Cell cycle regulation in Caulobacter: location, location, location

Erin D. Goley 0 Antonio A. Iniesta 0 Lucy Shapiro ) 0 0 Department of Developmental Biology, Beckman Center, Stanford University School of Medicine , 279 Campus Drive, Stanford, CA 94305 , USA - Summary Cellular reproduction in all organisms requires temporal and spatial coordination of crucial events, notably DNA replication, chromosome segregation and cytokinesis. Recent studies on the dimorphic bacterium Caulobacter crescentus (Caulobacter) highlight mechanisms by which positional information is integrated with temporal modes of cell cycle regulation. Caulobacter cell division is inherently asymmetric, yielding progeny with different fates: stalked cells and swarmer cells. Cell type determinants in stalked progeny promote entry into S ce phase, whereas swarmer progeny remain in G1 phase. en Moreover, initiation of DNA replication is allowed only ic once per cell cycle. This finite window of opportunity is S l l e C fo Introduction l The fundamental cell cycle events of a prokaryotic cell are the a n same as those of a eukaryotic cell: the genome is replicated, ru duplicated DNA is segregated into daughter compartments and Jo cytokinesis separates the cell into two, all of which are coordinated with cell growth. Accurate execution of each of these steps relative to the others in time and space is crucial for the survival of progeny, necessitating the existence of strict regulatory mechanisms that ensure their fidelity. In eukaryotes, the dramatic metaphase alignment of chromosomes and morphological changes accompanying cytokinesis convinced cell biologists early on that sophisticated programs of spatial regulation must be integrated with those that control the timing of cell cycle events. Analogous processes occur in prokaryotes; yet bacterial cells were long regarded as little more than living test tubes that could probably get by with timing mechanisms and relatively simple self-organizing capabilities. Classic research into cell cycle control in bacteria was focused, therefore, on defining the enzymology and genetic and chemical signals that promote DNA replication, chromosome partitioning and cytokinesis. The past decade or so has seen a renaissance in bacterial cell biology, with the recognition that the interior of the bacterial cell is highly organized: it is replete with specifically and dynamically localized proteins, DNA and other biomolecules, and possesses a surprisingly diverse cytoskeleton (Ebersbach and Jacobs-Wagner, 2007; Gitai, 2005; Lewis, 2004; MollerJensen and Lowe, 2005). This paradigm shift has been spurred on by advanced labeling and imaging technologies that make even the smallest bacterial cells amenable to cell biological probing. Within this context, we have begun to appreciate the imposed by coordinating spatially constrained proteolysis of CtrA, an inhibitor of DNA replication initiation, with forward progression of the cell cycle. Positional cues are equally important in coordinating movement of the chromosome with cell division site selection in Caulobacter. The chromosome is specifically and dynamically localized over the course of the cell cycle. As the duplicated chromosomes are partitioned, factors that restrict assembly of the cell division protein FtsZ associate with a chromosomal locus near the origin, ensuring that the division site is located towards the middle of the cell. importance of the physical choreography of molecules during the bacterial cell cycle. In this Commentary, we discuss cell cycle control in the context of the three-dimensional organization of the bacterium Caulobacter crescentus (referred to hereafter as Caulobacter), focusing on recent advances in our understanding of the elegant spatial mechanisms that govern the initiation of DNA replication and cell division site selection. The Caulobacter cell cycle Caulobacter is a Gram-negative, aquatic -proteobacterium that has emerged as the pre-eminent model for analysis of prokaryotic cell cycle regulation. It is easily cultured and manipulated genetically in the laboratory, and has the advantage that one can easily synchronize cells without perturbing their normal physiology: a simple density centrifugation procedure yields a relatively pure population of cells in G1 phase, which progress coordinately through the cell cycle (Evinger and Agabian, 1977). This allows precise monitoring of all aspects of Caulobacter cell cycle progression. Additionally, morphological changes that occur over the course of the Caulobacter life cycle and are intimately coupled to other cell cycle events serve as faithful visual identifiers of the cell cycle status of any given cell. Caulobacter undergo an asymmetric division each cell cycle to produce morphologically distinct progeny that have different fates: a motile, flagellated swarmer cell and a non-motile stalked cell (Fig. 1A). The swarmer cell is unable to enter S phase until it differentiates into a stalked cell, releasing its flagellum and building a stalk where the flagellum previously resided. Upon this swarmer-to-stalked cell transition (analogous to the G1-S transition in eukaryotes), the cell becomes competent to initiate DNA replication, an event that occurs exactly once per cell cycle. Replication initiates at a single origin and proceeds bidirectionally (Dingwall and Shapiro, 1989; Marczynski and Shapiro, 1992), with chromosomal loci being segregated to daughter compartments soon after they are duplicated (Viollier et al., 2004). Concurrently with DNA replication and segregation, the stalked cell elongates and, just as replication is completed, begins to constrict at the incipient cell division site (Jensen, 2006). A flagellum is assembled at the pole opposite the stalk in the predivisional cell, and the completion of cytokinesis yields a swarmer daughter and a stalked daughter. In addition to the differences in polar morphology between swarmer and stalked cells, they also differ in size, with swarmer cells being smaller than stalked cells. The stalked daughter immediately Stage in the cell cycle Fig. 1. The Caulobacter cell cycle. (A) Each Caulobacter cell division yields a swarmer cell and a stalked cell. Upon differentiating into a stalked cell, the swarmer cell sheds its flagellum, builds a stalk and initiates DNA replication (the chromosome is depicted as a circular black line and as a structure during replication). Just as DNA replication and segregation are concluding, the predivisional cell begins to constrict at the nascent division site. A flagellum is constructed at the pole opposite the stalk, and the completion of cytokinesis generates a new stalked cell and a new swarmer cell. (B) The forward progression of the cell cycle is driven by three master regulators: CtrA, DnaA and GcrA. The levels of each protein oscillate in time over the course of the cell cycle, as indicated graphically, and they successively regulate the transcription of ~200 genes. begins another round of DNA replication and cell di (...truncated)