JOUR. AL OF BACTERIOLOGY, Aug. 1979, p. 424431 0021-9193/79/08-0424/08$02.00/0

Vol. 139, No. 2

Negative Control of Octopine Degradation and Transfer Genes of Octopine Ti' Plasmids in Agrobacterium tumefaciens PIETER M. KLAPWIJKt* AND ROBBERT A. SCHILPEROORT Department of Biochemistry, State University of Leidert, 2333 AL Leiden, The Netherlands Received for publication 4 June 1979

The regulatory system that controls the expression of the Ti plasmid-borne octopine degradation (uad) and transfer (tra) genes in Agrobacterium tumefaciens was studied. A deletion mutant derived from the cointegrate plasmid R702::Ti-B6S3 was isolated, which was compatible with a wild-type Ti plasmid and which had retained the uad genes. By means of this mutant plasmid pAL116, it was possible to make cells diploid for the uad genes. pAL116 was introduced into Rec- strains that contained different types of regulation mutants for the uad and tra genes. The repression pattern that was found in this complementation analysis indicated that the uad and tra operons are controlled by a common repressor system. Several results indicated that there may be additional transcriptional relations between both operons. The corresponding genes of the nontumorigenic octopine plasmid pAt-AG60 appeared to be controlled by a repressor related to that of the octopine Ti plasmid.

Recently it has been demonstrated that part of the Ti plasmid harbored by the crown gall organism Agrobacterium tumefaciens is transferred into the plant cell during the process of tumor induction (2). This unique property of the Ti plasmid explains the interest of molecular biologists in the gene functions encoded by this extrachromosomal element. It is known that the octopine type Ti plasmids carry genes that control the tumor-specific synthesis of octopine [N2(D-1)-carboxyethyl-L-arginine] (1, 7, 16). Genes that code for an octopine-degrading system (i.e., a permease and an oxidase) are located elsewhere on the plasmid (13, 15, 16). These genes are expressed after induction by octopine (11, 14). Transmission of Ti plasmids by conjugation is also dependent on induction by octopine (6, 8, 12, 19). Results obtained by two groups suggest that octopine degradation (uad) genes and transfer (tra) genes are controlled by a common regulatory gene (15, 18). Although the types of regulatory mutants isolated thus far are readily explicable by a negative control mechanism (10), no conclusive evidence for this model could be given in earlier reports. To test the model it was essential to be able to make cells diploid for the uad and tra genes of the Ti plasmid. Replication of Ti plasmids is stringent, and incompatibility prevents the maintainance of two related Ti plasmids in the same cell (9). To avoid incom-

patibility phenomena, it was necessary to construct a replicon carrying the uad genes, but compatible with Ti plasmids. The use of a Recbackground facilitates such an approach because it prevents the recombinational rearrangement of plasmid genes. In this study a Rec strain (15a) was employed to isolate, by selecting for markers of two plasmids, a deletion mutant from a R702::Ti cointegrate plasmid that had lost the Ti-coded incompatibility. The mutant plasmid lacked a large piece of Ti DNA, but it had retained the uad genes. This plasmid was used to test the phenotypic effects of the Ti plasmid regulatory mutations isolated previously (15) on the wild-type uad alleles. The results support a negative con-

t Present address: Department of Radiation Genetics and Chemical Mutagenesis, Sylvius Laboratories, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands.

424

trol model. (This paper is part of a Ph.D. thesis submitted by P.M.K. at the State University of Leiden, AL Leiden, The Netherlands.) MATERIALS AND METHODS Bacterial strains. Bacterial strains are listed in Table 1. All incubations were carried out at 29°C. Media. Media were identical to those described by Klapwijk et al. (15). Conjugation conditions. Conjugation conditions were as described previously (15). All transfers of Ti plasmids were on SM medium containing 1 mg of MnSO4 per liter supplemented when necessary with octopine (2 g/liter). Transfer of pALlll and pAL116 was on rich medium (TY). In all experiments, mating was carried out on filters. Selection of octopine-utilizing bacteria. Selec-

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VOL. 139, 1979

425

TABLE 1. Strains Strain

Derivation

Plasmid sonChertiesprop- ViRelence propertpes

C58-C9

+

Ti-Ach5

Uad' Trac Uad' Tcr Kmr Smr Uad' Tcr Kmr Smr AInce Uad' Tra'

Rif' Rifr

+

pAL657d pAL104

Eryr Spcr

+ -

Cb' Uadc Trac Uadc Tra'

pAL202e

Cbr AUad- Tra-

pAL203d pAL2l8d pAL116

Cb' Uad- Trac

LBA634 LBA692a LBA949

Nal' Str' Nalr Strr

+ ±

pAL634

C58-C9

C58-C9

Nalr Strr

NDb

pAL116

LBA4001 LBA4011 LBA4014 LBA4017 LBA4020 LBA4023

(-Ach5)c Ach5-C3 LBA4011 Ach5 Ach5-C3 LBA4014

LBA4027 LBA4045 LBA4072

LBA4014 LBA4014 LBA4020

Rif r

Rifr Rif

+ +

Eryr SpCr

-

pAL111

Cb Uad- TraUad' Tcr KMr Smr AInce CbO Uad' Trac Cbr Uad' Tra' Cbr Uadc Trai

Reference/source

P.J.J. Hooykaas 15a This study R.H. Hamilton 15 15 15 P.M. Klapwijk Koekman et al., in press 15 15 This study

ND This study pAL121 LBA4073 LBA4020 Eryr Spcr ND pAL122 This study LBA4074 LBA4020 Eryr SpCr ND pAL123 This study LBA4075 LBA4020 Eryr Spcr LBA4301 LBA4011 Rif Rec15a + pAL203 This study LBA4304 LBA4301 Rifr RecCbr Uad- Trac ND Uad+ LBA4316 LBA4301 Rif r RecThis study pAt-AG60 This study ND LBA4317 LBA4301 Rif rRecpAL657 Cbr Uadc Trac a pALlil is a cointegrate plasmid consisting of IncPl-type R plasmid R702 conferring Tcr Kmr Smr and octopine Ti plasmid Ti-B6S3 (15a). b ND, Not determined. 'Based on restriction endonuclease fingerprints, the Ti plasmids from Ach5 and B6S3 are identical (6). d pAL657, pAL202, pAL203, and pAL218 all carry Tnl, which confers carbenicillin resistance (Cbr). eAInc- means loss of Ti directed incompatibility due to a deletion in the plasmid.

tion of octopine-utilizing bacteria was performed as described previously (15). Colonies were tested for octopine utilization by streaking them on octopinebromothymol blue indicator medium (8). Selection for antibiotic resistances. Selection was on the following concentrations (milligrams per liter of SM medium): rifampin, 20; spectinomycin, 500; erythromycin, 25; tetracycline, 1; carbenicillin, 10. When used in combination with erythromycin, the concentration of carbenicillin was 5 mg/liter. Homooctopine sensitivity. As reported by Petit and Tempe (18) and us (15), mutants constitutive for octopine degradation are sensitive to homooctopine. We found that this effect was reproducible and could be used as a measure for the level of expression of the uad genes. Octopine permease activity. Octopine permease activity was tested by the protocol described by Klapwijk et al. (14). Construction of regulatory mutant plasmids carrying INl. To isolate Ti plasmids that had, in addition to the Uad+ marker, another selectable trait, they were allowed to recombine with pAL202, which is harbored by LBA4023. pAL202 is a derivative of pAL657 that has a large deletion resulting in the loss of uad and tra functions, but has retained Tnl (Cb') (B. P. Koekman et al., Plasmid, in press). A donor strain harboring a plasmid with a particular uad regulatory mutation was crossed with LBA4023 in the presence of octopine. Selection was for Rif' Cbr Uad+

transconjugant colonies. In every case plasmids were isolated that exhibited the uad and tra properties of the donor plasmid. Transfer of the recombinant plasmids demonstrated an absolute linkage of Uad+ and Cb'; segregation was less than 1%. Isolation of plasmid DNA and restriction endonuclease treatment. The procedures employed were described by Koekman et al. (in press). Detection of plasmid DNA. Detection of plasmid DNA on agarose gels was carried out by the protocol described by Casse et al. (J. Gen. Microbiol., in press). Symbols. Uad- is used to designate strains that are unable to utilize octopine but are unaffected in the utilization of arginine (13). These mutants are also unable to degrade lysopine and octopinic acid. Tra- is used to indicate a strain that is unable to promote under appropriate conditions the transfer of its Ti plasmid. Uadc refers to constitutive octopine degradation; Uad' refers to inducible degradation. Trac means constitutive conjugative transfer of the Ti plasmid, Tra' represents inducibility by octopine. Recdesignates recombination deficiency. Rifr means resistance to rifampin; Spcr means resistance to spectinomycin, and EryF means resistance to erythromycin. The resistance markers Cbr (carbenicillin), Km' (kanamycin), Tcr (tetracycline), and Smr (streptomycin) are extrachromosomally located. Chemicals. All chemicals were of standard analytical grade. Homooctopine was synthesized from L-homoarginine (Koch Light) and bromopropionic acid

426

KLAPWIJK AND SCHILPEROORT

(Merck, zur Synthese) by the procedure of Bomhoff (Ph.D. thesis, University of Leiden, Leiden, The Netherlands, 1974). The stereoisomers were not separated. D(+)-Octopine was from Sigma, oxytetracycline was from Mycofarm Delft, and carbenicillin (Pyopen) was from Beecham. Rifampin was a generous gift from Ciba Geigy.

RESULTS Isolation and characterization of pAL116. As explained in the introduction, it is essential to work with compatible plasmids when performing complementation studies on plasmid-borne mutations. In A. tumefaciens, the cointegrate plasmid pALill (R702::Ti-B6S3) was incompatible with Ti plasmids (15a) and thus not directly applicable. We knew that the insertion of R702 had taken place in a site in SmaI restriction fragment 12 of the Ti plasmid (Fig. 1 and 3; G. Ooms and B. P. Koekman, unpublished data). This meant that R702 was located between on one side the uad genes and on the other side the Ti replicator region. This

J. BACTERIOL.

gave rise to the possibility of isolating deletion mutants from pALlll that had lost the Ti replicator, but that had retained the uad genes. In this respect, we hoped that loss of the replication genes would be accompanied by loss of Ti incompatibility. In a non-recombination-deficient background the isolation of compatible mutant plasmids by means of coselection on markers of two different but partially homologous plasmids was likely to be complicated by the recombinational rearrangement of plasmid markers. The introduction of a rec mutation into A. tumefaciens (15a) overcame this difficulty. When pALlil was introduced with selection for Rifr Tcr into the Rec+ strain LBA4027 harboring the Tnl (Cbr) carrying Ti plasmid pAL203, the CbF marker was retained as a result of plasmid recombination (rescued) in 5 to 10% of the cells. By using Rec- strain LBA40304 (carrying pAL203) as acceptor, the frequency of rescue of CbF was reduced by a factor of 500 or more (15a). It was anticipated that a significant number of Cbr Tcr transconjugants from such a

FIG. 1. Restriction map of octopine Ti plasmid adapted from Chilton et al. (3) showing some relevant regions. (A) Deletion mapping has shown that this region contains uad genes (Koekman et al., Plasmid, in press); (B) deleted plasmids always have retained this part of the octopine Ti plasmid; (C) the cointegrate plasmid pAL111 used in this study has R702 inserted in SmaI restriction fragment 12; (D) the deleted cointegrate plasmid pALl16, obtained during the present study, has suffered a deletion in the left part of the Ti plasmid. The dotted lines indicate that in all cases the exact boundaries are not known.

NEGATIVE CONTROL OF Ti PLASMID GENES

VOL. 139, 1979

cross would have a deletion in pALlll, resulting in the loss of incompatibility towards Ti plasmid pAL203. The development of Rif r Tc' Cb' transconjugants from a LBA692(pAL111) x LBA4304(pAL203) cross could be due to the following four different types of events: class i, in which pALlll had lost all of its Ti DNA before or after conjugation and was thus not able to exert incompatibility towards pAL203; class ii, in which Cbr was rescued from pAL203 onto pALlll by residual rec activity or by recindependent transposition of Tnl onto pALlll; class iii, in which CbF was retained in the recipient as a result of transposition of Tnl from pAL203 into the bacterial chromosome; or class iv, in which pALlll had lost the gene functions that control Ti incompatibility by mutation, either a deletion or a point mutation. Therefore, we analyzed 68 such CbF Tcr transconjugants to find members of class iv. The results are summarized in Table 2. Class i was easy to recognize, since these bacteria had to be Uad-, like LBA4304. The other bacteria could be divided in two groups: those which gave 100% linkage of Tcr and Cbr (class ii) and those which did not (classes iii and iv). The latter two classes could only be distinguished when purified Tcr Uad+ transconjugant clones, obtained from matings with the plasmid-free strain LBA4020, were crossed back with LBA4304. Bacteria with compatible plasmids should give 100% Cbr Tcr transconjugants, whereas bacteria of class iii would still give less than 1% Cbr Tcr transconjugants. In one case the bacteria gave 100% Cbr transconjugants when tested this way. This strain, LBA4072, showed a 100% linkage of Tcr and Uad+ in transfer experiments. When DNA was extracted from a transconjugant of a LBA4072 x LBA4304 cross and was subjected to agarose gel electrophoresis, two plasmid bands were visible. One of the plasmids had the same size as a Ti plasmid, whereas the other was slightly smaller (Fig. 2). The plasmid harbored by LBA4072 was called pAL116. When pAL116 DNA was treated with restriction TABLE 2. Analysis of CbF Tc' transconjugants from crosses LBA692(pAL11I) x LBA4304(pAL203) Degradation of octopine

Linkage with Tc' in crosses with LBA4020 Cbr

-

-

+ + + +

+ + -

a See text.

-

No. of

isol

tes

Class inferred'

Uad+ 28

+ + +

2 20 17 1

ii ii ii iv

1

2

427

3

FIG. 2. Agarose gel electrophoresis of plasmid DNA isolated according to Casse et al. (in press). Lane 1, DNA prepared from LBA692 carrying R702::Ti cointegrate plasmid pALIII. Lane 2, DNA prepared from a transconjugant strain carrying pAL116 and the Ti plasmid pAL203. Lane 3, DNA prepared from a strain harboring pAL2O3 and the R plasmid R702.

endonucleases and the fingerprints on agarose gels were examined in relation to the restriction map of the Ti plasmid (3), it turned out that pAL116 was a deletion mutant of pALlll. pAL116 was missing a large stretch of Ti DNA (55 x 106 to 60 x 106 daltons) from SmaI fragment 2 to SmaI fragment 3a, but all R702 DNA was retained (Fig. 3). The deleted DNA was that containing the Ti replicator, whereas the DNA known to contain the uad genes was still present (Fig. 1). The introduction of pAL116 into a pAL657-carrying strain did not give rise to the loss of pAL657 whether the recipient was Recor not. On the other hand, the parental plasmid was very efficient in this respect: 100 TcF transconjugants tested were all Cb8 in a Rec- background (Table 3). pAL116 had retained all resistance determinants located on R702. In strains harboring pAL116, the octopine permease was normally inducible by octopine (Table 4). No tumor induction on tomato plants could be observed. The results indicated that pAL116 had lost the genes that control Ti incompatibility and so could be of use in complementation studies with Ti plasmids. Mutations causing constitutivity for tra and uad functions. In a previous report we described the isolation of several regulatory mutants of the octopine Ti plasmid (15). The constitutive mutants could be distinguished as belonging to one of three classes: (i) constitutive

428

KLAPWIJK AND SCHILPEROORT

J. BACTERIOL.

for octopine enzymes (Uadc), (ii) constitutive for Ti transfer (Trac), and (iii) constitutive for both traits (Trac Uadc). The isolation of these classes was also reported by Petit and Tempe (18). In both publications a model was proposed that consisted of two operons (uad and tra) coordinately controlled by a common repressor gene. This model interpreted constitutivity of the separate traits as operator mutations and constitutivity of both traits as a repressor mutation. The

model predicts that in Uadc Trac mutants the mutation will be recessive towards wild-type uad genes, whereas in Uadc or Trac mutants the mutation will be dominant. We performed complementation experiments with our regulatory mutants and plasmid pAL116 to test whether the proposed model was adequate. The three regulatory mutant plasmids were as follows. (i) pAL657, octopine Ti plasmid derived from TiB6S3, conferred upon its host an Uadc Trac phenotype. The plasmid carried Tnl, conferring carbenicillin resistance (8). (ii) pAL121, also de4 3 1 2 rived from Ti-B6S3, carried Tnl and a mutation that caused a constitutive level of Ti transfer (Trac). The plasmid was constructed by recombination between the original mutant plasmid pAL634 and the Tnl-carrying deleted plasmid pAL202 (15) under coselection of CbF and Uad+. This provided a plasmid with a selectable marker besides Uad+. (iii) pAL123, derived from Ti-Ach5, carried Tnl in addition to a mutation causing an Uadc phenotype. It was constructed by recombination of pAL104 (15) and pAL202 in the same way and for the same reason as pAL121. To obtain a reference Ti plasmid that contained the normal regulatory system (i.e., repression of uad and tra genes), plasmid pAL122 was constructed by recombination of Ti-Ach5 and pAL202. To facilitate complementation analysis, the plasmids were transferred into the Recstrain LBA4301. Their properties were tested, and thereafter pALl 16 was introduced into them under selection for Tcr transconjugants after mating with LBA4072. The properties of the resulting strains are given in Table 4. The data show that the constitutive character of bacteria containing pAL657 was not maintained in the presence of pAL116. It is therefore evident that the normal regulation of pAL657 was restored by a gene product from pAL116. The Trac mutation carried by pAL121 was dominant: the transfer frequency stayed at a level of 10-2 in bacteria harboring both pAL116 here The Trac mutation FIG. 3. Restriction patterns after agarose gel elec- and pAL121. due to a mutation in present the operator was probably trophoresis of plasmid DNA treated with restriction endonuclease SmaI from Serratia marcescens. Lane for the tra genes. This seems to be a point 1, pAL116. Lane 2, Ti-Ach5. Lane 3, pAL111. Lane 4, mutation, since we found that the mutation on pAL121 reverted readily to inducibility. The reR702. TABLE 3. Transfer experiments demonstrating the absence of incompatibility between pAL116 and pAL657 Donor

Acceptor

Frequency of donor

LBA692(pAL111) LBA692(PAL111)

LBA4014(pAL657) LBA4317(pAL657) LBA4014(pAL657) LBA4317(pAL657)

5 x 10-2 2 x 10-4 12 x 10-2 8 x 10-3

LBA949(PAL116)

LBA949(PAL116)

plamid trafer

% Rif' Tc'

colonies that are Cb 7 0 100 100

Background of aacceptor

Rec+ ReC-

Rec+

ReC-

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429

TABLE 4. Properties of strains derived from LBA4301 harboring the plasmids used in this study [IH]octopine Transfer of Ti plas- Transfer of pAL116 Transfer of pAtPhenotype Plasmid con- uptake' (cpm) mid (Cbr) (T c') AG60 Sensitivity inferred +ind' -indd +inde ]indh +indc -indd -ind" +inde oto homo-

-ind +ind'

pAL116 pAL657 pAL121 pAL123 pAL122 pAL657 + pAL116 pAL121 + pAL116 pAL123 + pAL116 pAL122 + pAL116

indd

+ind"

-

indd

+ind'

1.7 x 1o-212.4 x 10-2 239" 3,166f 3,848 3,721 3 x 10-2 2 x 10-' 10-2 1.5 x 10-2 158 2,104 3,044 3,208