Energy dependent proteases ensure the timely removal of unwanted proteins in a highly selective fashion. In Caulobacter crescentus, protein degradation by the ClpXP protease is critical for cell cycle progression; however, only a handful of substrates are currently known. Here, we use a trapping approach to identify putative substrates of the ClpP associated proteases in C. crescentus. Biochemical validation of several of these targets reveals specific protease recognition motifs and suggests a need for ClpXP specific degradation beyond degradation of known cell cycle regulators. We focus on a particular instance of regulated proteolysis in Caulobacter by exploring the role of ClpXP in degrading the stalk synthesis transcription factor TacA. We show that TacA degradation is controlled during the cell cycle dependent on the ClpXP regulator CpdR and that stabilization of TacA increases degradation of another ClpXP substrate, CtrA, while restoring deficiencies associated with prolific CpdR activity. Together, our work reveals a number of new validated ClpXP substrates, clarifies rules of protease substrate selection, and demonstrates how regulated protein degradation is critical for Caulobacter development and cell cycle progression.

Proteolytic control of Caulobacter cell cycle proteins is primarily executed by ClpXP, a dynamically localized protease implicated in turnover of several factors critical for faithful cell cycle progression. Here, we show that the transient midcell localization of ClpXP that precedes cytokinesis requires the FtsZ component of the divisome. Although ClpAP does not exhibit subcellular localization, FtsZ is a substrate of both ClpXP and ClpAP in vivo and in vitro. A peptide containing the C-terminal portion of the FtsA divisome protein is a substrate of both ClpXP and ClpAP in vitro but is primarily degraded by ClpAP in vivo. Caulobacter carries out an asymmetric division in which FtsZ and FtsA are stable in stalked cells but degraded in the non-replicative swarmer cell where ClpAP alone degrades FtsA and both ClpAP and ClpXP degrade FtsZ. While asymmetric division in Caulobacter normally yields larger stalked and smaller swarmer daughters, we observe a loss of asymmetric size distribution among daughter cells when clpA is depleted from a strain in which FtsZ is constitutively produced. Taken together, these results suggest that the activity of both ClpXP and ClpAP on divisome substrates is differentially regulated in daughter cells.

Caulobacter crescentus, a Gram-negative alpha-proteobacterium, has surfaced as a powerful model system for unraveling molecular networks that control the bacterial cell cycle. A straightforward synchronization protocol and existence of many well-defined developmental markers has allowed the identification of various molecular circuits that control the underlying differentiation processes executed at the level of transcription, translation, protein localization and dynamic proteolysis. The oligomeric AAA+ protease ClpXP is a well-characterized example of an enzyme that exerts post-translational control over a number of pathways. Also, the proteolytic pathways of its candidate proteins are reported to play significant roles in regulating cell cycle and protein quality control. A detailed evaluation of the impact of its proteolysis on various regulatory networks of the cell has uncovered various significant cellular roles of this protease in C. crescentus. A deeper insight into the effects of regulatory proteolysis with emphasis on cell cycle progression could shed light on how cells respond to environmental cues and implement developmental switches. Perturbation of this network of molecular machines is also associated with diseases such as bacterial infections. Thus, research holds immense implications in clinical translation and health, representing a promising area for clinical advances in the diagnosis, therapeutics and prognosis.