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Analysis of global transcriptional changes in H. neptunium cells depleted of the cell cycle regulators CckA or ChpT

ABSTRACT: Within the bacterial phylum of the Alphaproteobacteria many species show complex life cycles. Proper regulation of these life cycles requires cell cycle regulation pathways, one of which is the so-called CtrA pathway. Key factors in the CtrA pathway are the histidine kinase CckA and the phosphotransferase ChpT, which are directly involved in cell cycle specific activation of the response regulator CtrA. Within the Alphaproteobacteria, stalked budding bacteria are even more asymmetric than most other representatives. How they regulate their cell cycle has not been studied before. Here, we investigated the transcriptional profiles of Hyphomonas neptunium cells depleted of CckA or ChpT and compared their profiles to that of wildtype cells. We identified that the essential proteins CckA and ChpT influence the transcription levels of multiple essential processes, such as cell division, as well as specific cell cycle regulated functions such as motility.

ORGANISM(S): Hyphomonas neptunium

PROVIDER: GSE134341 | GEO | 2022/01/01

REPOSITORIES: GEO

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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.

Project description:Investigation of whole genome expression changes in Magnetospririllum magneticum mutants, probing the role of the CtrA regulatory pathway. The mutants are further described in a manuscript submitted for publication at J. Bacteriology. Developmental events across the prokaryotic life cycle are highly regulated at the transcriptional and post-translational levels. Key elements of a few regulatory networks are conserved among phylogenetic groups of bacteria, although the features controlled by these conserved systems are as diverse as the organisms encoding them. In this work, we probe the role of the CtrA regulatory network, conserved throughout the Alphaproteobacteria, in the magnetotactic bacterium, Magnetospirillum magneticum strain AMB-1, which possesses unique intracellular organization and compartmentalization. While we show that CtrA in AMB-1 is not essential for viability, it is required for motility, and its putative phosphorylation state dictates the ability of CtrA to activate the flagella biosynthesis gene cascade. Gene expression analysis of strains expressing active and inactive CtrA alleles point to the composition of the extended CtrA regulon, including both direct and indirect targets. These results, combined with a bioinformatic study of the AMB-1 genome, enabled the prediction of an AMB-1 specific CtrA binding site. Further, phylogenetic studies comparing CtrA sequences from Alphaproteobacteria in which the role of CtrA has been experimentally examined reveals an ancestral role of CtrA in the regulation of motility and suggests that its essential functions in other Alphaproteobacteria were acquired subsequently. Total RNA was recovered from each of the wild-type and mutant strains, reverse transcribed to cDNA, fluorescently labeled, and hybridized to whole genome microarrays. The arrays contain 7 probes/gene, with the entire genome duplicated twice. In addition, the arrays contain 7 probes for 22 unannotated ORFs and tiling of a genomic region from coordinates 977403-1097027.

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Analysis of global transcriptional changes in H. neptunium cells depleted of the cell cycle regulators CckA or ChpT

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