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
Rhodobacter sphaeroides expresses a bb3-type quinol oxidase, and two cytochrome c oxidases: cytochrome aa3 and cytochrome cbb3. We report here the characterization of the genes encoding this latter oxidase. The ccoNOQP gene cluster of R. sphaeroides contains four open reading frames with high similarity to all ccoNOQP/fixNOQP gene clusters reported so far. CcoN has the six highly conserved histidines proposed to be involved in binding the low spin heme, and the binuclear center metals. ccoO and ccoP code for membrane bound mono- and diheme cytochromes c. ccoQ codes for a small hydrophobic protein of unknown function. Upstream from the cluster there is a conserved Fnr/FixK-like box which may regulate its expression. Analysis of a R. sphaeroides mutant in which the ccoNOQP gene cluster was inactivated confirms that this cluster encodes the cbb3-type oxidase previously purified. Analysis of proton translocation in several strains shows that cytochrome cbb3 is a proton pump. We also conclude that cytochromes cbb3 and aa3 are the only cytochrome c oxidases in the respiratory chain of R. sphaeroides.