RESUMEN
In Pseudomonas fluorescens biocontrol strain CHA0, the two-component system GacS/GacA positively controls the synthesis of extracellular products such as hydrogen cyanide, protease, and 2,4-diacetylphloroglucinol, by upregulating the transcription of small regulatory RNAs which relieve RsmA-mediated translational repression of target genes. The expression of the stress sigma factor sigmaS (RpoS) was controlled positively by GacA and negatively by RsmA. By comparison with the wild-type CHA0, both a gacS and an rpoS null mutant were more sensitive to H2O2 in stationary phase. Overexpression of rpoS or of rsmZ, encoding a small RNA antagonistic to RsmA, restored peroxide resistance to a gacS mutant. By contrast, the rpoS mutant showed a slight increase in the expression of the hcnA (HCN synthase subunit) gene and of the aprA (major exoprotease) gene, whereas overexpression of sigmaS strongly reduced the expression of these genes. These results suggest that in strain CHA0, regulation of exoproduct synthesis does not involve sigmaS as an intermediate in the Gac/Rsm signal transduction pathway whereas sigmaS participates in Gac/Rsm-mediated resistance to oxidative stress.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Estrés Oxidativo , Pseudomonas fluorescens/fisiología , Proteínas de Unión al ARN/metabolismo , Proteínas Represoras/metabolismo , Factor sigma/metabolismo , Proteínas Bacterianas/genética , Exopeptidasas/genética , Exopeptidasas/metabolismo , Peróxido de Hidrógeno/farmacología , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupos CH-NH2 , Pseudomonas fluorescens/genética , Pseudomonas fluorescens/crecimiento & desarrollo , Pseudomonas fluorescens/metabolismo , Proteínas de Unión al ARN/genética , Proteínas Represoras/genética , Factor sigma/genética , Transducción de SeñalRESUMEN
In the root-colonizing biocontrol strain CHA0 of Pseudomonas fluorescens, cell density-dependent synthesis of extracellular, plant-beneficial secondary metabolites and enzymes is positively regulated by the GacS/GacA two-component system. Mutational analysis of the GacS sensor kinase using improved single-copy vectors showed that inactivation of each of the three conserved phosphate acceptor sites caused an exoproduct null phenotype (GacS-), whereas deletion of the periplasmic loop domain had no significant effect on the expression of exoproduct genes. Strain CHA0 is known to synthesize a solvent-extractable extracellular signal that advances and enhances the expression of exoproduct genes during the transition from exponential to stationary growth phase when maximal exoproduct formation occurs. Mutational inactivation of either GacS or its cognate response regulator GacA abolished the strain's response to added signal. Deletion of the linker domain of the GacS sensor kinase caused signal-independent, strongly elevated expression of exoproduct genes at low cell densities. In contrast to the wild-type strain CHA0, the gacS linker mutant and a gacS null mutant were unable to protect tomato plants from crown and root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici in a soil-less microcosm, indicating that, at least in this plant-pathogen system, there is no advantage in using a signal-independent biocontrol strain.