Project description:Hyphomonas neptunium proliferates by a unique budding mechanism in which daughter cells emerge from the end of a stalk‐like extension emanating from the mother cell body. Studies of this species have so far been hampered by the lack of a genetic system and of molecular tools allowing the regulated expression of target genes. Based on microarray analyses, this work identifies two H. neptunium promoters that are specifically activated by copper and zinc, respectively. Functional analyses show that they have low basal activity and a high dynamic range, thus meeting the requirements for use as a multi‐purpose expression system.
Project description: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.
