Wargachuk Richard, Marczynski Gregory T
McGill University, Department of Microbiology and Immunology, Montreal, Quebec, Canada.
McGill University, Department of Microbiology and Immunology, Montreal, Quebec, Canada
J Bacteriol. 2015 Nov;197(22):3521-32. doi: 10.1128/JB.00460-15. Epub 2015 Aug 31.
It is not known how diverse bacteria regulate chromosome replication. Based on Escherichia coli studies, DnaA initiates replication and the homolog of DnaA (Hda) inactivates DnaA using the RIDA (regulatory inactivation of DnaA) mechanism that thereby prevents extra chromosome replication cycles. RIDA may be widespread, because the distantly related Caulobacter crescentus homolog HdaA also prevents extra chromosome replication (J. Collier and L. Shapiro, J Bacteriol 191:5706-5715, 2009, http://dx.doi.org/10.1128/JB.00525-09). To further study the HdaA/RIDA mechanism, we created a C. crescentus strain that shuts off hdaA transcription and rapidly clears HdaA protein. We confirm that HdaA prevents extra replication, since cells lacking HdaA accumulate extra chromosome DNA. DnaA binds nucleotides ATP and ADP, and our results are consistent with the established E. coli mechanism whereby Hda converts active DnaA-ATP to inactive DnaA-ADP. However, unlike E. coli DnaA, C. crescentus DnaA is also regulated by selective proteolysis. C. crescentus cells lacking HdaA reduce DnaA proteolysis in logarithmically growing cells, thereby implicating HdaA in this selective DnaA turnover mechanism. Also, wild-type C. crescentus cells remove all DnaA protein when they enter stationary phase. However, cells lacking HdaA retain stable DnaA protein even when they stop growing in nutrient-depleted medium that induces complete DnaA proteolysis in wild-type cells. Additional experiments argue for a distinct HdaA-dependent mechanism that selectively removes DnaA prior to stationary phase. Related freshwater Caulobacter species also remove DnaA during entry to stationary phase, implying a wider role for HdaA as a novel component of programed proteolysis.
Bacteria must regulate chromosome replication, and yet the mechanisms are not completely understood and not fully exploited for antibiotic development. Based on Escherichia coli studies, DnaA initiates replication, and the homolog of DnaA (Hda) inactivates DnaA to prevent extra replication. The distantly related Caulobacter crescentus homolog HdaA also regulates chromosome replication. Here we unexpectedly discovered that unlike the E. coli Hda, the C. crescentus HdaA also regulates DnaA proteolysis. Furthermore, this HdaA proteolysis acts in logarithmically growing and in stationary-phase cells and therefore in two very different physiological states. We argue that HdaA acts to help time chromosome replications in logarithmically growing cells and that it is an unexpected component of the programed entry into stationary phase.
尚不清楚不同细菌如何调节染色体复制。基于对大肠杆菌的研究，DnaA启动复制，而DnaA的同源物（Hda）利用RIDA（DnaA的调节性失活）机制使DnaA失活，从而防止额外的染色体复制周期。RIDA可能广泛存在，因为远缘相关的新月柄杆菌同源物HdaA也能防止额外的染色体复制（J. 科利尔和L. 夏皮罗，《细菌学杂志》191:5706 - 5715，2009，http://dx.doi.org/10.1128/JB.00525 - 09）。为了进一步研究HdaA/RIDA机制，我们构建了一个新月柄杆菌菌株，该菌株可关闭hdaA转录并快速清除HdaA蛋白。我们证实HdaA可防止额外复制，因为缺乏HdaA的细胞会积累额外的染色体DNA。DnaA结合核苷酸ATP和ADP，我们的结果与已确立的大肠杆菌机制一致，即Hda将活性DnaA - ATP转化为无活性的DnaA - ADP。然而，与大肠杆菌DnaA不同，新月柄杆菌DnaA也受选择性蛋白酶解调节。缺乏HdaA的新月柄杆菌细胞在对数生长期细胞中减少了DnaA的蛋白酶解，从而表明HdaA参与了这种选择性DnaA周转机制。此外，野生型新月柄杆菌细胞进入稳定期时会清除所有DnaA蛋白。然而，缺乏HdaA的细胞即使在营养耗尽的培养基中停止生长时仍保留稳定的DnaA蛋白，而在野生型细胞中这种培养基会诱导完全的DnaA蛋白酶解。额外的实验支持了一种独特的依赖HdaA的机制，该机制在稳定期之前选择性地去除DnaA。相关的淡水柄杆菌属物种在进入稳定期时也会去除DnaA，这意味着HdaA作为程序性蛋白酶解的一个新成分具有更广泛的作用。
细菌必须调节染色体复制，但其机制尚未完全了解，也未被充分用于抗生素开发。基于对大肠杆菌的研究，DnaA启动复制，DnaA的同源物（Hda）使DnaA失活以防止额外复制。远缘相关的新月柄杆菌同源物HdaA也调节染色体复制。在这里，我们意外地发现，与大肠杆菌Hda不同，新月柄杆菌HdaA还调节DnaA的蛋白酶解。此外，这种HdaA蛋白酶解作用于对数生长期和稳定期细胞，因此作用于两种非常不同的生理状态。我们认为HdaA有助于在对数生长期细胞中安排染色体复制时间，并且它是进入稳定期程序性过程中一个意想不到的成分。
