulfur source . These data indicated that the cloned gene encodes a protein that was a functional homolog of the CysM in S . typhimurium. J Environ Sci Health B, 1999 Nov, 34(6), 1083 - 99 Short-chain fatty acids affect cell-association and invasion of HEp-2 cells by Salmonella typhimurium; Durant JA et al.; This study demonstrates that the growth of S . typhimurium in Luria Bertani broth supplemented with acetate, propionate, butyrate, or a mixture of the three SCFA, affected cell-association and the ability to invade cultured HEp-2 cells . Cell-association and invasion was determined after growth for 4 h of growth in the presence of the SCFA at pH 6 and 7 . The results suggest that the growth rate of the culture may have affected cell-association and invasion since accompanying the significant decrease in growth rate in the presence of SCFA at pH 6 was a decrease in cell-association and invasion . However, the results also suggest that the individual SCFA may play a role in modulating cell-association and the invasion phenotype and the regulation of cell-association and invasion by the SCFA was dependent on the concentration and the pH of the medium . Although the growth rates were similar for S . typhimurium in the SCFA mixture, butyrate (100 mM) and propionate (50 mM) at pH 6, differences in cell-association and invasion were observed among these cultures . Also, at pH 7, differences were observed among the SCFA treatments even though the growth rates were similar. Mikrobiol Z, 1999 Jul-Aug, 61(4), 59 - 63 {The inhibition by interferon type I of the initial phase of experimental salmonellosis}; Fil'chakov IV et al.; Using the bacteriological methods the level of the genus Salmonella representative histadhesion to intestinal mucosa was evaluated . Balb/c mice and Salmonella typhimurium 415 strain were used for investigation . Native homologous I type interferon was injected to experimental animals 24 before the experiment in a dose of 1000 U/mice . "False" interferon was administered to the control group animals . It was established that the level of Salmonella histadhesion in mice of the experimental group was an order less than in control . Study of the parameters of the process of Salmonella interaction with intestinal mucosa showed that the challenge dose of Salmonella typhimurium 24 after interferon injection must be 7 times higher for the control mice than for experimental ones . Thus, the barrier function of intestinal mucosa after administration of the native I type interferon in vivo increased 7 times. Int J Pharm, 1999 Nov 30, 191(2), 141 - 9 A comparative study of Mono Mac 6 cells, isolated mononuclear cells and Limulus amoebocyte lysate assay in pyrogen testing; Moesby L et al.; Pyrogen induced secretion of interleukin 6 (IL-6) in Mono Mac 6 (MM6) cells was measured . The ability of the MM6 cell culture to detect pyrogens was compared to the Limulus amoebocyte lysate (LAL) test and isolated mononuclear cells (MNC) . The detection limit of MM6 for lipopolysaccharide (LPS) and Staphylococcus aureus was comparable to that of MNC . Aspergillus niger and Candida albicans induced IL-6 in isolated MNC, but not in MM6 . The detection limit for Salmonella typhimurium in the MM6 assay was comparable to that of the LAL assay . As expected, S . aureus and C . albicans did not show any LAL activity . A . niger and Influenza virus showed some activity in the LAL test, but could not be detected by MM6 cells . In conclusion, the MM6 assay is a good supplement to the current pyrogen assays for detection of LPS, S . aureus and S . typhimurium, but the MM6 assay could not detect A . niger, C . albicans and Influenza virus. Gene, 1999 Nov 15, 240(1), 99 - 106 A recombinase-based selection of differentially expressed bacterial genes; Altier C et al.; Bacterial genes are often differentially expressed in response to specific environmental conditions . We have devised a method to identify regulated bacterial promoters, such that transient promoter expression leads to a permanent and selectable change in bacterial phenotype . This system consists of a promoterless derivative of cre, the phage P1 recombinase, carried on a plasmid, and two chromosomal loxP sites, the targets of the Cre recombinase . The loxP sites flank npt, conferring kanamycin resistance, and sacB, which confers sensitivity to sucrose, allowing positive selection for both the presence and absence of this chromosomal cassette . Fusion of active promoters to cre induces recombination of the loxP sites and deletion of intervening DNA, allowing selection on media containing sucrose, while inactive promoters fail to induce recombination and so remain resistant to kanamycin . We tested the system in Salmonella typhimurium using a known regulated promoter, that from the araBAD operon, and found it to be a sensitive indicator of gene expression over a wide range of promoter induction . We then used this system to identify S . typhimurium genes that are specifically expressed when bacteria interact with cultured epithelial cells and identified a novel DNA fragment, not found in E . coli, which might represent part of a new pathogenicity island. Mol Microbiol, 1999 Nov, 34(4), 850 - 64 Salmonella typhimurium leucine-rich repeat proteins are targeted to the SPI1 and SPI2 type III secretion systems; Miao EA et al.; Salmonellae encode two virulence-associated type III secretion systems (TTSS) within Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2) . Two Salmonella typhimurium genes, sspH1 and sspH2, that encode proteins similar to the Shigella flexneri and Yersinia species TTSS substrates, IpaH and YopM, were identified . SspH1 and SspH2 are proteins containing leucine-rich repeats that are differentially targeted to the SPI1 and SPI2 TTSS . sspH2 transcription was induced within RAW264.7 macrophages, and was dependent upon the SPI2-encoded regulator ssrA/ssrB . In contrast, sspH1 transcription is independent of SPI2, and is not induced after bacterial phagocytosis by eukaryotic cells . Infection of eukaryotic cells with strains expressing a SspH2-CyaA fusion protein resulted in SPI2 TTSS-dependent cAMP increases . In contrast, SspH1-CyaA-mediated cAMP increases were both SPI1 and SPI2 TTSS dependent . sspH2-like sequences were found in most Salmonella serotypes examined, whereas sspH1 was detected in only one S . typhimurium isolate, indicating that the copy number of sspH genes can be variable within Salmonella serotypes . S . typhimurium deleted for both sspH1 and sspH2 was not able to cause a lethal infection in calves, indicating that these genes participate in S . typhimurium virulence for animals. Mol Microbiol, 1999 Nov, 34(4), 767 - 79 Flagellar proteins and type III-exported virulence factors are the predominant proteins secreted into the culture media of Salmonella typhimurium; Komoriya K et al.; We analysed all major proteins secreted into culture media from Salmonella typhimurium . Proteins in culture supernatants were collected by trichloroacetic acid precipitation, separated in SDS-polyacrylamide gels and analysed by amino acid sequencing . Wild-type strain SJW1103 cells typically gave rise to nine bands in SDS gels: 89, 67, 58, 52, 50, 42, 40, 35 and (sometimes) 28 kDa . A search of the sequences in the available databases revealed that they were either flagellar proteins or virulence factors . Six of them were flagella specific: FlgK or HAP1 (58 kDa), FliC or flagellin (52 kDa), FliD or HAP2 (50 kDa), FlgE or hook protein (42 kDa), FlgL or HAP3 (35 kDa) and FlgD or hook-cap protein (28 kDa) . The other four bands were specific for virulence factors: SipA (89 kDa), SipB (67 kDa), SipC (42 kDa) and InvJ (40 kDa) . The 42 kDa band was a mixture of FlgE and SipC . We also analysed secreted proteins from more than 30 flagellar mutants, and they were categorized into four groups according to their band patterns: wild type, mot type, polyhook type and master gene type . Virulence factors were constantly secreted at a higher level in all flagellar mutants except a deltamot (motAB deletion) mutant, in which the amounts were greatly reduced . A new morphological pathway of flagellar biogenesis including protein secretion is presented. Mol Microbiol, 1999 Oct, 34(2), 305 - 16 PhoP-PhoQ homologues in Pseudomonas aeruginosa regulate expression of the outer-membrane protein OprH and polymyxin B resistance; Macfarlane EL et al.; Rapid adaptation to environmental challenge is essential for the survival of many bacterial species, and is often effectively mediated by two-component regulatory systems . Part of the adaptive response of Pseudomonas aeruginosa to Mg2+ starvation is overexpression of the outer-membrane protein OprH and increased resistance to the polycationic antibiotic polymyxin B . Two overlapping open reading frames that encoded proteins with high similarities to the PhoP-PhoQ two-component regulatory system of Salmonella typhimurium were identified downstream of the oprH gene . A P . aeruginosa PhoP-null mutant, H851, was constructed by means of a phoP:xylE-GmR transcriptional fusion, and shown to be deficient in OprH expression . In contrast, an analogous PhoQ-null mutant, H854 (phoQ:xylE-GmR), exhibited constitutive overexpression of OprH . Normal Mg2+-regulated OprH expression could be restored in both mutants by complementation with a plasmid carrying the phoP and phoQ genes . Measurement of the catechol-2,3-dioxygenase activity, expressed from the xylE transcriptional fusion in strains H851 and H854, indicated that PhoP-PhoQ is involved in the regulation of phoP-phoQ as well as oprH . Reverse transcription polymerase chain reaction experiments and Northern blot analysis revealed linkage of oprH, phoP and phoQ into an operon that was demonstrated to be under the joint control of PhoP-PhoQ and Mg2+ ion concentration . In addition, studies of the polymyxin B resistance of the two mutant strains, H851 and H854, indicated that PhoP-PhoQ is involved in regulating P . aeruginosa polymyxin resistance in response to external Mg2+ concentrations. Biochim Biophys Acta, 1999 Sep 21, 1421(1), 5 - 18 Regulation of flavin dehydrogenase compartmentalization: requirements for PutA-membrane association in Salmonella typhimurium; Surber MW et al.; PutA is a multifunctional, peripheral membrane protein which functions both as an autogenous transcriptional repressor and the enzyme which catalyzes the two-step conversion of proline to glutamate in Salmonella typhimurium and Escherichia coli . To understand how PutA associates with the membrane, we determined the role of FAD redox and membrane components in PutA-membrane association . Reduction of the tightly bound FAD is required for both derepression of the put operon and membrane association of PutA . FADH(2) alters the conformation of PutA, resulting in an increased hydrophobicity . Previous studies used enzymatic activity as an assay for membrane association and concluded that electron transfer from the reduced FAD in PutA to the membrane is required for the PutA-membrane interaction . However, direct physical assays of PutA association with membrane vesicles from quinone deficient mutants demonstrated that although electron transfer is essential for proline dehydrogenase activity, it is not required for PutA-membrane association per se . Furthermore, PutA efficiently associated with liposomes, indicating that PutA-membrane association does not require interactions with other membrane proteins . PutA enzymatic activity can be efficiently reconstituted with liposomes containing ubiquinone and cytochrome bo, confirming that proline dehydrogenase can pass electrons directly to the quinone pool . These results indicate that PutA-membrane association is due strictly to a protein-lipid interaction initiated by reduction of FAD. J Bacteriol, 1999 Nov, 181(22), 6977 - 86 A novel lipolytic enzyme located in the outer membrane of Pseudomonas aeruginosa; Wilhelm S et al.; A lipase-negative deletion mutant of Pseudomonas aeruginosa PAO1 still showed extracellular lipolytic activity toward short-chain p-nitrophenylesters . By screening a genomic DNA library of P . aeruginosa PAO1, an esterase gene, estA, was identified, cloned, and sequenced, revealing an open reading frame of 1,941 bp . The product of estA is a 69.5-kDa protein, which is probably processed by removal of an N-terminal signal peptide to yield a 67-kDa mature protein . A molecular mass of 66 kDa was determined for (35)S-labeled EstA by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography . The amino acid sequence of EstA indicated that the esterase is a member of a novel GDSL family of lipolytic enzymes . The estA gene showed high similarity to an open reading frame of unknown function located in the trpE-trpG region of P . putida and to a gene encoding an outer membrane esterase of Salmonella typhimurium . Amino acid sequence alignments led us to predict that this esterase is an autotransporter protein which possesses a carboxy-terminal beta-barrel domain, allowing the secretion of the amino-terminal passenger domain harboring the catalytic activity . Expression of estA