DNA-binding Response Regulator Research Articles

Residue coevolution and mutational landscape for OmpR and NarL response regulator subfamilies

DNA-binding response regulators (DBRRs) are a broad class of proteins that operate in tandem with their partner kinase proteins to form two-component signal transduction systems in bacteria. Typical DBRRs are composed of two domains where the conserved N-terminal domain accepts transduced signals and the evolutionarily diverse C-terminal domain binds to DNA. These domains are assumed to be functionally independent, and hence recombination of the two domains should yield novel DBRRs of arbitrary input/output response, which can be used as biosensors. This idea has been proved to be successful in some cases; yet, the error rate is not trivial. Improvement of the success rate of this technique requires a deeper understanding of the linker-domain and inter-domain residue interactions, which have not yet been thoroughly examined. Here, we studied residue coevolution of DBRRs of the two main subfamilies (OmpR and NarL) using large collections of bacterial amino acid sequences to extensively investigate the evolutionary signatures of linker-domain and inter-domain residue interactions. Coevolutionary analysis uncovered evolutionarily selected linker-domain and inter-domain residue interactions of known experimental structures, as well as previously unknown inter-domain residue interactions. We examined the possibility of these inter-domain residue interactions as contacts that stabilize an inactive conformation of the DBRR where DNA binding is inhibited for both subfamilies. The newly gained insights on linker-domain/inter-domain residue interactions and shared inactivation mechanisms improve the understanding of the functional mechanism of DBRRs, providing clues to efficiently create functional DBRR-based biosensors. Additionally, we show the feasibility of applying coevolutionary landscape models to predict the functionality of domain-swapped DBRR proteins. The presented result demonstrates that sequence information can be used to filter out bioengineered DBRR proteins that are predicted to be nonfunctional due to a high negative predictive value.

Effects of uranium mining on soil bacterial communities and functions in the Qinghai-Tibet plateau

The microecological effects of plateau uranium mining are still unknown. In this study, we used 16S rRNA high-throughput sequencing to analyze the impact of plateau uranium mining on the microbial diversity and community structure of tailings soil, tunnel soil, and soil at different depths in an open pit. The results showed that uranium mining significantly reduced soil microbial community richness and diversity indicators, including Chao1, Pielou evenness, and Shannon index (P < 0.05). Uranium mining activities significantly reduced the abundance of RB41, Vicinamidactaceae, and Nitrospira (P < 0.05). Interestingly, the abundance of Thiobacillus, Sphingomonas, and Sulfuriferula significantly increased in the soil samples from various environments and depths during uranium mining (P < 0.05). Beta diversity analysis found that uranium mining resulted in the differentiation of soil microbial communities. Functional enrichment analysis found that uranium mining resulted in the functional enrichment of DNA binding response regulator, DNA helicase, methyl-accepting chemotaxis protein, and Helicase conserved C-terminal domain, whereas cell wall synthesis, nonspecific serine/threonine protein kinase, RNA polymerase sigma-70 factor, and ATP binding cassette transporter were significantly affected by uranium mining (P < 0.05). In addition, we also found that different uranium mining environments and soil depths enriched diverse microbial populations and functions to cope with the environmental pressures that were elicited by uranium mining, including Gaiella, Gemmatimonas, Lysobacter, Pseudomonas, signal transformation histidine kinase, DNA-directed DNA polymerase, and iron complex outer membrane receptor protein functions (P < 0.05). The results have enhanced our understanding of the impact of uranium mining on plateau soil microecological stability and the mechanism of microbial response to uranium mining activities for the first time and aided us in screening microbial strains that can promote the environmental remediation of uranium mining in plateaus.

In Silico Subtractive Proteomics and Molecular Docking Approaches for the Identification of Novel Inhibitors against Streptococcus pneumoniae Strain D39.

Streptococcus pneumoniae is a notorious Gram-positive pathogen present asymptomatically in the nasophayrnx of humans. According to the World Health Organization (W.H.O), pneumococcus causes approximately one million deaths yearly. Antibiotic resistance in S. pneumoniae is raising considerable concern around the world. There is an immediate need to address the major issues that have arisen as a result of persistent infections caused by S. pneumoniae. In the present study, subtractive proteomics was used in which the entire proteome of the pathogen consisting of 1947 proteins is effectively decreased to a finite number of possible targets. Various kinds of bioinformatics tools and software were applied for the discovery of novel inhibitors. The CD-HIT analysis revealed 1887 non-redundant sequences from the entire proteome. These non-redundant proteins were submitted to the BLASTp against the human proteome and 1423 proteins were screened as non-homologous. Further, databases of essential genes (DEGG) and J browser identified almost 171 essential proteins. Moreover, non-homologous, essential proteins were subjected in KEGG Pathway Database which shortlisted six unique proteins. In addition, the subcellular localization of these unique proteins was checked and cytoplasmic proteins were chosen for the druggability analysis, which resulted in three proteins, namely DNA binding response regulator (SPD_1085), UDP-N-acetylmuramate-L-alanine Ligase (SPD_1349) and RNA polymerase sigma factor (SPD_0958), which can act as a promising potent drug candidate to limit the toxicity caused by S. pneumoniae. The 3D structures of these proteins were predicted by Swiss Model, utilizing the homology modeling approach. Later, molecular docking by PyRx software 0.8 version was used to screen a library of phytochemicals retrieved from PubChem and ZINC databases and already approved drugs from DrugBank database against novel druggable targets to check their binding affinity with receptor proteins. The top two molecules from each receptor protein were selected based on the binding affinity, RMSD value, and the highest conformation. Finally, the absorption, distribution, metabolism, excretion, and toxicity (ADMET) analyses were carried out by utilizing the SWISS ADME and Protox tools. This research supported the discovery of cost-effective drugs against S. pneumoniae. However, more in vivo/in vitro research should be conducted on these targets to investigate their pharmacological efficacy and their function as efficient inhibitors.

The Sensory Histidine Kinase CusS of Escherichia coli Senses Periplasmic Copper Ions.

Two-component regulatory systems composed of a membrane-bound sensor/sensory histidine kinase (HK) and a cytoplasmic, DNA-binding response regulator (RR) are often associated with transenvelope efflux systems, which export transition metal cations from the periplasm directly out of the cell. Although much work has been done in this field, more evidence is needed for the hypothesis that the respective two-component regulatory systems are indeed sensing periplasmic ions. If so, a regulatory circuit between the concentration of periplasmic metal cations, sensing of these metals, and control of expression of the genes for transenvelope efflux systems that remove periplasmic cations can be assumed. Escherichia coli possesses only one transenvelope efflux system for metal cations, the Cus system for export of Cu(I) and Ag(I). It is composed of the transenvelope efflux system CusCBA, the periplasmic copper chaperone CusF, and the two-component regulatory system CusS (HK) and CusR (RR). Using phoA- and lacZ-reporter gene fusions, it was verified that an assumed periplasmic part of CusS is located in the periplasm. CusS was more important for copper resistance in E. coli under anaerobic conditions than under aerobic conditions and in complex medium more than in mineral salts medium. Predicted copper-binding sites in the periplasmic part of CusS were identified that, individually, were not essential for copper resistance but were in combination. In summary, evidence was obtained that the two-component regulatory system CusSR that controls expression of cusF and cusCBA does indeed sense periplasmic copper ions. IMPORTANCE Homeostasis of essential-but-toxic transition metal cations such as Zn(II) and Cu(II)/Cu(I) is an important contributor to the fitness of environmental bacteria and pathogenic bacteria during their confrontation with an infected host. Highly efficient removal of threatening concentrations of these metals can be achieved by the combined actions of an inner membrane with a transenvelope efflux system, which removes periplasmic ions after their export from the cytoplasm to this compartment. To understand the resulting metal cation homeostasis in the periplasm, it is important to know if a regulatory circuit exists between periplasmic metal cations, their sensing, and the subsequent control of the expression of the transenvelope efflux system. This publication adds evidence to the hypothesis that two-component regulatory systems in control of the expression of genes for transenvelope efflux systems do indeed sense metal cations in the periplasm.

Interplay between Two-Component Regulatory Systems Is Involved in Control of Cupriavidus metallidurans Metal Resistance Genes.

Metal resistance of Cupriavidus metallidurans is based on determinants that were acquired in the past by horizontal gene transfer during evolution. Some of these determinants encode transmembrane metal efflux systems. Expression of most of the respective genes is controlled by two-component regulatory systems composed of a membrane-bound sensor/sensory histidine kinase (HK) and a cytoplasmic, DNA-binding response regulator (RR). Here, we investigated the interplay between the three closely related two-component regulatory systems CzcRS, CzcR2S2, and AgrRS. All three systems regulate the response regulator CzcR, while the RRs AgrR and CzcR2 were not involved in czc regulation. Target promoters were czcNp and czcPp for genes upstream and downstream of the central czc gene region. The two systems together repressed CzcRS-dependent upregulation of czcP-lacZ at low zinc concentrations in the presence of CzcS but activated this signal transmission at higher zinc concentrations. AgrRS and CzcR2S2 interacted to quench CzcRS-mediated expression of czcNp-lacZ and czcPp-lacZ. Together, cross talk between the three two-component regulatory systems enhanced the capabilities of the Czc systems by controlling expression of the additional genes czcN and czcP. IMPORTANCE Bacteria are able to acquire genes encoding resistance to metals and antibiotics by horizontal gene transfer. To bestow an evolutionary advantage on their host cell, new genes must be expressed, and their expression should be regulated so that resistance-mediating proteins are produced only when needed. Newly acquired regulators may interfere with those already present in a host cell. Such an event was studied here in the metal-resistant bacterium Cupriavidus metallidurans. The results demonstrate how regulation by the acquired genes interacts with the host's extant regulatory network. This leads to emergence of a new system level of complexity that optimizes the response of the cell to periplasmic signals.

Improving prodigiosin production by transcription factor engineering and promoter engineering in Serratia marcescens.

Prodigiosin (PG), a red linear tripyrrole pigment produced by Serratia marcescens, has attracted attention due to its immunosuppressive, antimicrobial, and anticancer properties. Although many studies have been used to dissect the biosynthetic pathways and regulatory network of prodigiosin production in S. marcescens, few studies have been focused on improving prodigiosin production through metabolic engineering in this strain. In this study, transcription factor engineering and promoter engineering was used to promote the production of prodigiosin in S. marcescens JNB5-1. Firstly, through construing of a Tn5G transposon insertion library of strain JNB5-1, it was found that the DNA-binding response regulator BVG89_19895 (OmpR) can promote prodigiosin synthesis in this strain. Then, using RNA-Seq analysis, reporter green fluorescent protein analysis and RT-qPCR analysis, the promoter P17 (PRplJ) was found to be a strong constitutive promoter in strain JNB5-1. Finally, the promoter P17 was used for overexpressing of prodigiosin synthesis activator OmpR and PsrA in strain JNB5-1 and a recombinant strain PG-6 was obtained. Shake flask analysis showed that the prodigiosin titer of this strain was increased to 10.25 g/L, which was 1.62-times that of the original strain JNB5-1 (6.33 g/L). Taken together, this is the first well-characterized constitutive promoter library from S. marcescens, and the transcription factor engineering and promoter engineering can be also useful strategies to improve the production of other high value-added products in S. marcescens.

A Novel Regulation of K-antigen Capsule Synthesis in Porphyromonas gingivalis Is Driven by the Response Regulator PG0720-Directed Antisense RNA.

The periodontal pathogen Porphyromonas gingivalis strain W83 displays at least three different surface glycans, specifically two types of lipopolysaccharides (O-LPS and A-LPS) and K-antigen capsule. Despite the importance of K-antigen capsule to the virulence of P. gingivalis, little is known as to how expression of genes involved in the synthesis of this surface glycan is regulated. The genes required for K-antigen capsule synthesis are located in a locus that encodes a number of transcripts, including an operon (PG0104 to PG0121, generating ~19.4-kb transcript) which contains a non-coding 77-bp inverted repeat (77 bpIR) region near the 5'-end. Previously, we identified a 550-nucleotide antisense RNA molecule (designated asSuGR for antisense Surface Glycan Regulator) encoded within the 77-bpIR element that influences the synthesis of surface glycans. In this study, we demonstrate that the DNA-binding response regulator PG0720 can bind the promoter region of asSuGR and activate expression of asSuGR, indicating that PG0720 may indirectly influence transcript levels of the K-antigen capsule operon expressed from the sense strand. The data show that deletion of the PG0720 gene confers a defect in the presentation of surface polysaccharides compared with the parent strain and quantitative RT-PCR (qPCR) analysis determined that the overall expression of genes involved in K-antigen capsule synthesis were down-regulated in the PG0720 mutant. Furthermore, the defects of the PG0720 deletion mutant were restored by complementation. Importantly, the PG0720 deletion mutant showed reduced virulence. Altogether, our data show that the response regulator PG0720 regulates expression of asSuGR, a trans-acting antisense RNA molecule involved in modulating the production of surface polysaccharides in P. gingivalis strain W83. The data provide further evidence that surface glycans are key virulence determinants and significantly advances our understanding of the molecular mechanisms controlling the synthesis of P. gingivalis K-antigen capsule, a key virulence determinant.

Identification of the kinanthraquinone biosynthetic gene cluster by expression of an atypical response regulator.

Recent advances in genome sequencing have revealed a variety of secondary metabolite biosynthetic gene clusters in actinomycetes. Understanding the biosynthetic mechanism controlling secondary metabolite production is important for utilizing these gene clusters. In this study, we focused on the kinanthraquinone biosynthetic gene cluster, which has not been identified yet in Streptomyces sp. SN-593. Based on chemical structure, 5 type II polyketide synthase gene clusters were listed from the genome sequence of Streptomyces sp. SN-593. Among them, a candidate gene cluster was selected by comparing the gene organization with grincamycin, which is synthesized through an intermediate similar to kinanthraquinone. We initially utilized a BAC library for subcloning the kiq gene cluster, performed heterologous expression in Streptomyces lividans TK23, and identified the production of kinanthraquinone and kinanthraquinone B. We also found that heterologous expression of kiqA, which belongs to the DNA-binding response regulator OmpR family, dramatically enhanced the production of kinanthraquinones.

Generating asymmetry in a changing environment: cell cycle regulation in dimorphic alphaproteobacteria.

While many bacteria divide by symmetric binary fission, some alphaproteobacteria have strikingly asymmetric cell cycles, producing offspring that differs significantly in their morphology and reproductive state. To establish this asymmetry, these species employ a complex cell cycle regulatory pathway based on two-component signaling cascades. At the center of this network is the essential DNA-binding response regulator CtrA, which acts as a transcription factor controlling numerous genes with cell cycle-relevant functions as well as a regulator of chromosome replication. The DNA-binding activity of CtrA is controlled at the level of both protein phosphorylation and stability, dependent on an intricate network of regulatory proteins, whose function is tightly coordinated in time and space. CtrA is differentially activated in the two (developing) offspring, thereby establishing distinct transcriptional programs that ultimately determine their distinct cell fates. Phase-separated polar microdomains of changing composition sequester proteins involved in the (in-)activation and degradation of CtrA specifically at each pole. In this review, we summarize the current knowledge of the CtrA pathway and discuss how it has evolved to regulate the cell cycle of morphologically distinct alphaproteobacteria.

Quorum Sensing System-Regulated Proteins Affect the Spoilage Potential of Co-cultured Acinetobacter johnsonii and Pseudomonas fluorescens From Spoiled Bigeye Tuna (Thunnus obesus) as Determined by Proteomic Analysis.

Food spoilage by certain species of bacteria is reported to be regulated by quorum sensing (QS). Acinetobacter johnsonii and Pseudomonas fluorescens, the major specific spoilage organisms, are found to be limited in their QS and co-culture interactions. The aim of this study was to determine how QS-regulated proteins affect the spoilage potential of co-cultured A. johnsonii and P. fluorescens obtained from spoiled bigeye tuna (Thunnus obesus) using a proteomics approach. The A. johnsonii, P. fluorescens, and their co-culture tested the N-acyl-homoserine lactone (AHL) activities using reporter Chromobacterium violaceum CV026 and LC-MS/MS in qualitative and quantitative approaches, respectively. These latter showed that, of the 470 proteins and 444 proteins in A. johnsonii (A) and P. fluorescens (P), respectively, 80 were significantly up-regulated and 97 were significantly down-regulated in A vs. AP, whereas 90 were up-regulated and 65 were down-regulated in P vs. AP. The differentially expressed proteins included the AI-2E family transporter OS, 50S ribosomal protein L3, thioredoxin reductase OS, cysteine synthase CysM OS, DNA-binding response regulator, and amino acid ABC transporter ATPase OS. The cellular process (GO:0009987), metabolic process (GO:0008152), and single-organism process (GO:0044699) were classified into the gene ontology (GO) term. In addition, energy production and conversion, amino acid transport and metabolism, translation, ribosomal structure and biogenesis, post-translational modification, protein turnover, and chaperones were distributed into the clusters of orthologous groups of proteins (COG) terms. The KEGG pathways revealed that 84 and 77 differentially expressed proteins were divided into 20 KEGG pathways in A vs. AP and P vs. AP, respectively, and amino acid metabolism, carbohydrate metabolism, energy metabolism, and translation were significantly enriched. Proteins that correlated with the spoilage-related metabolic pathways, including thioredoxin reductase OS, cysteine synthase OS, and pyridoxal phosphate-dependent enzyme family protein OS, were identified. AI-2E family transporter OS and LuxR family transcriptional regulator OS were identified that related to the QS system. These findings provide a differential proteomic profile of co-culture in A. johnsonii and P. fluorescens, and have potential applications in QS and the regulation of spoilage potential.

Integrative and quantitative view of the CtrA regulatory network in a stalked budding bacterium.

The Alphaproteobacteria show a remarkable diversity of cell cycle-dependent developmental patterns, which are governed by the conserved CtrA pathway. Its central component CtrA is a DNA-binding response regulator that is controlled by a complex two-component signaling network, mediating distinct transcriptional programs in the two offspring. The CtrA pathway has been studied intensively and was shown to consist of an upstream part that reads out the developmental state of the cell and a downstream part that integrates the upstream signals and mediates CtrA phosphorylation. However, the role of this circuitry in bacterial diversification remains incompletely understood. We have therefore investigated CtrA regulation in the morphologically complex stalked budding alphaproteobacterium Hyphomonas neptunium. Compared to relatives dividing by binary fission, H. neptunium shows distinct changes in the role and regulation of various pathway components. Most notably, the response regulator DivK, which normally links the upstream and downstream parts of the CtrA pathway, is dispensable, while downstream components such as the pseudokinase DivL, the histidine kinase CckA, the phosphotransferase ChpT and CtrA are essential. Moreover, CckA is compartmentalized to the nascent bud without forming distinct polar complexes and CtrA is not regulated at the level of protein abundance. We show that the downstream pathway controls critical functions such as replication initiation, cell division and motility. Quantification of the signal flow through different nodes of the regulatory cascade revealed that the CtrA pathway is a leaky pipeline and must involve thus-far unidentified factors. Collectively, the quantitative system-level analysis of CtrA regulation in H. neptunium points to a considerable evolutionary plasticity of cell cycle regulation in alphaproteobacteria and leads to hypotheses that may also hold in well-established model organisms such as Caulobacter crescentus.

Phosphorylation systems in symbiotic nitrogen-fixing bacteria and their role in bacterial adaptation to various environmental stresses.

Symbiotic bacteria, commonly called rhizobia, lead a saprophytic lifestyle in the soil and form nitrogen-fixing nodules on legume roots. During their lifecycle, rhizobia have to adapt to different conditions prevailing in the soils and within host plants. To survive under these conditions, rhizobia fine-tune the regulatory machinery to respond rapidly and adequately to environmental changes. Symbiotic bacteria play an essential role in the soil environment from both ecological and economical point of view, since these bacteria provide Fabaceae plants (legumes) with large amounts of accessible nitrogen as a result of symbiotic interactions (i.e., rhizobia present within the nodule reduce atmospheric dinitrogen (N2) to ammonia, which can be utilized by plants). Because of its restricted availability in the soil, nitrogen is one of the most limiting factors for plant growth. In spite of its high content in the atmosphere, plants are not able to assimilate it directly in the N2 form. During symbiosis, rhizobia infect host root and trigger the development of specific plant organ, the nodule. The aim of root nodule formation is to ensure a microaerobic environment, which is essential for proper activity of nitrogenase, i.e., a key enzyme facilitating N2 fixation. To adapt to various lifestyles and environmental stresses, rhizobia have developed several regulatory mechanisms, e.g., reversible phosphorylation. This key mechanism regulates many processes in both prokaryotic and eukaryotic cells. In microorganisms, signal transduction includes two-component systems (TCSs), which involve membrane sensor histidine kinases (HKs) and cognate DNA-binding response regulators (RRs). Furthermore, regulatory mechanisms based on phosphoenolopyruvate-dependent phosphotranspherase systems (PTSs), as well as alternative regulatory pathways controlled by Hanks-type serine/threonine kinases (STKs) and serine/threonine phosphatases (STPs) play an important role in regulation of many cellular processes in both free-living bacteria and during symbiosis with the host plant (e.g., growth and cell division, envelope biogenesis, biofilm formation, response to stress conditions, and regulation of metabolism). In this review, we summarize the current knowledge of phosphorylation systems in symbiotic nitrogen-fixing bacteria, and their role in the physiology of rhizobial cells and adaptation to various environmental conditions.

Comparative Analysis of the Residue Co-Evolution of the DNA-Binding Response Regulator Subfamilies

Comparative Analysis of the Residue Co-Evolution of the DNA-Binding Response Regulator Subfamilies

Transcriptional regulator ArcA mediates expression of oligopeptide transport systems both directly and indirectly in Shewanella oneidensis

In γ-proteobacterial species, such as Escherichia coli, the Arc (anoxic redox control) two-component system plays a major role in mediating the metabolic transition from aerobiosis to anaerobiosis, and thus is crucial for anaerobic growth but dispensable for aerobic growth. In Shewanella oneidensis, a bacterium renowned for respiratory versatility, Arc (SoArc) primarily affects aerobic growth. To date, how this occurs has remained largely unknown although the growth defect resulting from the loss of DNA-binding response regulator SoArcA is tryptone-dependent. In this study, we demonstrated that the growth defect is in part linked to utilization of oligopeptides and di-tripeptides, and peptide uptake but not peptide degradation is significantly affected by the SoArcA loss. A systematic characterization of major small peptide uptake systems manifests that ABC peptide transporter Sap and four proton-dependent oligopeptide transporters (POTs) are responsible for transport of oligopeptides and di-tripeptides respectively. Among them, Sap and DtpA (one of POTs) are responsive to the SoarcA mutation but only dtpA is under the direct control of SoArcA. We further showed that both Sap and DtpA, when overproduced, improve growth of the SoarcA mutant. While the data firmly establish a link between transport of oligopeptides and di-tripeptides and the SoarcA mutation, other yet-unidentified factors are implicated in the growth defect resulting from the SoArcA loss.

Regulation of exploratory growth and antibiotic production in Streptomyces venezuelae by the two-component system CutRS

Antibiotic production and cellular development in bacteria are intimately linked to the extracellular environment. One key mechanism by which bacteria recognise and respond to these external cues is through two-component regulatory systems (2CS). Consisting of a membrane-bound sensor kinase and a cognate DNA-binding response regulator, these 2CSs are essential in the response to a myriad of signals including antibiotic attack, microbial interaction and nutrient availability. The soil-associated filamentous actinobacteria Streptomyces spp. are prolific antibiotic producers with a large number of 2CS. In addition to their complex life cycle a new developmental stage has recently been described termed exploration. When exploring, the streptomycetes rapidly expand via non-branching vegetative hyphae when contacted by fungi, a juxtaposition to the canonical lifecycle which ends in sporulation. Fifteen of the fifty-six 2CS in the model organism Streptomyces venezuelae are highly conserved throughout Streptomycetaceae. Having developed and screened a 2CS operon deletion library within S. venezuelae we determined that one of these, cutRS, is involved in co-ordinating exploration and antibiotic production. The cutRS deletion mutant displays unrepressed exploration and overproduction of chloramphenicol. With a greater understanding of how Streptomyces spp. identify and respond to external signals we can imitate and subvert these systems. Using this we aim to activate cryptic biosynthetic gene clusters enabling the discovery of novel antimicrobial products which may prove beneficial in the clinical setting.

Role of cell division protein divIVA in Enterococcus faecalis pathogenesis, biofilm and drug resistance: A future perspective by in silico approaches

Antibiotics resistance is the major problem in clinical settings which leads to the emergence of drug resistant bacteria. Biofilm formation is one of the grounds for the emergence of antibiotics resistant strains of Enterococcus faecalis. Our group previously reported in a comparative proteomic study of biofilm and planktonic state of E. faecalis that cell division protein divIVA was two folds overexpressed in biofilm state as compared to planktonic one and suggested its involvement in biofilm formation and antibiotics resistance. In this in silico study molecular docking showed that DNA bind to the conserved amino acid residues of divIVA domain and suggested that divIVA possibly secretes DNA into extra polymeric substance (EPS) which is the part of biofilm. We also performed the STRING analysis of cell division protein divIVA and predicted their interactive partners {cell division proteins/divisome complex (ftsZ, ftsA, divIV, ftsL, & gpsB), hypothetical proteins (sepF, EF_0261, EF_1000, EF_0998, EF_1006 & EF_1040), isoleucyl-tRNA synthetase (ileS), septation ring formation regulator (ezrA), S4 domain-containing protein (EF_1001), rod shape-determining protein (mreC), UDP-N-acetylmuramoyl-L-alanyl-d-glutamate synthetase (murD), UDP-diphospho-muramoyl-pentapeptide beta-N- acetylglucosaminyltransferase (murG), Lipoprotein signal peptidase (lspA), adenylate kinase (adk) and DNA-binding response regulator (vicR)}. We suggest that cumulatively divIVA and its interactive partners might be directly or indirectly involved in E. faecalis cell division, growth, biofilm formation, virulence and resistance.

Hanks-Type Serine/Threonine Protein Kinases and Phosphatases in Bacteria: Roles in Signaling and Adaptation to Various Environments.

Reversible phosphorylation is a key mechanism that regulates many cellular processes in prokaryotes and eukaryotes. In prokaryotes, signal transduction includes two-component signaling systems, which involve a membrane sensor histidine kinase and a cognate DNA-binding response regulator. Several recent studies indicate that alternative regulatory pathways controlled by Hanks-type serine/threonine kinases (STKs) and serine/threonine phosphatases (STPs) also play an essential role in regulation of many different processes in bacteria, such as growth and cell division, cell wall biosynthesis, sporulation, biofilm formation, stress response, metabolic and developmental processes, as well as interactions (either pathogenic or symbiotic) with higher host organisms. Since these enzymes are not DNA-binding proteins, they exert the regulatory role via post-translational modifications of their protein targets. In this review, we summarize the current knowledge of STKs and STPs, and discuss how these enzymes mediate gene expression in prokaryotes. Many studies indicate that regulatory systems based on Hanks-type STKs and STPs play an essential role in the regulation of various cellular processes, by reversibly phosphorylating many protein targets, among them several regulatory proteins of other signaling cascades. These data show high complexity of bacterial regulatory network, in which the crosstalk between STK/STP signaling enzymes, components of TCSs, and the translational machinery occurs. In this regulation, the STK/STP systems have been proved to play important roles.

Lvr, a Signaling System That Controls Global Gene Regulation and Virulence in Pathogenic Leptospira.

Leptospirosis is an emerging zoonotic disease with more than 1 million cases annually. Currently there is lack of evidence for signaling pathways involved during the infection process of Leptospira. In our comprehensive genomic analysis of 20 Leptospira spp. we identified seven pathogen-specific Two-Component System (TCS) proteins. Disruption of two these TCS genes in pathogenic Leptospira strain resulted in loss-of-virulence in a hamster model of leptospirosis. Corresponding genes lvrA and lvrB (leptospira virulence regulator) are juxtaposed in an operon and are predicted to encode a hybrid histidine kinase and a hybrid response regulator, respectively. Transcriptome analysis of lvr mutant strains with disruption of one (lvrB) or both genes (lvrA/B) revealed global transcriptional regulation of 850 differentially expressed genes. Phosphotransfer assays demonstrated that LvrA phosphorylates LvrB and predicted further signaling downstream to one or more DNA-binding response regulators, suggesting that it is a branched pathway. Phylogenetic analyses indicated that lvrA and lvrB evolved independently within different ecological lineages in Leptospira via gene duplication. This study uncovers a novel-signaling pathway that regulates virulence in pathogenic Leptospira (Lvr), providing a framework to understand the molecular bases of regulation in this life-threatening bacterium.

Regulation of potassium dependent ATPase (kdp) operon of Deinococcus radiodurans.

The genome of D. radiodurans harbors genes for structural and regulatory proteins of Kdp ATPase, in an operon pattern, on Mega plasmid 1. Organization of its two-component regulatory genes is unique. Here we demonstrate that both, the structural as well as regulatory components of the kdp operon of D. radiodurans are expressed quickly as the cells experience potassium limitation but are not expressed upon increase in osmolarity. The cognate DNA binding response regulator (RR) effects the expression of kdp operon during potassium deficiency through specific interaction with the kdp promoter. Deletion of the gene encoding RR protein renders the mutant D. radiodurans (ΔRR) unable to express kdp operon under potassium limitation. The ΔRR D. radiodurans displays no growth defect when grown on rich media or when exposed to oxidative or heat stress but shows reduced growth following gamma irradiation. The study elucidates the functional and regulatory aspects of the novel kdp operon of this extremophile, for the first time.

Direct interaction between the transcription factors CadC and OmpR involved in the acid stress response of Salmonella enterica.

In Salmonella enterica serovar Typhimurium, the acid-sensing regulator CadC activates transcription of the cadBA operon which contributes to the acid tolerance response. The DNA-binding response regulator OmpR in two-component regulatory system with EnvZ binds to its own promoter for autoinduction. We previously reported that CadC exerts a negative influence on ompR transcription during acid adaptation. However, its underlying mechanisms remain to be elucidated. Here we show that the level of OmpR protein is gradually reduced by a gradual increase in the CadC level using an arabinose-inducible expression system, indicating there exists a negative correlation between the expression levels of two transcription factors. To explore the molecular basis for OmpR repression by CadC, we performed in vitro binding assays and determined that CadC directly interacts with OmpR. We further show that inactivation of cadC inhibits transcription of the fliC gene, which encodes the major flagellar subunit, resulting in impaired flagellar motility under acid-adaptation conditions. Together, our findings suggest that CadC may repress autoinduction of the OmpR response regulator through their direct interaction.