- Email: [email protected]
Genetic map of an octopine TI-plasmid
PLASMID
2, 347-357 (1979)
Genetic
Map of an Octopine
TI-Plasmid
BERTUSP. KOEKMAN,GERT OOMS,PIETER M. KLAPWIJK, AND ROBBERTA. SCHILPEROORT Department
of Biochemistry, State University of Leiden, P.O. Box 9505, 2300 RA Leiden, The Netherlands
Received July 5, 1978 Several deletion mutants of an octopine TI-plasmid were mapped by digestion with the restriction enzyme Sma I. The T region, as it is defined on the B6-806 plasmid, does not appear to be an essential area for tumour induction on the plasmid of AchS. The genes for octopine breakdown, plasmid transfer, and the replicator were roughly localized. The possibility of using mutants with large deletions as a cloning vehicle in Agrobacrerium tumefaciens is discussed.
Crown gall tumours arise on many species of dicotyledonous plants after wounding and infection with Agrobacterium tumefaciens. A large plasmid (Zaenen et al., 1974) in the bacterial cells, the TI-plasmid, is responsible for the oncogenic properties (Van Larebeke et al., 1974, 1975; Watson et al., 1975). Crown gall tumours produce either octopine, or nopaline, or neither of these compounds (Menage and Morel, 1964; Goldmann et al., 1%9), depending on the bacterial strain that elicited the tumour. Generally, bacteria that induce octopine tumours catabolize octopine, whereas bacteria that induce nopaline tumours catabolize nopaline (Petit et al., 1970; Bomhoff, 1974). Breakdown of these arginine derivatives is encoded by the TIplasmid (Bon&off, 1974; Watson et al., 1975; Bomhoff et al., 1976). The presence in crown .gall tumour cells of a TI-plasmid fragment-called T-DNA-in multiple copies (Chilton et al ., 1977) and its transcription into RNA (Drummond et al., 1977; Ledeboer et al., 1978) might mean that T-DNA genes are responsible for the tumorous state of the crown gall cells as well as for the synthesis of lysopine dehydrogenase, the enzyme that catalyzes the synthesis of octopine and a few other crown gall-specific compounds (Goldmann et al., 1968; Kemp, 1977; Ottenet al., 1977). Direct evidence that TI-plasmid genes are actually
needed for tumorigenesis can be obtained from mutants affected in these genes. Among them, deletion mutants are particularly suitable. Recently, the TI-plasmid of octopine strain B6-806 has been physically mapped (Chilton et al., 1978a).Aside from the T region, which need not contain the virulence genes per se, the position of genetic markers on the restriction map remained unknown. These markers include genes for replication and conjugative transfer of the TI-plasmid, genes that code for octopine utilization by the bacterium, which are different from the genes that code for octopine synthesis by the plant (Klapwijk et al., 1976; Montoya et al., 1977), and genes for exclusion of bacteriophage AP-1 (Van Larebeke et al., 1975; Hooykaas et al., 1977). The approximate positions of these genes were deduced from Sma I restriction fingerprints of deleted TI-plasmids. Furthermore, we demonstrate that large parts of the Ach5 TI-plasmid, including the T region, as it is defined for pTi-B6-806 (Chilton et al., 1978b), can be deleted without loss of oncogenicity. MATERIALS Bacterial
AND METHODS
Strains
Bacterial strains are listed in Table 1. To isolate deletion mutants, we made use of the
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0147-619X/79/030347-11$02.00/0 Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
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ET AL.
TABLE 1 CHARACTERISTICS
OF BACTERIAL
STRAINS
Strain
Origin
Rasmid
Carbenicillin resistante
LBA 4017” LBA 4018 LBA 4019
LBA 4017 LBA 4017
PAL 104 PAL 105 PAL 106
-
+ +b +b
+ -
+ -e -c
+ + k
121 69 80
LBA 4014d LBA 4029
LBA 4014
pAL 657 PAL 204
+ +
+ +
+ +
+ -
+ +
125 114
LBA LBA LBA LBA LBA
LBA LBA LBA LBA LBA
PAL PAL PAL PAL PAL
+ + + + +
-
-
-
-
44 30 25 45 31
4023 4035 4040 4043 4052
4014 4014 4014 4014 4014
202 206 209 211 212
Virulence
Octopine in tumour
Octopine utilization and transfer
AP- 1 exclusion
Estimated MW of plasmid x 10-C
u LBA 4017 is Uadc derivative of Ach5. b Weakly virulent: induces tumours on Helianthus, Solarium, and Kalanchod, but not on Nicotiana (Klapwijk et al., 1978). c For lack of a marker that could be selected for, transfer could not be tested. d LBA 4014 is a rifampicin-resistant derivative of Ach5-C3 that contains TI-B6S3::Tnl (UadcTrac).
fact that mutants constitutive for octopine utilization (Klapwijk et al., 1977) are sensitive to homooctopine when their TIplasmid is in the chromosomal background of strain Ach5 (Petit and Tempe, 1976; Klapwijk et al., 1978). Mutants affected in octopine utilization grow well on SM medium containing 2 mg/ml homooctopine, whereas wild-type bacteria do not. These uad- l mutants, which arise at a frequency of 10-6-10-7, were purified and tested for reversion to Uad+ phenotype. The nonrevetting strains were used. Deletion mutants were derived from two Uad” strains, LBA 4014, carrying Tnl in its TI-plasmid (Hooykaas et al., submitted), and LBA 4017. The latter strain was selected on SM medium containing noroctopine as a sole source of carbon (Klapwijk et al., 1978). 1 Abbreviations: EtBr, ethidium bromide; SSC, 0.15 NaCl, 0.015 M Na, citrate; T,,E,,, 10 mM Tris, 0.1 mM EDTA; Tnl, transposon 1; tra, genes specifying conjugative transfer of the TI-plasmid; Trac, constitutive for transfer; uad, (unusual amino acid derivative); genes specifying octopine utilization by Agrobacterium; Uad’, constitutive for octopine degradation. M
Media LC broth contains per liter: 10 g Difco tryptone, 5 g Difco yeast extract, 8 g NaCl, 1 ml 1 M Tris-HCl, pH 7.2. SM medium was described by Klapwijk et al., (1977). Test for Phenotypic Traits Virulence tests. Overnight cultures in SM were inoculated on KulunchoL; duigremontiunu stems 24 h after wounding as described by Bomhoff et al. (1976). Octopine production by tumours. Octopine production by tumours was tested as described by Schilperoort and Bomhoff (1975) with a modification by Klapwijk et al. (1976). Octopine permeuse activity. Octopine permease activity was tested as described by Klapwijk et al. (1977). Octopine oxiduse activity. Conversion of [3H]octopine to [3H]arginine by toluenetreated cells was tested electrophoretically (Klapwijk et al., 1978). Conjugative transfer. Bacteria were mated to LBA 4009, a plasmidless derivative of
GENETIC
MAP OF AN OCTOPINE
strain Ach5, which is resistant to nalidixic acid and streptomycin. Log phase cells in SM containing 100pg/rnl octopine were concentrated to 2 x log/ml. From a 1: 1 mixture of donor and recipient, 0.1 ml was pipetted onto a Millipore filter placed on solid SM medium containing 2 mg/ml octopine (Klapwijket al., 1978) and incubated for 44 h at 29°C. The bacteria were resuspended on 0.9% NaCl and dilutions were plated on SM medium containing 10 pg/rnl carbenicillin, 50 pg/ml nalidixic acid, and 500 &ml streptomycin. AP-I exclusion. One drop of an overnight culture of bacteria in LC was mixed with 1 ml of LC medium containing 2 mM MgSO,. After 2 h shaking at 29”C, 50-500 plaqueforming units were added and the mixture was allowed to stand at room temperature for 10 min. Soft LC agar (3.5 ml) containing 20 mM MgSO, was added and the mixture was poured onto an LC plate containing 2 mM MgSO,. Plaques were scored after incubation overnight at 29°C (Van Larebeke et al., 1975;Hooykaas, personal communication). Isolation
of Plasmid DNA
Isolation of plasmid DNA was performed essentially as described by Currier and Nester (1976), with the following minor modifications: (1) bacteria were grown in LC broth till the A860 had reached 0.8 to 1.0; (2) cells were lysed at twice that concentration; washing steps were omitted; (3) 200 ml of lysate was sheared in a 400-ml beaker with a vibromixer (Ledeboer, 1976) at maximum speed for 15 to 60 s, depending on the decrease of viscosity; (4) CsCl-EtBr gradients were run for 60 h at 30,000 rpm in a Beckman 50 Ti rotor at 12°C; DNA from 400 ml of culture could be centrifuged per 50 Ti tube; (5) EtBr was removed from plasmid DNA by three extractions with isoamyl alcohol saturated with 20 x SSC, followed by exhaustive dialysis against TloEol, pH 8; (6) When necessary, plasmid DNA was concentrated by dialysis against 30% polyethylene glycol 6000 dissolved in TIOEOl,pH 8.
Restriction
TI-PLASMID
349
Enzyme Fragmentation
In a reaction mixture of 50 ~1, 1 to 1.5 pg of plasmid DNA was digested with 10 units of Sma I or 2 units of Hpa 1. The Sma mixture contained 30 mM Tris, pH 9.0, 15 mM KCl, 7 mM /3-mercaptoethanol, and 3 mM MgClz and was incubated 1 h at 29°C. The Hpa mixture contained 6 mM Tris, pH 7.5,50 mM NaCl, 6 mM P-mercaptoethanol, and 6 mM MgClz and was incubated 1 h at 37°C. After digestion, 10 ~1 of 0.1% bromophenol blue in 12.5% Ficoll (Pharmacia, Sweden) was added and the sample was applied to a gel. Sma was isolated according to Mulder and Delius (1972) and Hpa according to Bickle et al. (1977). Gel Electrophoresis
Gel electrophoresis was carried out on vertical 0.7% agarose (SeaKern) slab gels between sand-blasted glass plates in a buffer containing 40 mM Tris, pH 7.9, 5 mM NaOAc, 1 mM EDTA, and 1 pg/ml EtBr. The dimensions of the gel were 17 x 40 x0.5 cm. Samples were electrophoresed for 5 min at 200 V, and for 20-24 h at 100 V. After running, the gel was placed onto a Chromato-Vue transilluminator (Ultraviolet Products Inc.) and photographed through a red filter. RESULTS
The Sma fragmentation pattern of the TIplasmids from the two constitutive strains used are represented in slots 1 (PAL 657 from LBA 4014) and 2 (PAL 104 from LBA 4017) of Fig. 1. It is apparent that in PAL 657, which acquired Tnl , band number 5 is replaced by a band at a position above the Sma 3 doublet. The new band, designated 5*, has a molecular weight of approximately 10 x 106, corresponding to an increase of 3.4 x 106. This can be explained by the integration of Tnl, for which a molecular weight of 3 x lo6 has been reported (Heffron et al., 1975). Except for the Tnl insertion, the Sma
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KOEKMAN
1 2 5 3 4 6 7 8 9 10 11 12 13 14 A 14 B 15 16 17 18
ET AL.
1 2 5* 3 4 6 7 8 9 10 ,I1 ,I2 ,I3 14 A 14 B 15 16 17 18
FIG. 1. Sma I fingerprints of plasmids from virulent deletion mutants. From left to right: PAL 657, PAL 104, pAL 106, PAL 105, pAL 204,and PAL 657.
digests ofpAL 657 (a B6S3 plasmid) and PAL 104 (an Ach5 plasmid) are similar to the pattern of pT1 B6-806, used by Chilton et al.
(1978a). The only difference is that the PAL 104 and pAL 657 doublet 14 is resolved into two bands. Fragment 14a is slightly larger
GENETIC
MAP OF AN OCTOPINE
TI-PLASMID
351
(k 2.55 x 106)than the corresponding fragments from pTi-B6-806, whereas fragment 14b has the same molecular weight (2.5 x 106). As can be seen from Fig. 1, there is also some resolution of the Sma 16doublet: In slots 1, 2, and 6 both 16a and 16b are present, whereas in slots 3, 4, and 5 band 16b is missing from the doublet. The following is based on the assumption that the fragmentation maps of PAL 104, PAL 657, and pTi-B6-806 are the same. Virulent Deletion Mutants Deleted plasmids that still confer virulence upon the bacterium are derived from PAL 657 as well as from PAL 104. Their Sma digests are shown in Fig. 1. The positions of the deletions are summarized in Fig. 4; for molecular weights see Table 1. The molecular weights of the Sma fragments are listed in Table 2. PAL 204 (from LBA 4029). Visible are bands 1,2,5*, 3,4, a new band of molecular TABLE
2
MOLECULAR WEIGHTS OF SMA I DIGEST FRAGMENTS OF pTi-B6-806 PLASMID DNA” Sma I fragment number 1 2 3 4 5 6 7 8 9 10 11 12 13 14‘4 148 15 16 17 18 19 20 21 a Chilton et al. (1978 T(Y).
MW x 10-e
Multiplicity
14.9 14.0 9.2 7.5 6.6 6.05 6.0 5.7 5.5 4.0 3.78 3.22 2.65 2.55 2.5 2.28 1.58 1.23 1.17 0.88 0.67 0.64
1 1 2 1 1 1 1 1 1 3 1 1 1 1 1 1 2 1 1 1 1 1
14 \ 15 \
FIG. 2. From left to right: Hpa I fingerprints 657, pAL 106, and PAL 105.
of PAL
weight 7 x 106, 6, 7, 8, 10, 12, 14a, 14b, 15, 16a, 17, and 18. From the presence of fragments 7 and 12 and the absence of frag-
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KOEKMANETAL.
FIG. 3. Sma I fingerprints of plasmids from avirulent deletion mutants, From left to right: PALL 657 PAL 202, pAL 206, PAL 209, PAL 211, and PAL 212.
ments 9 and 11, we conclude that 9 and 11 Fusion of 9 and 21 is unlikely becausle the have fused to form the new 7 million. band sum of their molecular weights 1is less than and that the region between them is deleted. 7 x 106.
GENETIC MAP OF AN OCTOPINE TI-PLASMID
353
FIG. 4. Schematic representation of deleted plasmids from virulent strains. Conserved Sma I restriction fragments are shown. The molecular weight increase of fragment 5 due to Tnl insertion has been
ignored. Saw tooth lines have arbitrarily been drawn in the middle of the fragments in which the deletions are suspected to end. (*) The dotted lines give a more accurate indication of the end-points of PAL 105 and 106. Full details are given in the text.
PAL 105 Cfrom LBA 4018). Visible are bands 1,2,3,5,8, 10, 14b, 15, 16a, and, less clear, 17 and 18. From this, it can be concluded that, at the right side of the T-region, at least the DNA between fragments 4 and 7 is deleted. Because fragments 3b and 1Ocare part of multiple bands, their presence or absence cannot be established. From a digest with Hpa I (Fig. 2) fragment 13 is missing, whereas 14 is still present. Hpa 13 overlaps the termini of Sma 1Ocand 3b (Chilton et al., 1978a). If the deletion ended in the overlap of Sma 1Oc and Hpa 13, a new band with a molecular weight of at least 2.83 x lo6 (the remaining part of 1Oc)would be expected to appear in the Sma digest of PAL 105. Because no such fusion product is visible, it must be smaller than 106. Therefore, we conclude that the deletion ends in the overlap of Hpa 13 with Sma 3b (Fig. 6) and that Sma 3b has fused to Sma 4. PAL 106 Cfrom LBA 4019). From the Hpa
digest (Fig. 2) of this plasmid, both fragments 13 and 14 are absent, whereas 3 is still present. Therefore, the deletion ends in Hpa 14 (Fig. 6) and removes the T region. The Sma digest differs from that of PAL 105 in that band 4 is still present, and that a new band of 3.3 x lo6 (between 10 and 14b) appears. This band arises by fusion of Sma 1Oc and Sma 6. Avirulent Deletion Mutants Figure 3 shows the Sma digests of the TIplasmids from the avirulent class of deletion mutants, derived from PAL 657. Fragment 5* is missing from all of these plasmids, whereas fragment 14b is still present. Because of its small size, fragment 20, located between 5* and lib, can be visualized only when the gel is overloaded (not shown). Because all strains from the avirulent class re-
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KOEKMAN
ET AL.
FIG. 5. Schematic representation ofdeleted plasmids from avirulent strains. For further details see the legend to Fig. 3.
tain the carbenicillin resistance conferred by the insertion of Tnl into fragment 5, we conclude that the deletions start in fragment 5* and not in fragment 20. Indeed, in a number of cases (PAL 202,209,211) fragment 20 is too small to account for the molecular weights of the new bands that arise from the fusion of the two bands in which the deletion ends. We shall discuss the deletions in order of their size (see Fig. 5 for the location of the deletions and Table 1 for their molecular weights). PAL 211 (jiiom LBA 4043). Visible are bands 2, 4, 6, a new band of molecular weight 4.3 x 106, 10, 14a, 14b, and 15. By overloading the gel, also band 18 could be detected (not shown). Note that bands 6 and 14a are present, whereas number 12, which according to Chilton er a/.( 1978a)is situated in between, is absent. We infer that the correct order of these fragments must be . ..9. 12, 14a, 6.... The new band is a combination of the remnants of bands 5* and 12. PAL 202 Cfrom LBA 4023). Bands 2,4,6, a
new band almost the same size as band 9 (5.5 x 106), 10, 14b, 15, 18 (compare PAL 211), and 20 (see above) are present. The new band 9* arises from fusion of bands 5* and 14a. PAL 206 and 212 (from LBA 4035 and
4052). Bands 2, anovel band, 10,14b, 15,18, and 20 are left. The new band has a molecular weight of about 5.7 x lo6 in the case of PAL 206; for PAL 212 its weight is ca. 6.5 x 10”. Both are fusion products of fragments 4 and 5*. pAL 209 Cfrom LBA 4040). This has the largest deletion of all mutants studied. It has a new band l*, larger than 1, band 10, 14b, 15, and 20. Fragment 1* has a molecular weight of at least 15 x lo6 and is a fusion product of bands 2 and 5*. DISCUSSION
When the positions of the deletions (Figs. 4 and 5) on the described plasmids are related to the phenotypic properties of the
GENETIC MAP OF AN OCTOPINE TI-PLASMID
355
FIG. 6. Location of genetic markers on the physical map of the octopine TI-plasmid. For AP-1 exclusion see Discussion. The PAL numbers on the outside represent the end-points of deletions.
strains which contain them (Table l), the following conclusions, summarized in Fig. 6, can be drawn: (1) The strains containing PAL 202, 206, 209, 211, and 212 have lost their virulence. All have deletions that remove the T region. Strains LBA 4018 and 4019 (carrying PAL 105 and 106, respectively) have a much attenuated virulence (Klapwijk et al., 1978) and their tumours do not contain octopine. Both strains lack a large area-about 40% of the TI-plasmid-on the right side of the TIplasmid, including part of the T region (PAL 105) or the entire T region (PAL 106). Although the deleted DNA-especially the T region-probably contains functions that are involved in tumorigenesis it is not essential for virulence. Essential genes must be situated between the Tnl insertion in Sma fragment 5 and the T region, because this area is deleted in the avirulent mutants. Chilton et al., (1978b) have defined the T region on the plasmid pTi-B6-806. Since the deleted plasmids PAL 105 and 106 are derived from the Ach5 TI-plasmid, a minor
difference between the AchS- and B6-806 plasmids in this region could explain our result that the T region is not essential for virulence. However, a more likely explanation can be found in the uncertainty that still exists about the left boundary of the T-DNA (Chilton et al., 1978b). Because PAL 105 misses part of the T region, the latter is not transferred to the plant as a discrete “transposon-like” structure, as suggested by Schell and Van Montagu (1978), because both ends of a transposon are needed for transposition (Kleckner, 1977). (2) All avirulent mutants have become sensitive to AP-1. Among these mutants PAL 211 has the smallest deletion (Fig. 5), so the genes for exclusion map in the upper part of the TI-plasmid, between Sma 5 and 12. LBA 4018 and 4019 are less sensitive to AP-1 than the avirulent mutants: The number of plaques formed is about four times lower and these plaques are very tiny. Because the end-points of the deletions in pAL 105 and 106 are at least lo6 apart, it
356
KOEKMAN
is unlikely that the exclusion genes are partially deleted in these plasmids. These genes probably map in separate areas of the TIplasmid: between Sma 12 and 3b (deleted in both PAL 105and pAL 2 11)) and between Sma 5 and 1Oc(deleted in PAL 211, but not in PAL 106). (3) LBA 4029 (carrying PAL 204) is affected only in the utilization of octopine and in the transfer of its TI-plasmid, which functions are closely related (Genetello et al., 1977; Kerr et al., 1977; Klapwijk et al., 1978). Consequently, genes necessary for octopine degradation and plasmid transfer map between Sma fragments 9 and 11 and are physically separate from the genes coding for octopine synthesis in the tumour (Klapwijket al., 1977;Montoyaet al., 1977), which map probably within the T region. Since LBA 4029 is still virulent, a conjugative mechanism is not involved in the process of tumour induction, as has been suggested by TempC et al. (1977). (4) The deletion mutants derived from LBA 4014 were selected for on two markers: carbenicillin resistance and Uad- character. Out of nine nonreverting mutants isolated, five have deletions that originate in the Sma band containing Tn 1 (LBA 4023,4035,4040, 4043, and 4052, carrying PAL 202,206,209, 211, and 212, respectively). The deletions extend in clockwise direction from Sma band 5” and hit the virulence and uud genes. Their suspected common origin suggeststhat Tnl is involved in their generation. In LBA 4029 (carrying pAL 204)-one of the other four mutants derived from LBA 4014-only uud genes are deleted. Since this deletion does not originate in Sma fragment 5*, it is probably spontaneous, as is the case for the mutants derived from LBA 4017. (5) The largest deletion isolated is that of LBA 4040, carrying pAL 209. Therefore, the replicator must be located on the piece of DNA which constitutes PAL 209. Thus, with LBA 4040 we have obtained a smallsized (25 x 106) Agrobucterium-specific drug-resistance plasmid, that may provide a suitable vector on which, e.g., the virulence and octopine genes can be cloned. Possibly
ET AL.
the new Sma fragment I*, that consists of fused Sma bands 2 and 5*, contains the replicator in addition to the carbenicillin marker, and can, after ligation, serve this purpose. ACKNOWLEDGMENTS The authors are indebted to Dr. M. -D. Chilton (Seattle, Wash.) for providing them with a physical map of the TI-plasmid before publication. The skilful technical assistance of Miss J. Poulis is gratefully acknowledged. Phage AP-1 was a generous gift from Prof. Dr. J. Schell (Ghent, Belgium). This work was supported in part by the Netherlands Foundation of Biological Research (BION) with financial aid from the Netherlands Organization for Advancement of Pure Scientific Research (ZWO).
REFERENCES BICKLE, T. A., PIROTTA, V., AND IMBER, J. (1977). A simple, general procedure for purifying restriction endonucleases. Nucl. AC. Rcs. 4, 2561-2572. BOMHOFF, G. H. (1974). Studies on crown gall-a plant tumour. Investigations on protein composition and on the use of guanidine compounds as a marker for transformed cells. Thesis, University of Leiden, The Netherlands. BOMHOFF,~. H., KLAPWIJK,~. M., KESTER,H. C. M., SCHILPEROORT, R. A., HERNALSTEENS, J. P., AND SCHELL, J. (1976). Octopine and nopaline: Synthesis and breakdown genetically controlled by a plasmid of Agrobacterium tumefaciens. Mol. Gen. Genet. 145, 177- 181. CHILTON, M. -D., DRUMMOND, M. H., MERLO, D. J., SCIAKY, D., MONTOYA, A. L., GORDON, M. P., AND NESTER, E. W. (1977). Stable incorporation of plasmid DNA into higher plant cells: The molecular basis of crown gall tumorigenesis. Cell 11, 263-271. CHILTON, M. -D., MONTOYA, A. L., MERLO, D. J., DRUMMOND, M. H., NUTTER, R., GORDON, M. P., AND NESTER, E. W. (1978a). Restriction endonuclease mapping of a plasmid that confers oncogenicity upon Agrobacterium tumefaciens strain B6-806. Plasmid 1, 254-269. CHILTON, M. -D., DRUMMOND, M. H., MERLO, D. J., AND SCIAKY, D. (1978b). Highly conserved DNA of Ti plasmids overlaps T-DNA maintained in plant turnours. Nature (London) 275, 147- 149. CURRIER, T. C., AND NESTER, E. W. (1976). Isolation of covalently closed circular DNA of high molecular weight from bacteria. Anal. Biochem. 66, 43 1-441. DRUMMOND, M. H., GORDON, M. P., NESTER, E. W., AND CHILTON, M. -D. (1977). Foreign DNA of bacterial plasmid origin is transcribed in crown gall tumours. Nature (London) 269, 535-536. GENETELLO, C., VAN LAREBEKE, N., HOLSTERS, M., DE PICKER, A., VAN MONTAGU, M., AND SCHELL, J.
GENETIC
MAP OF AN OCTOPINE
(1977). The TI plasmids of Agrobacterium as sex factors. Nature (London) 265, 561-563. GOLDMANN, A., TEMPI?, J., AND MOREL, G. (1968). Quelques particularitts de diverses souches d’Agrobacterium tumefaciens. C. R. Sot. Biol. 162, 630631. GOLDMANN, A., THOMAS, D. W., AND MOREL, G. (1969). Sur la structure de la nopaline, mCtabolite anormal de certaines tumeurs de Crown-gall. C. R. Acad. Sci. (Paris) 268, 852-854. HEFFRON, F., SUBLETT, R., HEDGES, R. W., JACOB, A., AND FALKOW, S. (1975). Origin of the TEM beta-lactamase gene found on plasmids. J. Bacterial. 122, 250-256. HOOYKAAS, P. J. J., KLAPWIJK, P. M., NUTI, M. P., SCHILPEROORT,R. A., AND R~~RSCH,A. (1977). Transfer of the Agrobacterium tumefaciens TI plasmid to avirulent Agrobacteria and to Rhizobium ex planta. J. Gen. Microbial. 98, 477-484. KEMP, J. D. (1977). A new amino acid derivative present in crown gall tumour tissue. Biochem. Biophys. Res. Commun. 74, 862-868. KERR, A., MANIGAULT, P., AND TEMPT, J. (1977). Transfer of virulence in viva and in vitro in Agrobacterium. Nature (London) 265, 560-561. KLAPWIJK, P. M., HOOYKAAS, P. J. J., KESTER, H. C. M., SCHILPEROORT, R. A., AND R~RSCH, A. (1976). Isolation and characterization of Agrobacterium tumefaciens mutants affected in the utilization of octopine, octopinic acid and nopaline. J. Gen. Microbial. 96, 155- 163. KLAPWIJK, P. M., OUDSHOORN, M., AND SCHILPEROORT, R. A. (1977). Inducible permease involved in the uptake of octopine, lysopine, and octopinic acid by Agrobacterium tumefaciens strains carrying virulence-associated plasmids. J. Gen. Microbial. 102, l-11. KLAPWIJK, P. M., SCHEULDERMAN, T., AND SCHILPEROORT, R. A. (1978). Co-ordinated regulation of octopine degradation and conjugative transfer of TIplasmids in Agrobacterium tumefaciens: evidence for a common regulatory gene and separate operons. J. Bacterial., 136, 775-785. KLECKNER, N. (1977). Translocatable elements in procaryotes. Cell 11, 1 l-23. LEDEBOER, A. M., KROL, A. J. M., DONS, J. J. M., SPIER, F., SCHILPEROORT,
R. A., ZAENEN,
I., VAN
LAREBEKE, N., AND SCHELL, J. (1976). On the isolation of TI-plasmid from Agrobacterium tumefaciens. Nucl. AC. Res. 3, 449-463. LEDEBOER, A. M. (1978). Large plasmids in Rhizobiaceae. I. Studies on the transcription of the tumour inducing plasmid from Agrobacterium tumefaciens in sterile crown gall tumour cells. II. Studies on large plasmids in different Rhizobium species. Thesis, University of Leiden, The Netherlands. MENAGE, A., AND MOREL, G. (1964). Sur la prtsence d’octopine dans les tissus de Crown-gall. C. R. Acad. Sri. (Paris) 259, 4795-4796. MONTOYA, A. L., CHILTON, M.-D., GORDON, M. P.,
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SCIAKY, D., AND NESTER, E. W. (1977). Octopine and nopaline metabolism in Agrobacterium tumefaciens and crown gall tumour cells: Role of plasmid genes. J. Bacterial. 129, lOl- 107. MULDER, C., AND DELIUS, H. (1972). Specificity of the break produced by restricting endonuclease R, in Simian Virus 40 DNA, as revealed by partial denaturation mapping. Proc. Natl. Acad. Sci. USA 69, 3215-3219. OTTEN, L. A. B. M., VREUGDENHIL, D., AND SCHILPEROORT, R. A. (1977). Properties of D(+)lysopine dehydrogenase from crown gall tumour tissue. Biochim. Biophys. Acta 485, 268-277. PETIT, A., DELHAYE, S., TEMPT, J., AND MOREL, G. (1970). Recherches sur les guanidines des tissus de Crown-gall. Mise en Cvidence d’une relation biochimique spCcifique entre les souches d’Agrobacterium tumefaciens et les tumeurs qu’elles induisent. Physiol. Vkgetale 8, 205-213. PETIT, A., AND TEMPT, J. (1976). Etude du metabolisme de I’homooctopine et de l’un de ses analogues par I’Agrobacterium tumefaciens. C. R. Acad. Sci. (Paris) 282, 69-71. SCHELL, J., AND VAN MONTAGU, M. (1978). Transfer, maintenance and expression of bacterial TI plasmid DNA in plant cells transformed withA. tumefaciens. In “Brookhaven Symposia in Biology” (C. W. Anderson, ed.), Vol 29: Genetic Interactions and Gene Transfer, pp. 36-49. Brookhaven National Laboratory, Upton, New York. SCHILPEROORT, R. A., AND BOMHOFF, G. H. (1975). Crown gall: A model for tumour research and genetic engineering. In: “Genetic Manipulations with Plant Material” (L. Ledoux, ed.), pp. 163-181. Plenum Press, New York. TEMP& J., PETIT. A., HOLSTERS, M., VAN MONTAGU, M., AND SCHELL, 3. (1977). Thermosensitive step associated with transfer of the TI plasmid during conjugation: Possible relation to transformation in crown gall. Proc. Natl. Acad. Sci. USA 74, 28482849. VAN LAREBEKE,N., ENGLER, G., HOLSTERS, M., VAN DEN ELSACKER, S., ZAENEN, I., SCHILPEROORT, R. A., AND SCHELL, J. (1974). Large plasmid in Agrobacterium tumefaciens essential for crown gall inducing activity. Nature (London) 252, 169- 170. VAN LAREBEKE, N., GENETELLO, C., SCHELL, J., SCHILPEROORT, R. A., HERMANS, A. K., HERNALSTEENS, J. P., AND VAN MONTAGU, M. (1975). Acquisition of tumour-inducing ability by non-oncogenie Agrobacteria as a result of plasmid transfer. Nature (London) 255, 742-743. WATSON, B., CURRIER, T. C., GORDON, M. P., CHILTON, M.-D., AND NESTER, E. W. (1975). Plasmid required for virulence of Agrobacterium tumefaciens. J. Bacterial. 123, 255-264. ZAENEN, I., VAN LAREBEKE, N., TELJCHY, H., VAN MONTAGU, M., AND SCHELL, J..(1974). Supercoiled circular DNA in crown gall inducing Agrobacterium strains. J. Mol. Bio/. 86, 109-127.
2, 347-357 (1979)
Genetic
Map of an Octopine
TI-Plasmid
BERTUSP. KOEKMAN,GERT OOMS,PIETER M. KLAPWIJK, AND ROBBERTA. SCHILPEROORT Department
of Biochemistry, State University of Leiden, P.O. Box 9505, 2300 RA Leiden, The Netherlands
Received July 5, 1978 Several deletion mutants of an octopine TI-plasmid were mapped by digestion with the restriction enzyme Sma I. The T region, as it is defined on the B6-806 plasmid, does not appear to be an essential area for tumour induction on the plasmid of AchS. The genes for octopine breakdown, plasmid transfer, and the replicator were roughly localized. The possibility of using mutants with large deletions as a cloning vehicle in Agrobacrerium tumefaciens is discussed.
Crown gall tumours arise on many species of dicotyledonous plants after wounding and infection with Agrobacterium tumefaciens. A large plasmid (Zaenen et al., 1974) in the bacterial cells, the TI-plasmid, is responsible for the oncogenic properties (Van Larebeke et al., 1974, 1975; Watson et al., 1975). Crown gall tumours produce either octopine, or nopaline, or neither of these compounds (Menage and Morel, 1964; Goldmann et al., 1%9), depending on the bacterial strain that elicited the tumour. Generally, bacteria that induce octopine tumours catabolize octopine, whereas bacteria that induce nopaline tumours catabolize nopaline (Petit et al., 1970; Bomhoff, 1974). Breakdown of these arginine derivatives is encoded by the TIplasmid (Bon&off, 1974; Watson et al., 1975; Bomhoff et al., 1976). The presence in crown .gall tumour cells of a TI-plasmid fragment-called T-DNA-in multiple copies (Chilton et al ., 1977) and its transcription into RNA (Drummond et al., 1977; Ledeboer et al., 1978) might mean that T-DNA genes are responsible for the tumorous state of the crown gall cells as well as for the synthesis of lysopine dehydrogenase, the enzyme that catalyzes the synthesis of octopine and a few other crown gall-specific compounds (Goldmann et al., 1968; Kemp, 1977; Ottenet al., 1977). Direct evidence that TI-plasmid genes are actually
needed for tumorigenesis can be obtained from mutants affected in these genes. Among them, deletion mutants are particularly suitable. Recently, the TI-plasmid of octopine strain B6-806 has been physically mapped (Chilton et al., 1978a).Aside from the T region, which need not contain the virulence genes per se, the position of genetic markers on the restriction map remained unknown. These markers include genes for replication and conjugative transfer of the TI-plasmid, genes that code for octopine utilization by the bacterium, which are different from the genes that code for octopine synthesis by the plant (Klapwijk et al., 1976; Montoya et al., 1977), and genes for exclusion of bacteriophage AP-1 (Van Larebeke et al., 1975; Hooykaas et al., 1977). The approximate positions of these genes were deduced from Sma I restriction fingerprints of deleted TI-plasmids. Furthermore, we demonstrate that large parts of the Ach5 TI-plasmid, including the T region, as it is defined for pTi-B6-806 (Chilton et al., 1978b), can be deleted without loss of oncogenicity. MATERIALS Bacterial
AND METHODS
Strains
Bacterial strains are listed in Table 1. To isolate deletion mutants, we made use of the
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0147-619X/79/030347-11$02.00/0 Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
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ET AL.
TABLE 1 CHARACTERISTICS
OF BACTERIAL
STRAINS
Strain
Origin
Rasmid
Carbenicillin resistante
LBA 4017” LBA 4018 LBA 4019
LBA 4017 LBA 4017
PAL 104 PAL 105 PAL 106
-
+ +b +b
+ -
+ -e -c
+ + k
121 69 80
LBA 4014d LBA 4029
LBA 4014
pAL 657 PAL 204
+ +
+ +
+ +
+ -
+ +
125 114
LBA LBA LBA LBA LBA
LBA LBA LBA LBA LBA
PAL PAL PAL PAL PAL
+ + + + +
-
-
-
-
44 30 25 45 31
4023 4035 4040 4043 4052
4014 4014 4014 4014 4014
202 206 209 211 212
Virulence
Octopine in tumour
Octopine utilization and transfer
AP- 1 exclusion
Estimated MW of plasmid x 10-C
u LBA 4017 is Uadc derivative of Ach5. b Weakly virulent: induces tumours on Helianthus, Solarium, and Kalanchod, but not on Nicotiana (Klapwijk et al., 1978). c For lack of a marker that could be selected for, transfer could not be tested. d LBA 4014 is a rifampicin-resistant derivative of Ach5-C3 that contains TI-B6S3::Tnl (UadcTrac).
fact that mutants constitutive for octopine utilization (Klapwijk et al., 1977) are sensitive to homooctopine when their TIplasmid is in the chromosomal background of strain Ach5 (Petit and Tempe, 1976; Klapwijk et al., 1978). Mutants affected in octopine utilization grow well on SM medium containing 2 mg/ml homooctopine, whereas wild-type bacteria do not. These uad- l mutants, which arise at a frequency of 10-6-10-7, were purified and tested for reversion to Uad+ phenotype. The nonrevetting strains were used. Deletion mutants were derived from two Uad” strains, LBA 4014, carrying Tnl in its TI-plasmid (Hooykaas et al., submitted), and LBA 4017. The latter strain was selected on SM medium containing noroctopine as a sole source of carbon (Klapwijk et al., 1978). 1 Abbreviations: EtBr, ethidium bromide; SSC, 0.15 NaCl, 0.015 M Na, citrate; T,,E,,, 10 mM Tris, 0.1 mM EDTA; Tnl, transposon 1; tra, genes specifying conjugative transfer of the TI-plasmid; Trac, constitutive for transfer; uad, (unusual amino acid derivative); genes specifying octopine utilization by Agrobacterium; Uad’, constitutive for octopine degradation. M
Media LC broth contains per liter: 10 g Difco tryptone, 5 g Difco yeast extract, 8 g NaCl, 1 ml 1 M Tris-HCl, pH 7.2. SM medium was described by Klapwijk et al., (1977). Test for Phenotypic Traits Virulence tests. Overnight cultures in SM were inoculated on KulunchoL; duigremontiunu stems 24 h after wounding as described by Bomhoff et al. (1976). Octopine production by tumours. Octopine production by tumours was tested as described by Schilperoort and Bomhoff (1975) with a modification by Klapwijk et al. (1976). Octopine permeuse activity. Octopine permease activity was tested as described by Klapwijk et al. (1977). Octopine oxiduse activity. Conversion of [3H]octopine to [3H]arginine by toluenetreated cells was tested electrophoretically (Klapwijk et al., 1978). Conjugative transfer. Bacteria were mated to LBA 4009, a plasmidless derivative of
GENETIC
MAP OF AN OCTOPINE
strain Ach5, which is resistant to nalidixic acid and streptomycin. Log phase cells in SM containing 100pg/rnl octopine were concentrated to 2 x log/ml. From a 1: 1 mixture of donor and recipient, 0.1 ml was pipetted onto a Millipore filter placed on solid SM medium containing 2 mg/ml octopine (Klapwijket al., 1978) and incubated for 44 h at 29°C. The bacteria were resuspended on 0.9% NaCl and dilutions were plated on SM medium containing 10 pg/rnl carbenicillin, 50 pg/ml nalidixic acid, and 500 &ml streptomycin. AP-I exclusion. One drop of an overnight culture of bacteria in LC was mixed with 1 ml of LC medium containing 2 mM MgSO,. After 2 h shaking at 29”C, 50-500 plaqueforming units were added and the mixture was allowed to stand at room temperature for 10 min. Soft LC agar (3.5 ml) containing 20 mM MgSO, was added and the mixture was poured onto an LC plate containing 2 mM MgSO,. Plaques were scored after incubation overnight at 29°C (Van Larebeke et al., 1975;Hooykaas, personal communication). Isolation
of Plasmid DNA
Isolation of plasmid DNA was performed essentially as described by Currier and Nester (1976), with the following minor modifications: (1) bacteria were grown in LC broth till the A860 had reached 0.8 to 1.0; (2) cells were lysed at twice that concentration; washing steps were omitted; (3) 200 ml of lysate was sheared in a 400-ml beaker with a vibromixer (Ledeboer, 1976) at maximum speed for 15 to 60 s, depending on the decrease of viscosity; (4) CsCl-EtBr gradients were run for 60 h at 30,000 rpm in a Beckman 50 Ti rotor at 12°C; DNA from 400 ml of culture could be centrifuged per 50 Ti tube; (5) EtBr was removed from plasmid DNA by three extractions with isoamyl alcohol saturated with 20 x SSC, followed by exhaustive dialysis against TloEol, pH 8; (6) When necessary, plasmid DNA was concentrated by dialysis against 30% polyethylene glycol 6000 dissolved in TIOEOl,pH 8.
Restriction
TI-PLASMID
349
Enzyme Fragmentation
In a reaction mixture of 50 ~1, 1 to 1.5 pg of plasmid DNA was digested with 10 units of Sma I or 2 units of Hpa 1. The Sma mixture contained 30 mM Tris, pH 9.0, 15 mM KCl, 7 mM /3-mercaptoethanol, and 3 mM MgClz and was incubated 1 h at 29°C. The Hpa mixture contained 6 mM Tris, pH 7.5,50 mM NaCl, 6 mM P-mercaptoethanol, and 6 mM MgClz and was incubated 1 h at 37°C. After digestion, 10 ~1 of 0.1% bromophenol blue in 12.5% Ficoll (Pharmacia, Sweden) was added and the sample was applied to a gel. Sma was isolated according to Mulder and Delius (1972) and Hpa according to Bickle et al. (1977). Gel Electrophoresis
Gel electrophoresis was carried out on vertical 0.7% agarose (SeaKern) slab gels between sand-blasted glass plates in a buffer containing 40 mM Tris, pH 7.9, 5 mM NaOAc, 1 mM EDTA, and 1 pg/ml EtBr. The dimensions of the gel were 17 x 40 x0.5 cm. Samples were electrophoresed for 5 min at 200 V, and for 20-24 h at 100 V. After running, the gel was placed onto a Chromato-Vue transilluminator (Ultraviolet Products Inc.) and photographed through a red filter. RESULTS
The Sma fragmentation pattern of the TIplasmids from the two constitutive strains used are represented in slots 1 (PAL 657 from LBA 4014) and 2 (PAL 104 from LBA 4017) of Fig. 1. It is apparent that in PAL 657, which acquired Tnl , band number 5 is replaced by a band at a position above the Sma 3 doublet. The new band, designated 5*, has a molecular weight of approximately 10 x 106, corresponding to an increase of 3.4 x 106. This can be explained by the integration of Tnl, for which a molecular weight of 3 x lo6 has been reported (Heffron et al., 1975). Except for the Tnl insertion, the Sma
350
KOEKMAN
1 2 5 3 4 6 7 8 9 10 11 12 13 14 A 14 B 15 16 17 18
ET AL.
1 2 5* 3 4 6 7 8 9 10 ,I1 ,I2 ,I3 14 A 14 B 15 16 17 18
FIG. 1. Sma I fingerprints of plasmids from virulent deletion mutants. From left to right: PAL 657, PAL 104, pAL 106, PAL 105, pAL 204,and PAL 657.
digests ofpAL 657 (a B6S3 plasmid) and PAL 104 (an Ach5 plasmid) are similar to the pattern of pT1 B6-806, used by Chilton et al.
(1978a). The only difference is that the PAL 104 and pAL 657 doublet 14 is resolved into two bands. Fragment 14a is slightly larger
GENETIC
MAP OF AN OCTOPINE
TI-PLASMID
351
(k 2.55 x 106)than the corresponding fragments from pTi-B6-806, whereas fragment 14b has the same molecular weight (2.5 x 106). As can be seen from Fig. 1, there is also some resolution of the Sma 16doublet: In slots 1, 2, and 6 both 16a and 16b are present, whereas in slots 3, 4, and 5 band 16b is missing from the doublet. The following is based on the assumption that the fragmentation maps of PAL 104, PAL 657, and pTi-B6-806 are the same. Virulent Deletion Mutants Deleted plasmids that still confer virulence upon the bacterium are derived from PAL 657 as well as from PAL 104. Their Sma digests are shown in Fig. 1. The positions of the deletions are summarized in Fig. 4; for molecular weights see Table 1. The molecular weights of the Sma fragments are listed in Table 2. PAL 204 (from LBA 4029). Visible are bands 1,2,5*, 3,4, a new band of molecular TABLE
2
MOLECULAR WEIGHTS OF SMA I DIGEST FRAGMENTS OF pTi-B6-806 PLASMID DNA” Sma I fragment number 1 2 3 4 5 6 7 8 9 10 11 12 13 14‘4 148 15 16 17 18 19 20 21 a Chilton et al. (1978 T(Y).
MW x 10-e
Multiplicity
14.9 14.0 9.2 7.5 6.6 6.05 6.0 5.7 5.5 4.0 3.78 3.22 2.65 2.55 2.5 2.28 1.58 1.23 1.17 0.88 0.67 0.64
1 1 2 1 1 1 1 1 1 3 1 1 1 1 1 1 2 1 1 1 1 1
14 \ 15 \
FIG. 2. From left to right: Hpa I fingerprints 657, pAL 106, and PAL 105.
of PAL
weight 7 x 106, 6, 7, 8, 10, 12, 14a, 14b, 15, 16a, 17, and 18. From the presence of fragments 7 and 12 and the absence of frag-
352
KOEKMANETAL.
FIG. 3. Sma I fingerprints of plasmids from avirulent deletion mutants, From left to right: PALL 657 PAL 202, pAL 206, PAL 209, PAL 211, and PAL 212.
ments 9 and 11, we conclude that 9 and 11 Fusion of 9 and 21 is unlikely becausle the have fused to form the new 7 million. band sum of their molecular weights 1is less than and that the region between them is deleted. 7 x 106.
GENETIC MAP OF AN OCTOPINE TI-PLASMID
353
FIG. 4. Schematic representation of deleted plasmids from virulent strains. Conserved Sma I restriction fragments are shown. The molecular weight increase of fragment 5 due to Tnl insertion has been
ignored. Saw tooth lines have arbitrarily been drawn in the middle of the fragments in which the deletions are suspected to end. (*) The dotted lines give a more accurate indication of the end-points of PAL 105 and 106. Full details are given in the text.
PAL 105 Cfrom LBA 4018). Visible are bands 1,2,3,5,8, 10, 14b, 15, 16a, and, less clear, 17 and 18. From this, it can be concluded that, at the right side of the T-region, at least the DNA between fragments 4 and 7 is deleted. Because fragments 3b and 1Ocare part of multiple bands, their presence or absence cannot be established. From a digest with Hpa I (Fig. 2) fragment 13 is missing, whereas 14 is still present. Hpa 13 overlaps the termini of Sma 1Ocand 3b (Chilton et al., 1978a). If the deletion ended in the overlap of Sma 1Oc and Hpa 13, a new band with a molecular weight of at least 2.83 x lo6 (the remaining part of 1Oc)would be expected to appear in the Sma digest of PAL 105. Because no such fusion product is visible, it must be smaller than 106. Therefore, we conclude that the deletion ends in the overlap of Hpa 13 with Sma 3b (Fig. 6) and that Sma 3b has fused to Sma 4. PAL 106 Cfrom LBA 4019). From the Hpa
digest (Fig. 2) of this plasmid, both fragments 13 and 14 are absent, whereas 3 is still present. Therefore, the deletion ends in Hpa 14 (Fig. 6) and removes the T region. The Sma digest differs from that of PAL 105 in that band 4 is still present, and that a new band of 3.3 x lo6 (between 10 and 14b) appears. This band arises by fusion of Sma 1Oc and Sma 6. Avirulent Deletion Mutants Figure 3 shows the Sma digests of the TIplasmids from the avirulent class of deletion mutants, derived from PAL 657. Fragment 5* is missing from all of these plasmids, whereas fragment 14b is still present. Because of its small size, fragment 20, located between 5* and lib, can be visualized only when the gel is overloaded (not shown). Because all strains from the avirulent class re-
354
KOEKMAN
ET AL.
FIG. 5. Schematic representation ofdeleted plasmids from avirulent strains. For further details see the legend to Fig. 3.
tain the carbenicillin resistance conferred by the insertion of Tnl into fragment 5, we conclude that the deletions start in fragment 5* and not in fragment 20. Indeed, in a number of cases (PAL 202,209,211) fragment 20 is too small to account for the molecular weights of the new bands that arise from the fusion of the two bands in which the deletion ends. We shall discuss the deletions in order of their size (see Fig. 5 for the location of the deletions and Table 1 for their molecular weights). PAL 211 (jiiom LBA 4043). Visible are bands 2, 4, 6, a new band of molecular weight 4.3 x 106, 10, 14a, 14b, and 15. By overloading the gel, also band 18 could be detected (not shown). Note that bands 6 and 14a are present, whereas number 12, which according to Chilton er a/.( 1978a)is situated in between, is absent. We infer that the correct order of these fragments must be . ..9. 12, 14a, 6.... The new band is a combination of the remnants of bands 5* and 12. PAL 202 Cfrom LBA 4023). Bands 2,4,6, a
new band almost the same size as band 9 (5.5 x 106), 10, 14b, 15, 18 (compare PAL 211), and 20 (see above) are present. The new band 9* arises from fusion of bands 5* and 14a. PAL 206 and 212 (from LBA 4035 and
4052). Bands 2, anovel band, 10,14b, 15,18, and 20 are left. The new band has a molecular weight of about 5.7 x lo6 in the case of PAL 206; for PAL 212 its weight is ca. 6.5 x 10”. Both are fusion products of fragments 4 and 5*. pAL 209 Cfrom LBA 4040). This has the largest deletion of all mutants studied. It has a new band l*, larger than 1, band 10, 14b, 15, and 20. Fragment 1* has a molecular weight of at least 15 x lo6 and is a fusion product of bands 2 and 5*. DISCUSSION
When the positions of the deletions (Figs. 4 and 5) on the described plasmids are related to the phenotypic properties of the
GENETIC MAP OF AN OCTOPINE TI-PLASMID
355
FIG. 6. Location of genetic markers on the physical map of the octopine TI-plasmid. For AP-1 exclusion see Discussion. The PAL numbers on the outside represent the end-points of deletions.
strains which contain them (Table l), the following conclusions, summarized in Fig. 6, can be drawn: (1) The strains containing PAL 202, 206, 209, 211, and 212 have lost their virulence. All have deletions that remove the T region. Strains LBA 4018 and 4019 (carrying PAL 105 and 106, respectively) have a much attenuated virulence (Klapwijk et al., 1978) and their tumours do not contain octopine. Both strains lack a large area-about 40% of the TI-plasmid-on the right side of the TIplasmid, including part of the T region (PAL 105) or the entire T region (PAL 106). Although the deleted DNA-especially the T region-probably contains functions that are involved in tumorigenesis it is not essential for virulence. Essential genes must be situated between the Tnl insertion in Sma fragment 5 and the T region, because this area is deleted in the avirulent mutants. Chilton et al., (1978b) have defined the T region on the plasmid pTi-B6-806. Since the deleted plasmids PAL 105 and 106 are derived from the Ach5 TI-plasmid, a minor
difference between the AchS- and B6-806 plasmids in this region could explain our result that the T region is not essential for virulence. However, a more likely explanation can be found in the uncertainty that still exists about the left boundary of the T-DNA (Chilton et al., 1978b). Because PAL 105 misses part of the T region, the latter is not transferred to the plant as a discrete “transposon-like” structure, as suggested by Schell and Van Montagu (1978), because both ends of a transposon are needed for transposition (Kleckner, 1977). (2) All avirulent mutants have become sensitive to AP-1. Among these mutants PAL 211 has the smallest deletion (Fig. 5), so the genes for exclusion map in the upper part of the TI-plasmid, between Sma 5 and 12. LBA 4018 and 4019 are less sensitive to AP-1 than the avirulent mutants: The number of plaques formed is about four times lower and these plaques are very tiny. Because the end-points of the deletions in pAL 105 and 106 are at least lo6 apart, it
356
KOEKMAN
is unlikely that the exclusion genes are partially deleted in these plasmids. These genes probably map in separate areas of the TIplasmid: between Sma 12 and 3b (deleted in both PAL 105and pAL 2 11)) and between Sma 5 and 1Oc(deleted in PAL 211, but not in PAL 106). (3) LBA 4029 (carrying PAL 204) is affected only in the utilization of octopine and in the transfer of its TI-plasmid, which functions are closely related (Genetello et al., 1977; Kerr et al., 1977; Klapwijk et al., 1978). Consequently, genes necessary for octopine degradation and plasmid transfer map between Sma fragments 9 and 11 and are physically separate from the genes coding for octopine synthesis in the tumour (Klapwijket al., 1977;Montoyaet al., 1977), which map probably within the T region. Since LBA 4029 is still virulent, a conjugative mechanism is not involved in the process of tumour induction, as has been suggested by TempC et al. (1977). (4) The deletion mutants derived from LBA 4014 were selected for on two markers: carbenicillin resistance and Uad- character. Out of nine nonreverting mutants isolated, five have deletions that originate in the Sma band containing Tn 1 (LBA 4023,4035,4040, 4043, and 4052, carrying PAL 202,206,209, 211, and 212, respectively). The deletions extend in clockwise direction from Sma band 5” and hit the virulence and uud genes. Their suspected common origin suggeststhat Tnl is involved in their generation. In LBA 4029 (carrying pAL 204)-one of the other four mutants derived from LBA 4014-only uud genes are deleted. Since this deletion does not originate in Sma fragment 5*, it is probably spontaneous, as is the case for the mutants derived from LBA 4017. (5) The largest deletion isolated is that of LBA 4040, carrying pAL 209. Therefore, the replicator must be located on the piece of DNA which constitutes PAL 209. Thus, with LBA 4040 we have obtained a smallsized (25 x 106) Agrobucterium-specific drug-resistance plasmid, that may provide a suitable vector on which, e.g., the virulence and octopine genes can be cloned. Possibly
ET AL.
the new Sma fragment I*, that consists of fused Sma bands 2 and 5*, contains the replicator in addition to the carbenicillin marker, and can, after ligation, serve this purpose. ACKNOWLEDGMENTS The authors are indebted to Dr. M. -D. Chilton (Seattle, Wash.) for providing them with a physical map of the TI-plasmid before publication. The skilful technical assistance of Miss J. Poulis is gratefully acknowledged. Phage AP-1 was a generous gift from Prof. Dr. J. Schell (Ghent, Belgium). This work was supported in part by the Netherlands Foundation of Biological Research (BION) with financial aid from the Netherlands Organization for Advancement of Pure Scientific Research (ZWO).
REFERENCES BICKLE, T. A., PIROTTA, V., AND IMBER, J. (1977). A simple, general procedure for purifying restriction endonucleases. Nucl. AC. Rcs. 4, 2561-2572. BOMHOFF, G. H. (1974). Studies on crown gall-a plant tumour. Investigations on protein composition and on the use of guanidine compounds as a marker for transformed cells. Thesis, University of Leiden, The Netherlands. BOMHOFF,~. H., KLAPWIJK,~. M., KESTER,H. C. M., SCHILPEROORT, R. A., HERNALSTEENS, J. P., AND SCHELL, J. (1976). Octopine and nopaline: Synthesis and breakdown genetically controlled by a plasmid of Agrobacterium tumefaciens. Mol. Gen. Genet. 145, 177- 181. CHILTON, M. -D., DRUMMOND, M. H., MERLO, D. J., SCIAKY, D., MONTOYA, A. L., GORDON, M. P., AND NESTER, E. W. (1977). Stable incorporation of plasmid DNA into higher plant cells: The molecular basis of crown gall tumorigenesis. Cell 11, 263-271. CHILTON, M. -D., MONTOYA, A. L., MERLO, D. J., DRUMMOND, M. H., NUTTER, R., GORDON, M. P., AND NESTER, E. W. (1978a). Restriction endonuclease mapping of a plasmid that confers oncogenicity upon Agrobacterium tumefaciens strain B6-806. Plasmid 1, 254-269. CHILTON, M. -D., DRUMMOND, M. H., MERLO, D. J., AND SCIAKY, D. (1978b). Highly conserved DNA of Ti plasmids overlaps T-DNA maintained in plant turnours. Nature (London) 275, 147- 149. CURRIER, T. C., AND NESTER, E. W. (1976). Isolation of covalently closed circular DNA of high molecular weight from bacteria. Anal. Biochem. 66, 43 1-441. DRUMMOND, M. H., GORDON, M. P., NESTER, E. W., AND CHILTON, M. -D. (1977). Foreign DNA of bacterial plasmid origin is transcribed in crown gall tumours. Nature (London) 269, 535-536. GENETELLO, C., VAN LAREBEKE, N., HOLSTERS, M., DE PICKER, A., VAN MONTAGU, M., AND SCHELL, J.
GENETIC
MAP OF AN OCTOPINE
(1977). The TI plasmids of Agrobacterium as sex factors. Nature (London) 265, 561-563. GOLDMANN, A., TEMPI?, J., AND MOREL, G. (1968). Quelques particularitts de diverses souches d’Agrobacterium tumefaciens. C. R. Sot. Biol. 162, 630631. GOLDMANN, A., THOMAS, D. W., AND MOREL, G. (1969). Sur la structure de la nopaline, mCtabolite anormal de certaines tumeurs de Crown-gall. C. R. Acad. Sci. (Paris) 268, 852-854. HEFFRON, F., SUBLETT, R., HEDGES, R. W., JACOB, A., AND FALKOW, S. (1975). Origin of the TEM beta-lactamase gene found on plasmids. J. Bacterial. 122, 250-256. HOOYKAAS, P. J. J., KLAPWIJK, P. M., NUTI, M. P., SCHILPEROORT,R. A., AND R~~RSCH,A. (1977). Transfer of the Agrobacterium tumefaciens TI plasmid to avirulent Agrobacteria and to Rhizobium ex planta. J. Gen. Microbial. 98, 477-484. KEMP, J. D. (1977). A new amino acid derivative present in crown gall tumour tissue. Biochem. Biophys. Res. Commun. 74, 862-868. KERR, A., MANIGAULT, P., AND TEMPT, J. (1977). Transfer of virulence in viva and in vitro in Agrobacterium. Nature (London) 265, 560-561. KLAPWIJK, P. M., HOOYKAAS, P. J. J., KESTER, H. C. M., SCHILPEROORT, R. A., AND R~RSCH, A. (1976). Isolation and characterization of Agrobacterium tumefaciens mutants affected in the utilization of octopine, octopinic acid and nopaline. J. Gen. Microbial. 96, 155- 163. KLAPWIJK, P. M., OUDSHOORN, M., AND SCHILPEROORT, R. A. (1977). Inducible permease involved in the uptake of octopine, lysopine, and octopinic acid by Agrobacterium tumefaciens strains carrying virulence-associated plasmids. J. Gen. Microbial. 102, l-11. KLAPWIJK, P. M., SCHEULDERMAN, T., AND SCHILPEROORT, R. A. (1978). Co-ordinated regulation of octopine degradation and conjugative transfer of TIplasmids in Agrobacterium tumefaciens: evidence for a common regulatory gene and separate operons. J. Bacterial., 136, 775-785. KLECKNER, N. (1977). Translocatable elements in procaryotes. Cell 11, 1 l-23. LEDEBOER, A. M., KROL, A. J. M., DONS, J. J. M., SPIER, F., SCHILPEROORT,
R. A., ZAENEN,
I., VAN
LAREBEKE, N., AND SCHELL, J. (1976). On the isolation of TI-plasmid from Agrobacterium tumefaciens. Nucl. AC. Res. 3, 449-463. LEDEBOER, A. M. (1978). Large plasmids in Rhizobiaceae. I. Studies on the transcription of the tumour inducing plasmid from Agrobacterium tumefaciens in sterile crown gall tumour cells. II. Studies on large plasmids in different Rhizobium species. Thesis, University of Leiden, The Netherlands. MENAGE, A., AND MOREL, G. (1964). Sur la prtsence d’octopine dans les tissus de Crown-gall. C. R. Acad. Sri. (Paris) 259, 4795-4796. MONTOYA, A. L., CHILTON, M.-D., GORDON, M. P.,
TI-PLASMID
351
SCIAKY, D., AND NESTER, E. W. (1977). Octopine and nopaline metabolism in Agrobacterium tumefaciens and crown gall tumour cells: Role of plasmid genes. J. Bacterial. 129, lOl- 107. MULDER, C., AND DELIUS, H. (1972). Specificity of the break produced by restricting endonuclease R, in Simian Virus 40 DNA, as revealed by partial denaturation mapping. Proc. Natl. Acad. Sci. USA 69, 3215-3219. OTTEN, L. A. B. M., VREUGDENHIL, D., AND SCHILPEROORT, R. A. (1977). Properties of D(+)lysopine dehydrogenase from crown gall tumour tissue. Biochim. Biophys. Acta 485, 268-277. PETIT, A., DELHAYE, S., TEMPT, J., AND MOREL, G. (1970). Recherches sur les guanidines des tissus de Crown-gall. Mise en Cvidence d’une relation biochimique spCcifique entre les souches d’Agrobacterium tumefaciens et les tumeurs qu’elles induisent. Physiol. Vkgetale 8, 205-213. PETIT, A., AND TEMPT, J. (1976). Etude du metabolisme de I’homooctopine et de l’un de ses analogues par I’Agrobacterium tumefaciens. C. R. Acad. Sci. (Paris) 282, 69-71. SCHELL, J., AND VAN MONTAGU, M. (1978). Transfer, maintenance and expression of bacterial TI plasmid DNA in plant cells transformed withA. tumefaciens. In “Brookhaven Symposia in Biology” (C. W. Anderson, ed.), Vol 29: Genetic Interactions and Gene Transfer, pp. 36-49. Brookhaven National Laboratory, Upton, New York. SCHILPEROORT, R. A., AND BOMHOFF, G. H. (1975). Crown gall: A model for tumour research and genetic engineering. In: “Genetic Manipulations with Plant Material” (L. Ledoux, ed.), pp. 163-181. Plenum Press, New York. TEMP& J., PETIT. A., HOLSTERS, M., VAN MONTAGU, M., AND SCHELL, 3. (1977). Thermosensitive step associated with transfer of the TI plasmid during conjugation: Possible relation to transformation in crown gall. Proc. Natl. Acad. Sci. USA 74, 28482849. VAN LAREBEKE,N., ENGLER, G., HOLSTERS, M., VAN DEN ELSACKER, S., ZAENEN, I., SCHILPEROORT, R. A., AND SCHELL, J. (1974). Large plasmid in Agrobacterium tumefaciens essential for crown gall inducing activity. Nature (London) 252, 169- 170. VAN LAREBEKE, N., GENETELLO, C., SCHELL, J., SCHILPEROORT, R. A., HERMANS, A. K., HERNALSTEENS, J. P., AND VAN MONTAGU, M. (1975). Acquisition of tumour-inducing ability by non-oncogenie Agrobacteria as a result of plasmid transfer. Nature (London) 255, 742-743. WATSON, B., CURRIER, T. C., GORDON, M. P., CHILTON, M.-D., AND NESTER, E. W. (1975). Plasmid required for virulence of Agrobacterium tumefaciens. J. Bacterial. 123, 255-264. ZAENEN, I., VAN LAREBEKE, N., TELJCHY, H., VAN MONTAGU, M., AND SCHELL, J..(1974). Supercoiled circular DNA in crown gall inducing Agrobacterium strains. J. Mol. Bio/. 86, 109-127.