Our goal is to identify the signals and regulatory proteins that control DNA replication and chromosome segregation during the progression of a bacterial cell cycle. Caulobacter crescentus has a well-defined cell cycle which includes temporally and spatially constrained differentiation events. This bacterium is particularly well suited for an analysis of the control of chromosome replication and partition because cultures are easily synchronized and have a distinct G1-S transition, replication occurs in a specific cell type only once per cell cycle, and an asymmetric division yields separable progeny with different morphological features and replicative abilities. The annotated genome sequence of 3767 genes has been completed and we have generated DNA microarrays for the analysis of full genome transcription networks. In Caulobacter, members of the two-component signal transduction family of proteins play critical roles in many aspects of cell cycle control. In addition to serving as a repressor of replication initiation, the CtrA response regulator controls the expression of 30% of the 553 genes whose expression is under cell cycle control. Groups of genes involved in DNA replication that are expressed during the G1 -S transition are not among those controlled by CtrA. We will now identify the key regulatory factors that control these G1-S transition genes. At another level of regulation, the origin of replication is confined to the cell pole where the replisome assembles at the start of replication. We will determine the mechanisms by which the origin is dynamically localized to the cell pole, and the role of the SMC [Structural Maintenance of Chromosomes] protein in chromosome condensation and segregation. Finally, we have shown that newly replicated DNA remains in the hemi-methylated state until the end of the cell cycle. We will determine the role of the chromosome methylation state on cell cycle-regulated gene expression and chromosome segregation.