High-throughput proteogenomics of Ruegeria pomeroyi: seeding a better genomic annotation for the whole marine Roseobacter clade

Abstract

BackgroundThe structural and functional annotation of genomes is now heavily based on data obtained using automated pipeline systems. The key for an accurate structural annotation consists of blending similarities between closely related genomes with biochemical evidence of the genome interpretation. In this work we applied high-throughput proteogenomics to Ruegeria pomeroyi, a member of the Roseobacter clade, an abundant group of marine bacteria, as a seed for the annotation of the whole clade.ResultsA large dataset of peptides from R. pomeroyi was obtained after searching over 1.1 million MS/MS spectra against a six-frame translated genome database. We identified 2006 polypeptides, of which thirty-four were encoded by open reading frames (ORFs) that had not previously been annotated. From the pool of 'one-hit-wonders', i.e. those ORFs specified by only one peptide detected by tandem mass spectrometry, we could confirm the probable existence of five additional new genes after proving that the corresponding RNAs were transcribed. We also identified the most-N-terminal peptide of 486 polypeptides, of which sixty-four had originally been wrongly annotated.ConclusionsBy extending these re-annotations to the other thirty-six Roseobacter isolates sequenced to date (twenty different genera), we propose the correction of the assigned start codons of 1082 homologous genes in the clade. In addition, we also report the presence of novel genes within operons encoding determinants of the important tricarboxylic acid cycle, a feature that seems to be characteristic of some Roseobacter genomes. The detection of their corresponding products in large amounts raises the question of their function. Their discoveries point to a possible theory for protein evolution that will rely on high expression of orphans in bacteria: their putative poor efficiency could be counterbalanced by a higher level of expression. Our proteogenomic analysis will increase the reliability of the future annotation of marine bacterial genomes.

Proteogenomics of Gammarus fossarum to Document the Reproductive System of Amphipods

“…Data import filter options were set to 400 (minimum mass), 5000 (maximum mass), 0 (grouping tolerance), 0 (intermediate scans), and 1000 (threshold), as described previously by Christie-Oleza et al (30). MS/MS spectra were assigned to peptide sequences with the Mascot Daemon 2.3.2 search engine (Matrix Science) against the NCBInr protein sequence database or the GFOSS database.…”

Proteogenomic Analysis of Bradyrhizobium japonicum USDA110 Using Genosuite, an Automated Multi-algorithmic Pipeline

“…The operon has been shown to be under regulation of oxygenresponsive transcriptional activator protein NifA (38) and is Novel Proteins and Comparative Genomics-We extended newly identified annotations of B. japonicum in related rhizobial genomes based on orthology of NPCRs. Ortho-proteogenomic strategy was first applied on genus Mycobacterium by Gallien et al (1) and recently adopted by Christie-Oleza et al for annotating Roseobacter clad (10). For this analysis we included all the sequenced genomes in Bradyrhizobiaceae family along with Mesorhizobium loti, Sinorhizobium meliloti, and Rhizobium leguminosarum because these are widely studied rhizobial genomes.…”

N-Terminal-oriented Proteogenomics of the Marine Bacterium Roseobacter Denitrificans Och114 using N-Succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP) Labeling and Diagonal Chromatography

“…The error rate depends on the primary annotation system, but also on the organism, as reported for Halobacterium salinarum and Natromonas pharaonis (24), Deinococcus deserti (21), and Ruegeria pomeroyi (18), where the error rate is estimated above 10%. Identification of a correct translational start site is essential for the genetic and biochemical analysis of a protein because errors can seriously impact subsequent biological studies.…”

“…Because genome annotation is now fully automated, the need for accurate annotation for model organisms with experimental data is crucial. Many projects related to genome re-annotation of microorganisms with the help of proteomics have been recently reported, such as for Mycoplasma pneumoniae (9), Rhodopseudomonas palustris (10), Shewanella oneidensis (11), Thermococcus gammatolerans (12), Deinococcus deserti (13), Salmonella thyphimurium (14), Mycobacterium tuberculosis (15,16), Shigella flexneri (17), Ruegeria pomeroyi (18), and Candida glabrata (19), as well as for higher organisms such as Anopheles gambiae (20) and Arabidopsis thaliana (4,5).…”