## 3 Structure and Function of Alternative \(\bullet\) Factors
### _Group 4 (ECF) \(\sigma\) Factors_  
Group 4 is also known as the ExtraCytoplasmic Function (ECF) group on account of the frequent role of members in sensing and responding to signals that are generated outside of the cell or in the cell membrane [52]. The ECF group is numerically by far the largest and most diverse at the primary sequence level, consisting of at least 43 major phylogenetically distinct sub-groups [53]. Biological roles include envelope stress response (e.g., _E. coli_\(\sigma^{\mathrm{E}}\), _B. subtilis_\(\sigma^{\mathrm{W}}\)), iron transport (_E. coli_\(\sigma^{\mathrm{F}\mathrm{e}el}\), _Pseudomonas aeruginosa_\(\sigma^{\mathrm{PvdS}}\)), oxidative stress (_S. coelicolor_\(\sigma^{\mathrm{R}}\) and _Rhodobacter sphaeroides_\(\sigma^{\mathrm{E}}\)), and the general stress response (e.g., _Methylobacterium extorquens_\(\sigma^{\mathrm{EcfG1}}\)). The number of ECF \(\sigma\) factors encoded by a genome can vary enormously from zero in obligate intracellular bacteria such as Chlamydia to over 115 in the planktomycete _Gemmata obscuriglobus_[54]. Some ECF subgroups are widespread in bacterial phyla, indicating an ancient origin, while most tend to be phylum-specific. Indeed in their initial classification of 2700 ECF \(\sigma\) factors, [PERSON] and colleagues identified 24 minor groups and hundreds of proteins that could not be sub-classified [53]. Clearly, as genome sequencing technology is directed towards less studied phyla, many additional major and minor subgroups will be uncovered. The presence of multiple ECF \(\sigma\) factors raises the question of whether cross-talk occurs, such that some promoters are recognised by several \(\sigma\) factors, potentially allowing the integration of distinct signals towards a coordinated response. This is the case in _B. subtilis_ where cell envelope stress response is coordinated by several ECF \(\sigma\) factors with partially overlapping regulons [55]. Nonetheless, a study that tested for orthogonality among the broad spectrum of ECF \(\sigma\) factors concluded that there is generally little cross-talk between different subclasses, ensuring insulated transcriptional responses [56]. ECF \(\sigma\) genes are often clustered with the genes they regulate when the regulons are small, although some, such as \(\sigma^{\mathrm{R}}\) in _S. coelicolor_, target \(>\)100 genes [57, 58]. ECF \(\sigma\) factors lack both \(\sigma_{1.1}\) and \(\sigma_{3}\), which makes them the most minimal \(\sigma\) factors (Figure 1a). They also lack the first helix of \(\sigma\)R1.2 that is responsible for discriminator interactions in Group 1/2 \(\sigma\) factors [18, 20] (Figure 2). While most are small, there are exceptions including members of the widely distributed ECF41 subfamily that have \(\sim\)100 amino acid residue C-terminal extensions that are thought to modulate activity [59]. Promoters recognised by ECF \(\sigma\) factors include a \(-\)35 \"AAC\" motif in two-thirds of examples and more diverged sequences at \(-\)10 [53], suggesting that promoter specificity is derived primarily from interactions between \(\sigma_{2}\) and the \(-\)10 element (although \(-\)35 interactions are also important [56]). Consistent with this, key residues that interact with the \(-\)10 region are on a variable loop. The recent structure of _E. coli_\(\sigma^{E}_{2}\) in complex with non-template \(-\)10 ssDNA (G\({}_{12}\)TCAAA\(\cdot\)\(\tau\)) revealed that, unlike the case for Group 1 \(\sigma\) factors, only a single base (C\({}_{-\)10}\) was flipped out from the ssDNA base stack [23] (Figure 2). This nucleotide is wrapped by a helical hinge in \(\sigma\)R2.3 (\(\sigma\)3-loop L3\(-\)\(\alpha\)4) with the loop becoming ordered upon C\({}_{-\)10 binding. Remarkably, the replacement of this loop in \(\sigma^{E}\) with the equivalent regions from other ECF \(\sigma\) factors with altered \(-\)10 recognition switched promoter specificity.