Mechanisms of C. crescentus regulation of chromosome replication by a cell cycle regulator protein

Caulobacter crescentus divides asymmetrically to produce two different progeny, a swarmer progeny that is replication incompetent, and a stalked cell progeny that is replication competent. Upon cell division, a cell cycle regulator protein (CtrA) was identified only in the swarmer cells, and additional circumstantial evidence links this protein to being a repressor of chromosome replication. For example, this response regulator protein binds to five specific sites in the replication origin (Cori) designated [a-e]. We carefully studied the binding characteristics of both phosphorylated (CtrA~P) and unphosphorylated forms of the protein to the five binding sites [a-e] in the replication origin (Chapter 2) and upstream of the ctrA gene (Chapter 3). We showed that phosphorylation significantly enhances binding affinity in the replication origin (Chapter 2) but not upstream of ctrA (Chapter 3). In addition a "pseudo-active" protein form (CtrA D51E) that resembles the phosphorylated form in vivo did not improve the binding characteristics (Chapter 3). These results suggest that enhanced binding on phosphorylation is not the only signal achieved on phosphorylation. In fact, CtrA half-site mutation binding studies shows that phosphorylation stimulates protein/protein interaction and cooperative binding between sites [a] and [b] in the replication origin (Chapter 2). We show that CtrA binding site [b] is the major contributor in the cooperative CtrA binding between [a] and [b] (Chapter 4). We demonstrate that cooperative binding of CtrA~P to sites [a] and [b] repress transcription from a strong promoter (Ps), which in turn blocks plasmid replication. In addition, mutating site [b] to block CtrA binding to [a] and [b] has a deleterious effect on chromosome replication (Chapter 4).This cooperative CtrA binding at [a] and [b] is independent from the upstream binding sites [c-e] (Chapter 2). CtrA∼P binding in the origin is altered in the presence of the histone-like protein (IHF) that also binds and overlaps CtrA binding site [c] (Chapter 5). In-fact, IHF binds and overlaps binding site [c] (Chapter 5). We propose a replication model in the stalked cell were IHF binding hinders active CtrA binding in the replication origin and regulates cooperative transcription that coincides with replication initiation