RESUMEN
RraA is a protein inhibitor of RNase E, which degrades and processes numerous RNAs in Escherichia coli. Streptomyces coelicolor also contains homologs of RNase E and RraA, RNase ES and RraAS1/RraAS2, respectively. Here, we report that, unlike other RraA homologs, RraAS1 directly interacts with the catalytic domain of RNase ES to exert its inhibitory effect. We further show that rraAS1 gene deletion in S. coelicolor results in a higher growth rate and increased production of actinorhodin and undecylprodigiosin, compared with the wild-type strain, suggesting that RraAS1-mediated regulation of RNase ES activity contributes to modulating the cellular physiology of S. coelicolor.
Asunto(s)
Proteínas Bacterianas/metabolismo , Dominio Catalítico , Endorribonucleasas/antagonistas & inhibidores , Endorribonucleasas/metabolismo , Regulación Bacteriana de la Expresión Génica , Streptomyces coelicolor/genética , Streptomyces coelicolor/fisiología , Antraquinonas/metabolismo , Proteínas Bacterianas/genética , Endorribonucleasas/química , Eliminación de Gen , Prodigiosina/análogos & derivados , Prodigiosina/metabolismo , Streptomyces coelicolor/crecimiento & desarrolloRESUMEN
RraA is a protein inhibitor of RNase E (Rne), which catalyzes the endoribonucleolytic cleavage of a large proportion of RNAs in Escherichia coli. The antibiotic-producing bacterium Streptomyces coelicolor also contains homologs of RNase E and RraA, designated as RNase ES (Rns), RraAS1, and RraAS2, respectively. Here, we report that RraAS2 requires both scaffold domains of RNase ES for high-affinity binding and inhibitory action on the ribonucleolytic activity. Analyses of the steady-state level of RNase E substrates indicated that coexpression of RraAS2 in E. coli cells overproducing Rns effectively inhibits the ribonucleolytic activity of full-length RNase ES, but its inhibitory effects were moderate or undetectable on other truncated forms of Rns, in which the N- or/and C-terminal scaffold domain was deleted. In addition, RraAS2 more efficiently inhibited the in vitro ribonucleolytic activity of RNase ES than that of a truncated form containing the catalytic domain only. Coimmunoprecipitation and in vivo cross-linking experiments further showed necessity of both scaffold domains of RNase ES for high-affinity binding of RraAS2 to the enzyme, resulting in decreased RNA-binding capacity of RNase ES. Our results indicate that RraAS2 is a protein inhibitor of RNase ES and provide clues to how this inhibitor affects the ribonucleolytic activity of RNase ES.
Asunto(s)
Proteínas Bacterianas/metabolismo , Endorribonucleasas/antagonistas & inhibidores , Endorribonucleasas/metabolismo , Streptomyces coelicolor/metabolismo , Proteínas Bacterianas/genética , Dominio Catalítico , Endorribonucleasas/química , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Unión Proteica , Estabilidad del ARN , ARN Bacteriano/metabolismoRESUMEN
Resistance-nodulation-division (RND) efflux pumps are associated with multidrug resistance in many gram-negative pathogens. The genome of Vibrio vulnificus encodes 11 putative RND pumps homologous to those of Vibrio cholerae and Escherichia coli. In this study, we analyzed three putative RND efflux pumps, showing homology to V. cholerae VexAB and VexCD and to E. coli AcrAB, for their functional roles in multidrug resistance of V. vulnificus. Deletion of the vexAB homolog resulted in increased susceptibility of V. vulnificus to bile acid, acriflavine, ethidium bromide, and erythromycin, whereas deletion of acrAB homologs rendered V. vulnificus more susceptible to acriflavine only. Deletion of vexCD had no effect on susceptibility of V. vulnificus to these chemicals. Upon exposure to these antibacterial chemicals, expression of tolCV1 and tolCV2, which are putative outer membrane factors of RND efflux pumps, was induced, whereas expression levels of vexAB, vexCD, and acrAB homologs were not significantly changed. Our results show that the V. vulnificus homologs of VexAB largely contributed to in vitro antimicrobial resistance with a broad substrate specificity that was partially redundant with the AcrAB pump homologs.
Asunto(s)
Proteínas Portadoras/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Vibrio cholerae/genética , Vibrio vulnificus/genética , Vibrio vulnificus/metabolismo , Antibacterianos/farmacología , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/genética , Resistencia a Múltiples Medicamentos/genética , Eritromicina/farmacología , Proteínas de Transporte de Membrana/genética , Pruebas de Sensibilidad Microbiana , Análisis de Secuencia de Proteína , Vibrio cholerae/metabolismo , Vibrio vulnificus/efectos de los fármacos , Vibrio vulnificus/crecimiento & desarrolloRESUMEN
The biofilm-dependent modulation gene (bdm) has recently been shown to play a role in osmotic-induced formation of biofilm in Escherichia coli. In this study, we demonstrated that deletion of bdm results in down-regulation of flagella biosynthesis genes and, consequently, a defect in E. coli motility. In addition, we employed atomic force microscopy to confirm the absence of flagella-like structures on the surface of bdm-null cells. These findings indicate that bdm plays a key role in regulatory pathway for the formation of flagella.
