Competitiveness prediction for nodule colonization in Sinorhizobium meliloti through combined in vitro tagged strain characterization and genome-wide association analysis

Associations between leguminous plants and symbiotic nitrogen-fixing rhizobia are a classic example of mutualism between a eukaryotic host and a specific group of prokaryotic microbes. Although this symbiosis is in part species-specific, different rhizobial strains may colonise the same nodule. Some rhizobial strains are commonly known as better competitors than others, but detailed analyses that aim to predict rhizobial competitive abilities based on genomes are still scarce. Here, we performed a bacterial genome-wide association (GWAS) analysis to define the genomic determinants related to the competitive capabilities in the model rhizobial species Sinorhizobium meliloti. For this, 13 tester strains were GFP-tagged and assayed vs. 3 RFP-tagged reference competitor strains (Rm1021, AK83, and BL225C) in a Medicago sativa nodule occupancy test. Competition data and strain genomic sequences were employed to build a model for GWAS based on k-mers. Among the k-mers with the highest scores, 51 k-mers mapped on the genomes of four strains showing the highest competition phenotypes (> 60% single strain nodule occupancy; GR4, KH35c, KH46 and SM11) vs. BL225C. These k-mers were mainly located on the symbiosis-related megaplasmid pSymA, specifically on genes coding for transporters, proteins involved in the biosynthesis of cofactors and proteins related to metabolism (e.g., fatty acids). The same analysis was performed considering the sum of single and mixed nodules obtained in the competition assays vs. BL225C, retrieving k-mers mapped on the genes previously found and on vir genes. Therefore, the competition abilities seem to be linked to multiple genetic determinants and comprise several cellular components.

Methods
Methods description is available at DOI: 10.1128/mSystems.00550-21

豆科植物与共生固氮根瘤菌的共生关系，是真核宿主与特定原核微生物类群之间互利共生的经典范例。尽管该共生体系在一定程度上具有物种特异性，但不同根瘤菌菌株可共同定殖于同一根瘤。部分根瘤菌菌株通常被认为比其他菌株具有更优的竞争能力，但基于基因组预测根瘤菌竞争能力的详细分析仍较为匮乏。本研究以模式根瘤菌物种苜蓿根瘤菌（Sinorhizobium meliloti）为研究对象，通过细菌全基因组关联分析（genome-wide association study, GWAS）挖掘与其竞争能力相关的基因组决定因子。

为此，我们对13株测试菌株进行绿色荧光蛋白（green fluorescent protein, GFP）标记，并与3株红色荧光蛋白（red fluorescent protein, RFP）标记的参考竞争菌株（Rm1021、AK83及BL225C）开展紫花苜蓿（Medicago sativa）根瘤定殖竞争试验。结合竞争数据与菌株基因组序列，我们构建了基于k聚体（k-mers）的GWAS分析模型。在得分最高的k聚体中，有51个k聚体定位至4株竞争表型最优（单菌株根瘤定殖占比>60%；GR4、KH35c、KH46及SM11）且与BL225C竞争的菌株基因组上。这些k聚体主要位于共生相关巨型质粒pSymA上，具体涉及编码转运蛋白、辅因子生物合成相关蛋白以及代谢相关蛋白（如脂肪酸代谢相关蛋白）的基因。

我们还针对竞争试验中获得的单根瘤与混合根瘤的总和与BL225C的竞争数据开展了相同分析，检索到定位至前述基因以及毒力基因（vir genes）上的k聚体。综上，根瘤菌的竞争能力似乎与多个遗传决定因子相关，且涉及多种细胞组分。

方法
方法描述可参见DOI: 10.1128/mSystems.00550-21