Various unsaturated fatty acids (notably palmitoleic acid and oleic acid) interfered with plaque production by the lipid-containing bacteriophage PR4 on lawns of Escherichia coli. Addition of fatty acid to give 50 ,Lg/ml (-0.2 mM) at the time of infection prevented phage replication. If, however, the fatty acid was added after infection, normal amounts of phage were produced. If the fatty acid was added (to 50 ug/ml) to the host cell culture a long enough time before infection such that the

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... y acid concentration in the growth medium at the time of infection was reduced to E5 ,ug/ml (due to fatty acid incorporation by the host cells), normal phage replication occurred also. Neither palmitoleic acid nor oleic acid prevented PR4 attachment to E. coli. Several types of experiments indicated that it is the entry process of the virus that is inhibited by these fatty acids. Specifically, if the fatty acid was added at the time of infection, the host cells were not killed by the virus and no detectable amounts of viral protein were synthesized. In addition, experiments using purified radioisotope-labeled virions showed directly that entry is inhibited. Mutants of PR4 that did replicate in the presence of oleic acid arose spontaneously at a frequency of 106. Three of these mutants that have been further characterized have protein and phospholipid compositions indistinguishable from those of wild-type PR4. Lipid-containing bacterial viruses have been used in recent years as model systems for studying various aspects of enveloped viruses in general, including virion structures (1, 14), assembly processes (7, 12, 15), mechanisms of inactivation (8, 16, 17) , and the inhibition of virus replication (7). We are especially interested in bacteriophage PR4, which contains lipid and can replicate in gram-negative bacteria, including Escherichia coli, that carry an appropriate drug resistance plasmid (3-5). We are studying the structure of phage PR4 and its mode of replication in E. coli. We have characterized the lipids of the virion (10) and have determined the molecular weights of the six major proteins detectable in the virus (6). In the midst of a recent study on PR4 replication in various "membrane" mutants of E. coli (11), we found that unsaturated fatty acid auxotrophs of E. coli are not susceptible hosts for PR4. Unsaturated fatty acids such as palmitoleic acid (16:1, A9, cis) or oleic acid (18:1, A9, cis) must be supplied in the medium for growth of these cells. As we show in this paper, the presence of significant concentrations of either of these fatty acids in the growth medium prevents the replication of phage PR4 in even "normal" (i.e., non-fatty acid auxotrophic) E. coli cells. The characterization of this inhibition of phage PR4 replication by these fatty acids is described. MATERIALS AND METHODS General. The E. coli K-12 derivative used in these experiments has been previously described (11). Strain PS2R requires tryptophan (amber), arginine (ochre), and purines (amber) for growth and contains the RPl drug resistance plasmid. E. coli PS2R was grown in NBY, a rich nutrient broth-yeast extract medium previously described (10), or in supplemented low-sulfate medium (LS'). LS medium contains, per liter of distilled water: Tris, 12.1 g; KCl, 1.5 g; NaCl, 15 g; MgSO4 7H20, 25 mg; NH4Cl, 1 g; glucose, 5 g; KH2PO4, 25 mg; and K2HPO4, 75 mg (adjusted to pH 7.6). Appropriate supplements were added to LS to produce LS' to allow PS2R growth. Stocks of phages PR4 and T4 were grown in NBY or LS' medium in E. coli PS2R. Our procedures for growing and assaying both E. coli PS2R and phages have been previously described (10, 11). Assays for PFU in the presence of fatty acids were also performed, using only NBY top agar. Production and purification of labeled lvirus.