Proteolysis and the generation of cellular asymmetry [electronic resource] : new roles for the ClpXP and ClpAP proteases in the Caulobacter cell cycle
Keywords: Biology.

Spatially and temporally regulated proteolysis plays a vital role in cellular differentiation and progression through the bacterial cell cycle. For Caulobacter crescentus, differentiation begins with the transition from a swarmer cell to a replicationally-competent stalked cell. The stalked cell further differentiates into a predivisional that eventually gives rise to two morphologically distinct daughter progeny with different cell fates: a motile swarmer cell and a sessile stalked cell. This transformation requires the precise timing and coordination of multiple processes, including synthesis and removal of polar organelles (such as pili, the flagellum, the stalk and the chemotaxis machinery), DNA replication initiation, segregation and methylation of the newly replicated chromosome and finally, cytokinesis. Additionally, because Caulobacter differentiation is inherently asymmetric, factors controlling many of these processes must be regulated spatially as well. The work detailed in this thesis further explores new roles for the ClpXP and ClpAP proteases in the cell cycle-dependent regulation of components that comprise the divisome, a large macro-molecular protein complex that drives cytokinesis, and the development of the stalk apparatus, a long tubular apparatus that extends from the pole of the Caulobacter cell body, during Caulobacter differentiation. Following asymmetric cell division in Caulobacter, the FtsZ and FtsA divisome-associated components are maintained in the daughter stalked cell, but rapidly cleared from the daughter swarmer cell. The rates at which FtsZ and FtsA are cleared from the newly formed swarmer cell strongly suggest that stability is regulated via proteolysis. However, prior to this work, the protease(s) involved had not yet been identified. Previously, our lab has shown that the ClpXP protease transiently localizes to the incipient cell division site just prior to completion of cell division. In Chapter 2, I demonstrate that the localization of ClpX is concomitant with, and dependent on the formation of the FtsZ Z-ring. In vivo and intro proteolysis data demonstrate that FtsZ is substrate for the ClpXP and ClpAP proteases, while FtsA is a substrate for the ClpAP protease with proteolysis occurring primarily in the swarmer cell and just after completion of cell division. Modification to the very C-terminus of FtsZ or the N-terminus FtsA results reduced or abrogated proteolysis suggesting that Clp destruction motifs likely reside within these regions of the FtsZ and FtsA peptides. Normally, cell division in Caulobacter yields a smaller swarmer cell and larger stalked cell. The loss of size asymmetry in Caulobacter cells constitutively expressing FtsZ in the absence of a functional clpA allele demonstrate the requirement for precise regulation of FtsZ and FtsA levels during the course of the cell cycle for normal divisome functionality. Stalk formation in Caulobacter is a cell cycle-regulated event, suggesting that the factors governing stalk biogenesis are likely to be temporally and spatially regulated during the cell cycle. Recently, the TacA response regulator has been implicated in control of stalk development. During the swarmer-to-stalked cell transition, ClpXP localizes to the developing stalked pole where the protease forms a degradation complex with PopA, CpdR and RcdA. In chapter 3, I show that the clearance of TacA during the swarmer-to-stalked cell transition is commensurate with the localization of ClpXP to the incipient stalked pole. Fluorescent imaging also indicates that both CpdR and RcdA are required for the spatial regulation of TacA during the course of the cell cycle. Overexpression of a constitutively active mutant variant of TacA (TacAD54E) exhibits cell morphology defects and loss of stalk elongation control, which demonstrates the need for control of TacA activation. I also show that ShkA, a component of the phosphorelay system that phosphorylates and activates TacA, is a substrate for the ClpXP protease. Proteolysis of ShkA likely contributes to the regulation of TacA activation. Collectively, these data reveal a mechanism of temporal and spatial regulation for stalk biogenesis in Caulobacter. Thus, this thesis work has contributed to the understanding of how intracellular proteolysis coordinates cell cycle events (such as cytokinesis) with developmental events (such as stalk biosynthesis) during differentiation.