Hybrid Two-component System Research Articles

A mucin-responsive hybrid two-component system controls Bacteroides thetaiotaomicron colonization and gut homeostasis.

The mammalian intestinal tract contains trillions of bacteria. However, the genetic factors that allow gut symbiotic bacteria to occupy intestinal niches remain poorly understood. Here, we identified genetic determinants required for Bacteroides thetaiotaomicron colonization in the gut using transposon sequencing analysis. Transposon insertion in BT2391, which encodes a hybrid two-component system, increased the competitive fitness of B. thetaiotaomicron. The BT2391 mutant showed a growth advantage in a mucin-dependent manner and had an increased ability to adhere to mucus-producing cell lines. The increased competitive advantage of the BT2391 mutant was dependent on the BT2392-2395 locus containing susCD homologs. Deletion of BT2391 led to changes in the expression levels of B. thetaiotaomicron genes during gut colonization. However, colonization of the BT2391 mutant promoted DSS colitis in low-fiber diet-fed mice. These results indicate that BT2391 contributes to a sustainable symbiotic relationship by maintaining a balance between mucosal colonization and gut homeostasis.

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.

Hybrid liquid-membrane lenses made by one fixed and one or two active optical components.

The paper presents a methodology of calculation of the inner structure of two- and three-component hybrid liquid-membrane lenses with variable focal length that have corrected spherical aberration and coma. Specifically, the formulas for calculation of initial-design inner parameters (radii of curvatures of individual surfaces, axial thickness, and refractive indices of a material of the lens) of a thin-lens system are derived for a hybrid two-component system (doublet) made by one glass and one liquid-membrane lens, and a hybrid three-component lens (triplet) made by one glass lens and two liquid-membrane lenses, which both have variable focal length and corrected spherical aberration and coma for an object at infinity. As optimization during the optical design process requires the starting point be very close to the optimal solution, the presented approach can be successfully used for its calculation, as it is based on fundamental proven formulas of optical aberrations.

Structural insights into the histidine-containing phosphotransfer protein and receiver domain of sensor histidine kinase suggest a complex model in the two-component regulatory system inPseudomonas aeruginosa

In Pseudomonas aeruginosa, an important opportunistic pathogen that causes numerous acute and chronic infections, the hybrid two-component system (TCS) regulates the swarming ability and biofilm formation with a multistep phospho-relay, and consists of hybrid-sensor histidine kinase (HK), histidine-containing phospho-transfer protein (Hpt) and response regulator (RR). In this work, two crystal structures of HptB and the receiver domain of HK PA1611 (PA1611REC) of P. aeruginosa have been determined in order to elucidate their interactions for the transfer of the phospho-ryl group. The structure of HptB folds into an elongated four-helix bundle - helices α2, α3, α4 and α5, covered by the short N-terminal helix α1. The imidazole side chain of the conserved active-site histidine residue His57, located near the middle of helix α3, protrudes from the bundle and is exposed to solvent. The structure of PA1611REC possesses a conventional (β/α)5 topology with five-stranded parallel β-sheets folded in the central region, surrounded by five α-helices. The divalent Mg2+ ion is located in the negatively charged active-site cleft and interacts with Asp522, Asp565 and Arg567. The HptB-PA1611REC complex is further modeled to analyze the binding surface and interactions between the two proteins. The model shows a shape complementarity between the convex surface of PA1611REC and the kidney-shaped HptB with fewer residues and a different network involved in interactions compared with other TCS complexes, such as SLN1-R1/YPD1 from Saccharomyces cerevisiae and AHK5RD/AHP1 from Arabidopsis thaliana. These structural results provide a better understanding of the TCS in P. aeruginosa and could potentially lead to the discovery of a new treatment for infection.

Frequency modulation of the Raman spectrum at the interface DNA - ZrO2 nanoparticles

The development of modern bio-nanoelectronic technologies requires new hybrid organo-inorganic systems that have a specific functional purpose and allow work in the tissues of living organisms. The practical realization and scientific study of a hybrid two-component system in the form of DNA molecule fragments and nanoparticles of biocompatible ceramics was the goal of this work. The interaction of a DNA molecule with nanoparticles of a solid solution of the composition ZrO2–3mol% Y2O3 (YSZ) was studied using Raman scattering and transmission electron microscopy. Samples of YSZ oxide and hydroxide were investigated in triplicate at room temperature using LabRAM HR Evolution Horiba spectrometer and JEM 200A instrument. The effect of the force field of the surface of YSZ nanoparticles on the optical properties of the DNA molecule in the TRIS buffer solution was shown. The effects of increasing the intensity of spectral lines in the long-wavelength region 250 – 660 cm-1 (254 cm -1, 322 cm -1, 470 cm -1, 663 cm -1). suppressing the intensity of spectral lines in the shortwave region 600 – 3300 cm -1 (604 cm -1, 917 cm -1, 951 cm -1, 1256 cm -1, 1302 cm -1, 2767 cm -1, 3180 cm -1, 3241 cm -1, 3285 cm -1), as well as shifting lines 519 cm -1, 917 cm -1, 1048 cm -1, 1547 cm -1 and 1639 on 40-60 cm-1 in the shortwave region are established. From the standpoint of the theory of wave processes, a qualitative description of the detected Up-conversion effect was done. An assumption about the potential applicability of the YSZ nanoparticle surface – DNA nucleotide interfaces as a heterodyne frequency converter in molecular- and bio-electronic devices was made.

Sigma factor RpoN employs a dual transcriptional regulation for controlling twitching motility and biofilm formation in Lysobacter enzymogenes OH11.

Lysobacter is a Gram-negative genus comprising a group of environmental bacteria with abilities to produce abundant novel antibiotics, as well as adopting a unique type IV pilus (T4P)-mediated twitching motility (TM) that remains poorly understood. Here, we employ L. enzymogenes OH11 exhibiting significant antifungal activity as a working model to address this issue. Via mutating the 28 potential sigma factors in strain OH11, we have identified one protein RpoNOH11 (sigma 54) that is indispensable for T4P formation and TM. We further showed that RpoNOH11 not only regulates the transcription of pilA, but also another crucial gene chpA that encodes a hybrid two-component transduction system. The L. enzymogenes RpoNOH11 was found to directly bind to the promoter of chpA to control its transcription, which is found to be essential for the T4P-mediated TM. To our knowledge, such a transcriptional regulation performed by RpoN in control of bacterial TM has never been reported. Finally, we showed that L. enzymogenes OH11 could also produce biofilm that is likely employed by this strain to infect fungal pathogens. Mutation of rpoN OH11, pilA and chpA all led to a significant decrease in biofilm formation, suggesting that the dual transcriptional regulation of pilA and chpA by RpoNOH11 plays a key role for RpoNOH11 to modulate the biofilm formation in L. enzymogenes. Overall, this study identified chpA as a new target of RpoN for controlling the T4P-mediated twitching motility and biofilm formation in L. enzymogenes OH11.

Regulation and Molecular Basis of Environmental Muropeptide Uptake and Utilization in Fastidious Oral Anaerobe Tannerella forsythia.

Tannerella forsythia is a Gram-negative oral anaerobe associated with periodontitis. This bacterium is auxotrophic for the peptidoglycan amino sugar N-acetylmuramic (MurNAc) and likely relies on scavenging peptidoglycan fragments (muropeptides) released by cohabiting bacteria during their cell wall recycling. Many Gram-negative bacteria utilize an inner membrane permease, AmpG, to transport peptidoglycan fragments into their cytoplasm. In the T. forsythia genome, the Tanf_08365 ORF has been identified as a homolog of AmpG permease. In order to confirm the functionality of Tanf_08365, a reporter system in an Escherichia coli host was generated that could detect AmpG-dependent accumulation of cytosolic muropeptides via a muropeptide-inducible β-lactamase reporter gene. In trans complementation of this reporter strain with a Tanf_08365 containing plasmid caused significant induction of β-lactamase activity compared to that with an empty plasmid control. These data indicated that Tanf_08365 acted as a functional muropeptide permease causing accumulation of muropeptides in E. coli and thus suggested that it is a permease involved in muropeptide scavenging in T. forsythia. Furthermore, we showed that the promoter regulating the expression of Tanf_08365 was activated significantly by a hybrid two-component system regulatory protein, GppX. We also showed that compared to the parental T. forsythia strain a mutant lacking GppX in which the expression of AmpG was reduced significantly attenuated in utilizing free muropeptides. In summary, we have uncovered the mechanism by which this nutritionally fastidious microbe accesses released muropeptides in its environment, opening up the possibility of targeting this activity to reduce its numbers in periodontitis patients with potential benefits in the treatment of disease.

ChpA Controls Twitching Motility and Broadly Affects Gene Expression in the Biological Control Agent Lysobacter enzymogenes.

Lysobacter enzymogenes (L. enzymogenes) is an agriculturally important Gram-negative bacterium that employs T4P (type IV pili)-driven twitching motility to exhibit its antifungal function. Yet, it is still unclear how this bacterium regulates its twitching motility. Here, by using strain OH11 as the working model organism, we showed that a hybrid two-component system ChpA acts as a positive regulator in controlling twitching motility in L. enzymogenes. ChpA is a hybrid TCS (two-component transduction system) contains 7 domains including those for auto-phosphorylation and phosphate group transfer, as well as a phosphate receiver (REC) domain. Mutation of chpA completely abolished the wild-type twitching motility, as evidenced by the absence of mobile cells at the margin of the mutant colonies. Further studies of domain-deletion and phenotypic characterization reveal that domains responsible for phosphorylation and phosphotransfer, but not the REC domain, were indispensable for ChpA in regulating twitching motility. Transcriptome analyses of the chpA knockout strain indicated that ChpA was extensively involved in controlling expression of a wide variety of genes (totaling 243). The products of these differentially expressed genes were involved in multiple physiological and biological functions in L. enzymogenes. Thus, we have not only identified a new regulator controlling twitching motility in L. enzymogenes, but also provided the first report demonstrating the broad impact of the conserved ChpA in gene regulation in Gram-negative bacteria.

Degradation of cyclic diguanosine monophosphate by a hybrid two-component protein protects Azoarcus sp. strain CIB from toluene toxicity

Cyclic diguanosine monophosphate (c-di-GMP) is a second messenger that controls diverse functions in bacteria, including transitions from planktonic to biofilm lifestyles, virulence, motility, and cell cycle. Here we describe TolR, a hybrid two-component system (HTCS), from the β-proteobacterium Azoarcus sp. strain CIB that degrades c-di-GMP in response to aromatic hydrocarbons, including toluene. This response protects cells from toluene toxicity during anaerobic growth. Whereas wild-type cells tolerated a sudden exposure to a toxic concentration of toluene, a tolR mutant strain or a strain overexpressing a diguanylate cyclase gene lost viability upon toluene shock. TolR comprises an N-terminal aromatic hydrocarbon-sensing Per-Arnt-Sim (PAS) domain, followed by an autokinase domain, a response regulator domain, and a C-terminal c-di-GMP phosphodiesterase (PDE) domain. Autophosphorylation of TolR in response to toluene exposure initiated an intramolecular phosphotransfer to the response regulator domain that resulted in c-di-GMP degradation. The TolR protein was engineered as a functional sensor histidine kinase (TolRSK) and an independent response regulator (TolRRR). This classic two-component system (CTCS) operated less efficiently than TolR, suggesting that TolR was evolved as a HTCS to optimize signal transduction. Our results suggest that TolR enables Azoarcus sp. CIB to adapt to toxic aromatic hydrocarbons under anaerobic conditions by modulating cellular levels of c-di-GMP. This is an additional role for c-di-GMP in bacterial physiology.

Prioritization of a plant polysaccharide over a mucus carbohydrate is enforced by a Bacteroides hybrid two‐component system

Bacteroides is a dominant genus within the intestinal microbiota of healthy humans. Key adaptations of the Bacteroides to the dynamic intestinal ecosystem include a diverse repertoire of genes involved in sensing and processing numerous diet- and host-derived polysaccharides. One such adaptation is the carbohydrate-sensing hybrid two-component system (HTCS) family of signalling sensors, which has been widely expanded within the Bacteroides. Using Bacteroides thetaiotaomicron as a model, we have created a chimeric HTCS consisting of the well-characterized sensing domain of one HTCS, BT1754, and the regulatory domain of another HTCS, BT0366, to explore the regulatory capabilities of these molecules. We found that the BT0366 regulatory region directly binds to and mediates induction of the adjacent polysaccharide utilization locus (PUL) using whole-genome transcriptional profiling after inducing signalling through our chimeric protein. We also found that BT0366 activation simultaneously leads to repression of distal PULs involved in mucus carbohydrate consumption. These results suggest a novel mechanism by which an HTCS enforces a nutrient hierarchy within the Bacteroides via induction and repression of multiple PULs. Thus, hybrid two-component systems provide a mechanism for prioritizing consumption of carbohydrates through simultaneous binding and regulation of multiple polysaccharide utilization loci.

A scissor blade-like closing mechanism implicated in transmembrane signaling in a Bacteroides hybrid two-component system

Signaling across the membrane in response to extracellular stimuli is essential for survival of all cells. In bacteria, responses to environmental changes are predominantly mediated by two-component systems, which are typically composed of a membrane-spanning sensor histidine kinase and a cytoplasmic response regulator. In the human gut symbiont Bacteroides thetaiotaomicron, hybrid two-component systems are a key part of the bacterium's ability to sense and degrade complex carbohydrates in the gut. Here, we identify the activating ligand of the hybrid two-component system, BT4663, which controls heparin and heparan sulfate acquisition and degradation in this prominent gut microbe, and report the crystal structure of the extracellular sensor domain in both apo and ligand-bound forms. Current models for signal transduction across the membrane involve either a piston-like or rotational displacement of the transmembrane helices to modulate activity of the linked cytoplasmic kinases. The structures of the BT4663 sensor domain reveal a significant conformational change in the homodimer on ligand binding, which results in a scissor-like closing of the C-termini of each protomer. We propose this movement activates the attached intracellular kinase domains and represents an allosteric mechanism for bacterial transmembrane signaling distinct from previously described models, thus expanding our understanding of signal transduction across the membrane, a fundamental requirement in many important biological processes.

Two-Component Systems and Their Co-Option for Eukaryotic Signal Transduction

Two-component signaling pathways involve histidine kinases, response regulators, and sometimes histidine-containing phosphotransfer proteins. Prevalent in prokaryotes, these signaling elements have also been co-opted to meet the needs of signal transduction in eukaryotes such as fungi and plants. Here we consider the evolution of such regulatory systems, with a particular emphasis on the roles they play in signaling by the plant hormones cytokinin and ethylene, in phytochrome-mediated perception of light, and as integral components of the circadian clock.

A hybrid two-component system of Tannerella forsythia affects autoaggregation and post-translational modification of surface proteins

Tannerella forsythia is a Gram-negative oral anaerobe closely associated with both periodontal and periapical diseases. The ORF TF0022 of strain ATCC 43037 encodes a hybrid two-component system consisting of an N-terminal histidine kinase and a C-terminal response regulator. Disruption of the TF0022 locus enhanced autoaggregation of the broth-cultured cells. Comparative proteome analyses revealed that two S-layer proteins in the TF0022 mutant exhibited decreased apparent masses by denaturing gel electrophoresis, suggesting a deficiency in post-translational modification. Furthermore, the mutant decreased the production of a glycosyltransferase encoded by TF1061 that is located in a putative glycosylation-related gene cluster. Quantitative real-time PCR revealed reduced transcription of TF1061 and the associated genes in the TF0022 mutant. These results indicate that TF0022 upregulates the expression of the glycosylation-related genes and suggest modulation of the autoaggregation of T. forsythia cells by a possible post-translational modification of cell-surface components.

A hybrid two-component system protein from Azospirillum brasilense Sp7 was involved in chemotaxis

We here report the sequence and functional analysis of org35 of Azospirillum brasilense Sp7, which was originally identified to be able to interact with NifA in yeast-two-hybrid system. The org35 encodes a hybrid two-component system protein, including N-terminal PAS domains, a histidine kinase (HPK) domain and a response regulator (RR) domain in C-terminal. To determine the function of the Org35, a deletion–insertion mutant in PAS domain [named Sp7353] and a complemental strain Sp7353C were constructed. The mutant had reduced chemotaxis ability compared to that of wild-type, and the complemental strain was similar to the wild-type strain. These data suggested that the A. brasilense org35 played a key role in chemotaxis. Variants containing different domains of the org35 were expressed, and the functions of these domains were studied in vitro. Phosphorylation assays in vitro demonstrated that the HPK domain of Org35 possessed the autokinase activity and that the phosphorylated HPK was able to transfer phosphate groups to the RR domain. The result indicated Org35 was a phosphorylation-communicating protein.

Markov random fields reveal an N-terminal double beta-propeller motif as part of a bacterial hybrid two-component sensor system.

The recent explosion in newly sequenced bacterial genomes is outpacing the capacity of researchers to try to assign functional annotation to all the new proteins. Hence, computational methods that can help predict structural motifs provide increasingly important clues in helping to determine how these proteins might function. We introduce a Markov Random Field approach tailored for recognizing proteins that fold into mainly beta-structural motifs, and apply it to build recognizers for the beta-propeller shapes. As an application, we identify a potential class of hybrid two-component sensor proteins, that we predict contain a double-propeller domain.

Regulation of the Xylan-degrading Apparatus of Cellvibrio japonicus by a Novel Two-component System

The microbial degradation of lignocellulose biomass is not only an important biological process but is of increasing industrial significance in the bioenergy sector. The mechanism by which the plant cell wall, an insoluble composite structure, activates the extensive repertoire of microbial hydrolytic enzymes required to catalyze its degradation is poorly understood. Here we have used a transposon mutagenesis strategy to identify a genetic locus, consisting of two genes that modulate the expression of xylan side chain-degrading enzymes in the saprophytic bacterium Cellvibrio japonicus. Significantly, the locus encodes a two-component signaling system, designated AbfS (sensor histidine kinase) and AbfR (response regulator). The AbfR/S two-component system is required to activate the expression of the suite of enzymes that remove the numerous side chains from xylan, but not the xylanases that hydrolyze the beta1,4-linked xylose polymeric backbone of this polysaccharide. Studies on the recombinant sensor domain of AbfS (AbfS(SD)) showed that it bound to decorated xylans and arabinoxylo-oligosaccharides, but not to undecorated xylo-oligosaccharides or other plant structural polysaccharides/oligosaccharides. The crystal structure of AbfS(SD) was determined to a resolution of 2.6A(.) The overall fold of AbfS(SD) is that of a classical Per Arndt Sim domain with a central antiparallel four-stranded beta-sheet flanked by alpha-helices. Our data expand the number of molecules known to bind to the sensor domain of two-component histidine kinases to include complex carbohydrates. The biological rationale for a regulatory system that induces enzymes that remove the side chains of xylan, but not the hydrolases that cleave the backbone of the polysaccharide, is discussed.

Gut Bug's Sensors Bag Us Carbs

Bacteria make heavy use of two-component regulatory systems to sense external signals. The membrane-localized sensor proteins have histidine kinase and phosphoacceptor domains and communicate to intracellular response regulators, which often act as transcription factors. There are also hybrid two-component systems (HTCSs), which are really "one-component," because the sensor and response regulator sequences are combined. Sonnenburg et al. analyzed the function of an HTCS in Bacteroides thetaiotaomicron , a bacterium that inhabits the human gut. Such bacteria are required for digestion of dietary polysaccharides. The human genome has a meager supply of the required hydrolases and lyases for such digestion, whereas B. thetaiotaomicron' s genome encodes more than 200 of the former and more than a dozen of the latter. The gene encoding the HTCS BT3172 was detected in a screen for genes whose expression was enhanced in bacteria residing in the gut of rats eating a diet high in polysaccharides. Loss of BT3172 caused decreased expression of α-mannosidase genes, and expression of BT3172 was enhanced by exposure of bacteria to mannosides. The predicted structure of BT3172 has three transmembrane domains and would result in sensor regions on the periplasmic and cytoplasmic sides of the bacterial inner membrane. The periplasmic sensor might sense mannosides in the environment. The authors used a yeast two-hybrid screen to detect binding partners for the cytoplasmic sensor domain of BT3172 and detected two proteins involved in glucose metabolism--glucose 6-phosphate isomerase and glucose 6-phosphate dehydrogenase. Thus, the authors speculate that BT3172 may serve to coordinate carbohydrate use and availability. They propose that more complete understanding of gut bacteria could allow "personalized nutrition where diet is matched to the nutrient processing capacity of an individual's microbiota." E. D. Sonnenburg, J. L. Sonnenburg, J. K. Manchester, E. E. Hansen, H. C. Chiang, J. I. Gordon, A hybrid two-component system protein of a prominent human gut symbiont couples glycan sensing in vivo to carbohydrate metabolism. Proc. Natl. Acad. Sci. U.S.A. 103 , 8834-8839 (2006). [Abstract] [Full Text]

A hybrid two-component system protein of a prominent human gut symbiont couples glycan sensing in vivo to carbohydrate metabolism.

Bacteroides thetaiotaomicron is a prominent member of our normal adult intestinal microbial community and a useful model for studying the foundations of human-bacterial mutualism in our densely populated distal gut microbiota. A central question is how members of this microbiota sense nutrients and implement an appropriate metabolic response. B. thetaiotaomicron contains a large number of glycoside hydrolases not represented in our own proteome, plus a markedly expanded collection of hybrid two-component system (HTCS) proteins that incorporate all domains found in classical two-component environmental sensors into one polypeptide. To understand the role of HTCS in nutrient sensing, we used B. thetaiotaomicron GeneChips to characterize their expression in gnotobiotic mice consuming polysaccharide-rich or -deficient diets. One HTCS, BT3172, was selected for further analysis because it is induced in vivo by polysaccharides, and its absence reduces B. thetaiotaomicron fitness in polysaccharide-rich diet-fed mice. Functional genomic and biochemical analyses of WT and BT3172-deficient strains in vivo and in vitro disclosed that alpha-mannosides induce BT3172 expression, which in turn induces expression of secreted alpha-mannosidases. Yeast two-hybrid screens revealed that the cytoplasmic portion of BT3172's sensor domain serves as a scaffold for recruiting glucose-6-phosphate isomerase and dehydrogenase. These interactions are a unique feature of BT3172 and specific for the cytoplasmic face of its sensor domain. Loss of BT3172 reduces glycolytic pathway activity in vitro and in vivo. Thus, this HTCS functions as a metabolic reaction center, coupling nutrient sensing to dynamic regulation of monosaccharide metabolism. An expanded repertoire of HTCS proteins with diversified sensor domains may be one reason for B. thetaiotaomicron's success in our intestinal ecosystem.