The long-range goal of this project is to determine the mechanism used by bacteria to select the proper division site at midcell in preference to other potential division sites that are located elsewhere within the cell. Since the products of the three min genes, minC, minD and minE, are responsible for this site-selection process, we will attempt to define the mechanism of action of each of the gene products in the site-selection process. Previous work has shown that MinD localizes to potential division sites at poles and midcell in the absence of the other Min proteins, and that coexpression of MinE or domains of MinE causes redistribution of membrane-associated Gfp-MinD into structures whose position depends on the presence of the MinE topological specificity domain. The major immediate aims of the proposal will be: i. To define the determinants in MinD that are responsible for its localization to poles and midcell, its role in formation of the MinE ring, and its ability to activate the MinC division inhibitor; ii. To determine the molecular basis of the different membrane-associated MinD structures that are induced by coexpression of Gfp-MinD with MinE and with the N-terminal region of MinE, using a combination of fluorescence microscopy, molecular genetics, and membrane-biochemistry; iii. To determine the localization pattern of MinC and the effects of MinC on localization of MinD and MinE; iv. To complete the high-resolution three-dimensional structure of the MinE topological specificity domain and to map topological specificity mutations of MinE to the three-dimensional structure; v. To characterize new minicell mutants that map outside of the minCDE locus and determine whether any of them code for the topological targets for MinD and MinE localization.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM060632-03S1
Application #
6641895
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Program Officer
Deatherage, James F
Project Start
2000-02-01
Project End
2004-03-31
Budget Start
2002-04-01
Budget End
2003-03-31
Support Year
3
Fiscal Year
2002
Total Cost
$63,159
Indirect Cost
Name
University of Connecticut
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
City
Farmington
State
CT
Country
United States
Zip Code
06030
Taghbalout, Aziz; Ma, Luyan; Rothfield, Lawrence (2006) Role of MinD-membrane association in Min protein interactions. J Bacteriol 188:2993-3001
Drew, Donald A; Osborn, Mary J; Rothfield, Lawrence I (2005) A polymerization-depolymerization model that accurately generates the self-sustained oscillatory system involved in bacterial division site placement. Proc Natl Acad Sci U S A 102:6114-8
Shih, Yu-Ling; Kawagishi, Ikuro; Rothfield, Lawrence (2005) The MreB and Min cytoskeletal-like systems play independent roles in prokaryotic polar differentiation. Mol Microbiol 58:917-28
Ma, Luyan; King, Glenn F; Rothfield, Lawrence (2004) Positioning of the MinE binding site on the MinD surface suggests a plausible mechanism for activation of the Escherichia coli MinD ATPase during division site selection. Mol Microbiol 54:99-108
Ma, Lu-Yan; King, Glenn; Rothfield, Lawrence (2003) Mapping the MinE site involved in interaction with the MinD division site selection protein of Escherichia coli. J Bacteriol 185:4948-55
Shih, Yu-Ling; Le, Trung; Rothfield, Lawrence (2003) Division site selection in Escherichia coli involves dynamic redistribution of Min proteins within coiled structures that extend between the two cell poles. Proc Natl Acad Sci U S A 100:7865-70
Shih, Yu-Ling; Fu, Xiaoli; King, Glenn F et al. (2002) Division site placement in E.coli: mutations that prevent formation of the MinE ring lead to loss of the normal midcell arrest of growth of polar MinD membrane domains. EMBO J 21:3347-57
Szeto, Tim H; Rowland, Susan L; Rothfield, Lawrence I et al. (2002) Membrane localization of MinD is mediated by a C-terminal motif that is conserved across eubacteria, archaea, and chloroplasts. Proc Natl Acad Sci U S A 99:15693-8