Characterization of the CrbS/R Two-Component System in Pseudomonas fluorescens Reveals a New Set of Genes under Its Control and a DNA Motif Required for CrbR-Mediated Transcriptional Activation

Sepúlveda, Edgardo; Lupas, Andrei N.

doi:10.3389/fmicb.2017.02287

Cited by 13 publications

(48 citation statements)

References 65 publications

(77 reference statements)

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“…The presence of crbR significantly increased lacZ expression ( Fig. 5A), consistent with crbR directly activating acs expression, as observed in V. vulnificus and Pseudomonas (22,28).…”

supporting

confidence: 82%

“…5B and C), which may include the binding site targeted by CrbR. A previous study identified a putative CrbR binding site, with the primary consensus depicted as GAC(N 4 )GTC (28). Within the 15-bp region that we identified lies the sequence TCC(TAAA)GTCT (boldface indicates base pairs that match with the consensus CrbR binding site), which could act as the binding target.…”

mentioning

confidence: 82%

“…After transporting the signal, the pathway may then immediately convert this information into a response, by directly activating the expression of a suite of genes, including acs. This is borne out in other bacterial species; an ortholog of CrbR in Vibrio vulnificus, AcsR, directly binds to and activates the acs promoter (22), and a binding site has been defined in the promoters of genes targeted by CrbR homologs across the gammaproteobacteria (28). Thus, we hypothesize that a similar mechanism is operative in V. cholerae.…”

mentioning

confidence: 87%

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Modulation of CrbS-Dependent Activation of the Acetate Switch in Vibrio cholerae

et al. 2018

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controls the pathogenicity of interactions with arthropod hosts via the activity of the CrbS/R two component system. This signaling pathway regulates the consumption of acetate, which in turn, alters the relative virulence of interactions with arthropods, including CrbS is a histidine kinase that links a transporter-like domain to its signaling apparatus via putative STAC and PAS domains. CrbS and its cognate response regulator are required for expression of acetyl-CoA synthetase (), which converts acetate to acetyl-CoA. We demonstrate that the STAC domain of CrbS is required for signaling in culture; without it, transcription is reduced in LB medium, and cannot grow on acetate minimal media. However, the strain remains virulent towards and expresses similarly to wild-type during infection. This suggests that there exists a unique signal or environmental variable that modulates CrbS in the gastrointestinal tract of Secondly, we present evidence in support of CrbR, the response regulator that interacts with CrbS, binding directly to the promoter, and we identify a region of the promoter that CrbR may target. We further demonstrate that nutrient signals, together with the CRP-cAMP system, control transcription, but regulation may occur indirectly, as CRP-cAMP activates expression of the and genes. Lastly, we define the role of the Pta-AckA system in and identify redundancy built into acetate excretion pathways in this pathogen. CrbS is a member of a unique family of sensor histidine kinases, as its structure suggests that it may link signaling to transport of a molecule. However, mechanisms through which CrbS senses and communicates information about the outside world are unknown. In the , orthologs of CrbS regulate acetate metabolism, which can, in turn, affect interactions with host organisms. Here, we situate CrbS within a larger regulatory framework, demonstrating that is regulated by nutrient sensing systems. Furthermore, CrbS domains may play varying roles in signaling during infection and growth in culture, suggesting a unique mechanism of host recognition. Lastly, we define the roles of additional pathways in acetate flux, as a foundation for further studies of this metabolic nexus point.

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“…The presence of crbR significantly increased lacZ expression ( Fig. 5A), consistent with crbR directly activating acs expression, as observed in V. vulnificus and Pseudomonas (22,28).…”

supporting

confidence: 82%

mentioning

confidence: 82%

mentioning

confidence: 87%

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Modulation of CrbS-Dependent Activation of the Acetate Switch in Vibrio cholerae

et al. 2018

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“…Similar to the transcriptomics approach, targets for RRs may be identified by measuring protein abundances between WT and mutant strains. Proteomics studies are often accompanied by transcriptomics, metabolomics or ChIP‐seq analyses (Wang et al ., , ; Sepulveda and Lupas, ; Sun et al ., ; Reed et al ., ).…”

Section: Expression Profiling Methodsmentioning

confidence: 99%

Tools to map target genes of bacterial two‐component system response regulators

Rajeev

Garber

Mukhopadhyay

2020

Environ Microbiol Rep

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Studies on bacterial physiology are incomplete without knowledge of the signalling and regulatory systems that a bacterium uses to sense and respond to its environment. Two-component systems (TCSs) are among the most prevalent bacterial signalling systems, and they control essential and secondary physiological processes; however, even in model organisms, we lack a complete understanding of the signals sensed, the phosphotransfer partners and the functions regulated by these systems. In this review, we discuss several tools to map the genes targeted by transcriptionally acting TCSs. Many of these tools have been used for studying individual TCSs across diverse species, but systematic approaches to delineate entire signalling networks have been very few. Since genome sequences and high-throughput technologies are now readily available, the methods presented here can be applied to characterize the entire DNA-binding TCS signalling network in any bacterial species and are especially useful for non-model environmental bacteria.

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“…The STAC domain is suggested to mediate interactions with other proteins or control the transport cycle of the SLC5 domain 12 . It follows a PAS domain (about 115 amino acids) that may bind a yet to be identified signal molecule, a dimerization and histidine phosphotransfer domain (DHp, about 65 amino acids), and a catalytic ATP-binding domain CA (about 115 amino acids) 13 . The individual domains are connected by linker sequences (Fig.…”

mentioning

confidence: 99%

Transport and kinase activities of CbrA of Pseudomonas putida KT2440

Wirtz

Eder

Schipper

et al. 2020

Sci Rep

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the cbrA/cbrB system is a two-component signal transduction system known to participate in the regulation of the cellular carbon/nitrogen balance and to play a central role in carbon catabolite repression in Pseudomonas species. cbrA is composed of a domain with similarity to proteins of the solute/sodium symporter family (SLC5) and domains typically found in bacterial sensor kinases. Here, the functional properties of the sensor kinase cbrA and its domains are analyzed at the molecular level using the system of the soil bacterium P. putida KT2440 as a model. It is demonstrated that CbrA can bind and transport L-histidine. Transport is specific for L-histidine and probably driven by an electrochemical proton gradient. The kinase domain is not required for L-histidine uptake by the SLC5 domain of CbrA, and has no significant impact on transport kinetics. Furthermore, it is shown that the histidine kinase can autophosphorylate and transfer the phosphoryl group to the response regulator CbrB. The SLC5 domain is not essential for these activities but appears to modulate the autokinase activity. A phosphatase activity of CbrA is not detected. None of the activities is significantly affected by L-histidine. the results demonstrate that cbrA functions as a L-histidine transporter and sensor kinase. Transporters integral to cytoplasmic membranes usually catalyze the selective uptake of nutrients or the extrusion of metabolic end products and toxic solutes. However, some of these transporters play a central role also in signal transduction 1,2. In bacteria, so-called trigger transporters (temporarily) interact with membrane components of signal transduction systems and modulate their activity 2. For example, the lysine transporter LysP allows activation of a CadC-dependent acid stress response only when lysine can be taken up from the environment 3. The C 4-dicarboxylite transporter DcuB and the glucose-6-phosphate transporter UhpC interact with histidine kinases of specific two-component systems (TCSs) and stimulate phosphotransfer to the cognate response regulators when the respective substrate is present 1,4. While the interaction of transporters with separate signal transductions systems and the functional consequences are relatively well understood, little is known about the role of transporters that are covalently linked to domains typically found in bacterial signaling cascades. Prominent examples are members of the solute/ sodium symporter family (SLC5) 5,6. Besides sodium-motive force-dependent transporters for proline (PutP of archaea and bacteria 7), monosaccharides (SGLT of bacteria and higher eukaryotes 8) and others 9-11 , the family contains bacterial proteins in which a complete SLC5 domain is connected via a STAC (SLC5 and TCS Associated Component) domain to domains found in histidine kinases or diguanylate cyclase 5,12,13. SLC5 transporters fused via STAC to histidine kinase domains are usually associated with response regulators and resemble TCSs. CbrA/ CbrB represents such a histidine kinase/response r...

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Characterization of the CrbS/R Two-Component System in Pseudomonas fluorescens Reveals a New Set of Genes under Its Control and a DNA Motif Required for CrbR-Mediated Transcriptional Activation

Cited by 13 publications

References 65 publications

Modulation of CrbS-Dependent Activation of the Acetate Switch in Vibrio cholerae

Modulation of CrbS-Dependent Activation of the Acetate Switch in Vibrio cholerae

Tools to map target genes of bacterial two‐component system response regulators

Transport and kinase activities of CbrA of Pseudomonas putida KT2440

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