CA1192149A - Broad host range dna cloning system for gram-negative bacteria - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
Novel compositions and methods for cloning DNA in a broad range of Gram-negative bacteria. Relatively small vectors are prepared substantially free of DNA sequences coding for transfer functions, while having a broad host range replication function. While the plasmid is not self-transmissible, it is transmissible by means of a helper plasmid, which desirably has a narrow host range replication function. Exogenous DNA may be inserted into the first plasmid and conjugally transferred to the target host by means of a convenient bacterial host.

Description

This invention relates to a broad host range DNA
cloning system for Gram-negative bacteria.
Numerous plasmid elements have been developed as cloning vehicles in Escherichia coli. While there have been _ __ _ .
numerous developmen-ts with E. col:i, and numerous opportunities still remain, there will be many situations where other microorganisms will be of interest or be required for a particular purpose. In addi-tion, the ability to transform other microorganisms will be a powerful tool in the genetic analysis of species, particularly where other genetic systems are unavailable. Furthermore, vectors could be used to augment or modify desirable characteristics o~ the host bacterium, such as nitrogen fi~ation by Rhizobium species or hydrocarbon degradation of Pseudomonas species.
A suitable vector should have a wide range of desirable properties. These properties should provide for efficient introduction of exogenous DNA, allow for high efficiency of introduction of the plasmid into the host bacteria, provide stable maintenance in exconjugants and not confer hazardous properties to the host. In addition, the plasmid should not be readily transmissibleO
Meyer et al., DNA Insertion Elements, Plasmids, and Episomes, 1977, Cold Spring Harbor Laboratory, pp. 599-566, describes the properties of the plasmid RK2 as a cloning vehicle. The regions necessary ~or DNA replication of RK2 are described by Meyer and Helinski (1977), Biochem. Biophys.
Acta. 478, 109-113, and Thomas and Helinski (1979), J.
Bacteriol. 141, 213~ The site for conjugal mobilizability in RK2 is described by Guiney and Helinski (1979~, Mol. Gen.
GenetO 176, 183-1~9 According to the present invention novel cloning vehicles having broad host range specificity ~or Gram-negative bacteria are provided. The vehi.cles are relatively small having one or more convenient restriction sites, normally having a single res-triction site for one or more restriction enzymes. The vehicles are further characterized by being non-self-transmissible, but capable of transmission with a helper plasmid. The vehicles have a broad host range replication system and normally have a marker allowing for selection of transformants. The vehicle may be introduced into the target host either by transforming the conjugal mating host with the vehicle and the helper plasmid in a single cell or by transforming conjugal mating cells separately, so that one group of cells has the helper plasmid and another group the vehicle plasmid. A wide range of Gram-negative bacteria can be modified with exogenous DNA, while substantially ensuring the absence of transmissibility of the vehicle containing the exogenous DNA to other micro~
organisms.
Aspects of the invention are illustrated in the drawings, in which:
Figure 1 is a map of RK2, where Ap, Tc and Km refer to genes conferring resistance to ampicillin, tetra-cycline, and kanamycin, respectively. oriV is the origin of replication. trfA and trfB refer to transacting replication functions. rlx refers to the relaxation complex site. Tra refers to regions containing genes required for conjugal transfer.
Figure 2 is the construction of pRK290. Small arrows indicate cleavage sites for restriction enzyme~ used at each step; for HaeII, the approximate position of cleav-ages leading ultimately to pRK290 are indicated. BAP refersto treatment with bacterial alkaline phosphatase. Solid bars repre-~2~

sent conjugal transfer genes; open ~ars are essential repli-catioh regions. pRK2501 is a previously constructed RK2 deletion derivative containing a HaeII kanamycin fragment that had been inserted in vitro. K-KPnI; H-HlndIII;
Hae-HaeII; B-B~l; R-EcoRl.

Novel plasmid compositions are provided as vehicles for introduction of exogenous DNA in Gram-negative bac-teria, as well as methods for introduction of the plasmid vehicle into Gram-negative bacteria. The plasmid vehicles have a broad host range replication system and are therefore capable of being stably maintained in a wide range o~ Gram-negative bacteria. The plasmid vehicles also have a broad host range transmissibility in combination with a trans-complementing helper plasmid. Desirably, the plasmid vehicle is relatively small, retaining a complete replication function, while lacking a transfer function but retaining mobili~ability with a helper plasmid. In addition, the plasmid should have at least one marker allowing for selection and have unique restriction sites by at least one restriction enzyme, where insertion at the site does not r sult in the loss of a neces-sary or desirable function or does result in the loss of a function which allows for selection. The vehicle should not confer hazardous pxoperties to the target host or signifi-cantly extend the range of antibiotic resistance. The vehi-cle will be transmissible with a helper plasmid by biparental and desirably triparental conjugal transfer.
The subject vehicle has a broad host range compat-ibility. It is capable of being introduced into and replicat~
ing in at least 5, preferably at least about 8, more prefer-ably at least 10 different genera of ~ram-negati~e bacteria.
Furthermore, it is capable of replicating in a convenient donor microorganism. Particularly useful as the donor organ ism is Escherichia, more particularly E. col1. Of Gram-negative bacteria of interest as recipients are the gen~ra:Pseudomonas; Alcaali~enes; Neisseria; Klebsiella; Serratia;
Acinetobacter; Haemophilus; Rhizobium; ~zotobacter;
.
Xanthobacter; Salmon lla; Shigella; Vibrlo; Yersi~ia;
Erwinia; etc.

4~

Desirably, the plasmid vehicle should be relatively small, so as to be able to accep-t relatively large inseLtions of exogenous DNA. Usually, the plasmid will be substantially less than about 50 kbp (kilobase pairs), usually below about 40 kbp, more usually less than about 30 kbp and yenerally grea-ter than about 10 kbp, conveniently frorn about 10 to ~5 kbp, more usually from about 15 -to 25 kbp.
The vehicle will be incapable of self-transmis-sibility. That is, it will lack at leas-t a substantial portion of the DNA sequences involved wi-th conjugal transfer.
Normally, at least about 40% by number of nucleotides of the DNA sequences defining the transfer function will be absent, more usually at least about 50%. All of the DNA sequences involved with the transfer function need not be removed and in situations where o-ther essential functions of -the plasmid overlap a portion of the DNA sequence involved with the transfer function, this portion will be retained. The less of the transfer function present in the plasmid vehicle, the less likely there is to be recombination with the helper plasmid to recreate an intact transfer function.
While the transfer function must be inoperative, it is essential that the plasmid be capable of mobilization by means of a helper plasmid. That is, the vehicle must be capable of con]ugal transfer from a donor to the recipient Gram-negative bacteria. Therefore, the subject plasmid has the mobilizability function.
While not always essential, particularly where the exogenous DNA provides a means for selec-tion, it will norrnal-ly be desirable to provide a marker as part of the clonin~3 vehicle which allows for selection of transformants. Depend-ing upon the nature of the host, a wide and diverse variety of markers may be ernployed. Conveniently, antibiotic resis-tance may be employed which allows for selec-tion of trans formants by culturing the cells on a mediwn containing the particular antibiotic. Antibiotic resistance can be provided to ampicillin, penicillin, tetracycline, kanamycin, etc.
Resistance can also be provided to heavy metals. Al-terna-tively, prototrophy can be provided to ~n auxotrophic host.

a ~

That is, a host lacking the abili-ty to produce an essential metabolite is -transformed with -the vehicle which provides -the structural genes necessary for the enzymes for producing the me-taboli-te. By culturing the transformants in a cul-ture medium lacking the essential metabolite, the transformants can be selected. O-ther more sophisticated techniques include providing incompatibility to par-ticular bacteriophage strains, resistance to toxins, changes in morphology, and the like. In some ins-tances, i-t will be desirable to have a plurality of markers, where one of the markers has a restric-tion site for insertion of the exogenous DNA. The loss of the property provided by -the marker allo~s for detection of plasmids into which the exogenous DNA has been inserted.
Other alternatives exist for monitoring for plasmids having the desired exogenous DN~.
The cloning vehicle will normally have at least one unique restriction site for a restriction enzyme and may have a number of unique restriction sites for the equivalent number of restriction enzymes. The restriction sites will be in non-essential areas of the cloning vehicle, so as not to disturb the functioning of the plasmids. A wide variety of restriction enzymes are known, such as EcoRI, PstI, 8indI, II
and III, HaeII, ~I, SalI, ~I, XhoI, and SmaI, as illus-tra-tive but not exhaustive of restriction enzymes.
As already indicated, the vehicle must not be self-transmissible, but must be capable of conjugal transfer by trans~complementation with a helper plasmid. Desirably, the cloning vehicle plasmid should be capable of transmis-sion, where the helper plasmid and the ~ehicle are initially in different donor cells. The vehicle plasmid will have the necessary function ~or conjugal mobilizability, as well as a functioning replication system. The functioning replication system may involve one or more genes, which may be contiguous or widely separated.
3~ Th~ exogenous DNA which is introduced will vary depending upon the purpose of the -transformation, the nature of the host, the intended use of -the host, and -the like. The host may be used most simply for ampliflcation of the exogenous DNA, to provide a ready source of the DNA. For the most part, the exogenous DN~ will have one or more structural genes, which may be under individual control or polycistronic.
The structural genes may be part of an operon, with or with out the regulatory genes providing either repression or activation.
The exogenous DNA may be used to augment a natural function of the host, by providing enhanced production of a particular protein e.g. enzyme. The exogenous DNA may be used to modify a property of the host. For example, _9eudomo~as may be modified to expand ~the range of hydrocarbon substrates which can be utilized by the bacteria. Nitrogen fixing bacteria, such as Rhi~obium may be modified to ~ary their host specificity. Pathogenic microorganisms may be modified to attenuate or destroy their virulence.
Microorganisms may be modified to provide for modification of a product naturally produced by the microorganism. That is, enzymes may be expressed from the exogenous DNA, which will react with a metabolite or catabolite -to produce a product of ~0 interest. Regulatory functions may be introduced into the microorganism, so as to modify its characteristics or response to changes in its environment. The subject v~hicle therefore provides a wide variety of opportunities for pro-ducing DNA, augmenting properties of Gram-negative bacteria, modifying properties of Gram-negative bacteria, and producing compounds forei~n to a Gram-negative bacterium host.
At the insertion restriction site, it may be desir-~ble to introduce various regulatory signals recognized by the target host. The regulatory signals may include promoters, operator~, a CAP binding site, terminator sitP, a ribosomal start site or terminator site, or the like. Of course, the regulatory signals may ~e provided on a ~oreign DNA se~uence to be inserted, rather than having them orig-inally present on -the vehicle.
The subject vehicle will normally be prepared from an available plasmid, by modification of the plasmid to provide the desired properties. However, synthetic tech-niques can be used or a combination of synthetic techni~ues with DNA se~uences obtained from naturally occurring plas-mids. Eor broad hos-t range compa-tabili-ty, a convenien-t plasmid source are those plasmids having broad-host-ranyes, such as the P-incompa-tibility group. Of -these, the plasmid RK2 is exemplary. RK2 has undesirable fea-tures in being large, grea-ter -than 50 kbp, being self-transrnissible, and in conferring resis-tance -to several clinically important anti-biotics. RK2 is further characterized by having the transfer genes non-contiguous and dis-tributed in widely separated regions. The same is true for the genes involved with DNA
replication. RK2 also has the mobili~ibility gene over-lapping a transfer function gene.
A broad-host-range cloning vehicle lacking self-transmissibility, but being capable of mobilization by trans-complementation with a helper plasmid having a func-tion-ing transfer function, can be prepared as follows. I-t will be assumed that the transfer function genes are widely sepa-rated on -the plasmid.
~0 A plurality of restriction sites are cleaved by one or more restriction enzymes to provide a plurality of frag-ments. The restriction sites are chosen so as not to cleave at an essential gene, unless the two fragments are to be reunited at the cleavage site to recreate the intact gene.
Also, the cleavage sites are chosen to provide fragments lacking the essential genes, but containing all or par-t of a gene involving the transfer function. The minimum number of essential genes are the genes involved with replication and mobilizibility.
The fragments are allowe~ to randomly reunite and the resulting plasmids chosen for retaining the capability of replication and mobili~ibili-ty, but not self-transmissibil ity, as well as retaining at least one restriction site at other than a structural gene.
Since it is desirable to have as little of the transfer function D~A se~uences as reasonably feasible, the selec-ted plasmids may be further cleaved, where non-essen-tial DNA and transfer function ~NA may be further removed. One convenient techni~ue is to make a single restriction cleavage at or near a transfer function gene and remove the remaining phosphate at the site e.g. with alkaline phosphatase, -to prevent recirculation at the si-te. ey cleaving on both sides of -the single cleavage sit.e, addi-tional DNA may be removed to reduce the size of the plasmid and recreate liga-table ends for recirculariza-tion.
Alterna-tively, one can choose fragments which lack the transfer function, as well as an essential func-tion.
Where the original plasmid has been mapped, -the desired DNA
sequence defining the gene may be excised and introduced at an appropriate site into the appropriately rejoined fragments.
Fig. 1 shows the plasmid RK2 indicating the sites having the genes conferring antibiotic resistance, the restriction sites, the genes involved with the transmis-sibility function, which are the shaded areas and the genes involved with -the replication function which are the open areas. RK~ was used to prepare an exemplary cloning vehicle, fulfilling the requirements of having a broad host range, capable of mobilization at high frequency with a helper plasmid, conferring limited antibiotic resistance to the target host, having unique restriction sites with at least one restriction enzyme, and ~eing relatively small so as to accommodate large exogenous DNA sequences.
RK2 had been mapped with the DNA replication sites described by Meyer and Helinski (1977), su~ra, and Thomas and Helins]~i ~1979~, supra, while the conjugal mobilizability function was reported by Guiney and Melins~i (1979), supra.
~he manner in which the exemplary cloning vehicle was prepared is shown in Fig. 2. ~K2 is cleaved at four positions by ~I, resulting in four fragments, only two of which carried all the genetic information necessary for autonomous replication and tetracycline resistance. Trans-forma-tion of E. coli with a total Kpn diges-t of RK2 DNA and selection for tetracycline resistance yielded transformants containing the two fragments in either of the two possible orientations. DNA with the fragmen-ts aligned as in RK~ was 4~

treated to remove as much DN~ as possible from either side of the singlP HindIII site without removing rlx or trfA. After digesting the DNA to completion wi-th HlndIII followed by treatment with bacterial alkaline phospha-tase (BAP) to render it incapable of being covalently re~clrcularized by DNA
ligase, a partial digestion with HaeII was then used -to generate pse-udo-random cu-ts on either side of the BAP-treated HindIII site. Whenever at leas-t one cleavage occurred on both sides of the ~IindIII site, a molecule is generated that could be circularized by DNA ligase. So long as the resul-t-ing plasmid retained essen-tial replication regions, such molecules could be detected by transformation. The molecules were screened for retention of -the rlx site by monitoring mobilizabili-ty with pRK2013. (Figurski and Helinski (1979) 15 Proc. Natl. Acad. Sci. USA 76, 1648-1652). The smallest such derivative was selected, represen-ting a deletion of approxi-mately 1:l.5 kb of ~NA. The next step removed most oE the 12.1 kb of DNA between the single EcoRl si-te and the single ~II site of RK2. For convenience, pRK2501 was employed.
20 (Kahn et al. (1979) Methods in Enzymology, Vol. 68, Recom-binant DNA R. Wu, ed. Academic Press, New York). The dis-tance between the single EcoRl and BglII sites in pRK2501 is only 1.1 kb. Their two DNAs were digested jointly with E R1 and _~II, ligated and used to transform E. coli for te-tracycline resistance. Substitution of the appropriate fragment was monitored by screening transformants for sensi-tivity to ampicillin and kanamycin. The resulting plasmid was 20 kb in size, has two single restriction enzyme sites into which a variety of EcoRl and ~II generated DNA frag-ments can be cloned successfully and has other enzyme siteswithin the tetracycline sites, which sites include _maI an~
SalI. The molecules specifically lack sites for the enzymes BamHI, HindIII, PstI, KpnI, _~I, and XhoI, and may be less -subject to xestriction in those hosts. The copy number of pRK290 in E. coll was found to be similar -to that Gf RK2.
For convenience, since interruption of either cloning site does not lead to a detectible change in colony phenotype ~e.g. insertional inactivation), it is desira~le -to treat the restriction enzyme-cleaved vehicle with alkaline phosphatase prior to ligation.
EXPERIMENTAL
Materials and Methods S Bacterial strains E. coli HB101 pro leu thi lacy strr endoI
recA r m ; R. melilotl 102F34 and 104B5 are available from Nitragin Co.; Serratia marcescens MWl is a clinical isolate reported by D. Guiney; Pseudomonas aeruginosa PAO is also reported by D. Guiney; K. pneumoniae M5A1 is reported by W.
Brill; Acinetobacter calcoaceticus is reported by John Ingraham.
Enz~mes.
Restriction endonuclease EcoRl was purified by the 1~ inventors; ~II was provided by C. Yanofsky; all other restriction enzymes which were employed were obtained from Biolabs, Inc. T4 DNA ligase is available from Bethesda Research Laboratories, Inc. and is used at a concentration of lm/ml for ligations. Lacterial alkaline phosphatase is ob~
tained from Miles Laboratories and is dialyzed into 10mM
glycine, pH9.5 and 0.1mM ZnC1~ for storage. DNA was reacted with this enzyme at 65C for 90min in 10mM Tris, pH9.5. The reaction was terminated by phenol extraction.
Bacterial Matin~s.
Matings were performed by mixing 109 cells each of the donor and recipient and filtering the suspension onto 0.45~ Millipore*filters. The filters were incubated at 30C
on non~selective agar plates for 3 to 6 hours beEore the cells were resuspended and plated.
Isolation of R. meliloti DNA.
. . . ~
Total DNA from R. meliloti was obtained from 500ml of stationary-phase cells grown in yeastmannitol broth (Vincent (1970) In: A Manual for ~he Practical Study of the Root-Nodule Bacteria, I. B. P. ~andbook No. 15, Blackwell 5cientific Publications, Oxford, pp. 1-45). Washed cells were resuspended in ~0m~ Tris/20mM EDTA, pH8.0 and lysed with pre-digested pronase (500~g/ml) and Sarkosyl*[1~) for 60min at 37~C. DNA was purified by ~quilibrium centrifugation * Trade Mark firs-t in neutral CsCl (p = 1.70g/cc) and -then in CsCl-ethidium bromide (p - 1.55g/cc).
Size Frac 1onation of R. meliloti DNA .
Total R. meliloti DNA was par-tially digested with ~II to give fragments in the range 10-30 kb. 140~g of such DNA was hea-ted briefly at 65C and layered directly onto a 36ml 10-~0% sucrose yradient in 20mM Tris, pH8.0, lOmM EDTA, 50mM NaCl. Cen-trifugation was for 18hrs at 23000 rpm in an SW27 rotor at 25C. E'ractions were monitored for DNA size on a 0.5% agarose gel. Those containing DNA predominantly 12-25 kb in size were pooled and used for construction of the gene bank.
Construction of an R. meliloti Gene Bank.
. . . _ , _ pRK290 DNA was digested exhaustively with ~II and was then treated with bacterial alkaline phosphatase. A
small background of transformants was obtained from this DNA, with or without ligation, which probably represented residual uncleaved molecules. Size-fractionated R. melilot1 DNA, ligated to this vector, was used to transform ~IB 101 to tetracycline resistance. The expression time following heat-shock was kept short (approximately 40min) to avoid the generation of siblings.
The following -table, Table 1, shows the frequencies with which pRK2gO was transferred in-to a variety of Gram-negative bacteria as part of the binary plasmid system. TheE. col~ s-train HB101 was chosen as the plasmid host because it is recombination-deficien-t (rec A ), which is desirable because the vehicle and helper plasmid share regions of homology and HB101 lacks the normal restriction system which might otherwise inactivate unmodified foreign DNA carried as inserts.

Conju~al Transfer Frequencies of pRK2013/pRK290 Binary Plasmid Systems for Various-~ram Nega-tive Bac-teria TcR Km /Nm Conjugants Conjugan-ts Donor Recipient Recip en-ts ReciPients E.c.HB101 (pRK2013) E.c. HB101 rif - 8.5xlO
E.c.HB101 (pRK290) E.c. HB101 rif 0 E.c.EIB101 (pRK2013, pRK290) E.c. HB101 rif 4.0xlO 8.2xlO 1 E.c.~IB101 (pRK2013) R.m. 104B5 nal - 1.7xlO 7 E.c.HB101 (pRK290) ROm. 104B5 nal 0 E.c.HB101 (pRK2C13, pRK290) R.m. 104B5 nal 4.6xlO 2 8.4xlO 4 E.c.HB101 (pRK2013) + R.m. 104B5 nal 8.3xlO 2 5.6xlO 4 E.c. HB101 (pRK290) E.c. HB101 (pRK2013) + S.m. nal 6.6x10-2 2.2xlO-E.c. HB101 (pRK290) E.c. HB101 (pRK2013) + K.p. M5A11.4xlO 1 8.8xlO 1 E.c. HB101 (pRK290) E.c. E~101 (pRK2013) + P.a. PAO nal2.6xlO 1 8.4xlO 7 E.c. HB101 (pRK290) E.c. HB101 (pRK2013) + A.c. rif 8.3xlO 4 3.0xlO 4 E.c. HB101 (pRK290) Tc=Tetracycline; Km=Kanamycin. pRK290 is Tc resistan-t, pRK2013 is Km resistant.

E.c. = Escherichia coli R.m. = Rhizobium meliloti . _ _ S.m. = Serratia marcescens K.p. = Klebsiella pneumonlae P.a. = Pseudomonas aeru~inosa A.c. = Acinetobacter calcoace-ticus The first three lines show the high frequency of self-transmissibility displayed by the helper plasmid pRK2013, the absence of self-transmissibility for the vector pRK290, and the high frequency transfer of pRK290 in t:he binary plasmid system. While the majority of e~conjugants sel~c-ted on tetracycline were found -to carry both pRK2013 and pRK290, a sizable portion, approximately 15%, carried only pRK290.
The pattern observed for R. meliloti is ~ui-te different from that observed for E. coli. As shown on line 4, pRK2013 has a low rate of transfer indicating the relatively na.rrow host range of this plasmid. This property is particularly desirable in the helper plasmid to diminish the joint presence of the helper plasmid and the vehicle in the recipient cell. As shown on line 6, pRK290 as a compo-nent of the binary plasmid system shows a high rate of trans-fer into Rhizobium. Based on the observation of some .
neomycin resistant conjugants, homologous recombination between the helper plasmid and the vehicle is believed to have occurred in the recipient during binary sys-tem matings.
As shown in line 7 and subsequent lines, it is not necessary to have pRK2013 and pRK290 together in the same cell at the start of mating for efficient mobilization, it is equally efficient to have triparental matings. Cloned DNA
can thus be "stored" in suitable E. coli strains such as HB101 until the time for transfer without necessitating the prior introduction of pRK2013. With all of the Gram-negative bacteria studied, both vehicle and helper plasmid exerted mutual incompa-tibility leading to a rapi~ segregational loss of the non-selected plasmid.
~ [ restriction ~nzyme fragments of the cellular DN~ of R. melilotl 102F34 were sized frac-tionated on a 10-40%
sucrose gradient and fragments 15-20 kb in size were ligated to ~II diyested pRK290 DNA that had been pre-treated with bacterial alkaline phosphatase and the resultiny plasmids used to transform E. coli. Based on a restriction digest pattern of 300 transformants, it was estimated that 929 of 1285 transformants or approximately 72% carried DNA inser-Z~
1~
tions. The average size of the inserts was 19 kb. No instability of -the cloned Rhizobium DNAs in HB101 main-tained . .
under selected pressure was experienced. Even in the absence of selec-tion, the rate of plasmid loss was generally low, generally less than 1% per generation. Employing colony hybridization (Gruns-tein and Hogness (1975) Proc. Natl. Acad.
Sci. USA 72, 3961-3965) with a known plasmid having nitrogenase structural genes of K. pneumoniae, a single clone was identified which carries as a part of a 26 kb insert a 3.6 }~ ~II fragmen-t with strong homology -to the plasmid pSA30 ~Reidel et al. (19793 Proc. Natl. Acad. Sci. USA 76, 286~-2870; Cannon et al. (1979) Mol. Gen. Genet. 174, 59-66).
In accordance with the subject invention, novel cloning vehicles are provided having broad host range speci-ficity, without conferring hazardous properties or extendedantibiotic resistance to the Gram-negative host. In addi-tion, the vehicles have uni~ue restriction sites for inser-tion of exogenous DNA, while desirably lacking restriction sites for a wide variety of restriction enzymes endogenous to a number of Gram-negative bacteria. The vehicles are rela-tively small allowing for insertion of large DNA se~uences while still retaining a high frequency of conjugal trans-missibility.
Although the foregoing invention has been described in some detail by way of illustration and example for pur-poses of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims (23)

The embodiments of the invention in which an ex-clusive property or privilege is claimed are defined as follows:-

1. A plasmid cloning vehicle of less than about 40 kbp characterized by having a broad-host-range compatibil-ity by being capable of replication in a plurality of Gram-negative bacteria and lacking self-transmissibility but capable of conjugal transfer by means of a helper plasmid in the same or different donor cell used for said conjugal transfer.

2. A plasmid cloning vehicle according to Claim 1, wherein said vehicle is further characterized by having at least one marker allowing for selection of trans-formants.

3. A plasmid cloning vehicle according to Claim 2, wherein said marker is antibiotic resistance.

4. A plasmid cloning vehicle according to Claim 1, wherein said vehicle is of from about 10 to 30 kbp.

5. A plasmid cloning vehicle according to Claim 1, wherein said vehicle is capable of replication in E.
coli and capable of conjugal transfer to Gram-negative bacteria from said E. coli.

6. A plasmid cloning vehicle according to Claim 1, having at least one unique restriction site in a non-essential region.

7. A plasmid comprising a plasmid cloning vehicle according to Claim 6 and including a structural gene inserted into said unique restriction site.

8. A plasmid cloning vehicle of from about 10 to 30 kbp derived from a plasmid of the P-l incompatibility group characterized by having a broad-host-range compatibil-ity by being capable of replication in a plurality of Gram-negative bacteria, lacking self-transmissibility but capable of conjugal transfer by means of a helper plasmid in the same or different donor cell used for said conjugal transfer and having at least one marker allowing for selection of transformants.

9. A plasmid cloning vehicle according to Claim 8 wherein said marker is antibiotic resistance.

10. A plasmid cloning vehicle according to Claims 8 or 9 having at least one unique restriction site in a non-essential region.

11. A plasmid comprising a cloning vehicle according to Claim 10 and a structural gene inserted into said unique restriction site.

12. A plasmid cloning vehicle according to Claim 8, capable of replication in E. coli and capable of conjugal transfer from said E. coli to a plurality of Gram-negative bacteria.

13. A plasmid cloning vehicle having substantially the same composition as pRK290.

14. A method for transforming recipient Gram-negative bacteria which comprises:
mixing (1) donor bacteria containing plasmids comprising a cloning vehicle capable of replication in said donor and recipient bacteria, but incapable of self-transmissibility and having at least one marker allowing for selection of transformants; and (2) helper plasmids capable of replication in said donor bacteria and self-transmissibil-ity, with (3) said recipient bacteria under conjugal transfer conditions; and isolating said recipient Gram-negative bacteria and selecting for transformants by means of said marker.

15. A method according to Claim 14, wherein said plasmids are in the same cell.

16. A method according to Claim 14, wherein said plasmids are in different cells.

17. A method according to any of Claims 14, 15 or 16, wherein said donor bacteria are E. coli.

18. A method according to Claim 14, wherein said cloning vehicle is derived from a P-l incompatibility group plasmid.

19. A Gram-negative bacterial cell containing a plasmid comprising a cloning vehicle of less than about 40 kbp capable of replicating in said cell, incapable of self-transmissability but capable of conjugal transfer by means of a helping plasmid in the same or different donor cell, and having a marker allowing for selection for said Gram-negative bacteria containing said vehicle, and a structural gene exogenous to said gram-negative bacteria.

20. A cell according to claim 19, wherein said Gram-negative bacteria are Pseudomonas.

21. A cell according to claim 19, wherein said Gram-negative bacteria are Rhizoblum.

22. A cell according to claim 19, wherein said Gram-negative bacteria are Neisseria.

23. A Gram-negative bacterial cell containing a plasmid cloning vehicle according to claim 19, wherein said vehicle is of from about 10 to 30 kbp.