Marczynski G T
Department of Microbiology & Immunology, McGill University, Montreal, Quebec, Canada H3A 2B4.
J Bacteriol. 1999 Apr;181(7):1984-93. doi: 10.1128/JB.181.7.1984-1993.1999.
Caulobacter crescentus exhibits cell-type-specific control of chromosome replication and DNA methylation. Asymmetric cell division yields a replicating stalked cell and a nonreplicating swarmer cell. The motile swarmer cell must differentiate into a sessile stalked cell in order to replicate and execute asymmetric cell division. This program of cell division implies that chromosome replication initiates in the stalked cell only once per cell cycle. DNA methylation is restricted to the predivisional cell stage, and since DNA synthesis produces an unmethylated nascent strand, late DNA methylation also implies that DNA near the replication origin remains hemimethylated longer than DNA located further away. In this report, both assumptions are tested with an engineered Tn5-based transposon, Tn5Omega-MP. This allows a sensitive Southern blot assay that measures fully methylated, hemimethylated, and unmethylated DNA duplexes. Tn5Omega-MP was placed at 11 sites around the chromosome and it was clearly demonstrated that Tn5Omega-MP DNA near the replication origin remained hemimethylated longer than DNA located further away. One Tn5Omega-MP placed near the replication origin revealed small but detectable amounts of unmethylated duplex DNA in replicating stalked cells. Extra DNA synthesis produces a second unmethylated nascent strand. Therefore, measurement of unmethylated DNA is a critical test of the "once and only once per cell cycle" rule of chromosome replication in C. crescentus. Fewer than 1 in 1,000 stalked cells prematurely initiate a second round of chromosome replication. The implications for very precise negative control of chromosome replication are discussed with respect to the bacterial cell cycle.
新月柄杆菌表现出对染色体复制和DNA甲基化的细胞类型特异性控制。不对称细胞分裂产生一个正在复制的柄细胞和一个不进行复制的游动细胞。游动细胞必须分化为固着的柄细胞才能进行复制并执行不对称细胞分裂。这种细胞分裂程序意味着染色体复制在每个细胞周期中仅在柄细胞中启动一次。DNA甲基化仅限于细胞分裂前阶段，并且由于DNA合成产生未甲基化的新生链，后期DNA甲基化也意味着靠近复制起点的DNA比远离复制起点的DNA保持半甲基化的时间更长。在本报告中，使用基于Tn5的工程转座子Tn5Omega-MP对这两个假设进行了测试。这允许进行灵敏的Southern印迹分析，以测量完全甲基化、半甲基化和未甲基化的DNA双链体。Tn5Omega-MP被放置在染色体周围的11个位点，结果清楚地表明，靠近复制起点的Tn5Omega-MP DNA比远离复制起点的DNA保持半甲基化的时间更长。一个放置在复制起点附近的Tn5Omega-MP在正在复制的柄细胞中显示出少量但可检测到的未甲基化双链DNA。额外的DNA合成会产生第二条未甲基化的新生链。因此，未甲基化DNA的测量是对新月柄杆菌染色体复制“每个细胞周期仅一次”规则的关键测试。每1000个柄细胞中少于1个会过早启动第二轮染色体复制。关于细菌细胞周期，讨论了对染色体复制进行非常精确的负调控的意义。
