Gober 95-13222 The bacterium Caulobacter crescentus expresses polarity during its cell cycle that is generated by a simple developmental program. Cell division yields two dissimilar daughter cells: a motile swarmer cell and a sessile stalked cell. These two cell types differ in their relative programs of gene expression and DNA replication. The progeny stalked cell reinitiates DNA replication immediately after cell division, whereas replication is silenced for a defined period of time in the swarmer cell. The Caulobacter developmental cycle allows the experimental analysis of the basic mechanisms underlying cell cycle controlled gene expression, the regulation of DNA replication and the molecular basis of positional information. The objectives of this research are to genetically and biochemically determine the molecular basis of chromosomal partitioning and the coupling of this event to cell division in Caulobacter. The ease at which synchronized populations can be obtained plus, the intrinsic asymmetry of the Caulobacter predivisional cell make it an ideal system for tracking the subcellular location of newly replicated chromosomes and the proteins that direct them during the partitioning process. Specifically, the role of the cellular homologues of plasmid partitioning gene products (parA and parB) in chromosomal partitioning will be determined. In bacterial cells the newly replicated chromosomes efficiently partition to the poles of the predivisonal cell, ensuring that each daughter cell inherits its own copy of the chromosome Partitioning of unit- or low-copy number plasmids may provide an experimental framework with which to dissect chromosome partitioning. Plasmid partitiomng is accomplished in part, through the action of, ParA and ParB, two plasmid-specific gene products. Both ParA and ParB are necessary for efficient plasmid partitioning. ParB functions as a DNA binding protein and specifically binds the plasmid centromere, which is often located immediately downstream of the parB cod ing sequence. ParA is homologous to a large family of ATPases and plays an undefined role in partitioning. In several species of bacteria, the chromosomal homologues of parA and parB have been identified. The parA and parB homologues of Caulobacter have been cloned and sequenced. These two genes are expressed as an operon and immediately following the parB coding region lies a DNA sequence containing a number of inverted and direct repeats that could serve as the centromere during partitioning. Genetic experiments indicate that parA is essential for cell viability and parB is required for efficient partitioning of chromosomal DNA. The parAB operon is expressed under cell cycle control, with peal~ expression occurring at the latter stages of DNA replication. Furthermore, ParA is concentrated at the poles of the Caulobacter cell. ParA and ParB may have several possible roles in chromosome partitioning. First, they may directly participate in chromosome movement by functioning to tether or direct the chromosomes to the locomotor apparatus. Alternatively, they may function in regulating a cell cycle checkpoint that coordinates chromosome partitioning and cell division. In order to dissect the roles of ParA and ParB in chromosome partitioning in Caulobacter, the following experimental approaches are being taken: (1) Genetic analysis of the Caulobacter parA and parB genes will be performed to determine their role in chromosome partitioning and cell division. In addition, the proteins that specifically interact with ParA and ParB during cell division and chromosome partitioning will be identlfied using a genetic suppressor analysis and a two-hybrid screen. Par protein interactions will also be assayed utilizing in vitro techniques including co-immunoprecipitation (2) The function of ParB DNA binding activity and its role in chromosome partitioning will be determined. (3) The mechanisms responsible for the cell cycle regulation of parA andparB expression and the subcellular location of ParA and ParB du ring the cell division cycle will be defined. To determine whether ParA or ParB function in a cell cycle checkpoint, the effect of conditional parA and parB mutations on the coordination of cell division, the timing of initiation of DNA replication and the expression of developmentally regulated genes will be tested. This research will contribute to a basic understanding of an importnt component of bacterial reproduction.
