The long-term objective of this research is to gain a better understanding, at the molecular level, of how membrane associated multimeric protein structures assemble and interact to perform specific functions in the bacteria. Two systems will be studied using a combination of biochemical, molecular biological and electron microscopic techniques. The role of the TolQRA and B proteins in maintaining membrane integrity and facilitating the uptake of the group A colicins will be investigated. The location and topology of these proteins within the bacterial envelope will be determined using immunological and genetic techniques. Chemical crosslinking and affinity chromatography methods will be used to determine how the Tol proteins interact with each other and with other components of the bacterial envelope. Chimeric proteins containing regions of colicins E1 and E3 and reporter molecules will be used to help understand the mechanism of colicin uptake into the cell and the role that the Tol proteins play in this process. Attention will be paid to ways in which this information can be used to help design systems for delivering macromolecules into gram negative bacteria. Investigation will continue on the membrane associated assembly of the filamentous phage fl. Emphasis will be placed on understanding the role that the products of the phage gene I play in the formation of the assembly site as well as in the thioredoxin aided assembly of the phage capsid subunits. Mutational analysis will be used to determine whether the smaller product of gene I, resulting from an internal translation initiating event, is required for phage assembly. Chemical crosslinking and affinity chromatography methods will be used to determine the bacterial and phage components which the gene I protein interacts with during assembly. Further knowledge about fl phage assembly may lead to new insights about the mechanism of more complicated assembly processes such as that for conjugative pili. In addition, it may lead to better methods to efficiently assemble foreign proteins on the phage and increase the use of this phage in molecular biological research.
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