Purification and characterization of agrocin 84.

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.

Chemical nature of agrocin 84 and its effect on a virulent strain of Agrobacterium tumefaciens.

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.

Host-phage interaction on Agrobacterium tumefaciens. IV. Phage-directed protein synthesis.

Gel electrophoretic and autoradiographic techniques were used to detect the temporal sequence of protein synthesis after infection of the sensitive strain Agrobacterium tumefaciens with phage LV-1. Three classes of protein were detected: early proteins, class I, which include a protein capable of shutting off host protein synthesis; class II, proteins which are detected after 30 min; and late proteins, class III, which include the phage-directed endolysin and five additional proteins that appear 45 min after infection.

Host-bacteriophage interaction in Agrobacterium tumefaciens. 3. Phage-coded endolysins.

Endolysins were detected in a sensitive strain of Agrobacterium tumefaciens (B6) after infection with phage LV-1 and in the lysogen A. tumefaciens V-1 after induction with mitomycin C. A similar endolysin was found in mitomycin C-induced A. tumefaciens C-58, which apparently harbors a defective prophage.

Replication of bacteriophage SH-133 in the facultative autotroph Hydrogenomonas facilis. I. Bacteriophage synthesis under heterotrophic, autotrophic, and mixotrophic growth conditions.

The ability of bacteriophage SH-133 to replicate in heterotrophically (H-) and autotrophically (A-) grown Hydrogenomonas facilis was examined. Both the synthesis of infectious phage particles and the efficiency of plating (EOP) were reduced by 90% in A-grown cells. Adsorption of phage and lethal effects on H. facilis were identical in both systems. One-step growth experiments showed that cell lysis preceded the appearance of infectious particles in A-grown cells. Burst size studies with mixotrophically grown cells did not indicate the presence of an inhibitor of phage synthesis indigenous to autotrophic metabolism. DNA synthesis was identical in H- and A-grown infected cells; however, protein synthesis was significantly reduced in A-grown infected cells when compared with protein synthesis in H-grown infected cells. The data suggest that the reduction in EOP and phage synthesis in A-grown cells is caused by a defect in viral protein synthesis which results in the limited production of an essential viral protein at the time of cell lysis.

Replication of bacteriophage SH-133 in the facultative autotroph Hydrogenomonas facilis. II. Bacteriophage synthesis and hydrogenase induction under step-up and step-down growth conditions.

Autotrophically grown infected cells are able to replicate phage SH-133 after being switched to a heterotrophic environment (step-up growth). The effect of step-down growth on phage replication varies with the choice of organic substrate. Phage replication and the induction of cellular hydrogenase occur under step-down growth from acetate but not peptone broth. The requirement of a continued source of energy for phage replication in either heterotrophically or autotrophically grown cells could be uniquely demonstrated in this phage-host system by the deletion of hydrogen from the growth medium.

Bacteriophage release in a lysogenic strain of Agrobacterium tumefaciens.

Bacteriophage release in a lysogenic strain of Agrobacterium tumefaciens V-1 is temperature-sensitive. At 25 C and 30 C, phage was released in a ratio of 1 plaque-forming unit per 100 bacteria; at 35 C, although bacterial growth was not inhibited, phage release was suppressed. Phage synthesis was induced by heat shock, 42 C for 30 min, ultraviolet irradiation, and mitomycin C. Induction by ultraviolet light was unusual-an immediate rise in phage titer followed irradiation. A large increase occurred after a 90-min latent period. The lysogenic strain was cured of the phage by incubation at 37 C, and the cured strain produced plant tumors.

Isolation and characterization of Hydrogenomonas facilis bacteriophages under heterotrophic growth conditions.

Pootjes, Christine F. (The Pennsylvania State University, University Park), R. B. Mayhew, and B. D. Korant. Isolation and characterization of Hydrogenomonas facilis bacteriophages under heterotrophic growth conditions. J. Bacteriol. 92:1787-1791. 1966.-We have isolated five strains of bacteriophage specific for Hydrogenomonas facilis. The host range of the phage is limited to H. facilis. Morphologically, the phage particles consist of a head 580 A in diameter and a short tail 200 A in length. The particles share a common surface antigen, and all contain deoxyribonucleic acid. The five strains differ from each other in growth characteristics, heat stability, and neutralizing antigens.