Two-component Phosphorelay System Research Articles

A Survey of Two-Component Systems in Coxiella burnetii Reveals Redundant Regulatory Schemes and a Requirement for an Atypical PhoBR System in Mammalian Cell Infection.

Coxiella burnetii is an obligate intracellular bacterium and the etiological agent of Q fever in humans. C. burnetii transitions between a replicative, metabolically active large-cell variant (LCV) and a spore-like, quiescent small-cell variant (SCV) as a likely mechanism to ensure survival between host cells and mammalian hosts. C. burnetii encodes three canonical two-component systems, four orphan hybrid histidine kinases, five orphan response regulators, and a histidine phosphotransfer protein, which have been speculated to play roles in the signaling required for C. burnetii morphogenesis and virulence. However, very few of these systems have been characterized. By employing a CRISPR interference system for genetic manipulation of C. burnetii, we created single- and multigene transcriptional knockdown strains targeting most of these signaling genes. Through this, we revealed a role for the C. burnetii PhoBR canonical two-component system in virulence, regulation of [Pi] maintenance, and Pi transport. We also outline a novel mechanism by which PhoBR function may be regulated by an atypical PhoU-like protein. We also determined that the GacA.2/GacA.3/GacA.4/GacS orphan response regulators coordinately and disparately regulate expression of SCV-associated genes in C. burnetii LCVs. These foundational results will inform future studies on the role of C. burnetii two-component systems in virulence and morphogenesis. IMPORTANCE C. burnetii is an obligate intracellular bacterium with a spore-like stability allowing it to survive long periods of time in the environment. This stability is likely due to its biphasic developmental cycle, whereby it can transition from an environmentally stable small-cell variant (SCV) to a metabolically active large-cell variant (LCV). Here, we define the role of two-component phosphorelay systems (TCS) in C. burnetii's ability to survive within the harsh environment contained in the phagolysosome of host cells. We show that the canonical PhoBR TCS has an important role in C. burnetii virulence and phosphate sensing. Further examination of the regulons controlled by orphan regulators indicated a role in modulating gene expression of SCV-associated genes, including genes essential for cell wall remodeling.

Identification of Potential Cytokinin Responsive Key Genes in Rice Treated With Trans-Zeatin Through Systems Biology Approach.

Rice is an important staple food grain consumed by most of the population around the world. With climate and environmental changes, rice has undergone a tremendous stress state which has impacted crop production and productivity. Plant growth hormones are essential component that controls the overall outcome of the growth and development of the plant. Cytokinin is a hormone that plays an important role in plant immunity and defense systems. Trans-zeatin is an active form of cytokinin that can affect plant growth which is mediated by a multi-step two-component phosphorelay system that has different roles in various developmental stages. Systems biology is an approach for pathway analysis to trans-zeatin treated rice that could provide a deep understanding of different molecules associated with them. In this study, we have used a weighted gene co-expression network analysis method to identify the functional modules and hub genes involved in the cytokinin pathway. We have identified nine functional modules comprising of different hub genes which contribute to the cytokinin signaling route. The biological significance of these identified hub genes has been tested by applying well-proven statistical techniques to establish the association with the experimentally validated QTLs and annotated by the DAVID server. The establishment of key genes in different pathways has been confirmed. These results will be useful to design new stress-resistant cultivars which can provide sustainable yield in stress-specific conditions.

Crystal structure of the inactive state of the receiver domain of Spo0A from Paenisporosarcina sp. TG-14, a psychrophilic bacterium isolated from an Antarctic glacier.

The two-component phosphorelay system is the most prevalent mechanism for sensing and transducing environmental signals in bacteria. Spore formation, which relies on the two-component phosphorelay system, enables the long-term survival of the glacial bacterium Paenisporosarcina sp. TG-14 in the extreme cold environment. Spo0A is a key response regulator of the phosphorelay system in the early stage of spore formation. The protein is composed of a regulatory N-terminal phospho-receiver domain and a DNA-binding C-terminal activator domain. We solved the three-dimensional structure of the unphosphorylated (inactive) form of the receiver domain of Spo0A (PaSpo0A-R) from Paenisporosarcina sp. TG-14. A structural comparison with phosphorylated (active form) Spo0A from Bacillus stearothermophilus (BsSpo0A) showed minor notable differences. A molecular dynamics study of a model of the active form and the crystal structures revealed significant differences in the α4 helix and the preceding loop region where phosphorylation occurs. Although an oligomerization study of PaSpo0A-R by analytical ultracentrifugation (AUC) has shown that the protein is in a monomeric state in solution, both crosslinking and crystal-packing analyses indicate the possibility of weak dimer formation by a previously undocumented mechanism. Collectively, these observations provide insight into the mechanism of phosphorylation-dependent activation unique to Spo0A.

Genetic Dissection of the Regulatory Network Associated with High c-di-GMP Levels in Pseudomonas putida KT2440

Most bacteria grow in nature forming multicellular structures named biofilms. The bacterial second messenger cyclic diguanosine monophosphate (c-di-GMP) is a key player in the regulation of the transition from planktonic to sessile lifestyles and this regulation is crucial in the development of biofilms. In Pseudomonas putida KT2440, Rup4959, a multidomain response regulator with diguanylate cyclase activity, when overexpressed causes an increment in the intracellular levels of c-di-GMP that gives rise to a pleiotropic phenotype consisting of increased biofilm formation and crinkly colony morphology. In a broad genomic screen we have isolated mutant derivatives that lose the crinkly morphology, designed as cfc (crinkle free colony). A total of 19 different genes have been identified as being related with the emergence of the cfc phenotype either because the expression or functionality of Rup4959 is compromised, or due to a lack of transduction of the c-di-GMP signal to downstream elements involved in the acquisition of the phenotype. Discernment between these possibilities was investigated by using a c-di-GMP biosensor and by HPLC-MS quantification of the second messenger. Interestingly five of the identified genes encode proteins with AAA+ ATPase domain. Among the bacterial determinants found in this screen are the global transcriptional regulators GacA, AlgU and FleQ and two enzymes involved in the arginine biosynthesis pathway. We present evidences that this pathway seems to be an important element to both the availability of the free pool of the second messenger c-di-GMP and to its further transduction as a signal for biosynthesis of biopolimers. In addition we have identified an uncharacterized hybrid sensor histidine kinase whose phosphoaceptor conserved histidine residue has been shown in this work to be required for in vivo activation of the orphan response regulator Rup4959, which suggests these two elements constitute a two-component phosphorelay system.

The VieB auxiliary protein negatively regulates the VieSA signal transduction system in Vibrio cholerae.

BackgroundVibrio cholerae is a facultative pathogen that lives in the aquatic environment and the human host. The ability of V. cholerae to monitor environmental changes as it transitions between these diverse environments is vital to its pathogenic lifestyle. One way V. cholerae senses changing external stimuli is through the three-component signal transduction system, VieSAB, which is encoded by the vieSAB operon. The VieSAB system plays a role in the inverse regulation of biofilm and virulence genes by controlling the concentration of the secondary messenger, cyclic-di-GMP. While the sensor kinase, VieS, and the response regulator, VieA, behave similar to typical two-component phosphorelay systems, the role of the auxiliary protein, VieB, is unclear.ResultsHere we show that VieB binds to VieS and inhibits its autophosphorylation and phosphotransfer activity thus preventing phosphorylation of VieA. Additionally, we show that phosphorylation of the highly conserved Asp residue in the receiver domain of VieB regulates the inhibitory activity of VieB.ConclusionTaken together, these data point to an inhibitory role of VieB on the VieSA phosphorelay, allowing for additional control over the signal output. Insight into the function and regulatory mechanism of the VieSAB system improves our understanding of how V. cholerae controls gene expression as it transitions between the aquatic environment and human host.

HepK, a protein-histidine kinase from the cyanobacterium Anabaena sp. strain PCC 7120, binds sequence-specifically to DNA

Abstract Two-component phosphorelay systems are minimally consisted of a sensory protein-histidine kinase (HK) and a response regulator (RR). HK autophosphorylates its conserved histidine residue in response to stimulus from an

Role of Sodium in the RprY-Dependent Stress Response in Porphyromonas gingivalis

Porphyromonas gingivalis is a Gram-negative oral anaerobe which is strongly associated with periodontal disease. Environmental changes in the gingival sulcus trigger the growth of P. gingivalis and a concurrent shift from periodontal health to disease. Bacteria adjust their physiology in response to environmental changes and gene regulation by two-component phospho-relay systems is one mechanism by which such adjustments are effected. In P. gingivalis RprY is an orphan response regulator and previously we showed that the RprY regulon included genes associated with oxidative stress and sodium metabolism. The goals of the present study were to identify environmental signals that induce rprY and clarify the role of the regulator in the stress response. In Escherichia coli an RprY-LacZ fusion protein was induced in sodium- depleted medium and a P. gingivalis rprY mutant was unable to grow in similar medium. By several approaches we established that sodium depletion induced up-regulation of genes involved in oxidative stress. In addition, we demonstrated that RprY interacted directly with the promoters of several molecular chaperones. Further, both genetic and transcription data suggest that the regulator acts as a repressor. We conclude that RprY is one of the regulators that controls stress responses in P. gingivalis, possibly by acting as a repressor since an rprY mutant showed a superstress reponse in sodium-depleted medium which we propose inhibited growth.

Transcriptome analysis of candidate genes and signaling pathways associated with light-induced brown film formation in Lentinula edodes

High-throughput Illumina RNA-seq was used for deep sequencing analysis of the transcriptome of poly(A)+ RNA from mycelium grown under three different conditions: 30 days darkness (sample 118), 80 days darkness (313W), and 30 days darkness followed by 50 days in the light (313C), in order to gain insight into the molecular mechanisms underlying the process of light-induced brown film (BF) formation in the edible mushroom, Lentinula edodes. Of the three growth conditions, BF formation occurred in 313C samples only. Approximately 159.23 million reads were obtained, trimmed, and de novo assembled into 31,511 contigs with an average length of 1,746 bp and an N 50 of 2,480 bp. Based on sequence orientations determined by a BLASTX search against the NR, Swiss-Prot, COG, and KEGG databases, 24,246 (76.9 %) contigs were assigned putative descriptions. Comparison of 313C/118 and 313C/313W expression profiles revealed 3,958 and 5,651 significantly differentially expressed contigs (DECs), respectively. Annotation using the COG database revealed that candidate genes for light-induced BF formation encoded proteins linked to light reception (e.g., WC-1, WC-2, phytochrome), light signal transduction pathways (e.g., two-component phosphorelay system, mitogen-activated protein kinase pathway), and pigment formation (e.g., polyketide synthase, O-methyltransferase, laccase, P450 monooxygenase, oxidoreductase). Several DECs were validated using quantitative real-time polymerase chain reaction. Our report is the first to identify genes associated with light-induced BF formation in L. edodes and represents a valuable resource for future genomic studies on this commercially important mushroom.

Generic signal-specific responses: cytokinin and context-dependent cellular responses

The phytohormone cytokinin triggers numerous and diverse responses during the plant life cycle via a two-component phosphorelay signalling system. Each step of the signalling cascade is supported by a gene family comprising several members. While functional redundancy is observed among family members, additional gene-specific functions encoded by cis-regulatory and coding sequence of individual family members have been described and contribute to specificity in signalling output. In addition, the cellular context of the signal-receiving cell affects the response triggered. Recent studies in Arabidopsis have demonstrated how expression of cytokinin signalling components predefines a spatiotemporal map of signalling sensitivity, which causes local signal amplification and attenuation. In summary, the specific interpretation of cytokinin signalling is affected by an orchestrated interplay of signalling genes and cellular context.

The BLUF-EAL protein YcgF acts as a direct anti-repressor in a blue-light response of Escherichia coli

The blue light using FAD (BLUF)-EAL protein YcgF is a known blue-light sensor of Escherichia coli, but its direct regulatory output and physiological function have remained unknown. Here, we demonstrate that unlike other EAL domain proteins, YcgF does not degrade the signaling molecule c-di-GMP, but directly binds to and releases the MerR-like repressor YcgE from its operator DNA upon blue-light irradiation. As a consequence, a distinct regulon of eight small proteins (of 71-126 amino acids) is strongly induced. These include YmgA and YmgB, which, via the RcsC/RcsD/RcsB two-component phosphorelay system, activate production of the biofilm matrix substance colanic acid as well as acid resistance genes and the biofilm-associated bdm gene and down-regulate adhesive curli fimbriae. Thus, small proteins under YcgF/YcgE control seem to act as "connectors" that provide additional signal input into a two-component signaling pathway. Moreover, we found ycgF and ycgE expression to be strongly activated at low temperature, and we elucidate how blue light, cold, and starvation signals are integrated in the expression and activity of the YcgF/YcgE/small protein signaling pathway. In conclusion, this pathway may modulate biofilm formation via the two-component network when E. coli has to survive in an extrahost aquatic environment.

A novel regulation on developmental gene expression of fruiting body formation in Myxobacteria

Myxobacteria are Gram-negative soil microorganisms that prey on other microorganisms. Myxobacteria have significant potential for applications in biotechnology because of their extraordinary ability to produce natural products such as secondary metabolites. Myxobacteria also stand out as model organisms for the study of cell-cell interactions and multicellular development during their complex life cycle. Cellular morphogenesis during multicellular development in myxobacteria is very similar to that in the eukaryotic soil amoebae. Recent studies have started uncovering molecular mechanisms directing the myxobacterial life cycle. We describe recent studies on signal transduction and gene expression during multicellular development in the myxobacterium Myxococcus xanthus. We provide our current model for signal transduction pathways mediated by a two-component His-Asp phosphorelay system and a Ser/Thr kinase cascade.

RNA-binding protein Csx1 mediates global control of gene expression in response to oxidative stress.

Fission yeast Spc1 (Sty1), a stress-activated mitogen-activated protein kinase (MAPK) homologous to human p38, orchestrates global changes in gene expression in response to diverse forms of cytotoxic stress. This control is partly mediated through Atf1, a transcription factor homologous to human ATF2. How Spc1 controls Atf1, and how the cells tailor gene expression patterns to different forms of stress, are unknown. Here we describe Csx1, a novel protein crucial for survival of oxidative but not osmotic stress. Csx1 associates with and stabilizes atf1+ mRNA in response to oxidative stress. Csx1 controls expression of the majority of the genes induced by oxidative stress, including most of the genes regulated by Spc1 and Atf1. These studies reveal a novel mechanism controlling MAPK-regulated transcription factors and suggest how gene expression patterns can be customized to specific forms of stress. Csx1-like proteins in humans may perform similar tasks.

Crystal Structure of the CheA Histidine Phosphotransfer Domain that Mediates Response Regulator Phosphorylation in Bacterial Chemotaxis

The x-ray crystal structure of the P1 or H domain of the Salmonella CheA protein has been solved at 2.1-A resolution. The structure is composed of an up-down up-down four-helix bundle that is typical of histidine phosphotransfer or HPt domains such as Escherichia coli ArcB(C) and Saccharomyces cerevisiae Ypd1. Loop regions and additional structural features distinguish all three proteins. The CheA domain has an additional C-terminal helix that lies over the surface formed by the C and D helices. The phosphoaccepting His-48 is located at a solvent-exposed position in the middle of the B helix where it is surrounded by several residues that are characteristic of other HPt domains. Mutagenesis studies indicate that conserved glutamate and lysine residues that are part of a hydrogen-bond network with His-48 are essential for the ATP-dependent phosphorylation reaction but not for the phosphotransfer reaction with CheY. These results suggest that the CheA-P1 domain may serve as a good model for understanding the general function of HPt domains in complex two-component phosphorelay systems.

Characterization of the Cph1 holo-phytochrome from Synechocystis sp. PCC 6803.

The cph1 gene from the unicellular cyanobacterium Synechoycstis sp. PCC 6803 encodes a protein with the characteristics of plant phytochromes and histidine kinases of two-component phospho-relay systems. Spectral and biochemical properties of Cph1 have been intensely studied in vitro using protein from recombinant systems, but virtually nothing is known about the situation in the natural host. In the present study, His6-tagged Cph1 was isolated from Synechocystis cells. The cph1-his gene was expressed either under the control of the natural cph1 promoter or over-expressed using the strong promoter of the psbA2 gene. Upon purification with nickel affinity chromatography, the presence of Cph1 in extracts was confirmed by immunoblotting and Zn2+-induced fluorescence. The Cph1 extracts exhibited a red/far-red photoactivity characteristic of phytochromes. Difference spectra were identical with those of the phycocyanobilin adduct of recombinant Cph1, implying that phycocyanobilin is the chromophore of Cph1 in Synechocystis.

Substrate Sequestering Favors Phosphorylation in Bacteria

Prokaryotes can modulate gene expression in response to changes in external osmolarity through a two-component phosphorelay signaling system that involves a transmembrane histidine kinase called EnvZ. EnvZ also has phosphatase activity, and so it can both phosphorylate and dephosphorylate a transcription factor called OmpR. Qin et al. report that the presence of DNA fragments corresponding to the promoter regions of OmpR-regulated genes caused a reduction in the apparent phosphatase activity of EnvZ in vitro. Biochemical analysis of complexes formed in vitro suggests that DNA binds to phosphorylated OmpR and sequesters it from phosphatase action of EnvZ. This finding offers an alternative for earlier results interpreted to show that EnvZ kinase activity can be enhanced by DNA and leaves open the question of how the dual enzymatic activity of EnvZ is regulated in vivo. L. Qin, T. Yoshida, M. Inouye, The critical role of DNA in the equilibrium between OmpR and phosphorylated OmpR mediated by EnvZ in Escherichia coli . Proc. Natl. Acad. Sci. U.S.A. 98 : 908-913 (2001). [Abstract] [Full Text]

Structure-function relationships in the Bvg and Evg two-component phosphorelay systems

The unorthodox two-component phosphorelay systems BvgAS and EvgAS of Bordetella pertussis and E. coli, respectively, are suitable model systems to investigate the molecular basis of signalling specificity, because, despite their high relatedness on the sequence level, they do not cross-talk to each other. We could show that the two systems belong to the obligate type of phosphorelay systems and that signalling specificity is mediated by the HPt modules of the histidine kinases and the receiver domains of the effector proteins. To gain more insight into signalling specificity on the molecular level, we started a detailed structural analysis of the respective proteins using a combination of genetic and biochemical methods including limited proteolysis and chemical modification of purified proteins and their mass spectrometrical analysis.

Dimerization of signalling modules of the EvgAS and BvgAS phosphorelay systems

Biophysical and biochemical properties of signalling proteins or domains derived from the unorthodox EvgAS and BvgAS two-component phosphorelay systems of Escherichia coli and Bordetella pertussis were investigated. Oligomerization of the effector proteins EvgA and BvgA and of truncated EvgS and BvgS derived signalling proteins containing the receiver and histidine containing phosphotransfer (HPt) domains or comprising only the HPt domains were characterized by native gel electrophoresis, gel permeation experiments and analytical ultracentrifugation. The results obtained by the different methods are consistent with non-phosphorylated EvgA and BvgA proteins being dimers in solution with a dissociation constant significantly below 1 μM. In contrast, all sensor derived domains of EvgS and BvgS were observed to be monomers in vitro. No indications for a phosphorylation induced stimulation of oligomerization of the C-terminal histidine kinase domains could be detected. In agreement with these data, surface plasmon resonance studies revealed a 2:1 stoichiometry in the interaction of EvgA with the immobilized EvgS HPt domain and an affinity constant of 1.24×10 6 M −1.

Identification of YPD1, a gene of Candida albicans which encodes a two-component phosphohistidine intermediate protein.

We have identified the YPD1 phosphohistidine intermediate two-component gene of Candida albicans. YPD1 has an open reading frame of 552 bp. It is located on chromosome 1 and an mRNA specific for YPD1 is detected under both yeast and hyphal growth. YPD1 encodes a protein of 184 amino acids with an estimated molecular mass of 20.5 kDa. A search for similarities with other proteins in databases showed that CaYpd1p exhibits the greatest overall similarity with Ypd1p from Saccharomyces cerevisiae (34.2% identity; 49.4% similarity) as well as with the C-terminus half of a protein from Schizosaccharomyces pombe (Accession No. CAA22174). However, CaYpd1p also shows similarity with other eukaryotic and prokaryotic proteins which function as phosphohistidine intermediates in two-component phospho-relay systems. In these cases, similarity was restricted to the amino acid sequences which surround the conserved histidine residue that is phosphorylated. In addition, CaYPD1 (but not CaYPD1(H69Q)) complements the lack of YPD1 in S. cerevisiae. This observation supports the premise that CaYpd1p also may function as a phosphohistidine intermediate protein in C. albicans.