The activity of extracytoplasmic function σ-factors (ECFs) is typically regulated by anti-σ factors. In a number of highly abundant ECF groups, including ECF41 and ECF42, σ-factors contain fused C-terminal protein domains, which provide the necessary regulatory function instead. Here, we identified the contact interface between the C-terminal extension and the core σ-factor regions required for controlling ECF activity. We applied direct coupling analysis (DCA) to infer evolutionary covariation between contacting amino acid residues for groups ECF41 and ECF42. Mapping the predicted interactions to a recently solved ECF41 structure demonstrated that DCA faithfully identified an important contact interface between the SnoaL-like extension and the linker between the σ 2 and σ 4 domains. Systematic alanine substitutions of contacting residues support this model and suggest that this interface stabilizes a compact conformation of ECF41 with low transcriptional activity. For group ECF42, DCA supports a structural homology model for their C-terminal tetratricopeptide repeat (TPR) domains and predicts an intimate contact between the first TPR-helix and the σ 4 domain. Mutational analyses demonstrate the essentiality of the predicted interactions for ECF42 activity. These results indicate that C-terminal extensions indeed bind and regulate the core ECF regions, illustrating the potential of DCA for discovering regulatory motifs in the ECF subfamily.
Extracytoplasmic function σ factors (ECFs) represent the third most abundant fundamental principle of bacterial signal transduction, outranked only by one- and two-component systems. A recent census of ECFs revealed a large number of novel groups whose functions and regulatory mechanisms have not yet been elucidated. Here, we report the characterization of members of the novel group ECF42. ECF42 is a highly abundant and widely distributed ECF group that is present in 11 phyla but is predominantly found in Analysis of the genomic context conservation did not identify a putative anti-σ factor. Instead, ECF42 genes are cotranscribed with genes encoding a conserved DGPF protein. We have experimentally verified the target promoter of these ECFs (TGTCGA in the -35 region and CGA/TC in the -10 region), which was found upstream of the ECF42-encoding operons in, suggesting that ECF42s are positively autoregulated. RNA sequencing (RNA-seq) was performed to define the regulons of the three ECF42 proteins in , which identified mostly genes encoding DGPF proteins. In contrast to typical ECFs, ECF42 proteins harbor a long C-terminal extension, which is crucial for their activity. Our work provides the first analysis of the function and regulatory mechanism of this novel ECF group that contains a regulatory C-terminal extension. In contrast to the one- and two-component signal transduction systems in bacteria, the importance and diversity of ECFs have only recently been recognized in the course of comprehensive phylogenetic and comparative genomics studies. Thus, most of the ECFs still have not been experimentally characterized regarding their physiological functions and regulation mechanisms so far. The physiological roles, target promoter, and target regulons of a novel group of ECFs, ECF42, in have been investigated in this work. More importantly, members of this group are characterized by a C-terminal extension, which has been verified to harbor a regulatory role in ECF42s. Hence, our work provides an important source for further research on such C-terminal extension containing ECFs.
The σ subunit of the bacterial RNA polymerase determines promoter specificity. The extracytoplasmic function σ factors (ECFs) represent the most abundant and diverse group of alternative σ factors and are present in the vast majority of bacterial genomes. Typically, ECFs are regulated by anti-σ factors that sequester their cognate ECFs, thereby preventing their interaction with the RNA polymerase. Beyond these ECF paradigms, a number of distinct modes of regulation have been proposed and experimentally investigated. Regulatory extensions represent one such alternative mechanism of ECF regulation that can be found in 18 phylogenetically distinct ECF groups. Here, the σ factors contain additional domains that are fused to the ECF core domains and are involved in stimulus perception and modulation of σ factor activity. We will summarize the current state of knowledge on regulating ECF activity by C-terminal extensions. We will also discuss newly identified ECF groups containing either N-or C-terminal extensions and propose possible mechanisms by which these extensions have been generated and affect ECF σ factor activity. Based on their modular architecture and the resulting physical connection between stimulus perception and transcriptional output, these ECFs are analogous to one-component systems, the primary mechanism of bacterial signal transduction. contributed equally to the writing of this review.
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