PhosCalc: A tool for evaluating the sites of peptide phosphorylation from Mass Spectrometer data

© 2008 MacLean et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

A DNA element recognised by the molybdenum-responsive transcription factor ModE is conserved in Proteobacteria, green sulphur bacteria and Archaea

BACKGROUND: The transition metal molybdenum is essential for life. Escherichia coli imports this metal into the cell in the form of molybdate ions, which are taken up via an ABC transport system. In E. coli and other Proteobacteria molybdenum metabolism and homeostasis are regulated by the molybdate-responsive transcription factor ModE. RESULTS: Orthologues of ModE are widespread amongst diverse prokaryotes, but not ubiquitous. We identified probable ModE-binding sites upstream of genes implicated in molybdenum metabolism in green sulphur bacteria and methanogenic Archaea as well as in Proteobacteria. We also present evidence of horizontal transfer of nitrogen fixation genes between green sulphur bacteria and methanogenic Archaea. CONCLUSIONS: Whereas most of the archaeal helix-turn-helix-containing transcription factors belong to families that are Archaea-specific, ModE is unusual in that it is found in both Archaea and Bacteria. Moreover, its cognate upstream DNA recognition sequence is also conserved between Archaea and Bacteria, despite the fundamental differences in their core transcription machinery. ModE is the third example of a transcriptional regulator with a binding signal that is conserved in Bacteria and Archaea

Bioinformatic identification of novel regulatory DNA sequence motifs in Streptomyces coelicolor

BACKGROUND: Streptomyces coelicolor is a bacterium with a vast repertoire of metabolic functions and complex systems of cellular development. Its genome sequence is rich in genes that encode regulatory proteins to control these processes in response to its changing environment. We wished to apply a recently published bioinformatic method for identifying novel regulatory sequence signals to gain new insights into regulation in S. coelicolor. RESULTS: The method involved production of position-specific weight matrices from alignments of over-represented words of DNA sequence. We generated 2497 weight matrices, each representing a candidate regulatory DNA sequence motif. We scanned the genome sequence of S. coelicolor against each of these matrices. A DNA sequence motif represented by one of the matrices was found preferentially in non-coding sequences immediately upstream of genes involved in polysaccharide degradation, including several that encode chitinases. This motif (TGGTCTAGACCA) was also found upstream of genes encoding components of the phosphoenolpyruvate phosphotransfer system (PTS). We hypothesise that this DNA sequence motif represents a regulatory element that is responsive to availability of carbon-sources. Other motifs of potential biological significance were found upstream of genes implicated in secondary metabolism (TTAGGTtAGgCTaACCTAA), sigma factors (TGACN(19)TGAC), DNA replication and repair (ttgtCAGTGN(13)TGGA), nucleotide conversions (CTACgcNCGTAG), and ArsR (TCAGN(12)TCAG). A motif found upstream of genes involved in chromosome replication (TGTCagtgcN(7)Tagg) was similar to a previously described motif found in UV-responsive promoters. CONCLUSIONS: We successfully applied a recently published in silico method to identify conserved sequence motifs in S. coelicolor that may be biologically significant as regulatory elements. Our data are broadly consistent with and further extend data from previously published studies. We invite experimental testing of our hypotheses in vitro and in vivo

A Boolean Model of the Pseudomonas syringae hrp Regulon Predicts a Tightly Regulated System

The Type III secretion system (TTSS) is a protein secretion machinery used by certain gram-negative bacterial pathogens of plants and animals to deliver effector molecules to the host and is at the core of the ability to cause disease. Extensive molecular and biochemical study has revealed the components and their interactions within this system but reductive approaches do not consider the dynamical properties of the system as a whole. In order to gain a better understanding of these dynamical behaviours and to create a basis for the refinement of the experimentally derived knowledge we created a Boolean model of the regulatory interactions within the hrp regulon of Pseudomonas syringae pathovar tomato strain DC3000 Pseudomonas syringae. We compared simulations of the model with experimental data and found them to be largely in accordance, though the hrpV node shows some differences in state changes to that expected. Our simulations also revealed interesting dynamical properties not previously predicted. The model predicts that the hrp regulon is a biologically stable two-state system, with each of the stable states being strongly attractive, a feature indicative of selection for a tightly regulated and responsive system. The model predicts that the state of the GacS/GacA node confers control, a prediction that is consistent with experimental observations that the protein has a role as master regulator. Simulated gene “knock out” experiments with the model predict that HrpL is a central information processing point within the network

Protein domains and architectural innovation in plant-associated Proteobacteria

BACKGROUND: Evolution of new complex biological behaviour tends to arise by novel combinations of existing building blocks. The functional and evolutionary building blocks of the proteome are protein domains, the function of a protein being dependent on its constituent domains. We clustered completely-sequenced proteomes of prokaryotes on the basis of their protein domain content, as defined by Pfam (release 16.0). This revealed that, although there was a correlation between phylogeny and domain content, other factors also have an influence. This observation motivated an investigation of the relationship between an organism's lifestyle and the complement of domains and domain architectures found within its proteome. RESULTS: We took a census of all protein domains and domain combinations (architectures) encoded in the completely-sequenced proteobacterial genomes. Nine protein domain families were identified that are found in phylogenetically disparate plant-associated bacteria but are absent from non-plant-associated bacteria. Most of these are known to play a role in the plant-associated lifestyle, but they also included domain of unknown function DUF1427, which is found in plant symbionts and pathogens of the alpha-, beta- and gamma-Proteobacteria, but not known in any other organism. Further, several domains were identified as being restricted to phytobacteria and Eukaryotes. One example is the RolB/RolC glucosidase family, which is found only in Agrobacterium species and in plants. We identified the 0.5% of Pfam protein domain families that were most significantly over-represented in the plant-associated Proteobacteria with respect to the background frequencies in the whole set of available proteobacterial proteomes. These included guanylate cyclase, domains implicated in aromatic catabolism, cellulase and several domains of unknown function. We identified 459 unique domain architectures found in phylogenetically diverse plant pathogens and symbionts that were absent from non-pathogenic and non-symbiotic relatives. The vast majority of these were restricted to a single species or several closely related species and so their distributions could be better explained by phylogeny than by lifestyle. However, several architectures were found in two or more very distantly related phytobacteria but absent from non-plant-associated bacteria. Many of the proteins with these unique architectures are predicted to be secreted. In Pseudomonas syringae pathovar tomato, those genes encoding genes with novel domain architectures tended to have atypical GC contents and were adjacent to insertion sequence elements and phage-like sequences, suggesting acquisition by horizontal transfer. CONCLUSIONS: By identifying domains and architectures unique to plant pathogens and symbionts, we highlighted candidate proteins for involvement in plant-associated bacterial lifestyles. Given that characterisation of novel gene products in vivo and in vitro is time-consuming and expensive, this computational approach may be useful for reducing experimental search space. Furthermore we discuss the biological significance of novel proteins highlighted by this study in the context of plant-associated lifestyles

PolIVb influences RNA-directed DNA methylation independently of its role in siRNA biogenesis.

Comparative StudyJournal ArticleResearch Support, Non-U.S. Gov'tThis article contains supporting information online at www.pnas.org/cgi/content/full/0709632105/DC1. Freely available online through the PNAS open access option. © 2008 by The National Academy of Sciences of the USADNA-dependent RNA polymerase (Pol)IV in Arabidopsis exists in two isoforms (PolIVa and PolIVb), with NRPD1a and NRPD1b as their respective largest subunits. Both isoforms are implicated in production and activity of siRNAs and in RNA-directed DNA methylation (RdDM). Deep sequence analysis of siRNAs in WT Arabidopsis flowers and in nrpd1a and nrpd1b mutants identified >4,200 loci producing siRNAs in a PolIV-dependent manner, with PolIVb reinforcing siRNA production by PolIVa. Transposable element identity and pericentromeric localization are both features that predispose a locus for siRNA production via PolIV proteins and determine the extent to which siRNA production relies on PolIVb. Detailed analysis of DNA methylation at PolIV-dependent loci revealed unexpected deviations from the previously noted association of PolIVb-dependent siRNA production and RdDM. Notably, PolIVb functions independently in DNA methylation and siRNA generation. Additionally, we have uncovered siRNA-directed loss of DNA methylation, a process requiring both PolIV isoforms. From these findings, we infer that the role of PolIVb in siRNA production is secondary to a role in chromatin modification and is influenced by chromatin context

A comparison of Pfam and MEROPS: Two databases, one comprehensive, and one specialised.

BACKGROUND: We wished to compare two databases based on sequence similarity: one that aims to be comprehensive in its coverage of known sequences, and one that specialises in a relatively small subset of known sequences. One of the motivations behind this study was quality control. Pfam is a comprehensive collection of alignments and hidden Markov models representing families of proteins and domains. MEROPS is a catalogue and classification of enzymes with proteolytic activity (peptidases or proteases). These secondary databases are used by researchers worldwide, yet their contents are not peer reviewed. Therefore, we hoped that a systematic comparison of the contents of Pfam and MEROPS would highlight missing members and false-positives leading to improvements in quality of both databases. An additional reason for carrying out this study was to explore the extent of consensus in the definition of a protein family. RESULTS: About half (89 out of 174) of the peptidase families in MEROPS overlapped single Pfam families. A further 32 MEROPS families overlapped multiple Pfam families. Where possible, new Pfam families were built to represent most of the MEROPS families that did not overlap Pfam. When comparing the numbers of sequences found in the overlap between a MEROPS family and its corresponding Pfam family, in most cases the overlap was substantial (52 pairs of MEROPS and Pfam families had an intersection size of greater than 75% of the union) but there were some differences in the sets of sequences included in the MEROPS families versus the overlapping Pfam families. CONCLUSIONS: A number of the discrepancies between MEROPS families and their corresponding Pfam families arose from differences in the aims and philosophies of the two databases. Examination of some of the discrepancies highlighted additional members of families, which have subsequently been added in both Pfam and MEROPS. This has led to improvements in the quality of both databases. Overall there was a great deal of consensus between the databases in definitions of a protein family

Draft genome sequences of Phytophthora kernoviae and Phytophthora ramorum lineage EU2 from Scotland

Newly discovered Phytophthora species include invasive pathogens that threaten trees and shrubs. We present draft genome assemblies for three isolates of Phytophthora kernoviae and one isolate of the EU2 lineage of Phytophthora ramorum, collected from outbreak sites in Scotland

Multivariate analysis of morphological variation in enset (Ensete ventricosum (Welw.) Cheesman) reveals regional and clinal variation in germplasm from south and south western Ethiopia

Enset (Ensete ventricosum (Welw.) Cheesman) is cultivated by millions of people across Ethiopia in diverse agro-ecological and cultural settings, selecting for various agronomic traits. However, as for other underutilized crops, our understanding of the diversity and utilization of enset remains limited. This work sought to redress this limitation by estimating morphological diversity among enset accessions collected from major enset growing regions, including across altitudinal gradients. In total, landraces comprising 387 accessions originating from nine regions of Ethiopia were characterized using multivariate analysis of 15 quantitative traits. Cluster analysis grouped accessions in to five distinct classes with maximum number of accessions 338 in cluster (I) and minimum 1 in cluster (V). The clustering of accessions did not show grouping on the basis of region of origin. The first four principal components accounted for ~74% of the total variance. Linear discriminant analysis indicated that around 40.8% (160 accessions) and 45.2% (175 accession) of the studied accessions were correctly classified to their respective regions of origin altitude groups, respectively. The breadth of phenotypic differences in these 15 traits suggests significant degrees of genetic variation. These traits will be exploited to identify potential donors for future enset improvement efforts

A highly specific tool for identification of Xanthomonas vasicola pv. musacearum based on five Xvm-specific coding sequences

Xanthomonas vasicola pv. musacearum (Xvm) is a bacterial pathogen responsible for the economically important Xanthomonas wilt disease on banana and enset crops in Sub-Saharan Africa. Given that the symptoms are similar to those of other diseases, molecular diagnosis is essential to unambiguously identify this pathogen and distinguish it from closely related strains not pathogenic on these hosts. Currently, Xvm identification is based on polymerase chain reaction (PCR) with GspDm primers, targeting the gene encoding general secretory protein D. Experimental results and examination of genomic sequences revealed poor specificity of the GspDm PCR. Here, we present and validate five new Xvm-specific primers amplifying only Xvm strains