Developmental Control of DNA Replication in Caulobacter

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DESCRIPTION (provided by applicant): Our goal is to identify the mechanisms that integrate temporal and spatial signals to coordinate the initiation of DNA replication, chromosomal origin movement to the cell poles, and the mid-cell assembly of the cell division ring during a bacterial cell cycle. A small number of critical master transcriptional regulators, DnaA, GcrA, and CtrA, control cell cycle progression in Caulobacter. These three proteins are part of a regulatory circuit that together control the expression of genes required for chromosome replication and cytokinesis. We have discovered that DnaA controls both replication initiation and the transcription of cell cycle-regulated genes. DnaA turns on the transcription of GcrA, which, in turn, turns on the transcription of CtrA. GcrA and CtrA oscillate out of phase during the cell cycle to control approximately 150 temporally-regulated genes for polar morphogenesis, DNA methylation, and cell division. DnaA is a critical lynchpin in the regulatory cascade and we will now analyze the temporal control of DnaA availability and activity. We will characterize the genes controlled, in turn, by GcrA and identify a new factor that appears to negatively control the expression of a set of 5 replication enzymes. Caulobacter coordinates the transcription of ctrA with the progression of DNA replication using the differential methylation state of the replicating chromosome. We will now explore the role of DNA methylation in the control of transcription of multiple replication genes all of which have methylation sites in their promoters. Finally, an important question is how chromosome replication and segregation is coordinated with cell division. We have a discovered a unique mechanism that links the MreB actin-dependent movement of the newly replicated origin to the opposite cell pole and the mid-cell positioning of the FtsZ division ring. A ParA-family ATPase, MipZ, in complex with ParB, binds to the origin region and moves with the replicated origin as it transits the length of the cell. Because MipZ is an inhibitor of FtsZ polymerization, the Z-ring can form only at mid-cell once the origins and the accompanying MipZ complex are safely secured at the two cell poles. We will now define the mechanisms that coordinate these events. Defining the regulatory circuitry that drives the bacterial cell cycle has revealed methyltranferases as circuit nodes and as such, targets for antibiotic discovery. Based on this work, we have succeeded in designing a new small molecule antibiotic that is currently in clinical trials.