Non-canonical functions of two-component signaling proteins in the cell cycle of Caulobacter crescentus

University Of California-Berkeley, Berkeley CA

Investigators

Abstract

Two-component signal transduction is a mechanism used by nearly all bacteria to organize intracellular events and respond to changes in the environment. In a two-component signaling pathway, the upstream histidine kinase protein senses a signal and autophosphorylates on a conserved histidine residue. The phosphoryl group is then passed to a downstream response regulator protein, which generates a cellular response, such as a change in gene expression, metabolism, or motility. Although this linear paradigm is correct in most cases, some two-component proteins with important cellular roles appear to function by alternative mechanisms, or in branched networks. The project focuses on two proteins, DivK and DivL, that are essential for cell cycle progression in the aquatic bacterium Caulobacter crescentus. The aim is to determine how the response regulator DivK suppresses the activity of a histidine kinase, CckA, which functions in a separate two-component pathway. Suppression of CckA activity at the correct time is necessary for Caulobacter cells to begin chromosome replication. The other aim is to establish the function of an essential protein, DivL, which is homologous to histidine kinases, but which appears to act by a mechanism other than phosphoryl transfer. In particular, it is proposed that DivL participates in essential protein-protein interactions which are modulated by binding and hydrolysis of ATP. Experiments are proposed to measure the ATP binding and hydrolysis by DivL mutants that cause distinct cellular phenotypes, and to identify new proteins that interact with DivL to mediate its effects on the cell division cycle. This research will expand the repertoire of biochemical activities used by histidine kinases and response regulators to influence each other and cellular events. Broader Impacts This project will provide an intensive summer lab course in microbiology for 10 students per year. This course is different from many lab classes in that the students do experiments whose results are not known beforehand. In addition to fundamental techniques in microbiology and molecular biology, they learn how to design and troubleshoot experiments so that their results are interpretable and reliable. In past years, each student has deleted a previously unstudied gene in Caulobacter crescentus and characterized the resulting mutant strain. In upcoming years, the students will do projects closely related to the scientific goals of this research, such as generating specific mutations in divL and determining their cellular effects, or performing genetic screens to identify proteins that interact with DivL or DivK. Students learn how new scientific knowledge is generated, and they gain experience that prepares them for independent research projects. Women and students from underrepresented groups are encouraged to undertake research projects in the applicant's laboratory.