Transcriptional regulation by σ factor phosphorylation in bacteria

A major form of transcriptional regulation in bacteria occurs through the exchange of the primary σ factor of RNA polymerase (RNAP) with an alternative extracytoplasmic function (ECF) σ factor 1 . ECF σ factors are generally intrinsically active and are retained in an inactive state via the sequestration into σ factor–anti-σ factor complexes until their action is warranted 2 – 20 . Here, we report a previously uncharacterized mechanism of transcriptional regulation that relies on intrinsically inactive ECF σ factors, the activation of which and interaction with the β′-subunit of RNAP depends on σ factor phosphorylation. In Vibrio parahaemolyticus , the threonine kinase PknT phosphorylates the σ factor EcfP, which results in EcfP activation and expression of an essential polymyxin-resistant regulon. EcfP phosphorylation occurs at a highly conserved threonine residue, Thr63, positioned within a divergent region in the σ2.2 helix. Our data indicate that EcfP is intrinsically inactive and unable to bind the β′-subunit of RNAP due to the absence of a negatively charged DAED motif in this region. Furthermore, our results indicate that phosphorylation at residue Thr63 mimics this negative charge and licenses EcfP to interact with the β′-subunit in the formation of the RNAP holoenzyme, which in turn results in target gene expression. This regulatory mechanism is a previously unrecognized paradigm in bacterial signal transduction and transcriptional regulation, and our data suggest that it is widespread in bacteria. Alternative σ factors regulate the activity of RNA polymerases under specific conditions and are regulated through various mechanisms, most of which depend on anti-σ factors to regulate their on/off status. This study reports a new mode of σ factor regulation that does not require an anti-σ factor, but instead σ factor phosphorylation in response to the presence of an antibiotic.