The oriT Region of the Agrobacterium tumefaciens Ti - Journal of ...

Vol. 174, No. 19

JOURNAL OF BACTERIOLOGY, OCt. 1992, p. 6238-6246

0021-9193/92/196238-09$02.00/0 Copyright ©) 1992, American Society for Microbiology

The oriT Region of the Agrobacterium tumefaciens Ti Plasmid pTiC58 Shares DNA Sequence Identity with the Transfer Origins of RSF1010 and RK2/RP4 and with T-Region Borders DAVID M. COOK' AND STEPHEN K. FARRAND 12* Departments of Plant Pathology' and Microbiology,2 University of Illinois, Urbana, Illinois 61801 Received 30 March 1992/Accepted 5 August 1992 Ti plasmids of Agrobacterium tumefaciens are conjugal elements whose transfer is induced by certain opines secreted from crown galls. On transmissible plasmids, DNA transfer initiates within a cis-acting site, the origin of conjugal transfer, or oriT. We have localized an oriT on the A. tumefaciens plasmid pTiC58 to a region containing the conjugal transfer loci traI and traII and acc, which is the locus encoding catabolism of the two conjugal opines, agrocinopines A and B. The smallest functional oriT clone, a 65-bp BamHI-ApaI fragment in the recombinant plasmid pDCBA60-11, mapped within the traII locus. The nucleotide sequence for a 665-bp KpnI-EcoRI fragment with oriT activity was determined. DNA sequence alignments showed identities between the pTiC58 oriT and the transfer origins of RSF1010, pTF1, and RK2/RP4 and with the pTiC58 T-region borders. The RSF1010-like sequence on pTiC58 is located in the smallest active oriT clone of pTiC58, while the sequence showing identities with the oriT regions of RK2/RP4 and with T-region borders maps outside this region. Despite their sequence similarities, pTiC58 oriT clones were not mobilized by RP4; nor could vectors containing the RK2/RP4 oriT region or the oriT-mob region from RSF1010 be mobilized by pTiC58. In contrast, other Ti plasmids and a conjugally activeAgrobacterium opine catabolic plasmid, pAtK84b, efficiently mobilized pTiC58 oriT clones. In addition, the RSF1010 derivative, pDSK519, was mobilized at moderate frequencies by an Agrobacterium strain harboring only the cryptic plasmid pAtC58 and at very low frequencies by an Agrobacterium host that does not contain any detectable plasmids.

plasmid transfer initiates. DNA sequences contained within the oriT are recognized by plasmid-encoded transfer factors, one of which acts as a single-stranded, site-specific endonuclease. This endonuclease cleaves a single strand of DNA at the nic site. Thus, nicking at the oriT determines which strand of the duplex DNA will be transferred to the recipients (reviewed in reference 54). Processing and transfer of the Ti plasmid T-region to the plant cell share structural and functional characteristics with events occurring during plasmid conjugation (45). For example, the highly conserved T-region border sequences are the functional equivalents of the oriT, acting as recognition and cleavage sites for the site-specific nicking complexes (35, 51, 55). Furthermore, both transfer processes involve singlestranded DNA transfer intermediates (1, 12), and both require cell-to-cell contact (28, 39). Results from heterologous transfer experiments strengthen the idea that T-DNA transfer is a modified conjugal process. Derivatives of the mobilizable IncQ plasmid RSF1010 which carry an oriT and cognate mob genes can be mobilized to plant cells by the Ti plasmid-encoded vir system (7). Recent studies suggest that components of the vir system interact with the R plasmid transfer complex (52). We report here the localization of the origin of conjugal transfer for the A. tumefaciens Ti plasmid pTiC58 and present the nucleotide sequence of an approximately 0.7-kb region which contains the oriT. This region includes domains which are identical to sequences of other known plasmid transfer origins and to a conserved segment of the T-region borders of pTiC58. We also demonstrate that the pTiC58 oriT can be mobilized by other Agrobacterium Ti and opine catabolic plasmids but not by the broad-host-range plasmid

The gram-negative phytopathogen Agrobacterium tumefaciens induces crown gall tumors on susceptible plant species by a novel gene-transfer process. Tumorigenicity is dependent upon a large plasmid, called Ti, present in the bacterium. During infection, a portion of the Ti plasmid called the T-region is excised and exported from the agrobacterial cell to a plant cell. Once the T-region is integrated into the plant nuclear genome, expression of transfer DNA (T-DNA)-encoded genes leads to the synthesis of phytohormones and unique nutritional compounds collectively named opines. The overproduction of the plant growth factors causes tumor formation. Opines are secreted from the tumors into the rhizosphere, where they become available as a nutritional source to A. tumefaciens strains carrying an appropriate Ti plasmid encoding functions for opine uptake and catabolism (39). In addition to carrying pathogenicity determinants, Ti plasmids also encode conjugal transfer functions which allow for Ti plasmid transmission to Ti plasmid-less agrobacteria (31). Ti plasmid conjugal transfer is normally repressed but is inducible by an opine subset, the conjugal opines (23, 38). Once a strain of Agrobacterium has acquired a Ti plasmid, it is capable of utilizing opines as a sole nutritional substrate. Thus, the opines synthesized by the biologically engineered plant cells act as signals to stimulate processes that mediate the propagation and the spread of the Ti plasmid (20). In characterized conjugal plasmid systems, a cis-acting region on the plasmid is essential for transfer. This site, the origin of conjugal transfer, or oriT, is the place at which *

Corresponding author. 6238

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pTiC58 ORIGIN OF CONJUGAL TRANSFER

6239

TABLE 1. Bacterial strains and plasmids

DH5ot 1231(pRK2013) S17-1 A. tumefaciens C58ClRS UIA143 LBA4011(pTiC58Trac) UIA143(pTiC58Trac) T37

NT1(pTil5955Trac) NT1(pAtK84b) C58ClFC UIA5 Plasmids pCP13 pTHB58 pTHB55 pVK101 pVKE34 pSal52 pSa4AH pTHH204 pRK415 pDSK519 pRK415K pDCH4 pDCP4.4 pDCE20 pDCE20ASal pDCKP2.8 pDCKPl.5 pDCKE.7 pDCPE.8

pDCKE.7AB pDCKBam pDCBA60-11 pUC18 pUC19

Source or reference

Relavent genotype, phenotype, or characteristicsa

Strain or plasmid

Strains E. coli DH1

supE44 hsdRl 7 recAl gyrA96 thi-2 relAl supE44 AlacUl69(48olacZAM15) hsdRl7 recAl end4l gyrA96 thi-1 relAl Tra' Kmr Tpr Smr Mob'

41 41

Rif' Strr (pAtC58) Ery' recA143 (pAtC58) Rif AgrS' Trac Noc+ (pTiC58Trac) Eryr Agr"s Trac Noc+ recA143 (pTiC58Trac, pAtC58) AgrS Trac Noc+ (pTiT37) Trac Occc (pTil5955Trac, pAtC58) Noc+ Tra+ (pAtK84b, pAtC58) 5pFUr Cmr Rif' Strr; GM19017 derivative, pTi-, pAt-

49 21 11 This study Our collection Our collection Our collection This study 40; also our collection

IncPl cosmid vector, Tcr Kmr pCP13:-:BamHI-partial(pTiC58) pCP13:.BamHI-partial(pTiC58) IncPl cloning vector, Tcr Kmr pVK101::EcoRI-34(pTiC58) IncW cloning vector, Cmr Kmr Spr HindIII deletion derivative of pSal52, Cmr Kmr pSa4AH::HindIII-3(pTiC58), Cmr Kmr IncPl broad-host-range cloning vector, Tcr IncQ RSF1010-derived cloning vector, Kmr Kanamycin-resistant derivative of pRK415, Tcr Kmr pRK415K::HindIII-4(pTiC58), Tcr Kmr pRK415K::PstI-4.4-kb clone(pTiC58), Tcr Kmr pRK415K::EcoRI-20(pTiC58), Tcr Kmr SalI-digested deletion derivative of pDCE20 pRK415K:KpnI-PstI-2.8-kb clone(pTiC58), Tcr Kmr pRK415K::KpnI-PstI-1.5-kb clone(pTiC58), Tcr Kmr pRK415K::ApnI-EcoRI-665-bp clone(pTiC58), Tcr Kmr pRK415K:PstI-EcoRI-0.8-kb clone(pTiC58), Tcr Kmr BamHI deletion derivative of pDCKE.7, Tcr Kmr pRK415K:KpnI-BamHI-394-bp clone(pTiC58), Tcr Kmr pRK415K:BamHI-ApaI-65-bp clone(pTiC58), Tcr, Kmr

13 25 25 32 This study 47 S.-B. Hong Our collection 30 30 This study This study This study This study This study This study This study This study This study This study This study This study Our collection Our collection

Ampr Ampr

a Abbreviations: AgrS, agrocin 84 sensitivity; Agrl, agrocin 84 supersensitivity;

Ampr, ampicillin

resistance;

Cmr,

Our collection 44

chloramphenicol resistance;

5-FUr,

5-fluorouracil resistance; Eryr, erythromycin resistance; Kmn, kanamycin resistance; Mob', mobilizing; Noc+, nopaline catabolism; OceC, constitutive for octopine catabolism; pAt-, no pAtC58; pTi-, no Ti plasmid; Rif, rifampin resistance; sp', spectinomycin resistance; StrT, streptomycin resistance; Tpr, trimethoprim resistance; Tra+, self-conjugal; Trac, transfer constitutive.

RP4. Furthermore, an RSF1010 derivative plasmid is not mobilized by the Ti plasmid, but the R plasmid can be transferred from Agrobactenium strains harboring only the cryptic plasmid pAtC58 and, to a lesser extent, even from a plasmidless agrobacterial host.

MATERIALS AND METHODS Bacterial strains and plasmids. Strains of Agrobacterium and Escherichia coli, as well as the plasmids used in this study, are described in Table 1. E. coli DH5a was used to maintain recombinant plasmids. Strain UIA143, a recA mutant of A. tumefaciens NT1 (21), was used to construct most of the merodiploid donor strains. A. tumefaciens C58C1RS (49) and C58C1FC served as the recipients.

All pTiC58 oriT region subclones except pVKE34 and pTBH204 were constructed in pRK415K. To construct pRK415K, the kanamycin resistance cassette from the cosmid vector pCP13 (13) was excised as a BamHI fragment, the restriction sites were filled in with Klenow polymerase, and the DNA fragment was blunt-end ligated into the unique XmnI site of the broad-host-range vector pRK415 (30). Ti plasmid restriction fragment designations are those of Depicker et al. (14). Media and growth conditions. The rich media for the growth of E. coli and Agrobacterium strains included LB (GIBCO Laboratories) and nutrient agar (Difco Laboratories). The defined media used for culturing Agrobacterium strains included AT minimal medium (48), AB minimal medium (9), and Stonier's minimal medium (46). AT minimal

6240

COOK AND FARRAND

medium was supplemented with 0.15% (NH4)2SO4 (ATN). Glucose (0.2%), mannitol (0.2%), or 1 mM nopaline-9 mM arginine was added as the sole carbon source. Media were solidified by the addition of agar (Difco) to 1.5%. Selective media for E. coli contained the following antibiotics at the following concentrations: tetracycline, 10 ,ug/ml; ampicillin, 100 ,ug/ml; and kanamycin, 30 jig/ml. Selective media for Agrobacterium strains contained the following antibiotics at the following concentrations: tetracycline, 2 ,±g/ml; kanamycin, 50 or 100 ,ug/ml; erythromycin, 100 ,ug/ml; rifampin, 50 ,ug/ml; streptomycin, 200 j,g/ml; 5-fluorouracil, 50 ,ug/ml; and chloramphenicol, 50 ,ug/ml. Agrobacterium cultures were grown at 28°C; E. coli cells were cultured at 37°C. Plasmid isolation and characterization. Plasmid DNA was isolated by the alkaline lysis method (41) or by the technique of Hayman and Farrand (26). Restriction digests were performed by using reaction conditions recommended by the manufacturers. Restriction fragments were separated by electrophoresis in 0.7 or 1.0% agarose gels in Tris-borateEDTA buffer. Electrophoresis in polyacrylamide gels was used to resolve fragments of fewer than 250 bp, as outlined previously (41). Bacterial strain constructions. E. coli and Agrobacterium cells were transformed according to previously published procedures (27, 41). Cosmid clones maintained in E. coli were mobilized to agrobacteria by the pRK2013-based triparental mating system (17) or the S17-1-based biparental mating technique (44). Merodiploid strains of Agrobacterium were constructed as follows. pTiC58Trac was isolated from strain LBA4011 (11) and transformed into strain UIA143. The constitutive transfer ability of pTiC58Trac in UIA143 was confirmed by plate matings with C58C1RS as the recipient, as described previously by Beck von Bodman et al. (4). Cosmid clones of pTiC58 were mobilized to UIA143(pTiC58Trac), while oriT subclones in pRK415K, pSa152, or pSa4AH were introduced by transformation (27). Control strains with oriT clones but no Ti plasmid and derivatives of strains T37, NT1(pTil5955Trac), and NT1 (pAtK84b) with pTiC58 oniT clones were constructed by employing the same procedures. oriT mobilization assay. Mobilization of various recombinant clones by pTiC58Trac, pTiT37, or pTil5955Trac was determined as follows. Cultures of donors and recipients were grown overnight in 2 ml of ATN-mannitol with the appropriate antibiotics. Each culture was diluted 1:10 into 2 ml of the same medium. These subcultures were incubated for 7 to 8 h at 28°C and centrifuged at 6,000 rpm (Sorvall GLC-2B centrifuge, HL-4 rotor) for 10 min, and the cells pellets were washed three times with 1 x AT salts. Cell titers were determined by plating appropriately diluted samples onto nutrient agar. For all matings, 200-,ul volumes each of donor and recipient cultures were mixed in a 1.5-ml microcentrifuge tube and vacuum filtered onto a nitrocellulose membrane (Millipore; 25-mm thick, 0.45-,um pore size). The filters were placed bacteria side up on the surface of an ATN-mannitol agar plate and incubated for 2 h at 28°C. We determined previously that mobilization of oriT clones from a donor is maximal between 1 and 2 h after initiation of mating on minimal agar medium (12a). This allowed for the quantification of mobilization frequencies on the basis of initial transfer events. After the mating period, cells were removed from the filters by vortexing in a 1-ml volume of 1 x AT. Replicate 10-,ul samples of serially diluted mating suspensions were plated as discrete spots onto selective medium. Selection plates were incubated at 28°C for 2 to 3 days, at which time transconjugant colonies were counted.

J. BACTERIOL.

Random transconjugants from mobilization experiments were picked to selective medium and tested for coinheritance of the Ti or the opine catabolite plasmid. Transconjugants were also analyzed for resistance to erythromycin when UIA143 was used as the donor. Plasmid profiles of randomly selected transconjugants were analyzed to detect recombination events. Agrocinopine-induced mobilization by pAtK84b. Donor cells carrying pAtK84b and pTiC58 oriT clones and recipient cells were cultured overnight, collected, and washed as described above. For opine induction and matings, agar towers (7-mm diameter; separated from the remaining agar by a 3-mm-wide moat) were formed in ATN minimal plates. Five microliters of a solution of agrocinopines A and B (2.0 p.g/.ld) (a generous gift from Maarten Ryder) was spotted onto the surface of the tower and allowed to soak into the agar; likewise, a mannitol solution (0.4 ,ug/ml; 5 ,ul) was spotted to a second set of agar towers. Suspensions of donor strains and the recipient strain (10 ,ul) were spotted separately onto mannitol towers; 10-pl volumes of donor suspensions also were placed onto the agrocinopine-impregnated towers, and the cultures were incubated for 24 h at 280C. After being incubated, the towers were transferred to tubes containing 1 ml of AT minimal salts, and the cells were resuspended by vigorous vortexing. Twenty-microliter volumes of donor and recipient suspensions were mixed and spotted onto identical towers impregnated with agrocinopines or mannitol. Mating plates were incubated for 24 h at 28°C, the cells were again resuspended in 1 x AT and diluted appropriately, and samples were plated onto medium to select for transconjugants as described above. DNA sequencing and analysis. The dideoxy chain termination method (42) was used to sequence both strands of a 665-bp KpnI-EcoRI onT-active restriction fragment cloned into pUC18 and pUC19. Sequencing reactions were carried out with the Sequenase version 2.0 kit according to the manufacturer's instructions (US Biochemical). In some reaction mixtures, azobases supplied by US Biochemical were used to resolve areas in which band compression obscured the sequence. Sequencing reaction products were separated by electrophoresis in 0.2-mm-thick polyacrylamide gels as described by Ansorge and Barker (2). Autoradiographs were prepared, and the nucleotide sequences were analyzed with the DNASTAR software package (DNASTAR, Inc., Madison, Wis.). Nucleotide sequence accession number. The GenBank nucleotide sequence accession number for the 665-bp pTiC58 oriT region is M95646. RESULTS Identification of an oniT region on pTiC58. An oriT assay dependent upon pTiC58Trac-mediated trans mobilization was developed to screen a series of overlapping cosmid clones of pTiC58 for transfer origin activity. The recA strain, UIA143 (21), was used to construct donors to prevent homologous recombination between the Ti plasmid and the recombinant clones. One cosmid clone, pTHB58 (Fig. 1), was mobilized by pTiC58Trac at an average frequency of 2.7 x 10-2 per input donor. Under the same conditions, pTiC58Trac transfers at a frequency of 102 per input donor. All other cosmid clones tested showed no detectable mobilization (data not shown). This list includes cosmid clone pTHB55, whose insert DNA overlaps with that of pTHB58 in the region where oniT activity was localized (Fig. 1). pTHB55 encodes a wild-type repressor function, AccR, that

pTiC58 ORIGIN OF CONJUGAL TRANSFER

VOL. 174, 1992

6241

pTHB58(C58) pTHBS5(C58)

pTHH204(C 1

tidll

kb

8I Tra !

Tra 11

1| 261

21l

;12

21b |

Ilkb

I'

HindlIl, ,' ' E

E

)

PE

tl31l ,,

3

4

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(...)

E

"

p

1 )

pTIC58

Hildlll pDCH4

I

I I

~~~~~pDCP4.4

.... -I

l

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l

I-I

pDCE2O pVKE34 pDCKP2.8 pDCKPI.5 pDCPE.8 pDCKE.7

I (') I (+)

H

pDCKBon pDCE.7,&B

pDCBA6O.1l

FIG. 1. Physical map of the traII-traI-acc region of the A. tumefaciens plasmid pTiC58. The positions of two of the cosmid clones tested for oniT activity are shown above the HindIII restriction map. Although pTHB55 includes HindIII fragment 4, it was not mobilized, since this clone also encodes a wild-type copy of a repressor (R) which when placed in trans to pTiC58Trac prevents conjugal transfer in the absence of agrocinopines A and B (3). Subclones of HindIII fragment 4 are shown below its restriction map. All clones were derived from wild-type pTiC58 (C58). + + + +, + + +, + +, and +, relative mobilization frequencies, in decreasing order; -, no detectable mobilization (detection limit, 10-'); E, EcoRI; P, PstI; K, KpnI.

eliminates the constitutive transfer ability of pTiC58Trac (3). The cosmid vector pCP13 was not mobilized by pTiC58Trac. Likewise, donor strains containing the cosmid clones, but not pTiC58Trac, showed no measurable mobilization activity. Random transconjugants from all mobilization assays were screened for the presence of the mobilized plasmid and also for concomitant transfer of pTiC58Trac. Except when spontaneous Tetr mutants of C58C1RS arose, the plasmid DNA isolated from transconjugants gave restriction profiles corresponding to those of the donor oriT-containing clones. Between 6 and 56% of the Tcr or Kmr transconjugants also grew on medium containing nopaline as a sole carbon source, indicating that they had coinherited the mobilizing Ti plasmid. Subcloning the oriT region of pTiC58. Two subclones, one containing HindIII fragment 4 in pRK415K (pDCH4) and the second containing HindIII fragment 3 in pSa4AH (pTHH204), were isolated and screened for mobilization by TiC58 (Fig. 1). Plasmid pDCH4 was mobilized at an average frequency of 2.5 x 10-3 per input donor, while the adjacent HindITI fragment 3 showed no activity (Table 2). The vector pRK415K was not mobilized by pTiC58Trac, and neither was pDCH4 in the absence of pTiC58Trac. To exclude a vector-specific influence on the mobilization of oniT clones, HindIII fragment 4 also was cloned into the alternate vector pSal52 (IncW) (47). This construct was mobilized by pTiC58Trac at an average frequency of 4.7 x 10-2. The oriT-active region was progressively subcloned by using EcoRI, PstI, and KpnI sites (Fig. 1). A 4.4-kb PstI fragment, subcloned as pDCP4.4, was mobilized at an average fre-

quency of 9.4 x 10-2 (Table 2). Two smaller subclones, pDCE20 and pDCKP1.5, were mobilized at average frequencies of 1.1 x 10-2 and 3.5 x 10-3, respectively (Fig. 1; Table 2). These two plasmids contain overlapping DNA, and this segment was subcloned as a 665-bp KpnI-EcoRI fragment in TABLE 2. Mobilization frequencies for subclones of pTHB55, pTHB58, and pDCH4 Clone

pRK415K

pTHH204 pSal52 pDCH4 pDCP4.4 pDCE20 pVKE34 pDCKP1,5 pDCKP2.8 pDCKE.7 pDCPE.8 pDCKE.7AB pDCKBam pDCBA60-11

Mobilization frequency' With Without pTiC58Tra' pTiC58Tra'

NT NT NT