The long-term objective of this research is to gain a better understanding, at the molecular level, of how membrane associated protein structures interact to form specific functions in the bacteria. Two systems will be studied using genetic, biochemical and molecular biological techniques. The first system concerns the assembly of the filamentous bacteriophage, a process that occurs entirely at the bacterial membrane. The three capsid and three assembly proteins required for initiation and elongation of assembly will be systematically expressed in the presence or absence of the DNA packaging signal. Chemical and cysteine disulfide crosslinking methods will be used to see how the transmembrane regions of these proteins interact to initiate and elongate the assembling phage particle. The relevance of any identified interactions will be monitored by determining the effect that specific mutant phage proteins have on any observed interactions. Attempts will be made to specify the roles that the noncapsid gene I protein and its overlapping gene XI product play in the assembly process. Better knowledge of the filamentous phage assembly system will aid in our understanding of the apparent mechanistically similar export of many virulence factors from pathogenic bacteria as well as better understanding the production of the cholera toxin determining filamentous bacteriophage. This knowledge may also lead to better designed vectors for the combinational display of proteins on the bacteriophage surface. The second system studies the role of the membrane associated TolQRA and B proteins in maintaining the integrity of the outer membrane of gram negative bacteria as well as facilitating the uptake of the group A colicins. Genetics and affinity chromatography will be used to determine the cytoplasmic molecules that TolQ interacts with to allow the Tol system to function. Attempts will be made to determine if the long helical domain of TolA participates in coiled coil interactions for proper functioning. These studies should give a better understanding of how the outer membrane is maintained and perhaps help design systems for delivering macromolecules into gram negative bacteria.
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