HP0333, a member of the dprA family, is involved in natural transformation in Helicobacter pylori.
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Helicobacter pylori is naturally competent for DNA transformation, but the mechanism by which transformation occurs is not known. For Haemophilus influenzae, dprA is required for transformation by chromosomal but not plasmid DNA, and the complete genomic sequence of H. pylori 26695 revealed a dprA homolog (HP0333). Examination of genetic databases indicates that DprA homologs are present in a wide variety of bacterial species. To examine whether HP0333 has a function similar to dprA of H. influenzae, HP0333, present in each of 11 strains studied, was disrupted in two H. pylori isolates. For both mutants, the frequency of transformation by H. pylori chromosomal DNA was markedly reduced, but not eliminated, compared to their wild-type parental strains. Mutation of HP0333 also resulted in a marked decrease in transformation frequency by a shuttle plasmid (pHP1), which differs from the phenotype described in H. influenzae. Complementation of the mutant with HP0333 inserted in trans in the chromosomal ureAB locus completely restored the frequency of transformation to that of the wild-type strain. Thus, while dprA is required for high-frequency transformation, transformation also may occur independently of DprA. The presence of DprA homologs in bacteria known not to be naturally competent suggests a broad function in DNA processing. (+info)
Characterization of Enterococcus faecalis alkaline phosphatase and use in identifying Streptococcus agalactiae secreted proteins.
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We have identified and characterized an Enterococcus faecalis alkaline phosphatase (AP, encoded by phoZ). The predicted gene product shows homology with alkaline phosphatases from a variety of species; it has especially high similarity with two alkaline phosphatases from Bacillus subtilis. Expression of phoZ in Escherichia coli, E. faecalis, Streptococcus agalactiae (group B streptococcus [GBS]), or Streptococcus pyogenes (group A streptococcus [GAS]) produces a blue-colony phenotype on plates containing a chromogenic substrate, 5-bromo-4-chloro-3-indolylphosphate (XP or BCIP). Two tests were made to determine if the activity of the enzyme is dependent upon the enzyme's subcellular location. First, elimination of the signal sequence reduced AP activity to 3% of the wild-type activity (or less) in three species of gram-positive bacteria. Restoration of export, using the signal sequence from C5a peptidase, restored AP activity to at least 50% of that of the wild type. Second, we engineered two chimeric proteins in which AP was fused to either a periplasmic domain or a cytoplasmic domain of lactose permease (a membrane protein). In E. coli, the periplasmic fusion had 17-fold-higher AP activity than the cytoplasmic fusion. We concluded that AP activity is export dependent. The signal sequence deletion mutant, phoZDeltass, was used to identify random genomic fragments from GBS that encode exported proteins or integral membrane proteins. Included in this set of fragments were genes that exhibited homology with the Rib protein (a cell wall protein from GBS) or with DppB (an integral membrane protein from GAS). AP acts as a reporter enzyme in GBS, GAS, and E. faecalis and is expected to be useful in a variety of gram-positive bacteria. (+info)
In vivo oxidation-reduction kinetics of OxyR, the transcriptional activator for an oxidative stress-inducible regulon in Escherichia coli.
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The OxyR protein is a transcriptional activator for a subset of peroxide stress-inducible genes, most of which are involved in defense systems against oxidative stress. Recently, it was demonstrated that purified OxyR has one intramolecular disulfide bond, which led to the proposal that the reversible disulfide bond formation regulates the activity of OxyR as a transcription factor in response to peroxide stress. In this study, I demonstrated by SDS-PAGE under non-reducing conditions that an intramolecular disulfide bond is formed in OxyR upon exposure of the cells to hydrogen peroxide in vivo. Experiments using strains expressing mutant OxyR proteins with Cys to Ser single amino acids substitutions confirmed that the disulfide bond is formed between the Cys-199 and -208. Kinetic analyses indicated that the formation of the disulfide bond is rapid and transient, oxidized within 30 s and re-reduced within 5 min after the addition of hydrogen peroxide in the wild-type strain. These results provide evidence for the regulatory role of the reversible oxidation of dithiol to disulfide in sensing peroxide stress in vivo and signal transduction to the transcription apparatus by OxyR. (+info)
DNA repair in Bacillus subtilis: excision repair capacity of competent cells.
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Competent Bacillus subtilis were investigated for their ability to support the repair of UV-irradiated bacteriophage and bacteriophage DNA. UV-irradiated bacteriophage DNA cannot be repaired to the same level as UV-irradiated bacteriophage, suggesting a deficiency in the ability of competent cells to repair UV damage. However, competent cells were as repair proficient as noncompetent cells in their ability to repair irradiated bacteriophage in marker rescue experiments. The increased sensitivity of irradiated DNA is shown to be due to the inability of excision repair to function on transfecting DNA in competent bacteria. Furthermore, competent cells show no evidence of possessing an inducible BsuR restriction system to complement their inducible BsuR modification enzyme. (+info)
Plasmid copy number control: isolation and characterization of high-copy-number mutants of plasmid pE194.
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A plasmid, pE194, obtained from Staphylococcus aureus confers resistance to macrolide, lincosamide, and streptogramin type B ("MLS") antibiotics. For full expression, the resistance phenotype requires a period of induction by subinhibitory concentrations of erythromycin. A copy number in the range of 10 to 25 copies per cell is maintained during cultivation at 32 degrees C. It is possible to transfer pE194 to Bacillus subtilis by transformation. In B. subtilis, the plasmid is maintained at a copy number of approximately 10 per cell at 37 degrees C, and resistance is inducible. Tylosin, a macrolide antibiotic which resembles erythromycin structurally and to which erythromycin induces resistance, lacks inducing activity. Two types of plasmid mutants were obtained and characterized after selection on medium containing 10 microgram of tylosin per ml. One mutant class appeared to express resistance constitutively and maintained a copy number indistinguishable from that of the parent plasmid. The other mutant type had a 5- to 10-fold-elevated plasmid copy number (i.e., 50 to 100 copies per cell) and expressed resistance inducibly. Both classes of tylosin-resistant mutants were shown to be due to alterations in the plasmid and not to modifications of the host genome. (+info)
Molecular basis of rifampin resistance in Streptococcus pneumoniae.
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Rifampin resistance among South African clinical isolates of Streptococcus pneumoniae was shown to be due to missense mutations within the rpoB gene. Sequence analysis of 24 rifampin-resistant isolates revealed the presence of mutations within cluster I as well as novel mutations in an area designated pneumococcus cluster III. Of the 24 isolates characterized, only 1 resistant isolate did not contain any mutations in the regions sequenced. Either the cluster I or the cluster III mutations separately conferred MICs of 32 to 128 microg/ml. Clinical isolate 55, for which the MIC was 256 microg/ml, was noted to contain 9 of the 10 mutations identified, which included the cluster I and cluster III mutations. As in Escherichia coli, it is possible that cluster I (amino acids 406 to 434) and cluster III (amino acids 523 to 600) of S. pneumoniae interact to form part of the antibiotic binding site, thus accounting for the very high MIC observed for isolate 55. PCR products containing cluster I or cluster III mutations were able to transform rifampin-susceptible S. pneumoniae to resistance. Although many of the isolates studied displayed identical sequences, pulsed-field gel electrophoresis analysis revealed that the isolates were not of clonal origin. (+info)
Propagation of TEM- and PSE-type beta-lactamases among amoxicillin-resistant Salmonella spp. isolated in France.
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A survey conducted between 1987 and 1994 at the University Hospital of Besancon, France, demonstrated a dramatic increase (from 0 to 42. 5%) in the prevalence of amoxicillin resistance among Salmonella spp. Of the 96 resistant isolates collected during this period (including 77 Typhimurium), 54 were found to produce TEM-1 beta-lactamase, 40 produced PSE-1 (equivalent to CARB-2), one produced PSE-1 plus TEM-2, and one produced OXA-1 in isoelectric focusing and DNA hybridization experiments. Plasmids coding for these beta-lactamases were further characterized by (i) profile analysis, (ii) restriction fragmentation pattern analysis, (iii) hybridization with an spvCD-orfE virulence probe, and (iv) replicon typing. In addition, isolates of S. typhimurium were genotypically compared by pulsed-field gel electrophoresis of XbaI-macrorestricted chromosomal DNA. Altogether, these methods showed that 40 of the 41 PSE-1 producers were actually the progeny of a single epidemic S. typhimurium strain lysotype DT104. Isolates of that strain were found to harbor RepFIC virulence plasmids with somewhat different restriction profiles, but which all carried the bla(PSE-1) gene. Of these virulence/resistance plasmids, 15 were transmissible to Escherichia coli. TEM-1-producing S. typhimurium displayed much greater genotypic and plasmidic diversities, suggesting the acquisition of the bla(TEM-1) gene from multiple bacterial sources by individual strains. In agreement with this, 32 of the 35 S. typhimurium plasmids encoding TEM-1 were found to be conjugative. These data show that development of amoxicillin resistance among Salmonella, especially in serovar Typhimurium, results from both gene transfers and strain dissemination. (+info)
Decreased azithromycin susceptibility of Neisseria gonorrhoeae due to mtrR mutations.
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Single-dose azithromycin therapy has recently been used in Uruguay for the treatment of uncomplicated gonococcal infections. As part of an active surveillance study to monitor the emergence of antibiotic resistance in gonococcal isolates, we examined the levels of azithromycin susceptibility in 51 consecutive isolates obtained from males with uncomplicated gonococcal urethritis. Isolates with decreased susceptibility to azithromycin (MICs, 0.25 to 0.5 microg/ml) were common, and these isolates often displayed cross-resistance to hydrophobic antimicrobial agents (erythromycin and Triton X-100). Resistance to erythromycin and Triton X-100 is frequently due to overexpression of the mtrCDE-encoded efflux pump mediated by mutations in the mtrR gene, which encodes a transcriptional repressor that modulates expression of the mtrCDE operon. Accordingly, we questioned whether clinical isolates that express decreased azithromycin susceptibility harbor mtrR mutations. Promoter mutations that would decrease the level of expression of mtrR as well as a missense mutation at codon 45 in the mtrR-coding region that would result in a radical amino acid replacement within the DNA-binding motif of MtrR were found in these strains. When these mutations were transferred into azithromycin-susceptible strain FA19 by transformation, the susceptibility of gonococci to azithromycin was decreased by nearly 10-fold. The mtrCDE-encoded efflux pump system was responsible for this property since insertional inactivation of the mtrC gene resulted in enhanced susceptibility of gonococci to azithromycin. We conclude that the mtrCDE-encoded efflux pump can recognize azithromycin and that the emergence of gonococcal strains with decreased susceptibility to azithromycin can, in part, be explained by mtrR mutations. (+info)