Isolation and characterization of diverse nopaline type Ti plasmids of Agrobacterium tumefaciens from Japan

Ninety Agrobacterium strains were isolated from naturally appearing crown galls in Japan. They were classified into several groups based on opine type, biovars, tumorigenicity, and indigeneous plasmid profiles. Twenty-nine strains utilized nopaline, but none utilized octopine. Eighteen isolates were tumorigenic, nopaline type strains and thus classified as Agrobacterium tumefaciens. Some strains possessed anomalous traits such as lysine utilization, resistance to agrocin 84, and a lack of motility. Pathogenic strains contained Ti plasmids of either 200 kb or 260 kb, as identified by hybridization to T-DNA of the known Ti plasmid. However, the restriction enzyme cleavage patterns, arising from hybridization to the probe, were different from each other and indicated that nopaline type Ti plasmids possess more diverse T-DNA structures than previously reported. Five of 6 representative strains induced tumors on 6 plant species (tomato, petunia, poplar, kalanköe, apple, and grape). Among these, apple was notable, since only a few strains have been reported to be pathogenic to this plant. On petunia, 4 strains developed large tumors while 2 produced only small tumors. Teratomas were formed on poplar in a strain-dependent manner, but not on tomato. These results suggest that our isolates are wide host range strains, and that host-specificity of these strains is related to diverse T-DNA structures.     

Succinamopine: a new crown gall opine

Agrobacterium tumefaciens strains can incite plant tumors consisting of transformed cells that synthesize novel metabolites called opines. The pattern of opine synthesis is dictated by plasmid-borne genes in the pathogen; additional plasmid genes confer on the pathogen the ability to catabolize the same pattern of opines synthesized. One group of A. tumefaciens strains, AT181, EU6, and T10/73, contains closely related tumor-inducing (Ti) plasmids that encode the ability to degrade the opine nopaline; but tumors incited by these strains do not synthesize nopaline. We demonstrated by Southern blot hybridization that AT181(pTi) has no DNA homologous to the nopaline synthase gene of pTi T37, a nopaline Ti plasmid that appears to be most closely related to this group based on fingerprint analysis. Tumors incited by these seemingly anomalous strains contain a new opine that we designate succinamopine. Its structure is analogous to that of nopaline, with asparagine replacing arginine. Evidence for the structure of succinamopine, as well as those of two related metabolites, succinamopine lactam and succinopine lactam, will be published elsewhere. Ability to catabolize succinamopine, succinamopine lactam, and succinopine lactam is encoded by pTi AT181, pTi EU6, and pTi T10/73, but not by any of 15 other Ti and root-inducing plasmids tested. Three avirulent strains tested did not catabolize succinamopine, succinamopine lactam, or succinopine lactam. We propose that pTi AT181, pTi EU6, and pTi T10/73 be designated the succinamopine Ti plasmids.     

Broad-Host-Range Agrocin of Agrobacterium tumefaciens

Eighteen strains of Agrobacterium tumefaciens isolated from crown galls were tested for agrocin production. Of six agrocin-producing strains, one (D286) produced a broad-host-range agrocin active against strains carrying nopaline, octopine, and agropine type Ti plasmids. Sensitivity to agrocin D286 was found to map in the 11- to 18-megadalton region of the nopaline Ti plasmid pTiC58. The agrocin was partially purified, and its physical characteristics were consistent with its being a nucleotide, as is agrocin 84. Agrocin D286 was shown to inhibit DNA, RNA, and protein syntheses. Strain D286 spontaneously lost its pathogenicity, and its potential for use in the biological control of crown gall is discussed.     

Genetic analysis of an Agrobacterium tumefaciens strain producing an agrocin active against biotype 3 pathogens

Summary The successful biocontrol agent for crown gall disease, Agrobacterium radiobacter strain K84, is unable to protect grapevines from infection. We have identified a strain of Agrobacterium tumefaciens, J73, which produces an agrocin active both in vitro and in vivo against grapevine pathogens (Webster et al. 1986). We now report on the curing of this strain of its nopaline-type Ti plasmid and the location, by transposon mutagenesis, of the genes involved in the production of the agrocin. The Ti plasmid was cured by the introduction of selectable plasmids carrying the origins of replication of either the nopaline Ti plasmid, pTiC58, or the octopine Ti plasmid, pTi15955. Tn5 mutagenesis indicated that the genes responsible for agrocin production and/or export are located both on the chromosome and on a plasmid, pAgJ73, which co-migrated in agarose gels with pTiJ73. As the two plasmids were separable after transposon mutagenesis, we postulate that during or after mutagenesis of the agrocin plasmid, DNA rearrangements occurred between it and pTiJ73, resulting in an increase in size of pAgJ73. We provide evidence that the rearrangements involved the duplication of nopaline catabolism genes from pTiJ73 and their insertion into pAgJ73, which facilitated the resolution of the two plasmids. As expected pTiJ73 has homology with the nopaline Ti plasmid, pTiC58.     

Absolute stereochemistry of leucinopine,a crown gall opine

Crown gall tumors incited by Agrobacterium tumefaciens strain Bo542 have been reported to synthesize a tumor-specific substance identified as N-(1,3-dicarboxypropyl)-leucine (leucinopine), a compound with two centers of asymmetry. We report here evidence that leucinopine is indeed a crown gall opine, in that it is specifically catabolized by A. tumefaciens strains carrying the tumor-inducing plasmid pTi Bo542, as well as strains carrying closely related plasmids pTi AT1 and pTi AT4. We further report catabolism of leucinopine by the succinamopine-type strains A518, A519 and A532, carrying pTi EU6, pTi AT181 and pTi T10/73, respectively. Strains lacking any virulence plasmid, as well as those carrying octopine or nopaline type Ti plasmids or mannopine type Ri plasmids, did not catabolize leucinopine. On the basis of specificity of catabolism by bacteria carrying pTi Bo542, we conclude that the stereochemistry of natural leucinopine is l-threo, i.e. l^glu,l^leu. Such stereochemistry is novel in the opines known thus far: octopine, nopaline and succinamopine have d,l-stereochemistry: d^ala,l^arg (octopine), d^glu,l^arg (nopaline) and d^glu,l^asn (succinamopine).     

Characterization of an Unusual New Agrobacterium tumefaciens Strain from Chrysanthemum morifolium Ram

We characterized five isolates of Agrobacterium tumefaciens from naturally occurring galls on Chrysanthemum morifolium. The isolates are similar, possibly identical, members of a single strain of A. tumefaciens that we designate Chry5. The strain is a biotype I, as indicated by its response to both newly described and traditional biotype tests. Chry5 produces tumors on at least 10 plant species. It is unusual in its ability to form efficiently large tumors on soybean (Glycine max), a species normally refractory to transformation. Chry5 is unable to utilize octopine or mannopine as a carbon source. Although Chry5 can catabolize a single isomer each of nopaline and succinamopine, it differs from other known nopaline and succinamopine strains in its insensitivity to agrocin 84. This pattern of opine catabolism is unique among Agrobacterium strains examined to date. All five isolates of Chry5 contain at least two plasmids, one of which shares homology with pTiB6.     

Bacteriocin production inAgrobacterium tumefaciens

Agrobacterium tumefaciens C58 forms “plaques” during layer cultivation. The “plaques” were shown not to be caused by the presence of a temperate bacteriophage or by random contamination. The “plaques” and their central microcolonies were used to repeatedly isolate cultures producing an antibiotic substance against the original strainA. tumefaciens C58, other nopaline strains, some octopine strains ofA. tumefaciens and some strains of the related genusRhizobium. The substance is thus a bacteriocin; in analogy to agrocins 84 and D286 it was named agrocin C58. The agrocin is not inactivated by trypsin. Its production by strain C58 was found only on cultivation on solid but not liquid media. The producing isolate ofA. tumefaciens C58 (strain C58i2) contains neither plasmid pTiC58 nor the plasmid analogous to pAgK84 which controls the production of agrocin 84 inA. radiobacter K84.     

Genetic isolation and physical characterization of pAgK84, the plasmid responsible for agrocin 84 production

The plasmid responsible for agrocin 84 biosynthesis by Agrobacterium radiobacter strain K84 has been genetically isolated free from any opine-catabolic plasmids. This was accomplished by mobilizing the agrocin plasmid, pAgK84, into a Ti plasmid-free A. tumefaciens strain, A136. The mobilizing element, pAt84a, was then cured from such a transconjugant by cultivation at 37 °C. Derivatives of strain A136 harboring both plasmids or pAgK84 only produce agrocin 84. The agrocin plasmid isolated from these strains is indistinguishable by restriction endonuclease analysis from that in strain K84. A physical map of pAgK84 has been constructed with respect to six restriction endonucleases. The plasmid is cut only once by XbaI and twice by HpaI. Hybridization analysis shows that pAgK84 is closely related to pAtBo542a, a 25-Mdal plasmid from a virulent, agrocinogenic A. tumefaciens strain of European origin. Similar analyses indicate, however, that pAgK84 shows no detectable homology to octopine or nopaline-type Agrobacterium plasmids.     

L,L-Succinamopine: an epimeric crown gall opine

Alfalfa tumour incited by Agrobacterium tumefaciens strain A281, carrying the tumour inducing plasmid pTi Bo542, synthesizes agropine and related mannityl opines. In addition it contains a small amount of leucinopine and large quantities of a new opine here identified as N-[(1S)-1-carboxy 2-carbamoylethyl]-(S)-glutamic acid. This new opine, L,L-succinamopine, is the L^glu epimer of the succinamopine previously isolated from tumours incited by pTi AT181 and related strains. The latter opine should now be designated D,L-succinamopine. This is the first example of the natural occurrence of epimeric opine structures.     

Deletion derivatives of pAgK84 and their use in the analysis of Agrobacterium plasmid functions

The 47.7-kb plasmid pAgK84, present in Agrobacterium radiobacter strain K84, confers production of a novel, highly specific, antiagrobacterial antibiotic called agrocin 84. Strain K84 is used commercially to biocontrol crown gall caused by agrocin 84-susceptible strains of Agrobacterium tumefaciens. Efficient biocontrol is dependent upon production of agrocin 84 by strain K84. Starting with a derivative of pAgK84 containing a Tn5 insertion, a series of deletion derivatives of the plasmid were isolated. The smallest of these, pJS500, contains about 8 kb of the original agrocin plasmid and localized the replication functions to between 4 and 6 o'clock on the physical map. A smaller derivative, produced by clonal rescue of a Tn5 insertion in the 4 o'clock region, further localized the minimal replication functions to a 1.5-kb region mapping between coordinates 18.1 and 19.6. Analysis of plasmid stability indicated that functions required for maintenance of the plasmid under nonselective conditions are tightly linked to the minimal replication region. This region also encodes incompatibility functions; the deletion derivatives were all incompatible with the wild-type pAgK84. The stability/replication locus of pAgK84 maps just anticlockwise from the Tra region. This region is retained fully in pAgK1026, the directed Tra⁻ derivative of pAgK84 which is now in use as the primary crown gall biocontrol agent in Australia. One of the deletion derivatives, the 15-kb pJS400, was used as a vector to clone the KpnI fragments of an octopine-type Ti plasmid. Traits known to be encoded on these fragments were expressed and properly regulated in Agrobacterium hosts. One clone, encoding the Ti plasmid replication/incompatibility region, was used to cure IncRh1 Ti plasmids from their hosts. This clone also was found to be incompatible with pAtK84b, a large plasmid encoding opine catabolism present in A. radiobacter strain K84. This indicates that the opine catabolic plasmid is closely related to the IncRh1 Ti plasmids.     

Isolation of Different Agrobacterium Biovars from a Natural Oak Savanna and Tallgrass Prairie

Populations of agrobacteria in excess of 10⁵ CFU/g were recovered from 12 soil and root samples obtained from the Allison Savanna, Minn., a natural oak savanna and tallgrass prairie which has never been disturbed agriculturally. Of 126 strains picked randomly from selective media, 54 were identified as Agrobacterium spp. Biovar 2 strains predominated (35 of 54), but these strains were distributed into three phenotypically distinct subgroups. Of the remaining Agrobacterium strains, four were biovar 1-2, one was biovar 1, and none were biovar 3. The last 14 Agrobacterium strains formed a homogeneous group which differed biochemically from the hitherto reported biovars. Opine utilization (coded for by genes on the tumor-inducing plasmid in pathogenic Agrobacterium spp.) by these agrobacteria was limited to two biovar 2 strains. In contrast, 10 nonfluorescent gram-negative strains utilized either nopaline or octopine as the sole carbon and nitrogen source. There may be a need to reexamine the source and role of opines in the terrestrial environment because (i) all of these opine utilizers were isolated from an environment free of crown gall, the only known terrestrial source of opines, and (ii) 83% of the opine utilizers were not Agrobacterium spp.     

Aeromonas, a possible etiological agent in the formation of secondary tumors of crown gall

Abstract From a secondary tumor in a bean stem we have isolated a Gram-negative bacteria, named by us T.2. These bean stems had crown gall tumors induced by the ATV strain of Agrobacterium tumefaciens . This bacterium was classified as belonging to the genus Aeromonas and possesses the capacity of inducing overgrowths in plants, synthesizing indole acetic acid (IAA). The codified phenotypic characteristics of bacterium T.2. via the Ti-plasmid of A. tumefaciens , such as opine utilization and sensitivity to agrocin 84, have been studied. Neither octopine nor nopaline is utilized by T.2. and it is resistant to agrocin 84, whereas the strain ATV of A. tumefaciens utilizes nopaline, and is sensitive to agrocin 84.     

Biological Control of Agrobacterium tumefaciens, Colonization, and pAgK84 Transfer with Agrobacterium radiobacter K84 and the Tra- Mutant Strain K1026

The efficacies of Agrobacterium radiobacter K84 and K1026 in root colonization, crown gall control, and plasmid transfer were compared. Levels of root colonization by K84 and K1026 of Montclar and Nemaguard peach seedlings were similar during the 21 days of the experiment. Four strains of A. tumefaciens bv. 1 were used for soil inoculations in biological control experiments on GF677 and Adafuel peach × almond rootstocks; two were sensitive and two were resistant to agrocin 84. Both strains K84 and K1026 were very efficient in controlling the sensitive strains, but some tumors appeared with both treatments. In the biocontrol of resistant strains, no galls were observed in K1026-treated plants, but some K84-treated plants had galls. Recovery of agrobacteria from galls in experiments with sensitive and resistant strains showed that all of the isolates from the controls or K1026-treated plants and most of the isolates from K84-treated plants had the same characteristics as the inoculated strains. Nine isolates from the K84-treated plants growing in soil inoculated with one resistant strain were virulent and produced agrocin 84. These isolates had a plasmid that hybridized with a probe prepared with the BamHI C fragment from pAgK84. These results show the efficiency of K1026 in biocontrol of agrocin 84-sensitive and -resistant strains of A. tumefaciens and suggest the use of K1026 as a safer organism than K84 for biological control of crown gall.     

Tn5 insertions in the agrocin 84 plasmid: the conjugal nature of pAgK84 and the locations of determinants for transfer and agrocin 84 production

The kanamycin-resistance transposon Tn5 was randomly introduced into pAgK84, a 47.7-kb plasmid coding for agrocin 84 production in Agrobacterium. Using such marked plasmids, pAgK84 was found to be conjugal. It could be transferred to several Agrobacterium strains including those harboring octopine- or nopaline-type Ti plasmids. Its presence has no effect on Ti plasmid functions such as opine utilization and tumorigenicity, but it does confer agrocin 84 immunity upon previously sensitive strains. The plasmid could also be conjugally transferred to a Nod+ Fix+ strain of Rhizobium meliloti. The production of agrocin 84 is expressed in all Agrobacterium and Rhizobium transconjugants tested. The agrocin plasmid could not be introduced into restrictionless Escherichia coli or Pseudomonas aeruginosa recipients by conjugation or transformation. The sites of 92 independent Tn5 insertions were mapped on pAgK84. These insertions are dispersed over the entire length of the plasmid. Analysis of the sites and effects of the Tn5 insertions has allowed us to construct a functional map of pAgK84. Forty-three of these insertions, spanning a 20-kb segment of the plasmid, abolished or greatly reduced the production of agrocin 84. The presence of two insertions within this segment having an effect on agrocin production suggests that at least three regions of the plasmid are involved in agrocin 84 biosynthesis. Fourteen of the Tn5 insertion derivatives are no longer conjugally transferable. These insertions all map to a single region of the plasmid and define about 3.5-kb as being associated with transfer functions.     

New class of limited-host-range Agrobacterium mega-tumor-inducing plasmids lacking homology to the transferred DNA of a wide-host-range, tumor-inducing plasmid

Biotype 1 and 2 strains of Agrobacterium tumefaciens were isolated from crown gall tumors of Lippia canescens plants growing as ground cover in Arizona. The isolates were agrocin 84 sensitive, did not catabolize octopine, nopaline, agropine, or mannopine, and were limited in their tumorigenic host range. One biotype 2 strain, AB2/73, showed the most limited host range; it incited tumors only on Lippia strains, the cucurbit family of plants, and Nicotiana glauca. Megaplasmids were detected in the isolates by vertical agarose gel electrophoresis. The unusual host range, as well as sensitivity to agrocin 84, were plasmid specified since they were conjugally cotransferred with plasmids from donor strain AB2/73. Correlation of deletions with concomitant loss of virulence and agrocin 84 sensitivity identified the megaplasmid pAtAB2/73d as the virulence element in strain AB2/73. The estimated size of this tumor-inducing plasmid was 500 kilobases. Axenic growth of tumor tissue incited by strains carrying pAtAB2/73d was phytohormone independent. Although the limited-host-range megaplasmid pAtAB2/73d lacked any detectable homology to the phytohormone-biosynthetic genes in wide-host-range transferred DNA (tms-1, tms-2, tmr), it showed homology to the wide-host-range virB, virC, virD, and virG loci. Therefore, pAtAB2/73d represents a new class of tumor-inducing plasmids distinguished by its large size, the absence of determinants for the catabolism of several known opines, the presence of agrocin 84 sensitivity, and its lack of homology to wide-host-range transferred DNA contrasted with its conservation of sequences from the wise-host-range vir region.     

Occurrence of free and conjugated 12,13-epoxytrichothecenes and zearalenone in banana fruits infected with Fusarium moniliforme.

Agrobacterium tumefaciens J73, a biotype 2 strain harboring a nopaline Ti plasmid, was found to produce an agrocin active against a broad range of A. tumefaciens strains, including grapevine isolates. Sensitivity to J73 is not encoded by a Ti plasmid. Optimal conditions for the production of the agrocin were determined.     

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.     

Deletion derivatives of pAgK84 and their use in the analysis of Agrobacterium plasmid functions.

The 47.7-kb plasmid pAgK84, present in Agrobacterium radiobacter strain K84, confers production of a novel, highly specific, antiagrobacterial antibiotic called agrocin 84. Strain K84 is used commercially to biocontrol crown gall caused by agrocin 84-susceptible strains of Agrobacterium tumefaciens. Efficient biocontrol is dependent upon production of agrocin 84 by strain K84. Starting with a derivative of pAgK84 containing a Tn5 insertion, a series of deletion derivatives of the plasmid were isolated. The smallest of these, pJS500, contains about 8 kb of the original agrocin plasmid and localized the replication functions to between 4 and 6 o'clock on the physical map. A smaller derivative, produced by clonal rescue of a Tn5 insertion in the 4 o'clock region, further localized the minimal replication functions to a 1.5-kb region mapping between coordinates 18.1 and 19.6. Analysis of plasmid stability indicated that functions required for maintenance of the plasmid under nonselective conditions are tightly linked to the minimal replication region. This region also encodes incompatibility functions; the deletion derivatives were all incompatible with the wild-type pAgK84. The stability/replication locus of pAgK84 maps just anticlockwise from the Tra region. This region is retained fully in pAgK1026, the directed Tra- derivative of pAgK84 which is now in use as the primary crown gall biocontrol agent in Australia. One of the deletion derivatives, the 15-kb pJS400, was used as a vector to clone the KpnI fragments of an octopine-type Ti plasmid. Traits known to be encoded on these fragments were expressed and properly regulated in Agrobacterium hosts. One clone, encoding the Ti plasmid replication/incompatibility region, was used to cure IncRh1 Ti plasmids from their hosts. This clone also was found to be incompatible with pAtK84b, a large plasmid encoding opine catabolism present in A. radiobacter strain K84. This indicates that the opine catabolic plasmid is closely related to the IncRh1 Ti plasmids.