The general stress response in Alphaproteobacteria was recently described to depend on the alternative sigma factor 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-NepREcfG 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.

Significance Bacteria possess many regulatory systems to monitor their environment and adapt their physiology accordingly. Whereas most systems sense one specific signal, the general stress response (GSR) is activated by many signals and protects cells against a wide range of adverse conditions. In Alphaproteobacteria, the GSR is controlled by the response regulator PhyR, but little is known about the upstream pathways. Here, we establish the GSR as a complex regulatory network composed of a particular family of partially redundant sensor kinases and of additional response regulators that modulate PhyR activity in Sphingomonas melonis . Given the broad conservation of this kinase family, it is possible that it plays a general role in the GSR in Alphaproteobacteria.

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.

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. R egulated gene expression systems are a powerful tool to study physiology, allowing, for example, dosage-effect studies, conditional expression of toxic alleles, and depletion analysis of essential genes; accordingly, they are well developed for model organisms (1-7) but are missing for many non-model organisms. Most systems rely on a transcriptional repressor that tightly binds to operator sites in the promoter region of target genes in the absence of an inducer, thereby preventing transcription; when an inducer is present, it allosterically binds to and inactivates the transcriptional regulator, leading to derepression of promoters. Despite this simple concept, identification of inducible promoters, control elements, and inducing conditions in a particular organism is not always a trivial task. On the other hand, it is often difficult to exploit a particular system for use in organisms other than the original host because of the need for dedicated transporters for the inducer or a different promoter specificity of the RNA polymerase holoenzyme or the requirement of a coactivator for full promoter activity (8). Some of these obstacles can be circumvented by engineering artificial inducible promoters by placing a constitutive minimal promoter known to be active in a particular organism with operator sequences and the repressor of a heterologous system (2, 9, 10). We here describe such a system for sphingomonads, a phylogenetically diverse group of environmentally abundant bacteria comprising the genera Sphingomonas, Sphingobium, Novosphingobium, and Sphingopyxis (11). Members of this group are well known for their unusual ability to degrade a wide range of compounds, including pesticides, herbicides, xenobiotics, and many other aromatics. Due to this property, they are prospective candidates for bioremediation; in addition, several str...

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