A functional map of the replicator region of the octopine Ti plasmid

PLASMID

7,

119-132 (1982)

A Functional

Map of the Replicator

B. P. KOEKMAN,~.

Region of the Octopine

Ti Plasmid

J. J. HOOYKAAS,' AND R. A. SCHILPER~~RT

Deportment of Biochemistry, University of Leiden, Wassenaarseweg 64. 2333 AL Leidea The Netherlands Received May 27, 1981; revised September 3, 1981 Using in v&o-generated deletion mutants of the c&opine plasmid pTiB6, we locahzed a region specifying copy control and plasmid stability (stb) on the right side of the replication origin (ori). A region that prevents the establishment of nopaline Ti plasmids in strains carrying an o&opine Ti plasmid was found to be situated to the left of the replication origin. This function appeared to be unrelated to plasmid incompatibility and has been called ein (for establishment inhibition). Strains harboring an octopine Ti plasmid from which the ein region had been deleted were gocd recipients for nopaline Ti plasmids. The ein- o&opine Ti plasmids were themselves susceptible to establishment inhibition not only by nopaline Ti plasmids, but also by octopine Ti plasmids. Thus, octopine Ti plasmid derivatives that arc eirr- failed to establish-after introduction by transformation-in recipients carrying an octopine Ti plasrnid. Rare transformants that did arise contained cointegrates between the residing octopine plasmid and the incoming ein- plasmid. Integration had taken place at, or close to the replication origin and did not affect virulence of the residing plasmid. After cointegration of an octopine Ti plasmid with an incoming ein- stb- plasmid, the resultant cointegrate showed the selective “amplification” of the internal ein- stb- plasmid during growth of the host in the presence of carbenicillin.

Agrobacterium tumefaciens induces tumors at wound sites on dicotyledonous plants. A large plasmid, called the Ti plasmid, harbored by the bacterium, is responsible for virulence (Zaenen et al., 1974; Van Larebeke et al., 1974, 1975; Watson et al., 1975). Upon tumor induction, part of the plasmid, called the T region, becomes integrated into the plant genome (e.g., Chilton et al., 1977; Lemmers et al., 1980; Thomashow et al,. 1980a), where it is transcribed into poly(A)containing messenger RNAs (Drummond et al., 1977; Ledeboer, 1978; Gurley et al., 1979). Ti plasmids code for the synthesis of amino acid derivatives such as octopine and nopaline in the tumor, and for the breakdown of these tumor-specific compounds by the bacterium (Bomhoff et al., 1976; Hooykaas et al., 1977; Montoya et al., 1977). Aside from the limited host range octopine Ti plasmids that have been discovered recently (Thomashow et al., 1980b), the oc’ To whom correspondence and requests for reprints should be sent.

topine Ti plasmids are very similar. They differ from the various types of nopaline Ti plasmids (Currier and Nester, 1976a; Sciaky et al., 1978). Octopine and nopaline Ti plasmids share in their T region a small region of strong DNA homology-referred to as the common sequence-that is essential for oncogenicity on a variety of hosts (Chilton et al., 1978a; Depicker et al.. 1978; Ooms et al., 198 I). They also share extensive regions of weaker DNA homology (Drummond and Chilton, 1978; Hepburn and Hindley, 1979) including the replicator region (Koekman et al., 1979, 1980). Octopine and nopaline Ti plasmids belong to the same incompatibility group, incRh-1 (Hooykaas et al., 1980b). Even in the presence of a selective regimen, nopaline Ti plasmids fail to establish as independent replicons in recipients carrying an octopine Ti plasmid and vice versa. However, nopaline Ti plasmids may be rescued by cointegrate formation with the resident o&opine Ti plasmid (Hooykaas et al., 198Ob). The recombination event leading to cointegrate for119

0147-619X/82/0201 Copytigbt 0 1982 by AAI1 rights of reproduction

19-14$02.00/O Pms, Inc. in any form resmfcd.

120

KOEKMAN,

HOOYKAAS,

AND SCHILPEROORT

mation has been found to occur in the common sequence (Hooykaas et al., 1980b). In this paper we will show that the function which is responsible for establishment inhibition-called ein2-of nopaline Ti plasmids in octopine Ti plasmid carrying cells, is itself unrelated to incompatibility. In a previous paper (Koekman et al., 1980) we described the construction of deletion mutants of octopine plasmid pTiB6 that had lost establishment inhibition toward nopaline Ti plasmids and that were expelled by the incompatibility exerted by an incoming nopaline Ti plasmid. Here we demonstrate, that, in addition, these octopine Ti

plasmid derivatives are unable to establishafter having been introduced by transformation-in strains carrying a wild-type octopine Ti plasmid. In other words, they are susceptible to the ein function exerted by a residing octopine Ti plasmid. These einplasmids may form stable cointegrates with wild-type octopine Ti plasmids by recombination at, or close to, the replication origin. Further reduction of the size of the smallest octopine Ti plasmid derivative obtained thus far-the 4.2-Mdal plasmid pAL2821-has allowed the localization of a region specifying plasmid maintenance and copy number control. (This paper forms part of a thesis submitted by B.P.K. at the State University of Leiden).

’ Abbreviations used: crb, carbenicillin; ein, establishment inhibition; EtBr, ethidium bromide; inc, incompatibility; lean, kanamycin; occ, o&opine catabolism; ori, origin of replication; rif, rifampicin; spc, spectinomycin; stb, plasmid stability and copy control; str, streptomycin; Tn, transposon.

MATERIALS

AND METHODS

Bacteria. All strains used in this work have the chromosomal background of the Ti

TABLE

1

BACTERIA Relevant Strain LBA No. 288 661 677 1010 1509 1525 I546 2240 2803 2808 2809 2811 2818 2819 2821 2828 2829 2832 2838 2839 2862 2885

Chromcsomal markem rif, nal rif, nal id. id. id. id. gcn, no” rif, nal rif, nal id. rif, ml id. rif, nal id. id. id. id. id.

Plasmid

derivation

stb”

pTiB6 pTi4d pTiB6 pTiB6::Tn1831 id. id. pTiCSS::R702(pAL2229) pAL209, deleted pTiB6::pAL2803 pAL1509::pAL2803 pAL2803, deleted pTiB6::pALZBl I pAL1509::pAL2811 pAL2811, deleted pTiBb::pAL2821 pAL1509::pAL2821 pAL2821, delekd pTW::pAL2832 pAL1509::pAL2832 pAL2821::TnS, deleted pTiB6::Tnl. deleted (pAL209)

‘Symbols denote resirtanced to: rif, cifampicin; ’ stb. plasmid stability. oVirulence on tomato. ‘An o&pine Ti plasmid. ‘Not determined. ‘Due to d encoded by R702.

nal. nalidixic

occ

vif

plasmid

properties

crb

spc

kan

+

+

_ +

_ -

_ -

_ -

+ + + + + +

+ + + + + -

+ + + + -

-

-

-

+ + + + + + + + + -

+ + + + + + -

+ + + + + + -

+ +

+ +

+

-

+ + -

+ + + + + + + + + + + + + +

+ + + +’ -

acid; gen, gcntamycin;

+ + + + -

nav. novobiocin.

+ + -

M. (x10-6)

121 n.d.’ 121 132 132 132 178 13.2 135 146 6.6 128 139 4.2 126 137 2.5 124 135 4.7 25

Source J. Schell .I. Schcll Hooykaas This work Hooykaas id. id. Hooykaas Kockman This work id. Koekman This work id. Koekman This work id. id. id. id. Koekman Koekman

et 01. (1980b) et 01. (1980a)

( 1979) cf 01. (1980)

et a/. ( 1980)

ef rrl. ( 1980)

et 01. (unpublished) et al. (1980)

FUNCTIONAL

MAP OF OCTOPINE

plasmid cured nopaline strain C58-C9; further characteristics are listed in Table 1. Plasmids are referred to by the number of their host, prefixed by PAL, except for pTi4, pTiB6, pAL209, and pAL2229, which are carried by LBA677, LBA66 1/LBA 1010, LBA2885, and LBA2240, respectively. Bacteria were cultured at 29°C with shaking at 250 rpm. Bacteria carrying unstable Ti plasmid derivatives were cultured in the presence of 100 pg/ml carbenicillin. Media and plasmid isolation. These are described by Koekman et al. ( 1980). Enzyme reactions. Restriction enzymes HaeII and Hind111 were purchased from Miles Laboratories, Inc., BamHI from New England Biolabs, and T4 ligase from Bethesda Research Laboratories. HpaI was isolated according to Bickle et al. ( 1977). SmaI and XhoI were gifts from Dr. P. H. Pouwels (Medical Biological Laboratory TNO, Rijswijk, The Netherlands) and Dr. C. A. van Sluis from our laboratory, respectively. Digestion conditions for SmaI were as described by Koekman et al. (1979). Restriction with HaeII was carried out in 6 mM MgC12, 6 mrvr Tris-HCI, pH 7.4, 6 mM B-mercaptoethanol for 2 h at 37°C; HindIII, BamHI, and HpaI were incubated in the same buffer supplemented with 50 mM NaCl, and XhoI in the same buffer with 150 mM NaCI. Ligation of DNA fragments was carried out in 6.6 mM MgC&, 6 mM TrisHCl, pH 7.6, 10 mM dithiothreitol, and 0.4 mrvt ATP for 16 hr at 13°C followed by successive extractions with phenol and chloroform, and overnight precipitation with ethanol at -20°C. For the construction of plasmid pAL2832, we digested 1 pg of pAL2821 DNA with Ha&, and ligated the fragments in a final volume of 25 ~1, corresponding to a termini concentration of 80 nM. Gel electrophoresis. This was performed either in acetate buffer containing 0.7% agarose (Koekman et al., 1979) or in borate buffer with 0.8% agarose (Meyers et al., 1976). The former conditions were also used for molecular weight measurements. Virulence assays. Virulence was tested on

Ti PLASMID

REPLICATOR

REGION

121

two week-old tomato seedlings (Lycopersicum esculentum, var. Rutgers) by puncturing the hypocotyl with a sterile wooden toothpick dipped in a colony of the strain to be tested. Bacteria were transTransformation. formed with plasmid DNA as described by Koekman et al. (1980). In experiments involving many different recipient strains and/ or donor plasmids, the amounts of plasmid DNA solution (20-200 pg/ml) and concentrated bacteria (5 X 10” cells/ml) used were reduced to 4 and 20 ~1, respectively. After freezing and thawing, this mixture was supplemented with 80 ~1 YEB medium and plated in its totality. DNA concentrations of different plasmid preparations were adjusted so as to give comparable numbers of transformants. Conjugation. Conjugation conditions were as described by Hooykaas et al. (1980a). Stability tests. Plasmids were tested for stability by inoculating a colony from a selective plate into I ml of TY medium and shaking overnight. The 10e6 dilution was plated on TY medium without selection, and lo-50 colonies were tested on selective plates. Plasmids were scored as unstable when less than 20% of the colonies tested would grow. Alternatively, 1 ~1 of overnight culture was spotted on a selective plate and stability was judged by inspection after 2 days incubation. Total DNA isolation. Bacteria from 25 ml of culture were lysed with SDS and Pronase as described by Currier and Nester (1976b). The lysate was sheared by forcing it twice through a hypodermic needle, and extracted four times with phenol, chloroform, and ether. The DNA was dialyzed overnight vs 0.2 M NaOAc, pH 5, ethanol precipitated, and dissolved in sterile water. Radiolabeling and hybridization conditions. These were as described by Koekman et al. ( 1980). RESULTS

Mapping of Xhol and HaelI pAL2821

Sites on

Plasmid pAL2821 is the smallest derivative of the octopine plasmid pTiB6 obtained

122

KOEKMAN,

HOOYKAAS,

AND

SCHILPEROORT

FIG. 1. Physical map of pAL2821. The calibration on the inside is in megadaltons. The encircled numbers on the outside refer to Hoe11 fragments. The dashed circles divide the map into regions originating from different Hind111 (inner circle) and ZfpaI (middle circle) fragments of the octopine Ti plasmid. Except for the minute piece of DNA overlapping ZfpeI fragment 4, Hind111 fragment l* corresponds to base pairs 3574-4957 of Tnl. The arrow on the solid line defining crbR points at the inverted repeat. The solid sector between 0.95 and 2.65 Mdal is removed upon the formation of pAL2832.

thus far (Koekman et al., 1980). It encodes no known functions except for carbenicillin resistance ( CrbR) and carries the replication origin of the octopine Ti plasmid. In order to confine the position of this origin of replication further, elimination of nonessential DNA from this plasmid is necessary. For this reason, we had to know the cleavage sites of a number of restriction enzymes on this plasmid. A map of plasmid pAL2821, based on the map of its ancestor, pAL2803, that was published earlier (Koekman et al., 1980) is shown in Fig. 1. The molecular weight of pAL2821 has been slightly corrected (4.2 vs 4.4 Mdal in the previous paper, due to the reassessment of the molecular weight of the largest BumHI fragment from 1.95 to 1.75 Mdal). An unique XhoI site was mapped on pAL2821, 1 Mdal distant from the BamHI site at 3.25 Mdal. Upon diges-

tion with HueII, pAL282I was found to produce four fragments with molecular weights I .9, 0.9, 0.8, and 0.6 Mdal. The first fragment appeared to carry sites for the enzymes BamHI and HpaI, the second one for BumHI and Hi&II, and the third one for XhoI. From these data, the HaeII fragment order 1, 3,2,4 could be deduced. Considering that fragment 1 is cleaved by HpaI into frag ments of 1.7 and 0.2 Mdal, the HaeII map could be aligned with the known restriction map of pAL2821. Reduction

of the Size of PAL.2821

HueII-digested pAL2821 DNA was ligated and transformed into the plasmidless recipient strain LBA288. Three carbenicillin-resistant transformants were obtainednumbered LBA283 1 through LBA283 3and all contained plasmids with only HaeII

FUNCTIONAL

MAP OF OCTOPINE

1.90 1.75 1.55 1.50

0.95 0.90 0.80 0.60 I, x1( 1

2

3

L

FIG. 2. Restriction patterns of PAL2821 and pAL2832. (I) pAL2821 XHueII; (2) pAL2832 X HaeII; (3) pAL2832 X BumHI; (4) pAL2821 X BumHI.

fragments 1 and 4 (Fig. 2), which are contiguous in pAL2821 (Fig. 1). Because these fragments are cleaved eccentrically by EcoRI and HpaI, respectively (Fig. l), their relative orientation could be determined by double digestion with these enzymes. The orientation of the fragments appeared to be the same for pAL2831, pAL2832, and pAL2833 as for pAL2821. Plasmid pAL2832 was chosen for further experiments. Properties

Ti PLASMID

REPLICATOR

REGION

123

carbenicillin, the cells on an average carry at least 20 copies of the plasmid. Due to inevitable plasmid losses during isolation, this will be a minimum estimate. For the larger pTiB6 derivatives, differences in plasmid yield after growth of the host in the presence or absence of carbenicillin have never been observed. Growth of LBA2832 in the presence of 100 pg/ml carbenicillin is greatly retarded in comparison with growth in the absence of the antibiotic (Table 2). The viable count of such culture when plated on selective medium is at least 10 times less than is the viable count on nonselective medium. This suggests the possibility that maintenance of pAL2832 is often lethal to the host cell. Also the amount of plasmid DNA required to produce a given number of transformants is markedly (about five times) higher for pAL2832 DNA than it is for, e.g., pAL2821 DNA, and transformant colonies take about 3 instead of 2 days to appear. Recombination between the Octopine Ti Plasmid and Its Derivatives The small octopine Ti plasmid derivatives lack the establishment inhibition (ein) function toward nopaline plasmids, but they are still susceptible to incompatibility exerted by incRh-I plasmids (Koekman et al., 1980).

of pAL2832

In contrast to pAL2821 and the larger derivatives of pTiB6 (Koekman et al., 1980), which are all stable replicons, plasmid pAL2832 was found to be lost from its host in the absence of selection pressure. Moreover, the copy number of this plasmid depended on the antibiotic concentration ap plied in culture (Fig. 3). Although we have not carried out exact copy number calculations, it can be estimated from the amount of plasmid DNA isolated that, after growth of the host in the presence of 100 pg/ml

FIG. 3. Plasmid yields of LBA2832 grown with 0, 10, 30, and 100 fig/ml carbenicillin. CsCl/EtBr equilibrium density gradient centrifugation. Upper band: chromosomal DNA; lower band: plasmid DNA.

124

KOEKMAN, TABLE

HOOYKAAS,

AND SCHILPEROORT

2

of pTiB6 that are smaller than pAL209 transform LBAlOlO at a much lower frequency than they do transform LBA288. Generally, a decrease to about 2% of the frequency obtained with LBA288 was observed, although, depending on the plasmid DNA preparation, this figure may vary from 0.1% up to more than 10%. The reasons for this variation are unknown. All CrbR transformants were found to have remained OCC+. As the Occ+CrbR phenotype of the transformants was stable we investigated their plasmid content. Restriction enzyme profiles of plasmids from such Occ+CrbR strains revealed that they carried cointegrates consisting of the residing o&opine Ti plasmid and the incoming Ti plasmid derivative, whereby the recombination event had invariably occurred in HpaI fragment 11 and SmaI fragment 2 (Fig. 4), which contain the origin of replication of pTiB6 (Koekman et al., 1980). Since none of the small octopine Ti plasmid derivatives is cleaved by SmaI, the molecular weight increase of SmaI fragment 2 of pTiB6 equals the molecular weight of the inserted plasmid. Our results show that in contrast to the smaller derivatives of pTiB6, plasmid pAL209 does not-or at least not stablybecome integrated into pTiB6. Only one out of 500 CrbR colonies obtained after transformation of pAL209 into an octopine strain showed an Occ+ CrbR phenotype, and the Occf CrbR character of this strain was unstable. To investigate further the inability of pAL209 to form stable cointegrates with

GENERATION TIME ON TY MEDIUM Generation time (h) Strain LBA2803 LBA28 11 LBA2821 LBA2832 LBA2838 LBA2839 LBA2885 ’ Carbenicillin

-Carbenicillin

+Carbenicillin”

2 2 2 2 2 2 2

3 2.5 2.5 9 3 3.5 2.5

at 100 pg/ml.

tween establishment inhibition and incompatiblity, we tested the ability of these octopine Ti plasmid derivatives to exert incompatibility toward incRh- 1 plasmids. Therefore, we introduced them into LBA288 and LBAlOlO by transformation. Strain LBA288 is a plasmidless recipient, LBAlOlO an isogenic strain containing the octopine plasmid pTiB6. The results of a representative transformation experiment using different donor plasmids and recipient strains are shown in Table 3 and described in the next section. Plasmid pAL209, which is an Occ- deleted derivative of pTiB6 with M, = 25 Mdal (Koekman et al., 1979), transforms LBA288 and LBAlOlO with the same frequency. In the latter case, the residing o&opine plasmid is expelled due to the incompatibility between pAL209 and pTiB6. The derivatives TABLE NUMBERS

OF crbR COLONIES

Plasmid introduced

LBA288

pAL209

316

pAL2803 pAL28 11 pAL2821 pAL2832

139 359 940 331

OBTAINED

3

AFTER TRANSFORMATION

LBAlOlO

Phenotype of transformants

331

occ-

5

occ+ occ+ occ+ occ+

5

1 5

Note. LBA288 = Ti plasmid-free strain; LBAlOlO

OF DIFFERENT

RECIPIENT

LBAl509 1 211 151 341

516 230

= LBA288 (pTiB6); LBA1509

STRAINS

Phenotype of transformants occocc+ occ+ occ+ occ+ occ+

= LBA288 (pAL1509).

FUNCTIONAL

MAP OF OCTOPINE

Ti PLASMID

REPLICATOR

125

REGION

IL.0 (Sma12)

- 3.6 (Hpa Ill) -2.5

-6 IO

M,x

12

3L56789

FIG. 4. Restriction patterns of various cointegrates. (1) pAL2829 X HpaI; (2) pTiB6 X HpaI; (3) pAL2838 X ZfpaI; (4) id., host grown with 100 pg/ml carbenicillin; (5) pTiB6 X SmoI; (6) pAL2838 x SmaI; (7) id., host grown with 100 pg/ml carbenicillin; (8) pAL2808 X SmaI; (9) pAL2818 X SmoI.

pTiB6, we introduced pTiB6 by conjugation into strains LBA2885 (containing pAL209) and LBA2821 (containing pAL2821). The donor strain, LBA66 1, was counterselected by rifampicin, acceptor bacteria were selected on octopine or both o&opine and carbenicillin. The results are presented in Table 4, and show that the frequency of stable cointegration of pAL209 with pTiB6 is lower than 5 X 10P4, whereas this frequency for pAL2821 is 7.5 X 10e3. The markers CrbR and Occ+ were stably maintained in the latter case. Properties of the pTiB6/pAL2832 Cointegrate, pAL2838 The unstable plasmid pAL2832 regains stability when it integrates into pTiB6; the growth rate of hosts of pAL2838 in the presence of 100 pg/ml carbenicillin is normal

again in contrast with hosts of pAL2832 (Table 2). The integrated form of pAL2832 partially retains its capacity to form more TABLE

4

FREQUENCYOFCOINTEGRATEFORMATIONOF~T~B~ WITHpAL209OR pAL2821 Number of transconjugants

Recipient LBA2885 LBA2821 LBA288

Frequency of coupling Selection for Selection for of occ+ rifR occ+ rif” occ+ crbR and crbR 2100 1600 1600

0 12 0

<5 x lo+ 7.5 x lo-) -

Note. Plasmid pTiB6 (Occ+) was introduced into rif recipients by conjugation. LBA288 is Ti plasmid free; LBA2885 and LBA2821 carry the Cb’ Ti plasmid derivatives pAL209 and pAL2821, respectively. The figures in the right columns were obtained by dividing the figures in the middle columns.

126

KOEKMAN,

HOOYKAAS,

copies of itself than does the pTiB6 moiety of the cointegrate plasmid, when its host is grown in the presence of carbenicillin. For convenience, we shall refer to this as “amplification,” but it should be noted, that it is unrelated to the amplification of plasmids under relaxed control of replication, which is observed when protein synthesis of the host is inhibited. The phenomenon is illustrated in Fig. 4. Lane 5 shows the SmaI digestion pattern of pTiB6, and lane 6 that of pAL2838. As can be seen from this figure integration of pAL2832-which has no SmaI sites-into pTiB6 has occurred in SmaI fragment 2, resulting in an increase in size of the fragment of 2.5 Mdal. From lane 7 it can be seen that this SmaI fragment of PAL2832 increases significantly in size during growth of the host in the presence of carbenicillin. To find out which part of the SmaI fragment is “amplified” during growth in the presence of carbenicillin, plasmid pAL2838 was also digested with HpaI. Lane 2 of Fig. 4 shows the HpaI digestion pattern of pTiB6, lane 3 that of pAL2838. It can be concluded from the figure that integration of pAL28.32 into pTiB6 has occurred into HpaI fragment 11, which because of the presence of a HpaI site in pAL2832 falls apart in two new fragments of M, = 4.2 and 1.9 Mdal. The sum of these figures equals the sum of the molecular weights of pAL2832 (M, = 2.5 Mdal) and HpaI fragment 11 (M, = 3.6 Mdal; Chilton et al., 1978b). Lane 4 shows the HpaI digest of pAL2838 after growth of its host in the presence of carbenicillin (100 pg/ml). It can be seen from lane 4 in comparison with lane 3 that a new strong band of M, = 2.5 Mdal appears. This demonstrates that the enlargement in size of pTiB6 SmaI fragment 2 during growth of LBA2838 in the presence of carbenicillin is due to the selective amplification of the integrated 2.5Mdal plasmid pAL2832, resulting in a tandem arrangement of multiple copies of pAL2832 within the pAL2838 cointegrate. We estimate the number of copies to be six at this drug concentration. This is substantially lower than the minimum estimate for the free plasmid

AND SCHILPEROORT

under the same conditions, as is already suggested by the different growth rates of LBA2832 and LBA2838. Integration and amplification have no effect on the host strain’s ability to induce tumors (Table 1, Fig. 6). Transformation of the Small Plasmids into LBAI509. a Mutant Octopine Ti Plasmid with Altered Incompatibility Properties Three pTiB6 derivatives that carry an insertion of Tn183 1 close to the replication origin-PAL1 509, pALl525, pAL1546 (Hooykaas 1979; Ooms et al., 198 1j-were investigated for the possible lack of an Einf phenotype toward nopaline Ti plasmids and small pTiB6 derivatives. Therefore, strains LBA1509, 1525, and 1546 were used as recipients for a nopaline Ti plasmid (R702::pTiC58) as well as for plasmids pAL2811 and pAL209. The results of these experiments are shown in Table 5. It was found that LBA1525 and LBA1546 are transformed by pAL2811 as infrequently as LBAlOlO, which has a wild-type Ti plasmid; however, PAL 1546 inhibits the establishment of a nopaline Ti plasmid less well than wild-type pTiB6 and pAL1525. Most remarkably, in contrast with LBAlOlO strain LBA1509 appeared to be a good recipient for nopaline Ti plasmids as well as for plasmids pAL209, pAL2811, and the smaller derivatives of this plasmid. All transformants and transconjugants derived from LBAl509-except for one derivative obtained after transformation with pAL209 (see below)-were found to be still Occ+, indicating the presence of plasmid PAL 1509. Most of the transformants that had received small octopine Ti plasmid derivatives and transconjugants that had received a TraC derivative of nopaline plasmid pTiT37, lost the plasmids that had entered in the absence of selection pressure, due to incompatibility exerted by plasmid pALl509. Only in a few of them cointegrates were formed between the plasmid that had entered and pALl509. In case of transformation with pAL28 11 or

FUNCTIONAL

MAP OF OCTOPINE

Ti PLASMID

TABLE

REPLICATOR

127

REGION

5

PROPERTIES OF Tnl83 1 INSERTION MUTANT~ OF THE O~TOPINE Ti PLASMID AS RECIPIENTS FOR TRANSFORMATION WITH THE OC~OPINE PLA~MID DERIVATIVES pAL209 AND pAL28 11 AND FOR CONJUGATION WITH THE NOPALINE PLASMID pTiC58

Recipient strain, LBA No. 288 1509 1525 1546 1010

Number of transformants

Restriction fragment carrying insertion Virulence on tomato

SmaI

HpaI

Hi&III

+ + -I-

2 2 2 -

11 8 8 -

15 9 9 -

with pAL209

with pAL28 11

Acceptor frequency for pTiC58::R702”

316 278 320 221 331

534 227 2 2 5

3 x 1o-3 1.7 x 10-3 n.d. 2.5 X 1O-4 5.8 X 1O-6

a A cointegrate plasmid was used to obtain higher transfer frequencies.

its smaller derivatives the number of strains with cointegrates formed was comparable to that obtained after transformation of LBAlOlO with these same plasmids. For unknown reasons, transformants containing any of the small plasmids along with, but not integrated in pAL1509, grew markedly slower than the ones that contained cointegrates (3 vs 2 days required for colony formation on YEB medium containing 100 r(g/ ml carbenicillin). As opposed to what is observed with wild-type plasmid pTiB6, plasmid pALI is rarely displaced from its host by pAL209. Furthermore, a stable establishment of pAL209 in LBAl509 was not observed, and neither was cointegrate formation of pAL1509 with pAL209. These data show that there exists nonreciprocal incompatibility between pALl509 and (derivatives of) pTiB6 to the effect that pALl509 is capable of exerting incompatibility on pTiB6, but that it itself acts as being insensitive to incompatibility exerted by pTiB6. The one Occ- colony obtained after transformation of LBA1509 with pAL209 was SpcR. The SpcR marker shows that in this Occ- transformant recombination has occurred between incoming and resident plasmid, probably to the effect that the resident Occ+ plasmid (pAL1509) was converted into a (Spc”) derivative susceptible to incompatibility exerted by pAL209::Tn 183 1.

The similarity in behavior of nopaline Ti plasmids and small o&opine Ti plasmid derivatives when introduced into o&opine Ti plasmid carrying strains suggests a common mechanism underlying their aberrant incompatibility reaction and (forced) cointegration. The observation that pAL1546 inhibits the establishment of the small plasmids to the same extent as does pTiB6, but inhibits that of nopaline Ti plasmids to a lesser extent (Table 5) indicates that the processes of establishment inhibition after transformation and after conjugation may not be entirely identical. This may be due to the fact that in the former case the donor DNA probably enters the recipient cell in double-stranded form, whereas it is single stranded in the latter case. Mapping of pTiBd::Tn1831 Cointegrates of pALI Plasmids

Mutants and and the Small

Ooms et al., (198 1) have localized the insertion positions of Tn 1831 in strains LBA1509, LBA1525, and LBA1546 in SmaI fragment 2. These studies were now extended to show that for PAL 1509 the Tn 183 1 insertion is in HpaI fragment 11, and for pAL1525 and pAL1546 in HpaI fragment 8. To map the insertion sites more accurately, we hybridized a Hind111 digest of the total DNAs from the three strains with 32P-

128

KOEKMAN,

HOOYKAAS,

AND SCHILPEROORT

weights of HpaI fragment 11 (3.6 Mdal), pAL2821 (4.2 Mdal), and Tn1831 (11 Mdal), considering that pAL2821 contains one, and Tn1831 no HpaI sites (P. J. J. Hooykaas and G. Moolenaar, unpublished results). These results show that integration of small plasmids into pAL1509-as with wild-type pTiB6-occurs into the HpaI fragment (11) containing the origin of replication. Tumorigenic Properties Derivatives

FIG. 5. Southern transfers of HindIII-digested total DNAs from Tn 183 1 insertion mutants, hybridized with “P-labeled pAL2832 DNA. The Hind111 pattern of the complete octopine Ti plasmid is indicated with stripes.

labeled pAL2832 as a probe. By this procedure sequences homologous to parts of Hind111 fragments 9 and 15 will be visualized. From Fig. 5 it can be seen that in comparison with wild-type plasmid pTiB6 for pALI there is a shift in Hind111 fragment 15, and for pAL1525 and pAL1546 in Hind111 fragment 15. Because the probe contains only parts of both Hind111 fragments and because Tn 18 3 1 is cleaved several times by Hind111 (P. J. J. Hooykaas and G. Moolenaar, unpublished results), the increments are different, depending on the insertion position and the orientation of Tn183 1. Cointegrates derived from PAL 1509 and small plasmid were analyzed by restriction enzyme analysis. As an example the HpaI digest of pAL2829, a cointegrate of pALl509 with pAL2821, is shown in Fig. 4, lane 1. In comparison with plasmid pAL1509 (not shown) two new bands appear, one at the position of pTiB6 HpaI fragment 11 (3.6 Mdal) and the other just above pTiB6 HpaI fragment 4 ( 14.4 Mdal). Their position is consistent with the expected molecular weight, which is the sum of the molecular

of LBAl509

and

Strain LBA1509 is very weakly virulent on tomato and KulunchoC daigremontiunu (Hooykaas, 1979). Derivatives of LBAl509 carrying cointegrates of pALl509 with the small plasmids, however, were restored in tumorigenicity to some extent (Fig. 6). In comparison with LBAl509, derivatives with pAL2803 (LBA2809), pAL2821 (LBA2829), and pAL2832 (LBA2839) formed much larger tumors on tomato (Fig. 6) and Kalanchoe (not shown). However, tumors were still smaller than those induced by strains with a wild-type Ti plasmid or a cointegrate of the wild-type plasmid with a small plasmid (e.g., LBA2838, Fig. 6). The reason for the weak virulence of LBA1509 is unknown. The region covering the insertion position of Tn 183 1 can be deleted from R::Ti cointegrates without virulence being affected (J. Hille, personal communication). A possible explanation for the low virulence of LBA1509 might be that plasmid pAL1509 is tightly associated with the chromosome, whereas cointegrates derived from pAL1509 like wild-type pTiB6 are less tightly associated with it. This hypothesis comes from the observation that it is not possible to isolate plasmid DNA from strain LBA1509, whereas no problems are encountered in isolating other Ti plasmids or cointegrates derived from pALl509. Incompatibility between the Small Octopine Ti Plusmid Derivatives The results obtained with LBA1509 and LBAlOlO as recipients for small o&opine Ti

FUNCTIONAL

MAP OF OCTOPINE

Ti PLASMID

REPLICATOR

REGION

129

FIG. 6. Tomato hypocctyls infected with various strains. LBAlOlO carries wild-type pTiB6; LBA1509 mutant plasmid pALl509; LBA2809, LBA2829, and LBA2839 carry derivatives of pALI with an insertion of the small plasmids pAL2803, pAL2821, and pAL2832, respectively; LBA2838 carries a derivative of nTiB6 with an insertion of small plasmid pAL2832. Note the difference in tumorigenicity of LBA1509 and LBA2809, LBA2829, LBA2839.

plasmid derivatives are inconclusive with respect to the question, whether or not these small plasmids are able to exert incompatibility on incRh-1 plasmids. Therefore, we transformed a kanamycin-resistant derivative of pAL2821, plasmid pAL2862 to strain LBA2821 with selection for kanamycin resistance. As expected no establishment inhibition was observed. Agarose gel electrophoresis of plasmid DNA from the transformed cells revealed that after introduction of pAL2862, the resident plasmid was lost (data not shown), showing that the small plasmids are capable of exerting incompatibility toward each other. This result clearly demonstrates that the incompatibility behavior is normal when the establish-

ment inhibition function is absent from both donor and recipient plasmids. DISCUSSION

We have summarized the data obtained so far on a linear map of the replicator region of the octopine Ti plasmid (Fig. 7). The construction of plasmid pAL2832 makes it most likely that the origin of replication is located in the region between 4.5 and 5.5 Mdal on this map (arbitrarily assigning zero to the left terminus of SmaI fragment 2). In a previous paper, we reported on the failure to remove from the small plasmids the region defined by the Hind111 and BamHI sites at 5.95 and 6.4 Mdal, respectively. On account of this result, we proposed this region might

130

KOEKMAN,

I

HOOYKAAS,

AND SCHILF’EROORT lSma1 Hi dIII

2

0

9 I

I

I

15

I

11

L

I

t&I pAL

~

e-

em

I” 0

or1

’

f)

stb

‘I”

crb

” 5

2803 2811 2621 2832 2862

-

-

-

no.

209

-

I”” 10

I 15

FIG. 7. Linear map of the replicator region of pTiB6, showing the DNA present in the various derivatives of this plasmid. Thick lines represent DNA derived from Tnl. Only relevant restriction sites are shown. Abbreviations used: ein, establishment inhibition; ori, origin of replication; stb, plasmid stability and copy control; inc, plasmid incompatibility; crb, carbenicillin resistance. V Transposon insertion. The inc region is drawn so as to cover the length of pAL2821, because this plasmid is the smallest one that could thus far be shown to exert incompatibility on incRh-1 plasmids. The calibration is in megadaltons.

contain the origin of replication. It is proved here, that this DNA forms part of a region required for plasmid maintenance and copy control. We have designated this region stb, for stability. On the basis of the stability of pAL2821, and the instability of pAL2832 and pAL2862, it is localized between 5.5 and 7.8 Mdal on the map. When this region is deleted, the copy number of the plasmid is no longer strictly regulated and increases in direct relation to the antibiotic concentration in the environment. In addition to the gene(s) that regulate copy number, the stb region may contain a sequence comparable to the partitioning locus that has been postulated for plasmids pSCl0 I (Meacock and Cohen, 1980), and plasmids Rl and RlOO (Nordstrom et al., 1980; Miki et al., 1980). Loss of the partitioning locus also results in plasmid instability. Plasmids with deletions spanning a region at the left of the replication origin are able to form stable cointegrates with a wild-type octopine Ti plasmid. Such deleted plasmids are unable to expel a residing o&opine Ti plasmid from

the cell; however, they are able to expel plasmids that also lack the region at the left of the origin of replication. Furthermore, they are susceptible to elimination not only by octopine Ti plasmids but also by nopaline Ti plasmids, and they do not inhibit the establishment in the cell of nopaline Ti plasmids (Hooykaas et al., 1980b; Koekman et al., 1980). For this reason, we propose to designate the region specifying these properties ein, for establishment inhibition. It has been demonstrated earlier (Hooykaas et al., 1980b) that this inhibition phenomenon is distinct from surface exclusion. Generally, neither the o&opine Ti plasmid, nor its smaller derivatives, can be transformed into recipients carrying a nopaline plasmid. The only exception observed thus far is the nopaline plasmid pTiK27, whose behavior in a recipient is very similar to that, described for the Ein- mutant plasmid pALl509 (P. J. J. Hooykaas and B. Koekman, unpublished results); also, pTiK27 does not inhibit the establishment of an R::pTiB6 o&opine cointegrate plasmid after conju-

FUNCTIONAL

MAP OF OCTOPINE

gation (P. Hooykaas and B. Koekman, unpublished results). Interestingly, pTiK27 is the only nopaline plasmid that is reported to exhibit homology with HpaI fragment 8 of the octopine Ti plasmid (Drummond and Chilton, 1978). The ein function maps in this fragment (Fig. 7). The mechanism of action of the ein function still remains obscure, but the fact that the small ein- plasmids in comparison with the parental plasmid have become susceptible to establishment inhibition by the wild-type plasmids indicates that the region(s) encoding the ein function is physically separate from the region on which the ein product acts. The product encoded by the ein region may have restriction modification-like activity, so that the region at which the ein function acts may be protected to the restriction-like activity. The specificities of the ein functions of nopaline and octopine Ti plasmids are probably different. From the results presented it is clear, that the ein function prevents the establishment of (incompatible) pairs of octopine and/or nopaline plasmids in the same host cell, even in the presence of selection pressure, whereas incompatibility is defined as the inability to coexist stably in the same host cell in the absence of continued selection pressure (Novick et al., 1976). Plasmid pALl509, which does not express the ein function toward incoming plasmids, still exerts incompatibility, demonstrating again that ein and inc functions are not directly related. It has been reported for the incP-1 plasmid RK-2 (Meyer ef al., 1977) that susceptibility of the plasmid to elimination, but not its ability to eliminate, requires that its replication system be active (Meyer, 1979). Plasmid pALl509 is not sensitive to elimination by an incompatible plasmid, and may, therefore, be inactive in replication. This supports the view that this plasmid may be replicated along with the chromosome, as does the observation that its ability to confer virulence upon its host is restored by cointegration with pAL2832, which even lacks the region in which the Tnl831 insertion has taken place. This may be brought about by detachment of PAL 1509

Ti PLASMID

REPLICATOR

131

REGION

from the chromosome. It is noteworthy, that both this cointegrate (pAL2839) and pALl509 are stable in the cell. On account of the above considerations, it seems unlikely that the site where Tn1831 has inserted in pALI is per se involved in establishment inhibition, incompatibility, or virulence, particularly because substitution of the Ti replicator by an R plasmid replicator does not affect virulence (Hille et al., in preparation). ACKNOWLEDGMENTS We thank Drs. G. Ooms and J. Hille for stimulating discussions, T. Kartasova for the isolation of &XII, and H. den Dulk-Ras, G. Moolenaar, M. J. Vaessen, and R. van Veen for various experimental contributions. This work was supported in part by the Netherlands Foundation of Biological Research (BION), and the Netherlands Foundation of Chemical Research (SON), with financial aid from the Netherlands Organization for Advancement of Pure Scientific Research (ZWO).

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