Table_9_Phylogenomic Analysis Substantiates the gyrB Gene as a Powerful Molecular Marker to Efficiently Differentiate the Most Closely Related Genera Myxococcus, Corallococcus, and Pyxidicoccus.XLSX

Rapid and accurate strain identification of the most closely related genera Myxococcus, Corallococcus, and Pyxidicoccus can enhance the efficiency of the mining of novel secondary metabolites through dereplication. However, the commonly used 16S rRNA gene sequencing cannot accurately differentiate members of the three genera above, and the whole-genome sequencing is unable to rapidly and inexpensively provide species assignation toward a large number of isolates. To overcome the limitations, the gyrB gene was investigated as a candidate genetic marker for exploring the phylogenetic relationships of bacteria within the three genera and for developing the gyrB-based typing method. Here, the bacterial phylogeny and species affiliations of the three genera were determined based on the phylogenomic reconstruction and the analysis of digital DNA–DNA hybridization values among 90 genomes, further confirming nine novel taxa and assigning over one-third of genomes to defined species. The phylogenetic relationships of these strains based on the gyrB gene sequences were congruent with those based on their genome sequences, allowing the use of the gyrB gene as a molecular marker. The gyrB gene-specific primers for the PCR-amplification and sequencing of bacteria within the three genera were designed and validated for 31 isolates from our group collection. The gyrB-based taxonomic tool proved to be able to differentiate closely related isolates at the species level. Based on the newly proposed 98.6% identity threshold for the 966-bp gyrB gene and the phylogenetic inference, these isolates were assigned into two known species and eight additional putative new species. In summary, this report demonstrated that the gyrB gene is a powerful phylogenetic marker for taxonomy and phylogeny of bacteria within the closely related genera Myxococcus, Corallococcus, and Pyxidicoccus, particularly in the case of hundreds or thousands of isolates in environmental studies.

对亲缘关系紧密的黏球菌属（Myxococcus）、珊瑚球菌属（Corallococcus）和芽孢球菌属（Pyxidicoccus）菌株进行快速且精准的鉴定，可通过去重复筛选提升新型次级代谢产物的挖掘效率。然而，常规使用的16S rRNA基因测序无法精准区分上述三个属的菌株，而全基因组测序则难以快速且低成本地对大量分离株进行物种归属判定。为克服上述局限，本研究将DNA旋转酶B亚基基因（gyrB）作为候选遗传标记，用于探究这三个属细菌的系统发育关系，并开发基于gyrB的分型方法。本研究基于90个基因组的系统基因组重建及数字DNA-DNA杂交值分析，明确了这三个属的细菌系统发育关系与物种归属，进一步确认了9个新分类单元，并将超过三分之一的基因组归类至已定义物种。基于gyrB基因序列构建的菌株系统发育关系与基于全基因组序列的结果高度一致，证明gyrB基因可作为可靠的分子标记。本研究设计了针对上述三个属细菌的gyrB基因特异性引物，用于PCR扩增与测序，并利用本课题组保藏的31株分离株对引物进行了验证。基于gyrB的分类学工具可在物种水平上区分亲缘关系紧密的分离株。基于最新提出的966 bp长度gyrB基因98.6%的序列一致性阈值及系统发育推断，本研究将31株分离株划分为2个已知物种及8个推定新物种。综上，本研究证实，对于亲缘关系紧密的黏球菌属（Myxococcus）、珊瑚球菌属（Corallococcus）和芽孢球菌属（Pyxidicoccus）细菌而言，gyrB基因是极具应用价值的分类与系统发育分子标记，尤其适用于环境研究中数百乃至数千株分离株的鉴定工作。