Dokument

Zusammenfassung:
The cell cycle is driven by a highly ordered series of events that lead to the production of two cells each containing an exact copy of the parental chromosome. Studies of the molecular mechanisms coordinating the cell cycle have revealed that the mechanisms regulating chromosome replication and segregation are highly similar in all eukaryotic cells. Until recently, it appeared that prokaryotes and eukaryotes do not share much resemblance in the mechanisms governing cell cycle progression. However, advances in prokaryotic cell biology and the analysis of the machinery orchestrating the cell cycle of the α-proteobacterium Caulobacter crescentus unveiled surprising similarities in the regulatory processes employed. The cell division of C. crescentus gives rise to two distinct cell types whose morphology is tightly coupled to their developmental cell cycle phase. While the sessile stalked cell can immediately restart a new round of replication, the motile swarmer cell has to differentiate into a sessile cell before gaining competence for chromosome replication and subsequent proliferation. The DNA-binding response regulator CtrA has been identified as the master regulator driving the cell cycle of this strictly dimorphic organism. A complex two-component signaling network regulating the activity of CtrA is critical for proper cell cycle progression and maintenance of the cellular asymmetry. Genome comparison uncovered that CtrA and the signaling network regulating its activity are highly conserved within the clade of α proteobacteria. Here, we report the functional analysis of the conserved cell cycle regulatory network in Hyphomonas neptunium, a close relative of C. crescentus, which also displays a biphasic life cycle but generates its stalk at the opposite cell pole and proliferates by an unusual budding mechanism. By employing DNA-protein interaction studies coupled to RNA-seq-based transcriptomics of conditional mutants defective in factors involved in the regulation of CtrA we were able to globally identify genes directly regulated by CtrA in H. neptunium. The subsequent analysis of this regulon unveiled a conserved role of CtrA in the transcriptional regulation of essential processes such as cell division and DNA-segregation as well as in the establishment of cellular asymmetry. By applying in vivo localization and heterologous complementation studies coupled to biochemical analysis we were able to identify a high conservation of the signaling module responsible for translating CtrA activity into asymmetry in C. crescentus. Even though transcriptional profiling suggested a role of this conserved module in the regulation of CtrA activity in H. neptunium, the inactivation of candidate genes suprisingly did not result in a pronounced cell cycle defect or a loss of asymmetry. This finding suggests that alternative regulatory factors or mechanisms are involved in the regulation of CtrA in this organism. This study highlights that even though genes are conserved between organisms, high functional variability can arise even over short evolutionary distances. A global investigation of the two-component signaling pathway of H. neptunium by a comprehensive deletion analysis led to the identification of an uncharacterized conserved single domain response regulator. Inactivation of mocR (modulator of cell cycle R) led to a severe cell cycle defect suggesting a role in the cell cycle regulation of H. neptunium. Using transcriptional profiling we were able to identify a connection to the conserved cell cycle regulatory core module. The synteny of this gene paired with heterologous complementation experiments suggests a conserved role of this protein within the α-proteobacteria. Future in depth investigation of MocR will provide valuable insights into its role in cell cycle regulation and will contribute to our understanding of the mechanisms by which single domain response regulators, which lack a dedicated output domain, participate in two-component signaling.

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