Percentage Of Open Reading Frames Research Articles

Novel transaminases from thermophiles: from discovery to application.

SummaryTransaminases (TAs) are promising biocatalysts for chiral amine synthesis; however, only few thermophilic TAs have been described to date. In this work, a genome mining approach was taken to seek novel TAs from nine thermophilic microorganisms. TA sequences were identified from their respective genome sequences and their Pfam were predicted confirming that TAs class I–II are the most abundant (50%), followed by class III (26%), V (16%), IV (8%) and VI (1%). The percentage of open reading frames (ORFs) that are TAs ranges from 0.689% in Thermococcus litoralis to 0.424% in Sulfolobus solfataricus. A total of 94 putative TAs were successfully cloned and expressed into E. coli, showing mostly good expression levels when using a chemical chaperone media containing d‐sorbitol. Kinetic and end‐point colorimetric assays with different amino donors–acceptors confirmed TAs activity allowing for initial exploration of the substrate scope. Stereoselective and non‐stereoselective serine‐TAs were selected for the synthesis of hydroxypyruvate (HPA). Low HPA reaction yields were observed with four non‐stereoselective serine‐TAs, whilst two stereoselective serine‐TAs showed significantly higher yields. Coupling serine‐TA reactions to a transketolase to yield l‐erythrulose (Ery) substantially increased serine conversion into HPA. Combining both stereoselective serine‐TAs and transketolase using the inexpensive racemic D/L‐serine led to high Ery yield (82%). Thermal characterization of stereoselective serine‐TAs confirmed they have excellent thermostability up to 60°C and high optimum temperatures.

Re-assembly, quality evaluation, and annotation of 678 microbial eukaryotic reference transcriptomes.

Background De novo transcriptome assemblies are required prior to analyzing RNA sequencing data from a species without an existing reference genome or transcriptome. Despite the prevalence of transcriptomic studies, the effects of using different workflows, or “pipelines," on the resulting assemblies are poorly understood. Here, a pipeline was programmatically automated and used to assemble and annotate raw transcriptomic short-read data collected as part of the Marine Microbial Eukaryotic Transcriptome Sequencing Project. The resulting transcriptome assemblies were evaluated and compared against assemblies that were previously generated with a different pipeline developed by the National Center for Genome Research.ResultsNew transcriptome assemblies contained the majority of previous contigs as well as new content. On average, 7.8% of the annotated contigs in the new assemblies were novel gene names not found in the previous assemblies. Taxonomic trends were observed in the assembly metrics. Assemblies from the Dinoflagellata showed a higher number of contigs and unique k-mers than transcriptomes from other phyla, while assemblies from Ciliophora had a lower percentage of open reading frames compared to other phyla.ConclusionsGiven current bioinformatics approaches, there is no single “best” reference transcriptome for a particular set of raw data. As the optimum transcriptome is a moving target, improving (or not) with new tools and approaches, automated and programmable pipelines are invaluable for managing the computationally intensive tasks required for re-processing large sets of samples with revised pipelines and ensuring a common evaluation workflow is applied to all samples. Thus, re-assembling existing data with new tools using automated and programmable pipelines may yield more accurate identification of taxon-specific trends across samples in addition to novel and useful products for the community.

Genome variation in the symbiotic nitrogen-fixing bacteriumSinorhizobium meliloti

Differences in genome size and gene content are among the most important signatures of microbial adaptation and genome evolution. Here, we investigated the patterns of genome variation among 10 natural strains of the symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti, using pulse field gel electrophoresis (PFGE) and whole-genome microarray hybridizations. Our PFGE analysis showed a genome size range of 6.45-7.01 Mbp, with the greatest variation arising from the pSymA replicon, followed by pSymB; no size difference was evident among the chromosomes. Consistent with this pattern of size differences, 41.2% of open reading frames (ORFs) on pSymA were variably absent/present, followed by 12.7% on pSymB and 3.7% on the chromosome. However, the ORFs that were variably duplicated were more evenly distributed among the three replicons: 11.0%, 16.5%, and 15.3% of ORFs on pSymA, pSymB, and the chromosome, respectively. Among the 10 strains, the percentage of ORFs that were absent ranged from 1.51% to 6.35%, and the percentage of ORFs that were duplicated ranged from 0.27% to 8.56%. Our analyses showed that host plants, geographic origins, multilocus enzyme electrophoretic types, and replicon sizes had little influence on the distribution patterns of absent or duplicated ORFs. The proportions of ORFs that were either variably absent/present or variably duplicated differed greatly among the functional categories, for each of the three replicons as well as for the whole genome. Interestingly, we observed positive correlations among the three replicons in the number of absent ORFs as well as the number of duplicated ORFs, consistent with coordinated gene gains and losses in this important bacterium in nature.

Detecting the molecular scars of evolution in the Mycobacterium tuberculosis complex by analyzing interrupted coding sequences

BackgroundComputer-assisted analyses have shown that all bacterial genomes contain a small percentage of open reading frames with a frameshift or in-frame stop codon We report here a comparative analysis of these interrupted coding sequences (ICDSs) in six isolates of M. tuberculosis, two of M. bovis and one of M. africanum and question their phenotypic impact and evolutionary significance.ResultsICDSs were classified as "common to all strains" or "strain-specific". Common ICDSs are believed to result from mutations acquired before the divergence of the species, whereas strain-specific ICDSs were acquired after this divergence. Comparative analyses of these ICDSs therefore define the molecular signature of a particular strain, phylogenetic lineage or species, which may be useful for inferring phenotypic traits such as virulence and molecular relationships. For instance, in silico analysis of the W-Beijing lineage of M. tuberculosis, an emergent family involved in several outbreaks, is readily distinguishable from other phyla by its smaller number of common ICDSs, including at least one known to be associated with virulence. Our observation was confirmed through the sequencing analysis of ICDSs in a panel of 21 clinical M. tuberculosis strains. This analysis further illustrates the divergence of the W-Beijing lineage from other phyla in terms of the number of full-length ORFs not containing a frameshift. We further show that ICDS formation is not associated with the presence of a mutated promoter, and suggest that promoter extinction is not the main cause of pseudogene formation.ConclusionThe correlation between ICDSs, function and phenotypes could have important evolutionary implications. This study provides population geneticists with a list of targets, which could undergo selective pressure and thus alters relationships between the various lineages of M. tuberculosis strains and their host. This approach could be applied to any closely related bacterial strains or species for which several genome sequences are available.

Whole-genome analysis of transporters in the plant pathogen Xylella fastidiosa.

The transport systems of the first completely sequenced genome of a plant parasite, Xylella fastidiosa, were analyzed. In all, 209 proteins were classified here as constitutive members of transport families; thus, we have identified 69 new transporters in addition to the 140 previously annotated. The analysis lead to several hints on potential ways of controlling the disease it causes on citrus trees. An ADP:ATP translocator, previously found in intracellular parasites only, was found in X. fastidiosa. A P-type ATPase is missing-among the 24 completely sequenced eubacteria to date, only three (including X. fastidiosa) do not have a P-type ATPase, and they are all parasites transmitted by insect vectors. An incomplete phosphotransferase system (PTS) was found, without the permease subunits-we conjecture either that they are among the hypothetical proteins or that the PTS plays a solely metabolic regulatory role. We propose that the Ttg2 ABC system might be an import system eventually involved in glutamate import rather than a toluene exporter, as previously annotated. X. fastidiosa exhibits fewer proteins with > or =4 alpha-helical transmembrane spanners than any other completely sequenced prokaryote to date. X. fastidiosa has only 2.7% of all open reading frames identifiable as major transporters, which puts it as the eubacterium having the lowest percentage of open reading frames involved in transport, closer to two archaea, Methanococcus jannaschii (2.4%) and Methanobacterium thermoautotrophicum (2.4%).