RESUMO
Microcin J25 has two targets in sensitive bacteria, the RNA polymerase, and the respiratory chain through inhibition of cellular respiration. In this work, the effect of microcin J25 in E. coli mutants that lack the terminal oxidases cytochrome bd-I and cytochrome bo3 was analyzed. The mutant strains lacking cytochrome bo3 or cytochrome bd-I were less sensitive to the peptide. In membranes obtained from the strain that only expresses cytochrome bd-I a great ROS overproduction was observed in the presence of microcin J25. Nevertheless, the oxygen consumption was less inhibited in this strain, probably because the oxygen is partially reduced to superoxide. There was no overproduction of ROS in membranes isolated from the mutant strain that only express cytochrome bo3 and the inhibition of the cellular respiration was similar to the wild type. It is concluded that both cytochromes bd-I and bo3 are affected by the peptide. The results establish for the first time a relationship between the terminal oxygen reductases and the mechanism of action of microcin J25.
Assuntos
Bacteriocinas/farmacologia , Citocromos/biossíntese , Complexo de Proteínas da Cadeia de Transporte de Elétrons/biossíntese , Proteínas de Escherichia coli/biossíntese , Escherichia coli/enzimologia , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Oxirredutases/biossíntese , Grupo dos Citocromos b , Citocromos/genética , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Oxirredutases/genética , Espécies Reativas de Oxigênio/metabolismoRESUMO
Microcin J25 (MccJ25) is a cyclic peptide of 21 unmodified amino acid residues produced by a fecal strain of Escherichia coli. It has previously been shown that the antibiotic activity of this peptide is mainly directed to Enterobacteriaceae, including several pathogenic E. coli, Salmonella and Shigella strains. In this paper we show that MccJ25 acts on the cytoplasmic membrane of Salmonella newport cells producing alteration of membrane permeability, and the subsequent gradient dissipation, that initiate the inhibition of process, such as oxygen consumption. These results, taken together with our in vitro observations [Rintoul et al. (2000) Biochim. Biophys. Acta 1509, 65-72], strongly suggest that the disruption of the cytoplasmic membrane gradient is closely related to the bactericidal activity of MccJ25 in S. newport.
Assuntos
Antibacterianos/farmacologia , Bacteriocinas/farmacologia , Membrana Celular/efeitos dos fármacos , Escherichia coli/metabolismo , Peptídeos , Salmonella/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Humanos , Potenciais da Membrana/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Oxirredutases/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Salmonella/crescimento & desenvolvimento , Salmonella/patogenicidadeRESUMO
Escherichia coli microcin J25 (MccJ25) is a plasmid-encoded, cyclic peptide antibiotic consisting of 21 unmodified amino acid residues. It is primarily active on gram-negative bacteria related to the producer strain, inducing cell filamentation in an SOS-independent way. A mutation causing resistance to MccJ25 was isolated. Genetic analysis indicated that it resided in the rpoC gene, encoding the beta' subunit of RNA polymerase, at 90 min on the E. coli genetic map. The mutation was genetically crossed on to a plasmid containing the wild-type rpoC gene. The presence of the recombinant plasmid conferred complete resistance to otherwise sensitive strains. Nucleotide sequencing of the plasmid-borne, mutant rpoC gene revealed a ACC (Thr)-to-ATC (Ile) change at codon 931, within homology block G, an evolutionarily conserved region in the large subunits of all RNA polymerases. MccJ25 decreased RNA synthesis both in vivo and in vitro. These results point to the RNA polymerase as the target of microcin action. We favor the possibility that the filamentous phenotype induced by MccJ25 results from impaired transcription of genes coding for cell division proteins. As far as we know, MccJ25 is the first peptide antibiotic shown to affect RNA polymerase.
Assuntos
Antibacterianos/farmacologia , Bacteriocinas/farmacologia , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Escherichia coli/enzimologia , Peptídeos , Alelos , Sequência de Aminoácidos , Mapeamento Cromossômico , RNA Polimerases Dirigidas por DNA/genética , Resistência Microbiana a Medicamentos , Escherichia coli/efeitos dos fármacos , Escherichia coli/isolamento & purificação , Escherichia coli/fisiologia , Genes Bacterianos , Líquido Intracelular , Leucina , Dados de Sequência Molecular , Mutagênese , Análise de Sequência de DNA , Transcrição Gênica , UridinaRESUMO
Microcin J25 is a 2,107-Da, plasmid-encoded, cyclopeptide antibiotic produced by Escherichia coli. We have isolated lacZ fusions to mcjA (encoding the 58-amino-acid microcin precursor) and mcjB and mcjC (which are required for microcin maturation), and the regulation of these fusions was used to identify factors that control the expression of these genes. The mcjA gene was found to be dramatically induced as cells entered the stationary phase. Expression of mcjA could be induced by resuspending uninduced exponential-phase cells in spent supernatant obtained from an early-stationary-phase culture. Induction of mcjA expression was not dependent on high cell density, pH changes, anaerobiosis, or the buildup of some inducer. A starvation for carbon and inorganic phosphate induced mcjA expression, while under nitrogen limitation there was no induction at all. These results taken together suggest that stationary-phase induction of mcjA is triggered by nutrient depletion. The mcjB and mcjC genes were also regulated by the growth phase of the culture, but in contrast to mcjA, they showed substantial expression already during exponential growth. Induction of the microcin genes was demonstrated to be independent of RpoS, the cyclic AMP-Crp complex, OmpR, and H-NS. Instead, we found that the growth-phase-dependent expression of mcjA, mcjB, and mcjC may be explained by the concerted action of the positively acting transition state regulators ppGpp, Lrp, and integration host factor. Measurements of microcin J25 production by strains defective in these global regulators showed a good correlation with the reduced expression of the fusions in such mutant backgrounds.
Assuntos
Bacteriocinas/biossíntese , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Anaerobiose , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriocinas/genética , Bacteriocinas/metabolismo , Sequência de Bases , Meios de Cultivo Condicionados/química , Escherichia coli/metabolismo , Guanosina Pentafosfato/genética , Guanosina Pentafosfato/metabolismo , Concentração de Íons de Hidrogênio , Fatores Hospedeiros de Integração , Óperon Lac/genética , Óperon Lac/fisiologia , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Dados de Sequência Molecular , Oxigênio/farmacologia , Pirofosfatases/genética , Receptores Imunológicos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de DNARESUMO
A 4.8-kb plasmid region carrying the four genes mcjABCD necessary for production of and immunity to the cyclic peptide antibiotic microcin J25 (MccJ25) has been sequenced. mcjA encodes the primary structure of MccJ25 as a precursor endowed with an N-terminal extension of 37 amino acids. The products of mcjB and mcjC are thought to be involved in microcin maturation, which implies cleavage of McjA and head-tail linkage of the 21-residue pro-MccJ25. The predicted McjD polypeptide, which is highly similar to several ABC exporters, was found to be required for MccJ25 secretion, thus explaining its ability to confer immunity to MccJ25.
Assuntos
Antibacterianos/biossíntese , Bacteriocinas/biossíntese , Genes Bacterianos , Peptídeos Cíclicos/biossíntese , Plasmídeos/genética , Transportadores de Cassetes de Ligação de ATP/genética , Sequência de Aminoácidos , Bacteriocinas/genética , Sequência de Bases , Escherichia coli/genética , Dados de Sequência Molecular , Família Multigênica , Fases de Leitura Aberta , Precursores de Proteínas/genética , Homologia de Sequência de AminoácidosRESUMO
A Tn5 insertion in tolC eliminated microcin J25 production. The mutation had little effect on the expression of the microcin structural gene and presumably acted by blocking microcin secretion. The tolC mutants carrying multiple copies of the microcin genes were less immune to the microcin. TolC is thus likely a component of a microcin export complex containing the McjD immunity protein, an ABC exporter.
Assuntos
Antibacterianos/biossíntese , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Bacteriocinas/biossíntese , Bacteriocinas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Peptídeos , Transportadores de Cassetes de Ligação de ATP , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli , Expressão Gênica , Genes Bacterianos , Proteínas de Membrana Transportadoras , Mutagênese Insercional , Fenótipo , Plasmídeos/genética , Porinas/genéticaRESUMO
Microcin J25 (MccJ25) is a small peptide antibiotic produced by an Escherichia coli strain isolated from human feces. The genetic determinants for MccJ25 synthesis and immunity have been cloned from the low-copy-number wild-type plasmid pTUC1OO into the compatible vectors pBR322 and pACYC184. Physical and phenotypical analysis of insertion mutations and complementation tests defined three contiguous genes involved in MccJ25 production which span a region of about 2.2 kb. Immunity to the antibiotic is provided by an additional gene adjacent to the production region.
Assuntos
Bacteriocinas/genética , Escherichia coli/genética , Genes Bacterianos , Plasmídeos , Clonagem Molecular , Elementos de DNA Transponíveis , DNA Bacteriano/genética , Mutagênese Insercional , Mapeamento por RestriçãoRESUMO
Selection of spontaneous mutants for insensitivity to the peptide antibiotic microcin 25 led to the isolation of five categories of mutants. Phenotypic and mapping studies showed the mutations to be located in the fhuA, exb, tonB, and sbmA genes. The latter encodes a cytoplasmic membrane protein which is also required for the penetration of microcin B17.
Assuntos
Proteínas de Bactérias/metabolismo , Bacteriocinas/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Transporte Biológico , Teste de Complementação Genética , Receptores Virais/metabolismoRESUMO
The effect of the iron content of the medium on the yield of the peptide antibiotic microcin 25 was examined; synthesis was optimal in minimal media and was reduced by adding iron. Escherichia coli AY25, the wild-type producer of the antibiotic, showed a 95% decrease in microcin yield when grown in minimal medium containing 10 microM iron (high iron) as compared to 0.2 microM (low iron). Addition of chelators to Luria broth elicited microcin production, and there was a complete reversal of the effect of the chelators by adding iron. Studies with Escherichia coli mutants deficient in iron-regulated proteins (fur) suggested that factors other than Fur could mediate iron regulation of microcin synthesis.
Assuntos
Antibacterianos/biossíntese , Bacteriocinas/biossíntese , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Ferro/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Meios de Cultura , Escherichia coli/genética , Genes Bacterianos , Ferro/metabolismo , Quelantes de Ferro/farmacologia , Mutação , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismoRESUMO
A chromosomal Tn5 insertion resulting in complete resistance to the peptide antibiotic microcin 25 was mapped to the min 4 region of the Escherichia coli genetic map. Additional experiments showed that the insertion disrupted the fhuA gene, which encodes the multifunctional outer membrane receptor for ferrichrome, the antibiotic albomycin, colicin M, and bacteriophages T5, T1, and phi 80. Thus, microcin 25 and all of these agents share the same receptor.