CEA, DSV, IBEB, Lab Biochim System Perturb, Bagnols-sur-Cèze, F-30207, France.
BMC Genomics. 2012 Feb 15;13:73. doi: 10.1186/1471-2164-13-73.
The 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.
A 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.
By 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.
基因组的结构和功能注释现在主要基于使用自动化管道系统获得的数据。准确结构注释的关键在于融合密切相关基因组之间的相似性，以及对基因组解释的生化证据。在这项工作中，我们应用高通量蛋白质基因组学方法对 Ruegeria pomeroyi 进行研究，Ruegeria pomeroyi 是 Roseobacter 群的一个成员，Roseobacter 群是海洋细菌中丰富的一个群体，作为整个群注释的种子。
在对一个六框架翻译基因组数据库进行超过 110 万 MS/MS 光谱搜索后，我们获得了大量来自 Ruegeria pomeroyi 的肽数据集。我们鉴定了 2006 个多肽，其中 34 个由先前未注释的开放阅读框 (ORF) 编码。从“一击必杀”的 ORF 池中，即只有一个由串联质谱检测到的肽指定的 ORF，我们可以在证明相应的 RNA 被转录后，确认另外五个新基因的可能存在。我们还鉴定了 486 个多肽中最 N 端的肽，其中 64 个最初被错误注释。
通过将这些重新注释扩展到迄今为止测序的其他 36 个 Roseobacter 分离株（20 个不同的属），我们提出了在该群中校正 1082 个同源基因的分配起始密码子。此外，我们还报告了在编码重要三羧酸循环决定因素的操纵子中存在新基因的情况，这似乎是一些 Roseobacter 基因组的特征。大量检测到它们的相应产物提出了它们功能的问题。它们的发现为细菌中孤儿蛋白的高表达依赖的蛋白质进化提供了一个可能的理论：它们假定的低效率可以通过更高的表达水平来平衡。我们的蛋白质基因组学分析将提高未来海洋细菌基因组注释的可靠性。
