Biochemical characterization of the first essential two-component signal transduction system from Staphylococcus aureus and Streptococcus pneumoniae

The YYCFG two-component signal transduction system (TCSTS) has been shown to be essential to the viability of several gram-positive bacteria. However, the function of the gene pair remains unknown. Interestingly, while both components are essential to Staphylococcus aureus and Bacillus subtilis, only the response regulator (YYCF) is essential to Streptococcus pneumoniae. To study this essential TCSTS further, the S. pneumoniae and S. aureus truncated YycG histidine kinase and full-length YycF response regulator proteins were characterized at a biochemical level. The recombinant proteins from both organisms were expressed in Escherichia coli and purified. The YycG autophosphorylation activities were activated by ammonium. The apparent K(m )(ATP) of S. aureus YycG autophosphorylation was 130 microM and S. pneumoniae was 3.0 microM. Each had similar K(cat )values of 0.036 and 0.024 min(-1), respectively. Cognate phosphotransfer was also investigated indicating different levels of the phosphorylated YycG intermediates during the reaction. The S. pneumoniae YycG phosphorylated intermediate was not detectable in the presence of its cognate YycF, while phosphorylated S. aureus YycG and YycF were detected concurrently. In addition, noncognate phosphotransfer was demonstrated between the two species. These studies thoroughly compare the essential YycFG TCSTS from the two species at the biochemical level and also establish methods for assaying the activities of these antibacterial targets.     

Two-component signal transduction systems and regulation of virulence factors in <Emphasis Type="Italic">Xanthomonas</Emphasis>: a perspective

Two-component signal transduction systems (TCSTSs), consisting of a histidine kinase and a response regulator, play a critical role in regulating virulence gene expression in Gram-negative phytopathogenic bacteria Xanthomonas spp.. To date, 12 TCSTS genes have been identified, accounting for approximately 10% of the TCSTS genes in each genome that have been experimentally identified to be related to pathogenesis. These TCSTSs modulate the expression of a number of virulence factors through diverse molecular mechanisms such as interacting with DNA, protein-binding and involvement in second messenger metabolism, which generates a high level of regulatory versatility. Here we summarize the current knowledge in this field and discuss the emerging themes and remaining questions that are important in deciphering the signaling network of TCSTSs in Xanthomonas.     

Two-component signal transduction systems of Xanthomonas spp.: a lesson from genomics

The two-component signal transduction systems (TCSTSs), consisting of a histidine kinase sensor (HK) and a response regulator (RR), are the dominant molecular mechanisms by which prokaryotes sense and respond to environmental stimuli. Genomes of Xanthomonas generally contain a large repertoire of TCSTS genes (approximately 92 to 121 for each genome), which encode diverse structural groups of HKs and RRs. Among them, although a core set of 70 TCSTS genes (about two-thirds in total) which accumulates point mutations with a slow rate are shared by these genomes, the other genes, especially hybrid HKs, experienced extensive genetic recombination, including genomic rearrangement, gene duplication, addition or deletion, and fusion or fission. The recombinations potentially promote the efficiency and complexity of TCSTSs in regulating gene expression. In addition, our analysis suggests that a co-evolutionary model, rather than a selfish operon model, is the major mechanism for the maintenance and microevolution of TCSTS genes in the genomes of Xanthomonas. Genomic annotation, secondary protein structure prediction, and comparative genomic analyses of TCSTS genes reviewed here provide insights into our understanding of signal networks in these important phytopathogenic bacteria.     

Two-Component Signal Transduction Systems of Desulfovibrio vulgaris: Structural and Phylogenetic Analysis and Deduction of Putative Cognate Pairs

A large number of two-component signal transduction systems (TCSTS) including 59 putative sensory histidine kinases (HK) and 55 response regulators (RR) were identified from the Desulfovibrio vulgaris genome. In this study, the structural and phylogenetic analyses of all putative TCSTSs in D. vulgaris were performed. The results showed that D. vulgaris contained 21 hybrid-type HKs, implying that multiple-step phosphorelay may be a common signal transduction mechanism in D. vulgaris. Despite the low sequence similarity that restricted the resolution of the phylogenetic analyses, most TCSTS components of D. vulgaris were found clustered into several subfamilies previously recognized in Escherichia coli and Bacillus subtilis. An attempt was made in this investigation to identify the possible cognate HK-RR pairs not linked on the chromosome in D. vulgaris based on similar expression patterns in response to various environmental and genetic changes. Expression levels of all HK and RR genes were measured using whole-genome microarrays. Five groups of HK-RR genes not linked on the chromosome were identified as possible cognate pairs in D. vulgaris. The results provided a preliminary list of possible cognate HK-RR pairs and constitute a basis for further exploration of interaction and physiological function of TCSTSs in D. vulgaris. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Gail Simmons]     

The Campylobacter jejuni dccRS two-component system is required for optimal in vivo colonization but is dispensable for in vitro growth

A Campylobacter jejuni two-component signal transduction system (TCSTS), designated dccR-dccS (diminished capacity to colonize; Cj1223c-Cj1222c), has been found to be important for in vivo colonization but dispensable for in vitro growth. A DeltadccR response regulator mutant generated using the virulent strain 81-176 background exhibited significantly reduced colonization of immunocompetent limited flora (I-LF) mice, severe combined immunodeficient limited flora (SCID-LF) mice, and 1-day-old chicks. A DeltadccS sensor kinase mutant was likewise defective for colonization in the I-LF mouse model. DeltadccR-infected SCID-LF mice also exhibited dramatically reduced inflammation relative to wild type-infected SCID-LF mice. Despite this diminished colonization capacity, the DeltadccRS mutants were indistinguishable from wild type for growth under numerous in vitro conditions as well as for various phenotypes. Microarray analysis identified several genes encoding putative periplasmic and membrane proteins as being regulated by this two-component system; binding of purified His-tagged DccR to the promoter region of two of these genes supports a direct protein-DNA interaction. A conserved repeat sequence was identified in the promoter regions of these genes and in three other promoter regions in the genome, including that of an operon encoding a putative type I secretion system. Two of the regulated target genes were found to be essential for optimal colonization. Both the two-component system and the putative regulated genes have uncharacterized homologues in other Campylobacter and Helicobacter spp., suggesting that they may perform an important function in colonization among a variety of related pathogenic species.     

Genome-scale mutagenesis and phenotypic characterization of two-component signal transduction systems in Xanthomonas campestris pv. campestris ATCC 33913

The gram-negative bacterium Xanthomonas campestris pv. campestris is the causal agent of black rot disease of cruciferous plants. Its genome encodes a large repertoire of two-component signal transduction systems (TCSTSs), which consist of histidine kinases and response regulators (RR) to monitor and respond to environmental stimuli. To investigate the biological functions of these TCSTS genes, we aimed to inactivate all 54 RR genes in X. campestris pv. campestris ATCC 33913, and successfully generated 51 viable mutants using the insertion inactivation method. Plant inoculation identified two novel response regulator genes (XCC1958 and XCC3107) that are involved in virulence of this strain. Genetic complementation demonstrated that XCC3107, designated as vgrR (virulence and growth regulator), also affects bacterial growth and activity of extracellular proteases. In addition, we assessed the survival of these mutants under various stresses, including osmotic stress, high sodium concentration, heat shock, and sodium dodecyl sulfate exposure, and identified a number of genes that may be involved in the general stress response of X. campestris pv. campestris. Mutagenesis and phenotypic characterization of RR genes in this study will facilitate future studies on signaling networks in this important phytopathogenic bacterium.     

蓝藻的二元信号传导系统

二元系统是细菌中主要的信号传导途径 ,磷酸根转移介导的信号途径使细胞得以感受各种环境刺激并产生应答。组氨酸蛋白质激酶的自动磷酸化将磷酸基团传给反应调节蛋白 ,反过来作为分子开关控制不同的效应物活性。蓝藻是地球上最早出现的光合自养原核生物 ,在长期的生物进化过程中 ,它们发展了一系列独特的形态和生理代谢机制 ,使其能在各种不同生境中生长、繁殖和扩增。研究蓝藻信号传导途径为阐明其高度的环境适应性提供了理论基础。     

Partners in crime: phosphotransfer profiling identifies a multicomponent phosphorelay

The first multicomponent phosphorelay, regulating stalk biogenesis, has been identified in Caulobacter crescentus using a bioinformatic screen, targeted disruptions of each histidine kinase and response regulator, and a new technique called phosphotransfer profiling, in which a purified histidine kinase or histidine phosphotransferase is simultaneously assayed for the ability to phosphorylate each purified response regulator protein from one organism. This powerful combination of approaches will allow future researchers to map the interactions among all two-component signal transduction proteins in genetically tractable bacteria with sequenced genomes.     

Role of two-component regulatory systems in intracellular survival of Mycobacterium tuberculosis

The typical two-component regulatory systems (TCSs), consisting of response regulator and histidine kinase, play a central role in survival of pathogenic bacteria under stress conditions such as nutrient starvation, hypoxia, and nitrosative stress. A total of 11 complete paired two-component regulatory systems have been found in Mycobacterium tuberculosis, including a few isolated kinase and regulatory genes. Increasing evidence has shown that TCSs are closely associated with multiple physiological process like intracellular persistence, pathogenicity, and metabolism. This review gives the two-component signal transduction systems in M. tuberculosis and their signal transduction roles in adaption to the environment.     

Characterization of the genes and proteins of a two-component system from the hyperthermophilic bacterium Thermotoga maritima.

As a step towards studying representative members of the two-component family of signal transduction proteins, we have cloned genes encoding a histidine protein kinase and a response regulator from the hyperthermophilic bacterium Thermotoga maritima. The genes have been designated HpkA and drrA, respectively. The deduced HpkA sequence contains all five characteristic histidine protein kinase motifs with the same relative order and spacing found in the mesophilic bacterial proteins. A hydropathy profile indicates that HpkA possesses only one membrane-spanning segment located at the extreme N terminus. The N-terminal region of DrrA exhibits all of the characteristics of the conserved domains of mesophilic bacterial response regulators, and the C-terminal region shows high similarity to the OmpR-PhoB subfamily of DNA-binding proteins. Recombinant T. maritima proteins, truncated HpkA lacking the putative membrane-spanning N- terminal amino acids and DrrA, were expressed in Escherichia coli. Partial purification of T. maritima proteins was achieved by heat denaturation of E. coli host proteins. In an in vitro assay, truncated HpkA protein was autophosphorylated in the presence of ATP. Thus, the N-terminal hydrophobic region is not required for kinase activity. Phosphotransfer between truncated HpkA and DrrA was demonstrated in vitro with the partially purified proteins. The phosphorylation reactions were strongly temperature dependent. The results indicate that the recombinant T. maritima two-component proteins overexpressed in E. coli are stable as well as enzymatically active at elevated temperatures.     

Nitric oxide regulated two-component signaling in Pseudoalteromonas atlantica

Bacteria employ two-component signaling to detect and respond to environmental stimuli. In essence, two-component signaling relies on a protein called a response regulator that can elicit a change in gene expression or protein function in response to phosphoryl transfer from a histidine kinase. Phosphorylation of the associated histidine kinase is regulated by detection of an environmental signal, thus linking sensing to cellular response. Recently, it has been suggested that H-NOX (Heme-nitric oxide/oxygen binding) proteins may act as nitric oxide (NO) sensors in two-component signaling systems. NO/H-NOX regulated histidine kinases have been reported, but their cognate response regulators have yet to be identified. In this work we provide biochemical characterization of a complete NO/H-NOX-regulated two-component signaling pathway in the biofilm-dwelling marine bacterium, Pseudoalteromonas atlantica. In P. atlantica, as is typical for bacteria that code for H-NOX, an hnoX gene is found in the same operon as a gene coding for a two-component signaling histidine kinase (H-NOX-associated histidine kinase; HahK). We find that HahK is capable of autophosphorylation in vitro and that NO-bound H-NOX inhibits HahK activity, implicating H-NOX as a selective NO sensor. The cognate response regulator, a protein annotated as a cyclic-di-GMP processing enzyme that we have named HarR (H-NOX-associated response regulator), was identified using bioinformatics tools. Phosphoryl transfer from HahK to HarR has been established. This report reveals the first biochemical characterization of an H-NOX-associated response regulator and contributes to a deeper understanding of NO/H-NOX signaling in bacteria.     

Histidine Phosphotransfer Proteins in Fungal Two-Component Signal Transduction Pathways

The histidine phosphotransfer (HPt) protein Ypd1 is an important participant in the Saccharomyces cerevisiae multistep two-component signal transduction pathway and, unlike the expanded histidine kinase gene family, is encoded by a single gene in nearly all model and pathogenic fungi. Ypd1 is essential for viability in both S. cerevisiae and in Cryptococcus neoformans. These and other aspects of Ypd1 biology, combined with the availability of structural and mutational data in S. cerevisiae, suggest that the essential interactions between Ypd1 and response regulator domains would be a good target for antifungal drug development. The goal of this minireview is to summarize the wealth of data on S. cerevisiae Ypd1 and to consider the potential benefits of conducting related studies in pathogenic fungi.     

Systematic dissection and trajectory-scanning mutagenesis of the molecular interface that ensures specificity of two-component signaling pathways

Two-component signal transduction systems enable bacteria to sense and respond to a wide range of environmental stimuli. Sensor histidine kinases transmit signals to their cognate response regulators via phosphorylation. The faithful transmission of information through two-component pathways and the avoidance of unwanted cross-talk require exquisite specificity of histidine kinase-response regulator interactions to ensure that cells mount the appropriate response to external signals. To identify putative specificity-determining residues, we have analyzed amino acid coevolution in two-component proteins and identified a set of residues that can be used to rationally rewire a model signaling pathway, EnvZ-OmpR. To explore how a relatively small set of residues can dictate partner selectivity, we combined alanine-scanning mutagenesis with an approach we call trajectory-scanning mutagenesis, in which all mutational intermediates between the specificity residues of EnvZ and another kinase, RstB, were systematically examined for phosphotransfer specificity. The same approach was used for the response regulators OmpR and RstA. Collectively, the results begin to reveal the molecular mechanism by which a small set of amino acids enables an individual kinase to discriminate amongst a large set of highly-related response regulators and vice versa. Our results also suggest that the mutational trajectories taken by two-component signaling proteins following gene or pathway duplication may be constrained and subject to differential selective pressures. Only some trajectories allow both the maintenance of phosphotransfer and the avoidance of unwanted cross-talk.     

Probing the nucleotide binding and phosphorylation by the histidine kinase of a novel three-protein two-component system from Mycobacterium tuberculosis

The two-component signal transduction system from Mycobacterium tuberculosis bears a unique three-protein system comprising of two putative histidine kinases (HK1 and HK2) and one response regulator TcrA. By sequence analysis, HK1 is found to be an adenosine 5'-triphosphate (ATP) binding protein, similar to the nucleotide-binding domain of homologous histidine kinases, and HK2 is a unique histidine containing phosphotransfer (HPt)-mono-domain protein. HK1 is expected to interact with and phosphorylate HK2. Here, we show that HK1 binds 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate monolithium trisodium salt and ATP with a 1:1 stoichiometric ratio. The ATPase activity of HK1 in the presence of HK2 was measured, and phosphorylation experiments suggested that HK1 acts as a functional kinase and phosphorylates HK2 by interacting with it. Further phosphorylation studies showed transfer of a phosphoryl group from HK2 to the response regulator TcrA. These results indicate a new mode of interaction for phosphotransfer between the two-component system proteins in bacteria.