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CovS Inactivates CovR and Is Required for Growth under Conditions of General Stress in Streptococcus pyogenes. Tracy L. Dalton, 2004.The gram-positive human pathogen Streptococcus pyogenes (group A streptococcus [GAS]) causes diseases ranging from mild and often self-limiting infections of the skin or throat to invasive and life-threatening illnesses . To cause such diverse types of disease, the GAS must be able to sense adverse environments and regulate its gene expression accordingly . The CovR/S two-component signal transduction regulatory system in GAS represses about 15% of the GAS genome, including many genes involved in virulence, in response to the environment . We report that CovR is still able to repress transcription from several promoters in the absence of the putative histidine kinase sensor for this system, CovS . We also show that a phosphorylation site mutant (D53A) of CovR is unable to repress gene expression . In addition, we report that a strain with a nonpolar mutation in CovS does not grow at a low pH, elevated temperature, or high osmolarity . The stress-related phenotypes of the CovS mutant were complemented by expression of covS from a plasmid . Selection for growth of a CovS mutant under stress conditions resulted in isolation of second-site mutations that inactivated covR, indicating that CovR and CovS act in the same pathway . Also, at 40°C in the wild-type strain, CovR appeared to be less active on the promoter tested, which is consistent with the hypothesis that it was partially inactivated by CovS . We suggest that under mild stress conditions, CovS inactivates CovR, either directly or indirectly, and that this inactivation relieves repression of many GAS genes, including the genes needed for growth of GAS under stress conditions and some genes that are necessary for virulence . Growth of many gram-positive bacteria under multiple-stress conditions requires alteration of promoter recognition produced by RNA polymerase association with the general stress response sigma factor,  B .
We provide evidence that for GAS, which lacks a sigB ortholog,
growth under stress conditions requires the CovR/S two-component
regulatory system instead . This two-component system in GAS thus
appears to perform a function for which other gram-positive bacteria
utilize an alternative sigma factor .
Persistence of Vancomycin-Resistant Enterococci in New Zealand Broilers after Discontinuation of Avoparcin Use. Janet M. Manson, 2004.Large amounts of tylosin, zinc-bacitracin, and avilamycin are currently used as prophylactics in New Zealand broiler production . Avoparcin was also used from 1977 to 2000 . A total of 382 enterococci were isolated from 213 fecal samples (147 individual poultry farms) using enrichment broths plated on m-Enterococcus agar lacking antimicrobials . These isolates were then examined to determine the prevalence of antimicrobial resistance . Of the 382 isolates, 5.8% (22 isolates) were resistant to vancomycin, and 64.7% were resistant to erythromycin . The bacitracin MIC was  256 µg/ml for 98.7% of isolates, and the avilamycin MIC was
8 µg/ml for 14.9% of isolates . No resistance to ampicillin or gentamicin was detected . Of the 22 vancomycin-resistant enterococci (VRE) isolates, 18 (81.8%) were Enterococcus faecalis, 3 were Enterococcus faecium, and 1 was Enterococcus durans . However, when the 213 fecal enrichment broths were plated on m-Enterococcus agar containing vancomycin, 86 VRE were recovered; 66% of these isolates were E . faecium and the remainder were E . faecalis . Vancomycin-resistant E . faecium isolates were found to have heterogenous pulsed-field gel electrophoresis (PFGE) patterns of SmaI-digested DNA, whereas the PFGE patterns of vancomycin-resistant E . faecalis isolates were identical or closely related, suggesting that this VRE clone is widespread throughout New Zealand . These data demonstrate that vancomycin-resistant E . faecalis persists in the absence and presence of vancomycin-selective pressure, thus explaining the dominance of this VRE clone even in the absence of avoparcin .
pH-Dependent Expression of Periplasmic Proteins and Amino Acid Catabolism in Escherichia coli. Lauren M. Stancik, 2002.Escherichia coli grows over a wide range of pHs (pH 4.4 to 9.2), and its own metabolism shifts the external pH toward either extreme, depending on available nutrients and electron acceptors . Responses to pH values across the growth range were examined through two-dimensional electrophoresis (2-D gels) of the proteome and through lac gene fusions . Strain W3110 was grown to early log phase in complex broth buffered at pH 4.9, 6.0, 8.0, or 9.1 . 2-D gel analysis revealed the pH dependence of 19 proteins not previously known to be pH dependent . At low pH, several acetate-induced proteins were elevated (LuxS, Tpx, and YfiD), whereas acetate-repressed proteins were lowered (Pta, TnaA, DksA, AroK, and MalE) . These responses could be mediated by the reuptake of acetate driven by changes in pH . The amplified proton gradient could also be responsible for the acid induction of the tricarboxylic acid (TCA) enzymes SucB and SucC . In addition to the autoinducer LuxS, low pH induced another potential autoinducer component, the LuxH homolog RibB . pH modulated the expression of several periplasmic and outer membrane proteins: acid induced YcdO and YdiY; base induced OmpA, MalE, and YceI; and either acid or base induced OmpX relative to pH 7 . Two pH-dependent periplasmic proteins were redox modulators: Tpx (acid-induced) and DsbA (base-induced) . The locus alx, induced in extreme base, was identified as ygjT, whose product is a putative membrane-bound redox modulator . The cytoplasmic superoxide stress protein SodB was induced by acid, possibly in response to increased iron solubility . High pH induced amino acid metabolic enzymes (TnaA and CysK) as well as lac fusions to the genes encoding AstD and GabT . These enzymes participate in arginine and glutamate catabolic pathways that channel carbon into acids instead of producing alkaline amines . Overall, these data are consistent with a model in which E . coli modulates multiple transporters and pathways of amino acid consumption so as to minimize the shift of its external pH toward either acidic or alkaline extreme . Development of a Gene Knockout System for the Halophilic Archaeon Haloferax volcanii by Use of the pyrE Gene. Gili Bitan-Banin, 2003.So far, the extremely halophilic archaeon Haloferax volcanii has the best genetic tools among the archaea . However, the lack of an efficient gene knockout system for this organism has hampered further genetic studies . In this paper we describe the development of pyrE-based positive selection and counterselection systems to generate an efficient gene knockout system . The H . volacanii pyrE1 and pyrE2 genes were isolated, and the pyrE2 gene was shown to code for the physiological enzyme orotate phosphoribosyl transferase . A  pyrE2 strain was constructed and used to isolate deletion mutants by the following two steps: (i) integration of a nonreplicative plasmid carrying both the pyrE2 wild-type gene, as a selectable marker, and a cloned chromosomal DNA fragment containing a deletion in the desired gene; and (ii) excision of the integrated plasmid after selection with 5-fluoroorotic acid . Application of this gene knockout system is described .
Evaluation of the Redox Dye 5-Cyano-2,3-Tolyl-Tetrazolium Chloride for Activity Studies by Simultaneous Use of Microautoradiography and Fluorescence In Situ Hybridization. Jeppe Lund Nielsen, 2003.Three microscopic in situ techniques were used simultaneously to investigate viability and activity on a single-cell level in activated sludge . The redox dye 5-cyano-2,3-tolyl-tetrazolium chloride (CTC) was compared with microautoradiography (MAR) and fluorescence in situ hybridization (FISH) to indicate activity of cells in Thiothrix filaments and in single floc-forming bacteria . The signals from MAR and FISH correlated well, whereas only 65% of the active Thiothrix cells and 41% of all single cells were detectable by CTC reduction, which mainly targeted the most active cells . Nutritional Similarity between Leaf-Associated Nonpathogenic Bacteria and the Pathogen Is Not Predictive of Efficacy in Biological Control of Bacterial Spot of Tomato. Alexei C. Dianese, 2003.It has been demonstrated that for a nonpathogenic, leaf-associated bacterium, effectiveness in the control of bacterial speck of tomato is correlated with the similarity in the nutritional needs of the nonpathogenic bacterium and the pathogen Pseudomonas syringae pv . tomato . This relationship was investigated further in this study by using the pathogen Xanthomonas campestris pv . vesicatoria, the causal agent of bacterial spot of tomato, and a collection of nonpathogenic bacteria isolated from tomato foliage . The effects of inoculation of tomato plants with one of 34 nonpathogenic bacteria prior to inoculation with the pathogen X . campestris pv . vesicatoria were quantified by determining (i) the reduction in disease severity (number of lesions per square centimeter) in greenhouse assays and (ii) the reduction in leaf surface pathogen population size (log10 of the number of CFU per leaflet) in growth chamber assays . Nutritional similarity between the nonpathogenic bacteria and X . campestris pv . vesicatoria was quantified by using either niche overlap indices (NOI) or relatedness in cluster analyses based upon in vitro utilization of carbon or nitrogen sources reported to be present in tomato tissues or in Biolog GN plates . In contrast to studies with P . syringae pv . tomato, nutritional similarity between the nonpathogenic bacteria and the pathogen X . campestris pv . vesicatoria was not correlated with reductions in disease severity . Nutritional similarity was also not correlated with reductions in pathogen population size . Further, the percentage of reduction in leaf surface pathogen population size was not correlated with the percentage of reduction in disease severity, suggesting that the epiphytic population size of X . campestris pv . vesicatoria is not related to disease severity and that X . campestris pv . vesicatoria exhibits behavior in the phyllosphere prior to lesion formation that is different from that of P . syringae pv . tomato .
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