After infection ofEscherichia coli B by bacteriophage T5, a major new protein species, as indicated by polyacrylamide gel electrophoresis, appears in the cells' membranes. Phage mutants with amber mutations in the first-step-transfer portion of their DNA have been tested for their ability to induce membrane protein synthesis after they infect E. coli B. We have found that phage with mutations in the Al gene of T5 do not induce the synthesis of the T5-specific major membrane protein, whereas

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... e that are mutant in the A2 gene do induce its synthesis. We conclude that gene Al must function normally for T5specific membrane protein biosynthesis to occur and that only the first 8% (firststep-transfer piece) of the DNA need be present in the cell for synthesis to occur. We recently demonstrated that when T5 bacteriophage infects Escherichia coli a major new protein of an approximate molecular weight of 57,000 appears in the cell membrane (3a). This protein, found in both the inner and outer membrane, accounts for as much as 10% of the membrane proteins synthesized early after infection of E. coli B by T5. The membrane protein is not seen if the phage are UV irradiated prior to infection. The kinetics of synthesis of the T5 membrane protein are consistent with it being a pre-early protein, i.e., a protein coded for by the initial 8% piece of T5 DNA (first-step-transfer [FST] DNA) that is transferred spontaneously to the cell after adsorption of the phage. Transcription and translation of this 8% of the DNA are necessary for the transfer of the remaining 92% of the DNA to the cell, which is followed by early and late protein synthesis (8; D. J. McCorquodate, Crit. Rev. Microbiol., in press). Proteins coded for by the initial 8% segment of DNA are synthesized most rapidly between 3 and 9 min postinfection (10, 11), at is the T5-specific membrane protein. It is also synthesized in cells that have been infected in the presence of chloramphenicol and then blended prior to the removal of the chloramphenicol (and hence contain only the FST DNA), and in T5-infected cells containing the colicinogenic factor Col Ib. Infection of these latter cells results in little or no synthesis of early or late proteins (14). A variety of mutants that map in the FST region have been isolated, and their properties have been studied (2, 11). Mutants in gene Al do not transfer the whole phage DNA molecule, do not break down host DNA after infection, and do not shut off the synthesis of host and pre-early phage proteins. Some Al-mutants are also unable to grow in cells containing the X prophage (7). Mutants in gene A2 are defective only in the transfer of the whole phage DNA molecule. This paper presents evidence that mutants in gene A2 induce normal synthesis of the T5-specific membrane protein, whereas mutants in gene Al do not. We conclude that only the first 8% of the DNA is necessary for synthesis of the membrane protein and that gene Al may be the structural gene for it. MATERIALS AND METHODS Organisms and media. E. coli B was obtained from the laboratory of M. J. Bessman, and E. coli CR63 was from M. L. Dirksen. M9 medium was made as described by Herriot and Barlow (5). Wildtype T5 was originally obtained from S. E. Luria. T5 amH16d, carrying an amber mutation in pre-early gene Al, and T5 amH231, carrying an amber mutation in pre-early gene A2, were isolated by Hendrickson and McCorquodale (4) after in vivo mutagenesis with 5-bromodeoxyuridine. The amber mutants were grown on E. coli CR63 by the confluent lysis method (1). Isolation of bacterial membranes. The experiments empolyed a double-labeling procedure in which proteins made in uninfected cells were labeled with ['4C]amino acids, and proteins made in infected cells were labeled with [3H]amino acids. Cells were grown at 37 C in M9 medium containing 0.5% yeast extract, 5 x 10-4 M CaCl2, and 10 ug each of leucine and tyrosine per ml from a 5% overnight inoculum in the same medium. Cultures of 100 ml were routinely used. Infection was carried out when the cells reached a concentration of about 109 cells/ 542 on May 9, 2020 by guest