CtrA,a Global Response Regulator,Uses a Distinct Second Category of Weak DNA Binding Sites for Cell Cycle Transcription Control in Caulobacter crescentus

CtrA controls cell cycle programs of chromosome replication and genetic transcription. Phosphorylated CtrA∼P exhibits high affinity (dissociation constant [K_d], <10 nM) for consensus TTAA-N7-TTAA binding sites with “typical” (N = 7) spacing. We show here that ctrA promoters P1 and P2 use low-affinity (K_d, >500 nM) CtrA binding sites with “atypical” (N ≠ 7) spacing. Footprints demonstrated that phosphorylated CtrA∼P does not exhibit increased affinity for “atypical” sites, as it does for sites in the replication origin. Instead, high levels of CtrA (>10 μM) accumulate, which can drive CtrA binding to “atypical” sites. In vivo cross-linking showed that when the stable CtrAΔ3 protein persists during the cell cycle, the “atypical” sites at ctrA and motB are persistently bound. Interestingly, the cell cycle timing of ctrA P1 and P2 transcription is not altered by persistent CtrAΔ3 binding. Therefore, operator DNA occupancy is not sufficient for regulation, and it is the cell cycle variation of CtrA∼P phosphorylation that provides the dominant “activation” signal. Protein dimerization is one potential means of “activation.” The glutathione S-transferase (GST) protein dimerizes, and fusion with CtrA (GST-CtrA) creates a stable dimer with enhanced affinity for TTAA motifs. Electrophoretic mobility shift assays with GST-CtrA revealed cooperative modes of binding that further distinguish the “atypical” sites. GST-CtrA also binds a single TTAA motif in ctrA P1 aided by DNA in the extended TTAACCAT motif. We discuss how “atypical” sites are a common yet distinct category of CtrA regulatory sites and new implications for the working and evolution of cell cycle control networks.The Caulobacter crescentus transcription regulator CtrA provides a new paradigm of cell cycle control (4, 18, 24). CtrA belongs to the large OmpR/PhoB response regulator (RR) family. Bacterial stimulus-response mechanisms often use RR proteins, which switch between phosphorylated and unphosphorylated states by contact with the cognate histidine kinase and histidine phosphotransfer proteins (38). Despite the apparent simplicity of this paradigm, the regulatory tasks of CtrA are exceptionally varied and complex. C. crescentus uses CtrA to divide asymmetrically and to produce distinct swarmer and stalked cells (Fig. ​(Fig.1).1). Cell cycle control also involves differentiation of swarmer cells to stalked cells, asymmetric remodeling that creates a new flagellated swarmer cell pole, and the regulation of chromosome replication so that it occurs in the stalked cells but not in the swarmer cells. Accordingly, CtrA controls genetic transcription at key stages of the cell cycle. CtrA also controls chromosome replication by binding five sites inside the replication origin (25, 31).Open in a separate windowFIG. 1.CtrA protein activity during the C. crescentus cell cycle. A nonreplicating but motile swarmer cell (Sw) differentiates into a replicating stalked cell (St). Growth of a predivisional cell (Div) produces a new flagellated swarmer pole. Segregating chromosomes are positioned in both the nonreplicating swarmer cell (rep−) and the replication-competent stalked cell (rep+). Shading indicates the temporal and spatial presence of CtrA. In wild-type (WT) cells, CtrA “activity” is controlled by cell cycle-programmed synthesis, proteolysis, and phosphorylation. In CtrAΔ3-containing cells, CtrA “activity” is controlled by cell cycle-programmed phosphorylation alone.CtrA activity is controlled by cell cycle programs that adjust the CtrA concentration and phosphorylation state (5). In wild-type cells, cell cycle synthesis and proteolysis adjust the CtrA concentration (Fig. ​(Fig.1).1). CtrA is synthesized before cell division but after septum formation; the CtrA protein is degraded in a stalked cell while it is retained in a swarmer cell (Fig. ​(Fig.1).1). The CtrA protein is maintained in a swarmer cell until this cell differentiates into a stalked cell, where CtrA is rapidly degraded (5, 27). However, mutating the last three C-terminal amino acids blocks CtrA proteolysis. The resulting CtrAΔ3 protein is stable and maintained in all cell types. Surprisingly, the CtrAΔ3 protein fully complements a ctrA null allele (25), and CtrAΔ3-containing cells are indistinguishable from wild-type cells. Apparently, under laboratory conditions, CtrA synthesis and proteolysis are redundant controls for CtrA activity. In CtrAΔ3-containing cells (Fig. ​(Fig.1),1), the activity of CtrA is adjusted by cell cycle changes in its phosphorylation state alone (5). For example, when the CtrAΔ3 protein persists in stalked cells, it is poorly phosphorylated and presumably inactive. However, stable CtrAΔ3 becomes increasingly phosphorylated coincident with the requirement for it and coincident with the synthesis of wild-type CtrA protein in wild-type cells. An essential histidine kinase (10) and a phosphorelay system (4, 39) phosphorylate CtrA and thereby change its activity during the cell cycle.Our studies address the specific basis of CtrA “activity” and the relative contributions of CtrA synthesis, proteolysis, and phosphorylation to cell cycle control (Fig. ​(Fig.1).1). How does phosphorylated CtrA∼P differ from unphosphorylated CtrA? One established biochemical “activity” of the phosphorylated CtrA∼P protein is increased affinity for the promoters of the C. crescentus fliQ, ccrM, and pilA genes (26, 34) and for the C. crescentus replication origin (31). The CtrA binding sites have the consensus sequence TTAA-N7-TTAA, whose N = 7 spacing is required for maximum CtrA-directed transcription (22). Over 50 transcription promoters are bound and regulated by CtrA (13), and it is not certain that all of their binding sites conform to the consensus described above. Below, we describe a distinct second category of weak CtrA binding sites that change previously untested assumptions about cell cycle transcription control.