9506954 Molineux The specific goal of this research is to provide a complete understanding of the mechanism of F exclusion of T7 and related phages, an observation first made over 30 years ago. This exclusion system exhibits many physiological, and perhaps also mechanistic, similarities with other abortive infections, including rex and P22 sieB exclusion of other lambdoid phages, rex exclusion of T4 rII mutants, and Col Ib exclusion of T5 and related phages. These abortive infections are all characterized by a complex phenomenology with no underlying molecular model or rationale to describe their properties, and it has proven difficult to distinguish between the primary cause of the abortive infection and ancillary events that occur merely because the abortively infected cell is dying. At the physiological level, the dysfunctions that occur during an abortive phage infection also resemble those occurring during cell killing by some bacteriocins, during eukaryotic cell killing by some bacterial toxins, and during non-permissive infections of specific cell types by certain animal viruses. Many of these biological systems are only poorly understood at the molecular level, at least in part because the activities of the genes involved in causing cell death have not been defined. In order to fully understand the molecular basis of any exclusion system, it is necessary to know all of the genes involved. From previous work from this laboratory, the T7-coded targets of F exclusion are known to be genes 1.2 and 10; both genes interact with F pifA, the sole F determinant of exclusion, and in doing so kill the cell. Killing occurs in the absence of phage infection: cloning plasmids expressing either 1.2 or gene 10 as the only T7 gene is lethal to cells harboring pifA as their only F gene. A genetic selection for mutants of E. coli that tolerate co-expression of the T7 and F genes has been developed and it is expected that various different mutants will be isolated that do not support F exclusion of T7. An initial screen for such mutants resulted in the isolation of one strain that, when containing wild-type F or a multicopy plasmid expressing pifA, plates T7 at the same eop as an F- strain. Some other mutants isolated in this screen do not exclude (when containing F) T7 as efficiently as their isogenic parent strain and have acquired a phenotype of resistance to high levels of the antibiotic tetracycline, suggesting an involvement of cell membrane components in the abortive infection. A more detailed study of these mutants, and others to be obtained in a more extensive search, will provide the essential genetic framework necessary for a complete molecular understanding of this phage exclusion system. %%% The goal of this research is to provide a complete understanding of the mechanism where a bacterial conjugation factor (the F plasmid) prevents the successful infection of bacterial cells by the T7 phage, a bacterial virus. This study should impact the field of host-parasite interactions in general, since it is likely that the phage and plasmid genes involved in F exclusion of T7 interact with conserved cellular functions whose activities are compromised in many different biological settings. ***
