Temporal Regulation of Genes Encoding the Flagellar Proximal Rod in Caulobacter crescentus

A fundamental aspect of biology is the mechanism by which cells are able to control their progression from one stage of the cell cycle to the next in a manner that ensures that all necessary cellular functions are successfully completed. Study of the cell cycle in bacteria has proceeded slowly, primarily due to the difficulty of obtaining pure populations of cells at a particular point in the cell cycle. The gram-negative aquatic bacterium Caulobacter crescentus undergoes a life cycle which, upon division, results in two dissimilar daughter cells: a sessile stalked cell and a motile swarmer cell that possesses a single polar flagellum. The swarmer cell stage lasts for approximately one-third of the life cycle, after which the swarmer differentiates into a stalked cell by ejecting the flagellum and growing a stalk at the pole of the cell that previously harbored the flagellum. Initiation of chromosomal replication depends on this differentiation event, so that swarmer cells are unable to initiate replication. The two cell types are easily separable by differential density gradient centrifugation because stalked cells are less dense than swarmer cells, so a pure population of swarmer cells can be obtained which can be used to study cell cycle-dependent phenomena. The aspect of the C. crescentus cell cycle that has been most thoroughly studied is the biogenesis of the single polar flagellum (reviewed in references 5, 15, 16, and 66). The flagellar regulon in C. crescentus is arranged in a complex, trans-acting hierarchy composed of approximately 50 genes (11, 12, 31) organized into four levels of gene expression, in which a complete complement of gene products from the early levels is required for proper expression of genes later in the hierarchy (6, 38, 50, 52, 69). In addition, the order of expression of the levels of the hierarchy reflects their order of assembly from the cytoplasm of the cell outward. Cell-proximal components such as inner basal body proteins are expressed before more distal components such as the flagellar hook and filament (6, 7, 17, 19, 34, 41, 46–48). The first level of the hierarchy is occupied by the cell cycle response regulator CtrA, which in response to cell cycle cues activates the transcription of early flagellar genes (55, 59, 68). Class II genes encode proteins that make up the basal body of the flagellum, including the MS ring (encoded by fliF) (29, 52), which anchors the flagellum in the inner membrane of the cell and is the first component to be assembled; the flagellum-specific protein export apparatus (including fliO, fliP, fliQ, fliR, and flhA) (20, 58, 69, 63, 71); the switch components of the flagellum (fliG, fliM, and fliN) (52, 70); and transcription factors flbD (4, 49, 52, 57, 64, 67) and rpoN (2, 5), which are required for expression of class III and IV genes. All the promoters for class II genes share a distinctive motif (20, 52, 60, 63, 71) that includes the binding site for CtrA (55, 59, 68), which along with RNA polymerase holoenzyme containing ς70 (68) is thought to be responsible for transcription of genes in this level of the hierarchy. All of the class II components must be properly expressed before transcription of the class III level of the hierarchy can commence. The master regulator of the flagellar regulon, CtrA, apparently has multiple roles in the C. crescentus cell cycle. In response to a cell cycle cue, CtrA is thought to bind to 9-mer sequences (TTAA-N7-TTAA) at the origin and at many promoters (28, 40, 41). Binding to the origin has the effect of preventing replication initiation, while binding to the promoters of flagellar genes activates transcription. CtrA is proteolytically degraded in stalked cells following formation of the septum between two incipient daughter cells, so that following division replication can immediately commence in that cell type (10). The presence of CtrA in the swarmer cell ensures that replication is not initiated inappropriately in swarmer cells until the swarmer-to-stalked cell transition has occurred, at which time the CtrA that has remained in that cell type is degraded, thus relieving repression of DNA replication. In addition to its role in suppressing reinitiation of chromosomal replication and in flagellar gene regulation, CtrA also regulates transcription of the critical cell division gene ftsZ (33). Thus, CtrA has an important role in uniting three distinct morphological-developmental processes in C. crescentus cells. Class III genes encode proteins which compose both the outer components of the basal body and the flagellar hook, which is a protein structure that serves as a flexible universal joint connecting the rod to the flagellar filament. In gram-negative bacteria, the rod spans the periplasm and is composed of five proteins: FlgB, FlgC, FlgF, FlgG, and FliE (1, 22, 32, 45). The distal rod, which spans most of the distance between the inner and outer membranes, is composed of FlgF and FlgG. The proximal rod connects the distal rod to the flagellar motor and consists of FlgB and FlgC. FliE is required for assembly of the proximal rod, although it is itself a protein of unknown function; it may serve as an adapter between the radially symmetric components below the rod and the helically symmetric rod components of the flagellum (43, 45). The C. crescentus genome sequencing project was used to identify and clone the three components of the proximal rod. Here we report the timing and level of expression of flgB. Additionally, we have determined the start site of transcription of flgB and found that it is close (53 bases) to the start site of a divergent operon that contains the class II component fliP. Epistasis experiments demonstrate that the transcription of these genes does not require complete class II gene expression and that neither does class III gene expression depend on successful expression of the proximal rod genes, indicating that they lie outside the flagellar regulatory hierarchy. In addition, we show that both flgB and fliP are dependent on the transcriptional regulator CtrA for proper levels of expression.