Cell Division Control During Rhizobium Differentiation

Margolin, William

Abstract

THIS IS A SHANNON AWARD PROVIDING PARTIAL SUPPORT FOR THE RESEARCH PROJECTS THAT FALL SHORT OF THE ASSIGNED INSTITUTE'S FUNDING RANGE BUT ARE IN THE MARGIN OF EXCELLENCE. THE SHANNON AWARD IS INTENDED TO PROVIDE SUPPORT TO TEST THE FEASIBILITY OF THE APPROACH; DEVELOP FURTHER TESTS AND REFINE RESEARCH TECHNIQUES; PERFORM SECONDARY ANALYSIS OF AVAILABLE DATA SETS; OR CONDUCT DISCRETE PROJECTS THAT CAN DEMONSTRATE THE PI'S RESEARCH CAPABILITIES OR LEAD ADDITIONAL WEIGHT TO AN ALREADY MERITORIOUS APPLICATION. THE APPLICATION BELOW IS TAKEN FROM THE ORIGINAL DOCUMENT SUBMITTED BY THE PRINCIPAL INVESTIGATOR. One of the most fundamental yet unanswered biological problems is how cells grow, divide, and differentiate into specialized cell types. An attractive and genetically accessible organism in which to address this problem from the standpoints of both cell division and differentiation is the prokaryotic endosymbiont Rhizobium meliloti, which establishes a facultative nitrogen-fixing symbiosis within the roots of alfalfa plants (Medicago sativa). By communicating with chemical signals, both organisms develop complex structures specialized for nitrogen fixation: the plant roots form nodules, and extracellular, rod-shaped free-living bacteria, after entering the cytoplasm of nodule cells, differentiate into non- growing, branched """"""""bacteroids"""""""" that synthesize nitrogenase. The initial symbiotic events, including the requirement for specific host-bacterial recognition and bacterial proliferation within the host, are analogous to invasion by other bacterial pathogens. However, pathogenesis fails to ensue after bacterial entry into the plant cytoplasm - the bacteroids stop dividing. Why? The long-term objective is to identify the bacterial switch that causes bacterial cell division arrest and differentiation, and to--determine whether a plant-derived signal molecule is involved in the control of the endosymbiont's physiology. This proposed work seeks to characterize genes of R. meliloti that control its cell division and morphology, and to determine whether these genes or their products are controlled during differentiation and thus might be potential differentiation """"""""switches"""""""". The focus is on the tubulin-like cell division protein FtsZ, since it is required for initiation of cell division in other bacteria and is a known target of several endogenous cell division inhibitors. What makes FtsZ an even more likely participant in potentially novel cell cycle regulation in R. meliloti is the presence of two novel and divergent ftsZ genes, one of which appears to be nonessential in free-living R. meliloti cells. The role of the R. meliloti FtsZ proteins in cell division and cell differentiation will be addressed via three general approaches. (1) Genetic: The first specific aims will be to determine the regulation, importance, and effects of ftsZ gene expression in the two cell types. (2) Biochemical: The third specific aim is to determine the basic biochemical properties of the two FtsZ proteins, including their interaction with other cellular components, and to compare them to E. coli FtsZ. (3) Cellular: The final two specific aims propose to determine the subcellular localization and formation of subcellular structures by the FtsZ proteins, and to define the parameters that influence formation of cellular branches. This proposed work on the cell cycle arrest of an intracellular bacterium by a eukaryotic cell has the potential to yield new strategies for controlling proliferation of pathogenic bacteria. In addition, this work on naturally occurring variants of FtsZ should generate new ideas on the function of tubulin-like proteins in all cells and the evolution of the eukaryotic cytoskeleton.