Role of Sphingomonas sp. strain Fr1 PhyR-NepR-σEcfG cascade in general stress response and identification of a negative regulator of PhyR

The general stress response in Alphaproteobacteria was recently described to depend on the alternative sigma factor σ(EcfG), whose activity is regulated by its anti-sigma factor NepR. The response regulator PhyR, in turn, regulates NepR activity in a partner-switching mechanism according to which phosphorylation of PhyR triggers sequestration of NepR by the sigma factor-like effector domain of PhyR. Although genes encoding predicted histidine kinases can often be found associated with phyR, little is known about their role in modulation of PhyR phosphorylation status. We demonstrate here that the PhyR-NepR-σ(EcfG) cascade is important for multiple stress resistance and competitiveness in the phyllosphere in a naturally abundant plant epiphyte, Sphingomonas sp. strain Fr1, and provide evidence that the partner switching mechanism is conserved. We furthermore identify a gene, designated phyP, encoding a predicted histidine kinase at the phyR locus as essential. Genetic epistasis experiments suggest that PhyP acts upstream of PhyR, keeping PhyR in an unphosphorylated, inactive state in nonstress conditions, strictly depending on the predicted phosphorylatable site of PhyP, His-341. In vitro experiments show that Escherichia coli inner membrane fractions containing PhyP disrupt the PhyR-P/NepR complex. Together with the fact that PhyP lacks an obvious ATPase domain, these results are in agreement with PhyP functioning as a phosphatase of PhyR, rather than a kinase.     

A structural model of anti‐anti‐σ inhibition by a two‐component receiver domain: the PhyR stress response regulator

PhyR is a hybrid stress regulator conserved in α‐proteobacteria that contains an N‐terminal σ‐like (SL) domain and a C‐terminal receiver domain. Phosphorylation of the receiver domain is known to promote binding of the SL domain to an anti‐σ factor. PhyR thus functions as an anti‐anti‐σ factor in its phosphorylated state. We present genetic evidence that Caulobacter crescentus PhyR is a phosphorylation‐dependent stress regulator that functions in the same pathway as σ^T and its anti‐σ factor, NepR. Additionally, we report the X‐ray crystal structure of PhyR at 1.25 Å resolution, which provides insight into the mechanism of anti‐anti‐σ regulation. Direct intramolecular contact between the PhyR receiver and SL domains spans regions σ₂ and σ₄, likely serving to stabilize the SL domain in a closed conformation. The molecular surface of the receiver domain contacting the SL domain is the structural equivalent of α4‐β5‐α5, which is known to undergo dynamic conformational change upon phosphorylation in a diverse range of receiver proteins. We propose a structural model of PhyR regulation in which receiver phosphorylation destabilizes the intramolecular interaction between SL and receiver domains, thereby permitting regions σ₂ and σ₄ in the SL domain to open about a flexible connector loop and bind anti‐σ factor.     

Multiple σEcfG and NepR Proteins Are Involved in the General Stress Response in Methylobacterium extorquens

In Alphaproteobacteria, the general stress response (GSR) is controlled by a conserved partner switch composed of the sigma factor σ^EcfG, its anti-sigma factor NepR and the anti-sigma factor antagonist PhyR. Many species possess paralogues of one or several components of the system, but their roles remain largely elusive. Among Alphaproteobacteria that have been genome-sequenced so far, the genus Methylobacterium possesses the largest number of σ^EcfG proteins. Here, we analyzed the six σ^EcfG paralogues of Methylobacterium extorquens AM1. We show that these sigma factors are not truly redundant, but instead exhibit major and minor contributions to stress resistance and GSR target gene expression. We identify distinct levels of regulation for the different sigma factors, as well as two NepR paralogues that interact with PhyR. Our results suggest that in M. extorquens AM1, ecfG and nepR paralogues have diverged in order to assume new roles that might allow integration of positive and negative feedback loops in the regulatory system. Comparison of the core elements of the GSR regulatory network in Methylobacterium species provides evidence for high plasticity and rapid evolution of the GSR core network in this genus.     

Non-specific, general and multiple stress resistance of growth-restricted Bacillus subtilis cells by the expression of the σB regulon

Bacillus subtilis cells respond almost immediately to different stress conditions by increasing the production of general stress proteins (GSPs). The genes encoding the majority of the GSPs that are induced by heat, ethanol, salt stress or by starvation for glucose, oxygen or phosphate belong to the σ^B-dependent general stress regulon. Despite a good understanding of the complex regulation of the activity of σ^B and knowledge of a very large number of general stress genes controlled by σ^B, first insights into the physiological role of this non-specific stress response have been obtained only very recently. To explore the physiological role of this regulon, we and others identified σ^B-dependent general stress genes and compared the stress tolerance of wild-type cells with mutants lacking σ^B or general stress proteins. The proteins encoded by σ^B-dependent general stress genes can be divided into at least five functional groups that most probably provide growth-restricted B. subtilis cells with a multiple stress resistance in anticipation of future stress. In particular, sigB mutants are impaired in non-specific resistance to oxidative stress, which requires the σ^B-dependent dps gene encoding a DNA-protecting protein. Protection against oxidative damage of membranes, proteins or DNA could be the most essential component of σ^B-mediated general stress resistance in growth-arrested aerobic Gram-positive bacteria. Other general stress genes have both a σ^B-dependent induction pathway and a second σ^B-independent mechanism of stress induction, thereby partially compensating for a σ^B deficiency in a sigB mutant. In contrast to sigB mutants, null mutations in genes encoding those proteins, such as clpP or clpC, cause extreme sensitivity to salt or heat.     

Cross-talk between the general stress response and sporulation initiation in Bacillus subtilis - the σ(B) promoter of spo0E represents an AND-gate

The general stress response and the decision-making processes of sporulation initiation are interconnected pathways in the regulatory network of Bacillus subtilis. In a previous study we provided evidence for a mechanism capable of impairing sporulation by σ(B) -dependent induction of spo0E, encoding a phosphatase specifically inactivating the sporulation master regulator Spo0A～P. Here we show that the σ(B) promoter (Pσ(B) ) of spo0E is responsive to sub-inhibitory levels of ethanol stress, producing a σ(B) -dependent sporulation deficient phenotype. In addition to positive regulation by σ(B) , we identified Rok, the repressor of comK, to be a direct repressor of spo0E expression from Pσ(B) . This constellation provides the possibility to integrate signals negatively acting on sporulation initiation through the σ(B) branch as well as a positive feedback loop acting on Pσ(B) by Rok that is most likely a direct consequence of Spo0A～P activity. Thus, the molecular mechanism described here offers the opportunity for cross-talk between the general stress response and sporulation initiation in the adaptational gene expression network of B.?subtilis.     

Alternate promoters direct stress-induced transcription of the Bacillus subtilis clpC operon

clpC ofBacillus subtilis is part of an operon containing six genes. Northern blot analysis suggested that all genes are co-transcribed and encode stress-inducible proteins. Two promoters (P_A and P_B) were mapped upstream of the first gene. P_A resembles promoters recognized by the vegetative RNA polymerase Eσ^A. The other promoter (P_B) was shown to be dependent on σ^B, the general stress σ factor in B. subtilis, suggesting that clpC, a potential chaperone, is expressed in a σ^B-dependent manner. This is the first evidence that σ^B in B, subtilis is involved in controlling the expression of a gene whose counterpart, clpB, is subject to regulation by σ³² in Escherichia coli, indicating a new function of σ^B-dependent general stress proteins. P_B deviated from the consensus sequence of σ^B promoters and was only slightly induced by starvation conditions. Nevertheless, strong induction by heat, ethanol, and salt stress occurred at the σ^B-dependent promoter, whereas the vegetative promoter was only weakly induced under these conditions. However, in a sigB mutant, the σ^A-like promoter became inducible by heat and ethanol stress, completely compensating for sigB deficiency. Only the downstream σ^A-like promoter was induced by certain stress conditions such as hydrogen peroxide or puromycin. These results suggest that novel stress-induction mechanisms are acting at a vegetative promoter. Involvement of additional elements in this mode of induction are discussed.     

Back to log phase: σS as a global regulator in the osmotic control of gene expression in Escherichia coli

It is now well established that the σ^S subunit of RNA polymerase is a master regulator in a complex regulatory network that governs the expression of many stationary-phase-inducible genes in Escherichiacoli. In this review, more recent findings will be summarized that demonstrate that σ^S also acts as a global regulator for the osmotic control of gene expression, and actually does so in exponentially growing cells. Thus, many σ^S-dependent genes are induced during entry into stationary phase as well as in response to osmotic upshift. K⁺ glutamate, which accumulates in hyperosmotically stressed cells, seems to specifically stimulate the activity of σ^S-containing RNA polymerase at σ^S-dependent promoters. Moreover, osmotic upshift results in an elevated cellular σ^S level similar to that observed in stationary-phase cells. This increase is the result of a stimulation of rpoS translation as well as an inhibition of the turnover of σ^S, which in exponentially growing non-stressed cells is a highly unstable protein. Whereas the RNA-binding protein HF-I, previously known as a host factor for the replication of phage Qβ RNA, is essential for rpoS translation, the recently discovered response regulator RssB, and ClpXP protease, have been shown to be required for σ^S degradation. The finding that the histone-like protein H-NS is also involved in the control of rpoS translation and σ^S turnover, sheds new light on the function of this protein in osmoregulation. Finally, preliminary evidence suggests that additional stresses, such as heat shock and acid shock, also result in increased cellular σ^S levels in exponentially growing cells. Taken together, σ^S function is clearly not confined to stationary phase. Rather, σ^S may be regarded as a sigma factor associated with general stress conditions.