Two-component Signaling Systems Research Articles

Mapping the \u2018Two-component system\u2019 network in rice

Two-component system (TCS) in plants is a histidine to aspartate phosphorelay based signaling system. Rice genome has multifarious TCS signaling machinery comprising of 11 histidine kinases (OsHKs), 5 histidine phosphotransferases (OsHPTs) and 36 response regulators (OsRRs). However, how these TCS members interact with each other and comprehend diverse signaling cascades remains unmapped. Using a highly stringent yeast two-hybrid (Y2H) platform and extensive in planta bimolecular fluorescence complementation (BiFC) assays, distinct arrays of interaction between various TCS proteins have been identified in the present study. Based on these results, an interactome map of TCS proteins has been assembled. This map clearly shows a cross talk in signaling, mediated by different sensory OsHKs. It also highlights OsHPTs as the interaction hubs, which interact with OsRRs, mostly in a redundant fashion. Remarkably, interactions between type-A and type-B OsRRs have also been revealed for the first time. These observations suggest that feedback regulation by type-A OsRRs may also be mediated by interference in signaling at the level of type-B OsRRs, in addition to OsHPTs, as known previously. The interactome map presented here provides a starting point for in-depth molecular investigations for signal(s) transmitted by various TCS modules into diverse biological processes.

Linearmycins Activate a Two-Component Signaling System Involved in Bacterial Competition and Biofilm Morphology.

Bacteria use two-component signaling systems to adapt and respond to their competitors and changing environments. For instance, competitor bacteria may produce antibiotics and other bioactive metabolites and sequester nutrients. To survive, some species of bacteria escape competition through antibiotic production, biofilm formation, or motility. Specialized metabolite production and biofilm formation are relatively well understood for bacterial species in isolation. How bacteria control these functions when competitors are present is not well studied. To address fundamental questions relating to the competitive mechanisms of different species, we have developed a model system using two species of soil bacteria, Bacillus subtilis and Streptomyces sp. strain Mg1. Using this model, we previously found that linearmycins produced by Streptomyces sp. strain Mg1 cause lysis of B. subtilis cells and degradation of colony matrix. We identified strains of B. subtilis with mutations in the two-component signaling system yfiJK operon that confer dual phenotypes of specific linearmycin resistance and biofilm morphology. We determined that expression of the ATP-binding cassette (ABC) transporter yfiLMN operon, particularly yfiM and yfiN, is necessary for biofilm morphology. Using transposon mutagenesis, we identified genes that are required for YfiLMN-mediated biofilm morphology, including several chaperones. Using transcriptional fusions, we found that YfiJ signaling is activated by linearmycins and other polyene metabolites. Finally, using a truncated YfiJ, we show that YfiJ requires its transmembrane domain to activate downstream signaling. Taken together, these results suggest coordinated dual antibiotic resistance and biofilm morphology by a single multifunctional ABC transporter promotes competitive fitness of B. subtilisIMPORTANCE DNA sequencing approaches have revealed hitherto unexplored diversity of bacterial species in a wide variety of environments that includes the gastrointestinal tract of animals and the rhizosphere of plants. Interactions between different species in bacterial communities have impacts on our health and industry. However, many approaches currently used to study whole bacterial communities do not resolve mechanistic details of interspecies interactions, including how bacteria sense and respond to their competitors. Using a competition model, we have uncovered dual functions for a previously uncharacterized two-component signaling system involved in specific antibiotic resistance and biofilm morphology. Insights gleaned from signaling within interspecies interaction models build a more complete understanding of gene functions important for bacterial communities and will enhance community-level analytical approaches.

BsmR degrades c-di-GMP to modulate biofilm formation of nosocomial pathogen Stenotrophomonas maltophilia

c-di-GMP is a cellular second messenger that regulates diverse bacterial processes, including swimming, biofilm formation and virulence. However, in Stenotrophomonas maltophilia, a nosocomial pathogen that frequently infects immunodeficient or immunoincompetent patients, the regulatory function of c-di-GMP remains unclear. Here we show that BsmR is a negative regulator of biofilm development that degrades c-di-GMP through its EAL domain. Increasing BsmR expression resulted in significant increase in bacterial swimming and decrease in cell aggregation. BsmR regulates the expression of at least 349 genes. Among them, 34 involved in flagellar assembly and a flagellar-assembly-related transcription factor (fsnR) are positively regulated. Although BsmR is a response regulator of the two-component signaling system, its role in biofilm formation depends on the expression level of its respective gene (bsmR), not on the protein’s phosphorylation level. A transcription factor, BsmT, whose coding gene is located in the same tetra-cistronic operon as bsmR, was shown to directly bind to the promoter region of the operon and, through a positive regulatory loop, modulate bsmR transcription. Thus, our results revealed that the c-di-GMP signaling pathway controls biofilm formation and swimming in S. maltophilia, suggesting c-di-GMP signaling as a target in the development of novel antibacterial agents to resist this pathogen.

Novel Predators Reshape Holozoan Phylogeny and Reveal the Presence of a Two-Component Signaling System in the Ancestor of Animals

Our understanding of the origin of animals has been transformed by characterizing their most closely related, unicellular sisters: the choanoflagellates, filastereans, and ichthyosporeans. Together with animals, these lineages make up the Holozoa [1,2]. Many traits previously considered "animal specific" were subsequently found in other holozoans [3, 4], showing that they evolved before animals,although exactly when is currently uncertain because several key relationships remain unresolved [2, 5]. Here wereport the morphology andtranscriptome sequencing from three novel unicellular holozoans: Pigoraptor vietnamica and Pigoraptor chileana, which are related to filastereans, and Syssomonas multiformis, which forms a new lineage with Corallochytrium in phylogenomic analyses. All three species are predatory flagellates that feed on large eukaryotic prey, and all three also appear to exhibit complex life histories with several distinct stages, including multicellular clusters. Examination of genes associated with multicellularity in animals showed that the new filastereans contain a cell-adhesion gene repertoire similar to those of other species in this group. Syssomonas multiformis possessed a smaller complement overall but does encode genes absent from the earlier-branching ichthyosporeans. Analysis of the T-box transcription factor domain showed expansion of T-box transcription factors based on combination with a non-T-box domain (a receiver domain), which has not been described outside of vertebrates. Thisdomain and other domains we identified in all unicellular holozoans are part of the two-component signaling system that has been lost in animals, suggesting the continued use of this system in the closest relatives of animals and emphasizing the importance of studying loss of function as well as gain in major evolutionary transitions.

AHP2, AHP3, and AHP5 act downstream of CKI1 in Arabidopsis female gametophyte development.

Histidine phosphotransfer proteins (HPs) are key elements of the two-component signaling system, which act as a shuttle to transfer phosphorylation signals from histidine kinases (HKs) to response regulators (RRs). CYTOKININ INDEPENDENT 1 (CKI1), a key regulator of central cell specification in the Arabidopsis female gametophyte, activates the cytokinin signaling pathway through the Arabidopsis histidine phosphotransfer proteins (AHPs). There are five HP genes in Arabidopsis, AHP1-AHP5, but it remains unknown which AHP genes act downstream of CKI1 in Arabidopsis female gametophyte development. Promoter activity analysis of AHP1-AHP5 in embryo sacs revealed AHP1, AHP2, AHP3, and AHP5 expression in the central cell. Phenotypic studies of various combinations of ahp mutants showed that triple mutations in AHP2, AHP3, and AHP5 resulted in defective embryo sac development. Using cell-specific single and double markers in the female gametophyte, the ahp2-2 ahp3 ahp5-2/+ triple mutant ovules showed loss of central cell and antipodal cell fates and gain of egg cell or synergid cell attributes, resembling the cki1 mutant phenotypes. These data suggest that AHP2, AHP3, and AHP5 are the major factors acting downstream of CKI1 in the two-component cytokinin signaling pathway to promote Arabidopsis female gametophyte development.

Requirement of the CroRS Two-Component System for Resistance to Cell Wall-Targeting Antimicrobials in Enterococcus faecium

Enterococci are serious opportunistic pathogens that are resistant to many cell wall-targeting antibiotics. The CroRS two-component signaling system responds to antibiotic-mediated cell wall stress and is critical for resistance to cell wall-targeting antibiotics in Enterococcus faecalis Here, we identify and characterize an orthologous two-component system found in Enterococcus faecium that is functionally equivalent to the CroRS system of E. faecalis Deletion of croRS in E. faecium resulted in marked susceptibility to cell wall-targeting agents including cephalosporins and bacitracin, as well as moderate susceptibility to ampicillin and vancomycin. As in E. faecalis, exposure to bacitracin and vancomycin stimulates signaling through the CroRS system in E. faecium Moreover, the CroRS system is critical in E. faecium for enhanced beta-lactam resistance mediated by overexpression of Pbp5. Expression of a Pbp5 variant that confers enhanced beta-lactam resistance cannot overcome the requirement for CroRS function. Thus, the CroRS system is a conserved signaling system that responds to cell wall stress to promote intrinsic resistance to important cell wall-targeting antibiotics in clinically relevant enterococci.

A Novel RAYM_RS09735/RAYM_RS09740 Two-Component Signaling System Regulates Gene Expression and Virulence in Riemerella anatipestifer.

The Gram-negative bacterium Riemerella anatipestifer is an important waterfowl pathogen, causing major economic losses to the duck-producing industry. However, little is known of the virulence factors that mediate pathogenesis during R. anatipestifer infection. In this study, RAYM_RS09735 and RAYM_RS09740 were predicted to form a two-component signaling system (TCS) through bioinformatics analysis. This TCS was highly conserved across the Flavobacteriaceae. A mutant YMΔRS09735/RS09740 strain was constructed to investigate the role of the RAYM_RS09735/RAYM_RS09740 TCS in R. anatipestifer virulence and gene regulation. The median lethal dose (LD50) of YMΔRS09735/RS09740 was found to be >1011 CFU, equivalent to that of avirulent bacterial strains. The bacterial abundances of the YMΔRS09735/RS09740 strain in the heart, brain, liver, blood, and spleen were significantly lower than that of the wild-type R. anatipestifer YM strain. Pathological analysis using hematoxylin and eosin staining showed that, compared to the wild-type, the mutant YMΔRS09735/RS09740 strain caused significantly less virulence in infected ducklings. RNAseq and real-time PCR analysis indicated that the RAYM_RS09735/RAYM_RS09740 TCS is a PhoP/PhoR system. This is a novel type of TCS for Gram-negative bacteria. The TCS was also found to be a global regulator of expression in R. anatipestifer, with 112 genes up-regulated and 693 genes down-regulated in the YMΔRS09735/RS09740 strain (~33% genes demonstrated differential expression). In summary, we have reported the first PhoP/PhoR TCS identified in a Gram-negative bacterium and demonstrated that it is involved in virulence and gene regulation in R. anatipestifer.

Inhibition and Dispersion of Biofilms: Targeting Bacterial Response Regulators to Resensitize Multidrug Resistant Bacteria to Antibiotics

Biofilms provide cells with protection against numerous physical and chemical assaults. Bacteria utilize biofilms to avoid antibiotics, leaving the bacteria resistant to antibiotic treatments. The rise of antibiotic resistance and lack of new viable treatments threaten to usher in a post‐antibiotic era if new approaches towards drug development are not made. Many of the currently available antibiotics target a narrow spectrum of metabolic processes required for cellular growth. These traditional approaches no longer offer long‐term protection due to the acquisition rate of resistance; new mechanisms must be pursued. One such mechanism is through the two‐component systems (TCSs) used in bacterial communication. TCSs allow the cell to detect and respond to changes in its environment and are used to trigger quorum sensing and virulence factors. Drugs that target the TCS signaling process could inhibit pathogenic behavior, making them a potent new approach that has not yet been exploited. The TCS response regulator BfmR from Acinetobacter baumannii is the master controller of biofilm formation, making BfmR an attractive drug target. BfmR is targeted by 2‐aminoimidazoles (2AIs), compounds that inhibit and disperse biofilms in Gram‐negative and Gram‐positive bacteria. This work focuses on the interactions between 2AI based compounds and the response regulators BfmR and PmrA/QseB, from Fancisella tularensis, which also regulates biofilm development. Understanding the impact of 2AIs on response regulators and their mechanism of interaction will lead to the development of more potent inhibitors.

Lysobacter PilR, the Regulator of Type IV Pilus Synthesis, Controls Antifungal Antibiotic Production via a Cyclic di-GMP Pathway.

Lysobacter enzymogenes is a ubiquitous soil gammaproteobacterium that produces a broad-spectrum antifungal antibiotic, known as heat-stable antifungal factor (HSAF). To increase HSAF production for use against fungal crop diseases, it is important to understand how HSAF synthesis is regulated. To gain insights into transcriptional regulation of the HSAF synthesis gene cluster, we generated a library with deletion mutations in the genes predicted to encode response regulators of the two-component signaling systems in L. enzymogenes strain OH11. By quantifying HSAF production levels in the 45 constructed mutants, we identified two strains that produced significantly smaller amounts of HSAF. One of the mutations affected a gene encoding a conserved bacterial response regulator, PilR, which is commonly associated with type IV pilus synthesis. We determined that L. enzymogenes PilR regulates pilus synthesis and twitching motility via a traditional pathway, by binding to the pilA promoter and upregulating pilA expression. Regulation of HSAF production by PilR was found to be independent of pilus formation. We discovered that the pilR mutant contained significantly higher intracellular levels of the second messenger cyclic di-GMP (c-di-GMP) and that this was the inhibitory signal for HSAF production. Therefore, the type IV pilus regulator PilR in L. enzymogenes activates twitching motility while downregulating antibiotic HSAF production by increasing intracellular c-di-GMP levels. This study identifies a new role of a common pilus regulator in proteobacteria and provides guidance for increasing antifungal antibiotic production in L. enzymogenesIMPORTANCE PilR is a widespread response regulator of the two-component system known for regulating type IV pilus synthesis in proteobacteria. Here we report that, in the soil bacterium Lysobacter enzymogenes, PilR regulates pilus synthesis and twitching motility, as expected. Unexpectedly, PilR was also found to control intracellular levels of the second messenger c-di-GMP, which in turn inhibits production of the antifungal antibiotic HSAF. The coordinated production of type IV pili and antifungal antibiotics has not been observed previously.

Identification, classification, and transcription profiles of the B-type response regulator family in pear.

Type-B response regulators (B-RRs) are transcription factors that function in the final step of two-component signaling systems. In model plants, B-RRs have been shown to play important roles in cytokinin signal transduction. However, the functions of B-RRs in pear have not been well studied. In this report, we conducted a genome-wide analysis and identified 11 putative genes encoding B-PpRR proteins based on the published genome sequence of Pyrus bretschneideri. A phylogenetic tree of the B-PpRR family was constructed, and the motif distribution, chromosome localization, and gene structure of B-PpRR family genes were determined. Gene transcript profiles, which were determined from transcriptome data, indicated that B-PpRR genes potentially function during pear fruit development, bud dormancy, and light/hormone-induced anthocyanin accumulation. Treatment of the fruitlets of ‘Cuiguan’ pear (Pyrus pyrifolia), which never accumulates anthocyanin, with the cytokinin N-(2-chloro-4-pyridyl)- N′-phenylurea (CPPU) clearly induced anthocyanin accumulation. Anthocyanins accumulated in the skin of fruitlets by 16 days after CPPU treatment, along with the significant activation of most anthocyanin biosynthetic genes. Analyses of B-PpRR transcript levels suggested that B-PpRR genes mediated this accumulation of anthocyanins. These findings enrich our understanding of the function of B-PpRR genes in the physiological processes of pear.

The sil Locus in Streptococcus Anginosus Group: Interspecies Competition and a Hotspot of Genetic Diversity

The Streptococcus Invasion Locus (Sil) was first described in Streptococcus pyogenes and Streptococcus pneumoniae, where it has been implicated in virulence. The two-component peptide signaling system consists of the SilA response regulator and SilB histidine kinase along with the SilCR signaling peptide and SilD/E export/processing proteins. The presence of an associated bacteriocin region suggests this system may play a role in competitive interactions with other microbes. Comparative analysis of 42 Streptococcus Anginosus/Milleri Group (SAG) genomes reveals this to be a hot spot for genomic variability. A cluster of bacteriocin/immunity genes is found adjacent to the sil system in most SAG isolates (typically 6–10 per strain). In addition, there were two distinct SilCR peptides identified in this group, denoted here as SilCRSAG-A and SilCRSAG-B, with corresponding alleles in silB. Our analysis of the 42 sil loci showed that SilCRSAG-A is only found in Streptococcus intermedius while all three species can carry SilCRSAG-B. In S. intermedius B196, a putative SilA operator is located upstream of bacteriocin gene clusters, implicating the sil system in regulation of microbe–microbe interactions at mucosal surfaces where the group resides. We demonstrate that S. intermedius B196 responds to its cognate SilCRSAG-A, and, less effectively, to SilCRSAG-B released by other Anginosus group members, to produce putative bacteriocins and inhibit the growth of a sensitive strain of S. constellatus.

Distribution of β-lactamase genes of Klebsiella pneumoniae isolates in Zhejiang province, China, and regulation of gene expression

Klebsiella pneumoniae is a common causative agent of nosocomial infections with a high level of resistance toward ?-lactam antibiotics. Our previous study showed that TEM-1 and SHV-11 are the predominant ?-lactamase-encoding genes of K. pneumoniae isolates in the Zhejiangarea, China. In this study, more clinical K. pneumoniae isolates were collected for detecting their ?-lactamase-encoding gene profiles by PCR and sequencing. qRT-PCR was then performed to determine the role of cefotaxime or penicillin in low concentrations to induce the ?-lactamase gene expression of K. pneumoniae isolates. Moreover, the K. pneumoniae isolates were pretreated with closantel (CLO), a histidine kinase inhibitor, before antibiotic treatment, and qRT-PCR and the ?-lactamase phenotype confirmatory test were then applied to determine the effect of CLO on the expression of the ?-lactamase genes. The results showed that, except for KPC-2, the 1/4 MIC cefotaxime or penicillin induced significant mRNA elevation of the TEM-1, CTX-M-14, SHV-11 and OXA-1?-lactamase genes, but this induction could be inhibited by CLO. After pretreatment withCLO,78.4~81.4%of the ?-lactam-resistant isolates became sensitive and the positive rate of the ?-lactamase production phenotype in the isolates was decreased from 100% to 27.1%. The data indicate thatTEM-1 (70.7%), SHV-11 (64.2%) and CTX-M-14 (40.5%) are the predominant ?-lactamase genes of the K. pneumoniae isolates in Zhejiang and sublethal dosage of ?-lactam antibiotics can induce the ?-lactamase gene expression of K. pneumoniae through histidine kinase-mediated two-component signaling systems.

Two-Component Signaling System VgrRS Directly Senses Extracytoplasmic and Intracellular Iron to Control Bacterial Adaptation under Iron Depleted Stress.

Both iron starvation and excess are detrimental to cellular life, especially for animal and plant pathogens since they always live in iron-limited environments produced by host immune responses. However, how organisms sense and respond to iron is incompletely understood. Herein, we reveal that in the phytopathogenic bacterium Xanthomonas campestris pv. campestris, VgrS (also named ColS) is a membrane-bound receptor histidine kinase that senses extracytoplasmic iron limitation in the periplasm, while its cognate response regulator, VgrR (ColR), detects intracellular iron excess. Under iron-depleted conditions, dissociation of Fe3+ from the periplasmic sensor region of VgrS activates the VgrS autophosphorylation and subsequent phosphotransfer to VgrR, an OmpR-family transcription factor that regulates bacterial responses to take up iron. VgrR-VgrS regulon and the consensus DNA binding motif of the transcription factor VgrR were dissected by comparative proteomic and ChIP-seq analyses, which revealed that in reacting to iron-depleted environments, VgrR directly or indirectly controls the expressions of hundreds of genes that are involved in various physiological cascades, especially those associated with iron-uptake. Among them, we demonstrated that the phosphorylated VgrR tightly represses the transcription of a special TonB-dependent receptor gene, tdvA. This regulation is a critical prerequisite for efficient iron uptake and bacterial virulence since activation of tdvA transcription is detrimental to these processes. When the intracellular iron accumulates, the VgrR-Fe2+ interaction dissociates not only the binding between VgrR and the tdvA promoter, but also the interaction between VgrR and VgrS. This relieves the repression in tdvA transcription to impede continuous iron uptake and avoids possible toxic effects of excessive iron accumulation. Our results revealed a signaling system that directly senses both extracytoplasmic and intracellular iron to modulate bacterial iron homeostasis.

Transcriptomic analysis displays the effect of (-)-roemerine on the motility and nutrient uptake in Escherichia coli.

Among the different families of plant alkaloids, (-)-roemerine, an aporphine type, was recently shown to possess significant antibacterial activity in Escherichia coli. Based on the increasing demand for antibacterials with novel mechanisms of action, the present work investigates the potential of the plant-derived alkaloid (-)-roemerine as an antibacterial in E. coli cells using microarray technology. Analysis of the genome-wide transcriptional reprogramming in cells after 60min treatment with 100 μg/mL (-)-roemerine showed significant changes in the expression of 241 genes (p value <0.05 and fold change >2). Expression of selected genes was confirmed by qPCR. Differentially expressed genes were classified into functional categories to map biological processes and molecular pathways involved. Cellular activities with roles in carbohydrate transport and metabolism, energy production and conversion, lipid transport and metabolism, amino acid transport and metabolism, two-component signaling systems, and cell motility (in particular, the flagellar organization and motility) were among metabolic processes altered in the presence of (-)-roemerine. The down-regulation of the outer membrane proteins probably led to a decrease in carbohydrate uptake rate, which in turn results in nutrient limitation. Consequently, energy metabolism is slowed down. Interestingly, the majority of the expressional alterations were found in the flagellar system. This suggested reduction in motility and loss in the ability to form biofilms, thus affecting protection of E. coli against host cell defense mechanisms. In summary, our findings suggest that the antimicrobial action of (-)-roemerine in E. coli is linked to disturbances in motility and nutrient uptake.

The Impact of Protein Phosphorylation on Chlamydial Physiology.

Chlamydia are Gram negative bacterial pathogens responsible for disease in humans and economically important domesticated animals. As obligate intracellular bacteria, they must gain entry into a host cell where they propagate within a parasitophorous organelle that serves as an interactive interface between the bacterium and the host. Nutrient acquisition, growth, and evasion of host defense mechanisms occur from this location. In addition to these cellular and bacterial dynamics, Chlamydia differentiate between two morphologically distinct forms, the elementary body and reticulate body, that are optimized for either extracellular or intracellular survival, respectively. The mechanisms regulating and mediating these diverse physiological events remain largely unknown. Reversible phosphorylation, including classical two-component signaling systems, partner switching mechanisms, and the more recently appreciated bacterial Ser/Thr/Tyr kinases and phosphatases, has gained increasing attention for its role in regulating important physiological processes in bacteria including metabolism, development, and virulence. Phosphorylation modulates these events via rapid and reversible modification of protein substrates leading to changes in enzyme activity, protein oligomerization, cell signaling, and protein localization. The characterization of several conserved chlamydial protein kinases and phosphatases along with phosphoproteome analysis suggest that Chlamydia are capable of global and growth stage-specific protein phosphorylation. This mini review will highlight the current knowledge of protein phosphorylation in Chlamydia and its potential role in chlamydial physiology and, consequently, virulence. Comparisons with other minimal genome intracellular bacterial pathogens also will be addressed with the aim of illustrating the importance of this understudied regulatory mechanism on pathogenesis and the principle questions that remain unanswered.

The σ54-dependent two-component system regulating sulfur oxidization (Sox) system in Acidithiobacillus caldus and some chemolithotrophic bacteria.

The sulfur oxidization (Sox) system is the central sulfur oxidization pathway of phototrophic and chemotrophic sulfur-oxidizing bacteria. Regulation and function of the Sox system in the chemotrophic Paracoccus pantotrophus has been elucidated; however, to date, no information is available on the regulation of this system in the chemolithotrophic Acidithiobacillus caldus, which is widely utilized in bioleaching. We described the novel tspSR-sox-like clusters in A. caldus and other chemolithotrophic sulfur-oxidizing bacteria containing Sox systems. The highly homologous σ54-dependent two-component signaling system (TspS/R), upstream of the sox operons in these novel clusters, was identified by phylogenetic analyses. A typical σ54-dependent promoter, P1, was identified upstream of soxX-I in the sox-I cluster of A. caldus MTH-04. The transcriptional start site (G) and the -12/-24 regions (GC/GG) of P1 were determined by rapid amplification of cDNA ends (5'RACE), and the upstream activator sequences (UASs; TGTCCCAAATGGGACA) were confirmed by electrophoretic mobility shift assays (EMSAs) in vitro and by UAS-probe-plasmids assays in vivo. Sequence analysis of promoter regions in tspSR-sox-like clusters revealed that there were similar σ54-dependent promoters upstream of the soxX genes. Based on our results, we proposed a TspSR-mediated signal transduction and transcriptional regulation pathway for the Sox system in A. caldus. The regulation of σ54-dependent two-component systems (TCSs) for Sox pathways were explained for the first time in A. caldus, A. thiooxidans, T. tepidarius, and T. denitrificans, indicating the significance of modulating the sulfur oxidization in these chemolithotrophic sulfur oxidizers.

Mutation of the Streptococcus gordonii Thiol-Disulfide Oxidoreductase SdbA Leads to Enhanced Biofilm Formation Mediated by the CiaRH Two-Component Signaling System.

Streptococcus gordonii is a commensal inhabitant of human oral biofilms. Previously, we identified an enzyme called SdbA that played an important role in biofilm formation by S. gordonii. SdbA is thiol-disulfide oxidoreductase that catalyzes disulfide bonds in secreted proteins. Surprisingly, inactivation of SdbA results in enhanced biofilm formation. In this study we investigated the basis for biofilm formation by the ΔsdbA mutant. The results revealed that biofilm formation was mediated by the interaction between the CiaRH and ComDE two-component signalling systems. Although it did not affect biofilm formation by the S. gordonii parent strain, CiaRH was upregulated in the ΔsdbA mutant and it was essential for the enhanced biofilm phenotype. The biofilm phenotype was reversed by inactivation of CiaRH or by the addition of competence stimulating peptide, the production of which is blocked by CiaRH activity. Competition assays showed that the enhanced biofilm phenotype also corresponded to increased oral colonization in mice. Thus, the interaction between SdbA, CiaRH and ComDE affects biofilm formation both in vitro and in vivo.

Inferring interaction partners from protein sequences

Specific protein-protein interactions are crucial in the cell, both to ensure the formation and stability of multiprotein complexes and to enable signal transduction in various pathways. Functional interactions between proteins result in coevolution between the interaction partners, causing their sequences to be correlated. Here we exploit these correlations to accurately identify, from sequence data alone, which proteins are specific interaction partners. Our general approach, which employs a pairwise maximum entropy model to infer couplings between residues, has been successfully used to predict the 3D structures of proteins from sequences. Thus inspired, we introduce an iterative algorithm to predict specific interaction partners from two protein families whose members are known to interact. We first assess the algorithm's performance on histidine kinases and response regulators from bacterial two-component signaling systems. We obtain a striking 0.93 true positive fraction on our complete dataset without any a priori knowledge of interaction partners, and we uncover the origin of this success. We then apply the algorithm to proteins from ATP-binding cassette (ABC) transporter complexes, and obtain accurate predictions in these systems as well. Finally, we present two metrics that accurately distinguish interacting protein families from noninteracting ones, using only sequence data.

The small membrane protein MgrB regulates PhoQ bifunctionality to control PhoP target gene expression dynamics.

In Escherichia coli and other γ-proteobacteria, the PhoQ-PhoP two-component signaling system responds to low extracellular Mg++ and cationic antimicrobial peptides. On transition to inducing conditions, the expression of PhoP-dependent genes increases rapidly, but then decays to a new, intermediate steady-state level, a phenomenon often referred to as partial adaptation. The molecular basis for this partial adaptation has been unclear. Here, using time-lapse fluorescence microscopy to examine PhoP-dependent gene expression in individual E. coli cells we show that partial adaptation arises through a negative feedback loop involving the small protein MgrB. When E. coli cells are shifted to low Mg++ , PhoQ engages in multiple rounds of autophosphorylation and phosphotransfer to PhoP, which, in turn, drives the expression of mgrB. MgrB then feeds back to inhibit the kinase activity of PhoQ. PhoQ is bifunctional such that, when not active as a kinase, it can stimulate the dephosphorylation of PhoP. Thus, MgrB drives the inactivation of PhoP and the observed adaptation in PhoP-dependent gene expression. Our results clarify the source of feedback inhibition in the E. coli PhoQ-PhoP system and reveal how exogenous factors, such as MgrB, can combine with a canonical two-component signaling pathway to produce complex temporal dynamics in target gene expression.

Type IV-pili dependent motility is co-regulated by PilSR and PilS2R2 two-component systems via distinct pathways in Myxococcus xanthus.

Myxococcus xanthus is an environmental bacterium with two forms of motility. One type, known as social motility, is dependent on extension and retraction of Type-IV pili (T4P) and production of extracellular polysaccharides (EPS). Several signaling systems have been linked to regulation of T4P-dependent motility. In particular, expression of the pilin subunit pilA requires the PilSR two-component signaling system (TCS). A second TCS, PilS2R2, encoded within the same locus that encodes PilSR, has also been linked to M. xanthus T4P-dependent motility. We demonstrate that PilSR and PilS2R2 regulate M. xanthus T4P-dependent motility through distinct pathways. Consistent with known roles of PilSR, our results indicate that the primary function of PilSR is to regulate expression of pilA. In contrast, PilS2 and PilR2 have little to no affect on PilA protein levels. However, deletion of pilR2 resulted in a reduction of assembled pili, significant decreases in EPS production and loss of T4P-dependent motility. Furthermore, the pilR2 mutation led to increased production of outer membrane vesicles (OMV). Collectively, we propose that PilS2R2 is required for proper assembly of T4P and regulation of OMV production, and hypothesize that production of these vesicles is related to M. xanthus motility.