Feedback control of a master bacterial cell-cycle regulator.

The transcriptional regulator CtrA controls several key cell-cycle events in Caulobacter crescentus, including the initiation of DNA replication, DNA methylation, cell division, and flagellar biogenesis. CtrA is a member of the response regulator family of two component signal transduction systems. Caulobacter goes to great lengths to control the time and place of the activity of this critical regulatory factor during the cell cycle. These controls include temporally regulated transcription and phosphorylation and spatially restricted proteolysis. We report here that ctrA expression is under the control of two promoters: a promoter (P1) that is active only in the early predivisional cell and a stronger promoter (P2) that is active in the late predivisional cell. Both promoters exhibit CtrA-mediated feedback regulation: the early P1 promoter is negatively controlled by CtrA, and the late P2 promoter is under positive feedback control. The CtrA protein footprints conserved binding sites within the P1 and P2 promoters. We propose that the P1 promoter is activated after the initiation of DNA replication in the early predivisional cell. The ensuing accumulation of CtrA results in the activation of the P2 promoter and the repression of the P1 promoter late in the cell cycle. Thus, two transcriptional feedback loops coupled to cell cycle-regulated proteolysis and phosphorylation of the CtrA protein result in the pattern of CtrA activity required for the temporal and spatial control of multiple cell-cycle events.

Cell cycle-dependent polar localization of an essential bacterial histidine kinase that controls DNA replication and cell division.

The master CtrA response regulator functions in Caulobacter to repress replication initiation in different phases of the cell cycle. Here, we identify an essential histidine kinase, CckA, that is responsible for CtrA activation by phosphorylation. Although CckA is present throughout the cell cycle, it moves to a cell pole in S phase, and upon cell division it disperses. Removal of the membrane-spanning region of CckA results in loss of polar localization and cell death. We propose that polar CckA functions to activate CtrA just after the initiation of DNA replication, thereby preventing premature reinitiations of chromosome replication. Thus, dynamic changes in cellular location of critical signal proteins provide a novel mechanism for the control of the prokaryote cell cycle.

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

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.

Cell type-specific phosphorylation and proteolysis of a transcriptional regulator controls the G1-to-S transition in a bacterial cell cycle.

The global transcriptional regulator CtrA controls multiple events in the Caulobacter cell cycle, including the initiation of DNA replication, DNA methylation, cell division, and flagellar biogenesis. CtrA is a member of the response regulator family of two component signal transduction systems and is activated by phosphorylation. We report here that this phosphorylation signal enters the cell cycle at mid S phase. In addition, CtrA function is modulated by temporally and spatially controlled proteolysis. When an active CtrA protein is present at the wrong time in the cell cycle, owing to expression of a mutant CtrA derivative that is active in the absence of phosphorylation and is not turned over during the cell cycle, the G1-to-S transition is blocked and the cell cycle aborts. Thus, both phosphorylation and proteolysis are critical determinants of bacterial cell cycle control in a manner that is analogous to the control of the eukaryotic cell cycle.

The control of temporal and spatial organization during the Caulobacter cell cycle.

The Caulobacter cell cycle exhibits time-dependent expression of differentiation events. These include the morphological transition of a swarmer cell to a replication-competent stalked cell and the subsequent polarized distribution of specific gene products that results in an asymmetric predivisional cell. Cell division then yields a new swarmer cell and a stem-cell-like stalked cell. Two-component signal transduction proteins involved in cell cycle control and proteins required for cell division and flagellar biogenesis have been shown to be regulated temporally and spatially during the cell cycle. The mechanisms underlying this regulation include protein phosphorylation and proteolysis.

Assembly of the Photosystem II oxygen-evolving complex is inhibited in psbA site-directed mutants of Chlamydomonas reinhardtii. Aspartate 170 of the D1 polypeptide.

Photosystem II catalyzes the photooxidation of water to molecular oxygen, providing electrons to the photosynthetic electron transfer chain. The D1 and D2 chloroplast-encoded reaction center polypeptides bind cofactors essential for Photosystem II function. Transformation of the chloroplast genome of the eukaryotic green alga Chlamydomonas reinhardtii has allowed us to engineer site-directed mutants in which aspartate residue 170 of D1 is replaced by histidine (D170H), asparagine (D170N), threonine (D170T), or proline (D170P). Mutants D170T and D170P are completely deficient in oxygen evolution, but retain normal (D170T) or 50% (D170P) levels of Photosystem II reaction centers. D170H and D170N accumulate wild-type levels of PSII centers, yet evolve oxygen at rates approximately 45% and 15% those of control cells, respectively. Kinetic analysis of chlorophyll fluorescence in the mutants reveals a specific defect in electron donation to the reaction center. Measurements of oxygen flash yields in D170H show, however, that those reaction centers capable of evolving oxygen function normally. We conclude that aspartate residue 170 of the D1 polypeptide plays a critical role in the initial binding of manganese as the functional chloroplast oxygen-evolving complex is assembled.