Chromosome Partitioning and the Regulation of Cell Division in Caulobacter

University Of California-Los Angeles, Los Angeles CA

Investigators

Abstract

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 project are to genetically and biochemically determine the molecular basis of chromosomal partitioning and the coupling of this event to cell division in Caulobacter. The project will examine the roles of the conserved biochemical functions of ParA and ParB in partitioning, chromosome orientation and cell division by first employing site-directed mutagenesis to create mutants of ParA that possess altered ATPase activity, and mutant ParB derivatives that have defects in DNA binding. These mutants will be introduced into Caulobacter cells and their effect on partitioning and cell division will be determined. Furthermore, the biochemical activities of these mutants will be assayed. The mechanism of ParA and ParB polar localization will also be determined by examining the role of ParA ATPase activity and ParB DNA binding activity in bipolar localization, defining the role of ParA and ParB interaction in directing localization, identifying the proteins responsible for ParA and ParB localization by screening for interacting gene products using an expression library and/or by biochemical means, and by performing in vitro assays with interacting proteins. Lastly, the mechanism of regulation of cell division by ParA and ParB will be determined by assaying the transcription of critical cell division genes in response to ParB depletion and ParA overexpression, and identifying mutants that do not respond to ParA/ParB regulation. This project will yield important information regarding how bacteria segregate their chromosomal DNA. Since the proteins under investigation are conserved in most species of bacteria it is likely that the investigations accomplished by this project will possibly contribute to the design of new antimicrobic compounds that inhibit this critical cellular process.