Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, Hawaii, United States of America.
PLoS One. 2011 Apr 21;6(4):e18496. doi: 10.1371/journal.pone.0018496.
A DNA marker that distinguishes plant associated bacteria at the species level and below was derived by comparing six sequenced genomes of Xanthomonas, a genus that contains many important phytopathogens. This DNA marker comprises a portion of the dnaA replication initiation factor (RIF). Unlike the rRNA genes, dnaA is a single copy gene in the vast majority of sequenced bacterial genomes, and amplification of RIF requires genus-specific primers. In silico analysis revealed that RIF has equal or greater ability to differentiate closely related species of Xanthomonas than the widely used ribosomal intergenic spacer region (ITS). Furthermore, in a set of 263 Xanthomonas, Ralstonia and Clavibacter strains, the RIF marker was directly sequenced in both directions with a success rate approximately 16% higher than that for ITS. RIF frameworks for Xanthomonas, Ralstonia and Clavibacter were constructed using 682 reference strains representing different species, subspecies, pathovars, races, hosts and geographic regions, and contain a total of 109 different RIF sequences. RIF sequences showed subspecific groupings but did not place strains of X. campestris or X. axonopodis into currently named pathovars nor R. solanacearum strains into their respective races, confirming previous conclusions that pathovar and race designations do not necessarily reflect genetic relationships. The RIF marker also was sequenced for 24 reference strains from three genera in the Enterobacteriaceae: Pectobacterium, Pantoea and Dickeya. RIF sequences of 70 previously uncharacterized strains of Ralstonia, Clavibacter, Pectobacterium and Dickeya matched, or were similar to, those of known reference strains, illustrating the utility of the frameworks to classify bacteria below the species level and rapidly match unknown isolates to reference strains. The RIF sequence frameworks are available at the online RIF database, RIFdb, and can be queried for diagnostic purposes with RIF sequences obtained from unknown strains in both chromatogram and FASTA format.
一个能够区分植物相关细菌种及种下水平的 DNA 标记是通过比较 6 个黄单胞菌属（Xanthomonas）的测序基因组得到的，该属包含许多重要的植物病原菌。这个 DNA 标记由 DNAA 复制起始因子（RIF）的一部分组成。与 rRNA 基因不同，在绝大多数测序的细菌基因组中，dnaA 是一个单拷贝基因，而 RIF 的扩增需要属特异性引物。计算机分析显示，RIF 能够区分密切相关的黄单胞菌属种的能力与广泛使用的核糖体基因间区（ITS）相当，甚至更强。此外，在一组 263 株黄单胞菌、雷尔氏菌属和棒形杆菌属菌株中，RIF 标记的直接测序成功率比 ITS 高约 16%。使用代表不同种、亚种、致病变种、小种、宿主和地理区域的 682 个参考菌株构建了黄单胞菌属、雷尔氏菌属和棒形杆菌属的 RIF 框架，共包含 109 个不同的 RIF 序列。RIF 序列显示出亚种分组，但并未将野油菜黄单胞菌或丁香假单胞菌的菌株归入现有命名的致病变种，也未将茄科雷尔氏菌属的菌株归入各自的小种，这证实了先前的结论，即致病变种和小种的命名不一定反映遗传关系。还对肠杆菌科的 3 个属（果胶杆菌属、泛菌属和迪凯氏菌属）的 24 个参考菌株进行了 RIF 测序。70 个先前未鉴定的雷尔氏菌属、棒形杆菌属、果胶杆菌属和迪凯氏菌属菌株的 RIF 序列与已知参考菌株的序列匹配或相似，说明了该框架用于在种以下水平分类细菌并快速将未知分离株与参考菌株匹配的实用性。RIF 序列框架可在在线 RIF 数据库 RIFdb 中获得，并可用于通过未知菌株获得的 RIF 序列在图谱和 FASTA 格式下进行诊断查询。
