Regulated proteolysis of a transcription factor complex is critical to cell cycle progression in Caulobacter crescentus

Cell cycle transitions are often triggered by the proteolysis of key regulatory proteins. In Caulobacter crescentus, the G1‐S transition involves the degradation of an essential DNA‐binding response regulator, CtrA, by the ClpXP protease. Here, we show that another critical cell cycle regulator, SciP, is also degraded during the G1‐S transition, but by the Lon protease. SciP is a small protein that binds directly to CtrA and prevents it from activating target genes during G1. We demonstrate that SciP must be degraded during the G1‐S transition so that cells can properly activate CtrA‐dependent genes following DNA replication initiation and the reaccumulation of CtrA. These results indicate that like CtrA, SciP levels are tightly regulated during the Caulobacter cell cycle. In addition, we show that formation of a complex between CtrA and SciP at target promoters protects both proteins from their respective proteases. Degradation of either protein thus helps trigger the destruction of the other, facilitating a cooperative disassembly of the complex. Collectively, our results indicate that ClpXP and Lon each degrade an important cell cycle regulator, helping to trigger the onset of S phase and prepare cells for the subsequent programmes of gene expression critical to polar morphogenesis and cell division.     

Localizing the subunit pool for the temporally regulated polar pili of Caulobacter crescentus

The pili of the stalked bacterium Caulobacter crescentus are assembled at a specific time in the life cycle at one pole of the cell and are composed of the monomer protein, pilin. A previous study demonstrated that the onset of pilin synthesis occurs well before pili appear on the surface, suggesting that pilin accumulates within the cell. In the present study, an electron microscope immunocytochemistry assay was used to determine the subcellular location of this unassembled pilin and its fate during pilus assembly and cell division. Populations of synchronously growing cells were embedded in epoxy resin at selected times during the cell cycle. Ultrathin sections were treated with pilin-specific antibody, followed by protein A coupled to colloidal gold. It was determined that the cellular location for unassembled pilin was the cell cytoplasm. All cell membranes and regions of nuclear material were poorly labeled. Quantitation demonstrated that label density increased during the period of pilin synthesis and declined during the period of pilus assembly and maintenance. The pilin pool was not unequally segregated at division; e.g., to the daughter cell that is elaborating pili. Mutants which have simultaneously lost the ability to produce flagella, pili, and other polar organelles, possibly due to alterations in the specialized region of polar organelle assembly, were also examined by the immunocytochemistry technique. There was no significant difference in the pilin pool size relative to the wild type, indicating that pilin synthesis continues in the absence of a functioning assembly site. This pattern of synthesis and assembly for the pilus is significantly different from that of the polar flagellum which is produced at the same time and location on the cell surface. These findings are discussed in relation to the hypothesized organization center at the cell pole which may have a major role in directing the assembly of all the polar structures.     

Caulobacter crescentus fatty acid-dependent cell cycle mutant.

A fatty acid auxotroph of Caulobacter crescentus, AE6001, which displays a strict requirement for unsaturated fatty acids to grow on glucose as the carbon source has been isolated. Starvation of AE6001 for unsaturated fatty acids resulted in a block in the cell cycle. Starved cultures accumulated at the predivisional cell stage after a round of DNA replication had been completed and after a flagellum had been assembled at the pole of the cell. Cell division and cell growth failed to occur probably because the mutant was unable to synthesize a membrane. An analysis of double mutants containing the fatB503 allele and other mutations in membrane biogenesis demonstrated that the cell cycle of AE6001 blocked at a homeostatic state. The addition of oleic acid to starved cultures permitted cell division and the initiation of a new round of DNA replication. The coincident block in both the initiation of DNA replication and membrane assembly, exhibited by starved cultures of this mutant, suggests that the fatB503 gene product may be involved in the coordination of these events.     

ppGpp and polyphosphate modulate cell cycle progression in Caulobacter crescentus

Caulobacter crescentus differentiates from a motile, foraging swarmer cell into a sessile, replication-competent stalked cell during its cell cycle. This developmental transition is inhibited by nutrient deprivation to favor the motile swarmer state. We identify two cell cycle regulatory signals, ppGpp and polyphosphate (polyP), that inhibit the swarmer-to-stalked transition in both complex and glucose-exhausted media, thereby increasing the proportion of swarmer cells in mixed culture. Upon depletion of available carbon, swarmer cells lacking the ability to synthesize ppGpp or polyP improperly initiate chromosome replication, proteolyze the replication inhibitor CtrA, localize the cell fate determinant DivJ, and develop polar stalks. Furthermore, we show that swarmer cells produce more ppGpp than stalked cells upon starvation. These results provide evidence that ppGpp and polyP are cell-type-specific developmental regulators.     

The noncoding RNA CcnA modulates the master cell cycle regulators CtrA and GcrA in Caulobacter crescentus

Bacteria are powerful models for understanding how cells divide and accomplish global regulatory programs. In Caulobacter crescentus, a cascade of essential master regulators supervises the correct and sequential activation of DNA replication, cell division, and development of different cell types. Among them, the response regulator CtrA plays a crucial role coordinating all those functions. Here, for the first time, we describe the role of a novel factor named CcnA (cell cycle noncoding RNA A), a cell cycle–regulated noncoding RNA (ncRNA) located at the origin of replication, presumably activated by CtrA, and responsible for the accumulation of CtrA itself. In addition, CcnA may be also involved in the inhibition of translation of the S-phase regulator, GcrA, by interacting with its 5′ untranslated region (5′ UTR). Performing in vitro experiments and mutagenesis, we propose a mechanism of action of CcnA based on liberation (ctrA) or sequestration (gcrA) of their ribosome-binding site (RBS). Finally, its role may be conserved in other alphaproteobacterial species, such as Sinorhizobium meliloti, representing indeed a potentially conserved process modulating cell cycle in Caulobacterales and Rhizobiales.

During cell cycle progression in the bacterium Caulobacter crescentus, the master cell cycle regulator CtrA is controlled by CcnA, a cell cycle-regulated non-coding RNA transcribed from a gene located at the origin of replication.     

Fatty acid degradation in Caulobacter crescentus.

Fatty acid degradation was investigated in Caulobacter crescentus, a bacterium that exhibits membrane-mediated differentiation events. Two strains of C. crescentus were shown to utilize oleic acid as sole carbon source. Five enzymes of the fatty acid beta-oxidation pathway, acyl-coenzyme A (CoA) synthase, crotonase, thiolase, beta-hydroxyacyl-CoA dehydrogenase, and acyl-CoA dehydrogenase, were identified. The activities of these enzymes were significantly higher in C. crescentus than the fully induced levels observed in Escherichia coli. Growth in glucose or glucose plus oleic acid decreased fatty acid uptake and lowered the specific activity of the enzymes involved in beta-oxidation by 2- to 3-fold, in contrast to the 50-fold glucose repression found in E. coli. The mild glucose repression of the acyl-CoA synthase was reversed by exogenous dibutyryl cyclic AMP. Acyl-CoA synthase activity was shown to be the same in oleic acid-grown cells and in cells grown in the presence of succinate, a carbon source not affected by catabolite repression. Thus, fatty acid degradation by the beta-oxidation pathway is constitutive in C. crescentus and is only mildly affected by growth in the presence of glucose. Tn5 insertion mutants unable to form colonies when oleic acid was the sole carbon source were isolated. However, these mutants efficiently transported fatty acids and had beta-oxidation enzyme levels comparable with that of the wild type. Our inability to obtain fatty acid degradation mutants after a wide search, coupled with the high constitutive levels of the beta-oxidation enzymes, suggest that fatty acid turnover, as has proven to be the case fatty acid biosynthesis, might play an essential role in membrane biogenesis and cell cycle events in C. crescentus.     

ClpAP is an auxiliary protease for DnaA degradation in Caulobacter crescentus

The Clp family of proteases is responsible for controlling both stress responses and normal growth. In Caulobacter crescentus, the ClpXP protease is essential and drives cell cycle progression through adaptor‐mediated degradation. By contrast, the physiological role for the ClpAP protease is less well understood with only minor growth defects previously reported for ΔclpA cells. Here, we show that ClpAP plays an important role in controlling chromosome content and cell fitness during extended growth. Cells lacking ClpA accumulate aberrant numbers of chromosomes upon prolonged growth suggesting a defect in replication control. Levels of the replication initiator DnaA are elevated in ΔclpA cells and degradation of DnaA is more rapid in cells lacking the ClpA inhibitor ClpS. Consistent with this observation, ClpAP degrades DnaA in vitro while ClpS inhibits this degradation. In cells lacking Lon, the protease previously shown to degrade DnaA in Caulobacter, ClpA overexpression rescues defects in fitness and restores degradation of DnaA. Finally, we show that cells lacking ClpA are particularly sensitive to inappropriate increases in DnaA activity. Our work demonstrates an unexpected effect of ClpAP in directly regulating replication through degradation of DnaA and expands the functional role of ClpAP in Caulobacter.     

Changes in murein composition during the cell cycle of Caulobacter crescentus

The amino acid and muropeptide compositions of murein (peptidoglycan) isolated from populations of Caulobacter crescentus predominantly composed of swarmer or stalked cells were determined and compared with the structure of murein sacculi obtained from a population of unsegregated cells. It appears that in spite of vast morphological alterations in the course of the cell cycle, the murein composition of the various cell types is not markedly different.