Project description:We measured abundances of tRNAs by means of hydro-tRNA-seq (Gogakos et al., 2017), a method based on partial alkaline RNA hydrolysis that generates fragments suitable for sequencing, in the genome-reduced bacterium Mycoplasma pneumoniae.
Project description:Given the facilities for whole genome sequencing with next-generation sequencers, structural and functional gene annotation is now only based on automated prediction. However, errors in terms of gene structure are still frequently reported especially for the correct determination of initiation start codons. Here, we propose a strategy to enrich and detect protein N-termini by mass spectrometry in order to refine genome annotation. After selective protein N-termini derivatization using (N-Succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPPAc-OSu) as labeling reagent, protein digestion was performed with three proteases in parallel. TMPP-labeled N-terminal-most peptides were further resolved from internal peptides by the COmbined FRActional DIagonal Chromatography (COFRADIC) sorting methodology before analysis with tandem mass spectrometry. We refined the annotation of the genome of a model marine bacterium, Roseobacter denitrificans.
Project description: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 strains of the symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti. Using the sequenced strain Rm1021 as a reference, the genome size and gene content variations were analyzed among ten diverse natural strains, through pulse field gel electrophoresis (PFGE) and whole-genome microarray hybridizations. Our PFGE analysis showed a genome size range of 6.45-7.01Mbp, with the greatest variation arising from the pSymA replicon, followed by that of pSymB. No observable size difference was evident among the chromosomes. Consistent with this pattern of size differences, 41.2% of ORFs on pSymA were variably absent/present, followed by 12.7% on pSymB, and 3.7% on the chromosome. However, the percentages of ORFs that were variably duplicated were more evenly distributed among the three replicons, 11.0%, 16.5% and 15.3% respectively for ORFs on pSymA, pSymB and the chromosome. Among the 10 strains, the percentages of absent ORFs ranged from 1.51% to 6.35% and those of duplicated ORFs 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 their numbers of absent ORFs as well as the numbers of duplicated ORFs, consistent with coordinated gene gains/losses in this important bacterium in nature. microarray:Sm6kOligo
