Polymyxin resistance in Agrobacterium tumefaciens and its effect on crown gall tumor induction.

Polymyxin-resistant (PBLr) mutants of Agrobacterium tumefaciens A6, B6, and B6M were isolated from polymyxin-sensitive (PBLs) parent strains in a defined medium containing 600 microgram of polymyxin B sulfate per millilitre. The weight and number of tumors induced by PBLr mutants on a variety of host plants such as carrot, potato, and pinto bean were 45--75% less than those induced by PBLs wild types. The crude cell envelopes (CCE) prepared from both PBLs and PBLr bacteria were inhibitory for tumor initiation when they were applied before or during the inoculation of viable tumorigenic bacteria, but not when they were applied 30 min after the inoculation of infectious bacteria. The potency to inhibit the tumor initiation by the CCE prepared from PBLs cells was approximately 50% higher than that by the equal amount of the CCE prepared from PBLr cells. The concentration of CCE preparations required to reduce tumor induction 50% in carrot and pinto bean was determined to be 2.6 mg/mL and 4.0--6.2 mg/mL for the CCE derived from PBLs and PBLr cells, respectively. These data suggest that the envelope structure or composition of PBLs and PBLr cells is distinct, and that the acquisition of resistance to polymyxin by agrobacteria modifies envelope structure or components which are essential for tumor initiation.

Effect of gibberellic Acid on crown gall tumor induction in aging primary pinto bean leaves.

Gibberellic acid was tested for its effect on tumor induction by Agrobacterium tumefaciens in primary pinto bean (Phaseolus vulgaris) leaves in various stages of development. The hormone was found to promote tumor induction in partially aged leaves but did not effect tumor induction in very young leaves or in fully matured leaves. It is suggested that the natural loss of susceptibility to tumor induction in maturing pinto bean leaves is associated with a concomitant loss of endogenous gibberellins and/or a sensitivity to gibberellins.

On the question of crown-gall tumor initiation by DNA of bacteriophage PS8.

DNA of Agrobacterium tumefaciens bacteriophage PS8 was isolated by using several procedures. Whole phage and phage DNA were tested for tumor-inducing ability on 10 species of plants with various additions to assist such activity. The reported tumorigenicity of phage PS8 DNA could not be confirmed, and no evidence to implicate phage PS8 involvement in tumor initiation was obtained.

Transfection in Agrobacterium tumefaciens.

Intact cells of Agrobacterium tumefaciens were examined for ability to take up biologically active LR-4 phage deoxyribonucleic acid (DNA) from the surrounding medium. DNA incorporation as measured by subsequent plaque formation (transfection) failed to occur when the bacteria were grown in defined minimal salts media, and was restricted to a 4-hr period in the early log phase of growth in enriched media. In the latter case, maximal transfection frequencies were obtained after a 25- to 30-min incubation with 22.5 mug of phage DNA/ml. Higher DNA concentrations or longer incubation times were inhibitory. Transfection was completely inhibited by deoxyribonuclease but not by ribonuclease, trypsin, or phage-specific antisera.

Enhancement of Agrobacterium tumefaciens Infectivity by Chlorpromazine Hydrochloride.

The tumor-initiating ability of Agrobacterium tumefaciens was enhanced two- to threefold after a preinoculation exposure to the phenothiazine derivative chlorpromazine hydrochloride. The mean number of tumors initiated per viable bacterium on pinto bean leaves was greatest after a 1-hr exposure to 15 mug of the drug per ml. Chlorpromazine had no significant effect on tumor initiation when applied to previously inoculated leaves. Chlorpromazine at 10 mug/ml was bacteriostatic; concentrations of 20 mug/ml and higher were bactericidal. In contrast to mitomycin C, which also enhances A. tumefaciens infectivity, chlorpromazine failed to induce temperate phage in several lysogenic strains of A. tumefaciens. Replication of a virulent A. tumefaciens phage, however, was not inhibited by the presence of chlorpromazine.

Enhancement of Agrobacterium tumefaciens infectivity by mitomycin C.

The ability of Agrobacterium tumefaciens to induce pinto leaf tumors may be enhanced two- to threefold after treatment with mitomycin C. The enhancement may be obtained with either lethal or nonlethal concentrations. With 10-min treatments, an optimal response was obtained with 0.005 mug of mitomycin C per ml in the absence of any change in the number of viable cells. Both the tumor induction process and the tumors induced by treated cultures appear qualitatively the same as controls. To account for these results, the antibiotic must increase the proportion of viable cells that will subsequently initiate tumors. One, or at most a few, random lesions in the bacterial chromosome seem to be the necessary requirement for this promotion. At mitomycin concentrations of 1 and 5 mug/ml, the ability of A. tumefaciens to initiate tumors is rapidly lost, indicating that a fairly intact bacterial chromosome is one of the essentials for the tumor induction process.

Deoxyribonucleic acid homology and taxonomy of Agrobacterium, Rhizobium, and Chromobacterium.

Hybridization experiments were carried out between high molecular weight, denatured, agar-embedded deoxyribonucleic acid (DNA) and homologous, nonembedded, sheared, denatured (14)C-labeled DNA from a strain of Agrobacterium tumefaciens and Rhizobium leguminosarum (the reference strains) in the presence of sheared, nonembedded, nonlabeled DNA (competing DNA) from the same or different nomen-species of Agrobacterium, Rhizobium, Chromobacterium, and several other organisms. Percentage of DNA homology was calculated from the results. The findings are discussed in relation to previous taximetric studies, present classification schemes, and guanine-cytosine content of the DNA. Strains of A. tumefaciens, A. radiobacter, A. rubi, A. rhizogenes, R. leguminosarum, and R. meliloti exhibited a mean percentage of DNA homology greater than 50 with the two reference strains. A. tumefaciens, A. radiobacter, and A. rubi were indistinguishable on the basis of DNA homology, with strain variations for this group involving up to 30% of their base sequences. The remainder of the organisms studied fall into at least six distinct genetic groups: (i) R. (Agrobacterium) rhizogenes, which is more homologous to R. leguminosarum than to the A. tumefaciens-A. radiobacter group; (ii) R. leguminosarum; (iii) R. meliloti; (iv) R. japonicum, which has a mean DNA homology of some 38 to 45% with the reference strains; (v) Chromobacterium, which is as genetically remote from the reference strains as, for example, Pseudomonas; and (vi) A. pseudotsugae strain 180, which has a DNA homology with A. tumefaciens and R. leguminosarum of only about 10%. Since this latter homology value is similar to what was found after hybridizations between the reference strains and organisms such as Escherichia coli and Bacillus subtilis, A. pseudotsugae should definitely be removed from the genus.

Ultraviolet-induced changes in the infectivity of Agrobacterium tumefaciens.

The infectivity of Agrobacterium tumefaciens strain B6 irradiated with short-wavelength ultraviolet light was followed as a function of dose. Previously reported enhancements of B6 infectivity by ultraviolet irradiation, in samples inoculated after 1.75 hr of dark incubation at 27 C, or immediately following irradiation, were found to occur most frequently after losses in cell viability of 60% and of 90% or more, respectively. Changes in colony-forming ability and tumor-initiating ability with increasing dose showed no obvious correlation until the maximal infectivity promotion of samples inoculated immediately after irradiation was reached. Thereafter, both bacterial responses typically decreased in parallel. With low dose rates, infectivity promotions were obtained with less than 10% loss in cell viability. Data for tumor appearance and tumor growth resulting from inoculations with irradiated cultures showed no significant differences from controls, nor did the age of the bacterial culture or age of the host plant influence the response. The infectivity promotion appears to result from an increase in the proportion of viable cells that will subsequently initiate tumors. The characteristics of this ultraviolet infectivity promotion are shown to be most similar to those found in prophage and bacteriocin induction.