Gorbatyuk B, Marczynski G T
Department of Microbiology and Immunology, McGill University, 3775 University Street, Montreal, Quebec, Canada H3A 2B4.
Mol Microbiol. 2001 Apr;40(2):485-97. doi: 10.1046/j.1365-2958.2001.02404.x.
Caulobacter crescentus chromosome replication is precisely coupled to a developmental cell cycle. Like most eubacteria, C. crescentus has a DnaA homologue that is presumed to initiate chromosome replication. However, the C. crescentus replication origin (Cori) lacks perfect consensus Escherichia coli DnaA boxes. Instead, the Cori strong transcription promoter (Ps) may regulate chromosome replication through the CtrA cell cycle response regulator. We therefore created a conditional dnaA C. crescentus strain. Blocking dnaA expression immediately decreased DNA synthesis, which stopped after approximately one doubling period. Fluorescent flow cytometry confirmed that DNA synthesis is blocked at the initiation stage. Cell division also stopped, but not swarmer to stalked cell differentiation. All cells became stalked cells that grew as long filaments. Therefore, general transcription and protein synthesis continued, whereas DNA synthesis stopped. However, transcription was selectively blocked from the flagellar fliQ and fliL and methyltransferase ccrM promoters, which require CtrA and are blocked by different DNA synthesis inhibitors. Interestingly, transcription from Cori Ps continued unaltered. Therefore, Ps transcription is not sufficient for chromosome replication. Approximately 6-8 h after blocked dnaA expression, cells lost viability exponentially. Coincidentally, beta-galactosidase was induced from one transcription reporter, suggesting an altered physiology. We conclude that C. crescentus DnaA is essential for chromosome replication initiation, and perhaps also has a wider role in cell homeostasis.
新月柄杆菌的染色体复制与发育细胞周期精确偶联。与大多数真细菌一样，新月柄杆菌有一个假定可启动染色体复制的DnaA同源物。然而，新月柄杆菌的复制起点（Cori）缺乏完美一致的大肠杆菌DnaA框。相反，Cori的强转录启动子（Ps）可能通过CtrA细胞周期反应调节因子来调控染色体复制。因此，我们构建了一个条件性dnaA新月柄杆菌菌株。阻断dnaA的表达会立即降低DNA合成，大约经过一个倍增期后DNA合成停止。荧光流式细胞术证实DNA合成在起始阶段被阻断。细胞分裂也停止了，但游动细胞到柄细胞的分化没有停止。所有细胞都变成了以长丝形式生长的柄细胞。因此，一般转录和蛋白质合成继续进行，而DNA合成停止。然而，鞭毛fliQ和fliL以及甲基转移酶ccrM启动子的转录被选择性阻断，这些启动子需要CtrA且会被不同的DNA合成抑制剂阻断。有趣的是，来自Cori Ps的转录不受影响地继续进行。因此，Ps转录不足以启动染色体复制。阻断dnaA表达约6 - 8小时后，细胞活力呈指数下降。巧合的是，一个转录报告基因诱导了β-半乳糖苷酶的表达，这表明生理状态发生了改变。我们得出结论，新月柄杆菌的DnaA对于染色体复制起始至关重要，并可能在细胞稳态中也发挥更广泛的作用。
