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1.
Sci Rep ; 7: 42529, 2017 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-28205647

RESUMO

Microcin PDI inhibits a diversity of pathogenic Escherichia coli through the action of an effector protein, McpM. In this study we demonstrated that expression of the inhibitory phenotype is induced under low osmolarity conditions and expression is primarily controlled by the EnvZ/OmpR two-component regulatory system. Functional, mutagenesis and complementation experiments were used to empirically demonstrate that EnvZ is required for the inhibitory phenotype and that regulation of mcpM is dependent on binding of the phosphorylated OmpR to the mcpM promoter region. The phosphorylated OmpR may recognize three different binding sites within this promoter region. Site-directed mutagenesis revealed that the McpM precursor peptide includes two leader peptides that undergo sequential cleavage at positions G17/G18 and G35/A36 during export through the type I secretion system. Competition assays showed that both cleaved products are required for the PDI phenotype although we could not distinguish loss of function from loss of secretion in these assays. McpM has four cysteines within the mature peptide and site-directed mutagenesis experiments demonstrated that the first two cysteines are necessary for McpM to inhibit susceptible cells. Together these data combined with previous work indicate that MccPDI is unique amongst the microcins that have been described to date.


Assuntos
Bacteriocinas/genética , Bacteriocinas/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Bacteriocinas/química , Sequência de Bases , Sítios de Ligação , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Técnicas de Inativação de Genes , Teste de Complementação Genética , Complexos Multienzimáticos/metabolismo , Fragmentos de Peptídeos/metabolismo , Fenótipo , Fosforilação , Regiões Promotoras Genéticas , Ligação Proteica , Transporte Proteico , Proteólise , Transativadores/metabolismo
2.
Artigo em Inglês | MEDLINE | ID: mdl-27999769

RESUMO

Type III secretion systems (T3SSs) contribute to microbial pathogenesis of Vibrio species, but the regulatory mechanisms are complex. We determined if the classic ExsACDE protein-protein regulatory model from Pseudomonas aeruginosa applies to Vibrio alginolyticus. Deletion mutants in V. alginolyticus demonstrated that, as expected, the T3SS is positively regulated by ExsA and ExsC and negatively regulated by ExsD and ExsE. Interestingly, deletion of exsE enhanced the ability of V. alginolyticus to induce host-cell death while cytotoxicity was inhibited by in trans complementation of this gene in a wild-type strain, a result that differs from a similar experiment with Vibrio parahaemolyticus ExsE. We further showed that ExsE is a secreted protein that does not contribute to adhesion to Fathead minnow epithelial cells. An in vitro co-immunoprecipitation assay confirmed that ExsE binds to ExsC to exert negative regulatory effect on T3SS genes. T3SS in V. alginolyticus can be activated in the absence of physical contact with host cells and a separate regulatory pathway appears to contribute to the regulation of ExsA. Consequently, like ExsE from P. aeruginosa, ExsE is a negative regulator for T3SS gene expression in V. alginolyticus. Unlike the V. parahaemolyticus orthologue, however, deletion of exsE from V. alginolyticus enhanced in vitro cytotoxicity.


Assuntos
Regulação Bacteriana da Expressão Gênica , Proteínas Repressoras/metabolismo , Sistemas de Secreção Tipo III/metabolismo , Vibrio alginolyticus/genética , Vibrio alginolyticus/metabolismo , Animais , Aderência Bacteriana , Sobrevivência Celular , Células Cultivadas , Cyprinidae , Células Epiteliais/microbiologia , Deleção de Genes , Teste de Complementação Genética , Imunoprecipitação , Ligação Proteica , Proteínas Repressoras/genética
3.
Antimicrob Agents Chemother ; 54(6): 2666-9, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20368404

RESUMO

Bovine-origin Escherichia coli isolates were tested for resistance phenotypes using a disk diffusion assay and for resistance genotypes using a DNA microarray. An isolate with gentamicin and amikacin resistance but with no corresponding genes detected yielded a 1,056-bp DNA sequence with the closest homologues for its inferred protein sequence among a family of 16S rRNA methyltransferase enzymes. These enzymes confer high-level aminoglycoside resistance and have only recently been described in Gram-negative bacteria.


Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Genes Bacterianos , Animais , Sequência de Bases , Bovinos , Primers do DNA/genética , DNA Bacteriano/genética , Escherichia coli/isolamento & purificação , Escherichia coli/metabolismo , Técnicas In Vitro , Metiltransferases/classificação , Metiltransferases/genética , Análise de Sequência com Séries de Oligonucleotídeos , Filogenia , RNA Bacteriano/metabolismo , RNA Ribossômico 16S/metabolismo
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