Genome-wide analysis of myxobacterial two-component systems:Genome relatedness and evolutionary changes

BACKGROUND: Two-component systems (TCSs) are abundant prokaryotic signaling pathways, whose evolution is of particular importance because of their role in bacterial pathogenicity. Comparative genomics can provide important insights into the evolution of these genes, but inferences are dependent on the relatedness of the compared genomes. This study investigated the relationship between evolutionary distance and TCS evolution in myxobacterial genomes, of which there are several sequenced examples, of varying relatedness, and which encode large numbers of TCSs. METHODS: Myxobacterial TCS gene sets were compared, orthologues defined, and changes in TCS properties such as gene organisation, domain architecture and size identified. RESULTS: Genome relatedness/evolutionary distance was found to have a large effect on the apparent frequency of evolutionary events affecting TCS genes, but not on the relative dominance of different types of mutations. Large (≥1 gene) indels were the most common changes, often giving rise to gene organisation changes. Smaller indels were also common, sometimes changing domain architecture, and/or leading to pseudogene formation. Individuality of myxobacterial TCS gene sets seems primarily due to lineage specific gene loss. However, there is also evidence of extensive acquisition of genes by lateral transfer, with gene duplication also creating new TCS genes. CONCLUSIONS: This study provides catalogues of myxobacterial TCS gene sets and their orthology relationships, benchmarked against genome relatedness. It also provides insights into the relationship between evolutionary distance and the inference of TCS estudies of TCS evolution beyond the myxobacteriavolution, which may be important for studies of TCS evolutiThe online version of this articleon beyond the myxobacteria. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-015-2018-y) contains supplementary material, which is available to authorized users

Feedback and molecular interactions in the process of light-induced carotenogenesis in Myxococcus xanthus

Myxococcus xanthus is a soil-dwelling bacterium which produces carotenoids upon irradiation with blue light. Genetic analysis has allowed elucidation of transduction of the light signal to the carotenogenic machinery within the cell. The primary element within the carotenogenic regulon is the genetic switch manifested by CarR and CarQ. CarR is an integral membrane protein which binds to the sigma factor CarQ and holds it in an inactive state at the cell membrane. Illumination of the cell with blue light excites the photosensitiser protoporphyrin IX (PPIX) within the bacterial membrane, which then excites molecular oxygen to the excited singlet state. Both singlet oxygen and excited triplet state PPIX can cause large amounts of cellular damage. Carotenoids prevent this damage by absorbing the excess energy from these excited species and dissipating it harmlessly as heat. The presence of singlet oxygen within the bacterial membrane causes the inactivation/degradation of CarR. Removal of CarR releases CarQ from the membrane enabling it to mediate transcription from various promoters. CarQ causes transcription of the crtI gene and of the carQRS operon which produces further CarQ and CarS. CarS causes de-repression of the crtEBDC cluster. The carotenogenic enzymes encoded by crtI and the crtEBDC cluster catalyse the production of carotenoids which quench the initial signalling molecules, singlet oxygen and triplet PPIX. This causes down-regulation of the regulon as a whole as CarR is no longer degraded and once again carries nascent CarQ to the membrane in an inactive state. The negative feedback loop described above is an important consideration when assessing mutants which produce carotenoids either constitutively (Car c phenotype), or under no conditions (Car- phenotype). This work investigates the consequences of Carc and Car- mutations on the activity of promoters within the Car regulon in order to clarify the roles of various genetic loci. It is demonstrated that CarA has no regulatory role in expression of crtI or carQRS and that the expression of crtI has no regulatory consequences. Sequencing downstream of crtI revealed a novel gene gufB (gene of unknown function B) which has homologues of no known function. The critical event in the activation of the carotenogenic system is expression of the carQRS operon allowed by the release of CarQ from its complex with CarR at the membrane. Attempts were made to extract information about the interaction of CarQ with its cognate promoter at carQRS through a variety of in vivo and in vitro molecular and genetic techniques. Site-directed mutations within pcarQRS were assessed in vivo through the use of lacZ transcriptional fusions, enabling identification of important regions within the carQRS promoter. In vitro experiments provided information about the possibility of using molecular methods to assess interactions between CarQ and the pcarQRS promoter

Myxobacterial Predation:A Standardised Lawn Predation Assay Highlights Strains with Unusually Efficient Predatory Activity

Myxobacteria prey upon a broad range of microorganisms. Lawn assays are commonly used to quantify myxobacterial predation—myxobacterial suspensions are spotted onto prey lawns, and monitored via spot expansion. The diversity in motility behaviours of myxobacterial strains and differing assay protocols in myxobacteriology laboratories led us to develop a highly-specified assay, which was applied to 28 myxobacterial strains preying on seven phytopathogenic prey species. Generally, prey organisms showed no qualitative differences in their susceptibility/resistance to myxobacterial predation. For most myxobacteria, prey did not stimulate, and in ~50% of cases actively hindered colony expansion. Only ~25% of predator/prey strain combinations exhibited greater colony expansion than in the absence of nutrients. The activity of predatory strains against different prey correlated, implying effective predators may have relatively non-specific predation mechanisms (e.g., broad specificity proteases/lipases), but no correlation was observed between predatory activity and phylogeny. Predation on dead (but intact) or lysed prey cells gave greater colony expansion than on live prey. Occasional strains grew substantially faster on dead compared to lysed cells, or vice-versa. Such differences in accessing nutrients from live, dead and lysed cells indicates there are strain-specific differences in the efficiencies/machineries of prey killing and nutrient acquisition, which has important implications for the ecology of myxobacterial predators and their prey

P2RP:a Web-based framework for the identification and analysis of regulatory proteins in prokaryotic genomes

BACKGROUND: Regulatory proteins (RPs) such as transcription factors (TFs) and two-component system (TCS) proteins control how prokaryotic cells respond to changes in their external and/or internal state. Identification and annotation of TFs and TCSs is non-trivial, and between-genome comparisons are often confounded by different standards in annotation. There is a need for user-friendly, fast and convenient tools to allow researchers to overcome the inherent variability in annotation between genome sequences. RESULTS: We have developed the web-server P2RP (Predicted Prokaryotic Regulatory Proteins), which enables users to identify and annotate TFs and TCS proteins within their sequences of interest. Users can input amino acid or genomic DNA sequences, and predicted proteins therein are scanned for the possession of DNA-binding domains and/or TCS domains. RPs identified in this manner are categorised into families, unambiguously annotated, and a detailed description of their features generated, using an integrated software pipeline. P2RP results can then be outputted in user-specified formats. CONCLUSION: Biologists have an increasing need for fast and intuitively usable tools, which is why P2RP has been developed as an interactive system. As well as assisting experimental biologists to interrogate novel sequence data, it is hoped that P2RP will be built into genome annotation pipelines and re-annotation processes, to increase the consistency of RP annotation in public genomic sequences. P2RP is the first publicly available tool for predicting and analysing RP proteins in users’ sequences. The server is freely available and can be accessed along with documentation at http://www.p2rp.org

Within-species variation in OMV cargo proteins:The Myxococcus xanthus OMV pan-proteome

Extracellular membrane vesicles are produced by all domains of life (bacteria, archaea and eukaryotes). Bacterial extracellular vesicles (outer membrane vesicles or OMVs) are produced by outer membrane blebbing, and contain proteins, nucleic acids, virulence factors, lipids and metabolites. OMV functions depend on their internal composition, therefore understanding the proteome of OMVs, and how it varies between organisms, is imperative. Here, we report a comparative proteomic profiling of OMVs from strains of Myxococcus xanthus, a predatory species of Gram-negative myxobacteria whose secretions include secondary metabolites and hydrolytic enzymes, thought to be involved in prey lysis. Ten strains were chosen for study, of which seven had genome sequences available. The remaining three strains were genome sequenced allowing definition of the core and accessory genes and genome-derived proteins found within the pan-genome and pan-proteome respectively. OMVs were isolated from each strain and proteins identified using mass spectrometry. The M. xanthus OMV pan-proteome was found to contain tens of ‘core’ and hundreds of ‘accessory’ proteins. Properties of the OMV pan-proteome were compared with those of the pan-proteome deduced from the M. xanthus pan-genome. On average, 80% of ‘core’ OMV proteins are encoded by genes of the core genome, yet the OMV proteomes of individual strains contain subsets of core genome-derived proteins which only partially overlap. In addition, the distribution of characteristics of vesicle proteins does not correlate with the genome-derived proteome characteristic distribution. We hypothesize that M. xanthus cells package a personalized subset of proteins whose availability is only partially dictated by the presence/absence of encoding genes within the genome