RESUMO
Superoxide dismutase (SOD) is a nearly ubiquitous enzyme among organisms that are exposed to oxic environments. The single SOD of Helicobacter pylori, encoded by the sodB gene, has been suspected to be a virulence factor for this pathogenic microaerophile, but mutations in this gene have not been reported previously. We have isolated mutants with interruptions in the sodB gene and have characterized them with respect to their response to oxidative stress and ability to colonize the mouse stomach. The sodB mutants are devoid of SOD activity, based on activity staining in nondenaturing gels and quantitative assays of cell extracts. Though wild-type H. pylori is microaerophilic, the mutants are even more sensitive to O(2) for both growth and viability. While the wild-type strain is routinely grown at 12% O(2), growth of the mutant strains is severely inhibited at above 5 to 6% O(2). The effect of O(2) on viability was determined by subjecting nongrowing cells to atmospheric levels of O(2) and plating for survivors at 2-h time intervals. Wild-type cell viability dropped by about 1 order of magnitude after 6 h, while viability of the sodB mutant decreased by more than 6 orders of magnitude at the same time point. The mutants are also more sensitive to H(2)O(2), and this sensitivity is exacerbated by increased O(2) concentrations. Since oxidative stress has been correlated with DNA damage, the frequency of spontaneous mutation to rifampin resistance was studied. The frequency of mutagenesis of an sodB mutant strain is about 15-fold greater than that of the wild-type strain. In the mouse colonization model, only 1 out of 23 mice inoculated with an SOD-deficient mutant of a mouse-adapted strain became H. pylori positive, while 15 out of 17 mice inoculated with the wild-type strain were shown to harbor the organism. Therefore, SOD is a virulence factor which affects the ability of this organism to colonize the mouse stomach and is important for the growth and survival of H. pylori under conditions of oxidative stress.
Assuntos
Helicobacter pylori/enzimologia , Helicobacter pylori/patogenicidade , Mutação , Estresse Oxidativo , Superóxido Dismutase/deficiência , Animais , Clonagem Molecular , Feminino , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/fisiopatologia , Helicobacter pylori/genética , Peróxido de Hidrogênio/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Oxigênio/farmacologia , Análise de Sequência de DNA , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , VirulênciaRESUMO
During Bacillus subtilis endospore formation, a complex protein coat is assembled around the maturing spore. The coat is made up of more than two dozen proteins that form an outer layer, which provides chemical resistance, and an inner layer, which may play a role in the activation of germination. A third, amorphous layer of the coat occupies the space between the inner coat and the cortex, and is referred to as the undercoat. Although several coat proteins have been characterized, little is known about their interactions during assembly of the coat. We show here that at least two open reading frames of the cotJ operon (cotJA and cotJC) encode spore coat proteins. We suggest that CotJC is a component of the undercoat, since we found that its assembly onto the forespore is not prevented by mutations that block both inner and outer coat assembly, and because CotJC is more accessible to antibody staining in spores lacking both of these coat layers. Assembly of CotJC into the coat is dependent upon expression of cotJA. Conversely, CotJA is not detected in the coats of a cotJC insertional mutant. Co-immunoprecipitation was used to demonstrate the formation of complexes containing CotJA and CotJC 6 h after the onset of sporulation. Experiments with the yeast two-hybrid system indicate that CotJC may interact with itself and with CotJA. We suggest that interaction of CotJA with CotJC is required for the assembly of both CotJA and CotJC into the spore coat.
Assuntos
Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fator sigma , Esporos Bacterianos/química , Fatores de Transcrição , Proteínas de Bactérias/análise , Imunofluorescência , Expressão Gênica/genética , Expressão Gênica/fisiologia , Genes Bacterianos/genética , Genes Bacterianos/fisiologia , Óperon/genética , Óperon/fisiologiaRESUMO
We used potassium permanganate (KMnO4) to identify unpaired thymidine (T) residues in promoter complexes formed by RNA polymerase (RNAP) associated with sigma E (sigma E-RNAP) from Bacillus subtilis. We found that a region of the spoIIID promoter from at least -10 to +1 becomes melted in the presence of this polymerase. In promoter complexes formed by RNAP associated with a mutant sigma E that melts promoter DNA inefficiently, we noted additional KMnO4 sensitivity at the -11 position of the spoIIID promoter. We suggest that the base pair at -11 is unpaired in both mutant and wild type (wt) complexes; however, close proximity of wt sigma E-RNAP with the T at -11 may protect it from KMnO4 attack. The absence of a close contact between the mutant sigma E-RNAP and the base at -11 may explain why this polymerase uses promoters less efficiently than wt sigma E-RNAP.
Assuntos
RNA Polimerases Dirigidas por DNA/efeitos dos fármacos , Permanganato de Potássio/farmacologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Fator sigma/efeitos dos fármacos , Fatores de Transcrição/efeitos dos fármacos , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Fator sigma/metabolismo , Timidina/metabolismo , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Bacillus sphaericus 2362 strains transformed with the plasmid pUB110 (4.5 kb) and plasmids derived from it, pLDT103 (7.6 kb) and pLDT117 (9.3 kb), were able to recycle (spore germination, vegetative growth, sporulation) in larvae of Culex quinquefasciatus. During the course of recycling, the pUB110 vector and the recombinant plasmid pLDT103 were stable (100 and 99.2%, respectively). However, the recombinant plasmid pLDT117 exhibited 23% segregational instability. Isolates which lost pLDT117 during recycling retained the one large plasmid native to B. sphaericus 2362.