Multiple genes coding for octopine-degrading enzymes in Agrobacterium.

Most biotype 2 strains of Agrobacterium tumefaciens and A. radiobacter which utilize nopaline also degrade octopine. In all such strains studied, the ability to degrade octopine did not appear to be transferred to plasmidless recipient cells under conditions of plasmid transfer in which the ability to utilize nopaline was transferred. An octopine-degrading mutant was isolated in a strain cured of its plasmid, suggesting that genes of octopine degradation may have a chromosomal location in some strains. In strains in which octopine utilization is coded by plasmid genes, octopine degradation was always inducible, whereas in strains which degrade both octopine and nopaline, octopine utilization was constitutive although nopaline degradation was inducible. When plasmids coding for octopine-utilizing ability were transformed into a strain containing either a nopaline- or null-type plasmid, transformants able to degrade octopine were either not observed or were unstable upon purification. All of these data suggest that plasmids associated with virulence are incompatible with one another, and therefore imply that the major groups of plasmids associated with virulence have a common origin.     

Expression of an Agrobacterium Ti plasmid gene involved in cytokinin biosynthesis is regulated by virulence loci and induced by plant phenolic compounds.

The nopaline-type Ti plasmid T37 of Agrobacterium tumefaciens carries two distinct genes that encode enzymes involved in cytokinin biosynthesis. In this report, we show that the level of expression of one of these genes was increased dramatically by culture conditions that increased the expression of Ti plasmid virulence genes, including coculture with plant cells or treatment with acetosyringone, a plant phenolic compound. When this nopaline-type Ti plasmid gene was introduced into Agrobacterium strains containing an octopine-type Ti plasmid, similar induction of expression by culture conditions was observed, and analysis of virulence region mutants demonstrated that this induction was under the control of the virA and virG regulatory loci. We further show that induction was strongly pH dependent in octopine strains but, under the conditions examined, pH independent in nopaline strains.     

A diffusible compound can enhance conjugal transfer of the Ti plasmid in Agrobacterium tumefaciens.

Several octopine strains of Agrobacterium tumefaciens were tested for Ti plasmid (pTi) transfer after induction by 400 micrograms of octopine per ml for 24 h. The strains could be divided into two groups, transfer efficient (Trae) and transfer inefficient (Traie); the respective rates of transfer were 0.77 x 10(-2) to 1.14 x 10(-2) and 0.33 x 10(-6) to 9.8 x 10(-6) plasmid transconjugant per donor cell. Transfer efficiencies of Traie strains were greatly increased when the time of induction was 72 h. A diffusible conjugation factor (CF) that can enhance conjugal transfer of pTi in A. tumefaciens was discovered when both Trae and Traie donor strains were induced in the same plate. The evidence indicates that CF is a key factor affecting transfer efficiency of pTi but is not sufficient by itself to induce transfer. Trac mutants can produce CF constitutively, and Trae strains can produce it after induction by low octopine concentrations. The transfer efficiency of Traie strains was greatly increased by adding CF to the induction medium. The thermosensitive strain B6S, which normally cannot conjugate at temperatures above 30 degrees C, could transfer pTi efficiently at 32 and 34 degrees C in the presence of CF. Production of CF is dependent on the presence of pTi but appears to be common for different opine strains; it was first detected in octopine strains, but nopaline strains also produced the same or a similar compound. CF is very biologically active, affecting donor but not recipient bacterial cells, but CF does not promote aggregation. Data suggest that CF might be an activator or derepressor in the conjugation system of A. tumefaciens. CF is a dialyzable small molecule and is resistant to DNase, RNase, protease, and heating to 100 degrees C for 10 min, but autoclaving (121 degrees C for 15 min) and alkaline treatment removed all activity.     

vir genes influence conjugal transfer of the Ti plasmid of Agrobacterium tumefaciens.

Mutation of the genes virA, virB, virC, and virG of the Agrobacterium tumefaciens octopine-type Ti plasmid pTiR10 was found to cause a 100- to 10,000-fold decrease in the frequency of conjugal transfer of this plasmid between Agrobacterium cells. This effect was not absolute, however, in that it occurred only during early times (18 to 24 h) of induction of the conjugal transfer apparatus by octopine. Induction of these mutant Agrobacterium strains by octopine for longer periods (48 to 72 h) resulted in a normal conjugal transfer frequency. The effect of these vir gene mutations upon conjugation could be restored by the introduction of cosmids harboring wild-type copies of the corresponding disrupted vir genes into the mutant Agrobacterium strains. In addition, transfer of the self-mobilizable plasmid pPH1JI was not impaired in any of the mutant Agrobacterium strains tested. The effect of vir gene function on the conjugal transfer of the Ti plasmid suggests that a relationship may exist between the processes that control the transfer of the T-DNA from Agrobacterium to plant cells and the conjugal transfer of the Ti plasmid between bacterial cells.     

Transport of nonmetabolizable opines by Agrobacterium tumefaciens.

We have examined the uptake of [14C]octopine and [14C]nopaline by Agrobacterium tumefaciens strains containing the C58 chromosomal background in medium suitable for the induction of vir genes. All strains tested could transport both of these opines, regardless of the presence or type of Ti plasmid (octopine or nopaline) present in the bacterium. The transport of these opines required active cellular metabolism. Nonradioactive octopine, nopaline, and arginine competed effectively with [14C]octopine and [14C]nopaline for transport into A. tumefaciens A136, suggesting that the transport of these opines occurs via an arginine transport pathway not encoded by the Ti plasmid.     

Utilization of octopine and nopaline by Agrobacterium

Tests for utilization of d-octopine and nopaline in defined media containing a carbon and nitrogen source were made on 60 strains of Agrobacterium representing four species and on a representative of each of five species of Rhizobium. Among 46 virulent strains of Agrobacterium, only two strains were found which utilized neither compound, while three strains were found which could utilize both. Of the remaining virulent strains, 27 utilized octopine and 14 utilized nopaline. Each of six strains of A. rhizogenes tested utilized only octopine but at a slower rate relative to growth than most A. tumefaciens. All eight of the A. radiobacter strains failed to utilize either compound, as did four of six nonvirulent strains of A. tumefaciens. The rhizobia did not utilize octopine or, with the possible exception of R. japonicum, nopaline. Virulence in the genus Agrobacterium is concluded to be highly correlated with the ability to utilize one or both of these compounds.     

Construction of Agrobacterium strains by electroporation of genomic DNA and its utility in analysis of chromosomal virulence mutations.

We have extended the technique of electroporation as a genetic tool for manipulating the Agrobacterium tumefaciens chromosome. We used this technique to introduce chromosomal DNA into recipient A. tumefaciens strains by electroporation and constructed isogenic chvE mutants that share the same chromosomal background but differ in their types of pTi (octopine or nopaline). Both nopaline and octopine pTi-carrying chvE mutants were deficient in vir regulon induction and exhibited similar reductions in host range.     

根癌农杆菌对单子叶植物竹子的细胞转化

本实验证明:具有重要经济价值的禾本科植物——竹子(bamboo)可被根癌农杆菌转化。在经有毒性的根癌农杆菌C_(580、A_(348)感染的竹子材料中,分别测到了专性的胭脂碱和章鱼碱,而对照处理及无毒性的根癌农杆菌VirA~-_(211)mx、NTI感染的竹子材料中,测不到冠瘿碱。     

Structure and characterization of the crown gall opines heliopine, vitopine and ridéopine

The crown gall opines heliopine from tumors induced by octopine type Agrobacterium tumefaciens strains A6, A136(pTiB6-806), E9, A652 and 1590-1 and vitopine from tumor induced by grapevine strains S4 and T2 are identical to synthetic N2-(1'R-carboxyethyl)-L-glutamine. Tumors produced by strains S4 and T2 do not contain octopine or lysopine, but they do contain heliopine and the new opine ridéopine identified as N-(4'-aminobutyl)-D-glutamic acid. Grapevine strains S4 and T2 grow normally on tumor heliopine or synthetic heliopine and on tumor and synthetic ridéopine as well as on ridéopine lactam as sole carbon source. While octopine strains A6 and A136(pTiB6-806) do not grow on heliopine, mutant colonies do appear after a few weeks. Heliopine catabolism by octopine strains is not induced by octopine.     

Comparison of Ti plasmids from three different biotypes of Agrobacterium tumefaciens isolated from grapevines.

Twenty-six plasmids from grapevine isolates of Agrobacterium tumefaciens were analyzed by SmaI fingerprinting and by hybridization of nick-translated DNA to DNA of another plasmid. These experiments established that octopine Ti plasmids are not highly conserved, although octopine Ti plasmids from biotype 1 A. tumefaciens strains appeared to be very similar. Octopine Ti plasmids from biotype 3 strains are more variable in terms of host range and SmaI fingerprints, but share extensive DNA homology. Fingerprints of nopaline Ti plasmids from strains of a given biotype resemble each other but not fingerprints of Ti plasmids from strains of the other two biotypes. The wide host range octopine Ti plasmid from the biotype 3 strain Ag86 shares more DNA homology with narrow host range Ti plasmids, nopaline Ti plasmids, and octopine catabolism plasmids than with the wide host range octopine Ti plasmid from biotype 1 strain 20/1. pTiAg86 does share homology with the portion of pTi20/1 integrated and expressed in plant tumor cells. Since all wide host range Ti plasmids studied contain these sequences, we suggest that natural selection for a wide host range resulted in the presence of the common sequences in distantly related plasmids. The lack of homology between this "common DNA" and limited host range Ti plasmids shows that the DNA sequences per se are not required for tumorigenesis.     

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.     

Agrobacterium tumefaciens can obtain sulphur from an opine that is synthesized by octopine synthase using S-methylmethionine as a substrate

Agrobacterium tumefaciens incites plant tumours that produce nutrients called opines, which are utilized by the bacteria during host colonization. Various opines provide sources of carbon, nitrogen and phosphorous, but virtually nothing was previously known about how A. tumefaciens acquires sulphur during colonization. Some strains encode an operon required for the catabolism of the opine octopine. This operon contains a gene, msh, that is predicted to direct the conversion of S-methylmethionine (SMM) and homocysteine (HCys) to two equivalents of methionine. Purified Msh carried out this reaction, suggesting that SMM could be an intermediate in opine catabolism. Purified octopine synthase (Ocs, normally expressed in plant tumours) utilized SMM and pyruvate to produce a novel opine, designated sulfonopine, whose catabolism by the bacteria would regenerate SMM. Sulfonopine was produced by tobacco and Arabidopsis when colonized by A. tumefaciens and was utilized as sole source of sulphur by A. tumefaciens. Purified Ocs also used 13 other proteogenic and non-proteogenic amino acids as substrates, including three that contain sulphur. Sulfonopine and 11 other opines were tested for induction of octopine catabolic operon and all were able to do so. This is the first study of the acquisition of sulphur, an essential element, by this pathogen.     

Transformation of Soybean Cells Using Mixed Strains of Agrobacterium tumefaciens and Phenolic Compounds

Cotyledon explants from germinated 1-day-old soybean seedling were inoculated with single or mixed strains of Agrobacterium tumefaciens. Mixed-strain infections with the supervirulent L,L-succinamopine type strain A281 (pTiBo542) and strain LBA4404 carrying an octopine type virulence (vir) region and a binary vector (pBin6) with a chimeric gene for kanamycin detoxification gave rise to tumors of which 25% were both kanamycin resistant and capable of hormone-independent growth. Singlestrain inoculations with LBA4404 (pBin6) failed to give rise to kanamycin-resistant callus. Syringaldehyde, a compound which induces vir genes carried on the Ti plasmid, increased the number of galls incited on excised cotyledons by the weakly virulent octopine type strain A348 (pTiA6). Similar results were obtained with whole plants treated with this strain in the presence of the vir-inducing compound acetosyringone. Our results indicate that the recovery of transformed soybean cells can be enabled in some instances by coinfecting with a supervirulent strain or in other instances promoted by adding a phenolic compound to the inoculum.     

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.     

Diversity among B6 strains of Agrobacterium tumefaciens.

A total of 20 laboratory substrains of Agrobacterium tumefaciens strain B6 were compared with respect to six characteristics, including 3-ketolactose production, lysogeny, octopine catabolism, tumorigenic host range, and plasmid content. Within this group of strains diversity was found for all characteristics except 3-ketolactose production. Six substrains were lysogenized with an omega-type phage, whereas one substrain appeared neither sensitive to nor lysogenized with this bacteriophage. All but two substrains catabolized octopine and induced tumors on carrot disks. These 18 substrains harbor deoxyribonucleic acid sequences homologous to pTiB6-806. The two substrains unable to catabolize octopine were nontumorigenic and lacked detectable Ti plasmid sequences. Of the 20 substrains, 13 also contained sequences homologous to the cryptic plasmid pAtB6-806; 2 of the 18 substrains tumorigenic on carrots failed to induce tumors on Kalanchoe leaves. Their inability to induced tumors on this host, could not be correlated with lysogeny, with the presence or absence of pAtB6-806, or with the very large cryptic plasmid recently described. The Ti plasmids from these two strains were indistinguishable from pTiB6-806 by restriction enzyme analysis and could genetically convert a cured A. tumefaciens strain to tumorigenicity on both plant species. The results with these two strains suggest that parameters of tumorigenicity, such as host range, may be controlled by the bacterial chromosome.     

Evidence for diverse types of large plasmids in tumor-inducing strains of Agrobacterium.

Homology between the large plasmids of 15 pathogenic Agrobacterium strains isolated from various parts of the world has been measured and was found to vary over a wide range, from 3 to 100%. Two genetically distinct groups of plasmids can be identified: one closely related to the plasmid of A. tumefaciens A6, an octopine-utilizing strain, and the other closely related to the plasmid of A. tumefaciens C-58, a nopaline-utilizing strain. The plasmids of four Agrobacterium strains do not belong to either group. One of these four strains utilizes octopine, one utilizes nopaline, and two utilize neither. Three strains contained two large plasmids. In one of these strains, the two plasmids were not homologous to one another. Chromosomal homologies for the Agrobacterium strains surveyed also vary over a wide range, but do not correlate with plasmid homologies. Neither do plasmid homologies correlate with any numerical classification scheme. The significance of these plasmid homology studies for crown gall tumorigenesis is considered.     

Two octopine dehydrogenases in crown-gall tumor tissue

Extracts from four crown-gall tumor tissue culture lines, originally induced by two octopine-type strains of Agrobacterium on three plant species, converted l-arginine-[5-³H] to a compound which co-migrated with octopine on electrophoresis. Synthesis showed dependence on added pyruvate and reduced pyridine nucleotide. Both NADH and NADPH were active and mixtures of the two coenzymes, when tested with Vinca strain W1 tumor extracts, were more effective than either coenzyme at comparable concentrations. Addition of an NADH-consuming enzyme system to reaction mixtures containing NADPH had little effect on this activity. Products formed by Vinca rosea strain W1 tumor extracts and Phaseolus vulgaris strain B6 tumor extracts in reaction mixtures containing pyruvate plus NADH or NADPH co-eluted with unlabeled octopine on ion exchange chromatography. The product from the Vinca reaction mixtures co-migrated with an octopine standard in three TLC systems. Permanganate treatment of the enzymatically formed tritiated product and of unlabeled octopine gave compounds with R_f, similar to arginine and γ-guanidinobutyric acid, the products expected from permanganate degradation of octopine. The Vinca W1 extracts catalyzed the oxidative cleavage of octopine, with the formation of arginine, in the presence of NAD or NADP. Two octopine dehydrogenases were concluded to be present in these tissues, one dependent on NAD, the second on NADP.     

Transposition of Tn904 encoding streptomycin resistance into the octopine Ti plasmid of Agrobacterium tumefaciens.

A transfer-deficient derivative of plasmid RP1-pMG1 was isolated after insertion of Mu cts62. The Tra- R plasmid was used to donate Tn904, encoding streptomycin resistance, to Ti plasmid pAL102 harbored by Agrobacterium tumefaciens Ach5. Under conditions promoting high Ti transfer frequencies, 155 strains were isolated in which the streptomycin marker coupled with Ti plasmid in further transfer experiments. These isolates represent stable insertions of Tn904 into the Ti plasmid. In addition, 19 strains were isolated in which the insertion of Tn904 was apparently unstable. The frequency of stable Tn904 transpositions was estimated to be 3 x 10(4-) per transferred Ti plasmid. Evidence was obtained that Tn904 readily may transpose from the Ti plasmid into the bacterial chromosome. The strains carrying Ti plasmids with stable insertions were characterized with respect to virulence, octopine degradation, octopine synthesis in induced tumors, and Ti plasmid transfer. Thirteen of the strains were found to be affected in tumor-inducing ability.