Dokument

Summary:
A major form of bacterial transcriptional regulation occurs by the exchange of the primary σ factor of the RNA polymerase with alternative ECF σ factors, which generally are retained in an inactive state by sequestration into σ/anti-σ factor complexes (until they are needed). Using Vibrio parahaemolyticus as a model organism, we report a novel mechanism of transcriptional regulation, which instead relies on intrinsically inactive ECF σ factors that in turn rely on σ factor phosphorylation for interaction with the RNA polymerase. Particularly, we show that upon polymyxin stress, the threonine kinase PknT phosphorylates the σ factor EcfP, resulting in EcfP activation and expression of an essential polymyxin resistance regulon. EcfP phosphorylation occurs at a highly conserved threonine residue, Thr63, positioned within a divergent region in the σ2.2 helix. EcfP is intrinsically inactive and unable to bind RNA polymerase due to the absence of a negatively charged DAED motif in this region. Our results indicate that phosphorylation at residue Thr63 mimics this negative charge and licenses EcfP for interaction with RNA polymerase and activation of target gene expression. Regulation of gene expression by phosphorylation of ECF σ factor is likely a widespread mechanism in bacteria, presenting a new paradigm in transcriptional regulation. One of the unique features of V. parahaemolyticus is that it exhibits a dual lifestyle. In liquid environments, where the bacteria are free-living, they exist as short swimmer cells with a single polar flagellum. However, upon encountering solid surfaces, the bacteria differentiate into highly elongated swarmer cells that are characterized by the presence of numerous peritrichous lateral flagella. Here, we report the involvement of the aforementioned threonine kinase/ECF σ factor system, namely PknT/EcfP, in regulation of swarming behavior. Strikingly, our findings indicate that this regulatory role depends on a phosphorylation-driven mechanism. We also provide evidence for the role of two other proteins encoded by genes present within the same operon as that of pknT and ecfP, namely VP0054 and VP0056, in regulation of swarming behavior. Our findings also reveal several key targets such as transporters and proteins involved in certain biosynthetic processes that are regulated by PknT. This is the first time that STKs have been implicated in swarming behavior in bacteria.

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