Allen Kerr

Agrobacterium vitis sp. nov. for strains of Agrobacterium biovar 3 from grapevines.

Agrobacterium isolates from grapevines that were previously characterized as biovar 3 strains were compared with the type strains of the four previously described species of the genus Agrobacterium, Agrobacterium tumefaciens, Agrobacterium radiobacter, Agrobacterium rhizogenes, and Agrobacterium rubi. Ten grapevine isolates were distinguished from other Agrobacterium species phenotypically by a number of biochemical tests and by their reaction with a monoclonal antibody raised to biovar 3. Levels of DNA binding between the type strains of previously described species and three grapevine isolates were estimated by optical measurement of initial DNA renaturation rates. The levels of DNA binding between grapevine isolates were 78 to 92%, and the levels of DNA binding with other type strains ranged from 7 to 47%. Our results indicate that the grapevine isolates are distinct from the type strains of the four previously described Agrobacterium species. A new species, Agrobacterium vitis, is proposed; the type strain is strain NCPPB 3554.

Biological control of crown gall through bacteriocin production

Abstract Crown gall caused by Agrobacterium radiobacter var. tumefaciens can be controlled biologically by a closely related non-pathogenic bacterium, A. radiobacter var. radiobacter . The control mechanism operates through the production of a bacteriocin by the controlling organism. With one exception, pathogenic strains sensitive to the bacteriocin are effectively controlled. The exception is a strain which produces another bacteriocin that inhibits the controlling organism. Pathogenic strains insensitive to the bacteriocin produced by the controlling organism are not subject to biological control. When a bacteriocin-sensitive pathogenic strain is exposed to bacteriocin, resistant colonies develop but these are no longer pathogenic.

Conjugal Transfer of Nopaline and Agropine Ti-Plasmids- The Role of Agrocinopines

SummaryThe conjugative behaviour of nopaline and agropine Ti-plasmids has been investigated. Using a technique which avoids enrichment of transconjugants on a mating medium we have shown that preculture in the presence of agrocinopines A or B of donor strains harbouring nopaline Ti plasmids promotes plasmid transfer whereas preculture of the same strains in the presence of nopaline has no such effect. Similarly, preculture in the presence of agrocinopines C or D promotes Ti-plasmid transfer from strains harbouring agropine Ti-plasmids.

Acquisition of virulence by non-pathogenic isolates of Agrobacterium radiobacter

Abstract Tumours developed on tomato plants inoculated with Agrobacterium radiobacter var. tumefaciens biotype 2. When tumours were contaminated with the saprophyte, Agrobacterium radiobacter var. radiobacter biotype 1, the previously saprophytic bacterium became virulent. Acquisition of virulence was dependent on the efficiency of the recipients, on the duration of contact between saprophyte and tumour and on the stage of tumour development. A difference in the efficiency of the donors was not detected. There was no evidence for acquisition of virulence when the bacteria were mixed in liquid culture.

Construction of a Tra− deletion mutant of pAgK84 to safeguard the biological control of crown gall

SummaryAgrobacterium radiobacter strain K84 is used commercially for the biological control of crown gall. It contains the conjugative plasmid pAgK84, which encodes the synthesis of agrocin 84, an antibiotic that inhibits many pathogenic agrobacteria. A breakdown of control is threatened by the transfer of pAgK84 to pathogens, which then become resistant to agrocin 84. A mutant of pAgK84 with a 5.9-kb deletion overlapping the transfer (Tra) region was constructed using recombinant DNA techniques. The BamHI fragment B1 which covers most of the Tra region was cloned in pBR325 and its internal EcoRI fragments D1 and H, which overlap the Tra region, were removed, leaving 3.7 kb and 0.5 kb of pAgK84 on either side of the deletion. The latter was increased to 3.3 kb by adding EcoRI fragment D2 from a BamHI fragment C clone. The modified pBR325 clone was mobilized into Agrobacterium strain NT1 harbouring pAgK84 with a Tn5 insertion just outside the Tra region but covered by the deletion. A Tra+ cointegrate was formed between the Tn5-insertion derivative and the pBR325-based deletion construct by homologous recombination. The cointegrate was transferred by conjugation to a derivative of strain K84 lacking pAgK84, in which a second recombination event generated a stable deletion-mutant by deletion-marker exchange. The resultant new strain of A. radiobacter, designated K1026, shows normal agrocin 84 production. Mating experiments show that the mutant plasmid, designated pAgK1026, is incapable of conjugal transfer at a detectable frequency.

Agrobacterium: genetic studies on agrocin 84 production and the biological control of crown gall☆

Abstract The role of the bacteriocin, agrocin 84, in the biological control. of crown gall was investigated by constructing new bacteriocinogenic strains. A small plasmid which codes for the ability to produce agrocin 84 has been identified and transferred to recipient strains by conjugation. Two donor strains were used; the avirulent strain 84 and the virulent strain 398. Agrocin production and virulence in the latter strain have been separated by genetic manipulation and avirulent, agrocin producing derivatives have been produced. Avirulent transconjugants which produced agrocin 84 varied in their effectiveness as biological control gents. Evidence is presented that this was probably due to variation in rate of growth of the different strains in the inoculation sites.

Regeneration of flax plants transformed by Agrobacterium rhizogenes

Regeneration of flax (Linum usitatissimum) following transformation by either Agrobacterium tumefaciens carrying a disarmed Ti-plasmid vector, or Agrobacterium rhizogenes carrying an unmodified Ri plasmid, was examined. Hypocotyl and cotyledon explants inoculated with A. tumefaciens formed transformed callus, but did not regenerate transformed shoots either directly or via callus. However, cotyledon explants inoculated with A. rhizogenes formed transformed roots which did regenerate transformed shoots. Ri T-DNA encoded opines were detected in the transformed plantlets and Southern hybridization analysis confirmed the presence of T-DNA from the Ri plasmid in their DNA. Transformed plantlets had curled leaves, short internodes and some had a more developed root system characterized by plagiotropic behaviour.

Agropine: A revised structure

Abstract The product known as agropine from crown-gall tumour is shown to be indistinguishable in all of its properties (physical, chemical, and biological) from N 2 -(1′-deoxy- d -mannitol-l′-yl)- l -glutamine, 1,2′-lactone, a C 11 H 20 N 2 O 7 compound produced by cyclisation of N 2 -(l′-deoxy- d -mannitol-l′-yl)- l -glutamine. Two prior assignments of a C 11 H 17 NO 7 formula obtained by high-resolution electron-impact mass spectrometry have been shown by field-desorption mass spectrometry to correspond to a fragment produced by the rapid loss of ammonia from the parent molecular species.

Arginine catabolism: A new function of both octopine and nopaline Ti-plasmids of Agrobacterium

SummaryThe oncogenic plasmids of Agrobacterium, the Ti-plasmids, carry genes that enable their bacterial host to catabolize opines. Opines are unusual amino acid derivatives that are only produced in crown gall tumours incited by oncogenic strains of Agrobacterium. The 2 opines, octopine and nopaline, are degraded by Agrobacterium strains carrying the octopine or the nopoline Ti-plasmid, respectively, to arginine and pyruvic acid, and to arginine and α-ketoglutaric acid. In this paper it is shown that the Ti-plasmids carry gene(s) involved in the utilisation of arginine as a carbon source. Strains harbouring wild type octopine or nopaline Ti-plasmids in the chromosomal context of strain C58C1 do not grow on arginine as a carbon source. However, they are able to grow on arginine provided that they are induced, or constitutive for opine catabolism. The features of ornithine utilisation are identical. The gene(s) involved in arginine and ornithine utilization in C58C1 (pTi-oct) or C58C1 (pTi-nop) are under the control of the regulator gene that controls octopine or nopaline catabolism. A tentative pathway of octopine utilization is proposed, in which at least two steps are Ti-plasmid coded, and probably belong to the same operon: 1-scission of octopine into arginine and pyruvic acid 2-transformation of an arginine derivative (GSA?) to glutamic acid.Arginine utilization as a carbon source is therefore a new function of the Ti-plasmid. As this function is not inducible by arginine but by opines, it provides a method for selecting regulatory mutants of opine catabolism in the genetic background of strain C58.

Agrobacterium: correlations between and transfer of pathogenicity, octopine and nopaline metabolism and bacteriocin 84 sensitivity

Abstract Seventy strains of Agrobacterium radiobacter were tested for pathogenicity, for their ability to utilize octopine and nopaline and for sensitivity to bacteriocin 84. There were many exceptions to the previously reported correlations between these characters. For example, many non-pathogenic strains were able to utilize octopine and/or nopaline and some were sensitive to bacteriocin 84. Nineteen pathogenic strains were tested for the ability to induce the synthesis of octopine and nopaline in crown gall tissue. As previously reported, biotype 1 strains which could utilize octopine, induced octopine synthesis in galls; the nopaline-utilizing strains induced nopaline synthesis. Although all biotype 2 pathogenic strains could utilize both octopine and nopaline, only nopaline was detected in galls induced by these strains. Studies on transfer of pathogenicity were used to determine which characters were transmitted with pathogenicity. Depending on which strains were used as donors, all the characters under study could be transmitted to an efficient recipient strain. The results are discussed in the context of the recent finding that the genetic information controlling pathogenicity in Agrobacterium is located on a large plasmid.