Evolution of plant RNA polymerase IV/V genes: evidence of subneofunctionalization of duplicated NRPD2/NRPE2-like paralogs in Viola (Violaceae)

<p>Abstract</p> <p>Background</p> <p>DNA-dependent RNA polymerase IV and V (Pol IV and V) are multi-subunit enzymes occurring in plants. The origin of Pol V, specific to angiosperms, from Pol IV, which is present in all land plants, is linked to the duplication of the gene encoding the largest subunit and the subsequent subneofunctionalization of the two paralogs (<it>NRPD1 </it>and <it>NRPE1</it>). Additional duplication of the second-largest subunit, <it>NRPD2/NRPE2</it>, has happened independently in at least some eudicot lineages, but its paralogs are often subject to concerted evolution and gene death and little is known about their evolution nor their affinity with Pol IV and Pol V.</p> <p>Results</p> <p>We sequenced a ~1500 bp <it>NRPD2/E2</it>-like fragment from 18 <it>Viola </it>species, mostly paleopolyploids, and 6 non-<it>Viola </it>Violaceae species. Incongruence between the <it>NRPD2/E2</it>-like gene phylogeny and species phylogeny indicates a first duplication of <it>NRPD2 </it>relatively basally in Violaceae, with subsequent sorting of paralogs in the descendants, followed by a second duplication in the common ancestor of <it>Viola </it>and <it>Allexis</it>. In <it>Viola</it>, the mutation pattern suggested (sub-) neofunctionalization of the two <it>NRPD2/E2</it>-like paralogs, <it>NRPD2/E2-a </it>and <it>NRPD2/E2-b</it>. The <it>d</it>_<it>N</it>/<it>d</it>_{<it>S </it>}ratios indicated that a 54 bp region exerted strong positive selection for both paralogs immediately following duplication. This 54 bp region encodes a domain that is involved in the binding of the Nrpd2 subunit with other Pol IV/V subunits, and may be important for correct recognition of subunits specific to Pol IV and Pol V. Across all <it>Viola </it>taxa 73 <it>NRPD2/E2</it>-like sequences were obtained, of which 23 (32%) were putative pseudogenes - all occurring in polyploids. The <it>NRPD2 </it>duplication was conserved in all lineages except the diploid MELVIO clade, in which <it>NRPD2/E2-b </it>was lost, and its allopolyploid derivates from hybridization with the CHAM clade, section <it>Viola </it>and section <it>Melanium</it>, in which <it>NRPD2/E2-a </it>occurred in multiple copies while <it>NRPD2/E2-b </it>paralogs were either absent or pseudogenized.</p> <p>Conclusions</p> <p>Following the relatively recent split of Pol IV and Pol V, our data indicate that these two multi-subunit enzymes are still in the process of specialization and each acquiring fully subfunctionalized copies of their subunit genes. Even after specialization, the <it>NRPD2/E2</it>-like paralogs are prone to pseudogenization and gene conversion and <it>NRPD2 </it>and <it>NRPE2 </it>copy number is a highly dynamic process modulated by allopolyploidy and gene death.</p

Recombination and selectional forces in cyanopeptolin NRPS operons from highly similar, but geographically remote Planktothrix strains

<p>Abstract</p> <p>Background</p> <p>Cyanopeptolins are nonribosomally produced heptapetides showing a highly variable composition. The cyanopeptolin synthetase operon has previously been investigated in three strains from the genera <it>Microcystis</it>, <it>Planktothrix </it>and <it>Anabaena</it>. Cyanopeptolins are displaying protease inhibitor activity, but the biological function(s) is (are) unknown. Cyanopeptolin gene cluster variability and biological functions of the peptide variants are likely to be interconnected.</p> <p>Results</p> <p>We have investigated two cyanopeptolin gene clusters from highly similar, but geographically remote strains of the same genus. Sequencing of a nonribosomal peptide synthetase (NRPS) cyanopeptolin gene cluster from the Japanese strain <it>Planktothrix </it>NIES 205 (205-<it>oci</it>), showed the 30 kb gene cluster to be highly similar to the <it>oci </it>gene cluster previously described in <it>Planktothrix </it>NIVA CYA 116, isolated in Norway. Both operons contained seven NRPS modules, a sulfotransferase (S) and a glyceric acid loading (GA)-domain. Sequence analyses showed a high degree of conservation, except for the presence of an epimerase domain in NIES 205 and the regions around the epimerase, showing high substitution rates and Ka/Ks values above 1. The two strains produce almost identical cyanopeptolins, cyanopeptolin-1138 and oscillapeptin E respectively, but with slight differences regarding the production of minor cyanopeptolin variants. These variants may be the result of relaxed adenylation (A)-domain specificity in the nonribosomal enzyme complex. Other genetic markers (16S rRNA, <it>ntc</it>A and the phycocyanin <it>cpc</it>BA spacer) were identical, supporting that these geographically separated <it>Planktothrix </it>strains are closely related.</p> <p>Conclusion</p> <p>A horizontal gene transfer event resulting in exchange of a whole module-encoding region was observed. Nucleotide statistics indicate that both purifying selection and positive selection forces are operating on the gene cluster. The positive selection forces are acting within and around the epimerase insertion while purifying selection conserves the remaining (major) part of the gene cluster. The presence of an epimerase in the gene cluster is in line with the D-configuration of Htyr, determined experimentally in oscillapeptin E in a previous study.</p

Cross-species amplification of 36 cyprinid microsatellite loci in Phoxinus phoxinus (L.) and Scardinius erythrophthalmus (L.)

<p>Abstract</p> <p>Background</p> <p>To conduct phylogeographic or population genetic studies, an adequate number of DNA markers for the focal species are required. Due to severe unavailability of genotype markers of any kind for the species Eurasian minnow (<it>Phoxinus phoxinus </it>L.) and rudd (<it>Scardinius erythrophthalmus </it>L.), we set out to attempt cross-amplification of a set of microsatellite loci from related species.</p> <p>Findings</p> <p>We tested 36 cyprinid microsatellite loci for cross-species amplification in minnow and rudd. Fifteen species-locus combinations produced amplifications in minnow, seven being polymorphic, while 18 combinations amplified in rudd, nine of these being polymorphic.</p> <p>Conclusions</p> <p>The positive cross-species amplifications present potential contributions to the establishment of genetic marker sets for population genetics studies of the two focal species.</p

Telonemia-specific environmental 18S rDNA PCR reveals unknown diversity and multiple marine-freshwater colonizations

<p>Abstract</p> <p>Background</p> <p>Recent surveys of eukaryote 18S rDNA diversity in marine habitats have uncovered worldwide distribution of the heterotrophic eukaryote phylum Telonemia. Here we investigate the diversity and geographic distribution of Telonemia sequences by in-depth sequencing of several new 18S rDNA clone libraries from both marine and freshwater sites by using a Telonemia-specific PCR strategy.</p> <p>Results</p> <p>In contrast to earlier studies that have employed eukaryote-wide PCR design, we identified a large and unknown diversity of phylotypes and the first rigorous evidence for several freshwater species, altogether comprising 91 unique sequences. Phylogenies of these and publicly available sequences showed 20 statistically supported sub-clades as well as several solitary phylotypes with no clear phylogenetic affiliation. Most of these sub-clades were composed of phylotypes from different geographic regions.</p> <p>Conclusions</p> <p>By using specific PCR primers we reveal a much larger diversity of Telonemia from environmental samples than previously uncovered by eukaryote-wide primers. The new data substantially diminish the geographic structuring of clades identified in earlier studies. Nevertheless, since these clades comprise several distinct phylotypes we cannot exclude endemicity at species level. We identified two freshwater clades and a few solitary phylotypes, implying that Telonemia have colonized freshwater habitats and adapted to the different environmental and ecological conditions at independent occasions.</p

Metagenomic and geochemical characterization of pockmarked sediments overlaying the Troll petroleum reservoir in the North Sea

Background Pockmarks (depressions in the seabed) have been discovered throughout the world’s oceans and are often related to hydrocarbon seepage. Although high concentrations of pockmarks are present in the seabed overlaying the Troll oil and gas reservoir in the northern North Sea, geological surveys have not detected hydrocarbon seepage in this area at the present time. In this study we have used metagenomics to characterize the prokaryotic communities inhabiting the surface sediments in the Troll area in relation to geochemical parameters, particularly related to hydrocarbon presence. We also investigated the possibility of increased potential for methane oxidation related to the pockmarks. Five metagenomes from pockmarks and plain seabed sediments were sequenced by pyrosequencing (Roche/454) technology. In addition, two metagenomes from seabed sediments geologically unlikely to be influenced by hydrocarbon seepage (the Oslofjord) were included. The taxonomic distribution and metabolic potential of the metagenomes were analyzed by multivariate analysis and statistical comparisons to reveal variation within and between the two sampling areas. Results The main difference identified between the two sampling areas was an overabundance of predominantly autotrophic nitrifiers, especially Nitrosopumilus, and oligotrophic marine Gammaproteobacteria in the Troll metagenomes compared to the Oslofjord. Increased potential for degradation of hydrocarbons, especially aromatic hydrocarbons, was detected in two of the Troll samples: one pockmark sample and one from the plain seabed. Although presence of methanotrophic organisms was indicated in all samples, no overabundance in pockmark samples compared to the Oslofjord samples supports no, or only low level, methane seepage in the Troll pockmarks at the present time. Conclusions Given the relatively low content of total organic carbon and great depths of hydrocarbon containing sediments in the Troll area, it is possible that at least part of the carbon source available for the predominantly autotrophic nitrifiers thriving in this area originates from sequential prokaryotic degradation and oxidation of hydrocarbons to CO2. By turning CO2 back into organic carbon this subcommunity could play an important environmental role in these dark oligotrophic sediments. The oxidation of ammonia to nitrite and nitrate in this process could further increase the supply of terminal electron acceptors for hydrocarbon degradation

A genome-wide analysis of nonribosomal peptide synthetase gene clusters and their peptides in a Planktothrix rubescens strain

<p>Abstract</p> <p>Background</p> <p>Cyanobacteria often produce several different oligopeptides, with unknown biological functions, by nonribosomal peptide synthetases (NRPS). Although some cyanobacterial NRPS gene cluster types are well described, the entire NRPS genomic content within a single cyanobacterial strain has never been investigated. Here we have combined a genome-wide analysis using massive parallel pyrosequencing ("454") and mass spectrometry screening of oligopeptides produced in the strain <it>Planktothrix rubescens </it>NIVA CYA 98 in order to identify all putative gene clusters for oligopeptides.</p> <p>Results</p> <p>Thirteen types of oligopeptides were uncovered by mass spectrometry (MS) analyses. Microcystin, cyanopeptolin and aeruginosin synthetases, highly similar to already characterized NRPS, were present in the genome. Two novel NRPS gene clusters were associated with production of anabaenopeptins and microginins, respectively. Sequence-depth of the genome and real-time PCR data revealed three copies of the microginin gene cluster. Since NRPS gene cluster candidates for microviridin and oscillatorin synthesis could not be found, putative (gene encoded) precursor peptide sequences to microviridin and oscillatorin were found in the genes <it>mdn</it>A and <it>osc</it>A, respectively. The genes flanking the microviridin and oscillatorin precursor genes encode putative modifying enzymes of the precursor oligopeptides. We therefore propose ribosomal pathways involving modifications and cyclisation for microviridin and oscillatorin. The microviridin, anabaenopeptin and cyanopeptolin gene clusters are situated in close proximity to each other, constituting an oligopeptide island.</p> <p>Conclusion</p> <p>Altogether seven nonribosomal peptide synthetase (NRPS) gene clusters and two gene clusters putatively encoding ribosomal oligopeptide biosynthetic pathways were revealed. Our results demonstrate that whole genome shotgun sequencing combined with MS-directed determination of oligopeptides successfully can identify NRPS gene clusters and the corresponding oligopeptides. The analyses suggest independent evolution of all NRPS gene clusters as functional units. Our data indicate that the <it>Planktothrix </it>genome displays evolution of dual pathways (NRPS and ribosomal) for production of oligopeptides in order to maximize the diversity of oligopeptides with similar but functional discrete bioactivities.</p

Assessing diversity of the female urine microbiota by high throughput sequencing of 16S rDNA amplicons

<p>Abstract</p> <p>Background</p> <p>Urine within the urinary tract is commonly regarded as "sterile" in cultivation terms. Here, we present a comprehensive in-depth study of bacterial 16S rDNA sequences associated with urine from healthy females by means of culture-independent high-throughput sequencing techniques.</p> <p>Results</p> <p>Sequencing of the V1V2 and V6 regions of the 16S ribosomal RNA gene using the 454 GS FLX system was performed to characterize the possible bacterial composition in 8 culture-negative (<100,000 CFU/ml) healthy female urine specimens. Sequences were compared to 16S rRNA databases and showed significant diversity, with the predominant genera detected being <it>Lactobacillus</it>, <it>Prevotella </it>and <it>Gardnerella</it>. The bacterial profiles in the female urine samples studied were complex; considerable variation between individuals was observed and a common microbial signature was not evident. Notably, a significant amount of sequences belonging to bacteria with a known pathogenic potential was observed. The number of operational taxonomic units (OTUs) for individual samples varied substantially and was in the range of 20 - 500.</p> <p>Conclusions</p> <p>Normal female urine displays a noticeable and variable bacterial 16S rDNA sequence richness, which includes fastidious and anaerobic bacteria previously shown to be associated with female urogenital pathology.</p

A metagenomic study of methanotrophic microorganisms in Coal Oil Point seep sediments

Background Methane oxidizing prokaryotes in marine sediments are believed to function as a methane filter reducing the oceanic contribution to the global methane emission. In the anoxic parts of the sediments, oxidation of methane is accomplished by anaerobic methanotrophic archaea (ANME) living in syntrophy with sulphate reducing bacteria. This anaerobic oxidation of methane is assumed to be a coupling of reversed methanogenesis and dissimilatory sulphate reduction. Where oxygen is available aerobic methanotrophs take part in methane oxidation. In this study, we used metagenomics to characterize the taxonomic and metabolic potential for methane oxidation at the Tonya seep in the Coal Oil Point area, California. Two metagenomes from different sediment depth horizons (0-4 cm and 10-15 cm below sea floor) were sequenced by 454 technology. The metagenomes were analysed to characterize the distribution of aerobic and anaerobic methanotrophic taxa at the two sediment depths. To gain insight into the metabolic potential the metagenomes were searched for marker genes associated with methane oxidation. Results Blast searches followed by taxonomic binning in MEGAN revealed aerobic methanotrophs of the genus Methylococcus to be overrepresented in the 0-4 cm metagenome compared to the 10-15 cm metagenome. In the 10-15 cm metagenome, ANME of the ANME-1 clade, were identified as the most abundant methanotrophic taxon with 8.6% of the reads. Searches for particulate methane monooxygenase (pmoA) and methyl-coenzyme M reductase (mcrA), marker genes for aerobic and anaerobic oxidation of methane respectively, identified pmoA in the 0-4 cm metagenome as Methylococcaceae related. The mcrA reads from the 10-15 cm horizon were all classified as originating from the ANME-1 clade. Conclusions Most of the taxa detected were present in both metagenomes and differences in community structure and corresponding metabolic potential between the two samples were mainly due to abundance differences. The results suggests that the Tonya Seep sediment is a robust methane filter, where taxa presently dominating this process could be replaced by less abundant methanotrophic taxa in case of changed environmental conditions

Evidence for positive selection acting on microcystin synthetase adenylation domains in three cyanobacterial genera

<p>Abstract</p> <p>Background</p> <p>Cyanobacteria produce a wealth of secondary metabolites, including the group of small cyclic heptapeptide hepatotoxins that constitutes the microcystin family. The enzyme complex that directs the biosynthesis of microcystin is encoded in a single large gene cluster (<it>mcy</it>). <it>mcy </it>genes have a widespread distribution among cyanobacteria and are likely to have an ancient origin. The notable diversity within some of the Mcy modules is generated through various recombination events including horizontal gene transfer.</p> <p>Results</p> <p>A comparative analysis of the adenylation domains from the first module of McyB (McyB1) and McyC in the microcystin synthetase complex was performed on a large number of microcystin-producing strains from the <it>Anabaena</it>, <it>Microcystis </it>and <it>Planktothrix </it>genera. We found no decisive evidence for recombination between strains from different genera. However, we detected frequent recombination events in the <it>mcyB </it>and <it>mcyC </it>genes between strains within the same genus. Frequent interdomain recombination events were also observed between <it>mcyB </it>and <it>mcyC </it>sequences in <it>Anabaena </it>and <it>Microcystis</it>. Recombination and mutation rate ratios suggest that the diversification of <it>mcyB </it>and <it>mcyC </it>genes is driven by recombination events as well as point mutations in all three genera. Sequence analysis suggests that generally the adenylation domains of the first domain of McyB and McyC are under purifying selection. However, we found clear evidence for positive selection acting on a number of amino acid residues within these adenylation domains. These include residues important for active site selectivity of the adenylation domain, strongly suggesting selection for novel microcystin variants.</p> <p>Conclusion</p> <p>We provide the first clear evidence for positive selection acting on amino acid residues involved directly in the recognition and activation of amino acids incorporated into microcystin, indicating that the microcystin complement of a given strain may influence the ability of a particular strain to interact with its environment.</p

A phylogenetic mosaic plastid proteome and unusual plastid-targeting signals in the green-colored dinoflagellate Lepidodinium chlorophorum

Background Plastid replacements through secondary endosymbioses include massive transfer of genes from the endosymbiont to the host nucleus and require a new targeting system to enable transport of the plastid-targeted proteins across 3-4 plastid membranes. The dinoflagellates are the only eukaryotic lineage that has been shown to have undergone several plastid replacement events, and this group is thus highly relevant for studying the processes involved in plastid evolution. In this study, we analyzed the phylogenetic origin and N-terminal extensions of plastid-targeted proteins from Lepidodinium chlorophorum, a member of the only dinoflagellate genus that harbors a green secondary plastid rather than the red algal-derived, peridinin-containing plastid usually found in photosynthetic dinoflagellates. Results We sequenced 4,746 randomly picked clones from a L. chlorophorum cDNA library. 22 of the assembled genes were identified as genes encoding proteins functioning in plastids. Some of these were of green algal origin. This confirms that genes have been transferred from the plastid to the host nucleus of L. chlorophorum and indicates that the plastid is fully integrated as an organelle in the host. Other nuclear-encoded plastid-targeted protein genes, however, are clearly not of green algal origin, but have been derived from a number of different algal groups, including dinoflagellates, streptophytes, heterokonts, and red algae. The characteristics of N-terminal plastid-targeting peptides of all of these genes are substantially different from those found in peridinin-containing dinoflagellates and green algae. Conclusions L. chlorophorum expresses plastid-targeted proteins with a range of different origins, which probably arose through endosymbiotic gene transfer (EGT) and horizontal gene transfer (HGT). The N-terminal extension of the genes is different from the extensions found in green alga and other dinoflagellates (peridinin- and haptophyte plastids). These modifications have likely enabled the mosaic proteome of L. chlorophorum