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Two Arginine Repressors Regulate Arginine Biosynthesis in Lactobacillus plantarum. Hervé Nicoloff, 2004.The repression of the carAB operon encoding carbamoyl phosphate synthase leads to Lactobacillus plantarum FB331 growth inhibition in the presence of arginine . This phenotype was used in a positive screening to select spontaneous mutants deregulated in the arginine biosynthesis pathway . Fourteen mutants were genetically characterized for constitutive arginine production . Mutations were located either in one of the arginine repressor genes [argR1 or argR2] present in L . plantarum or in a putative ARG operator in the intergenic region of the bipolar carAB-argCJBDF operons involvedin arginine biosynthesis . Although the presence of two ArgR regulators is commonly found in gram-positive bacteria, only single arginine repressors have so far been well studied in Escherichia coli or Bacillus subtilis . In L . plantarum, argininerepression was abolished when ArgR1 or ArgR2 was mutated in the DNA binding domain, or in the oligomerization domain or when an A123D mutation occurred in ArgR1 . A123, equivalent tothe conserved residue A124 in E . coli ArgR involved in arginine binding, was different in the wild-type ArgR2 . Thus, corepressor binding sites may be different in ArgR1 and ArgR2, which haveonly 35% identical residues . Other mutants harbored wild-typeargR genes, and 20 mutants have lost their ability to grow innormal air without carbon dioxide enrichment; this revealeda link between arginine biosynthesis and a still-unknown CO2-dependent metabolic pathway . In many gram-positive bacteria, the expressionand interaction of different ArgR-like proteins may imply acomplex regulatory network in response to environmental stimuli. Effect of pH on the In Vitro Activities of Amphotericin B, Itraconazole, and Flucytosine against Aspergillus Isolates. D. T. A. Te Dorsthorst, 2004.The in vitro susceptibilities of 21 Aspergillus isolates were tested against three antifungal agents in RPMI 1640 and yeast nitrogen base at pH 5.0 and 7.0 by a broth microdilution format of the NCCLS method . The MICs of amphotericin B and itraconazole were higher, while those of flucytosine were lower, at pH 5.0 than at pH 7.0 . The poor correlation between in vitro results and clinical outcome could be due to a difference in pH between the in vitro susceptibility test and at the site of infection . Control of Listeria monocytogenes in a Biofilm by Competitive-Exclusion Microorganisms. Tong Zhao, 2004.Biofilms from drains in food processing facilities with a recent history of no detectable Listeria monocytogenes in floor drains were cultured for microorganisms producing antilisterial metabolites . A total of 413 microbial isolates were obtained from 12 drain biofilm samples and were assayed at 15 and 37°C for activities that were bactericidal or inhibitory to L . monocytogenes, by two agar plate assays . Twenty-one of 257 bacterial isolates and 3 of 156 yeast isolates had antilisterial activity . All 24 isolates which produced metabolites inhibitory to L . monocytogenes were assayed for antilisterial activity in coinoculated broth cultures containing tryptic soy broth with yeast extract (TSB-YE) . A five-strain mixture of 103 CFU of L . monocytogenes/ml and 105 CFU of the candidate competitive-exclusion microorganism/ml was combined in TSB-YE and incubated at 37°C for 24 h, 15°C for 14 days, 8°C for 21 days, and 4°C for 28 days . Substantial inhibition of L . monocytogenes growth (4 to 5 log CFU/ml) was observed for nine bacterial isolates at 37°C, two at 15 and 8°C, and three at 4°C . The inhibitory isolates were identified as Enterococcus durans (six isolates), Lactococcus lactis subsp . lactis (two isolates), and Lactobacillus plantarum (one isolate) . The anti-L . monocytogenes activity of these isolates was evaluated in biofilms of L . monocytogenes on stainless steel coupons at 37, 15, 8, and 4°C . Results revealed that two isolates (E . durans strain 152 and L . lactis subsp . lactis strain C-1-92) were highly inhibitory to L . monocytogenes (growth inhibition of >5 log10 CFU of L . monocytogenes/cm2) . These two bacterial isolates appear to be excellent competitive-exclusion candidates to control L . monocytogenes in biofilms at environmental temperatures of 4 to 37°C . Nitric Oxide-Induced Homologous Recombination in Escherichia coli Is Promoted by DNA Glycosylases. Erik J. Spek, 2002.Nitric oxide (NO.) is involved in neurotransmission, inflammation, and many other biological processes . Exposure of cells to NO . leads to DNA damage, including formation of deaminated and oxidized bases . Apurinic/apyrimidinic (AP) endonuclease-deficient cells are sensitive to NO. toxicity, which indicates that base excision repair (BER) intermediates are being generated . Here, we show that AP endonuclease-deficient cells can be protected from NO . toxicity by inactivation of the uracil (Ung) or formamidopyrimidine (Fpg) DNA glycosylases but not by inactivation of a 3-methyladenine (AlkA) DNA glycosylase . These results suggest that Ung and Fpg remove nontoxic NO.-induced base damage to create BER intermediates that are toxic if they are not processed by AP endonucleases . Our next goal was to learn how Ung and Fpg affect susceptibility to homologous recombination . The RecBCD complex is critical for repair of double-strand breaks via homologous recombination . When both Ung and Fpg were inactivated in recBCD cells, survival was significantly enhanced . We infer that both Ung and Fpg create substrates for recombinational repair, which is consistent with the observation that disrupting ung and fpg suppressed NO.-induced recombination . Taken together, a picture emerges in which the action of DNA glycosylases on NO.-induced base damage results in the accumulation of BER intermediates, which in turn can induce homologous recombination . These studies shed light on the underlying mechanism of NO.-induced homologous recombination . Postgenomic Analysis of Four Novel Antigens of Group A Streptococcus: Growth Phase-Dependent Gene Transcription and Human Serologic Response. Sean D. Reid, 2002.Analysis of three group A Streptococcus genomes (serotypes M1, M3, and M18) recently identified four previously undescribed genes that encode extracellular proteins . Each of these genes encode proteins with an LPXTG amino acid motif that covalently links many virulence factors produced by gram-positive bacteria to the cell surface . Western immunoblot analysis of serum samples obtained from 80 patients with invasive infections, noninvasive soft tissue infections, pharyngitis, and rheumatic fever indicated that these four proteins are expressed in vivo . However, the level of gene transcript and the time of maximal gene transcription varied in representative serotype M1, M3, and M18 strains . Surface expression of two proteins was confirmed by flow cytometry . Studies using a mouse infection model suggest that antibodies specific for one of the proteins (Spy0843) may contribute to a protective host immune response against a serotype M1 infection . These results are additional evidence that postgenomic strategies provide new ways to identify and investigate novel bacterial proteins that may participate in host-pathogen interactions or serve as targets for therapeutics research . Mycothiol Is Essential for Growth of Mycobacterium tuberculosis Erdman. Dipti Sareen, 2003.Mycothiol (MSH) is the major low-molecular-mass thiol in mycobacteria and is associated with the protection of Mycobacterium tuberculosis from toxic oxidants and antibiotics . The biosynthesis of MSH is a multistep process, with the enzymatic reaction designated MshC being the ligase step in MSH production . A targeted disruption of the native mshC gene in M . tuberculosis Erdman produced no viable clones possessing either a disrupted mshC gene or reduced levels of MSH . However, when a second copy of the mshC gene was incorporated into the chromosome prior to the targeted disruption, multiple clones having the native gene disrupted and the second copy of mshC intact were obtained . These clones produced normal levels of MSH . These results demonstrate that the mshC gene and, more generally, the production of MSH are essential for the growth of M . tuberculosis Erdman under laboratory conditions . Adaptive Acid Tolerance Response of Streptococcus sobrinus. Marcelle M. Nascimento, 2004.Streptococcus mutans and Streptococcus sobrinus are the bacteria most commonly associated with human dental caries . A major virulence attribute of these and other cariogenic bacteria is acid tolerance . The acid tolerance mechanisms of S . mutans have begun to be investigated in detail, including the adaptive acid tolerance response (ATR), but this is not the case for S . sobrinus . An analysis of the ATR of two S . sobrinus strains was conducted with cells grown to steady state in continuous chemostat cultures . Compared with cells grown at neutral pH, S . sobrinus cells grown at pH 5.0 showed an increased resistance to acid killing and were able to drive down the pH through glycolysis to lower values . Unlike what is found for S . mutans, the enhanced acid tolerance and glycolytic capacities of acid-adapted S . sobrinus were not due to increased F-ATPase activities . Interestingly though, S . sobrinus cells grown at pH 5.0 had twofold more glucose phosphoenolpyruvate:sugar phosphotransferase system (PTS) activity than cells grown at pH 7.0 . In contrast, glucose PTS activity was actually higher in S . mutans grown at pH 7.0 than in cells grown at pH 5.0 . Silver staining of two-dimensional gels of whole-cell lysates of S . sobrinus 6715 revealed that at least 9 proteins were up-regulated and 22 proteins were down-regulated in pH 5.0-grown cells compared with cells grown at pH 7.0 . Our results demonstrate that S . sobrinus is capable of mounting an ATR but that there are critical differences between the mechanisms of acid adaptation used by S . sobrinus and S . mutans .
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