Orderly Replication and Segregation of the Four Replicons of Burkholderia cenocepacia J2315

Bacterial genomes typically consist of a single chromosome and, optionally, one or more plasmids. But whole-genome sequencing reveals about ten per-cent of them to be multipartite, with additional replicons which by size and indispensability are considered secondary chromosomes. This raises the questions of how their replication and partition is managed without compromising genome stability and of how such genomes arose. Vibrio cholerae, with a 1 Mb replicon in addition to its 3 Mb chromosome, is the only species for which maintenance of a multipartite genome has been investigated. In this study we have explored the more complex genome of Burkholderia cenocepacia (strain J2315). It comprises an extra replicon (c2) of 3.21 Mb, comparable in size to the3.87Mb main chromosome (c1), another extra replicon(c3) of 0.87 Mb and a plasmid of 0.09 Mb. The replication origin of c1 is typically chromosomal and those of c2 and c3 are plasmid-like; all are replicated bidirectionally. Fluorescence microscopy of tagged origins indicates that all initiate replication at mid-cell and segregate towards the cell quarter positions sequentially, c1-c2-p1/c3. c2 segregation is as well-phased with the cell cycle as c1, implying that this plasmid-like origin has become subject to regulation not typical of plasmids; in contrast, c3 segregates more randomly through the cycle. Disruption of individual Par systems by deletion of parAB or by addition of parS sites showed each Par system to govern the positioning of its own replicon only. Inactivation of c1, c2 and c3 Par systems not only reduced growth rate, generated anucleate cells and compromised viability but influenced processes beyond replicon partition, notably regulation of replication, chromosome condensation and cell size determination. In particular, the absence of the c1 ParA protein altered replication of all three chromosomes, suggesting that the partition system of the main chromosome is a major participant in the choreography of the cell cycle.细菌基因组通常以单条染色体为基础基因组，并可附带一条或多条质粒。但全基因组测序研究表明，约10%的细菌基因组为多分区结构，其额外携带的复制子（replicon）可根据分子大小与必需性被界定为次级染色体。这引出了两个核心科学问题：其一，这类多分区基因组如何在不破坏基因组稳定性的前提下实现复制与分离调控；其二，此类基因组的演化起源机制是怎样的。 霍乱弧菌（Vibrio cholerae）除拥有3 Mb的染色体外，还携带一条1 Mb的复制子，是目前唯一被研究过多分区基因组维持机制的物种。本研究针对洋葱伯克霍尔德菌（Burkholderia cenocepacia）J2315菌株的复杂基因组展开了探究。该菌株的基因组包含一条3.21 Mb的额外复制子（c2），其大小与3.87 Mb的主染色体（c1）相当；另有一条0.87 Mb的额外复制子（c3）以及一条0.09 Mb的质粒。 c1的复制起始位点具有典型的染色体特征，而c2与c3的复制起始位点则类似质粒；所有复制子均以双向方式进行复制。对带有标记的复制起始位点进行荧光显微镜观测结果显示，所有复制子均在细胞中部启动复制，并依次向细胞四等分位置进行分离，顺序为c1→c2→p1/c3。c2的分离过程与细胞周期的相位关联程度与c1相当，这意味着该类质粒样的复制起始位点已受到非质粒典型的调控机制作用；与之相反，c3的分离过程在细胞周期中更为随机。 通过敲除parAB基因或添加parS位点来破坏单个Par系统的实验表明，每个Par系统仅负责调控其对应复制子的定位。失活c1、c2与c3的Par系统不仅会降低菌株生长速率、产生无核细胞并削弱细胞活力，还会影响复制调控、染色体凝缩与细胞大小确定等复制子分离之外的生理过程。尤为关键的是，c1的ParA蛋白缺失会改变三个复制子的复制过程，这表明主染色体的分离系统是细胞周期调控程序中的核心参与者之一。