The object of this research is to gain a better understanding of how specific proteins which can be assembled into membrane associated bacterial structures are synthesized, interact with, and are assembled within the bacterial membrane. Three basic approaches will be used. The first is a continuation of our studies on the membrane events associated with the assembly of the filamentous bacteriophage f1. The phage specific proteins which have not been identified in the bacteria, will be isolated, characterized and their location within the bacteria determined using genetic, biochemical and immunological techniques. Attempts will be made to isolate membrane associated intermediates in bacteriophage assembly and characterize them using biochemical and electron microscope techniques. We will try to define an in vitro system in which DNA from the gene V-DAN intermediate will be assembled into a mature phage particle. The second approach will be to examine F-like sex pili using some of the technique we have developed in the study of the f1 bacteriophage. F pili will be analyzed for the presence of a specific protein or structure at the tip which specifically interacts with f1 phage or F- cells. Attempts will be made to isolate the protein and use it to examine the receptor on the F- cells. The orientation of F pilin will be analyzed in the inner membrane and the intact pilus as was done for the major coat protein of the f1 phage. Last, different group specific reagents will be tested for their ability to label proteins on the outside of spheroplasts. Promising techniques will be used to ascertain the orientation of various inner membrane proteins.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM018305-15
Application #
3269239
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1978-01-01
Project End
1987-12-31
Budget Start
1986-01-01
Budget End
1986-12-31
Support Year
15
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Duke University
Department
Type
Schools of Medicine
DUNS #
071723621
City
Durham
State
NC
Country
United States
Zip Code
27705
Vianney, A; Lewin, T M; Beyer Jr, W F et al. (1994) Membrane topology and mutational analysis of the TolQ protein of Escherichia coli required for the uptake of macromolecules and cell envelope integrity. J Bacteriol 176:822-9
Guy-Caffey, J K; Webster, R E (1993) The membrane domain of a bacteriophage assembly protein. Membrane insertion and growth inhibition. J Biol Chem 268:5496-503
Levengood-Freyermuth, S K; Click, E M; Webster, R E (1993) Role of the carboxyl-terminal domain of TolA in protein import and integrity of the outer membrane. J Bacteriol 175:222-8
Guy-Caffey, J K; Webster, R E (1993) The membrane domain of a bacteriophage assembly protein. Transmembrane-directed proteolysis of a membrane-spanning fusion protein. J Biol Chem 268:5488-95
Muller, M M; Vianney, A; Lazzaroni, J C et al. (1993) Membrane topology of the Escherichia coli TolR protein required for cell envelope integrity. J Bacteriol 175:6059-61
Rapoza, M P; Webster, R E (1993) The filamentous bacteriophage assembly proteins require the bacterial SecA protein for correct localization to the membrane. J Bacteriol 175:1856-9
Schandel, K A; Muller, M M; Webster, R E (1992) Localization of TraC, a protein involved in assembly of the F conjugative pilus. J Bacteriol 174:3800-6
Guy-Caffey, J K; Rapoza, M P; Jolley, K A et al. (1992) Membrane localization and topology of a viral assembly protein. J Bacteriol 174:2460-5
Levengood, S K; Beyer Jr, W F; Webster, R E (1991) TolA: a membrane protein involved in colicin uptake contains an extended helical region. Proc Natl Acad Sci U S A 88:5939-43
Webster, R E (1991) The tol gene products and the import of macromolecules into Escherichia coli. Mol Microbiol 5:1005-11

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