Negative control of bacterial DNA replication by a cell cycle regulatory protein that binds at the chromosome origin

Abstract

Caulobacter crescentus divides asymmetrically generating two distinct cell types at each cell division: a stalked cell competent for DNA replication, and a swarmer cell that is unable to initiate DNA replication until it differentiates into a stalked cell later in the cell cycle. The CtrA protein, a member of the response regulator family of the two-component signal transduction system, controls multiple cell cycle processes in Caulobacter and is present in swarmer cells but absent from stalked cells. We report that CtrA binds five sites within the chromosome replication origin in vitro. These sites overlap an essential DnaA box and a promoter in the origin that is essential for replication initiation. Analysis of mutant alleles of ctrA and point mutations in one of the CtrA binding sites in the origin demonstrate that CtrA represses replication in vivo. CtrA-mediated repression at the origin thus restricts replication to the stalked cell type. Thus, the direct coupling of chromosome replication with the cell cycle is mediated by the ubiquitous two-component signaling proteins.

Figure 1

Initiation of chromosome replication during the Caulobacter cell cycle negatively correlates with CtrA protein abundance. The flagellated swarmer cell (Sw) differentiates into a stalked cell (St) that initiates chromosome replication and asymmetric cell division yielding replicating (rep+) and nonreplicating (rep−) progeny (4, 5). The shading shows that CtrA is restricted to the swarmer cells and to the swarmer compartment of the predivisional cell (17). The graphs present the analysis of synchronized cells assayed simultaneously for CtrA protein abundance and the percentage of the chromosome replicated as the swarmer cells differentiate into stalked cells. The relative abundance of CtrA was determined by Western blot analysis of samples taken at the indicated times in a synchronous cell cycle, and replication state was determined by an assay of the relative amount of fully methylated and hemimethylated DNA as described in the text.

Figure 2

CtrA protein binds five sites within the Caulobacter chromosome replication origin (Cori). (A) Schematic of Cori. The thick horizontal line shows the minimal DNA sequences that support autonomous Cori plasmid replication (7). The dumbbell-shaped symbols denote the 9-mer motif GTTAA-N7-TTAA sequences (a–e), and short horizontal arrows mark DnaA boxes (6, 8). Bent arrows mark the RNA start sites for the “weak” (+201) and “strong” (+251) promoters (7). Restriction endonuclease sites: B, BamHI; Bg, BglII; E, EcoRI; Hp, HpaI; P, PstI. (B) DNase I protection footprinting experiments. + or − indicates that His6–CtrA protein (50 μg/ml maximum) was added or omitted from the footprinting reactions.

Figure 3

CtrA is necessary and sufficient to repress chromosome replication in vivo. (A) Results of decreasing and increasing CtrA activity on transcription from the Cori strong promoter (Fig. 1A) measured as relative β-galactosidase units (normalized to wild-type cells) from a lacZ reporter plasmid as described (9). Transcription was assayed in wild-type cells (ctrA⁺), in a temperature-sensitive mutant (ctrA401ts) and in a strain (PxylX::CtrΔ3) capable of oversupplying a nonproteolyzable CtrAΔ3 protein (17) controlled by a xylose-inducible promoter (16). (B) Loss of CtrA activity derepresses chromosome replication. Wild-type (ctrA⁺) and mutant ctrA401ts strains were grown at 30°C and then shifted to 37°C for the times indicated; DNA content (N chromosomes per cell) was then measured by fluorescent flow cytometry. Phase contrast photomicrographs correspond to the shaded histograms. (C) Oversupplying CtrA represses chromosome replication. Wild-type (ctrA⁺) and PxylX::ctrAΔ3 cultures were shifted to 0.2% xylose minimal medium for 10 h and analyzed as in B.

Figure 4

Directed mutagenesis at CtrA binding site d alters protein binding and increases Cori plasmid copy number in vivo. (A) Wild-type Cori sequence at site d showing the clustered base pair changes in Mut-d. (B) DNase I protection footprinting experiments were performed as in Fig. 2B (third panel). Outside lanes show the 5′-³²P end-labeled DNA fragments cut with EcoRI or HpaI. (C) Cori plasmids per chromosome, as determined by Southern blot hybridization. Caulobacter strain CB15N Δbla was electroplated with pBluescriptII plasmids (Stratagene) bearing otherwise identical wild-type (Wt) or mutant (Mut-d) Cori DNA between BamHI sites at −680 and +998 (see Fig. 1A) and analyzed as described (7). Radioactivity was measured with a PhosphorImager (Molecular Dynamics), and the ratio between Cori plasmid and chromosome band intensities is shown.