The goal of this research project is to characterize the Pif region of the F sex-factor. This region plays a role in the control of initiation of F replication at oriV1, (included in the region) and is involved in the abortive infection of certain virulent bacteriophages. The following topics will be investigated: I. The Genetics of the Pif Region and the Control of Pif Gene Expression - a) determine the number of Pif genes; b) identify pif protein products; c) study the location of pif proteins in uninfected and phage infected cells; d) determine the location and function of pif regulatory genes; e) determine the DNA sequence of the pif region; f) map transcription start points for pif expression. II. The Relationship of the Pif Region to F Factor Replication at oriV1. A portion of the Pif region overlaps the origin of F factor replication, oriV1. We have found a negative control gene, pifC, which regulates the synthesis of pif gene products in this region. In addition, positive control proteins (1 and 2) which play a role in the initiation of plasmid replication, appear to be located near or within the pif region. We will determine whether the pif genes and the positive control protein form and operon with a common regulatory region; b) establish the relationship of the pifC protein to the positive control protein reported by Eichenlaub (1) and to other proteins assigned to this region by Matsubara's work (2). III. Determination of the Mechanism by which Host Mutations Affect Pif Gene Expression. Mutations in the pimA gene prevent F factor inhibition of T7 development. We will a) establish whether the pimA mutation inteferes with the expression or activity of the Pif region; b) identify the pimA protein project after cloning the pimA structural gene; c) study the interaction of pimA with other components of the host cell; d) isolate pimA mutants to determine the mechanism of pimA; e) study the affect of pimA mutations on other plasmid and chromosomal operons. IV. Characterization of T7 mRNA Synthesis after Infection of Cells Containing Pif Region DNA - we will reinvestigate the level of certain T7 late mRNA species after T7 infection of F- cells and cells containing Pif region DNA. Quantitation will be performed by liquid hybridization of labeled RNA samples to specific regions of T7 DNA cloned onto plasma vectors.
|Miller, J F; Malamy, M H (1986) Mutational and in vivo methylation analysis of F-factor PifC protein binding to the pif operator and the region containing the primary origin of mini-F replication. Proc Natl Acad Sci U S A 83:1433-7|
|Miller, J F; Lanka, E; Malamy, M H (1985) F factor inhibition of conjugal transfer of broad-host-range plasmid RP4: requirement for the protein product of pif operon regulatory gene pifC. J Bacteriol 163:1067-73|