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Ralstonia solanacearum, the bacterium that causes bacterial wilt, is a destructive phytopathogen that can infect over 450 different plant species. Several agriculturally significant crop plants, including eggplant, tomato, pepper, potato, and ginger, are highly susceptible to this plant disease, which has a global impact on crop quality and yield. There is currently no known preventive method that works well for bacterial wilt. Bacteria use two-component systems (TCSs) to sense their environment constantly and react appropriately. This is achieved by an extracellular sensor kinase (SK) capable of sensing a suitable signal and a cytoplasmic response regulator (RR) which gives a downstream response. Moreover, our investigation revealed that R. solanacearum GMI1000 possesses a substantial count of TCSs, specifically comprising 36 RRs and 27 SKs. While TCSs are known targets for various human pathogenic bacteria, such as Salmonella, the role of TCSs in R. solanacearum remains largely unexplored in this context. Notably, numerous inhibitors targeting TCSs have been identified, including GHL (Gyrase, Hsp, and MutL) compounds, Walk inhibitors, and anti-TCS medications like Radicicol. Consequently, the investigation into the involvement of TCSs in virulence and pathogenesis has gained traction; however, further research is imperative to ascertain whether TCSs could potentially supplant conventional anti-wilt therapies. This review delves into the prospective utilization of TCSs as an alternative anti-wilt therapy, focusing on the lethal phytopathogen R. solanacearum. [ABSTRACT FROM AUTHOR]

The Integrated Stress Response (ISR) is an essential homeostatic signaling network that controls the cell’s biosynthetic capacity. Four ISR sensor kinases detect multiple stressors and relay this information to downstream effectors by phosphorylating a common node: the alpha subunit of the eukaryotic initiation factor eIF2. As a result, general protein synthesis is repressed while select transcripts are preferentially translated, thus remodeling the proteome and transcriptome. Mounting evidence supports a view of the ISR as a dynamic signaling network with multiple modulators and feedback regulatory features that vary across cell and tissue types. Here, we discuss updated views on ISR sensor kinase mechanisms, how the subcellular localization of ISR components impacts signaling, and highlight ISR signaling differences across cells and tissues. Finally, we consider crosstalk between the ISR and other signaling pathways as a determinant of cell health. [ABSTRACT FROM AUTHOR]

The Integrated Stress Response (ISR) is an essential homeostatic signaling network that controls the cell’s biosynthetic capacity. Four ISR sensor kinases detect multiple stressors and relay this information to downstream effectors by phosphorylating a common node: the alpha subunit of the eukaryotic initiation factor eIF2. As a result, general protein synthesis is repressed while select transcripts are preferentially translated, thus remodeling the proteome and transcriptome. Mounting evidence supports a view of the ISR as a dynamic signaling network with multiple modulators and feedback regulatory features that vary across cell and tissue types. Here, we discuss updated views on ISR sensor kinase mechanisms, how the subcellular localization of ISR components impacts signaling, and highlight ISR signaling differences across cells and tissues. Finally, we consider crosstalk between the ISR and other signaling pathways as a determinant of cell health.

A large number of small membrane proteins have been uncovered in bacteria, but their mechanism of action has remained mostly elusive. Here, we investigate the mechanism of a physiologically important small protein, MgrB, which represses the activity of the sensor kinase PhoQ and is widely distributed among enterobacteria. The PhoQ/PhoP two-component system is a master regulator of the bacterial virulence program and interacts with MgrB to modulate bacterial virulence, fitness, and drug resistance. A combination of cross-linking approaches with functional assays and protein dynamic simulations revealed structural rearrangements due to interactions between MgrB and PhoQ near the membrane/periplasm interface and along the transmembrane helices. These interactions induce the movement of the PhoQ catalytic domain and the repression of its activity. Without MgrB, PhoQ appears to be much less sensitive to antimicrobial peptides, including the commonly used C18G. In the presence of MgrB, C18G promotes MgrB to dissociate from PhoQ, thus activating PhoQ via derepression. Our findings reveal the inhibitory mechanism of the small protein MgrB and uncover its importance in antimicrobial peptide sensing. [ABSTRACT FROM AUTHOR]

The signal transduction paradigm in bacteria involves two-component systems (TCSs). Asgardarchaeota are archaea that may have originated the current eukaryotic lifeforms. Most research on these archaea has focused on eukaryotic-like features, such as genes involved in phagocytosis, cytoskeleton structure, and vesicle trafficking. However, little attention has been given to specific prokaryotic features. Here, the sequence and predicted structural features of TCS sensor kinases analyzed from two metagenome assemblies and a genomic assembly from cultured Asgardian archaea are presented. The homology of the sensor kinases suggests the grouping of Lokiarchaeum closer to bacterial homologs. In contrast, one group from a Lokiarchaeum and a meta-genome assembly from Candidatus Heimdallarchaeum suggest the presence of a set of kinases separated from the typical bacterial TCS sensor kinases. AtoS and ArcB homologs were found in meta-genome assemblies along with defined domains for other well-characterized sensor kinases, suggesting the close link between these organisms and bacteria that may have resulted in the metabolic link to the establishment of symbiosis. Several kinases are predicted to be cytoplasmic; some contain several PAS domains. The data shown here suggest that TCS kinases in Asgardian bacteria are witnesses to the transition from bacteria to eukaryotic organisms. [ABSTRACT FROM AUTHOR]

SUMMARY Legionella pneumophila is a Gram-negative environmental bacterium, which survives in planktonic form, colonizes biofilms, and infects protozoa. Upon inhalation of Legionella -contaminated aerosols, the opportunistic pathogen replicates within and destroys alveolar macrophages, thereby causing a severe pneumonia termed Legionnaires' disease. Gram-negative bacteria employ low molecular weight organic compounds as well as the inorganic gas nitric oxide (NO) for cell-cell communication. L. pneumophila produces, secretes, and detects the α-hydroxyketone compound Legionella autoinducer-1 (LAI-1, 3-hydroxypentadecane-4-one). LAI-1 is secreted by L. pneumophila in outer membrane vesicles and not only promotes communication among bacteria but also triggers responses from eukaryotic cells. L. pneumophila detects NO through three different receptors, and signaling through the volatile molecule translates into fluctuations of the intracellular second messenger cyclic-di-guanylate monophosphate. The LAI-1 and NO signaling pathways are linked via the pleiotropic transcription factor LvbR. In this review, we summarize current knowledge about inter-bacterial and inter-kingdom signaling through LAI-1 and NO by Legionella species., Competing Interests: The authors declare no conflict of interest.

The signal transduction paradigm in bacteria involves two-component systems (TCSs). Asgardarchaeota are archaea that may have originated the current eukaryotic lifeforms. Most research on these archaea has focused on eukaryotic-like features, such as genes involved in phagocytosis, cytoskeleton structure, and vesicle trafficking. However, little attention has been given to specific prokaryotic features. Here, the sequence and predicted structural features of TCS sensor kinases analyzed from two metagenome assemblies and a genomic assembly from cultured Asgardian archaea are presented. The homology of the sensor kinases suggests the grouping of Lokiarchaeum closer to bacterial homologs. In contrast, one group from a Lokiarchaeum and a meta-genome assembly from Candidatus Heimdallarchaeum suggest the presence of a set of kinases separated from the typical bacterial TCS sensor kinases. AtoS and ArcB homologs were found in meta-genome assemblies along with defined domains for other well-characterized sensor kinases, suggesting the close link between these organisms and bacteria that may have resulted in the metabolic link to the establishment of symbiosis. Several kinases are predicted to be cytoplasmic; some contain several PAS domains. The data shown here suggest that TCS kinases in Asgardian bacteria are witnesses to the transition from bacteria to eukaryotic organisms.

Signal transduction systems are the key players of bacterial adaptation and survival. The orthodox two-component signal transduction systems perceive diverse environmental stimuli and their regulatory response leads to cellular changes. Although rarely described, the unorthodox three-component systems are also implemented in the regulation of major bacterial behavior such as the virulence of clinically relevant pathogen P. aeruginosa. Previously, we described a novel three-component system in P. capeferrum WCS358 (RclSAR) where the sensor kinase RclS stimulates the intI1 transcription in stationary growth phase. In this study, using rclS knock-out mutant, we identified RclSAR regulon in P. capeferrum WCS358. The RNA sequencing revealed that activity of RclSAR signal transduction system is growth phase dependent with more pronounced regulatory potential in early stages of growth. Transcriptional analysis emphasized the role of RclSAR in global regulation and indicated the involvement of this system in regulation of diverse cellular activities such as RNA binding and metabolic and biocontrol processes. Importantly, phenotypic comparison of WCS358 wild type and ΔrclS mutant showed that RclS sensor kinase contributes to modulation of antibiotic resistance, production of AHLs and siderophore as well as host cell adherence and cytotoxicity. Finally, we proposed the improved model of interplay between RclSAR, RpoS and LasIR regulatory systems in P. capeferrum WCS358. [ABSTRACT FROM AUTHOR]

ABSTRACT Signaling of two-component systems by phosphoryl transfer requires interaction of the sensor kinase with the response regulator. Interaction of the C4-dicarboxylate-responsive and membrane-integral sensor kinase DcuS with the response regulator DcuR was studied. In vitro, the cytoplasmic part of DcuS (PASC-Kin) was employed. Stable complexes were formed, when either DcuS or DcuR were phosphorylated (Kd 22 ± 11 and 28 ± 7 nM, respectively). The unphosphorylated proteins produced a more labile complex (Kd 1380 ± 395 nM). Bacterial two-hybrid studies confirm interaction of DcuR with DcuS (and PASC-Kin) in vivo. The absolute contents of DcuR (197-979 pmol mg−1 protein) in the bacteria exceeded those of DcuS by more than 1 order of magnitude. According to the Kd values, DcuS exists in complex, with phosphorylated but also unphosphorylated DcuR. In live cell imaging, the predominantly freely diffusing DcuR becomes markedly less mobile after phosphorylation and activation of DcuS by fumarate. Portions of the low mobility fraction accumulated at the cell poles, the preferred location of DcuS, and other portions within the cell, representing phosphorylated DcuR bound to promoters. In the model, acitvation of DcuS increases the affinity toward DcuR, leading to DcuS-P × DcuR formation and phosphorylation of DcuR. The complex is stable enough for phosphate-transfer, but labile enough to allow exchange between DcuR from the cytosol and DcuR-P of the complex. Released DcuR-P diffuses to target promoters and binds. Uncomplexed DcuR-P in the cytosol binds to nonactivated DcuS and becomes dephosphorylated. The lower affinity between DcuR and DcuS avoids blocking of DcuS and allows rapid exchange of DcuR. IMPORTANCE Complex formation of membrane-bound sensor kinases with the response regulators represents an inherent step of signaling from the membrane to the promoters on the DNA. In the C4-dicarboxylate-sensing DcuS-DcuR two-component system, complex formation is strengthened by activation (phosphorylation) in vitro and in vivo, with trapping of the response regulator DcuR at the membrane. Single-molecule tracking of DcuR in the bacterial cell demonstrates two populations of DcuR with decreased mobility in the bacteria after activation: one at the membrane, but a second in the cytosol, likely representing DNA-bound DcuR. The data suggest a model with binding of DcuR to DcuS-P for phosphorylation, and of DcuR-P to DcuS for dephosphorylation, allowing rapid adaptation of the DcuR phosphorylation state. DcuR-P is released and transferred to DNA by 3D diffusion.

The solute binding proteins (SBPs) of prokaryotes are present in the extracytosolic space. Although their primary function is providing substrates to transporters, SBPs also stimulate different signaling proteins, including chemoreceptors, sensor kinases, diguanylate cyclases/phosphodiesterases and Ser/Thr kinases, thereby causing a wide range of responses. While relatively few such systems have been identified, several pieces of evidence suggest that SBP-mediated receptor activation is a widespread mechanism. (1) These systems have been identified in Gram-positive and Gram-negative bacteria and archaea. (2) There is a structural diversity in the receptor domains that bind SBPs. (3) SBPs belonging to thirteen different families interact with receptor ligand binding domains (LBDs). (4) For the two most abundant receptor LBD families, dCache and four-helix-bundle, there are different modes of interaction with SBPs. (5) SBP-stimulated receptors carry out many different functions. The advantage of SBP-mediated receptor stimulation is attributed to a strict control of SBP levels, which allows a precise adjustment of the systeḿs sensitivity. We have compiled information on the effect of ligands on the transcript/protein levels of their cognate SBPs. In 87 % of the cases analysed, ligands altered SBP expression levels. The nature of the regulatory effect depended on the ligand family. Whereas inorganic ligands typically downregulate SBP expression, an upregulation was observed in response to most sugars and organic acids. A major unknown is the role that SBPs play in signaling and in receptor stimulation. This review attempts to summarize what is known and to present new information to narrow this gap in knowledge.

The sensor kinase DcuS of Escherichia coli perceives extracellular fumarate by a periplasmic PASP sensor domain. Transmembrane (TM) helix TM2, present as TM2-TM2′ homo-dimer, transmits fumarate activation in a piston-slide across the membrane. The second TM helix of DcuS, TM1, is known to lack piston movement. Structural and functional properties of TM1 were analyzed. Oxidative Cys-crosslinking (CL) revealed homo-dimerization of TM1 over the complete membrane, but only the central part showed α-helical +3/+4 spacing of the CL maxima. The GALLEX bacterial two-hybrid system indicates TM1/TM1′ interaction, and the presence of a TM1-TM1′ homo-dimer is suggested. The peripheral TM1 regions presented CL in a spacing atypical for α-helical arrangement. On the periplasmic side the deviation extended over 11 AA residues (V32-S42) between the α-helical part of TM1 and the onset of PASP. In the V32-S42 region, CL efficiency decreased in the presence of fumarate. Therefore, TM1 exists as a homo-dimer with α-helical arrangement in the central membrane region, and non-α-helical arrangement in the connector to PASP. The fumarate induced structural response in the V32-S42 region is suggested to represent a structural adaptation to the shift of TM2 in the TM1-TM1′/TM2-TM2′ four-helical bundle. [ABSTRACT FROM AUTHOR]

ObjectivesTo predict the novel vaccine peptide candidates against gacS protein involved with the citrate utilization in the two-component system of A. baumannii-associated virulence as an alternative strategy to combat the multi-drug resistant strains using an immuno-informatic approach.MethodsThe study is designed as an observational in silico study design with the application of BepiPred, AlgPred, VaxiJen, AntigenPro, SolPro, Expasy ProtParam server, IEDB database, and MHC cluster analytical tools and servers to predict the immuno-dominant B-cell and T-cell epitopes from gacS FASTA sequences retrieved from UNIPROT database. Further peptide interactions with TLR-4 was assessed based on the number of hydrogen bonds.ResultsNine peptides (20aa) with the highest score of 1 were selected from the 137 epitopes, and five were predicted as antigenic epitopes (E1–E5). E3 was selected as the potent antigen (score: 0.939537) and E1 as the best vaccine candidate (score: 0.9803) under AntigenPro and Vaxijen server, respectively. SolPro predicted all epitopes as soluble peptides. ProtParam predictions showed E3 and E5 as stable proteins with a shelf life of 3.5 and 1.9 h and possessed negative GRAVY values. PsortB server predicted a final localization score of 7.88 for the gacS protein sequence as a cytoplasmic membrane protein. IEDB conservancy analysis showed 100% conserved sequences within the gacS sequence, and class I conservancy yielded positive values for all epitopes. Cluster analysis showed strong interactions, and the protein-peptide interactions with TLR-2 finally detected E5 as the best interacting peptide (H bonds = 14) followed by E3 (H bonds = 12).ConclusionThe study suggests five antigenic peptides as promiscuous vaccine candidates to target the gacS of A. baumannii using immuno-informatic approach toward the peptide synthesis and in vitro analysis. However, the study recommends further experimental validation for immunological response and memory through in vivo studies.

Objectives: To predict the novel vaccine peptide candidates against gacS protein involved with the citrate utilization in the two-component system of A. baumannii -associated virulence as an alternative strategy to combat the multi-drug resistant strains using an immuno-informatic approach. Methods: The study is designed as an observational in silico study design with the application of BepiPred, AlgPred, VaxiJen, AntigenPro, SolPro, Expasy ProtParam server, IEDB database, and MHC cluster analytical tools and servers to predict the immuno-dominant B-cell and T-cell epitopes from gacS FASTA sequences retrieved from UNIPROT database. Further peptide interactions with TLR-4 was assessed based on the number of hydrogen bonds. Results: Nine peptides (20aa) with the highest score of 1 were selected from the 137 epitopes, and five were predicted as antigenic epitopes (E1–E5). E3 was selected as the potent antigen (score: 0.939537) and E1 as the best vaccine candidate (score: 0.9803) under AntigenPro and Vaxijen server, respectively. SolPro predicted all epitopes as soluble peptides. ProtParam predictions showed E3 and E5 as stable proteins with a shelf life of 3.5 and 1.9 h and possessed negative GRAVY values. PsortB server predicted a final localization score of 7.88 for the gacS protein sequence as a cytoplasmic membrane protein. IEDB conservancy analysis showed 100% conserved sequences within the gacS sequence, and class I conservancy yielded positive values for all epitopes. Cluster analysis showed strong interactions, and the protein-peptide interactions with TLR-2 finally detected E5 as the best interacting peptide (H bonds = 14) followed by E3 (H bonds = 12). Conclusion: The study suggests five antigenic peptides as promiscuous vaccine candidates to target the gacS of A. baumannii using immuno-informatic approach toward the peptide synthesis and in vitro analysis. However, the study recommends further experimental validation for immunological response and memory through in vivo studies. [ABSTRACT FROM AUTHOR]

The water‐borne bacterium Legionella pneumophila replicates in environmental protozoa and upon inhalation destroys alveolar macrophages, thus causing a potentially fatal pneumonia termed 'Legionnaires' disease'. L. pneumophila employs the Legionella quorum sensing (Lqs) system to control its life cycle, pathogen–host cell interactions, motility and natural competence. Signaling through the Lqs system occurs through the α‐hydroxyketone compound Legionella autoinducer‐1 (LAI‐1) and converges on the prototypic response regulator LqsR, which dimerizes upon phosphorylation of the conserved aspartate, D108. In this study, we determine the high‐resolution structure of monomeric LqsR. The structure reveals a receiver domain adopting a canonical (βα)5 fold, which is connected through an additional sixth helix and an extended α5‐helix to a novel output domain. The two domains delineate a mainly positively charged groove, and the output domain adopts a five‐stranded antiparallel β‐sheet fold similar to nucleotide‐binding proteins. Structure‐based mutagenesis identified amino acids critical for LqsR phosphorylation and dimerization. Upon phosphorylation, the LqsRD172A and LqsRD302N/E303Q mutant proteins dimerized even more readily than wild‐type LqsR, and no evidence for semi‐phosphorylated heterodimers was obtained. Taken together, the high‐resolution structure of LqsR reveals functionally relevant amino acid residues implicated in signal transduction of the prototypic response regulator. [ABSTRACT FROM AUTHOR]

Xanthomonas campestris pv. campestris (Xcc) controls virulence and plant infection mechanisms via the activity of the sensor kinase and response regulator pair HpaS/hypersensitive response and pathogenicity G (HrpG). Detailed analysis of the regulatory role of HpaS has suggested the occurrence of further regulators besides HrpG. Here we used in vitro and in vivo approaches to identify the orphan response regulator VemR as another partner of HpaS and to characterize relevant interactions between components of this signalling system. Bacterial two‐hybrid and protein pull‐down assays revealed that HpaS physically interacts with VemR. Phos‐tag SDS‐PAGE analysis showed that mutation in hpaS reduced markedly the phosphorylation of VemR in vivo. Mutation analysis reveals that HpaS and VemR contribute to the regulation of motility and this relationship appears to be epistatic. Additionally, we show that VemR control of Xcc motility is due in part to its ability to interact and bind to the flagellum rotor protein FliM. Taken together, the findings describe the unrecognized regulatory role of sensor kinase HpaS and orphan response regulator VemR in the control of motility in Xcc and contribute to the understanding of the complex regulatory mechanisms used by Xcc during plant infection. [ABSTRACT FROM AUTHOR]

ABSTRACT We have identified and characterized the AccS multidomain sensor kinase that mediates the activation of the AccR master regulator involved in carbon catabolite repression (CCR) of the anaerobic catabolism of aromatic compounds in Azoarcus sp. CIB. A truncated AccS protein that contains only the soluble C-terminal autokinase module (AccS′) accounts for the succinate-dependent CCR control. In vitro assays with purified AccS′ revealed its autophosphorylation, phosphotransfer from AccS′∼P to the Asp60 residue of AccR, and the phosphatase activity toward its phosphorylated response regulator, indicating that the equilibrium between the kinase and phosphatase activities of AccS′ may control the phosphorylation state of the AccR transcriptional regulator. Oxidized quinones, e.g., ubiquinone 0 and menadione, switched the AccS′ autokinase activity off, and three conserved Cys residues, which are not essential for catalysis, are involved in such inhibition. Thiol oxidation by quinones caused a change in the oligomeric state of the AccS′ dimer resulting in the formation of an inactive monomer. This thiol-based redox switch is tuned by the cellular energy state, which can change depending on the carbon source that the cells are using. This work expands the functional diversity of redox-sensitive sensor kinases, showing that they can control new bacterial processes such as CCR of the anaerobic catabolism of aromatic compounds. The AccSR two-component system is conserved in the genomes of some betaproteobacteria, where it might play a more general role in controlling the global metabolic state according to carbon availability. IMPORTANCE Two-component signal transduction systems comprise a sensor histidine kinase and its cognate response regulator, and some have evolved to sense and convert redox signals into regulatory outputs that allow bacteria to adapt to the altered redox environment. The work presented here expands knowledge of the functional diversity of redox-sensing kinases to control carbon catabolite repression (CCR), a phenomenon that allows the selective assimilation of a preferred compound among a mixture of several carbon sources. The newly characterized AccS sensor kinase is responsible for the phosphorylation and activation of the AccR master regulator involved in CCR of the anaerobic degradation of aromatic compounds in the betaproteobacterium Azoarcus sp. CIB. AccS seems to have a thiol-based redox switch that is modulated by the redox state of the quinone pool. The AccSR system is conserved in several betaproteobacteria, where it might play a more general role controlling their global metabolic state.

Abstract Two-component signal transduction (TCS) cascades involve stimulus-dependent activation and phosphorylation of a sensor kinase (SK), which then transfers the phosphoryl moiety to the response regulator (RR) protein. The fidelity of this phosphotransfer reaction from the SK to the RR provides specificity to TCS signaling. In the present study, we show that for TcrX, a transcriptionally autoregulated RR of Mycobacterium tuberculosis , acetylation enhances its net phosphorylation from cognate SK TcrY and lowers it from a non-cognate SK MtrB. Similar acetylation mediated increase in phosphorylation was also observed for another RR MtrA from cognate SK MtrB. Thus, we establish a novel TCS signaling design wherein acetylation of RRs results in enhanced cognate phosphorylation and suppresses non-cognate phosphorylation. Using wild-type or acetylation-deficient TcrX proteins in M. tuberculosis H37Ra, we demonstrate that non-acetylated TcrX acts as a "phosphate sink" for MtrB and suppressing signal propagation from MtrB to MtrA in vivo , linking metabolism to TCS signaling. Overall, we report that acetylation of RRs shields TCSs from crosstalk, modulates the phosphatase activities and alters the DNA-binding activities of RRs, all of which are non-intuitive behavior of TCS systems. Graphical Abstract Unlabelled Image Highlights • Two-component systems of bacteria can be specific or participate in crosstalk. • Phosphotransfer ability between SK and RR is tuned by acetylation of response regulator proteins. • Acetylated RRs are more specific, while non-acetylated RR are amenable to crosstalk. • Acetylation of RR also alters their DNA-binding ability and dephosphorylation rates • Design links signaling fidelity to nutritional status in the bacterium. [ABSTRACT FROM AUTHOR]

Two-component signaling circuits (TCS) allow bacteria to detect environmental cues and to produce adaptive responses. These signaling systems are based on autophosphorylation and phosphoryl-group transfers between histidine and aspartate containing sensor kinase and response regulator proteins. Upon reception of a specific stimulus the sensor kinase protein autophosphorylates, by either an inter- or intra-molecular reaction, and transphosphorylates its cognate response regulator, which, typically, acts as a transcriptional regulator, thereby triggering physiological responses. Frequently, in the absence of the stimulus, the sensor kinase proteins are responsible for the dephosphorylation of their cognate response regulators. Furthermore, a group of sensor kinases have additional functional domains that are involved in a forward phosphorelay for signal transmission and in a reverse phosphorelay for signal decay. As is the case of the autophosphorylation reaction, the phosphoryl-group transfers involved in the forward and reverse phosphorelay can occur either intra- or inter-molecularly. In this review, we highlight some important features of bacterial TCS, with special emphasis on the autophosphorylation and phosphoryl-group transfer events. [ABSTRACT FROM AUTHOR]

A large number of small membrane proteins have been uncovered in bacteria, but their mechanism of action has remained mostly elusive. Here, we investigate the mechanism of a physiologically important small protein, MgrB, which represses the activity of the sensor kinase PhoQ and is widely distributed among enterobacteria. The PhoQ/PhoP two-component system is a master regulator of the bacterial virulence program and interacts with MgrB to modulate bacterial virulence, fitness, and drug resistance. A combination of cross-linking approaches with functional assays and protein dynamic simulations revealed structural rearrangements due to interactions between MgrB and PhoQ near the membrane/periplasm interface and along the transmembrane helices. These interactions induce the movement of the PhoQ catalytic domain and the repression of its activity. Without MgrB, PhoQ appears to be much less sensitive to antimicrobial peptides, including the commonly used C18G. In the presence of MgrB, C18G promotes MgrB to dissociate from PhoQ, thus activating PhoQ via derepression. Our findings reveal the inhibitory mechanism of the small protein MgrB and uncover its importance in antimicrobial peptide sensing.

Biofilms, complex communities of microorganisms surrounded by a selfproduced matrix, facilitate attachment and provide protection to bacteria. A natural model used to study biofilm formation is the symbiosis between Vibrio fischeri and its host, the Hawaiian bobtail squid, Euprymna scolopes. Host-relevant biofilm formation is a tightly regulated process and is observed in vitro only with strains that have been genetically manipulated to overexpress or disrupt specific regulators, primarily two-component signaling (TCS) regulators. These regulators control biofilm formation by dictating the production of the symbiosis polysaccharide (Syp-PS), the major component of the biofilm matrix. Control occurs both at and below the level of transcription of the syp genes, which are responsible for Syp-PS production. Here, we probed the roles of the two known negative regulators of biofilm formation, BinK and SypE, by generating double mutants. We also mapped and evaluated a point mutation using natural transformation and linkage analysis. We examined traditional biofilm formation phenotypes and established a new assay for evaluating the start of biofilm formation in the form of microscopic aggregates in shaking liquid cultures, in the absence of the known biofilm-inducing signal calcium. We found that wrinkled colony formation is negatively controlled not only by BinK and SypE but also by SypF. SypF is both required for and inhibitory to biofilm formation. Together, these data reveal that these three regulators are sufficient to prevent wildtype V. fischeri from forming biofilms under these conditions. [ABSTRACT FROM AUTHOR]

Abstract: Tuberculosis is a global health problem, with a third of the world's population infected with the bacillus, Mycobacterium tuberculosis. The problem is exacerbated by the emergence of multidrug resistant and extensively drug resistant strains. The search for new drug targets is therefore a priority for researchers in the field. The two‐component systems (TCSs) are central to the ability of the bacterium to sense and to respond appropriately to its environment. Here we summarize current knowledge on the paired TCSs of M. tuberculosis. We discuss what is currently understood regarding the signals to which each of the sensor kinases responds, and the regulons of each of the cognate response regulators. We also discuss what is known regarding attempts to inhibit the TCSs by small molecules and project their potential as pharmacological targets for the development of novel antimycobacterial agents. © 2018 IUBMB Life, 70(8):710–717, 2018 [ABSTRACT FROM AUTHOR]

Cytokinins are an important group of plant hormones that are also found in other organisms, including cyanobacteria. While various aspects of cytokinin function and metabolism are well understood in plants, the information is limited for cyanobacteria. In this study, we first experimentally confirmed a prenylation of tRNA by recombinant isopentenyl transferase NoIPT2 from Nostoc sp. PCC 7120, whose encoding gene we previously identified in Nostoc genome along with the gene for adenylate isopentenyl transferase NoIPT1. In contrast to NoIPT2, the transcription of NoIPT1 was strongly activated during the dark period and was followed by an increase in the cytokinin content several hours later in the light period. Dominant cytokinin metabolites detected at all time points were free bases and monophosphates of isopentenyladenine and cis-zeatin, while N-glucosides were not detected at all. Whole transcriptome differential expression analysis of cultures of the above Nostoc strain treated by cytokinin compared to untreated controls indicated that cytokinin together with light trigger expression of several genes related to signal transduction, including two-component sensor histidine kinases and two-component hybrid sensors and regulators. One of the affected histidine kinases with a cyclase/histidine kinase-associated sensory extracellular domain similar to the cytokinin-binding domain in plant cytokinin receptors was able to modestly bind isopentenyladenine. The data show that the genetic disposition allows Nostoc not only to produce free cytokinins and prenylate tRNA but also modulate the cytokinin biosynthesis in response to light, triggering complex changes in sensing and regulation. [ABSTRACT FROM AUTHOR]

The marine bacterium Vibrio fischeri initiates symbiotic colonization of its squid host, Euprymna scolopes , by forming and dispersing from a biofilm dependent on the sy mbiosis p olysaccharide locus ( syp ). Historically, genetic manipulation of V. fischeri was needed to visualize syp -dependent biofilm formation in vitro , but recently, we discovered that the combination of two small molecules, para -aminobenzoic acid (pABA) and calcium, was sufficient to induce wild-type strain ES114 to form biofilms. Here, we determined that these syp -dependent biofilms were reliant on the positive syp regulator RscS, since the loss of this sensor kinase abrogated biofilm formation and syp transcription. These results were of particular note because loss of RscS, a key colonization factor, exerts little to no effect on biofilm formation under other genetic and medium conditions. The biofilm defect could be complemented by wild-type RscS and by an RscS chimera that contains the N-terminal domains of RscS fused to the C-terminal HPT domain of SypF, the downstream sensor kinase. It could not be complemented by derivatives that lacked the periplasmic sensory domain or contained a mutation in the conserved site of phosphorylation, H412, suggesting that these cues promote signaling through RscS. Lastly, pABA and/or calcium was able to induce biofilm formation when rscS was introduced into a heterologous system. Taken together, these data suggest that RscS is responsible for recognizing pABA and calcium, or downstream consequences of those cues, to induce biofilm formation. This study thus provides insight into signals and regulators that promote biofilm formation by V. fischeri. IMPORTANCE Bacterial biofilms are common in a variety of environments. Infectious biofilms formed in the human body are notoriously hard to treat due to a biofilm's intrinsic resistance to antibiotics. Bacteria must integrate signals from the environment to build and sustain a biofilm and often use sensor kinases that sense an external signal, which triggers a signaling cascade to elicit a response. However, identifying the signals that kinases sense remains a challenging area of investigation. Here, we determine that a hybrid sensor kinase, RscS, is crucial for Vibrio fischeri to recognize para -aminobenzoic acid and calcium as cues to induce biofilm formation. This study thus advances our understanding of the signal transduction pathways leading to biofilm formation., Competing Interests: The authors declare no conflict of interest.

Bacteria synthesize and sense low molecular weight signaling molecules, termed autoinducers, to measure their population density and community complexity. One class of autoinducers, the α-hydroxyketones (AHKs), is produced and detected by the water-borne opportunistic pathogens Legionella pneumophila and Vibrio cholerae, which cause Legionnaires’ disease and cholera, respectively. The “Legionella quorum sensing” (lqs) or “cholera quorum sensing” (cqs) genes encode enzymes that produce and sense the AHK molecules “Legionella autoinducer-1” (LAI-1; 3-hydroxypentadecane-4-one) or cholera autoinducer-1 (CAI-1; 3-hydroxytridecane-4-one). AHK signaling regulates the virulence of L. pneumophila and V. cholerae, pathogen-host cell interactions, formation of biofilms or extracellular filaments, expression of a genomic “fitness island” and competence. Here, we outline the processes, wherein AHK signaling plays a role, and review recent insights into the function of proteins encoded by the lqs and cqs gene clusters. To this end, we will focus on the autoinducer synthases catalysing the biosynthesis of AHKs, on the cognate trans-membrane sensor kinases detecting the signals, and on components of the down-stream phosphorelay cascade that promote the transmission and integration of signaling events regulating gene expression.

Salmonella PhoQ is a histidine kinase with a periplasmic sensor domain (PD) that promotes virulence by detecting the macrophage phagosome. PhoQ activity is repressed by divalent cations and induced in environments of acidic pH, limited divalent cations, and cationic antimicrobial peptides (CAMP). Previously, it was unclear which signals are sensed by salmonellae to promote PhoQ-mediated virulence. We defined conformational changes produced in the PhoQ PD on exposure to acidic pH that indicate structural flexibility is induced in α-helices 4 and 5, suggesting this region contributes to pH sensing. Therefore, we engineered a disulfide bond between W104C and A128C in the PhoQ PD that restrains conformational flexibility in α-helices 4 and 5. PhoQW104C-A128C is responsive to CAMP, but is inhibited for activation by acidic pH and divalent cation limitation. phoQW104C-A128C Salmonella enterica Typhimurium is virulent in mice, indicating that acidic pH and divalent cation sensing by PhoQ are dispensable for virulence.

Background Two component signalling involves interaction between sensor kinase (SK) and response regulator (RR) proteins which depends on their phosphorylation status. Methods In this study we report the development of an in vitro FRET assay for studying interaction between fluorescently tagged SK and RR proteins. Results Using TCS proteins of Mycobacterium tuberculosis , we demonstrate that phosphorylation status of SK affects the SK–RR interaction, which varies from one TCS to another. The observation was strengthened by recordings from mutant SK and RR proteins. The assay retained the specificity/crosstalk potential of the participating proteins and reflected the inherent phosphotransfer potentials. Conclusions SK and RR proteins interact with each other in unphosphorylated state and the phosphorylation affects the interaction between SK and RR, which was reflected as reduction in FRET ratio. General significance A non-radioactive, in vitro FRET based assay is reported, which can be utilized for studying genome-wide partner screening, identifying crosstalk or specificity in TCSs. [ABSTRACT FROM AUTHOR]

Two-component signaling systems (TCSs) are composed of two proteins, sensor kinases and response regulators, which can cross-talk and integrate information between them by virtue of high-sequence conservation and modular nature, to generate concerted and diversified responses. However, TCSs have been shown to be insulated, to facilitate linear signal transmission and response generation. Here, we discuss various mechanisms that confer specificity or cross-talk among TCSs. The presented models are supported with evidence that indicate the physiological significance of the observed TCS signaling architecture. Overall, we propose that the signaling topology of any TCSs cannot be predicted using obvious sequence or structural rules, as TCS signaling is regulated by multiple factors, including spatial and temporal distribution of the participating proteins. [ABSTRACT FROM AUTHOR]

Bacteria have evolved to adapt to various conditions and respond to certain stress conditions. The ability to sense and efficiently reply to these environmental effects involve versatile array of sensors and global or specific regulators. Interestingly, modulation of the levels of active global regulators enables bacteria to respond to diverse signals via a single central transcriptional regulator and to activate or repress certain differentiation pathways at a spatio-temporal manner. The Gram-positive Bacillus subtilis is an ideal bacterium to study how membrane bound and cytoplasmic sensor kinases affect the level of phosphorylated global regulator, Spo0A which in response activates genes related to sliding, biofilm formation, and sporulation. In addition, other global regulators, including the two-component system DegS-DegU, modulate overlapping and complementary genes in B. subtilis related to surface colonization and biofilm formation. The intertwinement of global regulatory systems also allows the accurate modulation of differentiation pathways. Studies in the last decade enable us to get a deeper insight into the role of global regulators on the smooth transition of developmental processes in B. subtilis. [ABSTRACT FROM AUTHOR]

Many membrane-bound sensor kinases require accessory proteins for function. The review describes functional control of membrane-bound sensors by transporters. The C4-dicarboxylate sensor kinase DcuS requires the aerobic or anaerobic C4-dicarboxylate transporters DctA or DcuB, respectively, for function and forms DctA/DcuS or DcuB/DcuS sensor complexes. Free DcuS is in the permanent (ligand independent) ON state. The DctA/DcuS and DcuB/DcuS complexes, on the other hand, control expression in response to C4-dicarboxylates. In DctA/DcuS, helix 8b of DctA and the PASC domain of DcuS are involved in interaction. The stimulus is perceived by the extracytoplasmic sensor domain (PASP) of DcuS. The signal is transmitted across the membrane by a piston-type movement of TM2 of DcuS which appears to be pulled (by analogy to the homologous citrate sensor CitA) by compaction of PASP after C4-dicarboxylate binding. In the cytoplasm, the signal is perceived by the PASC domain of DcuS. PASC inhibits together with DctA the kinase domain of DcuS which is released after C4-dicarboxylate binding. DcuS exhibits two modes for regulating expression of target genes. At higher C4-dicarboxylate levels, DcuS is part of the DctA/DcuS complex and in the C4-dicarboxylate-responsive form which stimulates expression of target genes in response to the concentration of the C4-dicarboxylates (catabolic use of C4-dicarboxylates, mode I regulation). At limiting C4-dicarboxylate concentrations (= 0.05 mM), expression of DctA drops and free DcuS appears. Free DcuS is in the permanent ON state (mode II regulation) and stimulates low level (C4-dicarboxylate independent) DctA synthesis for DctA/DcuS complex formation and anabolic C4-dicarboxylate uptake. [ABSTRACT FROM AUTHOR]