RESUMEN
RpoS-dependent promoters require ppGpp for induction in the stationary phase. This has been thought to be a simple consequence of final sigma(S) itself requiring ppGpp for its production. By using four model promoters requiring final sigma(S) for normal induction in the stationary phase, we demonstrate that final sigma(S)-dependent promoters require ppGpp even in the presence of high levels of final sigma(S) produced ectopically. Similar to final sigma(70)-dependent promoters under positive control by ppGpp, the requirement of final sigma(S)-dependent promoters for this alarmone is bypassed by specific "stringent" mutations in the beta-subunit of RNA polymerase. The results suggest that stationary phase induction of both final sigma(S)- and final sigma(70)-dependent genes requires the stringent control modulon and that stringency confers dual control on the RpoS regulon by affecting promoter activity and the levels of the required final sigma-factor.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Guanosina Tetrafosfato/metabolismo , Proteínas de la Membrana , Regiones Promotoras Genéticas , Factor sigma/metabolismo , Proteínas Bacterianas/genética , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/metabolismo , Genotipo , Cinética , Mutagénesis , Plásmidos , Transducción Genética , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
The uspA promoter, driving production of the universal stress protein A in response to diverse stresses, is demonstrated to be under dual control. One regulatory pathway involves activation of the promoter by the alarmone guanosine 3',5'-bisphosphate, via the beta-subunit of RNA polymerase, whereas the other consists of negative control by the FadR repressor. In contrast to canonical dual control by activation and repression circuits, which depends on concomitant activation and derepression for induction to occur, the ppGpp-dependent activation of the uspA promoter overrides repression by an active FadR under conditions of severe cellular stress (starvation). The ability of RNA polymerase to overcome repression during stringency depends, in part, on the strength of the FadR operator. This emergency derepression is operative on other FadR-regulated genes induced by starvation and is argued to be an essential regulatory mechanism operating during severe stress.
Asunto(s)
Proteínas Bacterianas/genética , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Proteínas de Choque Térmico/genética , Proteínas Represoras/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Coenzima A Ligasas/genética , Coenzima A Ligasas/metabolismo , AMP Cíclico/metabolismo , Electroforesis en Gel Bidimensional , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Guanosina Tetrafosfato/genética , Guanosina Tetrafosfato/metabolismo , Proteínas de Choque Térmico/metabolismo , Datos de Secuencia Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plásmidos/genética , Regiones Promotoras Genéticas , Proteínas Represoras/metabolismo , Transducción Genética , Transformación Bacteriana , beta-GalactosidasaRESUMEN
The open reading frame immediately upstream of uspA is demonstrated to encode a 14-kDa protein which we named UspB (universal stress protein B) because of its general responsiveness to different starvation and stress conditions. UspB is predicted to be an integral membrane protein with at least one and perhaps two membrane-spanning domains. Overexpression of UspB causes cell death in stationary phase, whereas mutants of uspB are sensitive to exposure to ethanol but not heat in stationary phase. In contrast to uspA, stationary-phase induction of uspB requires the sigma factor sigmaS. The expression of uspB is modulated by H-NS, consistent with the role of H-NS in altering sigmaS levels. Our results demonstrate that a gene of the RpoS regulon is involved in the development of stationary-phase resistance to ethanol, in addition to the regulon's previously known role in thermotolerance, osmotolerance, and oxidative stress resistance.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Etanol/farmacología , Genes Bacterianos , Proteínas de la Membrana , Factor sigma/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Secuencia de Bases , Cartilla de ADN/genética , ADN Bacteriano/genética , Farmacorresistencia Microbiana/genética , Escherichia coli/fisiología , Expresión Génica , Interfase , Datos de Secuencia Molecular , Peso Molecular , Mutación , Regulón , Homología de Secuencia de AminoácidoRESUMEN
A mutation in the Escherichia coli gene encoding the stationary phase-inducible sigma factor (sigmaS, RpoS) not only abolishes transcription of some genes in stationary phase, but also causes superinduction of other stationary phase-induced genes. We have examined this phenomenon of repression by sigmaS using as a model system the divergently transcribed stationary phase-inducible genes, uspA and uspB. uspA is transcribed by sigma70-programmed RNA polymerase and is superinduced in an rpoS mutant, while uspB induction is sigmaS dependent. The data suggest that the superinduction of uspA is caused by an increased amount of sigma70 bound to RNA polymerase in the absence of the competing sigmaS. Increasing the ability of sigma70 to compete against sigmaS by overproducing sigma70 mimics the effect of an rpoS mutation by causing superinduction of sigma70-dependent stationary phase-inducible genes (uspA and fadD), silencing of sigmaS-dependent genes (uspB, bolAp1 and fadL) and inhibiting the development of sigmaS-dependent phenotypes, such as hydrogen peroxide resistance in stationary phase. In addition, overproduction of sigmaS markedly reduced stationary phase expression of a sigma70-dependent promoter. Thus, we conclude that sigma factors compete for a limiting amount of RNA polymerase during stationary phase. The implications of this competition in the passive control of promoter activity is discussed.