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PubMed1976

Octopine and nopaline synthesis and breakdown genetically controlled by a plasmid of Agrobacterium tumefaciens.

Several nopaline degrading strains and one octopine degrading strain are shown to loose oncogenicity as well as the ability to utilize these guanidine compounds when they are cured of their TI plasmid. To investigate whether the specific genes involved in the utilization of one or the other compound are located on the plasmid, plasmid-transfer experiments have been performed. The plasmid from a nopaline degrading strain has been transferred to a naturally non oncogenic Agrobacterium namely A. radiobacter. Furthermore, the plasmid from an octopine degrading strain has been transferred to a plasmid-cured strain which originally had the capacity to utilize nopaline. Both kinds of experiments prove that the TI plasmid determines the strain specificity with regard to the utilization of either octopine or nopaline. They also demonstrate that the synthesis of either octopine or nopaline in crown gall cells is also determined by genes located on the TI plasmid harboured by the transforming A. tumefaciens strains.

Extrachromosomal Inheritance↗
PubMed1978

In vivo transfer of the ti-plasmid of Agrobacterium tumefaciens to Escherichia coli.

The Ti-plasmids are naturally self-transmissible from their normal host Agrobacterium to E. coli. They are however unable to stably establish themselves as a replicon in E. coli. It is nevertheless possible to study the Ti-plasmids in E. coli with the help of Ti::RP4 cointegrate plasmids that transfer and maintain themselves very efficiently in E. coli. An E. coli harbouring such a Ti::RP4 plasmid is unable to catabolize octopine and unable to induce crown-gall tumours on plants.

Conjugation, Genetic↗
PubMed1979

Isolation of a recombination deficient Agrobacterium tumefaciens mutant.

The isolation of a recombination deficient (Rec-) strain of Agrobacterium tumefaciens is described. Strain LBA 4011 was mutagenized with nitrosoguanidine and after segregation 18,000 colonies were replica plated and UV irradiated. Twentytwo UV sensitive strains were isolated and tested for methylmethanesulphonate (MMS) sensitivity. Six of these strains were more MMS-sensitive than LBA 4011. A Ti plasmid that was genetically marked with Tn 1 (CbR) was introduced in these strains and the rescue of the CbR marker during superinfection with an incompatible cointegrate plasmid Ti::R 702 was determined. One strain exhibited a large reduction in rescue frequency. It is concluded that the latter strain was recombination deficient. This property did not influence the induction of plant tumours.

Methyl Methanesulfonate↗
PubMed1979

A new site-specific endonuclease showing phenotypical crypticity in a tumorigenic strain of Agrobacterium tumefaciens.

AtuBVI, an endonuclease showing new site-specificity, has been isolated from the tumorigenic strain IIBV7 of Agrobacterium tumefaciens, and is undetectable in the non-tumorigenic sister strain IIBNV6. AtuBVI degrades IIBV7 DNA in vitro and should, therefore, be regarded as being phenotypically cryptic in the bacterial cell; it also shows anomalous behavior under cerain incubation conditions. These properties point to a possible role for this enzyme in the insertion of exogenous Ti-plasmid DNA into plant tissues during tumorigenesis.

Coliphages↗
PubMed1978

Production of adventitious root primordia on hypocotyls of castor bean seedling, infected with Agrobacterium tumefaciens.

The development of sterile secondary tumours on hypocotyls of castor bean seedlings, inoculated with any of the ten isolates of Agrobacterium tumefaciens tested, were observed below the site of the primary tumours. Histopathological studies performed in the present work indicate that the observed secondary tumours were adventitious root primordia and not the ordinary type of secondary tumours. According to the available literature such findings are reported here for the first time. All the tested isolates of the present study were also able to initiate well defined roots in conjunction with crown gall (teratomas) on Bryophyllum crenata and Kalanchoe marmorata, but not in tomato.

Ricinus communis↗
PubMed1978

DNA modifying enzymes of Agrobacterium tumefaciens: effect of DNA topoisomerase, restriction endonuclease, and unique DNA endonuclease on plasmid and plant DNA.

Extracts from Agrobacterium tumefaciens strain ID135 contain three enzymes that have been characterized and partially purified. The first enzyme, a DNA topoisomerase, appeared to relax only negatively twisted DNA. The second enzyme, Atu I, a type II restriction endonuclease, generated the identical DNA digestion pattern as EcoRII when several DNAs were used. The third enzyme, endonuclease A, showed a preference for superhelical DNAs as substrates. When plasmid pCK135DNA, obtained from the virulent strain IDI135 of A. tumefaciens, or plant DNA was exposed to the three enzymes, changes in DNA patterns were observed due to either conformational changes or digestion of the DNAs. These enzymes may function in vivo in the processing and incorporation of bacterial DNA in plant cells.

Animals↗
PubMed1976

RP4 promotion of transfer of a large Agrobacterium plasmid which confers virulence.

Introduction of RP4 plasmid into Agrobacterium tumefaciens promotes the transfer on solid medium of large virulence-associated plasmids from virulent donor strains to a plasmidless avirulent recipient. Exconjugants were selected for the ability to utilize octopine or nopaline as the sole source of arginine, traits which are coded for by virulence-associated plasmids in the strains employed here. All exconjugants retained the arginine auxotrophy of the recipient strain, and were resistant to ampicillin and kanamycin, drugs to which RP4 confers resistance. Five exconjugant clones from one cross were shown by alkaline sucrose gradient analysis to contain both RP4 plasmid and the large virulence-associated plasmid of the donor strain. All five exconjugants exhibited virulence on carrot, sunflower and kalanchoe plants. These results indicate that virulence and the ability to degrade octopine are plasmid-borne traits in A. tumefaciens strains 15955 and A6, and extend the evidence that large plasmids in A. tumefaciens are vectors of virulence genes.

Arginine↗
PubMed1979

Rapid mapping of transposon insertion and deletion mutations in the large Ti-plasmids of Agrobacterium tumefaciens.

A procedure is presented, that has allowed the rapid assignment of transposon Tn1 and Tn7 insertion sites in the large (130 Md) nopaline Ti-plasmid pTiC58, to specific restriction enzyme fragments. Total bacterial DNA is isolated from Agrobacterium tumefaciens strain C58 mutants that carry a transposon in their Ti-plasmid, and digested with an appropriate restriction endonuclease. The fragments are separated on an agarose gel, denatured and transferred to nitrocellulose filters. These are hybridized against purified wild type pTiC58, or against segments of PTiC58, cloned in E. coli using pBR322 as a vector plasmid. DNA sequences homologous to the probe are detected by autoradiography, thus generating a restriction enzyme pattern of the plasmid from a digest of total bacterial DNA. Mutant fragments can be readily identified by their different position compared to a wild type reference. This protocol eliminates the need to separate the large plasmid from chromosomal DNA for every mutant. In principle, it can be applied to the restriction enzyme analysis of insertion or deletion mutants in any plasmid that has no extensive homology with the chromosome.

Chromosome Deletion↗
PubMed1976

Isolation and characterization of Agrobacterium tumefaciens mutants affected in the utilization of octopine, octopinic acid and lysopine.

Using an enrichment procedure, mutant strains of Agrobacterium tumefaciens were isolated that lacked the ability to utilize octopine as a nitrogen source. Of 55 such isolates, 44 were unable to utilize several amino acids; the remaining 11 strains were altered solely in their ability to utilize octopine, octopinic acid and lysopine. It is concluded that only the latter were plasmid mutations. Among them, there was a high, but no absolute, correlation with avirulence. All strains contained the T1 plasmid. All virulent strains showed active transport of octopine when they had previously been grown in medium containing octopine, whereas the avirulent strains failed to show such transport. All the virulent mutants induced tumours containing octopine. The results are discussed in relation to the hypothesis that the genes which code for the octopine synthesizing enzymes in the tumour are of bacterial origin.

Amino Acids, Dicarboxylic↗
PubMed1976

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.

Adenine Nucleotides↗
PubMed1979

Low-intensity microwave radiation and the virulence of Agrobacterium tumefaciens strain B6.

When virulent cells of Agrobacterium tumefaciens strain B6 were exposed to low-level microwave radiation at a frequency of 10,000 MHz and an intensity of 0.58 mW/cm2 for 30 to 120 min, a 30 to 60% decrease in their ability to produce tumors on potato and turnip disks was observed. This microwave exposure did not affect the viability of these bacteria or their ability to attach to a tumor-binding site nor did it induce thermal shock. This loss of virulence was reversible within 12 h.

Microwaves↗
PubMed1975

Carbohydrate catabolism of selected strains in the genus Agrobacterium.

Radiorespirometric and enzyme analyses were used to reveal the glucose-catabolizing mechanisms functioning in single strains of seven presumed Agrobacterium species. The Entner-Doudoroff and pentose cycle pathways functioned in A. radiobacter, A. tumefaciens, A. rubi, and A. rhizogenes. Whereas both catabolic pathways were utilized to an almost equal degree in the A. radiobacter and A. tumefaciens strains, use of the Entner-Doudoroff pathway predominated in the A. rubi and A. rhizogenes strains. A stellulatum catabolized glucose almost solely through the Entner-Doudoroff pathway. In A. pseudotsugae and A. gypsophilae, glucose was metabolized mainly through the Emden-Meyerhof-Parnas pathway; the pentose phosphate pathway was also utilized.

Aldehyde-Lyases↗
PubMed1979

Tumor induction by Agrobacterium tumefaciens prevented in Vigna sinensis seedlings systemically infected by ribonucleic acid viruses.

Cowpea (Vigna sinensis) seedlings failed to develop tumors after being inoculated with crown gall bacteria (Agrobacterium tumefaciens) if, at times earlier than 1 day later, they were inoculated on the primary leaves with a cowpea mosaic virus that systemically infects them. Inoculation with buffer or with a virus that is restricted to a localized infection, or to which the cowpea is immune, did not interfere with the subsequent development of tumors. The virus infection did not appear to affect directly the titer of A. tumefaciens in the inoculation sites. Nor did mixing of virus particles with A. tumefaciens prevent subsequent appearance of tumors. The influence of virus infection extended across grafts (into tissue that is not susceptible to the virus) and there prevented tumor formation. The sap from infected plants, but not purified virus, decreased tumor formation on carrot disks. Systemic virus infection may induce in cowpeas a translocated substance that prevents tumor induction by A. tumefaciens.

Mosaic Viruses↗
PubMed1977

Zeatin ribonucleosides in the transfer ribonucleic acid of Rhizobium leguminosarum, Agrobacterium tumefaciens, Corynebacterium fascians, and Erwinia amylovora.

Until recently, the presence in transfer ribonucleic acid (tRNA) of the hydroxylated cytokinin ribosylzeatin [N6-(4-hydroxy-3-methylbut-2-enyl)adenosine]was thought to be unique to higher plants. This extension of work from several laboratories indicates the presence of 2-methylthioribosylzeatin in the tRNA of the plant-associated bacteria Rhizobium leguminosarum, Agrobacterium tumefaciens, and Corynebacterium fascians, but not in that of Erwinia amylovora. This cytokinin has the cis configuration, as is normally found in the tRNA's of plants. The tRNA thionucleotide patterns in these bacteria are different from those of Escherichia coli, Bacillus subtilis, and Salmonella typhimurium, which contain the unhydroxylated analogs of ribosylzeatin or 2-methylthioribosylzeatin.

Corynebacterium↗
PubMed1978

Transfer of nitrogen fixation genes from a bacterium with the characteristics of both Rhizobium and Agrobacterium.

Strain T1K, reported to be Rhizobium trifolii strain T1 carrying the drug resistance plasmid RU-1drd, was able to transfer a cluster of nif+ genes to Escherichia coli K-12. Additional genetic material, resembling the gal-chlA region of E. coli, was also transferred from strain T1K. The segregation pattern of these transferred genes suggested that they were on a plasmid. Although strain TIK was able to nodulate red and white clover, it also formed very slow-growing galls on tomato stems and shared many physiological properties with Agrobacterium tumefaciens, to which it seemed more closely related than to R. trifolii. The R. trifolii hybrid T1 (R1-19drd), constructed by conjugation, did not share any of these properties of both A. tumefaciens. Thus, strain T1K appears to be a bacterium with properties of both A. tumefaciens and R. trifolii and with the capacity to transfer nif+ genes and other functions which it may have "cloned" from another bacterium such as Klebsiella.

Chromosomes, Bacterial↗
PubMed1978

Coordinated regulation of octopine degradation and conjugative transfer of Ti plasmids in Agrobacterium tumefaciens: evidence for a common regulatory gene and separate operons.

By using the analog noroctopine, mutants of agrobacterium tumefaciens were isolated with altered regulation patterns for the Ti plasmid-borne octopine utilization genes. These could be divided into three classes: (i) strains with a constitutive level of octopine enzymes and a high degree of spontaneous Ti transfer; (ii) one strain with constitutive octopine enzymes but no spontaneous Ti transfer; and (iii) strains with an altered inducibility in which, contrary to the wild-type Ti plasmid, conjugation and octopine utilization were induced by noroctopine. These results are best explained by the activity of a common regulatory gene. In a second step, using homo-octopine, mutants were isolated with lesions preventing the utilization of octopine. All mutations were plasmid borne and did not prevent the induction of tumors. Plasmids of two isolates were characterized by large deletions resulting in a decreased virulence and the absence of octopine in the tumor. With a plasmid carrying an inserted transposon Tn1, a significant number of strains were isolated which were unable both utilize octopine and to transfer the Ti plasmid. This suggests that there may be another common factor--presumably positive--between these traits. Transfer-negative mutants were still virulent. This seems to exclude a role for the conjugative transfer during the process of plant tumor induction. A way to test octopine oxidase by the use of permeable cells is described.

Arginine↗