Abstract：
Multipartite bacterial genomes pose challenges for genome engineering and the establishment of additional replicons. We simplified the tripartite genome structure (3.65 Mbp chromosome, 1.35 Mbp megaplasmid pSymA, 1.68 Mbp chromid pSymB) of the nitrogen-fixing plant symbiont Sinorhizobium meliloti. Strains with bi- and monopartite genome configurations were generated by targeted replicon fusions. Our design preserved key genomic features such as replichore ratios, GC skew, KOPS, and coding sequence distribution. Under standard culture conditions, the growth rates of these strains and the wild type were nearly comparable, and the ability for symbiotic nitrogen fixation was maintained. Spatiotemporal replicon organization and segregation were maintained in the triple replicon fusion strain. Deletion of the replication initiator-encoding genes, including the oriVs of pSymA and pSymB from this strain, resulted in a monopartite genome with oriC as the sole origin of replication, a strongly unbalanced replichore ratio, slow growth, aberrant cellular localization of oriC, and deficiency in symbiosis. Suppressor mutation R436H in the cell cycle histidine kinase CckA and a 3.2 Mbp inversion, both individually, largely restored growth, but only the genomic rearrangement recovered the symbiotic capacity. These strains will facilitate the integration of secondary replicons in S. meliloti and thus be useful for genome engineering applications, such as generating hybrid genomes.
摘要：
多部分细菌基因组对基因组工程和其他复制子的建立提出了挑战。我们简化了三方基因组结构(3.65Mbp染色体，1.35Mbp大质粒pSymA，固氮植物共生体中华根瘤菌的1.68Mbp染色体pSymB）。通过靶向复制子融合产生具有双-和单片段基因组构型的菌株。我们的设计保留了关键的基因组特征，如复制率，GC歪斜,KOPS,和编码序列分布。在标准培养条件下,这些菌株的生长速率和野生型几乎相当，并保持了共生固氮的能力。在三复制子融合菌株中保持时空复制子组织和分离。复制起始编码基因的缺失，包括该菌株的psyma和psymb的oriVs，产生了一个以oriC为唯一复制起点的单克隆基因组，强烈不平衡的复制率，生长缓慢,oriC的异常细胞定位，和共生不足。细胞周期组氨酸激酶CckA中的抑制突变R436H和3.2Mbp倒置，两者都是单独的，基本上恢复了增长，但只有基因组重排恢复了共生能力。这些菌株将促进二级复制子在Meliloti中的整合，因此可用于基因组工程应用。比如产生杂交基因组。
