Integrative and quantitative analysis reveals proper CtrA-dependent cell cycle regulation in the absence of the DivJ/PleC asymmetry module in the stalked budding bacterium Hyphomonas neptunium

Alphaproteobacteria stand out for their complex cell cycles, which are often regulated by the DivJ/PleC-DivK-DivL-CckA-ChpT-CtrA pathway. DivJ and PleC set up the polarity of the cell, thereby eventually leading to differential activation of the DNA-binding response regulator CtrA in the two nascent daughter cells. CtrA regulates replication and transcription of many genes, thereby ensuring that processes such as motility and cell division take place at the appropriate cell cycle stage. The cell cycle of the stalked budding alphaproteobacterium Hyphomonas neptunium culminates in an asymmetric cell division at the stalk-bud junction. Here, we investigate the role of the pathway from DivJ and PleC down to CtrA in this recently established model organism. Even though DivJ and PleC are localized to opposite poles, suggesting they are involved in polarity establishment inH. neptunium, DivJ, PleC and the other components of the upstream pathway (DivK and PleD) are not essential for cell cycle regulation. In contrast, the downstream part of the pathway starting from DivL is essential and involved in the regulation of important functions such as replication inhibition, cell division and motility, as shown by the identification of the (direct) regulon of CtrA. The overlap between the regulons of DivJ and PleC, DivK and CtrA is only partial, demonstrating that additional factors feeding into the pathway must be present in H. neptunium. Furthermore, unlike in other alphaproteobacteria, the regulation of CtrA throughout the cell cycle does not take place at the level of CtrA abundance in H. neptunium. All in all, the DivL-CckA-ChpT-CtrA pathway plays an essential role in the regulation of the complicated cell cycle ofH. neptunium, but several proteins feeding into CtrA remain undiscovered. The in-depth analysis of CtrA regulation in this stalked budding organism leads to hypotheses that might also hold in well-established model organisms such as Caulobacter crescentus. Examination of CtrA and GcrA whole genome binding/occupency (ChIP-Seq) in WT Hyphomonas neptunium LE670 (ATCC15444).

α变形菌门（Alphaproteobacteria）以其复杂的细胞周期为显著特征，这类周期通常由DivJ/PleC-DivK-DivL-CckA-ChpT-CtrA通路调控。DivJ与PleC可确立细胞极性，最终使两个新生子细胞中的DNA结合应答调节因子CtrA产生差异性激活。CtrA可调控众多基因的复制与转录，从而确保运动、细胞分裂等过程在恰当的细胞周期阶段顺利进行。柄芽生α变形菌（stalked budding alphaproteobacterium）海王星生丝杆菌（Hyphomonas neptunium）的细胞周期以在柄芽连接处发生不对称细胞分裂为最终环节。本研究针对这一新近建立的模式生物，探究了从DivJ、PleC直至CtrA的通路所发挥的作用。尽管DivJ与PleC定位于相反的细胞极，提示二者参与了H. neptunium的极性建立，但DivJ、PleC以及上游通路的其他组分（DivK与PleD）对于细胞周期调控并非必需。与之相反，以DivL为起点的通路下游部分是必需的，其参与调控复制抑制、细胞分裂与运动等重要功能，这一结论可通过对CtrA的直接调控组（regulon）的鉴定得到证实。DivJ、PleC、DivK与CtrA的调控组之间仅存在部分重叠，这表明H. neptunium中必然存在其他可汇入该通路的调控因子。此外，与其他α变形菌门物种不同，H. neptunium中CtrA在整个细胞周期中的调控并非通过CtrA的蛋白丰度水平实现。综上而言，DivL-CckA-ChpT-CtrA通路在H. neptunium复杂的细胞周期调控中发挥着关键作用，但仍有若干汇入CtrA调控通路的蛋白质尚未被探明。对这一株柄芽生生物的CtrA调控所开展的深入分析，可推导出或许也适用于新月柄杆菌（Caulobacter crescentus）这类成熟模式生物的相关假说。本研究对野生型（WT）海王星生丝杆菌LE670（ATCC15444）开展了CtrA与GcrA的全基因组结合/占据（ChIP-Seq，染色质免疫沉淀测序）分析。