Isolation and morphology of temperate Agrobacterium tumefaciens bacteriophage. J. Bacteriol. 92:746-750. 1966.-Lysogeny was detected in 14 strains of Agrobacterium tumefaciens among 130 bacterial strains tested with strain B-6 used as the host. Partial lysis was observed with 13 additional bacterial strains. Morphological studies of five strains showed that the phage had similar features. A typical phage (Lv-1) had a polyhedralshaped head, approximately 71 by 63 my, and a tail, approximately 211 m,t by 9.5 m,u. The phage nucleic acid was found to be deoxyribonucleic acid. The bacteriophage have been designated L (for lysogenic) followed by the bacterial strain designation in lower case letters. A temperate bacteriophage (w) was isolated from Agrobacterium tumefaciens (strain B-6) by Beardsley in 1955 (2). He used a phage-sensitive isolate of B-6, which had been obtained by ultraviolet irradiation, as the host. Other strains of A. tumefaciens tested, by Beardsley, as hosts for the w phage, however, failed to show zones of lysis (3). Recently J. McSharry and T. Stonier (personal communication) detected a temperate phage in A. tumefaciens B-2. This phage (PB-2) caused lysis of strains B-6, B6-G6, and a derivative of B-2.

A procedure for the rapid purification of milligram quantities of agrocin 84, a bacteriocin-like compound produced by Agrobacterium radiobacter strain K-84, has been developed. This procedure, which employs charcoal adsorption, ion-exchange, sieving chromatography, and continuous-flow electrophoresis, can yield agrocin 84 which is 65% pure on a dry weight basis. The purest preparations were strongly ultraviolet absorbing, with a maximum at 264 nm (epsilon 7.0/264 = 22,675 cm2 - M-1) and a minimum at 227 nm (ratio of 264 to 227 nm, 6.00). As has been reported, agrocin 84 contains an unusual phosphoramidate or 6-N-acyl linkage to adenine. Adenine, glucose, and phosphate are present in a 1:1:2 molar ratio. The molecular weight was estimated to be 1,350.

Agrocin 84, produced by Agrobacterium radiobacter K84, inhibited ribonucleic acid, deoxyribonucleic acid, and protein synthesis and amino acid transport in a susceptible, virulent strain of A. tumefaciens H-38-9. Cell motility was immediately stopped by action of the agrocin, 50% of the cells were killed within 15 min of contact, and the-remainder were inhibited. Agrocin 84 is trypsin and pepsin resistant, but chemical analysis indicated a small peptide with a molecular weight of 2,500 containing six different amino acids, including nine molecules of glutamine or glutamic acid and seven molecules of serine.

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SUMMARYFive temperate Agrobacterium phages, two of which are defective, and one presumed temperate, phage PS8, isolated from crown gall tissue, were prepared, purified and compared.The four phages, omega, PS8, PB2A and LV-I, were indistinguishable. They possess a hexagonal head of about 7o nm. with a flexible tail about 2oo nm. long. Their DNA had a T,, of 92.7 ° _+ oq °, a ~o GC of 56"7 + 0-2, a mol. wt of 34"1 × IO 6 and comprised the four normal bases A, T, G and C. No evidence was found for repetitive units in PBzA. These four genome DNA types gave IOO % hybridization (limit of error 6 %). The electrophoretic protein profiles on SDS polyacrylamide gels are essentially identical: four major bands with similar tool. wt. About half of the phage protein has a mol. wt of 48,ooo (perhaps a major head protein), about one-third has a mol. wt of 30,000, one sixth has a mol. wt of 15,5oo and 4% has a mol. wt of 69,0oo. No omega-type prophage DNA was detectable in the cured bacteria v-I c by DNA: DNA hybridization. The latter bacteria were as pathogenic as the prophage-containing parent strain v-I, which throws some doubt on the role of the omega phage group in crown gall induction. The phage-like particle Po362, isolated from the pathogenic Agrobacterium tumefaciens o362 had a 60 to 7o nm. head and a straight I3o nm. tail. Its DNA was double-stranded with a slightly lower T,~ of 92-4 °, a mol. wt of 25 × lO 6. The electrophoretic profiles of coat proteins differed from those of the other phages. Its DNA is less than 17 % homologous with the omega group. The eventual role of this phage in crown gall induction remains to be established. The phage-like particle P8149, isolated from the non-pathogenic Agrobaeterium radiobacter NCm 8149, was quite different. The bipyramidal head was much smaller (40 nm. diameter) and had a very short bud-like tail. The DNA was doublestranded, with a Tm of 93.9 °, a mol. wt of Io × Io 6. The electrophoretic profile of the coat proteins differed from those of the other phages. The DNA does not hybridize with the omega group. As this phage was isolated from a non-pathogenic A.radiobacter, it probably has no function in crown gall. A. radiobacter 8149 is bilysogenic.