Classification of Plant Associated Bacteria Using RIF, a Computationally Derived DNA Marker

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.

本研究通过比对黄单胞菌属（Xanthomonas）的6个已测序基因组，开发出一种可在物种及亚种水平区分植物伴生细菌的DNA分子标记（DNA marker）。黄单胞菌属包含诸多重要植物病原菌（phytopathogens）。该DNA分子标记对应dnaA复制起始因子（dnaA replication initiation factor，简称RIF）的一段序列。与核糖体RNA基因（rRNA genes）不同，在绝大多数已测序的细菌基因组中，dnaA均为单拷贝基因（single copy gene），且RIF的扩增需使用属特异性引物（genus-specific primers）。计算机模拟分析（in silico analysis）结果显示，相较于广泛使用的核糖体基因间区（ribosomal intergenic spacer region，简称ITS），RIF对黄单胞菌近缘物种的区分能力相当甚至更强。此外，在涵盖263株黄单胞菌属、青枯菌属（Ralstonia）与棒杆菌属（Clavibacter）菌株的实验中，RIF标记的双向直接测序成功率较ITS高约16%。研究团队基于682株代表不同物种、亚种、致病型（pathovar）、生理小种（race）、宿主及地理来源的参考菌株（reference strains），构建了黄单胞菌属、青枯菌属与棒杆菌属的RIF序列框架，共包含109种不同的RIF序列。RIF序列呈现出亚种水平的聚类分组，但无法将野油菜黄单胞菌（X. campestris）与菜豆黄单胞菌（X. axonopodis）的菌株归入目前已命名的致病型，也无法将青枯雷尔氏菌（R. solanacearum）的菌株归入其对应的生理小种，这验证了此前的结论：致病型与生理小种的命名未必能反映菌株间的遗传亲缘关系。研究人员还对肠杆菌科（Enterobacteriaceae）3个属的24株参考菌株进行了RIF序列测序，这3个属分别为果胶杆菌属（Pectobacterium）、泛菌属（Pantoea）与迪基氏菌属（Dickeya）。70株此前未被鉴定的青枯菌属、棒杆菌属、果胶杆菌属与迪基氏菌属菌株的RIF序列，与已知参考菌株的RIF序列相匹配或高度相似，这证明了该序列框架可用于菌株的亚种水平分类，并能快速将未知分离株与参考菌株进行匹配。RIF序列框架可通过在线RIF数据库（RIFdb）获取，支持以色谱图与FASTA格式（FASTA format）提交的未知菌株RIF序列进行诊断查询。