Association of the rITA Protein with the Bacterial Membrane ( bacteriophage T 4 / E . coli / mutants )

Cell membrane proteins synthesized after infection of Escherichia coli B with wild-type phage T4 and rIIA mutants were analyzed by dodecyl sulfatepolyacrylamide gel electrophoresis. A protein with an approximate molecular weight of 74,000 is present in membranes isolated from T4r+-infected cells, but is not found in membranes prepared from cells infected with an rIA mutant in which the major part of the rIIA cistron is deleted. In addition, infection of E. coli B with different rIIA amber mutants and deletions gives peptides, which are associated with the bacterial membrane, of molecular weights consistent with the location of the respective mutations in the cistron. The rIIA protein is synthesized with delayed early kinetics. The synthesis of the rIIB protein, which is also located in the membrane, is not affected by mutations in the A cistron; conversely, synthesis of the rIIA protein is not affected by mutations in the B cistron. A mutant (rI1 1589) contains a deletion that originates in the A cistron and extends into the adjacent B cistron. This mutant directs the synthesis of a compound membrane protein consisting of the undeleted portions of the A and B cistrons. The synthesis of the compound protein appears to be under the control of the A promoter. The Escherichia coli cell envelope is modified after infection with wild-type phage T4. For example, the cells become resistant to lysis from without (1), can be lysis inhibited (2), exclude superinfecting T4 phage (3) and ghosts (4), become increasingly permeable to ions and other compounds (5-7), and increase their synthesis and hydrolysis of phospholipids (8-10). At least some of these functions are coded for by the phage, because specific phage mutants, such as r (11), spackle (12), ac (7), and imm (13) affecting these properties of the membrane, have been isolated. Furthermore, some of these changes require protein synthesis for their elucidation, indicating that phage-coded proteins are involved in membrane modification (13, 14). One of these membrane changes is coded for by the rnI locus, which is composed of two cistrons, A and B (15). The r mutants do not elicit the phenomenon of lysis inhibition (11) and are incapable of growing in E. coli cells lysogenic for phage X (15). It has already been shown (16, 17) that the rIIB protein is an inner membrane protein made after phage T4 infection. In the present investigation, we show that the rIIA protein is also associated with the bacterial envelope. MATERIALS AND METHODS Bacteria and Phage. E. coli H150 is a mutant of E. coli B derived from strain B207 (18) that requires high levels of K+ in the medium for growth and is sensitive to rifampicin. Wild-type phage T4, the deletions r1I H88, r1I A105, rII 1589, and the amber mutants r1I EM64, rII HB32, and rII HB35 were used. The rII mutants were obtained from Dr. Sewell P. Champe and Dr. Peter Natale. Media and Growth Conditions. Cells were cultured in a glucose and salts medium supplemented with 0.3% casamino acids (19). Infected cells were labeled in medium containing 0.012% casamino acids. Growth of the bacteria and infection by phage T4 were described (19). All infections were monitored and, in all cases, less than 1% of the cells survived the infections. A 400-ml culture of cells at 5 X 108 cells per ml was infected at a multiplicity of 5 phage per cell. Phage-infected cells were labeled after infection for 10 min, with 0.063 MACi/ml of 14C or 0.19 ,Ci/ml of 3H with a mixture of amino acids (New England Nuclear Corp.). For 4 or 5 min of labeling, 0.13 ,Ci/ml of 14C and 0.38 ,Ci/ml of 3H were used. The cells were rapidly cooled, collected by centrifugation, and subjected to the appropriate fractionation. Membranes Were Isolated according to the method of Kaback (20). Analysis of Proteins by Dodecyl S04-Polyacrylamide Gel Electrophoresis. The preparation of gels and electrophoresis were done according to Laemmli (21). 10% Acrylamide-0.27% bis-acrylamide gels were used. Frozen gels were sliced with a commercially available lateral gel slicer (Diversified Scientific Instruments, San Leandro, Calif.). The sliced gels were transferred to counting vials and 0.5 ml of NCS solubilizer (Amersham-Searle) or Protosol (New England Nuclear Corp.) and 0.05 ml of distilled water were added. The vials were then held at 45-600 for 2 hr. To the cooled vials, 0.02 ml of freshly prepared 4% ascorbic acid was added followed by 10