How hydrophilic nucleic acids pass through a hydrophobic lipid bilayer, as during phage or viral infection, or as eukaryotic RNA transport through the nuclear membrane is not understood. They are using infection by phage T7 as a model to study DNA translocation across the bacterial cytoplasmic membrane. Cellular internalization of the complete T7 genome is a slow process, taking 10 minutes under normal conditions of infection, and they have developed an assay that monitors the rate and extent of genome entry. Current projects include identification of the E. coli and phage proteins that are involved in the passage of phage DNA from the phage head into the bacterial cytoplasm and to determine the source of energy for the process. They can also stop translocation of the phage DNA at a defined position on the genome during its entry into the cell, and intend to characterize the membrane channel through which the DNA is passing.
T7 grows well on most strains of bacteria to which it can adsorb; however, if the host cell contains the F plasmid, phage development is suddenly aborted midway through the infection. This abortive infection is a model for a common, but unexplained, biological phenomenon where the presence of one parasite in a cell protects from invasion by a second, unrelated parasite. The T7 - F interaction has become a more experimentally tractable model system than most other abortive infections. They have shown that expression of either of two separate phage genes, gene 1.2 and gene 10, and the single F gene pifA are responsible for the abortive infection. Ongoing projects in the laboratory include determination of the biochemical and physiological functions of these genes in the abortive infection. Plasmids expressing either gene 1.2 or gene 10 are normally lethal to cells expressing pifA; induction of a repressed phage gene in the presence of PifA leads to immediate and complete inhibition of all macromolecular synthesis and active transport in the cell. No other phage gene causes, or is necessary for, these physiological defects, which are identical to the defects seen during an abortive phage infection. The lab has selected for host mutants that tolerate the presence of each phage protein together with F PifA and are presently identifying the mutant E. coli genes by classical and molecular genetic techniques. One of these genes has not been defined previously and likely codes for an integral membrane protein. Some other mutants afford unprecedented levels of resistance to the antibiotic tetracycline and may thus be altered in novel E. coli functions.
Bacteriophage f1 and its close relative M13 are parasites that do not kill their host: progeny phage are continually extruded through the cell membrane. Phage production does, however, slow the growth rate of the bacterium. By selecting for faster growing phage-infected cells, they have discovered that the phage can integrate into the bacterial chromosome and exist as a stable lysogen or pseudolysogen. This result was totally unexpected, since f1 only contains 10 genes, none of which are known to function like the phage l repressor. The chromosomal and phage attachment sites by DNA sequencing are being identified and a genetic analysis of both the phage and host genes that are required for stable integration are beginning.
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