Cell cycle timing and developmental checkpoints in Caulobacter crescentus

Development in Caulobacter reflects a level of complexity once thought only to exist in eukaryotic cells. The cell cycle and development are not isolated from each other, but are interdependent processes. Checkpoints are in place to ensure that both cell cycle and developmental processes are completed accurately before the next stage is initiated. The timing of these processes is regulated by signal transduction networks that integrate signals from DNA replication, cell division and development. These signal transduction networks achieve precise timing of the cell cycle and development by regulating temporal gene expression, and protein activity by dynamic spatial localization within the cell and timed proteolysis.     

Cell cycle arrest of a Caulobacter crescentus secA mutant.

Cell differentiation is an inherent component of the Caulobacter crescentus cell cycle. The transition of a swarmer cell, with a single polar flagellum, into a sessile stalked cell includes several morphogenetic events. These include the release of the flagellum and pili, the proteolysis of chemotaxis proteins, the biogenesis of the polar stalk, and the initiation of DNA replication. We have isolated a group of temperature-sensitive mutants that are unable to complete this process at the restrictive temperature. We show here that one of these strains has a mutation in a homolog of the Escherichia coli secA gene, whose product is involved in protein translocation at the cell membrane. This C. crescentus secA mutant has allowed the identification of morphogenetic events in the swarmer-to-stalked cell transition that require SecA-dependent protein translocation. Upon shift to the nonpermissive temperature, the mutant secA swarmer cell is able to release the polar flagellum, degrade chemoreceptors, and initiate DNA replication, but it is unable to form a stalk, complete DNA replication, or carry out cell division. At the nonpermissive temperature, the cell cycle blocks prior to the de novo synthesis of flagella and chemotaxis proteins that normally occurs in the predivisional cell. Although interactions between the chromosome and the cytoplasmic membrane are believed to be a functional component of the temporal regulation of DNA replication, the ability of this secA mutant to initiate replication at the nonpermissive temperature suggests that SecA-dependent events are not involved in this process. However, both cell division and stalk formation, which is analogous to a polar division event, require SecA function.     

Synchronous Cell Differentiation in Caulobacter crescentus

The growth of a stalked bacterium, Caulobacter crescentus, has been synchronized easily and reproducibly by a new method. When this bacterium is grown to a late log phase in nutrient broth at 30 C with aeration, swarmer cells are accumulated in the culture to 80% of the whole cell population. When this culture is inoculated into fresh pre-warmed broth at twentyfold dilution, it immediately initiates synchronous cell growth. Simultaneously, synchronous cell differentiation is monitored by the susceptibility of the cells to RNA phage infection. The swarmer cells accumulated in the late log phase of growth possess nearly the same susceptibility to RNA phage infection as those in the early log phase of growth while RNA phage-adsorbing capacity is lower in such swarmer cells. It is suggested that the swarmer cells accumulated in the late log phase of growth have lost some pili.     

Screen for localized proteins in Caulobacter crescentus

Precise localization of individual proteins is required for processes such as motility, chemotaxis, cell-cycle progression, and cell division in bacteria, but the number of proteins that are localized in bacterial species is not known. A screen based on transposon mutagenesis and fluorescence activated cell sorting was devised to identify large numbers of localized proteins, and employed in Caulobacter crescentus. From a sample of the clones isolated in the screen, eleven proteins with no previously characterized localization in C. crescentus were identified, including six hypothetical proteins. The localized hypothetical proteins included one protein that was localized in a helix-like structure, and two proteins for which the localization changed as a function of the cell cycle, suggesting that complex three-dimensional patterns and cell cycle-dependent localization are likely to be common in bacteria. Other mutants produced localized fusion proteins even though the transposon has inserted near the 5' end of a gene, demonstrating that short peptides can contain sufficient information to localize bacterial proteins. The screen described here could be used in most bacterial species.     

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.     

Cell cycle control of cell morphogenesis in Caulobacter.

In Caulobacter crescentus, morphogenic events, such as cytokinesis, the establishment of asymmetry and the biogenesis of polar structures, are precisely regulated during the cell cycle by internal cues, such as cell division and the initiation of DNA replication. Recent studies have revealed that the converse is also true. That is, differentiation events impose regulatory controls on other differentiation events, as well as on progression of the cell cycle. Thus, there are pathways that sense the assembly of structures or the localization of complexes and then transduce this information to subsequent biogenesis or cell cycle events. In this review, we examine the interplay between flagellar assembly and the C. crescentus cell cycle.     

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.     

Utilization of histidine by Caulobacter crescentus

Caulobacter crescentus has an inducible pathway which is responsible for the degradation of histidine. Induction of this pathway occurs in the presence of both glucose and ammonia. Growth yield experiments indicate that only two of the three available nitrogens are used for growth suggesting that formamide may be produced as a waste product. However, formamide was not detected in the culture fluid and formate was formed instead. These results suggest that histidine may be degraded in a novel pathway which results in the production of 1 mol each of ammonia, glutamate and formate per mol of histidine. The third nitrogen from histidine appears to be sequestered in some kind of secondary metabolite.     

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.     

Biomineralization of Uranium by PhoY Phosphatase Activity Aids Cell Survival in Caulobacter crescentus

Caulobacter crescentus is known to tolerate high levels of uranium [U(VI)], but its detoxification mechanism is poorly understood. Here we show that C. crescentus is able to facilitate U(VI) biomineralization through the formation of U-P_i precipitates via its native alkaline phosphatase activity. The U-P_i precipitates, deposited on the cell surface in the form of meta-autunite structures, have a lower U/P_i ratio than do chemically produced precipitates. The enzyme that is responsible for the phosphatase activity and thus the biomineralization process is identified as PhoY, a periplasmic alkaline phosphatase with broad substrate specificity. Furthermore, PhoY is shown to confer a survival advantage on C. crescentus toward U(VI) under both growth and nongrowth conditions. Results obtained in this study thus highlight U(VI) biomineralization as a resistance mechanism in microbes, which not only improves our understanding of bacterium-mineral interactions but also aids in defining potential ecological niches for metal-resistant bacteria.     

Cell cycle and positional constraints on FtsZ localization and the initiation of cell division in Caulobacter crescentus

Swarmer cells of Caulobacter crescentus are devoid of the cell division initiation protein FtsZ and do not replicate DNA. FtsZ is synthesized during the differentiation of swarmer cells into replicating stalked cells. We show that FtsZ first localizes at the incipient stalked pole in differentiating swarmer cells. FtsZ subsequently localizes at the mid-cell early in the cell cycle. In an effort to understand whether Z-ring formation and cell constriction are driven solely by the cell cycle-regulated increase in FtsZ concentration, FtsZ was artificially expressed in swarmer cells at a level equivalent to that found in predivisional cells. Immunofluorescence microscopy showed that, in these swarmer cells, simply increasing FtsZ concentration was not sufficient for Z-ring formation; Z-ring formation took place only in stalked cells. Expression of FtsZ in swarmer cells did not alter the timing of cell constriction initiation during the cell cycle but, instead, caused additional constrictions and a delay in cell separation. These additional constrictions were confined to sites close to the original mid-cell constriction. These results suggest that the timing and placement of Z-rings is tightly coupled to an early cell cycle event and that cell constriction is not solely dependent on a threshold level of FtsZ.     

RNA-controlled regulation in Caulobacter crescentus

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Asymmetric segregation of heat-shock proteins upon cell division in Caulobacter crescentus

Three Caulobacter crescentus heat-shock proteins were shown to be immunologically related to the Escherichia coli heat-shock proteins GroEL, Lon and DnaK. A fourth heat-shock protein was detected with antibody to the C. crescentus RNA polymerase. This 37,000 Mr heat-shock protein might be related to the E. coli 32,000 Mr heat-shock sigma subunit. The synthesis of the major C. crescentus RNA polymerase sigma factor was not induced by heat shock. The E. coli GroEL protein and the related protein from C. crescentus were also induced by treatment with hydrogen peroxide. Like some of the proteins in the heat-shock protein families of Drosophila and yeast, the four heat-shock proteins in C. crescentus were found to be regulated developmentally under normal conditions. All four proteins were synthesized in the predivisional cell, but the progeny showed cell type-specific bias in the level of enhanced synthesis after heat shock. The 92,000 Mr Lon homolog and the 37,000 Mr RNA polymerase subunit were preferentially synthesized in the stalked cell, whereas the synthesis of the 62,000 Mr GroEL homolog was enhanced in the progeny swarmer cell. Furthermore, the four heat-shock proteins synthesized in the predivisional cell were partitioned in a specific manner upon cell division. The stalked cell, which initiates chromosome replication immediately upon division, received the Lon homolog, the DnaK homolog and the 37,000 Mr RNA polymerase subunit. The GroEL homolog, however, was distributed equally to both the stalked cell and the swarmer cell. These results provide access to the functions of C. crescentus heat-shock proteins under both normal and stress conditions. They also allow an investigation of the regulatory signals that modulate the asymmetric distribution of proteins and their subsequent cell type-specific expression in the initial stages of a developmental program.