Pepper Novel Pseudo Response Regulator Protein CaPRR2 Modulates Drought and High Salt Tolerance

Plants modify their internal states to adapt to environmental stresses. Under environmental stress conditions, plants restrict their growth and development and activate defense responses. Abscisic acid (ABA) is a major phytohormone that plays a crucial role in the osmotic stress response. In osmotic stress adaptation, plants regulate stomatal closure, osmoprotectant production, and gene expression. Here, we isolated CaPRR2 – encoding a pseudo response regulator protein – from the leaves of pepper plants (Capsicum annuum). After exposure to ABA and environmental stresses, such as drought and salt stresses, CaPRR2 expression in pepper leaves was significantly altered. Under drought and salt stress conditions, CaPRR2-silenced pepper plants exhibited enhanced osmotic stress tolerance, characterized by an enhanced ABA-induced stomatal closing and high MDA and proline contents, compared to the control pepper plants. Taken together, our data indicate that CaPRR2 negatively regulates osmotic stress tolerance.

Acetylation of Response Regulator Protein MtrA in M. tuberculosis Regulates Its Repressor Activity

MtrA is an essential response regulator (RR) protein in M. tuberculosis, and its activity is modulated after phosphorylation from its sensor kinase MtrB. Interestingly, many regulatory effects of MtrA have been reported to be independent of its phosphorylation, thereby suggesting alternate mechanisms of regulation of the MtrAB two-component system in M. tuberculosis. Here, we show that RR MtrA undergoes non-enzymatic acetylation through acetyl phosphate, modulating its activities independent of its phosphorylation status. Acetylated MtrA shows increased phosphorylation and enhanced interaction with SK MtrB assessed by phosphotransfer assays and FRET analysis. We also observed that acetylated MtrA loses its DNA-binding ability on gene targets that are otherwise enhanced by phosphorylation. More interestingly, acetylation is the dominant post-translational modification, overriding the effect of phosphorylation. Evaluation of the impact of MtrA and its lysine mutant overexpression on the growth of H37Ra bacteria under different conditions along with the infection studies on alveolar epithelial cells further strengthens the importance of acetylated MtrA protein in regulating the growth of M. tuberculosis. Overall, we show that both acetylation and phosphorylation regulate the activities of RR MtrA on different target genomic regions. We propose here that, although phosphorylation-dependent binding of MtrA drives its repressor activity on oriC and rpf, acetylation of MtrA turns this off and facilitates division in mycobacteria. Our findings, thus, reveal a more complex regulatory role of RR proteins in which multiple post-translational modifications regulate the activities at the levels of interaction with SK and the target gene expression.

A response regulator protein with antar domain, AvnR, in Acinetobacter baumannii ATCC 17978 impacts its virulence and amino acid metabolism

Acinetobacter baumannii, a Gram-negative coccobacillus, is notorious for its involvement in opportunistic infections around the world. Its resistance to antibiotics makes treatment of infections challenging. In this study, we describe a novel response regulator protein, AvnR (A1S_2006) that regulates virulence-related traits in A. baumannii ATCC17978. Sequence analysis suggests that AvnR is a CheY-like response regulator and contains the RNA-binding ANTAR (AmiR and NasR transcription anti-termination regulators) domain. We show that AvnR plays a role in regulating biofilm formation (on glass and plastic surfaces), surface motility, adhesion to A549 cells as well as in nitrogen metabolism in A. baumannii . RNA-Seq analysis revealed that avnR deletion results in altered expression of more than 150 genes (116 upregulated and 42 downregulated). RNA-Seq data suggest that altered biofilm formation and surface motility observed in the avnR deletion mutant is likely mediated by previously unknown pathways. Of note, was the altered expression of genes predicted to be involved in amino acid transport and metabolism in avnR deletion mutant. Biolog phenotypic array showed that deletion of avnR hampered A. baumannii ATCC17978’s ability to metabolize various nitrogen sources, particularly that of glutamic acid, serine, histidine, aspartic acid, isoleucine and arginine. Taken together our data show that AvnR, the first ANTAR protein described in A. baumannii, affects virulence phenotypes as well as its ability to metabolize nitrogen sources.

ArlCDE form the archaeal switch complex

Cells require a sensory system and a motility structure to achieve directed movement. Bacteria and archaea both possess rotating filamentous motility structures that work in concert with the sensory chemotaxis system. This allows microorganisms to move along chemical gradients. The central response regulator protein CheY can bind to the motor of the motility structure, the flagellum in bacteria and the archaellum in archaea. Both motility structures have a fundamentally different protein composition and structural organization. Yet, both systems receive input from the chemotaxis system. We applied a fluorescent microscopy approach in the model euryarchaeon Haloferax volcanii, and shed light on the sequence order in which signals are transferred from the chemotaxis system to the archaellum. Our findings indicate that the euryarchaeal specific ArlCDE are part of the archaellum motor and that they directly receive input from the chemotaxis system via the adaptor protein CheF. Hence, ArlCDE are an important feature of the archaellum of euryarchaea, are essential for signal transduction during chemotaxis and represent the archaeal switch complex.

In Silico Characterization and Screening for Inhibitors of Sdia: A Protein Involved in Quorom Sensing in Escherichia Coli

Bacteria use quorum sensing as a way of inter and intra- species communication. Quorum sensing was found to be important for bacteria for various processes including establishing an infection through virulence and biofilm formation. This is mediated autoinducers, which are usually produced by one group of bacteria and recognized by another group through a response regulator protein. LuxR is a response regulator protein first discovered in Vibreo fischeri and it recognizes autoinducers produced by the same species of bacteria. E. coli also has a response regulator called SdiA which is a homolog of LuxR, originally found to be involved in transcription and cell division. SdiA was later reported to regulate quorum sensing by binding to autoinducers called Acyl Homoserine Lactones (AHLs). SdiA is also reported to be involved in enhancing the multidrug resistance and virulence in pathogenic E. coli. Though many studies elaborate the functional aspects of SdiA, sequence and structural level analysis of this protein is missing in the literature. The current work aims at the in silico analysis and targeting of SdiA with structural analogs of AHLs. 7 compounds were found to be promising molecules to inhibit quorum sensing in E. coli.

Multikinase Networks: Two-Component Signaling Networks Integrating Multiple Stimuli

Bacteria depend on two-component systems to detect and respond to threats. Simple pathways comprise a single sensor kinase (SK) that detects a signal and activates a response regulator protein to mediate an appropriate output. These simple pathways with only a single SK are not well suited to making complex decisions where multiple different stimuli need to be evaluated. A recently emerging theme is the existence of multikinase networks (MKNs) where multiple SKs collaborate to detect and integrate numerous different signals to regulate a major lifestyle switch, e.g., between virulence, sporulation, biofilm formation, and cell division. In this review, the role of MKNs and the phosphosignaling mechanisms underpinning their signal integration and decision making are explored.

Single-molecule imaging of electroporated dye-labelled CheY in live Escherichia coli

For the past two decades, the use of genetically fused fluorescent proteins (FPs) has greatly contributed to the study of chemotactic signalling in Escherichia coli including the activation of the response regulator protein CheY and its interaction with the flagellar motor. However, this approach suffers from a number of limitations, both biological and biophysical: for example, not all fusions are fully functional when fused to a bulky FP, which can have a similar molecular weight to its fused counterpart; they may interfere with the native interactions of the protein and the chromophores of FPs have low brightness and photostability and fast photobleaching rates. A recently developed technique for the electroporation of fluorescently labelled proteins in live bacteria has enabled us to bypass these limitations and study the in vivo behaviour of CheY at the single-molecule level. Here we show that purified CheY proteins labelled with organic dyes can be internalized into E. coli cells in controllable concentrations and imaged with video fluorescence microscopy. The use of this approach is illustrated by showing single CheY molecules diffusing within cells and interacting with the sensory clusters and the flagellar motors in real time. This article is part of the themed issue ‘The new bacteriology’.

Crystal structure of the response regulator VraR fromStaphylococcus aureus

Staphylococcus aureusis one of the major causes of nosocomial infections today. Infections caused byS. aureusare a growing cause of concern owning to the widespread development of multiple antibiotic-resistant strains, particularly methicillin and vancomycin-intermediate resistant strains (MRSA and VISA). The VraSR two component system is reported to be highly related to the development of vancomycin resistance ofS. aureus. VraS is a membrane-bound sensor histidine kinase that received the signal stimulation from environment to regulate the phosphorylation status of its cognate response regulator protein VraR to control target genes transcription to result in the development of vancomycin resistance ofS. aureus.Here, we report the crystal structure of unphosphorylated VraR fromS. aureusin a dimeric form at 1.8 Å resolution. The crystals of VraR belong to the monoclinic space group C2 containing two protein molecules as a dimer in the asymmetric unit. The first β-strand at N-terminal end of each VraR subunit was inserted into the other subunit to form an intertwining dimer structure. Finally, according to previous reports and our crystal structure, we propose a regulation model for VraR. The feedback control ofvraSR operon would be the unphosphorylated dimeric VraR rather than the unphosphorylated monomeric VraR.

Subcellular Clustering of the Phosphorylated WspR Response Regulator Protein Stimulates Its Diguanylate Cyclase Activity

ABSTRACT WspR is a hybrid response regulator-diguanylate cyclase that is phosphorylated by the Wsp signal transduction complex in response to growth of Pseudomonas aeruginosa on surfaces. Active WspR produces cyclic di-GMP (c-di-GMP), which in turn stimulates biofilm formation. In previous work, we found that when activated by phosphorylation, yellow fluorescent protein (YFP)-tagged WspR forms clusters that are visible in individual cells by fluorescence microscopy. Unphosphorylated WspR is diffuse in cells and not visible. Thus, cluster formation is an assay for WspR signal transduction. To understand how and why WspR forms subcellular clusters, we analyzed cluster formation and the enzymatic activities of six single amino acid variants of WspR. In general, increased cluster formation correlated with increased in vivo and in vitro diguanylate cyclase activities of the variants. In addition, WspR specific activity was strongly concentration dependent in vitro, and the effect of the protein concentration on diguanylate cyclase activity was magnified when WspR was treated with the phosphor analog beryllium fluoride. Cluster formation appears to be an intrinsic property of phosphorylated WspR (WspR-P). These results support a model in which the formation of WspR-P subcellular clusters in vivo in response to a surface stimulus is important for potentiating the diguanylate cyclase activity of WspR. Subcellular cluster formation appears to be an additional means by which the activity of a response regulator protein can be regulated. IMPORTANCE Bacterial sensor proteins often phosphorylate cognate response regulator proteins when stimulated by an environmental signal. Phosphorylated response regulators then mediate an appropriate adaptive cellular response. About 6% of response regulator proteins have an enzymatic domain that is involved in producing or degrading cyclic di-GMP (c-di-GMP), a molecule that stimulates bacterial biofilm formation. In this work, we examined the in vivo and in vitro behavior of the response regulator-diguanylate cyclase WspR. When phosphorylated in response to a signal associated with surface growth, WspR has a tendency to form oligomers that are visible in cells as subcellular clusters. Our results show that the formation of phosphorylated WspR (WspR-P) subcellular clusters is important for potentiating the diguanylate cyclase activity of WspR-P, making it more active in c-di-GMP production. We conclude that oligomer formation visualized as subcellular clusters is an additional mechanism by which the activities of response regulator-diguanylate cyclases can be regulated.