Extra cytoplasmic function σ factor activation

The bacterial cell envelope is essential for cell viability and is a target for numerous antibiotics and host immune defenses. Thus bacteria must sense and respond to damage to the cell envelope. Many bacteria utilize alternative σ factors such as extracytoplasmic function (ECF) σ factors to respond to cell envelope stress. Although ECF σ factors are utilized by both Gram negative and Gram positive bacteria to respond to cell envelope stress, the mechanisms of sensing differ. In this review, we examine the events and proteins that are required for activation of two model extracytoplasmic function σ factors, σ(E) in E. coli and σ(W) in B. subtilis.     

Discovery of the extracytoplasmic function σ factors

This special issue of Molecular Microbiology marks the 25^th anniversary of the discovery of the extracytoplasmic function (ECF) σ factors, proteins that subsequently emerged as the largest group of alternative σ factors and one of the three major pillars of signal transduction in bacteria, alongside one‐ and two‐component systems. A single bacterial genome can encode > 100 ECF σ factors, and combined with their cognate anti‐σ factors, they represent a modular design that primarily functions in transmembrane signal transduction. Here, we first describe the immediate events that led to the 1994 publication in the Proceeding of the National Academy of Sciences USA, and then set them in the broader context of key events in the history of σ biology research.     

Analysis of the role of Bacillus subtilis σ(M) in β-lactam resistance reveals an essential role for c-di-AMP in peptidoglycan homeostasis

The Bacillus subtilis extracytoplasmic function (ECF) σ factor σ(M) is inducible by, and confers resistance to, several cell envelope-acting antibiotics. Here, we demonstrate that σ(M) is responsible for intrinsic β-lactam resistance, with σ(X) playing a secondary role. Activation of σ(M) upregulates several cell wall biosynthetic enzymes including one, PBP1, shown here to be a target for the beta-lactam cefuroxime. However, σ(M) still plays a major role in cefuroxime resistance even in cells lacking PBP1. To better define the role of σ(M) in β-lactam resistance, we characterized suppressor mutations that restore cefuroxime resistance to a sigM null mutant. The most frequent suppressors inactivated gdpP (yybT) which encodes a cyclic-di-AMP phosphodiesterase (PDE). Intriguingly, σ(M) is a known activator of disA encoding one of three paralogous diadenylate cyclases (DAC). Overproduction of the GdpP PDE greatly sensitized cells to β-lactam antibiotics. Conversely, genetic studies indicate that at least one DAC is required for growth with depletion leading to cell lysis. These findings support a model in which c-di-AMP is an essential signal molecule required for cell wall homeostasis. Other suppressors highlight the roles of ECF σ factors in counteracting the deleterious effects of autolysins and reactive oxygen species in β-lactam-treated cells.     

Planar substrate-binding site dictates the specificity of ECF-type nickel/cobalt transporters

The energy-coupling factor (ECF) transporters are multi-subunit protein complexes that mediate uptake of transition-metal ions and vitamins in about 50% of the prokaryotes, including bacteria and archaea. Biological and structural studies have been focused on ECF transporters for vitamins, but the molecular mechanism by which ECF systems transport metal ions from the environment remains unknown. Here we report the first crystal structure of a NikM, TtNikM2, the substrate-binding component (S component) of an ECF-type nickel transporter from Thermoanaerobacter tengcongensis. In contrast to the structures of the vitamin-specific S proteins with six transmembrane segments (TSs), TtNikM2 possesses an additional TS at its N-terminal region, resulting in an extracellular N-terminus. The highly conserved N-terminal loop inserts into the center of TtNikM2 and occludes a region corresponding to the substrate-binding sites of the vitamin-specific S components. Nickel binds to NikM via its coordination to four nitrogen atoms, which are derived from Met1, His2 and His67 residues. These nitrogen atoms form an approximately square-planar geometry, similar to that of the metal ion-binding sites in the amino-terminal Cu²⁺- and Ni²⁺-binding (ATCUN) motif. Replacements of residues in NikM contributing to nickel coordination compromised the Ni-transport activity. Furthermore, systematic quantum chemical investigation indicated that this geometry enables NikM to also selectively recognize Co²⁺. Indeed, the structure of TtNikM2 containing a bound Co²⁺ ion has almost no conformational change compared to the structure that contains a nickel ion. Together, our data reveal an evolutionarily conserved mechanism underlying the metal selectivity of EcfS proteins, and provide insights into the ion-translocation process mediated by ECF transporters.     

Regulatory overlap and functional redundancy among Bacillus subtilis extracytoplasmic function sigma factors

Bacillus subtilis encodes seven extracytoplasmic function (ECF) sigma factors that regulate partially overlapping regulons related to cell envelope homeostasis and antibiotic resistance. Here, we investigated their physiological role by constructing a mutant set of single, double, triple, and quadruple ECF sigma factor deletions in the undomesticated B. subtilis strain NCIB3610. This mutant set was subsequently screened for defects in motility, multicellular differentiation, and sensitivity to more than 200 chemicals by using Phenotype MicroArrays. A quadruple mutant strain, harboring deletions of the sigV, sigY, sigZ, and ylaC gene, behaved indistinguishably from the wild-type strain, indicative of either regulatory redundancy or very specific functions of these four ECF sigma factors. In contrast, a triple mutant, inactivated for the sigM, sigW, and sigX genes (but none of the corresponding double mutants), showed a biphasic growth behavior and a complete loss of multicellular differentiation, as judged by both colony formation and the inability to form a pellicle. This triple mutant also displayed a greatly increased sensitivity to detergents and several cell wall antibiotics including beta-lactams, polymyxin B, and d-cycloserine. In several cases, these antibiotic-sensitive phenotypes are significantly enhanced in the triple mutant strain relative to strains lacking only one or two sigma factors.     

Effects of deficiency and overdose of group 2 sigma factors in triple inactivation strains of Synechocystis sp. strain PCC 6803

Acclimation of cyanobacteria to environmental changes includes major changes in the gene expression patterns partly orchestrated by the replacement of a particular σ subunit with another in the RNA polymerase holoenzyme. The cyanobacterium Synechocystis sp. strain PCC 6803 encodes nine σ factors, all belonging to the σ(70) family. Cyanobacteria typically encode many group 2 σ factors that closely resemble the principal σ factor. We inactivated three out of the four group 2 σ factors of Synechocystis simultaneously in all possible combinations and found that all triple inactivation strains grow well under standard conditions. Unlike the other strains, the ΔsigBCD strain, which contains SigE as the only functional group 2 σ factor, did not grow faster under mixotrophic than under autotrophic conditions. The SigB and SigD factors were important in low-temperature acclimation, especially under diurnal light rhythm. The ΔsigBCD, ΔsigBCE, and ΔsigBDE strains were sensitive to high-light-induced photoinhibition, indicating a central role of the SigB factor in high-light tolerance. Furthermore, the ΔsigBCE strain (SigD is the only functional group 2 σ factor) appeared to be locked in the high-fluorescence state (state 1) and grew slowly in blue but not in orange or white light. Our results suggest that features of the triple inactivation strains can be categorized as (i) direct consequences of the inactivation of a particular σ factor(s) and (ii) effects resulting from the higher probability that the remaining group 2 σ factors associate with the RNA polymerase core.     

The single extracytoplasmic-function sigma factor of Xylella fastidiosa is involved in the heat shock response and presents an unusual regulatory mechanism

Genome sequence analysis of the bacterium Xylella fastidiosa revealed the presence of two genes, named rpoE and rseA, predicted to encode an extracytoplasmic function (ECF) sigma factor and an anti-sigma factor, respectively. In this work, an rpoE null mutant was constructed in the citrus strain J1a12 and shown to be sensitive to exposure to heat shock and ethanol. To identify the X. fastidiosa sigma(E) regulon, global gene expression profiles were obtained by DNA microarray analysis of bacterial cells under heat shock, identifying 21 sigma(E)-dependent genes. These genes encode proteins belonging to different functional categories, such as enzymes involved in protein folding and degradation, signal transduction, and DNA restriction modification and hypothetical proteins. Several putative sigma(E)-dependent promoters were mapped by primer extension, and alignment of the mapped promoters revealed a consensus sequence similar to those of ECF sigma factor promoters of other bacteria. Like other ECF sigma factors, rpoE and rseA were shown to comprise an operon in X. fastidiosa, together with a third open reading frame (XF2241). However, upon heat shock, rpoE expression was not induced, while rseA and XF2241 were highly induced at a newly identified sigma(E)-dependent promoter internal to the operon. Therefore, unlike many other ECF sigma factors, rpoE is not autoregulated but instead positively regulates the gene encoding its putative anti-sigma factor.     

CorE from Myxococcus xanthus is a copper-dependent RNA polymerase sigma factor

The dual toxicity/essentiality of copper forces cells to maintain a tightly regulated homeostasis for this metal in all living organisms, from bacteria to humans. Consequently, many genes have previously been reported to participate in copper detoxification in bacteria. Myxococcus xanthus, a prokaryote, encodes many proteins involved in copper homeostasis that are differentially regulated by this metal. A σ factor of the ECF (extracytoplasmic function) family, CorE, has been found to regulate the expression of the multicopper oxidase cuoB, the P1B-type ATPases copA and copB, and a gene encoding a protein with a heavy-metal-associated domain. Characterization of CorE has revealed that it requires copper to bind DNA in vitro. Genes regulated by CorE exhibit a characteristic expression profile, with a peak at 2 h after copper addition. Expression rapidly decreases thereafter to basal levels, although the metal is still present in the medium, indicating that the activity of CorE is modulated by a process of activation and inactivation. The use of monovalent and divalent metals to mimic Cu(I) and Cu(II), respectively, and of additives that favor the formation of the two redox states of this metal, has revealed that CorE is activated by Cu(II) and inactivated by Cu(I). The activation/inactivation properties of CorE reside in a Cys-rich domain located at the C terminus of the protein. Point mutations at these residues have allowed the identification of several Cys involved in the activation and inactivation of CorE. Based on these data, along with comparative genomic studies, a new group of ECF σ factors is proposed, which not only clearly differs mechanistically from the other σ factors so far characterized, but also from other metal regulators.     

The CarD/CarG regulatory complex is required for the action of several members of the large set of Myxococcus xanthus extracytoplasmic function σ factors

Extracytoplasmic function (ECF) σ factors are critical players in signal transduction networks involved in bacterial response to environmental changes. The Myxococcus xanthus genome reveals ～45 putative ECF‐σ factors, but for the overwhelming majority, the specific signals or mechanisms for selective activation and regulation remain unknown. One well‐studied ECF‐σ, CarQ, binds to its anti‐σ, CarR, and is inactive in the dark but drives its own expression from promoter P_QRS on illumination. This requires the CarD/CarG complex, the integration host factor (IHF) and a specific CarD‐binding site upstream of P_QRS. Here, we show that DdvS, a previously uncharacterized ECF‐σ, activates its own expression in a CarD/CarG‐dependent manner but is inhibited when specifically bound to the N‐terminal zinc‐binding anti‐σ domain of its cognate anti‐σ, DdvA. Interestingly, we find that the autoregulatory action of 11 other ECF‐σ factors studied here depends totally or partially on CarD/CarG but not IHF. In silico analysis revealed possible CarD‐binding sites that may be involved in direct regulation by CarD/CarG of target promoter activity. CarD/CarG‐linked ECF‐σ regulation likely recurs in other myxobacteria with CarD/CarG orthologous pairs and could underlie, at least in part, the global regulatory effect of the complex on M. xanthus gene expression.