RESUMO
The Arc (anoxic redox control) two-component signal transduction system, consisting of the ArcB sensor kinase and the ArcA response regulator, allows adaptive responses of Escherichia coli to changes of O(2) availability. The arcA gene was previously known as the dye gene because null mutants were growth sensitive to the photosensitizer redox dyes toluidine blue and methylene blue, a phenotype whose molecular basis still remains elusive. In this study we report that the toluidine blue O (TBO) effect on the arc mutants is light independent and observed only during aerobic growth conditions. Moreover, 16 suppressor mutants with restored growth were generated and analyzed. Thirteen of those possessed insertion elements upstream of the cydAB operon, rendering its expression ArcA independent. Also, it was found that, in contrast to cythocrome d, cythocrome o was not able to confer toluidine blue resistance to arc mutants, thereby representing an intriguing difference between the two terminal oxidases. Finally, a mechanism for TBO sensitivity and resistance is discussed.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Grupo dos Citocromos b/metabolismo , Grupo dos Citocromos d/metabolismo , Citocromos/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Proteínas Repressoras/genética , Cloreto de Tolônio/farmacologia , Anaerobiose , Proteínas da Membrana Bacteriana Externa/metabolismo , Sequência de Bases , Carotenoides/metabolismo , Catalase/metabolismo , Corantes/farmacologia , Grupo dos Citocromos b/genética , Grupo dos Citocromos d/genética , Citocromos/genética , Escuridão , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/efeitos da radiação , Glucose/farmacologia , Luz , Dados de Sequência Molecular , Mutação/genética , Oxirredutases/genética , Regiões Promotoras Genéticas/genética , Espécies Reativas de Oxigênio/metabolismo , Proteínas Repressoras/metabolismo , Homologia de Sequência do Ácido Nucleico , Superóxido Dismutase/metabolismoRESUMO
Escherichia coli possesses a two-domain flavohemoglobin, Hmp, implicated in nitric oxide (NO) detoxification. To determine the contribution of each domain of Hmp toward NO detoxification, we genetically engineered the Hmp protein and separately expressed the heme (HD) and the flavin (FD) domains in a defined hmp mutant. Expression of each domain was confirmed by Western blot analysis. CO-difference spectra showed that the HD of Hmp can bind CO, but the CO adduct showed a slightly blue-shifted peak. Overexpression of the HD resulted in an improvement of growth to a similar extent to that observed with the Vitreoscilla hemeonly globin Vgb, whereas the FD alone did not improve growth. Viability of the hmp mutant in the presence of lethal concentrations of sodium nitroprusside was increased (to 30% survival after 2 h in 5 mM sodium nitroprusside) by overexpressing Vgb or the HD. However, maximal protection was provided only by holo-Hmp (75% survival under the same conditions). Cellular respiration of the hmp mutant was instantaneously inhibited in the presence of 13.5 microM NO but remained insensitive to NO inhibition when these cells overexpressed Hmp. When HD or FD was expressed separately, no significant protection was observed. By contrast, overexpression of Vgb provided partial protection from NO respiratory inhibition. Our results suggest that, despite the homology between the HD from Hmp and Vgb (45% identity), their roles seem to be quite distinct.