Genes directly controlled by CtrA, a master regulator of the Caulobacter cell cycle

Abstract

Studies of the genetic network that controls the Caulobacter cell cycle have identified a response regulator, CtrA, that controls, directly or indirectly, one-quarter of the 553 cell cycle-regulated genes. We have performed in vivo genomic binding site analysis of the CtrA protein to identify which of these genes have regulatory regions bound directly by CtrA. By combining these data with previous global analysis of cell cycle transcription patterns and gene expression profiles of mutant ctrA strains, we have determined that CtrA directly regulates at least 95 genes. The total group of CtrA-regulated genes includes those involved in polar morphogenesis, DNA replication initiation, DNA methylation, cell division, and cell wall metabolism. Also among the genes in this notably large regulon are 14 that encode regulatory proteins, including 10 two-component signal transduction regulatory proteins. Identification of additional regulatory genes activated by CtrA will serve to directly connect new regulatory modules to the network controlling cell cycle progression.

Figure 1

Temporal coordination of Caulobacter cell cycle events. The swarmer cell has a nonreplicating chromosome and a polar flagellum and pili. At the swarmer-to-stalked cell transition, the pili and the flagellum are lost, and a stalk is formed at that same pole, coincident with the initiation of DNA replication. Construction of a new flagellum at the pole opposite the stalk occurs during S phase. The cell then divides asymmetrically, yielding two distinct daughter cells: a stalked cell that initiates a new round of DNA replication and a smaller swarmer cell that cannot replicate its chromosome until after it differentiates into a stalked cell. Timing of several key cell cycle-regulated events is indicated by the black and gray bars below. CtrA is present in cell types shaded gray and controls genes or events involved in the cell cycle processes with gray timing bars.

Figure 2

Genome-wide analysis of in vivo CtrA-binding locations using IP and microarrays. (A) Schematic of the procedure for identifying intergenic regions to which CtrA is bound in vivo, as described in the text. Histograms are shown for the mean percentile ranks of the 1,549 intergenic regions in the CtrA-IP experiments (B) and in the mock-IP experiments (C). Intergenic regions enriched in the CtrA-IP experiments relative to the mock-IP experiments with a confidence level >95% are shown in orange in the CtrA-IP histogram (B).

Figure 3

Intersections of genomic data sets. The diagram summarizes data for the 116 (of 197) genes whose upstream regulatory regions were enriched by the IP-based binding analysis and which had valid data from expression profiling of wild-type and ctrA mutant strains. Of 116 gene, 88 were identified as CtrA-dependent for normal expression levels, and 69 were identified as cell cycle-regulated. The 55 genes within the overlap of all three data sets are identified here as members of the CtrA cell cycle regulon, as described in the text.

Figure 4

Expression profiles for genes in the CtrA regulon. Wild-type and ctrA^ts expression data, along with CC number, are shown for the 55 genes satisfying all three criteria for members of the CtrA regulon (Fig. 3). Other genes potentially in the same operon are listed to the right, separated by slashes. For wild-type profiles, expression ratios of RNA from each time point have been compared with a common reference and are encoded using the scale at the bottom (4). The column labeled “change in ctrA^ts ” indicates, with either a green or red box, genes that were significantly decreased or increased, respectively, in response to loss of CtrA function in the ctrA401^ts strain. Regulatory genes are listed in orange (see also Fig. 5).

Figure 5

Regulatory genes directly controlled by CtrA. The 14 regulatory genes in the CtrA cell cycle regulon are listed below the cell cycle timeline according to their approximate time of peak expression in wild-type cells. Numbering of the histidine kinases (HK), response regulators (RR), and hybrid histidine kinase (HK-RR) follows that in ref. . Hybrid histidine kinases are those kinases with a fused response regulator domain at their C-terminal ends.