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

PLASMID

8,

175-186 (1982)

Genetic Isolation and Physical Characterization of pAgK84, the Plasmid Responsible for Agrocin 84 Production JOHNE.SLOTAANDSTEPHENK.FARRAND Department of Microbiology, Stritch School of Medicine, Loyola University of Chicago, 2160 South First Avenue, Maywood, Illinois 60153 Received May 24, 1982 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, A 136. The mobilizing element, pAt84a, was then cured from such a transconjugant by cultivation at 37°C. Derivatives of strain Al36 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 Xbal 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.

Agrobacterium tumefaciensis the etiologic agent of crown gall, a neoplastic disease affecting many speciesof dicotyledenous plants. The diseaseis of considerable economic importance. Agrobacterium tumefaciens has been described as one of the top three agricultural pathogens in California (Schroth et al., 1971) and a major stone fruit pathogen in the mid-Atlantic states (Alconero, 1980). A recent analysis over the years 1975-1977 ranks A. tumefaciensas the third most significant bacterial plant pathogen in the United States(Kennedy and Alcorn, 1980). The disease as an economic entity is not confined to the United States. Crown gall is of major concern to grape growers in Continental Europe (Lehoczky, 1978) and to olive growers in Greece (Kerr and Panagopoulos, 1977). The organism also contributes to substantial economic losses in Australia and New Zealand. The pathogenic potential ofA. tumefaciens has led to a search for systems to control crown gall in the field. The most promising of these involves biological control of the 175

etiologic agent mediated by a specific bacteriocin. The bacteriocin is produced by a nontumorigenic A. radiobacter strain, K84 (Htay and Kerr, 1974; Dhanvantari, 1976; Kerr and Panagopoulos, 1977). This agency of biological control is considered a model system for antagonistic agriculture pest management (Schroth and Hancock, 1981). The active bacteriocin, called agrocin 84, has been identified as a disubstituted fraudulent analog of adenine (Roberts et al., 1977; Tate et al., 1979). Initial work indicated that agrocin 84 affected both protein synthesis and DNA replication in sensitive strains (McCardell and Pootjes, 1976). More recent studies suggest that the bacteriocin, which lacks a free 3’ - OH, acts as a terminator of DNA chain growth (P. J. Murphy, M. E. Tate, and A. Kerr, personal communication). All current evidence indicates that the genetic determinants for agrocin 84 production reside on a 30-Mdal plasmid present in strain K84. Thus when Cooksey and Moore (198 1) cured strain K84 of this element, the ability 0147-619X/82/050175-12%02.00/0 Copyright d 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.

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to produce agrocin 84 was lost. Further, Ellis et al. (1979), have shown that the 30-Mdal plasmid can be transferred by mobilization from one strain ofAgrobacterium to another. Transconjugants inheriting this element now produce agrocin 84. A particularly striking feature of the agrotin control system is its specificity. Sensitive A. tumefuciensstrains all harbor Ti plasmids of the nopaline type (Engler et al., 1975; Watson et al., 1975; Kerr and Roberts, 1976). The selectivity seemscorrelated with uptake, a trait apparently encoded by the nopalinetype Ti plasmids (Holsters et al., 1980). The locus controlling agrocin sensitivity, presumably by coding for uptake functions, has been mapped on the C58 Ti plasmid and is located well away from regions involved in tumorigenesis (Holsters et al., 1980). Recent evidence now suggeststhat agrocin 84 sensitivity may be related to the ability of sensitive strains to catabolize a newly discovered class of opines called agrocinopines (Ellis and Murphy, 1981). Unfortunately, such selectivity implies limited effectiveness and this indeed appears to be the case. In fields contaminated with innately resistant strains, crown gall is not controlled by agrocin 84 (Sule and Kado, 1980; Panagopoulos et al., 1978). The system is further compromised by the fact that sensitive strains can undergo modifications resulting in resistance to the agrocin without affecting tumorigenicity. This can occur by mutations in the Ti plasmid affecting agrocin sensitivity functions (Engler et al., 1975; Holsters et al., 1980) or by acquisition of the agrocin 84 plasmid and its immunity functions (Ellis et al., 1979). The development of an agrocin 84 based control system less prone to failure requires a better understanding of the genetic basis for agrocin 84 synthesis. With this as our goal we have begun a study of pAgK84, the 30Mdal plasmid specifying agrocin 84 production. We report here the genetic isolation and restriction enzyme mapping of this plasmid and also studies concerned with its relationship to other Agrobacterium plasmids.

MATERIALS

AND METHODS

Bacterial strains. Agrobacterium radiobatter strain K84 was obtained from Dr. James Lippincott, Northwestern University. Agrobacterium tumefaciens strains A 136 (rifR, nalR) and C58 (Watson et al., 1975) have been maintained in our laboratory since their receipt in 1975 from Dr. Eugene Nester, University of Washington. Agrobacterium tumefaciens strain 542TC2 (Guyon et al., 1980) was obtained from Dr. Mary-Dell Chilton, Washington University. Medium and bu&s. L broth and AB minimal medium have been previously described (Fan-and et al., 1981). Agrocin 84 assayswere performed on the mannitol-glutamate (MG)’ medium of Kerr and Htay (1974). SSPE buffer, used in the Southern hybridizations, is 0.18 M NaCl, 0.0 1 M NaP04, pH 7.0, 1 mM EDTA. Bacterial matings. Conjugal transfer of pAt84a, the nopaline-specifying plasmid of strain K84, was performed essentially as described by Ellis et al. (1979). The donor was pregrown in medium containing 1 pg of nopaline (Calbiochem Inc.)/ml and 500 pg of octopine (Sigma Chemical Inc.)/ml. Strain Al36 transconjugants were selected on the medium of Kerr et al. (1977) containing nopaline and octopine as sole sources of carbon and nitrogen and rifampin (15 pg/ml) to counterselect the donors. Agrocin 84 assays.Strains to be tested for agrocin 84 production were spotted on to the surface of solid MG medium. As many as eight strains could be tested on a single lOOmm-diameter plate. Strain K84 was always included in the center of each plate as a positive control. The cultures were allowed to grow at 28°C for 2 to 4 days. They were then exposed to chloroform vapors for 10 min, aired for an additional 10 min, and overlayed with 3.0 ml molten MG medium containing approximately 10’ colony-forming units of midexponential phase strain C58 as the in1 Abbreviations used: MC medium, mannitol-ghtamate; SSPE buffer, 0.18 M NaCl, 0.0 1 M NaPO,, pH 7.0, 1 mM EDTA; SDS, sodium dodecyl sulfate.

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OF THE AGROCIN

dicator. The cultures were incubated an additional 18-24 h at 28°C before being examined for agrocin production. Isolation of plasmid DNA. Plasmid DNA was isolated by the procedure of Casseet al. (1979) and purified, when necessary by centrifugation to equilibrium in CsCl gradients containing 300 pg ethidium bromide (EtBr)/ml. Plasmid analysis. Intact plasmid DNA molecules, partially purified from strains to be assayed,were analyzed by agarosegel electrophoresis in the horizontal mode as previously described (Farrand et al., 1981). Samples in the gels were subjected to electrophoresis at 50-60 mA constant current. Restriction endonuclease analysis. Plasmid DNA, purified by two cycles of centrifugation in CsCl-EtBr gradients was digested with restriction endonucleases under conditions described by the manufacturers (New England Biolabs and Bethesda Research Laboratories). Partial digestions were performed by incubating the DNA samples with limiting concentrations of enzymes for the standard length of time (1 h). When double digests were required, the DNA samples were treated first with the enzyme requiring the lowest salt concentration. Following the first digestion, the buffer solution was adjusted to higher salt concentrations and treated with the second enzyme. Fragments were separated by electrophoresis in 0.7 or 1.2% agarose gels run in the horizontal mode. Gels were stained, the DNA fragments visualized, and photographs prepared as previously described (Fart-and et al., 1981). Fragments produced by Hind111 cleavage of XDNA were used as the size standards. Restriction enzyme mapping. The majority of the restriction enzyme mapping was performed by redigestion of isolated restriction fragments in a manner similar to that described by Herrmann et al. (1980). Low melting temperature agarose(LGT, SeaKern) was used to separatefragments resulting from digestion with the first enzyme. Nick translations. Plasmid DNA was labeled to high specific activity with [a-32P]TTP

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(New England Nuclear) by nick translation essentially as described by Rigby et al. (1977). Southern transfersand hybridizations. Restriction enzyme-digested DNA samples, separated in 0.7% agarose gels, were denatured and transferred to nitrocellulose filter sheets by the technique of Southern (1975). Hybridizations were carried out using probe DNA in 15-20 ml of hybridization buffer (5X SSPE, 0.3% SDS, 100 rg sonicated calf thymus DNA/ml). The filters were incubated at 65°C for 12 h and washed four times at 45°C with 2X SSPE containing 0.2% SDS. Autoradiograms of the washed filters were prepared using Kodak XAR-5 X-ray film and intensifying screens. RESULTS

GeneticIsolation of pAgK84 In order to perform detailed molecular analyses on pAgK84, it was first necessaryto isolate it genetically from other A. radiobatter or A. tumefaciensTi-type plasmids. As a first step, pAgK84 was transferred by nopaline plasmid mobilization (Ellis et al., 1979) from A. radiobacter strain K84 to the Ti plasmid-free A. tumefaciensstrain, Al 36. Nopaline-catabolizing transconjugants were recovered at a frequency of approximately 1O5per input donor. Of these, approximately 10% were observed to produce agrocin 84. All tested transconjugants were resistant to rifampin and nalidixic acid and produced 3-ketoglycosides from lactose, traits identifying them as strain A 136 derivatives (Watson et al., 1975). Plasmid DNA was partially purified from randomly chosen transconjugants and analyzed by horizontal agarosegel electrophoresis. The agrocin-producing, nopaline-utilizing transconjugants (Fig. 1, lanes C and D) had inherited plasmids of two size classes.One, with a mass estimated at 125 Mdal, corresponds to pAt84a the nopalinespecifying plasmid in strain K84 (lane B). The other, approximately 30 Mdal in size, corresponds to the agrocin plasmid (Merlo and Nester, 1977) present in strain K84.

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A

B

C

D

E

F

G

H

FIG. 1. Agarose gel electrophoresis of partially purified plasmid DNA. Plasmid DNA was partially purified from A. tumefaciens strains as described by Casse ef al., (1979). The DNA was analyzed by horizontal agarose gel electrophoresis (Farrand et a/., 1981). Samples represent DNA isolated from (A) strain A 136; (B) strain K84, (C and D) two nopaline-catabolizing (nop’) agrocin-producing (Ag+) A 136 transconjugants; (E and F) two nop’ agrocin-nonproducing (Ag-) A 136 transconjugants. Lane G represents DNA isolated from a 37”Ggrown nopaline-noncatabolizing (nap-), Ag+ derivative unable to grow on AB minimal medium. Lane H shows the plasmid profile of an ABf revertant of the strain shown in lane G.

Lanes E and F show DNA isolated from two nopaline-catabolizing transconjugants which failed to produce detectable agrocin. These strains have all inherited only a single, 12% Mdal plasmid identical in size to pAt84a. It is known that certain noplaine-catabolic Ti plasmids are temperature sensitive for maintenance and can therefore be eliminated from their bacterial hosts by repeated culturing at 37°C (Watson et al., 1975). Although we had no successin even growing strain K84 at this temperature, we reasoned that it might be possible to cure the agrocinproducing A 136 transconjugants of pAt84a by this method. We would thereby expect to isolate derivatives harboring only the smaller plasmid. In such an experiment, after three passagesat 37°C on nutrient agar 8 out of

50 tested clones were found to be unable to catabolize nopaline. Curiously, unlike strain A 136 or their immediate transconjugant parent, none of these 50 clones were able to grow on AB minimal medium. However, when approximately 10’ cells from several 37°C grown isolates were spread on AB medium, all gave rise to AB-positive subclones at frequencies of about 1O-‘. All of these AB-positive subclones, including those still unable to catabolize nopaline, continued to produce agrocin 84. Plasmid profiles show that a nopaline noncatabolizing AB-negative isolate (Fig. 1, lane G) and its AB-positive derivative (lane H) no longer harbor the 125-Mdal plasmid present in strain K84 and its A 136 transconjugants. Both do, however, continue to maintain the 30-Mdal element.

CHARACTERIZATION

OF THE AGROCIN 84 PLASMID

Restriction EndonucleaseAnalysis

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isolated from an AB-negative strain (lane E) yields a digestion pattern indistinguishable We used restriction enzyme analysis to from that isolated from an AB-positive deconfirm the genetic isolation of pAgK84 in rivative (lane F). This would suggestthat the strain A136. Digestion with Hind111 gener- inability to grow on AB medium following ates 12 identifiable fragments including two culture at 37°C and subsequent reversion of doublets from the 30-Mdal plasmid in strain this trait cannot be attributed to any major Al36 (Fig. 2, lanes E and F). Plasmid DNA structural alteration of the 30-Mdal species.

FIG. 2. Endonuclease Hind111and EcoRI digests of plasmid DNA samples. Plasmid DNA was isolated and purified from A. tumefuciens strains as described under Materials and Methods. The DNA samples were digested with Hind111 (A-F) or EcoRI (G-K) and the fragments separated by electrophoresis in a 0.7% agarosegel (Farrand et al., 1981). The lanes contain DNA from (A) lambda phage; (B and G) strain K84; (C and H) strain A136(pAt84a, pAgK84); (D and I) strain A136(pAt84a); (E and J) strain A136(pAgK84) unable to grow on AB minimal medium; (F and K) the AB+ derivative of the isolate shown in lanes E and J.

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FIG. 3. Digestion of pAgK84 with various restriction endonucleases. Plasmid pAgK84 was isolated from an AB+ revertant of A 136(pAgK84). The DNA was digested with (A) EarnHI; (B) PSI: (C) &/II; (D) HaeIII; (E) Him-II; (F) SacI; (G) X6aI; (H) HpaI. Lane I contains lambda DNA digested with HindIII. The digestion fragments were separated by electrophoresis in a 0.7% agarose gel.

It is also clear from comparing lanes B, C, and D with E or F that all Hind111 fragments in the digest of total plasmid DNA from strain K84 (lane B) can be accounted for in digestsof plasmid DNA from A 136 (pAT84a) (lane D) and Al36 (pAgK84) (lanes E and F). Thus, strain K84 contains only one plasmid species of 30-Mdal size. These digests also indicate that pAgK84 in strain K84 is indistinguishable from that in the Al 36 transconjugants and also in their nopaline

plasmid-cured derivatives. The sum of the molecular weights of the Hind111 fragments produced from pAgK84 totals 47.6 kbp, equivalent to a size of 29.9 Mdal. This value is in good agreement with electron microscopic determinations reported in the literature (Merlo and Nester, 1977). Similar results were obtained from EcoRI digests (Fig. 2, lanes G-K). Thus no detectable alterations have been induced in the smaller element by any of the genetic or culture manipulations.

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OF THE AGROCIN 84 PLASMID TABLE 1

DIGESTIONPRODUCTSOFpAgK84 Fragment size (kbp) Fragment A B, B2

C DI D2

E F, F2

G H1 H2 I, I2

J K Total

EcoRI

Hind111

KpnI

PstI

sac1

BamHI

17.0 4.2 3.6 3.0 3.0 2.9 2.6 2.5 2.4 1.8 1.8 1.5 1.3

13.4 7.7 7.3 6.6 2.4 1.8 1.8 1.5 1.4 1.4 1.2 1.1

29.5 7.6 4.9 2.2 1.8 0.7 0.6 0.4

16.7 13.3 8.2 3.6 2.8 2.3 0.7

16.2 11.0 8.8 4.9 3.6 2.0 1.1

47.6

47.6

47.4

47.6

47.6

These results, taken as a whole, indicate that presence of a nopaline specifying plasmid is not required for agrocin 84 production and further establish the 30-Mdal plasmid as being responsible for this trait.

HpaI

XbuI

15.0 11.2 10.0 6.7 2.9 1.6

32.1 15.6

47.6

47.4

47.7

47.6

spect to BarnHI, SacI, PstI, and KpnI (Fig. 4). Each permutation of enzyme pairs was performed. For example we first determined the order of the Sac1 fragments with respect to the PstI fragments. Next we determined

Physical Map of pAgK84 To select enzymes with which to map pAgK84, we digested purified plasmid DNA with a number of different restriction endonucleases. Most yielded between 2 and 12 identifiable fragments (Fig. 3) while some, such as HaeIII (lane D) generated a large number of low-molecular-weight pieces. However, endonuclease%a1 (lane G) yielded only one fragment indicating that the plasmid has a single recognition sequencefor this enzyme. The sizesof the fragments produced by eight of these enzymes are presented in Table 1. Using the technique of Herrmann et al. (1980) whereby the plasmid was digested with one enzyme and then each fragment was isolated and recut with a second endonuclease, we developed a restriction map with re-

FIG. 4. Restriction endonuclease map of pAgK84. The map was constructed as described under Materials and Methods and Results. The single XbaI site has been arbitrarily designated the O/47.6-kbp position at 12 o’clock on the map.

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Sac1to BamHI. This was finally followed by determining the order of PstI fragments with respect to BarnHI. In all casesthe map was entirely consistent. The single XbaI site and the two HpaI sites were then mapped using standard double digest techniques. The order and placement of PstI fragments D, E, and G was confirmed by partial PstI digestions of the entire plasmid. The map is presented with the single XbaI site at the 0 kbp position.

Relationship to Other Agrobucterium Plusmids Inspection of the data of Guyon et al. (1980, see Fig. 4) showed an apparent similarity between SmuI digestion patterns of pAgK84 and pAtBo542a, a 25-Mdal cryptic plasmid from A. tumefaciensstrain Bo542. We then learned that strain Bo542 produces an inhibitory factor similar in its specificity to agrocin 84 (M.-D. Chilton, personal communication). Furthermore, a transconjugant strain, harboring both pTiBo542 and pAtBo542a (Guyon et al., 1980) gives a positive reaction in our agrocin assay (data not shown). Plasmid DNA isolated from this transconjugant strain, 542TC2, was treated with several restriction endonucleases and the cleavage patterns compared to those of pAgK84 (Fig. 5A). It can be seen that the intensly fluorescing bands, corresponding to fragments of cleaved pAtBo542a, comigrate for the most part with the digestion products of pAgK84. However, it appearsthat pAgK84 yields fragments which are not present in digests of pAtBo542a. For example, BumHI digestion of pAgK84 yields six fragments including the B doublet. Digestion of pAtBo542a yields a pattern differently only in that BumHI fragment A has been replaced by a new 7-kbp digestion product. This presumption of relatedness was confirmed by Southern transfer hybridizations. Figure 5B shows that radiolabeled pAgK84 specifically and strongly hybridizes to each of the restriction fragments of pAtBo542a generated by KpnI, PstI, and SucI. To assessits relationship to other Agro-

bacterium plasmids, radiolabeled pAgK84 was hybridized against Southern transfers of several different types of Ti plasmids digested with SmuI or KpnI. As can be seen in Fig. 6, strong hybridization bands were present in samples containing pAgK84 alone (lanes A and J) or mixtures of pAgK84 and pAt84a plasmid DNAs (lanes D and G). However, the probe showed no detectable homology when hybridized against digested octopinetype (lanes B and I), or nopaline-type (lanes C and H) Ti plasmids. Nor was any homology detected between the agrocin plasmid and pAt84a, the nopaline-specifying plasmid coresident in strain K84 (lanes E and F). DISCUSSION

Previous work in other laboratories implicated a 30-Mdal plasmid as the genetic agent of agrocin 84 biosynthesis (Ellis et al., 1979, Cooksey and Moore, 1980). We confirm this by showing that transconjugants harboring only the 30-Mdal plasmid synthesize the antibiotic (Fig. 1). We propose that this plasmid be called pAgK84 to identify its role in agrocin 84 production. Agrocin 84 biosynthesis appears to be determined by pAgK84. It is clear from the genetic isolation from strain K84 that its biosynthesis does not require the presenceof any second plasmid. The point at which the agrotin 84 biosynthetic pathway branches from chromosomaily coded nucleoside pathways is presently unknown. As pAgK84 shows no detectable homology with pAt84a, the nopaline-specifying plasmid coresident in strain K84 (Fig. 6), it would appear that no sequences involved in agrocin biosynthesis are shared by both plasmids. Merlo and Nester (1977) noted that the entire plasmid complement of strain K84 showed 38% sequence homology to pTiC58. Our results show no homology between pAgK84 and pTiC58. This would suggest that the 38% homology observed by these workers exists between pTiC58 and the large nopaline-specifying plasmid of strain K84. We present evidence showing strong re-

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OF THE AGROCIN 84 PLASMID

1 2 3 4 5 6 7 8 9101112131415

123

FIG. 5. Restriction endonuclease digestion and blot-hybridization analysis of pAgBo542. (A) Plasmids pAgK84 and pAgBo542 (also containing pTiBo542) were isolated, purified, and cleaved with various restriction endonucleasesas described under Materials and Methods. Fragments were separated by electrophoresis on a 0.7% agarose gel. Lane 1 contains lambda DNA digested with HindIII. The remaining 14 lanes are arranged in pairs and contain pAgK84 (first lane) and pAgBo542 (second lane) cleaved with 2-3, XbaI; 4-5, HpuI; 6-7, PstI; 8-9, SacI; 10-l 1, BumHI; 12-13, KpnI; and 14-15, SmaI. (B) The mixture of pAgBo542 and pTiBo542 was cleaved with KpnI (lane I), PstI (lane 2), or SmaI (lane 3) and the fragments separated by electrophoresis in a 0.7% agarose gel. The DNA was denatured transferred to a nitrocellulose sheet and hybridized against [‘*P]pAgK84 as described under Materials and Methods.

latedness between pAgK84 and pAtBo542a (Fig. 5). Consistent with this strain 542TC2, the C58-Cl transconjugant harboring pAtBo542a, produces an antibiotic-like substance showing a specificity similar, if not identical, to that of agrocin 84 (M.-D. Chilton, personal communication and our data not shown). For these reasons we, along with Dr. Nester and Dr. Chilton (personal communication), propose that the Bo542 plasmid be renamed pAgBo542 to reflect its

agrocinogenic nature. Assuming that pAgBo542 and pAgK84 share the same physical organization, analysis of restriction profiles (Fig. 5A) indicates that pAgBo542 lacks the entire region corresponding to coordinants 4.5 kbp through 13 kbp on the pAgK84 map. Whether this represents a deletion in a pAgK8Clike plasmid to form one like pAgBo542 or an insertion into a pAgBo542like plasmid to form one like pAgK84 cannot be determined. This relatedness is remark-

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ABCDEFGHIJ

FIG. 6. Relatedness of pAgK84 and opine catabolic Agrobacterium plasmids. Plasmid DNAs, isolated as described under Materials and Methods were digested with SmaI (lanes A-E) or KpnI (lanes F-J). Fragments were separated by electrophoresis in a 0.7% agarosegel. The DNA was denatured, transferred to a sheet of nitrocellulose paper, and hybridized against [32P]pAgK84 as described under Materials and Methods. Lanes contain pAgK84 (A and J); pTi15955 (B and I); pTiC58 (C and H); pAt84a+pAgK84 (D and G); pAt84a (E and F).

able in that the two original producer strains differ considerably in their characteristics and origins. Strain K84 is an avirulent biotype 2 strain isolated in, and apparently indigenous to Australia (Kerr and Htay, 1974). As noted above, it is a nopaline catabolizer and its large plasmid most likely shows some relatedness to pTiC58. Strain Bo542, on the

other hand, is a tumorigenic biotype 1 strain of European origin (Sciaky et al., 1978). Furthermore, its Ti plasmid codes for neither octopine nor nopaline catabolism. It does, however, code for agropine utilization (Guyon et al., 1980) and is closely related by sequencehomology to octopine-type Ti plasmids (Currier and Nester 1976b).

CHARACTERIZATION

OF THE AGROCIN 84 PLASMID

Plasmid pAgK84 does not contain sequences detectably homologous to pTi 15955, an octopine-specifying plasmid or to pTiC58 or pAt84a, two nopaline-specifying plasmids (Fig. 6). This would indicate that the agrocinogenic element has evolved independently from the Ti-type plasmids. On the other hand, its presence in two natural Agrobacterium isolates of rather different lineage and geographical location would suggestthat pAgK84 has been associated with the agrobacteria through their dissemination to various parts of the world. The apparent high degree of specificity shown by agrocin 84 toward the agrobacteria is consistant with this hypothesis. Furthermore, the ability of pAgK84 to be mobilized amongst the agrobacteria by various Ti plasmids (Ellis et al., 1979) furnishes a mechanism to account for its appearance in at least two rather dissimilar natural isolates. There remains the interesting observation that when strain A 136 derivatives harboring both pAt84a and pAgK84 are grown at 37°C they quickly and reversably lose their ability to grow on AB minimal medium. We have no explanation for this reproducible phenomenon. It does appear to be plasmid associated since strain Al 36, following cultivation at 37”C, remains capable of growth on the minimal medium. However, it is not related to any major alterations in pAgK84 or pAt84a. The plasmids in such AB-negative strains are indistinguishable by restriction endonuclease analysis from those in strain K84 and the AB-positive “revertants” (Fig. 2). The phenomenon may be a function solely of the nopaline plasmid, pAt84b. Strain A 136 transconjugants harboring only this large plasmid also exhibit the rapid loss of ability to grow on AB minimal medium following growth at 37°C. We are currently investigating this phenomenon more closely. The genetic isolation of pAgK84 and the development of a restriction endonuclease map constitute our first steps toward a detailed study of this plasmid. These results will provide the basis for the characterization and mapping of mutations affecting key plasmid

185

functions such as agrocin production and immunity. This in turn may allow us to construct better agrocin 84-mediated field control agents. ACKNOWLEDGMENT This work was supported by Grant CA 19402 to S. K. Farrand from the National Cancer Institute.

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COOKSEY,D. A., AND MOORE,L. W. (198 I ). An agrocin mutant of Agrobacferium rudiobacfer K84 and biological control of crown gall. Phytopathoiogy 71, 104. CURRIER, T. C., AND NESTER,E. W. (I 976a). Isolation of covalently closed circular DNA of high molecular weight from bacteria. Anal. Biochem. 66, 43 I-441. CURRIER, T. C., AND NESTER, E. W. (1976b). Evidence for diverse types of large plasmids in tumor inducing strains of Agrobacterium. J. Bacterial 126, I57- 165. DHANVANTARI, B. N. (1976). Biological control of crown gall of peach in Southwestern Ontario. Phznt Dis. Rep. 60, 549-551. ELLIS, J. G., AND MURPHY, P. J. (1981). Four new opines from crown gall tumors, their detection and properties. Mol. Gen. Genet. 181, 36-43. ELLIS, J. G., KERR, A., VAN MONTAGLJ, M., AND SCHELL,J. (1979). Agrobacferium: Genetic studies on agrocin 84 production and the biological control of crown gall. Physiol. Plant Putho/. 15, 311-319. ENGLER,G., HOLSTERS,M., VAN MONTAGU,M..SCHELL, J., HERNALSTEENS, J. P., AND SCHILPEROORT, R. (1975). Agrocin 84 sensitivity: A plasmid determined property in Agrobacterium tumefaciens. Mol. Gen. Genet. 138, 345-349.

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GUYON, P., CHILTON, M.-D., PETIT, A., AND TEMPT, J. ( 1980). Agropine in “null-type” crown gall tumors: Evidence for generality of the opine concept. Proc. Nat. Acad. Sci. USA 7, 2693-2697.

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C., DEBLOCK, M., DHAESE,P., DEPICKER,A., INZE, D., ENGLER,G., VILLARROEL,R., VAN MONTAGU, M., AND SCHELL,J. (1980). The functional organization of the nopaline A. tumefuciensplasmid pTiC58. Plasmid 3, 2 12-230.

HTAY, K., AND KERR, A. (1974). Biological control of crown gall: Seed and root inoculation. J. Apl. Bacterial. 31, 525-530.

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