The object of this research is to gain a better understanding of how membrane associated protein structures such as the F conjugative pili or complex transport systems are synthesized and assembled in the bacterial membrane and how they function to transport large macromolecules, such as protein or DNA, into or across the bacterial membranes. Many conjugative plasmids which require pili for transmission of their DNA, also contain genes which encode antibiotic resistance. Bacterial pathogens which contain these plasmids are a major problem in infectious diseases. Understanding the role of pili in conjugation may lead to better way to control the spread of these plasmids through the population. Similarly, understanding how proteins can interact with each other in the membrane to promote transport of macromolecules may aid in the design of systems to transport desired macromolecules to specific cells or organisms. Three different systems will be studied. Investigations will be continued on the assembly of the filamentous bacterophage fl, a process where the single stranded viral DNA is extruded through the membrane as capsid proteins are assembled around it. Emphasis will be placed on the role that the phage gene I protein plays in this process. Using various molecular biological, immunological and genetic techniques, attempts will be made to analyze how the internal signal sequence of this protein allows it to insert into the membrane and form the assembly site where the outer and inner membrane of the bacteria are fused. The second system will be the examination of the structure and the assembly and disassembly process of the F-conjugative pili. The structure at the tip of the pilus will be isolated and characterized and the orientation of the F-pilin protein in both the pilus and the inner membrane will be analyzed. Last, we will analyze the products of the tol QRAB gene cluster which are required for macromolecules such as the filamentous phage DNA or the E colicins to enter the bacterium. The DNA sequence analysis of these genes will be completed, the regulation of their expression studied and attempts made to isolate and characterize the location and function of these proteins.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Microbial Physiology and Genetics Subcommittee 2 (MBC)
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Duke University
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United States
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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. 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
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
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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