Two-component System Proteins Research Articles

Elizabethkingia anophelis MSU001 Isolated from Anopheles stephensi: Molecular Characterization and Comparative Genome Analysis.

Elizabethkingia anophelis MSU001, isolated from Anopheles stephensi in the laboratory, was characterized by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-ToF/MS), biochemical testing, and genome sequencing. Average nucleotide identity analysis revealed 99% identity with the type species E. anophelis R26. Phylogenetic placement showed that it formed a clade with other mosquito-associated strains and departed from a clade of clinical isolates. Comparative genome analyses further showed that it shared at least 98.6% of genes with mosquito-associated isolates (except E. anophelis As1), while it shared at most 88.8% of common genes with clinical isolates. Metabolites from MSU001 significantly inhibited growth of E. coli but not the mosquito gut symbionts Serratia marcescens and Asaia sp. W12. Insect-associated E. anophelis carried unique glycoside hydrolase (GH) and auxiliary activities (AAs) encoding genes distinct from those of clinical isolates, indicating their potential role in reshaping chitin structure and other components involved in larval development or formation of the peritrophic matrix. Like other Elizabethkingia, MSU001 also carried abundant genes encoding two-component system proteins (51), transcription factor proteins (188), and DNA-binding proteins (13). E. anophelis MSU001 contains a repertoire of antibiotic resistance genes and several virulence factors. Its potential for opportunistic infections in humans should be further evaluated prior to implementation as a paratransgenesis agent (by transgenesis of a symbiont of the vector).

OmpC-Dependent Bile Tolerance Contributes to E. coli Colonization of the Mammalian Intestine.

Escherichia coli persistently colonizes the mammalian intestine by mechanisms that are not fully understood. Previously, we found when streptomycin-treated mice were fed E. coli MG1655, the intestine selected for envZ missense mutants that outcompeted the wild type. The better-colonizing envZ mutants had a higher level of OmpC and reduced OmpF. This suggested the EnvZ/OmpR two-component system and outer membrane proteins play a role in colonization. In this study, we show that wild-type E. coli MG1655 outcompetes an envZ-ompR knockout mutant. Moreover, ompA and ompC knockout mutants are outcompeted by the wild type, while an ompF knockout mutant colonizes better than the wild type. Outer membrane protein gels show the ompF mutant overproduces OmpC. An ompC mutant is more sensitive to bile salts than the wild type and ompF mutant. The ompC mutant initiates colonization slowly because it is sensitive to physiological concentrations of bile salts in the intestine. Overexpression of ompC under the control of a constitutive promoter confers a colonization advantage only when ompF is deleted. These results indicate that fine-tuning of OmpC and OmpF levels is needed to maximize competitive fitness in the intestine. RNA sequencing reveals the EnvZ/OmpR two-component system is active in the intestine: ompC is upregulated and ompF is downregulated. While other factors could also contribute to the advantage provided by OmpC, we provide evidence that OmpC is important for E. coli to colonize the intestine because its smaller pore size excludes bile salts or other unknown toxic substances, while OmpF is deleterious because its larger pore size allows bile salts or other unknown toxic substances to enter the periplasm. IMPORTANCE Every mammalian intestine is colonized with Escherichia coli. Although E. coli is one of the most studied model organisms, how it colonizes the intestine is not fully understood. Here, we investigated the role of the EnvZ/OmpR two-component system and outer membrane proteins in colonization of the mouse intestine by E. coli. We report that an ompC mutant is a poor colonizer, while an ompF mutant, which overproduces OmpC, outcompetes the wild type. OmpF has a larger pore size that allows toxic bile salts or other toxic compounds into the cell and is deleterious for colonization of the intestine. OmpC has a smaller pore size and excludes bile salts. Our findings provide insights into why E. coli fine-tunes the levels of OmpC and OmpF during colonization via the EnvZ/OmpR two-component system.

Comparative Omics-Based Identification and Expression Analysis of a Two-Component System in Vigna radiata in Drought Stress

Two-component system (TCS) genes regulate a wide range of biological activities in prokaryotes and eukaryotes, including plants. TCS plays an important role in cellular responses to external stimuli, such as biotic and abiotic factors. In plants, this system supports cell division, leaf senescence, stress response, chloroplast division, and nutrient signaling. There are three kinds of proteins responsible for the appropriate functioning of the TCS system: histidine kinases (HKs), histidine phosphotransfer proteins (HPs), and response regulators (RRs). The results of the current study revealed that Vigna radiata has 54 genes encoding potential TCS proteins, which were divided into three subgroups: 18 HKs, 9 HPs (seven true and two pseudos), and 27 RRs (8 type-A, 8 type-B, 3 type-C, and 8 PRRS). The anticipated TCS genes were widely dispersed across all eleven chromosomes and had family-specific intron/exon structures. After investigating TCS genes in a variety of plant species, we determined that Vigna HK (L)s, HPs, and RRs have closer evolutionary relationships with other legume genes. Gene duplication, including segmental and tandem types, is the most frequent source of gene family expansion. Multiple stress-related cis-elements were predicted in the promoter sequences of the VrTCS genes. RNA-seq data analysis demonstrated that VrTCS genes were expressed in clusters of upregulated and downregulated groups in response to drought stress. Moreover, these clusters were differentially expressed as early or late responses to drought stress. Real-time qPCR showed that VrHK2, VrHK3, VrPHYE, VrHP4.1, VrRR5.2, and VrRR10 genes were upregulated, while VrRR3 and VrHP6.1 genes were downregulated in response to drought stress. The current study highlights the architecture of V. radiata TCS and provides a robust framework for subsequent functional evaluation.

Protein Therapeutic Target Candidates Against Acinetobacter baumannii, a Pathogen of Concern to Planetary Health: A Network-Based Integrative Omics Drug Discovery Approach.

Acinetobacter baumannii, an opportunistic gram-negative pathogen responsible for several nosocomial infections, has developed resistance to various antibiotics. Proteins involved in the two-component system (TCS), virulence, and antibiotic resistance (AR), help this pathogen in regulating antibiotic susceptibility and virulence mechanisms. The present study reports a network-based integrative omics approach to drug discovery to identify key regulatory proteins as therapeutic candidates against A. baumannii. We collected data on the TCS, virulence, and AR proteins from various databases (P2CS, VFDB, ARDB, and PAIDB), which were subjected to network, host-pathogen, and gene expression data analysis. Network analysis identified 43 hubs, and 10 proteins were found to be interacting with human proteins associated with vital pathways. Of the 53 (43 + 10) pathogen proteins, 46 had no orthologs in the human host. Twelve proteins, namely, RpfC, Wzc, OmpR, EnvZ, BfmS, PilG, histidine kinase, ABC 3 transport family protein, outer membrane porin OprD family, CsuD, Pgm, and LpxA, were differentially expressed in the resistant strain. We propose these proteins as key regulators that warrant evaluation as therapeutic target candidates in the future. Furthermore, structure prediction of ABC 3 transport family protein was performed as a case study. The findings from this study are poised to facilitate and inform drug discovery and development against A. baumannii.

Exploring the mono-/bistability range of positively autoregulated signaling systems in the presence of competing transcription factor binding sites.

Binding of transcription factor (TF) proteins to regulatory DNA sites is key to accurate control of gene expression in response to environmental stimuli. Theoretical modeling of transcription regulation is often focused on a limited set of genes of interest, while binding of the TF to other genomic sites is seldom considered. The total number of TF binding sites (TFBSs) affects the availability of TF protein molecules and sequestration of a TF by TFBSs can promote bistability. For many signaling systems where a graded response is desirable for continuous control over the input range, biochemical parameters of the regulatory proteins need be tuned to avoid bistability. Here we analyze the mono-/bistable parameter range for positively autoregulated two-component systems (TCSs) in the presence of different numbers of competing TFBSs. TCS signaling, one of the major bacterial signaling strategies, couples signal perception with output responses via protein phosphorylation. For bistability, competition for TF proteins by TFBSs lowers the requirement for high fold change of the autoregulated transcription but demands high phosphorylation activities of TCS proteins. We show that bistability can be avoided with a low phosphorylation capacity of TCSs, a high TF affinity for the autoregulated promoter or a low fold change in signaling protein levels upon induction. These may represent general design rules for TCSs to ensure uniform graded responses. Examining the mono-/bistability parameter range allows qualitative prediction of steady-state responses, which are experimentally validated in the E. coli CusRS system.

Quantitative proteomics reveals the antibiotics adaptation mechanism of Aeromonas hydrophila under kanamycin stress

Aeromonas hydrophila is a widespread opportunistic pathogen of aquatic fishes in freshwater habitats. The current emergence of antimicrobial-resistant A. hydrophila has been reported in the world while the bacterial antibiotics adaptive mechanism remains poorly explored. In this study, using quantitative proteomics technology, the behavior of A. hydrophila was investigated by comparing the differentially expression proteins between with and without kanamycin (KAN) treatment. A total of 374 altered proteins including 184 increasing and 190 proteins decreasing abundances were quantified when responding to KAN stress. The bioinformatics analysis showed that stress related proteins were hub proteins that significantly increased to reduce the pressure from the misreading of mRNA caused by KAN. Moreover, several metallic pathways, such as oxidative phosphorylation and TCA cycle pathways may affect KAN resistance. Finally, eight selected genes were deleted and their antibiotics susceptibilities to kanamycin were valued, respectively. Results showed that OmpA II family protein A0KI26, and two-component system protein AtoC may involve in the KAN resistance in this study. In general, our results provide an insight into the behaviors of bacterial responding to KAN stress, and demonstrate the intrinsic antibiotics adaptive mechanism of A. hydrophila. Biological significanceIn this study, the differentially expressed proteins (DEPs) of A. hydrophila strain between with and without kanamycin (KAN) were compared by using a data-independent acquisition (DIA) - based quantitative proteomics method. Bioinformatics analysis showed that stress - related proteins are hub proteins that significantly increased under KAN stress. Moreover, several metallic pathways, such as oxidative phosphorylation and citrate cycle (TCA cycle) pathways, can affect KAN resistance. Finally, our antibiotics susceptibility assay showed that the protein A0KI26 of the OmpA II family, and the AtoC of the two-component system may involve in KAN resistance in this study. These results provide insights into the antibiotics adaptation mechanism of A. hydrophila when responding to KAN stress.

Mutation of MtrA at the Predicted Phosphorylation Site Abrogates Its Role as a Global Regulator in Streptomyces venezuelae.

ABSTRACTThe global regulator MtrA controls development and primary and secondary metabolism in Streptomyces species. However, residues critical for its function have not yet been characterized. In this study, we identified residue D53 as the potential phosphorylation site of MtrA from Streptomyces venezuelae, a model Streptomyces strain. MtrA variants with amino acid substitutions at the D53 site were generated, and the effects of these substitutions were evaluated in vitro and in vivo. We showed that, although substitutions at D53 did not alter MtrA’s secondary structure, the MtrA D53 protein variants lost the ability to bind known MtrA recognition sequences (MtrA sites) in electrophoretic mobility shift assays. Complementation of the ΔmtrA strain with MtrA D53 protein variants did not affect overall strain growth. However, in comparison to the wild-type strain, chloramphenicol and jadomycin production were aberrant in the D53 variant strains, with levels similar to the levels in the ΔmtrA strain. Transcriptional analysis showed that the expression patterns of genes were also similar in the ΔmtrA strain and the D53 variant strains. Although the D53 protein variants and wild-type MtrA were produced at similar levels in S. venezuelae, chromatin immunoprecipitation-quantitative PCR results indicated that replacing the D53 residue rendered the altered proteins unable to bind MtrA sites in vivo, including MtrA sites that regulate genes involved in nitrogen metabolism and in chloramphenicol and jadomycin biosynthesis. In conclusion, our study demonstrates that the predicted phosphorylation site D53 is critical for the role of MtrA in regulation and suggests that MtrA functions in a phosphorylated form in the genus Streptomyces.IMPORTANCE Although phosphorylation has been shown to be essential for the activation of many response regulator proteins of two-component systems, the role of the phosphorylation site in the function of the global regulator MtrA in the genus Streptomyces has not been reported. In this study, we generated Streptomyces mutants that had amino acid substitutions at the predicted phosphorylation site of MtrA, and the effects of the substitutions were investigated by comparing the phenotypes of the resulting strains and their gene expression patterns with those of the wild-type strain and an MtrA deletion mutant. The ability of the altered proteins to bind known promoter targets in vitro was also evaluated. Our analyses showed that the predicted phosphorylation site D53 is critical for MtrA binding in vitro and for the normal functioning of MtrA in vivo. These studies further demonstrate the importance of MtrA as a global regulator in the genus Streptomyces.

Characterisation of the E. coli and Salmonella qseC and qseE mutants reveals a metabolic rather than adrenergic receptor role.

Catecholamine stress hormones (norepinephrine, epinephrine, and dopamine) are signals that have been shown to be used as environmental cues, which affect the growth and virulence of normal microbiota as well as pathogenic bacteria. It has been reported that Escherichia coli and Salmonella use the two-component system proteins QseC and QseE to recognise catecholamines and so act as bacterial adrenergic receptors. In this study, we mutated the E. coli O157:H7 and Salmonella enterica serovar Typhimurium genes encoding QseC and QseE and found that this did not block stress hormone responsiveness in either species. Motility, biofilm formation, and analysis of virulence of the mutants using two infection models were similar to the wild-type strains. The main differences in phenotypes of the qseC and qseE mutants were responses to changes in temperature and growth in different media particularly with respect to salt, carbon, and nitrogen salt sources. In this physiological respect, it was also found that the phenotypes of the qseC and qseE mutants differed between E. coli and Salmonella. These findings collectively suggest that QseC and QseE are not essential for E. coli and Salmonella to respond to stress hormones and that the proteins may be playing a role in regulating metabolism.

Expression, Purification, and Characterization of the Recombinant, Two-Component, Response Regulator ArlR from Fusobacterium nucleatum.

Fusobacterium nucleatum is associated with the incidence and development of multiple diseases, such as periodontitis and colorectal cancer (CRC). Until now, studies have proved only a few proteins to be associated with such pathogenic diseases. The two-component system is one of the most prevalent forms of bacterial signal transduction related to intestinal diseases. Here, we report a novel, recombinant, two-component, response regulator protein ArlR from the genome of F. nucleatum strain ATCC 25,586. We optimized the expression and purification conditions of ArlR; in addition, we characterized the interaction of this response regulator protein with the corresponding histidine kinase and DNA sequence. The full-length ArlR was successfully expressed in six E. coli host strains. However, optimum expression conditions of ArlR were present only in E. coli strain BL21 CodonPlus (DE3) RIL that was later induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) for 8h at 25°C. The SDS-PAGE analysis revealed the molecular weight of the recombinant protein as 27.3kDa with approximately 90% purity after gel filtration chromatography. Because ArlR was biologically active after its purification, it accepted the corresponding phosphorylated histidine kinase phosphate group and bound to the analogous DNA sequence. The binding constant between ArlR and the corresponding histidine kinase was about 2.1μM, whereas the binding constant between ArlR and its operon was 6.4μM. Altogether, these results illustrate an effective expression and purification method for the novel two-component system protein ArlR.

Pseudomonas Aeruginosa Responds to Altered Membrane Phospholipid Composition by Adjusting the Production of Two-Component Systems, Proteases and Iron Uptake Proteins

Pseudomonas Aeruginosa Responds to Altered Membrane Phospholipid Composition by Adjusting the Production of Two-Component Systems, Proteases and Iron Uptake Proteins

The predatory soil bacterium Lysobacter reprograms quorum sensing system to regulate antifungal antibiotic production in a cyclic-di-GMP-independent manner

Soil bacteria often harbour various toxins to against eukaryotic or prokaryotic. Diffusible signal factors (DSFs) represent a unique group of quorum sensing (QS) chemicals that modulate interspecies competition in bacteria that do not produce antibiotic-like molecules. However, the molecular mechanism by which DSF-mediated QS systems regulate antibiotic production for interspecies competition remains largely unknown in soil biocontrol bacteria. In this study, we find that the necessary QS system component protein RpfG from Lysobacter, in addition to being a cyclic dimeric GMP (c-di-GMP) phosphodiesterase (PDE), regulates the biosynthesis of an antifungal factor (heat-stable antifungal factor, HSAF), which does not appear to depend on the enzymatic activity. Interestingly, we show that RpfG interacts with three hybrid two-component system (HyTCS) proteins, HtsH1, HtsH2, and HtsH3, to regulate HSAF production in Lysobacter. In vitro studies show that each of these proteins interacted with RpfG, which reduced the PDE activity of RpfG. Finally, we show that the cytoplasmic proportions of these proteins depended on their phosphorylation activity and binding to the promoter controlling the genes implicated in HSAF synthesis. These findings reveal a previously uncharacterized mechanism of DSF signalling in antibiotic production in soil bacteria.

Screening of binding proteins that interact with two components of the HOG MAPK pathway by the yeast two-hybrid method in Colletotrichum siamense

In phytopathogenic fungi, the Hog1 MAPK pathway plays roles in osmoregulation, oxidation responses, fungicide sensitivity, among others. However, how fungi depend on the HOG MAPK pathway to regulate their functions is poorly understood. To further elucidate the molecular mechanisms of the HOG MAPK pathway, a yeast two-hybrid (Y2H) system was adopted to screen interacting proteins of two key components (CsPbs2 and CsHog1) of the HOG MAPK pathway in a cDNA library of Colletotrichum siamense. The results showed that 19 proteins may interact with CsPbs2, including cytochrome P450 4F5, ceramide very-long-chain fatty acid hydroxylase SCS7, the oxidative stress response two-component system protein SSK1, and other proteins. Twenty-one proteins may interact with CsHog1, including the cell wall protein PhiA, acriflavine sensitivity control protein acr-2, arachidonate 15-lipoxygenase, respiratory supercomplex factor 2-like protein, and others. Five proteins interact with both CsHog1 and CsPbs2: antigenic thaumatin-like protein, aspirochlorine biosynthesis protein N, secreted beta-glucosidase sun 1, alcohol dehydrogenase 1, and hypothetical protein GQ607_005106. Furthermore, the interaction of CsSCS7 and CsCytP450 4F5 with CsPbs2 was verified by His pull-down. qPCR revealed significantly upregulated CsPbs2, CsHog1, CsSCS7 and CsCytP450 4F5 expression in Colletotrichum treated with 50 μg/mL fludioxonil for 2 h. This research identified candidate CsHog1- and CsPbs2-interacting proteins via a Y2H system and confirmed the interactions of CsSCS7 and CsCytP450 4F5 with CsPbs2. The findings lay the foundation for further studies of HOG MAPK pathway function and regulatory mechanisms, especially regarding fungicide sensitivity.

Calcium-responsive kinase LadS modulates type I–F CRISPR-Cas adaptive immunity

The CRISPR-Cas systems are recently discovered adaptive immune strategies in bacteria and archaea against foreign genetic elements. Although gene-editing enabled by CRISPR-Cas9 has shown great promise for clinical application, little is known about potential mechanisms of CRISPR-Cas systems for regulating their own gene expression and altering the virulence within bacteria. Here, Gram-negative bacterium Pseudomonas aeruginosa PA14 that contains a Type I–F CRISPR-Cas system was used to study the mechanism endogenous CRISPR-Cas of regulation mechanism. We delineated the role of calcium as a positive regulator of the transcription of cas/csy complex and CRISPR-Cas immunity through the two-component system (TCS) protein kinase LadS. Furthermore, we identified a LadS downstream post-transcriptional regulator, RsmA, which targeted translation region of cas mRNA via A(N)GGA motif. Importantly, calcium-mediated influencing of CRISPR-Cas system was dependent on LadS and RsmA. Altogether, our findings uncover the previously unrecognized role of LadS/RsmA in modulating Type I–F CRISPR-Cas system via sensing calcium.

ELIHKSIR Web Server: Evolutionary Links Inferred for Histidine Kinase Sensors Interacting with Response Regulators

Two-component systems (TCS) are signaling machinery that consist of a histidine kinases (HK) and response regulator (RR). When an environmental change is detected, the HK phosphorylates its cognate response regulator (RR). While cognate interactions were considered orthogonal, experimental evidence shows the prevalence of crosstalk interactions between non-cognate HK–RR pairs. Currently, crosstalk interactions have been demonstrated for TCS proteins in a limited number of organisms. By providing specificity predictions across entire TCS networks for a large variety of organisms, the ELIHKSIR web server assists users in identifying interactions for TCS proteins and their mutants. To generate specificity scores, a global probabilistic model was used to identify interfacial couplings and local fields from sequence information. These couplings and local fields were then used to construct Hamiltonian scores for positions with encoded specificity, resulting in the specificity score. These methods were applied to 6676 organisms available on the ELIHKSIR web server. Due to the ability to mutate proteins and display the resulting network changes, there are nearly endless combinations of TCS networks to analyze using ELIHKSIR. The functionality of ELIHKSIR allows users to perform a variety of TCS network analyses and visualizations to support TCS research efforts.

Structural characterization of two solute-binding proteins for N,N′-diacetylchitobiose/N,N′,N′′-triacetylchitotoriose of the gram-positive bacterium, Paenibacillus sp. str. FPU-7

The chitinolytic bacterium Paenibacillus sp. str. FPU-7 efficiently degrades chitin into oligosaccharides such as N-acetyl-D-glucosamine (GlcNAc) and disaccharides (GlcNAc)2 through multiple secretory chitinases. Transport of these oligosaccharides by P. str. FPU-7 has not yet been clarified. In this study, we identified nagB1, predicted to encode a sugar solute-binding protein (SBP), which is a component of the ABC transport system. However, the genes next to nagB1 were predicted to encode two-component regulatory system proteins rather than transmembrane domains (TMDs). We also identified nagB2, which is highly homologous to nagB1. Adjacent to nagB2, two genes were predicted to encode TMDs. Binding experiments of the recombinant NagB1 and NagB2 to several oligosaccharides using differential scanning fluorimetry and surface plasmon resonance confirmed that both proteins are SBPs of (GlcNAc)2 and (GlcNAc)3. We determined their crystal structures complexed with and without chitin oligosaccharides at a resolution of 1.2 to 2.0 Å. The structures shared typical SBP structural folds and were classified as subcluster D-I. Large domain motions were observed in the structures, suggesting that they were induced by ligand binding via the “Venus flytrap” mechanism. These structures also revealed chitin oligosaccharide recognition mechanisms. In conclusion, our study provides insight into the recognition and transport of chitin oligosaccharides in bacteria.

A Comparative Genomics Approach for Shortlisting Broad-Spectrum Drug Targets in Nontuberculous Mycobacteria.

Many members of nontuberculous mycobacteria (NTM) are opportunistic pathogens causing several infections in animals. The incidence of NTM infections and emergence of drug-resistant NTM strains are rising worldwide, emphasizing the need to develop novel anti-NTM drugs. The present study is aimed to identify broad-spectrum drug targets in NTM using a comparative genomics approach. The study identified 537 core proteins in NTM of which 45 were pathogen specific and essential for the survival of pathogens. Furthermore, druggability analysis indicated that 15 were druggable among those 45 proteins. These 15 proteins, which were core proteins, pathogen-specific, essential, and druggable, were considered as potential broad-spectrum candidates. Based on their locations in cytoplasm and membrane, targets were classified as drug and vaccine targets. The identified 15 targets were different enzymes, carrier proteins, transcriptional regulator, two-component system protein, ribosomal, and binding proteins. The identified targets could further be utilized by researchers to design inhibitors for the discovery of antimicrobial agents.

Revealing the key point of the temperature stress response of Arthrospira platensis C1 at the interconnection of C- and N- metabolism by proteome analyses and PPI networking

BackgroundGrowth-temperature stress causes biochemical changes in the cells and reduction of biomass yield. Quantitative proteome of Arthrospira platensis C1 in response to low- and high temperature stresses was previously analysed to elucidate the stress response mechanism. The data highlighted the linkage of signaling proteins and proteins involved in nitrogen and ammonia assimilation, photosynthesis and oxidative stress.ResultsAfter phosphoproteome analysis was carried out in this study, the tentative temperature response cascade of A. platensis C1 was drawn based on data integration of quantitative proteome and phosphoproteome analysis and protein-protein interaction (PPI) networks. The integration revealed 31 proteins regulated at the protein-expression and post-translational levels; thus, this group of proteins was designated bi-level regulated proteins. PPI networks were then constructed based on A. platensis C1 gene inference from publicly available interaction data. The key two-component system (TCS) proteins, SPLC1_S082010 and SPLC1_S230960, were identified as bi-level regulated proteins and were linked to SPLC1_S270380 or glutamate synthase, an important enzyme in nitrogen assimilation that synthesizes glutamate from 2-oxoglutarate, which is known as the signal compound that regulates the carbon/nitrogen (C/N) balance of cells. Moreover, the role of the p-site in the PPIs of some phosphoproteins of interest was determined using site-directed mutagenesis and a yeast two-hybrid system. Evidence showing the critical role of the p-site in the PPI was observed for the multi-sensor histidine kinase SPLC1_S041070 (Hik28) and glutamate synthase. PPI subnetwork also showed that the Hik28 involved with the enzymes in fatty acid desaturation and nitrogen metabolism. The effect of Hik28-deletion was validated by fatty acid analysis and measurement of photosynthetic activity under nitrogen depletion.ConclusionsTaken together, the data clearly represents (i) the multi-level regulation of proteins involved in the stress response mechanism and (ii) the key point of the temperature stress response at the interconnection of C- and N- metabolism.

Role of a Two-Component Signal Transduction System RspA1/A2 in Regulating the Biosynthesis of Salinomycin in Streptomyces albus.

The two-component system "AfsQ1/Q2" plays a crucial role to activate the production of antibiotics ACT, RED, and CDA through directly binding the promoters of pathway-specific activator genes actII-ORF4, redZ, and cdaR respectively when grown under glutamate-supplemented minimal medium in Streptomyces coelicolor. In this report, we demonstrated that the RspA1/A2 (a homologous protein of two-component system AfsQ1/Q2) plays a regulatory role in salinomycin biosynthesis in Streptomyces albus. Gene deletion and complementation experiments showed that the RspA1/A2 promoted salinomycin production but inhibited cell growth when cultured in YMG medium supplemented with 3% soybean oil. More importantly, RspA1/A2 strengthens salinomycin biosynthesis by directly affecting the transcription of the pathway-specific activator gene slnR. Meanwhile, RspA1/A2 plays a negative role in the regulation of nitrogen assimilation and urea decarboxylation by interacting with the promoters of genes gdhA, glnA, amtB, and SLNWT_1828/1829. Gene sigW is located downstream of rspA1/A2 and encodes an extracytoplasmic function sigma factor. Moreover, it negatively regulates the salinomycin biosynthesis and promotes cell growth, which antagonizes the function of RspA1/A2. In short, these useful findings are proved helpful to enrich the understanding of the regulatory pathways of antibiotic biosynthesis by an ECF σ factor-TCS signal transduction system in Streptomyces.

Pseudomonas aeruginosa biofilm formation on endotracheal tubes requires multiple two-component systems

Introduction. Indwelling medical devices such as endotracheal tubes (ETTs), urinary catheters, vascular access devices, tracheostomies and feeding tubes are often associated with hospital-acquired infections. Bacterial biofilm formed on the ETTs in intubated patients is a significant risk factor associated with ventilator-associated pneumonia. Pseudomonas aeruginosa is one of the four frequently encountered bacteria responsible for causing pneumonia, and the biofilm formation on ETTs. However, understanding of biofilm formation on ETT and interventions to prevent biofilm remains lagging. The ability to sense and adapt to external cues contributes to their success. Thus, the biofilm formation is likely to be influenced by the two-component systems (TCSs) that are composed of a membrane-associated sensor kinase and an intracellular response regulator.Aim. This study aims to establish an in vitro method to analyse the P. aeruginosa biofilm formation on ETTs, and identify the TCSs that contribute to this process.Methodology. In total, 112 P. aeruginosa PA14 TCS mutants were tested for their ability to form biofilm on ETTs, their effect on quorum sensing (QS) and motility.Results. Out of 112 TCS mutants studied, 56 had altered biofilm biomass on ETTs. Although the biofilm formation on ETTs is QS-dependent, none of the 56 loci controlled quorum signal. Of these, 18 novel TCSs specific to ETT biofilm were identified, namely, AauS, AgtS, ColR, CopS, CprR, NasT, KdpD, ParS, PmrB, PprA, PvrS, RcsC, PA14_11120, PA14_32580, PA14_45880, PA14_49420, PA14_52240, PA14_70790. The set of 56 included the GacS network, TCS proteins involved in fimbriae synthesis, TCS proteins involved in antimicrobial peptide resistance, and surface-sensing. Additionally, several of the TCS-encoding genes involved in biofilm formation on ETTs were found to be linked to flagellum-dependent swimming motility.Conclusions. Our study established an in vitro method for studying P. aeruginosa biofilm formation on the ETT surfaces. We also identified novel ETT-specific TCSs that could serve as targets to prevent biofilm formation on indwelling devices frequently used in clinical settings.

Genome-wide characterization of two-component system (TCS) genes in melon (Cucumis melo L.)

To better understand cytokinin signaling in melon (Cucumis melo L.), one of the most important fruit crops in the Cucurbitaceae family, we identified and characterized melon two-component system (TCS) genes in this study. The results showed that there were 51 genes encoding putative TCS proteins in melon, and these TCS genes were classified into 3 subgroups, with 17 HK(L)s (histidine kinase/histidine-kinase like; 9 HKs and 8 HKLs), 9 HPs (histidine phosphotransfer proteins; 6 authentic and 3 pseudo), and 25 RRs (response regulators; 8 Type-A, 11 Type-B and 6 pseudo). The identity values of these cytokinin signaling proteins were revealed by analyzing their conserved motifs, domains and amino acid sequences. By analyzing TCS genes in different plant species, we found that melon HK(L)s, HPs and RRs had closer phylogenetic relationships with cucumber genes than with the genes of other plants, and the expansion of melon cytokinin signaling genes might be attributed to segmental duplication events. Analysis of the putative promoter regions (2-kb upstream regions of the start codon) revealed the enrichment of stress- and hormone-response cis-elements. The involvement of these putative TCS genes in melon cytokinin signaling was further supported by qRT-PCR data.