The taxonomic assignment of uncultured prokaryotes to known taxa is a major challenge in microbial systematics. This relies usually on the phylogenetic analysis of the ribosomal small subunit RNA or a few housekeeping genes. Recent works have disclosed ribosomal proteins as valuable markers for systematics and, due to the boom in complete genome sequencing, their use has become widespread. Yet, in the case of uncultured strains, for which complete genome sequences cannot be easily obtained, sequencing many markers is complicated and time consuming. Taking the advantage of the organization of ribosomal protein coding genes in large gene clusters, we amplified a 32 kb conserved region encompassing the spectinomycin (spc) operon using long range PCR from isolated and from uncultured nodular endophytic Frankia strains. The phylogenetic analysis of the 27 ribosomal protein genes contained in this region provided a robust phylogenetic tree consistent with phylogenies based on larger set of markers, indicating that this subset of ribosomal proteins contains enough phylogenetic signal to address systematic issues. This work shows that using long range PCR could break down the barrier preventing the use of ribosomal proteins as phylogenetic markers when complete genome sequences cannot be easily obtained.

中文翻译：

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未培养原核生物的分类学分配，采用针对壮观霉素操纵子的长距离PCR。

未培养原核生物向已知生物分类的分类学分配是微生物系统学的主要挑战。这通常依赖于核糖体小亚基RNA或一些管家基因的系统发育分析。最近的工作已经公开了核糖体蛋白作为系统学的有价值的标志物，并且由于在完整的基因组测序中的蓬勃发展，它们的使用已变得广泛。然而，在不能容易获得完整基因组序列的未培养菌株的情况下，许多标记的测序是复杂且费时的。利用大基因簇中核糖体蛋白编码基因的组织优势，我们使用长距离PCR从分离的和未培养的结节内生Frankia菌株中扩增了一个32kb的保守区域，其中包含壮观霉素（spc）操纵子。对这个区域中包含的27个核糖体蛋白基因的系统发育分析提供了一个强大的系统树，与基于较大标记集的系统发育相一致，表明该核糖体蛋白子集包含足够的系统发育信号来解决系统性问题。这项工作表明，当不能轻易获得完整的基因组序列时，使用长距离PCR可以打破障碍，阻止使用核糖体蛋白作为系统发育标记。