Single-domain response regulator involved in the general stress response of Methylobacterium extorquens

Metzger, Lisa C.; Francez‐Charlot, Anne; Vorholt, Julia A.

doi:10.1099/mic.0.066068-0

Cited by 20 publications

(23 citation statements)

References 46 publications

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“…5). Putative SdrG orthologs are found in other Alphaproteobacteria, and, interestingly, a previous genetic study in M. extorquens PA1 showed that its SdrG ortholog, encoded by Mext_0407, is similarly involved in the GSR (23). These observations suggest that SdrG might be central to the GSR in several Alphaproteobacteria.…”

Section: Discussionmentioning

confidence: 72%

“…In other Alphaproteobacteria, no genes coding for histidine kinases are found at the phyR locus, but several HWE and/or HisKA2 kinases are encoded elsewhere in the genome (22). For example, M. extorquens PA1 encodes 21 kinases of the HWE and/or HisKA2 type, but there is no clear indication from sequence homology or genomic context for a prime candidate as PhyR kinase (23).…”

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confidence: 99%

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Complex two-component signaling regulates the general stress response in Alphaproteobacteria

Kaczmarczyk¹,

Hochstrasser²,

Vorholt³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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117

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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.

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Section: Discussionmentioning

confidence: 72%

mentioning

confidence: 99%

Complex two-component signaling regulates the general stress response in Alphaproteobacteria

Kaczmarczyk¹,

Hochstrasser²,

Vorholt³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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117

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“…For M. extorquens, luciferase activity was measured in 96-well black/white isoplates (PerkinElmer) on a Victor 3 multilabel plate reader (PerkinElmer) essentially as described previously (27) except that the measured values were corrected for the initial OD 600 .…”

Section: Methodsmentioning

confidence: 99%

“…For all species except M. extorquens, P Q5 activity was monitored using plasmid pQF-lacZ; for M. extorquens, a fragment containing P Q5 and cymR* was subcloned in a previously described bacterial luciferase (luxCDABE) transcriptional reporter plasmid (27). Whereas P Q5 was essentially inactive in A. tumefaciens (Ͻ2 Miller units with or without cumate; data not shown), the promoter was inducible in the other species tested with induction ratios of approximately 1,000-to 5,000-fold (Table 2 and Fig.…”

Section: Fig 4 (A)mentioning

confidence: 99%

Cumate-Inducible Gene Expression System for Sphingomonads and Other Alphaproteobacteria

Kaczmarczyk

Vorholt

Francez‐Charlot

2013

Appl Environ Microbiol

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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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“…Thus, in C. crescentus and M. extorquens, mutation of a single-domain response regulator unrelated to the EcfG system (LovR and Mext_0407, respectively) resulted in up-or downregulation of the EcfG response, respectively (35,44). Even though the biological relevance of these findings is not always clear, we cannot rule out that such additional levels of regulation also exist in S. meliloti.…”

Section: Fig 5 Rsic Inhibits Rsib1 Phosphorylation In Vivomentioning

confidence: 59%

A Putative Bifunctional Histidine Kinase/Phosphatase of the HWE Family Exerts Positive and Negative Control on the Sinorhizobium meliloti General Stress Response

Sauviac

Bruand

2014

J Bacteriol

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The EcfG-type sigma factor RpoE2 is the regulator of the general stress response in Sinorhizobium meliloti. RpoE2 activity is negatively regulated by two NepR-type anti-sigma factors (RsiA1/A2), themselves under the control of two anti-anti-sigma factors (RsiB1/B2) belonging to the PhyR family of response regulators. The current model of RpoE2 activation suggests that in response to stress, RsiB1/B2 are activated by phosphorylation of an aspartate residue in their receiver domain. Once activated, RsiB1/B2 become able to interact with the anti-sigma factors and release RpoE2, which can then associate with the RNA polymerase to transcribe its target genes. The purpose of this work was to identify and characterize proteins involved in controlling the phosphorylation status of RsiB1/B2. Using in vivo approaches, we show that the putative histidine kinase encoded by the rsiC gene (SMc01507), located downstream from rpoE2, is able to both positively and negatively regulate the general stress response. In addition, our data suggest that the negative action of RsiC results from inhibition of RsiB1/B2 phosphorylation. From these observations, we propose that RsiC is a bifunctional histidine kinase/phosphatase responsible for RsiB1/B2 phosphorylation or dephosphorylation in the presence or absence of stress, respectively. Two proteins were previously proposed to control PhyR phosphorylation in Caulobacter crescentus and Sphingomonas sp. strain FR1. However, these proteins contain a Pfam: HisKA_2 domain of dimerization and histidine phosphotransfer, whereas S. meliloti RsiC harbors a Pfam:HWE_HK domain instead. Therefore, this is the first report of an HWE_HK-containing protein controlling the general stress response in Alphaproteobacteria. Bacteria naturally live in constantly changing environments, where they are exposed to many stressful conditions, including nutrient limitation and biotic or abiotic stresses. The capacity to sense and adapt to these stresses is essential for the survival of the bacteria, which have evolved various types of stress responses. A number of these responses function by eliminating the inducing stress and/or repairing the associated cell damage. In parallel to these stress-specific responses, a so-called general stress response is activated under numerous different stress conditions and confers multiple stress resistances to the bacteria.It has been known for a long time that sigma factors play a central role in the control of the general stress response of both Gram-positive and Gram-negative bacteria. In Bacillus subtilis and other firmicutes, this response is controlled by B (1, 2), whereas in Escherichia coli and related Gammaproteobacteria, as well as in several Beta-and Deltaproteobacteria, it is controlled by S (3, 4). However, in the alphaproteobacterial group, the prominent role of extracytoplasmic-function sigma factors was uncovered recently with the finding that RpoE2 controls a general stress response in Sinorhizobium meliloti, the nitrogen-fixing symbiont of alfalfa (5). RpoE2 i...

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Single-domain response regulator involved in the general stress response of Methylobacterium extorquens

Cited by 20 publications

References 46 publications

Complex two-component signaling regulates the general stress response in Alphaproteobacteria

Complex two-component signaling regulates the general stress response in Alphaproteobacteria

Cumate-Inducible Gene Expression System for Sphingomonads and Other Alphaproteobacteria

A Putative Bifunctional Histidine Kinase/Phosphatase of the HWE Family Exerts Positive and Negative Control on the Sinorhizobium meliloti General Stress Response

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