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At least 19 recordsLinked to original sources
PubMed1975

Particulate cytochrome c in Agrobacterium tumefaciens.

In Agrobacterium tumefaciens the main part of c-type cytochromes is tightly bound to the bacterial cell envelope structures. Several techniques were attempted to solubilize these cytochromes. The highest yield of cytochromes released is obtained by treatment of particle suspensions with 5% Triton X-100. Further purification confirms that the proteins are not really solubilized, but still aggregated in small heterogeneous complexes. Chromatography on a CM-cellulose column demonstrates that at least three different c-type cytochromes are present: cyt c-550, cyt c-552 and cyt c-556.

Cytochrome c Group↗
PubMed1978

Effect of sublethal heat injury on tumour induction and RNA synthesis in Agrobacterium tumefaciens.

When cells of Agrobacterium tumefaciens are subjected to sublethal heat injury at 45 degrees C for 20 min, less than 5% of the viable population retain their ability to initiate tumour formation on Kalanchöe daigremontiana. If the cells are then incubated in phosphate buffer at 27 degrees C for 2 h, tumour initiation returns to control levels. Inhibitors of DNA and protein synthesis had little effect on the recovery of tumour initiation after heat injury. Rifamycin, a specific inhibitor of RNA synthesis, dramatically reduced recovery of tumour formation. During the heating process large amounts of RNA leaked from the cells which correlated with a degradation of rRNA. The addition of Mg to the heating buffer reduced the loss of RNA into the medium and the degradation of rRNA and tumour initiation was retained at control levels. It is concluded that while current evidence indicates that DNA is probably the transforming agent, RNA synthesis is an important component of the induction process.

Bacterial Proteins↗
PubMed1979

Host range conferred by the virulence-specifying plasmid of Agrobacterium tumefaciens.

The host range of Agrobacterium tumefaciens 1D1109, known to induce crown gall only on grapevine (Vitis spp.), was extended to include many plant species by transferring a tumor-inducing plasmid (pTi) from strain 1D1, a broad-host-range pathogen. The pTi plasmid was mobilized by the conjugative plasmid pRK2, which was inserted into 1D1 by mating with Escherichia coli J53(pRK2). The resulting transconjugants were screened for their ability to induce crown gall tumors on hosts other than grapevine by inoculation into sunflower. Transconjugants that were virulent on sunflower were then tested on 36 different host plants and compared with host-limited strain 1D1109 and the donor strain. Two transconjugants induced tumors on the same 28 plant species as those of the original plasmid donor 1D1(pRK2) (pTi). These results show that pRK2 promoted transfer of the pTi plasmid and suggest that the pTi plasmid rather than the A. tumefaciens chromosome determined the host range of the pathogen. Insertion of pRK2 alone did not extend the host range of strain 1D1109. Insertion of pS-a into A. tumefaciens 1D1 by mating with E. coli J53-1 (pS-a) resulted in the concomitant loss of pTi and virulence. There appears to be incompatibility between pTi and pS-a.

Conjugation, Genetic↗
PubMed1979

Interactions and DNA transfer between Agrobacterium tumefaciens, the Ti-plasmid and the plant host.

Agrobacterium tumefaciens is a gram-negative bacterium with the unique capacity to induce neoplasmic transformations in dicotyledonous plants. Recently, both the mechanism and the biological significance of this transformation have been elucidated. Agrobacterium tumefaciens strains contain a large extrachromosomal DNA plasmid (the Ti-plasmid). This Ti-plasmid is responsible for the oncogenic properties of Agrobacterium strains. A particular segment of the Ti-plasmid, containing information determining the tumorous growth pattern and the synthesis of so-called 'opines', e.g. octopine (N-alpha-(D-1-carboxyethyl)-L-arginine) and nopaline (N-alpha-(1,3-dicarboxypropyl)-L-argine), is transferred and stably maintained and expressed in the transformed plant cells. This phenomenon can be understood as a 'genetic colonization' of the plant cells by bacterial plasmid DNA so that the transformed plant cells will produce and secrete into the medium amino acid derivatives (the opines) that Ti-plasmid carrying agrobacteria can selectively use as carbon and nitrogen sources.

DNA Restriction Enzymes↗
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↗
PubMed1979

Isolation of a non-tumor-inducing mutant of the Ti plasmid of Agrobacterium tumefaciens strain B6.

A nonpathogenic mutant of Agrobacterium tumefaciens strain B6 was isolated and its properties compared with the parental strain in an effort to localize the mutation. Both B6 and its mutant (B6-95) had similar colony color and morphology, were ketolactose positive, utilized octopine, and contained plasmid DNA. Kinetic analysis of DNA reannealing showed that total DNA homology and plasmid DNA homology between B6 and B6-95 was at least 90%. The length of both plasmids was found to be 58 micrometer. Plasmid DNA from both B6 and the mutant was digested with endonucleases and the fragments separated by agarose gel electrophoresis. In all cases the pattern for B6 was identical with that of B6(-95). The Ti plasmid from B6 and the mutant was transferred to an avirulent, plasmidless strain of A. tumefaciens by in vitro conjugation and transformation. All of the B6 transconjugants and transformants were virulent, whereas all of the mutant transconjugants and transformants were avirulent. Electrophoretic patterns of endonuclease-digested plasmid DNA from transformants were identical to those of plasmid DNA from B6. Therefore, we conclude that the virulence mutation lies on the Ti plasmid.

Conjugation, Genetic↗
PubMed1976

[A RNA extract from oncogenic and non oncogenic strains of Agrobacterium tumefaciens is an indispensable element for the induction of tumors in Datura stramomium].

An RNA bound to the reverse transcriptase of Agrobacterium tumefaciens has been isolated and shown to be oncogenic for stem tissues of Datura stramonium grown under axenic conditions. The tumorous nature of the cellular change induced by the infectious rna was demonstrated by serial grafts of tumors on Datura stems and by cultivation of tumorous tissue in vitro on a medium without supplemental auxins and cytokinins. Active cellular proliferation within tissues of Datura stems was a prerequisite for expression of the oncogenic potential of the RNA. Further, infectious RNA was isolated from avirulent and attenuated strains of Agrobacterium tumefaciens including attenuated derivatives of strain AC58 which have been "heat-cured" of the plasmid associated with virulence. It is proposed that the infectious RNA is an essential but not the sole component of the tumor-inducing mechanism of the crown-gall bacterium.

Cell-Free System↗
PubMed1978

Proteins conferred by the virulence-specifying plasmid of Agrobacterium tumefaciens C-58.

Membrane-associated and periplasmic proteins of Agrobacterium tumefaciens C-58 were compared with those from avirulent (nontumrigenic) derivative strains by slab and two-dimensional gel electrophoresis. Two proteins (Per-I and Per-2), with a molecular weight of 37,500 and 37,300, respectively, were detected in the supernatant fraction of cells of strain C-58 treated with EDTA and lysozyme in which a 117-megadalton plasmid confers virulence on the organism. The same proteins are missing in an avirulent plasmid-free derivative of C-58. When this derivative is mated with C-58, the resulting transconjugants regain the large C-58 plasmid together with the restoration of virulence and the expression of Per-1 and Per-2 proteins. When the transconjugants were cured of their plasmid, they concomitantly lost their virulence and Per-1 and Per-2. The functional roles of these proteins are unknown, but they are associated with the outer membrane and periplasmic fraction of the Agrobacterium cell. If directly involved in tumorigenesis, these proteins are not the sole determinants of tumorigenicity because they are synthesized in an avirulent derivative of C-58 that carries a deletion in the plasmid in the region conferring the tumorigenic phenotype. These results strongly suggest that the Per-1 and Per-2 proteins are plasmid-coded gene products. The possible roles of these proteins in specifying host range and host-cell attachment are also discussed.

Bacterial Proteins↗
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

Agrobactin, a siderophore from Agrobacterium tumefaciens.

A siderophore (microbial iron transport compound) was isolated from low iron cultures of Agrobacterium tumefaciens B6. The substance was characterized as a threonyl peptide of spermidine acylated with 3 residues of 2,3-dihydroxybenzoic acid, the carbonyl group of 1 residue of the latter participating in an oxazoline ring with the beta-hydroxyl of the threonine moiety. The compound, N-[3-(2,3-dihydroxybenzamido)propyl]-N-[4-(2,3-dihydroxybenzamido)butyl]-2-(2,3-dihydroxyphenyl)-trans-5-methyl-oxazoline-4-carboxamide, was given the trivial name agrobactin. Exposure to acid opened the oxazoline ring to afford agrobactin A. Ferric agrobactin A and agrobactin A itself, but not agrobactin or its ferric complex, had some capacity to feed iron to enterobactin-deficient strains of Escherichia coli and Salmonella typhimurium. Agrobactin was produced by A. tumefaciens in response to iron deficiency and was able to reverse the iron starvation in this organism precipitated by the presence of a ferric complexing agent not utilized by the cells.

Biological Transport↗
PubMed1979

R-plasmid-mediated chromosomal gene transfer in Agrobacterium tumefaciens.

Although several techniques are available for transferring the Ti plasmids from one strain of agrobacterium tumefaciens to another, there are no reproducible methods for analysis of chromosomal markers in this phytopathogen. The R plasmid, R68.45, is known to show chromosomal mobilizing ability in several bacterial genera including the closely related Rhizobia. R68.45 was transferred into the prototrophic A. tumefaciens strain 15955. Ten kanamycin-resistant transconjugant clones were tested for chromosomal mobilizing ability by mating with strain SA10, rifampin- and streptomycin-resistant histidine auxotroph of strain 15955. Of the 10 donor clones, 2 showed high chromosomal mobilizing ability. Between 1,000 and 2,000 His+ colony-forming units per ml were obtained, a value 10 to 20 times greater than can be accounted for by spontaneous reversion. Sequential recloning and matings resulted in the isolation of relatively stable donor cultures. Chromosome gene transfer is dependent upon the presence in the donor of R68.45. Donors lacking an R plasmid or harboring the closely related plasmid RP4 failed to yield His+ transconjugants. With strain SA11, a methionine auxotroph of strain SA10, coinheritance of histidine and methionine independence could be demonstrated. Approximately half of the transconjugants also inherited R68.45. These results indicate that A. tumefaciens 15955 is capable of undergoing host chromosomal genetic exchange.

Chromosomes, Bacterial↗
PubMed1979

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.

Cell Wall↗
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↗
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

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↗