Role of core promoter sequences in the mechanism of swarmer cell-specific silencing of gyrB transcription in Caulobacter crescentus

Background  

Each Caulobacter crescentus cell division yields two distinct cell types: a flagellated swarmer cell and a non-motile stalked cell. The swarmer cell is further distinguished from the stalked cell by an inability to reinitiate DNA replication, by the physical properties of its nucleoid, and its discrete program of gene expression. Specifically, with regard to the latter feature, many of the genes involved in DNA replication are not transcribed in swarmer cells.     

Chromosomes segregration and development in Caulobacter crescentus

The pattern of genome segregation to progeny stalked and swarmer cells of Caulobacter crescentus has been determined in a study of the localization of information in developing cells. The genome of stalked cells was labeled with [³H]deoxy-guanosine to mark one of the two DNA strands preferentially. The segregation of this labeled strand after one or more rounds of replication and division in non-radioactive medium was determined by (a) the rate of accumulation of radio-activity during three successive generations of swarmer cells released from labeled stalked cells which were attached to glass plates, and (b) electron microscopy autoradiography of stalked and swarmer cell progeny of labeled stalked cells. The results indicate that most of the DNA of a given age in C. crescentus segre-gates randomly to the two cell types at division, and that the genome probably segregates as a single chromosomal unit.     

Chromosome segregation in an asymmetrically dividing bacterium,Caulobacter crescentus

The mode of chromosome segregation in an asymmetrically dividing bacterium, Caulobacter crescentus, was studied by examining the fate of labeled DNA strands. Swarmer cells (one type of Caulobacter daughter cell), in which single strands of DNA had been labeled with [³H]thymidine during the previous round of chromosome replication, were grown synchronously in a non-radioactive medium for two generations. The distribution of radioactivity among the cells was visualized by autoradiography under a phase-contrast microscope. The labeled DNA strands in each cell were found to consist of two conserved units. From this, we propose a model in which the swarmer cell has two identical chromosomes, which are segregated into the progeny swarmer cell and the progeny stalked cell after chromosome replication.     

Effects of (p)ppGpp on the Progression of the Cell Cycle of Caulobacter crescentus

Bacteria must control the progression of their cell cycle in response to nutrient availability. This regulation can be mediated by guanosine tetra- or pentaphosphate [(p)ppGpp], which are synthesized by enzymes of the RelA/SpoT homologue (Rsh) family, particularly under starvation conditions. Here, we study the effects of (p)ppGpp on the cell cycle of Caulobacter crescentus, an oligotrophic bacterium with a dimorphic life cycle. C. crescentus divides asymmetrically, producing a motile swarmer cell that cannot replicate its chromosome and a sessile stalked cell that is replication competent. The swarmer cell rapidly differentiates into a stalked cell in appropriate conditions. An artificial increase in the levels of (p)ppGpp in nonstarved C. crescentus cells was achieved by expressing a truncated relA gene from Escherichia coli, encoding a constitutively active (p)ppGpp synthetase. By combining single-cell microscopy, flow cytometry approaches, and swarming assays, we show that an increase in the intracellular concentration of (p)ppGpp is sufficient to slow down the swarmer-to-stalked cell differentiation process and to delay the initiation of chromosome replication. We also present evidence that the intracellular levels of two master regulators of the cell cycle of C. crescentus, DnaA and CtrA, are modulated in response to (p)ppGpp accumulation, even in the absence of actual starvation. CtrA proteolysis and DnaA synthesis seem indirectly inhibited by (p)ppGpp accumulation. By extending the life span of the motile nonreproductive swarmer cell and thus promoting dispersal and foraging functions over multiplication under starvation conditions, (p)ppGpp may play a central role in the ecological adaptation of C. crescentus to nutritional stresses.     

Synthesis of specific membrane proteins is a function of DNA replication and phospholipid synthesis in Caulobacter crescentus

Analysis of the effects on membrane function and protein composition of altering phospholipid synthesis in Caulobacter crescentus showed that, like other bacteria, C. crescentus continues to induce a lactose transport system and to synthesize most membrane proteins. However, we show that the incorporation of a set of outer membrane proteins primarily synthesized in stalked cells is dependent on DNA replication which, in turn, is dependent on membrane phospholipid synthesis. Furthermore, the incorporation of another set of membrane proteins, two of which are synthesized primarily in the swarmer cell, appears to be independent of the replication of the chromosome but to be directly dependent on phospholipid synthesis. We have also found that when phospholipid synthesis is blocked, the synthesis of the flagellar proteins is inhibited and that this effect may be mediated by the primary inhibition of DNA replication. Newton has presented evidence that the synthesis of flagellar proteins is dependent on specific execution points in DNA replication and that this connection serves as a temporal regulator of differential protein synthesis (Osley et al., 1977; Sheffery & Newton, 1981). We suggest here that a direct link between the replicating chromosome and the growing membrane might serve, in turn, to dictate the site of membrane assembly of newly synthesized gene products.     

Chromosome methylation and measurement of faithful, once and only once per cell cycle chromosome replication in Caulobacter crescentus

Caulobacter crescentus exhibits cell-type-specific control of chromosome replication and DNA methylation. Asymmetric cell division yields a replicating stalked cell and a nonreplicating swarmer cell. The motile swarmer cell must differentiate into a sessile stalked cell in order to replicate and execute asymmetric cell division. This program of cell division implies that chromosome replication initiates in the stalked cell only once per cell cycle. DNA methylation is restricted to the predivisional cell stage, and since DNA synthesis produces an unmethylated nascent strand, late DNA methylation also implies that DNA near the replication origin remains hemimethylated longer than DNA located further away. In this report, both assumptions are tested with an engineered Tn5-based transposon, Tn5Omega-MP. This allows a sensitive Southern blot assay that measures fully methylated, hemimethylated, and unmethylated DNA duplexes. Tn5Omega-MP was placed at 11 sites around the chromosome and it was clearly demonstrated that Tn5Omega-MP DNA near the replication origin remained hemimethylated longer than DNA located further away. One Tn5Omega-MP placed near the replication origin revealed small but detectable amounts of unmethylated duplex DNA in replicating stalked cells. Extra DNA synthesis produces a second unmethylated nascent strand. Therefore, measurement of unmethylated DNA is a critical test of the "once and only once per cell cycle" rule of chromosome replication in C. crescentus. Fewer than 1 in 1,000 stalked cells prematurely initiate a second round of chromosome replication. The implications for very precise negative control of chromosome replication are discussed with respect to the bacterial cell cycle.     

Plasmid and chromosomal DNA replication and partitioning during the Caulobacter crescentus cell cycle

Cell division in Caulobacter crescentus yields a swarmer and a stalked cell. Only the stalked cell progeny is able to replicate its chromosome, and the swarmer cell progeny must differentiate into a stalked cell before it too can replicate its chromosome. In an effort to understand the mechanisms that limit chromosomal replication to the stalked cell, plasmid DNA synthesis was analyzed during the developmental cell cycle of C. crescentus, and the partitioning of both the plasmids and the chromosomes to the progeny cells was examined. Unlike the chromosome, plasmids from the incompatibility groups Q and P replicated in all C. crescentus cell types. However, all plasmids tested showed a ten- to 20-fold higher replication rate in the stalked cells than the swarmer cells. We observed that all plasmids replicated during the C. crescentus cell cycle with comparable kinetics of DNA synthesis, even though we tested plasmids that encode very different known (and putative) replication proteins. We determined the plasmid copy number in both progeny cell types, and determined that plasmids partitioned equally to the stalked and swarmer cells. We also reexamined chromosome partitioning in a recombination-deficient strain of C. crescentus, and confirmed an earlier report that chromosomes partition to the progeny stalked and swarmer cells in a random manner that does not discriminate between old and new DNA strands.     

Caulobacter crescentus pili: analysis of production during development

The pili of Caulobacter crescentus are structures whose appearance is regulated during the development of the swarmer cell pole. Pili are assembled during the predivisional and swarmer cell stages, at the same time as the flagellum, and disappear as the swarmer cell differentiates into a stalked cell. Pilin is the protein which polymerizes to form the pilus. An immune precipitation assay, developed to examine the periodicity of pilin synthesis during the cell cycle, demonstrated that pilin synthesis begins in the early stalked cell and is probably completed before cell division. Thus, the entire period of synthesis occurs before the pili are clearly visible at the differentiated cell pole. Likewise, the functional stability of the pilin mRNA is relatively short, further suggesting that the protein monomers accumulate prior to assembly. Unlike the case of the flagellins, experiments with the DNA replication inhibitor hydroxyurea did not establish a correlation between the DNA replication and the onset of pilin synthesis. In addition to pilin, several other developmentally regulated proteins, including the flagellins, are reproducibly precipitated in the pilin immunoassay. Their presence in the precipitate is a specific consequence of the antipilin antibody. Analysis of the antibody preparations yielded conflicting results; electron microscopic studies with ferritin-coupled antibody and double diffusion analysis indicated no binding activity to any cell components other than pilin. However, an assay based on filter transferred preparations of electrophoresed cell proteins indicated that at least one additional class of proteins in the immune precipitate may bind pilin antibody. These results are discussed in the context of the possible formation of a discrete membrane complex in the polar region of the cell which may be involved in the regulation of spatial development in Caulobacter.     

SpoT regulates DnaA stability and initiation of DNA replication in carbon-starved Caulobacter crescentus

Cell cycle progression and polar differentiation are temporally coordinated in Caulobacter crescentus. This oligotrophic bacterium divides asymmetrically to produce a motile swarmer cell that represses DNA replication and a sessile stalked cell that replicates its DNA. The initiation of DNA replication coincides with the proteolysis of the CtrA replication inhibitor and the accumulation of DnaA, the replication initiator, upon differentiation of the swarmer cell into a stalked cell. We analyzed the adaptive response of C. crescentus swarmer cells to carbon starvation and found that there was a block in both the swarmer-to-stalked cell polar differentiation program and the initiation of DNA replication. SpoT is a bifunctional synthase/hydrolase that controls the steady-state level of the stress-signaling nucleotide (p)ppGpp, and carbon starvation caused a SpoT-dependent increase in (p)ppGpp concentration. Carbon starvation activates DnaA proteolysis (B. Gorbatyuk and G. T. Marczynski, Mol. Microbiol. 55:1233-1245, 2005). We observed that SpoT is required for this phenomenon in swarmer cells, and in the absence of SpoT, carbon-starved swarmer cells inappropriately initiated DNA replication. Since SpoT controls (p)ppGpp abundance, we propose that this nucleotide relays carbon starvation signals to the cellular factors responsible for activating DnaA proteolysis, thereby inhibiting the initiation of DNA replication. SpoT, however, was not required for the carbon starvation block of the swarmer-to-stalked cell polar differentiation program. Thus, swarmer cells utilize at least two independent signaling pathways to relay carbon starvation signals: a SpoT-dependent pathway mediating the inhibition of DNA replication initiation, and a SpoT-independent pathway(s) that blocks morphological differentiation.     

Dynamical Modeling of the Cell Cycle and Cell Fate Emergence in Caulobacter crescentus

The division of Caulobacter crescentus, a model organism for studying cell cycle and differentiation in bacteria, generates two cell types: swarmer and stalked. To complete its cycle, C. crescentus must first differentiate from the swarmer to the stalked phenotype. An important regulator involved in this process is CtrA, which operates in a gene regulatory network and coordinates many of the interactions associated to the generation of cellular asymmetry. Gaining insight into how such a differentiation phenomenon arises and how network components interact to bring about cellular behavior and function demands mathematical models and simulations. In this work, we present a dynamical model based on a generalization of the Boolean abstraction of gene expression for a minimal network controlling the cell cycle and asymmetric cell division in C. crescentus. This network was constructed from data obtained from an exhaustive search in the literature. The results of the simulations based on our model show a cyclic attractor whose configurations can be made to correspond with the current knowledge of the activity of the regulators participating in the gene network during the cell cycle. Additionally, we found two point attractors that can be interpreted in terms of the network configurations directing the two cell types. The entire network is shown to be operating close to the critical regime, which means that it is robust enough to perturbations on dynamics of the network, but adaptable to environmental changes.     

Cell-cycle control of a cloned chromosomal origin of replication from Caulobacter crescentus.

Caulobacter crescentus cell division is asymmetric and yields distinct swarmer cell and stalked cell progeny. Only the stalked cell initiates chromosomal replication, and the swarmer cell must differentiate into a stalked cell before chromosomal DNA replication can occur. In an effort to understand this developmental control of replication, we employed pulsed-field gel electrophoresis to localize and to isolate the chromosomal origin of replication. The C. crescentus homologues of several Escherichia coli genes are adjacent to the origin in the physical order hemE, origin, dnaA and dnaK,J. Deletion analysis reveals that the minimal sequence requirement for autonomous replication is greater than 430 base-pairs, but less than 720 base-pairs. A plasmid, whose replication relies only on DNA from the C. crescentus origin of replication, has a distinct temporal pattern of DNA synthesis that resembles that of the bona fide C. crescentus chromosome. This implies that cis-acting replication control elements are closely linked to this origin of replication. This DNA contains sequence motifs that are common to other bacterial origins, such as five DnaA boxes, an E. coli-like 13-mer, and an exceptional A + T-rich region. Point mutations in one of the DnaA boxes abolish replication in C. crescentus. This origin also possesses three additional motifs that are unique to the C. crescentus origin of replication: seven 8-mer (GGCCTTCC) motifs, nine 8-mer (AAGCCCGG) motifs, and five 9-mer (GTTAA-n7-TTAA) motifs are present. The latter two motifs are implicated in essential C. crescentus replication functions, because they are contained within specific deletions that abolish replication.     

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.     

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.     

Pattern of unequal cell division and development in Caulobacter crescentus

The pattern of asymmetric division has been examined in Caulobacter crescentus (gram-negative aquatic bacteria) by determining the position of the “division site” on cells of different ages. Measurements of cell width and length at this site, which corresponds to the point of eventual cell separation, were made on electron micrographs of cells stained with phosphotungstic acid. The results show that (i) the division site is formed early in the cell cycle and it constitutes the first visible feature on the growing stalked cell to differentiate the incipient swarmer cell, (ii) the division site is located asymmetrically (closer to the swarmer pole than the stalked pole) on the dividing cell, (iii) its position relative to the stalked and swarmer poles does not change during the cell cycle, and (iv) division is consequently unequal, with the swarmer cell always smaller than the stalked cell. The implications of these findings for general models of unequal cell division and stem cell development are discussed.