# Extracytoplasmic Function \(\sigma\) Factors as Tools for Coordinating Stress Responses
## 5 Regulation of the ECF Activity and Production  
From the perspective of the regulation of their activity, the functional control of ECFs can be grouped into  we general mechanisms, although each type of ECF may present distinctive regulatory features [59]. These include regulation by membrane-anchored anti-  factors, by soluble anti-  factors, through serine/threonine protein kinases (STPKs) and via C- and N-terminal extensions. Additionally, some ECFs may be regulated exclusively by controlling their production at the transcriptional level. All these regulatory mechanisms are represented in Figure 4. Of these, membrane-anchored anti-  factors represent the ancestral regulatory mechanism for ECFs from which the rest of them emerged [59], and is the most widespread (74% of the ECFs with a known regulatory mechanism). Soluble anti-  factors arose from the ancestor membrane-anchored proteins by means of gene truncations that led to a separation of the cytosolic anti-  domain from the transmembrane helices [59]. Anti-  factors are usually encoded by genes located in the vicinity of the gene encoding the target ECF, or even within the same transcriptional unit. They prevent the ECF from functioning by titration, while it is not needed. To release ECFs from inhibition, anti-  factors may undergo proteolysis, the most frequent mechanism for membrane-bound anti-  factors [59], or they may undergo conformational changes (or partner-switching events in the case of examples related to the regulation of the alphaproteobacterial general stress response, explained in detail below) [22]. Crystallography and homology modelling  
Figure 3: Diagram of the different groups of ECF  factors according to their possible functional role. Note that most of the groups of ECFs do not have a single experimentally characterized representative. analyses show a coevolution between the contact surfaces of ASDIs and their respective ECF pair [59]. Within ASDIs, structural studies have reported that in the EcfG-NepR and CnrH-CnrY pairs from S. melonisand Cupriavidus metalliduransrespectively [22, 64], the anti- factor embraces the outer surface of the ECF, preventing the interaction with the RNAP. Finally, a structural study of the ECF-anti- interaction of the BldN-RsbN pair from Streptomyces werezuelak60] shows that RsbN is able to prevent BldN from binding the RNAP, by directly blocking access of the \({}_{4}\) domain to the -35 box and by sequestering BldN to the membrane, as the anti- is a membrane-anchored protein. By comparing the structure of ASDIs and ASDIs, RsbN served to de ne the ASDIII anti- factor class for the rst time. STPKs were initially thought to have a minor role in signal transduction in Bacteria compared to their major regulatory roles in Eukarya However, the presence of coding genes for STPKs in bacterial genomes revealed through the numerous genome annotation projects developed in recent years has attracted the attention of microbiologists. In particular, ECFs classi ed into groups ECF43, ECF59 and ECF60 (the latter no longer appearing in the latest classi cation, with its original members not clustering in any of the current ones [56]) are encoded by genes located near genes encoding an STPK. Further analyses of this synteny in Xanthomonas cirtrevealed that a neighboring STPK (PKnS) is essential for the activation of its cognate ECF (EcfK), which regulates the Type VI Secretion System of this strain [65]. Recent studies have shed light on the mechanistic aspects of this regulation in the pair formed by EcfP and the STPK PknT in Vibrio parahaemolyticu66], which controls polymxin resistance. One key contact between the negatively charged DAED motif located in the \({}_{2}\) domain of factors and the positively charged helices located in the '-clamp of the core RNAP is essential for the formation of the holoenzyme [66, 67]. In STPK-regulated ECFs, the DAED motif is substituted by non-charged residues (STTA in EcfP).