Determination of Cell Shape in Caulobacter

University Of California-Los Angeles, Los Angeles CA

Investigators

Abstract

Intellectual Merit: In many organisms a major determinant of cellular morphology is the shape of the cell wall. In bacteria, the cell wall, or peptidoglycan layer, serves as a scaffold that both dictates cell shape and reinforces the cell against internal osmotic pressure. A fundamental problem in bacterial cell biology is understanding the molecular mechanisms that coordinate peptidoglycan assembly to generate the diverse forms exhibited by bacteria. In rod-shaped bacteria, MreB, MreC, MreD and RodA, encoded by the highly conserved rod shape-determining genes probably play a critical role in directing peptidoglycan assembly. One of these, MreB, has been shown to be the bacterial actin homolog, suggesting that a cytoskeletal-like structure is responsible for determining rod shape. MreB has been shown to form helical cables traversing the length of the cell that are required for spatially coordinating enzyme complexes involved in cell wall assembly. MreC, another cell shape-determining protein encoded by the gene adjacent to mreB is located in the periplasmic space and has been shown to directly interact with a complex of peptidoglycan-synthesizing enzymes (penicillin binding proteins (PBPs)). The overall objective of this project is to genetically and biochemically define the molecular mechanisms that coordinate morphogenesis during cell growth and cytokinesis in the bacterium Caulobacter crescentus. The ease at which synchronized populations can be obtained, plus the intrinsic asymmetry of the predivisional cell make C. crescentus an ideal organism for tracking the subcellular location of the proteins that orchestrate cell wall synthesis during the course of the cell cycle. The working hypothesis that the MreB cytoskeleton functions as an organizer or tracking device for the PBP peptidoglycan biosynthesis complex during cell elongation, septum formation, and biogenesis of the stalk, an appendage comprised of peptidoglycan located at a single pole of the cell. The experiments focus on the roles of rod-shape-determining proteins in organizing cell wall assembly complexes during both cell elongation, stalk biogenesis and division. The project consists of three major research goals: (a) determining the role of proteins encoded by the Caulobacter rod-shape operon in coordinating cell wall morphogenesis by examining the subcellular localization of RodA and MreD, defining how each of these protein products influences the formation of MreC/PBP complexes, and determining the role of cell shape proteins in influencing the spatial localization pattern of lytic transglycosylases and their interactions with PBPs, (b) determining the factors regulating the dynamic pattern of MreB localization during the cell cycle and (c) defining the role of the cytoskeletal proteins in directing peptidoglycan synthesis and stalk biogenesis. Broader Impacts: Among the broader impacts of this project is the inclusion of undergraduate science majors in many aspects of the research. These students will be enrolled in an established course involving their own independent research projects. This involves writing a proposal and quarterly research reports, as well as presenting their findings at the annual undergraduate research meeting. Dr. Gober is an active training faculty member of several outreach programs designed to foster and encourage the beginnings of a research career in undergraduates who are not traditionally exposed to a research environment. In addition, to helping jump-start careers in research, these programs also place a special emphasis on the training of young scientists from traditionally underrepresented groups.