Sentra: a database of signal transduction proteins for comparative genome analysis

Sentra (), a database of signal transduction proteins encoded in completely sequenced prokaryotic genomes, has been updated to reflect recent advances in understanding signal transduction events on a whole-genome scale. Sentra consists of two principal components, a manually curated list of signal transduction proteins in 202 completely sequenced prokaryotic genomes and an automatically generated listing of predicted signaling proteins in 235 sequenced genomes that are awaiting manual curation. In addition to two-component histidine kinases and response regulators, the database now lists manually curated Ser/Thr/Tyr protein kinases and protein phosphatases, as well as adenylate and diguanylate cyclases and c-di-GMP phosphodiesterases, as defined in several recent reviews. All entries in Sentra are extensively annotated with relevant information from public databases (e.g. UniProt, KEGG, PDB and NCBI). Sentra's infrastructure was redesigned to support interactive cross-genome comparisons of signal transduction capabilities of prokaryotic organisms from a taxonomic and phenotypic perspective and in the framework of signal transduction pathways from KEGG. Sentra leverages the PUMA2 system to support interactive analysis and annotation of signal transduction proteins by the users

MiST: a microbial signal transduction database

Signal transduction pathways control most cellular activities in living cells ranging from regulation of gene expression to fine-tuning enzymatic activity and controlling motile behavior in response to extracellular and intracellular signals. Because of their extreme sequence variability and extensive domain shuffling, signal transduction proteins are difficult to identify, and their current annotation in most leading databases is often incomplete or erroneous. To overcome this problem, we have developed the microbial signal transduction (MiST) database (), a comprehensive library of the signal transduction proteins from completely sequenced bacterial and archaeal genomes. By searching for domain profiles that implicate a particular protein as participating in signal transduction, we have systematically identified 69 270 two- and one-component proteins in 365 bacterial and archaeal genomes. We have designed a user-friendly website to access and browse the predicted signal transduction proteins within various organisms. Further capabilities include gene/protein sequence retrieval, visualized domain architectures, interactive chromosomal views for exploring gene neighborhood, advanced querying options and cross-species comparison. Newly available, complete genomes are loaded into the database each month. MiST is the only comprehensive and up-to-date electronic catalog of the signaling machinery in microbial genomes

Relating gene expression data on two-component systems to functional annotations in Escherichia coli

<p>Abstract</p> <p>Background</p> <p>Obtaining physiological insights from microarray experiments requires computational techniques that relate gene expression data to functional information. Traditionally, this has been done in two consecutive steps. The first step identifies important genes through clustering or statistical techniques, while the second step assigns biological functions to the identified groups. Recently, techniques have been developed that identify such relationships in a single step.</p> <p>Results</p> <p>We have developed an algorithm that relates patterns of gene expression in a set of microarray experiments to functional groups in one step. Our only assumption is that patterns co-occur frequently. The effectiveness of the algorithm is demonstrated as part of a study of regulation by two-component systems in <it>Escherichia coli</it>. The significance of the relationships between expression data and functional annotations is evaluated based on density histograms that are constructed using product similarity among expression vectors. We present a biological analysis of three of the resulting functional groups of proteins, develop hypotheses for further biological studies, and test one of these hypotheses experimentally. A comparison with other algorithms and a different data set is presented.</p> <p>Conclusion</p> <p>Our new algorithm is able to find interesting and biologically meaningful relationships, not found by other algorithms, in previously analyzed data sets. Scaling of the algorithm to large data sets can be achieved based on a theoretical model.</p

Different Evolutionary Modifications as a Guide to Rewire Two-Component Systems

Two-component systems (TCS) are short signalling pathways generally occurring in prokaryotes. They frequently regulate prokaryotic stimulus responses and thus are also of interest for engineering in biotechnology and synthetic biology. The aim of this study is to better understand and describe rewiring of TCS while investigating different evolutionary scenarios. Based on large-scale screens of TCS in different organisms, this study gives detailed data, concrete alignments, and structure analysis on three general modification scenarios, where TCS were rewired for new responses and functions: (i) exchanges in the sequence within single TCS domains, (ii) exchange of whole TCS domains; (iii) addition of new components modulating TCS function. As a result, the replacement of stimulus and promotor cassettes to rewire TCS is well defined exploiting the alignments given here. The diverged TCS examples are non-trivial and the design is challenging. Designed connector proteins may also be useful to modify TCS in selected cases

A census of membrane-bound and intracellular signal transduction proteins in bacteria: Bacterial IQ, extroverts and introverts

BACKGROUND: Analysis of complete microbial genomes showed that intracellular parasites and other microorganisms that inhabit stable ecological niches encode relatively primitive signaling systems, whereas environmental microorganisms typically have sophisticated systems of environmental sensing and signal transduction. RESULTS: This paper presents results of a comprehensive census of signal transduction proteins – histidine kinases, methyl-accepting chemotaxis receptors, Ser/Thr/Tyr protein kinases, adenylate and diguanylate cyclases and c-di-GMP phosphodiesterases – encoded in 167 bacterial and archaeal genomes, sequenced by the end of 2004. The data have been manually checked to avoid false-negative and false-positive hits that commonly arise during large-scale automated analyses and compared against other available resources. The census data show uneven distribution of most signaling proteins among bacterial and archaeal phyla. The total number of signal transduction proteins grows approximately as a square of genome size. While histidine kinases are found in representatives of all phyla and are distributed according to the power law, other signal transducers are abundant in certain phylogenetic groups but virtually absent in others. CONCLUSION: The complexity of signaling systems differs even among closely related organisms. Still, it usually can be correlated with the phylogenetic position of the organism, its lifestyle, and typical environmental challenges it encounters. The number of encoded signal transducers (or their fraction in the total protein set) can be used as a measure of the organism's ability to adapt to diverse conditions, the 'bacterial IQ', while the ratio of transmembrane receptors to intracellular sensors can be used to define whether the organism is an 'extrovert', actively sensing the environmental parameters, or an 'introvert', more concerned about its internal homeostasis. Some of the microorganisms with the highest IQ, including the current leader Wolinella succinogenes, are found among the poorly studied beta-, delta- and epsilon-proteobacteria. Among all bacterial phyla, only cyanobacteria appear to be true introverts, probably due to their capacity to conduct oxygenic photosynthesis, using a complex system of intracellular membranes. The census data, available at , can be used to get an insight into metabolic and behavioral propensities of each given organism and improve prediction of the organism's properties based solely on its genome sequence

The Role of MSA in the Global Regulation of Virulence in \u3ci\u3eStaphylococcus aureus\u3c/i\u3e

Staphylococcus aureus is an important pathogen causing life threatening diseases in humans. Previously we showed that msa modulates the activity of sarA (Staphylococcal accessory regulator), which is one of a major global regulator of virulence in S. aureus. The objective of this study is to characterize the role of msa (Modulator of SarA) in the global regulation of virulence in S. aureus. Structure and function predictions were done using several computational tools and approaches to understand the nature of msa. A novel S. aureus microarray meta-database (SAMMD) was designed and developed to compare and contrast other transcriptomes with msa transcriptome. msa and sarA transcriptomes were generated using the microarray technology. Phenotypic and molecular assays were performed to support microarray results. The results show that msa is a putative transmembrane protein, with three transmembrane segments, a distinct N-terminal cleavable signal peptide, four phophorylation sites (two outside and two inside the membrane) and a binding site in the cytoplasmic region. Microarray results and comparative transcriptome analysis using SAMMD showed that several genes regulated by msa are also regulated by sarA. Based on these results I hypothesize that msa is a novel signal transducer, which modulates the activity of genes involved in virulence in a sar/\-dependent manner, while modulating the activity of genes involved in metabolism in a sar-4-independent manner

Integration of large datasets for plant model organisms

This dissertation is concerned with bioinformatics data integration. The first chapter illustrates the current state of biological pathway databases in general, and in particular, plant pathway databases. Key studies are cited to illustrate the potential benefits that may come from further research into integration methods. Different models are explored to interface with the various stakeholders of biological data repositories. A public website (http://www.metnetonline.org) was built to address the role of a bioinformatics data warehouse as a server for external third parties. A dedicated API (MetNetAPI: http://www.metnetonline.org/api) accommodates bioinformaticians (and software developers in general) who wish to build advanced applications on top of MetNet. The API (implemented as .NET and Java libraries) was designed to be as user-friendly to programmers, as the public website is to end-users. Finally, a hybrid model is examined: the use of XML as a repository for information integration, downstream processing, and data manipulation. An overview of the use of XML in biological applications is included. MetNetAPI functions according to certain principles; a subset of the API is abstracted and implemented to interface with a range of other public databases. This results in a new bioinformatics toolkit that can be used to mix and match data from heterogeneous sources in a transparent manner. An example would be the grafting of protein-protein interaction data on top of araCyc pathways. Biological network data is often distributed over a variety of independently modeled databases. This dissertation makes two contributions to the field of bioinformatics: A new service - MetNet Online - is now operating which offers access to the earlier created and integrated MetNetDB data repository. The service is geared toward end-users, students and researchers alike, as well as seasoned bioinformatics software developers who wish to build their own applications on top of an already integrated datasource. Furthermore, integrated databases are only useful when they can be synchronized with their respective external sources. Thus, a framework was created that allows for a systematic approach to such integration efforts. In closing, this work provides a roadmap to maintain current as well as prepare for future integrated biological database projects

Effect of osmolytes on regulating the activities of the SSK1 response regulator from Saccharomyces cerevisiae

The multi-step His-Asp phosphorelay system in Saccharomyces cerevisiae allows cells to adapt to osmotic, oxidative and other environmental stresses. The pathway consists of a hybrid histidine kinase SLN1, a histidine-containing phosphotransfer (HPt) protein YPD1 and two response regulator proteins, SSK1 and SKN7. Under non-osmotic stress conditions, the SLN1 sensor kinase is active and phosphoryl groups are shuttled through YPD1 to SSK1, therefore maintaining the response regulator protein in a constitutively phosphorylated state. The cellular response to hyperosmotic stress involves rapid efflux of water and changes in intracellular ion and osmolyte concentration. To address the effect of osmolytes on the regulation of this signaling pathway, the individual and combined effects of NaCl and glycerol on phosphotransfer rates within the SLN1-YPD1-SSK1 phosphorelay were examined. In addition, the effect of osmolyte concentration on the half-life of the phosphorylated SSK1 receiver domain in the presence/absence of YPD1 was evaluated. The results show that the combined effects of glycerol and NaCl on the phosphotransfer reaction rates are different from the individual effects of glycerol and NaCl. The combinatory effect is likely more representative of the in vivo changes that occur during hyperosmotic stress. The results revealed that increasing osmolyte concentrations negatively affects the YPD1*SSK1~P interaction thereby facilitating dephosphorylation of SSK1 and activating the HOG1 MAP kinase cascade. At high osmolyte concentrations, the kinetics of the phosphorelay favors production of SSK1~P and inhibition of the HOG1 pathway.A similar multi-step signaling pathway is also utilized by Candida albicans, which is known for adaptation to oxidative stress, morphogenesis, cell wall biosynthesis and virulence in this opportunistic pathogenic yeast. To biochemically characterize major components of this pathway, studies were focused on in vitro reconstitution of the multi-step phosphorelay from C. albicans and biochemical characterization of the CaYPD1 (HPt protein) and CaSSK1 (response regulator protein). The heterologous phosphoryl transfer system SLN1-HK-RR &#8594; CaYPD1&#8594; CaSSK1 (or SSK1) was established and examined. The CaYPD1 histidine phosphotransfer protein exhibited similar phosphotransfer specificity in vitro towards the response regulator domain of CaSSK1-RR and SSK1-RR. The half-life of the phosphorylated regulatory domain of CaSSK1-RR was also measured and was approximately 9 min with a corresponding rate constant of 0.078 min-1. This result demonstrates a similar rate of CaSSK1-RR dephosphorylation compared to SSK1-RR suggesting possible functional similarities between these two response regulator proteins.Mutational analysis of the CaSSK1 response regulator domain was also performed. Mutants were expressed, purified and their activity was analyzed using an in vitro phosphorylation assay. Little or no phosphorylation was observed for the CaSSK1-RR D556N mutant. The radiolabel primarily resided with the CaYPD1 protein and did not get transferred to the D556N mutant. Likewise, the D513K mutant was also severely impaired in its ability to be phosphorylated by CaYPD1. The receiver domains of the D556N and the D513K mutants could not be appreciably phosphorylated in vitro indicating that constitutive activation of HOG1 occurs in vivo due to the inability of CaSSK1 to be phosphorylated

The Sinorhizobium meliloti ExoS/ChvI two-component regulatory system

Exopolysaccharides are essential for the establishment of the symbiosis between Sinorhizobium meliloti and Medicago sativa (alfalfa). The ExoS/ChvI two-component regulatory system is known as a regulator of succinoglycan production but the genes that are directly regulated by ChvI have not been determined. Difficulty isolating exoS and chvI null mutants has prompted the suggestion that these genes are essential for S. meliloti viability. We have successfully isolated exoS and chvI null mutants using a merodiploid facilitated strategy. We present evidence that the S. meliloti ExoS/ChvI two-component regulatory system is essential for symbiosis with alfalfa. Phenotypic analyses of exoS and chvI null mutant strains demonstrate that ExoS/ChvI controls both succinoglycan and galactoglucan production and is required for growth on over 21 different carbon sources. These new findings suggest that the ExoS/ChvI regulatory targets might not be the exo genes that are specific for succinoglycan biosynthesis but rather genes that have common influence on both succinoglycan and galactoglucan production. To obtain further insight into the nature of the ChvI regulon, we obtained a purified His•Tag-ChvI and used it to perform modified electrophoretic mobility shift assays. These assays were done using genomic DNA and were followed by cloning of DNA fragments having the highest affinity for ChvI. Sequencing of these fragments revealed that ChvI has a diverse regulon, it affects transcription of genes encoding enzymes that are involved in different pathways. Transcriptional gene fusion assays confirmed that ChvI is important for the activation of the transcription of the msbA2 operon, as well as repression of the transcription of the rhizobactin 1021 operon and genes SMc00262-61. ChvI-regulation of genes that are part of the connected thiamine and histidine biosynthesis pathways suggest that ChvI could act in a concerted manner to avoid limitation of important intermediates in these pathways. This study presents for the first time genes directly regulated by ChvI and this includes none of the exo genes. This work opens new avenues in the understanding of the global regulatory role of the symbiotically important ExoS/ChvI two-component regulatory system