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 factor¹. 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^{2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20}. 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.

^{细菌转录调节的主要形式是通过 RNA 聚合酶 (RNAP) 的主要 σ 因子与替代胞质外功能 (ECF) σ 因子1}的交换而发生。ECF σ 因子通常本质上是活跃的，并通过隔离到 σ 因子-抗 σ 因子复合物中而保留在非活性状态，直到其作用得到保证^{2,3,4,5,6,7,8,9,10,11 ,12,13,14,15,16,17,18,19,20}。在这里，我们报告了一种先前未表征的转录调节机制，该机制依赖于本质上不活跃的 ECF σ 因子，其激活以及与 RNAP β' 亚基的相互作用取决于 σ 因子磷酸化。在副溶血弧菌中，苏氨酸激酶 PknT 磷酸化 σ 因子 EcfP，从而导致 EcfP 激活并表达重要的多粘菌素抗性调节子。EcfP 磷酸化发生在高度保守的苏氨酸残基 Thr63 处，该残基位于 σ2.2 螺旋的发散区域内。我们的数据表明，EcfP 本质上是不活跃的，并且由于该区域不存在带负电荷的 DAED 基序，因此无法结合 RNAP 的 β' 亚基。此外，我们的结果表明，残基 Thr63 处的磷酸化模拟了这种负电荷，并允许 EcfP 与 β' 亚基相互作用形成 RNAP 全酶，进而导致靶基因表达。这种调节机制是细菌信号转导和转录调节中以前未被认识的范例，我们的数据表明它在细菌中广泛存在。