Laboratory of Genetics
Adjunct Associate Professor
Analysis of filamentous bacterial virus, f1.
This laboratory uses genetics, biochemistry, and molecular biology in an attempt to derive a complete analysis of the filamentous bacterial virus, f1, and of its interactions with its host, Escherichia coli. Other studies relate to protein-DNA recognition, membrane anchoring, and questions of protein structure.
Filamentous phage assembly, which initiates at the inner face of the E. coli cytoplasmic membrane, involves coating the cytoplasmic ssDNA genome with five membrane proteins coded by the virus. The assembling virus is secreted across both the cytoplasmic and outer membrane without killing or rupturing the host. We have identified E. coli thioredoxin as an essential component in assembly. Two phage-encoded proteins which are not part of the virus particle itself (pI, pIV) are also essential, and we are trying to understand their roles. Genetic experiments indicate that a nucleotide binding motif located in the cytoplasmic domain of the membrane-spanning protein pI is essential for function and suggest that pI interacts with: (a) the viral packaging signal (to initiate assembly); (b) thioredoxin (to elongate the particle); (c) one or more coat proteins; (d) pIV, the other morphogenetic protein. We have shown that pIV is secreted into the periplasm and subsequently integrates into the outer membrane where it forms a large multimer. This led us to propose that the multimer forms a channel through which the assembling phage passes. pIV is homologous to a family of bacterial proteins required for the export of specific proteins involved in pathogenesis. We have shown that some of these homologs form mixed multimers with pIV, suggesting that the assembly and/or export of filamentous phage and virulence factors may occur by related mechanisms. We have isolated and begun to characterize gene IV mutants defective in multimerization, others that alter the permeability properties of the outer membrane, and chimeric genes (varying portions of pIV and a bacterial homolog). We are also purifying the pIV multimer and, in collaboration with Sanford Simon, will look for channel activity directly.