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Summary The CckA-ChpT-CtrA phosphorelay is central to the regulation of the cell cycle in Caulobacter crescentus. The three proteins are conserved in Alphaproteobacteria, but little is known about their roles in most members of this class. Here, we characterized the system in Sphingomonas melonis. We found that the transcription factor CtrA is the master regulator of flagella synthesis genes, the hierarchical transcriptional organization of which is herein described. CtrA also regulates genes involved in exopolysaccharide synthesis and cyclic-di-GMP signaling, and is important for biofilm formation. In addition, the ctrA mutant exhibits an aberrant morphology, suggesting a role for CtrA in cell division. An analysis of the regulation of CtrA indicates that the phosphorelay composed of CckA and ChpT is conserved and that the absence of the bifunctional kinase/phosphatase CckA apparently results in overactivation of CtrA through ChpT. Suppressors of this phenotype identified the hybrid histidine kinase CcsA. Phosphorelays initiated by CckA or CcsA were reconstituted in vitro, suggesting that in S. melonis, CtrA phosphorylation is controlled by a branched pathway upstream of ChpT. This study thus suggests that signals can directly converge at the level of ChpT phosphorylation through multiple hybrid kinases to coordinate a number of important physiological processes.

Tunable promoters represent a pivotal genetic tool for a wide range of applications. Here we present such a system for sphingomonads, a phylogenetically diverse group of bacteria that have gained much interest for their potential in bioremediation and their use in industry and for which no dedicated inducible gene expression system has been described so far. A strong, constitutive synthetic promoter was first identified through a genetic screen and subsequently combined with the repressor and the operator sites of the Pseudomonas putida F1 cym / cmt system. The resulting promoter, termed P Q5 , responds rapidly to the inducer cumate and shows a maximal induction ratio of 2 to 3 orders of magnitude in the different sphingomonads tested. Moreover, it was also functional in other Alphaproteobacteria , such as the model organisms Caulobacter crescentus , Paracoccus denitrificans , and Methylobacterium extorquens . In the noninduced state, expression from P Q5 is low enough to allow gene depletion analysis, as demonstrated with the essential gene phyP of Sphingomonas sp. strain Fr1. A set of P Q5 -based plasmids has been constructed allowing fusions to affinity tags or fluorescent proteins.

The genes involved in flagellum synthesis, motility and chemotaxis in Escherichia coli are expressed in a hierarchical fashion. At the top of the hierarchy lies the master regulator FlhDC, required for the expression of the whole set of genes. The operon flhDC is controlled by numerous regulators including H-NS, CRP, EnvZ/OmpR, QseBC and LrhA. In the present work, we report that the flhDC operon is also negatively regulated by the His-Asp phosphorelay system RcsCDB. The regulation is potentiated by the RcsB cofactor RcsA. Genetic analysis indicates that an RcsAB box, located downstream of the promoter, is required for the regulation. The binding of RcsB and RcsA to this site was demonstrated by gel retardation and DNase I protection assays. In addition, mutation analysis suggests that RcsA-specific determinants lie in the right part of the 'RcsAB box'.

Two-component systems are major signal transduction pathways, which consist of histidine kinases and response regulators that communicate through phosphorylation. Here, we highlight a distinct class of single-domain response regulators containing the PFXFATG[G/Y] motif that are activated by a mechanism distinct from the Y-T coupling described for prototypical receiver domains. We first solved the structures of inactive and active SdrG, a representative of the FAT GUY family, and then biochemically and genetically characterized variants in which residues in this motif were mutated. Our results support a model of activation mainly driven by a conserved lysine and reveal that the rotation of the threonine induces the reorganization of several aromatic residues in and around the PFXFATG[G/Y] motif to generate intermediates resembling those occurring during classical Y-T coupling. Overall, this helps define a new subfamily of response regulators that emerge as important players in physiological adaptation.

Here we suggest that natural streptomycin resistance of many sphingomonads resides within rpsL. We constructed a dominant streptomycin sensitive rpsL allele and demonstrated its use as a counterselection marker in several sphingomonads. An rpsL based markerless gene deletion system was developed and validated by deleting four genes in Sphingomonas sp. strain Fr1.

Bacteria have evolved regulatory traits to rapidly adapt to changing conditions. Two principal regulatory mechanisms to modulate gene expression consist of regulation via alternative sigma factors and phosphorylation-dependent response regulators. PhyR represents a recently discovered protein family combining parts of both systems: a sigma factor-like domain of the extracytoplasmic function (ECF) subfamily linked to a receiver domain of a response regulator. Here we investigated the mode of action of this key regulator of general stress response in Methylobacterium extorquens . Our results indicate that PhyR does not act as a genuine sigma factor but instead controls gene expression indirectly through protein-protein interactions. This is evident from the analysis of additional proteins involved in PhyR-dependent gene regulation. We demonstrated that the ECF sigma factor-like domain of PhyR interacts with a protein, designated NepR, upon phosphorylation of the PhyR receiver domain. Using transcriptome analysis and phenotypic assays, we showed that NepR is a negative regulator of PhyR response. Furthermore, we provide biochemical and genetic evidence that NepR exerts this inhibitory effect through sequestration of the ECF sigma factor σ EcfG1 . Our data support an unprecedented model according to which PhyR acts as a mimicry protein triggering a partner-switching mechanism. Such a regulation of general stress response clearly differs from the two known models operating via σ S and σ B . Given the absence of these master regulators and the concomitant conservation of PhyR in Alphaproteobacteria, the novel mechanism presented here is most likely central to the control of general stress response in this large subclass of Proteobacteria.

The CckA-ChpT-CtrA phosphorelay is central to the regulation of the cell cycle in Caulobacter crescentus. The three proteins are conserved in Alphaproteobacteria, but little is known about their roles in most members of this class. Here, we characterized the system in Sphingomonas melonis. We found that the transcription factor CtrA is the master regulator of flagella synthesis genes, the hierarchical transcriptional organization of which is herein described. CtrA also regulates genes involved in exopolysaccharide synthesis and cyclic-di-GMP signaling, and is important for biofilm formation. In addition, the ctrA mutant exhibits an aberrant morphology, suggesting a role for CtrA in cell division. An analysis of the regulation of CtrA indicates that the phosphorelay composed of CckA and ChpT is conserved and that the absence of the bifunctional kinase/phosphatase CckA apparently results in overactivation of CtrA through ChpT. Suppressors of this phenotype identified the hybrid histidine kinase CcsA. Phosphorelays initiated by CckA or CcsA were reconstituted in vitro, suggesting that in S. melonis, CtrA phosphorylation is controlled by a branched pathway upstream of ChpT. This study thus suggests that signals can directly converge at the level of ChpT phosphorylation through multiple hybrid kinases to coordinate a number of important physiological processes.

The general stress response (GSR) allows bacteria to monitor and defend against a broad set of unrelated, adverse environmental conditions. In Alphaproteobacteria , the key step in GSR activation is phosphorylation of the response regulator PhyR. In Sphingomonas melonis Fr1, seven PhyR-activating kinases (Paks), PakA to PakG, are thought to directly phosphorylate PhyR under different stress conditions, but the nature of the activating signals remains obscure. PakF, a major sensor of NaCl and heat shock, lacks a putative sensor domain but instead harbors a single receiver (REC) domain (PakF REC ) N-terminal to its kinase catalytic core. Such kinases are called “hybrid response regulators” (HRRs). How HRRs are able to perceive signals in the absence of a true sensor domain has remained largely unexplored. In the present work, we show that stresses are actually sensed by another kinase, KipF (kinase of PakF), which phosphorylates PakF REC and thereby activates PakF. KipF is a predicted transmembrane kinase, harboring a periplasmic CHASE3 domain flanked by two transmembrane helices in addition to its cytoplasmic kinase catalytic core. We demonstrate that KipF senses different salts through its CHASE3 domain but is not a sensor of general osmotic stress. While salt sensing depends on the CHASE3 domain, heat shock sensing does not, suggesting that these stresses are perceived by different mechanisms. In summary, our results establish a two-tiered histidine kinase pathway involved in activation of the GSR in S. melonis Fr1 and provide the first experimental evidence for the so far uncharacterized CHASE3 domain as a salt sensor. IMPORTANCE Hybrid response regulators (HRRs) represent a particular class of histidine kinases harboring an N-terminal receiver (REC) domain instead of a true sensor domain. This suggests that the actual input for HRRs may be phosphorylation of the REC domain. In the present study, we addressed this question by using the HRR PakF. Our results suggest that PakF is activated through phosphorylation of its REC domain and that this is achieved by another kinase, KipF. KipF senses heat shock and salt stress, with the latter requiring the periplasmic CHASE3 domain. This work not only suggests that HRRs work in two-tiered histidine kinase pathways but also provides the first experimental evidence for a role of the so far uncharacterized CHASE3 domain in salt sensing.

Transcription of the Escherichia coli osmB gene is induced by several stress conditions. osmB is expressed from two promoters, osmBp1 and osmBp2 . The downstream promoter, osmBp2 , is induced after osmotic shock or upon entry into stationary phase in a σ S -dependent manner. The upstream promoter, osmBp1 , is independent of σ S and is activated by RcsB, the response regulator of the His-Asp phosphorelay signal transduction system RcsCDB. RcsB is responsible for the induction of osmBp1 following treatment with chlorpromazine. Activation of osmBp1 by RcsB requires a sequence upstream of its −35 element similar to the RcsB binding site consensus, suggesting a direct regulatory role. osmB appears as another example of a multistress-responsive gene whose transcription involves both a σ S -dependent promoter and a second one independent of σ S but controlled by stress-specific transcription factors.

Sinorhizobium meliloti is an α-proteobacterium that alternates between a free-living phase in bulk soil or in the rhizosphere of plants and a symbiotic phase within the host plant cells, where the bacteria ultimately differentiate into nitrogen-fixing organelle-like cells, called bacteroids. As a step toward understanding the physiology of S. meliloti in its free-living and symbiotic forms and the transition between the two, gene expression profiles were determined under two sets of biological conditions: growth under oxic versus microoxic conditions, and in free-living versus symbiotic state. Data acquisition was based on both macro- and microarrays. Transcriptome profiles highlighted a profound modification of gene expression during bacteroid differentiation, with 16% of genes being altered. The data are consistent with an overall slow down of bacteroid metabolism during adaptation to symbiotic life and acquisition of nitrogen fixation capability. A large number of genes of unknown function, including potential regulators, that may play a role in symbiosis were identified. Transcriptome profiling in response to oxygen limitation indicated that up to 5% of the genes were oxygen regulated. However, the microoxic and bacteroid transcriptomes only partially overlap, implying that oxygen contributes to a limited extent to the control of symbiotic gene expression.

Summary The genes involved in flagellum synthesis, motility and chemotaxis in Escherichia coli are expressed in a hierarchical fashion. At the top of the hierarchy lies the master regulator FlhDC, required for the expression of the whole set of genes. The operon flhDC is controlled by numerous regulators including H-NS, CRP, EnvZ/OmpR, QseBC and LrhA. In the present work, we report that the flhDC operon is also negatively regulated by the His-Asp phosphorelay system RcsCDB. The regulation is potentiated by the RcsB cofactor RcsA. Genetic analysis indicates that an RcsAB box, located downstream of the promoter, is required for the regulation. The binding of RcsB and RcsA to this site was demonstrated by gel retardation and DNase I protection assays. In addition, mutation analysis suggests that RcsA-specific determinants lie in the right part of the ‘RcsAB box’.

The general stress response (GSR) in Alphaproteobacteria was recently shown to be controlled by a partner-switching mechanism that is triggered by phosphorylation of the response regulator PhyR. Activation of PhyR ultimately results in release of the alternative extracytoplasmic function sigma factor σ(EcfG), which redirects transcription toward the GSR. Little is known about the signal transduction pathway(s) controlling PhyR phosphorylation. Here, we identified the single-domain response regulator (SDRR) SdrG and seven histidine kinases, PakA to PakG, belonging to the HWE/HisKA2 family as positive modulators of the GSR in Sphingomonas melonis Fr1. Phenotypic analyses, epistasis experiments, and in vitro phosphorylation assays indicate that Paks directly phosphorylate PhyR and SdrG, and that SdrG acts upstream of or in concert with PhyR, modulating its activity in a nonlinear pathway. Furthermore, we found that additional SDRRs negatively affect the GSR in a way that strictly requires PhyR and SdrG. Finally, analysis of GSR activation by thermal, osmotic, and oxidative stress indicates that Paks display different degrees of redundancy and that a specific kinase can sense multiple stresses, suggesting that the GSR senses a particular condition as a combination of, rather than individual, molecular cues. This study thus establishes the alphaproteobacterial GSR as a complex and interlinked network of two-component systems, in which multiple histidine kinases converge to PhyR, the phosphorylation of which is, in addition, subject to regulation by several SDRRs. Our finding that most HWE/HisKA2 kinases contribute to the GSR in S. melonis Fr1 opens the possibility that this notion might also be true for other Alphaproteobacteria.

The general stress response (GSR) is a widely conserved response that allows bacteria to cope with a multitude of stressful conditions. In the past years the PhyR-NepR-σ(EcfG) cascade was identified as the core pathway regulating the GSR in Alphaproteobacteria, in which it also plays an important role in bacteria-host interactions. The regulatory system is composed of the extracytoplasmic function sigma factor σ(EcfG), its anti-sigma factor NepR (for negative regulator of the PhyR response), and the anti-sigma factor antagonist PhyR (phyllosphere regulator). The three proteins function via a partner-switching mechanism that is triggered by PhyR phosphorylation, termed 'sigma factor mimicry'. This review will cover core features of the pathway, its physiological role, and summarize recent advances towards understanding of the partner-switching mechanism and of the two-component signaling pathways controlling the GSR.

Regulated promoters are an important basic genetic tool allowing, for example, gene-dosage and gene depletion studies. We have previously described a cumate-inducible promoter (PQ5) that is functional in diverse Alphaproteobacteria. This promoter has been engineered by combining a synthetic minimal promoter, Psyn2, and operator sites and the repressor of the Pseudomonas putida F1 cym/cmt system. In the present study, we engineered a vanillate-regulated promoter using Psyn2 and the regulatory elements of the Caulobacter crescentus vanR-vanAB system. We show that the resulting promoter, which we called PV10, responds rapidly to the inducer vanillate with an induction ratio of about two orders of magnitude in Sphingomonas melonis Fr1. In contrast to the switch-like behavior of PQ5, PV10 shows a linear dose-response curve at intermediate vanillate concentrations, allowing graded gene expression. PV10 is functionally compatible with and independent of PQ5 and cumate, and vice versa, suggesting that both systems can be used simultaneously., Scientific Reports, 4, ISSN:2045-2322

The general stress response of alphaproteobacteria is regulated by a partner-switching mechanism that involves the alternative sigma factor σ(EcfG), the anti-sigma factor NepR and the anti-sigma factor antagonist PhyR. To address the question of how the PhyR-NepR-σ(EcfG) cascade is activated and modulated in Methylobacterium extorquens, a forward genetic screen was applied. The screen identified the single-domain response regulator Mext_0407 as a novel regulatory element in the general stress response of M. extorquens. Analysis of phenotypes and of transcriptional fusions of PhyR-dependent genes shows that the mext_0407 deletion mutant fails to respond to various stresses. Mext_0407 requires the putative phosphorylatable aspartate-64 for its activity in vivo and genetic evidence indicates that Mext_0407 operates upstream of the PhyR-NepR-σ(EcfG) cascade.

Reprogramming gene expression is an essential component of adaptation to changing environmental conditions. In bacteria, a widespread mechanism involves alternative sigma factors that redirect transcription toward specific regulons. The activity of sigma factors is often regulated through sequestration by cognate anti-sigma factors; however, for most systems, it is not known how the activity of the anti-sigma factor is controlled to release the sigma factor. Recently, the general stress response sigma factor in Alphaproteobacteria, σ EcfG , was identified. σ EcfG is inactivated by the anti-sigma factor NepR, which is itself regulated by the response regulator PhyR. This key regulator sequesters NepR upon phosphorylation of its PhyR receiver domain via its σ EcfG sigma factor-like output domain (PhyR SL ). To understand the molecular basis of the PhyR-mediated partner-switching mechanism, we solved the structure of the PhyR SL –NepR complex using NMR. The complex reveals an unprecedented anti-sigma factor binding mode: upon PhyR SL binding, NepR forms two helices that extend over the surface of the PhyR SL subdomains. Homology modeling and comparative analysis of NepR, PhyR SL , and σ EcfG mutants indicate that NepR contacts both proteins with the same determinants, showing sigma factor mimicry at the atomic level. A lower density of hydrophobic interactions, together with the absence of specific polar contacts in the σ EcfG –NepR complex model, is consistent with the higher affinity of NepR for PhyR compared with σ EcfG . Finally, by reconstituting the partner switch in vitro, we demonstrate that the difference in affinity of NepR for its partners is sufficient for the switch to occur.

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.

PhyR is an unusual type of response regulator consisting of a receiver domain and an extracytoplasmic function (ECF) sigma factor-like domain. It was recently described as a master regulator of general stress response in Methylobacterium extorquens. Orthologues of this regulator are present in essentially all free-living Alphaproteobacteria. In most of them, phyR is genetically closely linked to a gene encoding an ECF sigma factor. Here, we investigate the role of these two regulators in the soybean symbiont Bradyrhizobium japonicum USDA110. Using deletion mutants and phenotypic assays, we showed that PhyR and the ECF sigma factor sigma(EcfG) are involved in heat shock and desiccation resistance upon carbon starvation. Both mutants had symbiotic defects on the plant hosts Glycine max (soybean) and Vigna radiata (mungbean). They induced fewer nodules than the wild type and these nodules were smaller, less pigmented, and their specific nitrogenase activity was drastically reduced 2 or 3 weeks after inoculation. Four weeks after infection, soybean nodule development caught up to a large extent whereas most mungbean nodules remained defective even 5 weeks after infection. Remarkably, both mutants triggered aberrant nodules on the different host plants with ectopically emerging roots. Microarray analysis revealed that PhyR and sigma(EcfG) control congruent regulons suggesting both regulators are part of the same signalling cascade. This finding was further substantiated by in vitro protein-protein interaction studies which are in line with a partner-switching mechanism controlling gene regulation triggered by phosphorylation of PhyR. The large number of genes of unknown function present in the PhyR/sigma(EcfG) regulon and the conspicuous symbiotic phenotype suggest that these regulators are involved in the Bradyrhizobium-legume interaction via yet undisclosed mechanisms.

PhyR represents a novel alphaproteobacterial family of response regulators having a structure consisting of two domains; a predicted amino-terminal extracytoplasmic function (ECF) sigma factor-like domain and a carboxy-terminal receiver domain. PhyR was first described in Methylobacterium extorquens AM1, in which it has been shown to be essential for plant colonization, probably due to its suggested involvement in the regulation of a number of stress proteins. Here we investigated the PhyR regulon using microarray technology. We found that the PhyR regulon is rather large and that most of the 246 targets are under positive control. Mapping of transcriptional start sites revealed candidate promoters for PhyR-mediated regulation. One of these promoters, an ECF-type promoter, was identified upstream of one-third of the target genes by in silico analysis. Among the PhyR targets are genes predicted to be involved in multiple stress responses, including katE , osmC , htrA , dnaK , gloA , dps , and uvrA . The induction of these genes is consistent with our phenotypic analyses which revealed that PhyR is involved in resistance to heat shock and desiccation, as well as oxidative, UV, ethanol, and osmotic stresses, in M. extorquens AM1. The finding that PhyR is involved in the general stress response was further substantiated by the finding that carbon starvation induces protection against heat shock and that this protection is at least in part dependent on PhyR.

The RcsCDB His-Asp phosphorelay is shown to positively regulate the bdm (biofilm-dependent modulation) and sra (stationary-phase-induced ribosome-associated protein) genes in Escherichia coli . The regulation is direct and requires an RcsB box next to the bdm −35 element. In addition, bdm is shown to be activated by osmotic shock in an Rcs-dependent way.

The FixLJ two-component system ofSinorhizobium melilotiis a global regulator, turning on nitrogen-fixation genes in microaerobiosis. Up to now,nifAandfixKwere the only genes known to be directly regulated by FixJ. We used a genomic SELEX approach in order to isolate new FixJ targets in the genome. This led to the identification of 22 FixJ binding sites, including the known sites in thefixK1andfixK2promoters. FixJ binding sites are unevenly distributed among the three replicons constituting theS. melilotigenome: a majority are carried either by pSymA or by a short chromosomal region of non-chromosomal origin. Thus FixJ binding sites appear to be preferentially associated with the pSymA replicon, which carries thefixJgene. Functional analysis of FixJ targets led to the discovery of two new FixJ-regulated genes,smc03253andproB2. This FixJ-dependent regulation appears to be mediated by a duplication of the wholefixKpromoter region, including the beginning of thefixKgene. Similar duplications were previously reported for thenifHpromoter. By systematic comparison of all promoter regions we found 17 such duplications throughout the genome, indicating that promoter duplication is a common mechanism for the evolution of regulatory pathways inS. meliloti.

The genes involved in flagellum synthesis, motility and chemotaxis in Escherichia coli are expressed in a hierarchical fashion. At the top of the hierarchy lies the master regulator FlhDC, required for the expression of the whole set of genes. The operon flhDC is controlled by numerous regulators including H-NS, CRP, EnvZ/OmpR, QseBC and LrhA. In the present work, we report that the flhDC operon is also negatively regulated by the His-Asp phosphorelay system RcsCDB. The regulation is potentiated by the RcsB cofactor RcsA. Genetic analysis indicates that an RcsAB box, located downstream of the promoter, is required for the regulation. The binding of RcsB and RcsA to this site was demonstrated by gel retardation and DNase I protection assays. In addition, mutation analysis suggests that RcsA-specific determinants lie in the right part of the 'RcsAB box'.