Primers used in this work.

Rhizobia are Gram-negative bacteria known for their ability to fix atmospheric N2 in symbiosis with leguminous plants. Current evidence shows that rhizobia carry in most cases a variable number of plasmids, containing genes necessary for symbiosis or free-living, a common feature being the presence of several plasmid replicons within the same strain. For many years, we have been studying the mobilization properties of pSmeLPU88b from the strain Sinorhizobium meliloti LPU88, an isolate from Argentina. To advance in the characterization of pSmeLPU88b plasmid, the full sequence was obtained. pSmeLPU88b is 35.9 kb in size, had an average GC % of 58.6 and 31 CDS. Two replication modules were identified in silico: one belonging to the repABC type, and the other to the repC. The replication modules presented high DNA identity to the replication modules from plasmid pMBA9a present in an S. meliloti isolate from Canada. In addition, three CDS presenting identity with recombinases and with toxin-antitoxin systems were found downstream of the repABC system. It is noteworthy that these CDS present the same genetic structure in pSmeLPU88b and in other rhizobial plasmids. Moreover, in all cases they are found downstream of the repABC operon. By cloning each replication system in suicide plasmids, we demonstrated that each of them can support plasmid replication in the S. meliloti genetic background, but with different stability behavior. Interestingly, while incompatibility analysis of the cloned rep systems results in the loss of the parental module, both obtained plasmids can coexist together.

根瘤菌（Rhizobia）是一类因可与豆科植物共生并固定大气氮而闻名的革兰氏阴性菌。现有研究证实，绝大多数根瘤菌菌株携带数量可变的质粒，这些质粒包含共生或自由生存所需的基因，其共同特征为同一菌株内存在多种质粒复制子。多年来，我们一直针对来自阿根廷分离株的苜蓿中华根瘤菌（Sinorhizobium meliloti）LPU88菌株中的pSmeLPU88b质粒的可移动特性开展研究。为深入解析pSmeLPU88b质粒，我们完成了其全序列测定。该质粒全长35.9 kb，平均GC含量为58.6%，共编码31个CDS（编码序列，Coding Sequence）。通过生物信息学分析，我们在该质粒上鉴定出两个复制模块：其一属于repABC型，另一属于repC型。这两个复制模块与来自加拿大苜蓿中华根瘤菌分离株的pMBA9a质粒的复制模块具有高度的DNA序列一致性。此外，我们在repABC系统下游发现了3个编码序列，分别与重组酶及毒素-抗毒素系统具有同源性。值得注意的是，这些编码序列在pSmeLPU88b与其他根瘤菌质粒中拥有完全一致的基因结构，且均位于repABC操纵子的下游区域。我们将两个复制系统分别克隆至自杀质粒中，实验证实二者均可在苜蓿中华根瘤菌的遗传背景下支持质粒复制，但二者的稳定性表现存在差异。有趣的是，尽管针对克隆得到的复制系统进行不相容性分析时会导致亲本复制模块丢失，但这两个质粒可稳定共存。