Ψ, a Temperate Phage of Agrobacterium tumefaciens , Is Mutagenic

Abstract

A new temperate bacteriophage (phage '1) of Agrobacterium tumefaciens strain B91 is described. Many octopine-utilizing strains of A. tumefaciens harbor prophage 4 or a phage that is similar if not identical to it. This phage has a very narrow host range, and we found that its growth is strongly reduced in strains which carry an octopine pTi plasmid. When sensitive bacteria are infected with 4, 1 to 3% of the survivors carry mutations on the chromosome, as well as on the pTi plasmid. This phenomenon appears to be a direct consequence of lysogenization. The possible mechanisms whereby such 14-induced mutations occur are discussed. The first lysogenic system described in Agrobacterium tumefaciens involved temperate phage omega, which was isolated from strain B6 (1). Later, several other reports (3, 16, 25) described the occurrence of temperate phages in a wide variety of A. tumefaciens strains. All of these phages had similar morphological, biological, and biophysical properties and the same immunity.In 1975, using electron microscopy, Vervliet et al. (22) demonstrated the occurrence of "phage-like particles" or "defective phages" in mitomycin C-treated cultures of strains B6-806, B6S3, B2As, and CV1. All of these particles were morphologically identical. The particles released by strain B6-806 were shown to contain a DNA molecule which had a molecular weight of 25.2 x 106. Hybridization by the heteroduplex method revealed no homology between this DNA and that of phage Ql.We report here that the phage released by the strains mentioned above is not defective and is harbored by several other octopine-utilizing strains of A. tumefaciens. This phage is not related to Ql, and we have called it phage 4. We describe the general characteristics of this temperate phage and demonstrate that it has two unusual properties. First, it frequently induces mutations in its host during lysogenization; and second, its growth is inhibited in strains which carry an octopine pTi plasmid. MATERIALS AND METHODSStrains and media. The bacterial strains used in this work are described in Tables 1, 2, and 5. Phages Ql and * were obtained from lysates produced by UV induction of strains B6 and B91 (Table 2), respectively. The following media were used. Nutrient broth (NB) contained 8 g of Merieux nutrient broth per liter, 5 g of NaCl per liter, and 5 g of glucose per liter. Minimal medium (MM) contained 2 g of (NH4)2SO4 per liter, 6 g of KH2PO4 per liter, 14 g of K2HPO4 per liter, 1 g of trisodium citrate per liter, 0.2 g of MgSO4-7H2O per liter, and 5 g of glucose per liter. When necessary, Lamino acids (100 ,ug/ml), bases (100 ,ug/ml), or vitamins (50 ,ug/ml) were added. The synthetic media used for assays of catabolic pTi functions (19) contained 13.6 g of KH2PO4 per liter, 0.2 g of MgSO4-7H2O per liter, 0.005 g of FeSO4 per liter, 0.01 g of CaCl2 per liter, 0.002 g of MnCl2 per liter, and 2 g of glucose per liter; the nitrogen source was (NH4)2SO4 (2 g/liter) for SM medium, arginine (0.1 g/liter) for ASM medium, or octopine (0.3 g/liter; ...

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