JOURNAL OF BACTERIOLOGY, July 1979, p. 280-286 0021-9193/79/07-0280/07$02.00/0
Vol. 139, No. 1
R-Plasmid-Mediated Chromosomal Gene Transfer in Agrobacterium tumefaciens SUZANNE E. HAMADA, JOAN P. LUCKEY, AND STEPHEN K. FARRAND* Department of Microbiology, Stritch School ofMedicine, Loyola University of Chicago, Maywood, Illinois 60153
Received for publication 13 February 1979
Although several techniques are available for transferring the Ti plasmids from strain of Agrobacterium tumefaciens to another, there are no reproducible methods for analysis of chromosomal markers in this phytopathogen. The R plasmid, R68.45, is known to show chromosomal mobilizing ability in several bacterial genera including the closely related Rhizobia. R68.45 was transferred into the prototrophic A. tumefaciens strain 15955. Ten kanamycin-resistant transconjugant clones were tested for chromosomal mobilizing ability by mating with strain SA10, a rifampin- and streptomycin-resistant histidine auxotroph of strain 15955. Of the 10 donor clones, 2 showed high chromosomal mobilizing ability. Between 1,000 and 2,000 His' colony-forming units per ml were obtained, a value 10 to 20 times greater than can be accounted for by spontaneous reversion. Sequential recloning and matings resulted in the isolation of relatively stable donor cultures. Chromosome gene transfer is dependent upon the presence in the donor of R68.45. Donors lacking an R plasmid or harboring the closely related plasmid RP4 failed to yield His' transconjugants. With strain SA11, a methionine auxotroph of strain SA10, coinheritance of histidine and methionine independence could be demonstrated. Approximately half of the transconjugants also inherited R68 45. These results indicate that A. tumefaciens 15955 is capable of undergoing host chromosomal genetic exchange. one
Since 1907 (21) Agrobacterium tumefaciens has been recognized as the etiological agent of crown gall, a neoplastic disease of dicotyledonous plants. The mechanism whereby this bacterium induces this proliferative disease has yet to be elucidated. However, it has been found that all tumorigenic strains of A. tumefaciens harbor one or more large plasmids (23, 24). One of these elements, the Ti plasmid, appears to be essential for tumorigenicity. Recent results from hybridization experiments indicate that DNA from crown gall tumor cells, but not normal plant cells, contains sequences homologous to a specific portion of the Ti plasmid (4). However, there is also some evidence suggesting involvement of bacterial chromosomal genes in tumor induction. For example, strain 5GlyFe, a derivative of strain A6, is avirulent but contains a large Ti-type plasmid. When this plasmid is transferred into strain NT1, a plasmidless derivative of strain C58 (23), resulting transconjugants are tumorigenic (19). This would suggest that strain 5GlyFe lacks some chromosomal determinant necessary for tumorigenicity. The Ti plasmids can be transferred from one strain of A. tumefaciens to another by R plasmid-mediated mobilization (5), direct matings
(8, 13), and DNA-mediated transformation (7). However, at the present time there are no methods available for chromosomal genetic analysis in this phytopathogen. Although there are several reports in the literature describing DNAmediated transformation of chromosomal markers (12, 14) and DNA-mediated transfection (18) of A. tumefaciens, none of these methodologies has been confirmed. A method for the analysis and mapping of chromosomal genes in A. tumefaciens may be of value in determining the role of such bacterial genes in the induction of crown gall tumors. The P-1 incompatibility (IncP1) group R plasmids are unique in showing a very broad conjugal host range among gram-negative bacteria including A. tumefaciens (15). Furthermore, these R plasmids, most notably RP1, RP4, and R68, have been shown to mobilize the transfer of host chromosomal genes in a number of bacterial species including Pseudomonas aeruginosa (10), Pseudomonas putida (16), Acinetobacter calcoaceticus (22), Rhodopseudomonas sphaeroides (20) and Rhizobium leguminosarum (2). This last organism has been shown by nucleic acid homology studies to be related to A. tumefaciens (9). We therefore undertook a
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CHROMOSOME TRANSFER IN AGROBACTERIUM
study to determine whether R68 45, a derivative of R68 (10), is capable of mobilizing the transfer of chromosomal genes in this organism. (Portions of this work were presented at the 78th Annual Meeting of the American Society for Microbiology [S. E. Hamada, J. P. Luckey, and S. K. Farrand, Abstr. Annu. Meet. Am. Soc. Microbiol., 1978, H86, p. 118]). MATERIALS AND METHODS Bacteria. Strains and their relevant characteristics listed in Table 1. Strains harboring R plasmids are designated by the R plasmid number in parentheses. Strains SA10 and SAl were derived in two steps from strains A3 and A25, respectively. Rifampin-resistant strains were isolated as spontaneous mutants arising on nutrient agar containing rifampin at 15 yg/ml. After purification and analysis of the auxotrophic markers, streptomycin-resistant derivatives were isolated in a similar fashion on nutrient agar supplemented with streptomycin (500 ,ig/ml). Media. Nutrient agar (Difco) was prepared by the manufacturer's directions. L broth (15) contained, in grams per liter: tryptone (Difco), 10.0; NaCl, 5.0; and yeast extract (Difco), 5.0. Before autoclaving the pH was adjusted to 7.0 to 7.2. Solid and liquid AB minimal media containing 0.5% glucose were prepared as previously described (3). AB buffer was AB minimal medium lacking glucose. Matings and selections. Matings on solid media were performed essentially as described by Sistrom (20). Equal volumes of mid-exponential-phase donor and recipient cultures (approximately 109 colony-forming units [CFU] per ml each) were mixed, and 10 0.02ml portions were immediately spotted on the surface of nutrient agar plates. After incubation at 290C for 12 to 48 h, growth was resuspended in 4 ml of AB buffer. Donor and recipient titers after resuspension ranged are
from 4 x 10i to 1.2 x 1010 CFU/ml. Transconjugants selected on AB agar supplemented with rifampin (15 ,g/ml), streptomycin (500,ug/ml), and appropriate amino acids (25,ug/ml) as required. Strains were tested for resistance to kanamycin (Km) by streaking on nutrient agar containing this aminoglycoside at 20 Ag/
Plasmid isolation. Partially purified plasmid DNA prepared by a modification of the Currier and Nester technique (6). After phenol extraction, the aqueous phase was extracted once with chloroformisoamyl alcohol (24:1, vol/vol). The reextracted aqueous phase was made 0.3 M sodium acetate and mixed with two volumes of ice cold 95% ethanol. After overnight precipitation at -20°C, the DNA was collected by centrifugation (12,000 rpm, 20 min, -5C) and redissolved in 100 p1 of TES buffer (0.05 M NaCl, 0.03 M Tris, 0.005 M Na2-EDTA, pH 8.0 ). Agarose gel electrophoresis. Partially purified plasmid DNA preparations were analyzed by electrophoresis in 0.7% horizontal agarose slab gels essentially as described by Meyers et al (17). Molecular weights were determined from standard curves constructed from the relative mobilities of six plasmids of known mass (17).
Transfer of R68.45 to A. tumefaciens. The ability of R68.45 to mobilize transfer of host chromosomal genes is somewhat unstable (10). Before transferring this R plasmid to A. tumefaciens, a Pseudomonas aeruginosa donor was selected which showed good chromosomal mobilizing ability (CMA). Twenty clones of strain PA025(R68.45) were mated on solid medium with strain PA02 as described by Haas and Holloway (10). Of the 20 donor clones, 4 gave
TABLE 1. Bacterial strainsa Characteristicc
Derivation or source
leulO argFlO (Ap/Cb, Km/Nm, Tc) ser-2
B. W. Holloway R. Olsen
E. coli K-12 J53(RP4)
met pro (Ap/Cb, Km/Nm, Tc)
A. tumefaciens 15955 A3
E. W. Nester From 15955 after treatment with NTG, E. W. Nester From A3 following treatment His- MetA25 with NTG, E. W. Nester From A3, this paper His- Rifr Strr SA10 From A25, this paper His- Met- Rifr Strr SAl a Abbreviations: argF10, requires arginine; His-, requires histidine; leulO, requires leucine; met and Metrequires methionine; pro, requires proline; ser-2, requires serine. Rifr and Strr, resistant to rifampin and streptomycin respectively. Ap, ampicillin; Cb, carbenicillin; Nm, neomycin; Tc, tetracycline; NTG, N-methyl-
N'-nitro-N-nitrosoguanidine. b Parenthetical designations refer to the R plasmids harbored by the standard strains. Parenthetical designations refer to resistance traits conferred by the R plasmids present in the strains.
HAMADA, LUCKEY, AND FARRAND
rise to serine-independent recombinants. One of these donor clones was mated with A. tunefaciens 15955 in L broth as previously described (15). Transconjugants resistant to 20 ug of Km per ml were recovered at a frequency of approximately 10-3 per input donor cell. One strain 15955 transconjugant was selected for further study. The presence of R68.45 in the strain 15955 transconjugant was determined by agarose gel electrophoretic analysis of partially purified plasmid DNA. Figure 1 shows that strain 15955(R68.45) contains two plasmid species. One, with an estimated mass of 110 megadaltons, migrates with the same electrophoretic mobility as the Ti plamid present in the recipient strain. The other plasmid shows essentially the same mobility as RP4 in the marker preparation and R68.45 in strain PA025(R68.45). Demonstration of chromosomal gene transfer. To assess the CMA of strain 15955(R68-45), 10 subclones were mated on solid medium with strain SA10. Equal volumes of mid-exponential-phase donor and recipient L-
broth cultures were mixed. Ten 0.02-ml portions of each mating mixture were immediately spotted on the surface of a nutrient agar plate. As controls, each donor subclone and 10 replicates of the recipient were independently inoculated on nutrient agar plates. All plates were incubated at 290C for 48 h, after which growth from each plate was resuspended in 4.0 ml of AB buffer. Portions (0.1 ml) from serial 10-fold dilutions were plated on AB minimal agar containing glucose, rifampin, and streptomycin. After 3 to 4 days at 290C, colonies were large enough to be counted. Two donor clones showed high CMA (Table 2), whereas the other eight donors yielded lower numbers of His' colonies. In no case was the donor observed to grow on the selective medium, and the numbers of revertant colonies of SA10 were always lower than the numbers of His' colonies appearing on plates spread with the mixed cultures. This experiment was repeated three times. Although absolute numbers of colonies differed with each experiment, the pattern remained constant. Higher numbers of His' colonies appeared on plates spread with the mixed cultures than appeared on plates spread with strain SA10 alone. To determine whether CMA was stable in those clones yielding high numbers of His' progeny, a single donor strain, 15955(R68845) clone 2 (Table 2), was subcloned, and 10 inidividual sister clones were mated with strain SA10. Unlike the results presented in Table 2, high levels of CMA were observed with all donor sister clones tested (Table 3). When strain 15955(R68.45) clone 3, originally showing low CMA, was recloned and mated with strain SA10, 3 of the 10 subclones yielded substantial numbers of His' colonies. As above, TABLE 2. CMA' among different clones of strain 15955(R68-45) His+ CFU/rnlb
Donor clone Donor
Donor + recipient 1,210
40 110 2,360 FIG. 1. Agarosegel electrophoresis ofpartialypu- 15955(R68-45)-3 200 390 rified plasmid DNA from P. aeruginosa and A. tu- 15955(R68-45)4 180 640 mefaciens strains. Plasmid DNA, partially purified 15955(R68.45)-5 160 630 as described in the text, was electrophoresed through 15955(R68.45)-6 80 660 0. 7% horizontal agarose gels by the method of Meyers 100 730 et al. (17). Slot A: marker mixture containing (from 15955(R68.45)-7 15955(R68.45)-8 40 390 top to bottom) Rldrdl9 (62 megadaltons); open cir- 15955(R68.45)-9 20 860 cular Sa; RP4 (38 megadaltons); covalently closed 30 410 circular Sa (26 megadaltons); chromosomal frag- 15955(R68.45)-10 a Determined by mating on nutrient agar with strain ments; RSF103 (5.5 megadaltons); pMB8 duner (3.74 megadaltons). Slot B: P. aeruginosa strain SA10. b Selected on AB minimal agar supplemented with PA025(R 68.45). Slot C: A. tumefaciens strain 15955. Slot D: A. tumefaciens strain 15955(R68.45). rifampin (15 yg/ml) and streptomycin (500 tg/ml). 0 0 0 0 0 0 0 0 0 0
CHROMOSOME TRANSFER IN AGROBACTERIUM
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TABLE 3. CMA' of subclones of strain 15955(R68. 45) clone 2b His+ CFU/ml Donor subclone
Donor + reDonor Reclplent cipient
10 0 15955(R68.45) clone 2-1 30 0 15955(R68-45) clone 2-2 60 0 15955(R68-45) clone 2-3 0 0 15955(R68-45) clone 2-4 20 0 15955(R68.45) clone 2-5 40 0 15955(R68.45) clone 2-6 80 0 15955(R68.45) clone 2-7 40 0 15955(R68-45) clone 2-8 0 20 15955(R68.45) clone 2-9 0 0 15955(R68-45) clone 2-10 aDetermined as described in Table 2. b See Table 2.
2,110 3,130 1,940 2,490 3,050 2,680 1,640 1,840 2,060 2,240
further subcloning gave rise to a majority of donors manifesting high CMA (data not shown). Dependence upon R68.45. Plasmids RP1, RP4, and R68.45 have all been shown to mobilize the transfer of chromosomal genes in several genera of gram-negative bacteria (2, 10, 11, 16, 20, 22). However, there is some evidence for species and even strain specificity among these closely related R plasmids. For example, whereas RP1, RP4, and R68 all show CMA in P. aeruginosa PAT strains, only R68-45 (a derivative of R68) is capable of mobilizing the transfer of chromosomal markers in the PAO strains of this organism (10). To investigate plasmid requirements for donor ability, strains 15955, 15955(RP4), and 15955(R68-45) were mated on solid media with strain SA10. The results (Table 4) indicate that only R68-45 shows detectable chromosomal mobilization in this strain of A. tumefaciens. No His' transconjugants of strain SA10 were detected when strains 15955 and 15955(RP4) were used as donors. Mating protocols. It has been reported that the efficiency of chromosomal gene moblization by R68.45 is dependent upon the method by which the cells are mated. By and large, matings performed on solid surfaces are much more efficient than those performed in liquid media (2, 10). To investigate this parameter with A. tumefaciens, agar plate matings were compared with broth mating and with matings on nitrocellulose filters. Donor strain 15955(R68 45) clone 2 and strain SA10 were grown independently in L broth to approximately 109 CFU/ml. In the first experiment, 2.1 ml of each was mixed, and 10 0.02-ml portions were spotted on the surface of nutrient agar plates. The plates were incubated at 290C overnight. After incubation, growth from the plates was resuspended in 4 ml of AB buffer,
serially diluted, and plated on selective medium. The remainder of the mating mixture (4 ml) was incubated in a 150-ml growth flask overnight at 290C with gentle shaking. After incubation, the cells were collected by centrifugation, washed once, and resuspended in 4 ml of AB buffer. The resuspended cells were serially diluted and plated on selective medium. In the second experiment, 2.1 ml each of donor and recipient cultures were mixed, and 0.2 ml was removed and plated as described above. The remaining 4 ml was gently vacuum fitered onto the surface of a sterile 0.45-,m nitrocellulose membrane (Millipore Corp., 25-mm diameter). Filters were placed, bacteria up, on nutrient agar plates and incubated at 290C overnight. The filters were then placed in 4 ml of AB buffer and vigorously agitated to remove and resuspend the cells. Serial dilutions were made and plated on selective medium. In all cases, the suspensions were adjusted to approximately the same total cell concentration before dilution and plating. The results, shown in Table 5, indicate that plate matings and Millipore filter matings are comparable in efficiency and appear to be much more efficient than those perforned in broth. Effect of donor-recipient ratio. To determine whether the donor-to-recipient ratio has any influence on the frequency of chromosomal gene transfer, constant numbers of recipient cells were mated on solid medium with variable numbers of donor cells. Donor-recipient ratios were varied over a range from 5:1 to 0.03:1. Input donor and recipient titers were determined directly after mixing of the two cultures before TABLE 4. Plasmid requirement for CMAa His+ CFU/ml Donor Recipient 0 None 15955 0 None 15955(RP4) 0 None 15955(R68-45) 60 SA10 None 40 SA10 15955 50 SA10 15955(RP4) SA10 3,870 15955(R68.45) aDetermined by mating on nutrient agar with approximately 5 x 108 CFU each of donor and recipient per ml. TABLE 5. Comparison of mating methods HiS+ CFU/mlb Method' Expt Plate 1 1,900 10 Broth Plate 2 4,450
Filter 3,750 text. aPerformed as described in the bNumbers have been corrected for reversion of strain SA10 to histidine independence.
HAMADA, LUCKEY, AND FARRAND
spotting on the nutrient agar plates. It is clear from the results presented in Table 6 that the frequency of conjugation, expressed as the number of His' CFU per milliliter divided by the input donor titer, is highest at donor-to-recipient ratios of 0.1. As the ratio is increased, both the frequency and the absolute number of recombinants declined. Cotransfer of markers. To determine whether cotransfer of independent markers could be detected, strain 15955(R68-45) was mated with strain SAll (His- Met-). After 12-h matings, bacteria were resuspended and transconjugants inheriting His+, Met' and His' Met' were selected. The results, presented in Table 7, show that each marker can be independently transferred. In addition, simultaneous selection for both markers gave rise to a small but significant number of prototrophic recombinants. To assess inheritance of unselected second markers, transconjugant clones from each single marker selection were assayed by streaking onto AB minimal agar supplemented with glucose only. Six percent of the His+ transconjugants were Met', whereas 12% of the Met' recombinants were also His+ (Table 8). Between 36 and 62% of the transconjugants were Km resistant, a trait conferred by R68-45. The frequency of TABLE 6. Effect of donor-recipient ratio on the frequency of chromosomal gene transfera F Ratio Input titer (CFU/ml) His
inheritance of Km resistance among singlemarker-selected transconjugants also inheriting the second marker was approximately that of those inheriting only the selected marker. The appearance of transconjugants remaining sensitive to Km suggests that R68.45 is not necessarily coinherited with transferred chromosomal genes. Inheritance of the extrachromosomal form of this R plasmid was assessed by agarose gel electrophoresis of partially purified DNA from sensitive and resistant transconjugants. Figure 2 demonstrates that transconjugants of each nutritional class which are resistant to Km have inherited a second plasmid species with an electrophoretic mobility identical to that of R68 -45. In a similar fashion, recombinants of these nutritional classes remaining sensitive to the antibiotic appear to lack the R plasmid in its autonomous form. TABLE 8. Acquisition of unselected markers Frequencyb Cross'
His+ 15955(R68. Met+ 45) x His+ Met+ SAll a See text and Table 7. Based on the analysis selected phenotype.
Met' Km His+ Km Km
0.067 0.62 0.12 0.36 1.00 0.40
of 200 colonies of each
1.2 x 109 3,000 5.0 x 10-' 1.2 x 109 4,000 1.6 x 10-6 1.2 x 109 3,900 9.3 x 10-6 x 108 1.2 x 109 4,100 3.4 x 10-5 490 1.4 x 10-5 x 107 1.2 x 109 a Determined by mating on nutrient agar as described in the text. bNumbers have been corrected for reversion of strain SA10 to histidine independence. c Expressed as the number of His' CFU per milliliter divided by the input donor titer.
5.0 2.0 0.35 0.10 0.03
6.0 2.5 4.2 1.2 3.6
x 109 x 108
TABLE 7. Cotransfer of independent markersa
0 0 0 None 15955 (R68-45) 0 40 60 SAl None 520 7,200 8,900 SAl 15955 (R68.45) a Determined by matings on nutrient agar with approximately 5 x 108 CFU each of donor and recipient per ml.
FIG. 2. Agar gel electrophoresis of partially purified plasmid DNA from transconjugants of A. tumefaciens strain SAlI. Conditions were as described in the text and the legend to Fig. 1. Slot A: marker mixture containing (from top to bottom) Rldrdi9; open circular Sa; RP4; covalently closed circular Sa; chromosomal fragments; RSF1010::AplO3 (8.7 megadaltons); RS1030; pMB8 dimer; pMB8 monomer (1.87 megadaltons). Slot B: strain SA11. Slot C: strain 15955(R68.45). Slot D: strain SAl His'. Slot E: strain SA11 His' Km. Slot F: strain SA11 Met'. Slot G: strain SAl Met' Km. Slot H: strain SAl His' Met'. Slot I: strain SA11 His' Met' Km.
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CHROMOSOME TRANSFER IN AGROBACTERIUM
DISCUSSION Cumulative results presented here indicate that the IncPl plasmid R68.45 is capable of mobilizing the transfer of chromosomal genes from one strain of A. tumefaciens to another. Four sets of experiments show this phenomenon to be true chromosomal gene transfer rather than marker reversion. First, although the recipient, strain SA10, occasionally shows reversion to histidine prototrophy (Table 2), the magnitude of this event is well below the frequency of His' conversion obtained in mixed matings (Table 3). Second, a possible influence of the prototrophic donor strain upon the reversion frequency of strain SA10 can be ruled out by the observation that not all donor R68.45-containing clones yield a high number of His' progeny. Supporting this point is the finding that conversion to histidine prototrophy is dependent upon the presence of R68-45 in the donor strain. Strain 15955 lacking an R plasmid or harboring the closely related plasmid, RP4, appears to lack any donor ability (Table 4). Thirdly, results indicating that donor strains showing high CMA can be obtained by recloning indicates the frequency of the chromosome transfer event is dependent upon some property of the donor strain. Finally, the fact that coinheritance of two independent auxotrophic markers can be demonstrated (Tables 7 and 8) indicates that the conversion phenomenon is not restricted to the mutation conferring histidine auxotrophy. Such cotransfer aLso suggests that chromosome mapping by marker frequency analysis (1) may prove feasible in A. tumefaciens. High CMA in donor strains appears to be a somewhat unstable trait. We occasionally find that a good donor clone has lost its ability to transfer chromosomal markers. This appears to be a general characteristic of R68.45-mediated chromosomal mobilization and has been observed with P. aeruginosa (10) and R. leguminosarum (J. E. Beringer, personal communication). We have found that donors showing high CMA can be reisolated by sequential recloning and test matings. This instability would suggest that, if integration of the R plasmid into the host chromosome is required for CMA, this association is highly reversible. The relatively low frequency of marker transfer supports this and further suggests that only a portion of the donor cell population is capable of transferring chromosomal markers. In this respect the A. tumefaciens system closely resembles that of P. aeruginosa (10), R. leguminosarum (2), and R. sphaeroides (20). Such low chromosomal transfer frequencies cannot be explained by a low level of R plasmid-mediated conjugation. In data
not shown, matings on solid media between strains 15955(R68.45) and SA10 or SAll yielded Km-resistant transconjugants containing R68. 45 at frequencies approaching 102 per input donor cell. Results of experiments presented here indicate that A. tumefaciens strain 15955 is capable of chromosomal gene exchange if provided with a suitable mobilizing element. Refinement of the system should allow analysis and mapping of the A. tumefaciens chromosome (11). ACKNOWLEDGMENTS We thank Marvin Stodolaky for his suggestions during the course of this work and for his critical review of the manu-
script. This research was supported by Public Health Service grant CA19402 from the National Cancer Institute.
6. 7. 8.
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