Improved broad-host-range plasmids for DNA cloning in Gram-negative bacteria

191

Gene. 70 (1988) 191-197 Elsevier GEN 02578

Short Communications Improved brood-host-range plasmids for DNA cloning in Gram-negative bacteria (Recombin~t DNA; mobilizable vectors; gene libraries; polylinker cloning sites; antibiotic resistance; cosmid; plant pathogens; phage ;1 packaging)

N.T. Keen, S. Tamaki, D. Kobayashi and D. Troilinger

Received Revised

10 March

1988

15 April 1988

Accepted

29 April 1988

Received

by publisher

10 June 1988

SUMMARY

Improved broad-boot-range plasmid vectors were constructed based on existing plasmids RSFlOlO and RK404. The new plasmids pDSK.509, pDSK519, and pRK415, have several additional cloning sites and improved ~tibio~~“resist~ce genes which facilitate sub~loni~g and mobi~zatiou into various Gram-negative bacteria. Several new polylinker sites were added to the Escherichia coli plasmids pUCl18 and pUC119, resulting in the new plasmids, pUC128 and pUC129. These plasmids facilitate the transfer of cloned DNA fragments to the broad-host-range vectors. Finally, the broad-host-range cosmid cloning vector pLAFR3 was improved by the addition of a double cos casette to generate the new plied, pLAFR5. This latter cosmid simplifies vector preparation and has permitted the rapid cloning of genomic DNA fragments generated with Sau3A. The resulting clones may be introduced into other Gram-negative bacteria by conjugation.

I~TRODU~iON

We have worked with several bacterial plant pathogens in the genera P~eud~~on~s and Elixir. DNA can be introduced into these bacteria via triparental conjugational matings with cells containing

Corvespondence ogy, University Riverside,

fo: Dr. N.T. Keen, Department of California,

CA 92521 (U.S.A.)

Room

of Plant Patbol-

2401 Webber

Hall East,

Tel. (714) 787-4134.

plasmids and cosmids based on’ the broad-hostrange vectors RK2 and RSFlOlO (Ditta et al., 1980; Frey et al., 1983). However, the available derivatives of these plasmids suffer from several deficiencies, such as antibiotic resistances, which are not useable in some of the bacteria noted above, and an in-

thiogalactopyranoside; moter, promoter DNA replication; cycline;

Abbreviations:

Ap, ampicillin;

bp, base pair(s); cos, cohesive site

of phage 2 DNA; ExoIII, exonuclease

side.

III; IPTG, isopropyl-&D-

0378-t 119/88JK!3.50 D 1988 Elsevier Science Publishers B.V. (Biomedical Division)

XGal,

kb, 1000 bp; Km, kanamycin;

of the E. coli j%gaIactosidase R, resistance;

lac pro-

gene; ori, origin of

ss, single strand(ed);

Tc, tetra-

5-bromo-4-chloro-3-indolyl-&D-galactopyrano-

192

plJC128

/--NotI--\ /

/EcoRI\

----BstXJ____-\

/SacI-\

/-Sacll\

/-Eagl-\

ATGACCATGATTACGAATTCGAGCTCCACCGCGGTGGCGGCCGCTCTA ile met thr met thr asn ser ser ser thr vu1 ala

BamHIvSmal-\

/-Xbal-v-Spel-\/

ala

ala

ala

let4

GAACTAGTG leu val g/u

leu

ser

ile

/-Pstl-vEcoRI\/EcoRV\/HindlllV_Clal-\

GATCCCCCGGGCTGCAGGAATTCGATATCAAGCTTATCGATA asp pro pro gly cys arg asn ile ser ser ser

I-Nsil-\ /-Sall-v-Xhol-\

/Apal-\I-Kpnl-\

/-Sphl-vHindlll\

CCGTCGACCTCGAGGGGGGGCCCGGTACCGATGCATGCAAGCTTC... pro ser thr ser gly pro val pro met his arg gb

puc129

ala

ser

phe

..,

/-Nsil-\ /HindIll\/-Sphl\

/-Kpnl\

ATGACCATGATTACGCCAAGCTTGCATGCATCGGTACCGGGCCCC met thr met ile thr pro ser leu his aEn ser val

I-Xhol\ CCC TCG pro ser

/-SalI-\

/-Apal\ pro

gb

pro

/-Clal-vHindlllvEcoRVvEcoRlv-Pstl-VSmal-\/

AGG TCG ACG GTA TCG ATA AGC TTG ATA TCG AAT TCC TGC AGC CCG GGG ser thr val ser ile ser 2eu ile ser asn ser ser pro gly arg cys

/ ---Notl--\

-BamHIv-Spel-\

/Xbal-\

/-Eagl\

/ ---BstXI---\ /-Sacll\

/-Sacl-vEcoRI\

GAT CCA CTA GTT CTA GAG CGG CCG CCA CCG CGG TGG AGC TCG asp pro leu vu1 leu g1f.h arg pro pro pro arg trp ser ser

AAT asn

TCA ... ser ...

Fig. 1. Polylinker regions of pIJC128 and pUCl29 with several new cloning sites and clusters of sites yielding 3’-ss termini at both ends for use with ExoIIIjSl deletions. Remaining plasmid sequences are the same as in pUCll8 and pUC119, respectively (Vieira and Messing, 1987). For the construction of pUC129, DNA of pUCl19 was digested with Hind111 and E;pnI and the resulting large DNA fragment was ligated with the synthetic ss oljgodeox~u~leo~de 5’-AGC~~AT~ATCGGTAC-3’ which had been phospho~lated with ATP and T4 DNA kinase. This DNA was transformed directly into E. c&i DH5a (Bethesda Research Laboratories). Blue transformants were analysed and 5 of 30 colonies yielded piasmids which contained au introduced NsiI site but lacked the normal restriction sites occurring between KpaI and Hind111 in the pUCll9 polylinker. One of these plasmids was retained and called pUC127. This plasmid was digested with KpnI -t-SstI and the large fragment was isolated from a soft agarose gel (Crouse et al., 1983). It was ligated with DNA of pBluescript KS (Stratagene) which had been digested with KpnI + SstI to release the internal portion of the polylinker (encompassing sites for Kpd, ApaI, XhoI, SalI, CZaI, HindIII, EcoRV, EcoRI, PstI, SmaI, BamHI, SpeI, XbaI, EugI, NotI, SucII, &XI and SstI). One of the transformed plasmids was selected which contained the internal polylinker, as evidenced by an SstII site not present in pUCl19 or pUC127, but also contained SphI andNsi1 sites, present in pUC127 but not in pBluescript KS. This plasmid was called pUCl29 and its polylinker sequence was confirmed by nucleotide sequencing (see sequence shown in the figure). For construction of pUC128, DNA of pUC129 was digested with SphI + &I, and the polylinker was ligated with pUCl18 DNA digested with the same enzymes. The resulting plasmid was called pUC128 and its polylinker sequence was confirmed to be that shown in the figure (by nucleotide sequencing).

I93

suf&ient cosmid

number vectors

of useable

pLAFR1

et al., 1982; Staskawicz

cloning

and pLAFR3

sites.

1987) were rn~~~

The

pUC12X

(Friedman

polylinker

et al., 1987) have also been

useful in our work but require several manipulations

batteries

for vector

preparation

linkers

packaging

of concatemerized

in order

to prevent

We

therefore constructed new plasmids and cosmids which are useful for cloning in E. coli as well as transfer to several other Gram-negative bacteria.

EXPERI~E~A~

cloning

in Fig. 1 to create

In addition

to the added

sites in the new plasmids,

of restriction

the

sites at either end of the poly-

yielding 3’ -termini are especially useful for unidirectional ExoIII/S 1 nuclease deletions (Henikoff, 1984). Thus, following deletions of cloned DNA segments and plasmid religation, inserts can readily be sequenced in the same plasmids (Vieira and Messing, 1987) and transferred to the broadhost-range plasmids with EcoRI and/or Hind111 or with other remaining poiylinker sites. All the usual advantages of the pUC plasmids such as high copy number and XGal color screening are available with pUC128 and pUC129. Thus, the plasmids produce a functional fl-galactosidase CIfragment, so that inserts into the polylinker sites may readily be detected

phage

vector molecules.

as described

and pUC129.

AND DISCUSSION

(a) Construction of pUC128 and pUC129 In order to increase the available polylinker cloning sites, pUC118 and pUCll9 (Vieira and Messing,

AccI Hind

HincII Sph I, Pst I, Sol I, XboI,

Born HI,

Smo I, Kpn I, Sst I, EcoRI

Y

Act I HinclI (PstI, Sal I, XbaI,

BornHI, SmaI, KpnI, SstI, Ecc RI>

Act

Act

m,

I

I

maps ofpDSK509 and pDSK5 19,plasmids with improved cloning sites derived from pRSF1010. For the construction ofpDSK509 (left), RSFIOIO DNA was digested at the PvuII sites, and the large DNA fragment was purified and ligated with an approx. 1.7-kb PvuIl fragment from Tn903 encoding Km resistance (Oka et al., 1981). This plasmid was then digested with PstI +EcoRI and the large fragment was ligated with pUC19 DNA (Norrander et al., 1983)that had been digested with the same enzymes. Km-resistant, Ap-sensitive E. coli transformant colonies were selected and their plasmids characterized. Several were found to contain X&I, KpnI and BarnHI sites not present in pRSF1010. After further confirmatory mapping, one ofthese plasmids was retained and called pDSK509. Fig. 2. Restriction

For construction

of pDSK519

followed by Sl nuclease sites of pDSK509

was electroeluted

I to insure the presence region and polylinker

XGal + IPTG plates were characte~zed was retained

and designated

DNA was digested

1984) and a DNA fragment,

had been deleted,

E. coli DNA polymerase La~Z~-peptid~coding

(right), pDSK509

(Henikoff,

pDSK519.

with PstI + BarnHI.

in which approx.

from an agarose

The large fragment

1.4 kb of DNA between

gel. This fragment

was then treated

of blunt ends and ligated to the 454-bp Hue11 fragment

(shown

by cross hatching

by restriction

enzyme mapping

in the figure). The plasmids

was treated

the &I

with ExoIII

and proximal

with Klenow

from pUCl19

containing

in blue tr~sfo~~t

and one of them with the inserted

fragment

PvuII

fragment

oriented

colonies

of the on

as shown

194

by loss of blue color on XGal media (Norrander et al., 1983). The color intensity of E. coedJM109 cells carrying the new plasmids appeared to be somewhat less on XGal plates than with pUC118 and pUC119, but this problem has not caused difficulties in routine cloning exercises. (b) Construction

of pDSK509 and pDSK519

The sulfanilamide and streptomycin resistances conferred by the broad-host-range plasmid RSF1010 are not generally usefti because many bacteria are relatively insensitive to these antibiotics and the plasmid has a limited number of unique cloning sites (e.g., see Frey et al., 1983). In order to construct a more useful vector, pDSK509 was constructed from RSFlOlO as indicated in Fig. 2. This plasmid should have utility as a broad-host-range vector because KmR is a widely useful marker and the plasmid has single polylinker sites for PstI, SalI, XbaI, BumHI, KptzI, EcoRI, HpaI and SstII. SstI sites occur twice in pDSK509 and the Hind111 site of the pUC19 polylinker was not retained because it occurs in the KmR gene. The H&II and SmaI bluntend restriction sites of the polyl~ker are also not readily useful for cloning because they occur elsewhere in the plasmid, but the unique HpaI site constitutes a convenient cloning site for blunt-ended restriction fragments. pDSIS509 was also modified to utilize XGal color screening for detection of recombinant plasmids, yielding pDSK519 (Fig. 2). This plasmid has a relatively high copy number in E. cob’ and Pseudomonas syringae and the lac promoter may be used to express genes cloned in the proper orientation. Yields of pDSK509 and pDSK519 DNA were approx. live times higher when extracted from E. coIi cells with the preparative rapid boiling method (Eisenberg and Holmes, 1982) as compared to the alkaline extraction method (Maniatis et al., 1982).

(Fig. 31, permits cloning into all of the polyIinker restriction sites of pRK404 as well as the additional unique EcoRI, XbaI, KpnI and SstI sites. The &&I site of the pUC19 polylinker is not generally useful because an SphI site occurs elsewhere in the plasmid. The unique DraI, ApaI, SmaI and EcoRV sites are convenient for mapping the orientation of inserted DNA fragments into the polylinker sites. Since pRK415 retains the Euc promoter of pRK404, bacterial genes inserted in the proper orientation into the polylinker should be expressed in E. coli. XGai color screening can also be used for plasmid cons~ctions in E. co& pRK415 has proven useful for subcloning and maintaining small DNA fragments in field isolates of P. syringae pv. glycinea and other P. syringae pathovars. If fragments larger than approx. 5 kb are cloned, however, from a few to more than 50 y0 of the P. syringae exconjugants have been observed to suffer deletions in the inserted DNA.

'Hin~~.S~hI,PstI,SolI,XboI, HincU

Fig. 3. Map ofpRK415, polylinker

of pRK415

digested

with EcoRI

BAL 31. Fragments were religated

Ditta et al. (1985) constructed the moderatelysized cloning vector pRK404 from pRK290. In order to increase the cloning usefulness of this plasmid, the EcaRI site outside the polylinker was deleted and the polylinker, derived from pUC9, was replaced by the pUC19 polylinker. The resulting construct, pRK415

plasmid

a derivative

ofpRK404

cloning sites. Plasmid pRK404

partially

(c) Construction

BarnHI,SrnoI,i(pn~,~st~,~~~~~'

AccI

and the DNA then treated

in which approx.

and transformed

with several new

(Ditta et al., 1985) was

into E. coli JM109. The resulting

from a blue colony on an XGal plate was digested

EcoRI + HindIII,

and the large DNA fragment

the small polylinker enzymes.

fragment

of pUC19 digested

This DNA was transformed

colony on XGal medium was retained designated Restriction

with

400 bp had been deleted with

was ligated with with the same

into strain JM-101, a blue and the resuitant

plasmid

pRK415. The deleted EcoRI site is shown in brackets. sites separated

by a slash occur close together.

195

Partial

;

Hint II

isolate

singly

restricted

Restrict

fragments

and

Restrict clone

into

clone

pBR322

into pBR322

pLAFR-5 -21.5 kb

6.0

t

Restrict

Restrict

with

BglIt

Pst I ;

Restrict

EcoRI;

with

Bst EII

t

with

EcoRI and Sco I ; isolate

EcoRI Sco I

- 400 bp fragment

- 4.7 kb fragmeni

;

Apa I

16.0

and isolate

Born HI,

Smo I,

Eco RI

Restrict

with and

ligate,

EcoRI

transform

/

Eco RI

HI Restrict with Hint II; EamHI linker; isolate -8OObp

fragment

.SalI

Fig. 4. Construction packaging

ofvector

of pLAFR5, concatamers.

a cosmid

vector

containing

tandem

cos sequences

which facilitate

An approx. 400-bp HincII fragment

containing

the cos sequence

with a ScaI linker at one but not both H&c11 termini, and the resulting

fragments

(in pCosle

to yield pCos2. The double cos insert was then removed to ligation BamHI

with pBR322

DNA to yield pCos2BB.

and ligated to pLAFR3

HB-101 transformants of this plasmid

was constructed

Finally, the approx.

for plasmids

800-bp tandem

and pcoslp)

cos fragment

and prevent

1983) was ligated

were both cloned into pBR322

from pCos2BB

linker prior

was removed

1.6-kb cos sequence.

of the double cos sequence

was retained

and cdled

was determined

pLAFR5.

using DvaI.

with

Tc-resistant

in which the double cos casette was ligated into the BglII site of pLAFR3,

and vector Bg111 sites. One of these plasmids and the orientation

preparation

with HincII and both ends ligated with a BamHI

DNA which had been digested with BglII to remove its single approx.

were screened

in loss of both the insert BamHI

from this plasmid

vector

from pCos0 (Hohn,

resulting

A restriction

map

196

(d) Construction

of pLAFR5

The cosmid vector pLAFR3 (Staskawicz et al., 1987) permits the cloning of Sau3A generated insert DNA fragments into a polylinker BamHI site. Vector religation can also be minimized by prior digestion of the vector in two batches, one with EcoRI and the other with HindHI, phosphatasing each and phenolextracting before a second digestion of the pooled DNAs with BarnHI and ligation with insert DNA. This procedure is unnecessary, however, if a plasmid with tandem cos sequences separated by a unique blunt-end restriction site is used, as was shown by Bates and Swift (1983). Accordingly, we const~cted the new cosmid vector pLAFR5 by modifying pLAFR3 to include tandem cos sequences with a unique ScaI site between them (Fig. 4). Plasmid pLAFR5 is used for cloning by digesting with ScaI + BamHI and, following phenol extraction and ethanol precipitation, ligating to unsized but phosphatased or to size-fractionated (on sucrose gradient; approx. 30 kb) DNA fragments generated via partial digestion with Sau3A according to IshHorowitz and Burke (198 1). The ligations are conducted in 5 mM ATP to suppress blunt-end ligation (Feretti and Sg~~ella, 198 1) so that vector dimers lacking inserts are not packaged in phage 2 heads. E. coZz’ DH-1or HB-101 cells are transduced according to Staskawicz et al. (1984) and TcR colonies screened for growth on L agar cont~ning 25 pg Tc/ml and mitomycin C at 1 gg/ml. For bacteria containing a recA gene capable of complementing the recA - mutations in the E. co& strains, this permits a ready test of library adequacy (Hicks et al., 1987). In our experiments with pLAFR5 DNA libraries from several Gram-negative bacteria, the number of mitomycin resistant colonies varied from one in 200 to one in 550 colonies. Since the small arm of pLAFR5 contains the oriY region essential for replication in E. coli (Smith et al., 1984), ligation and packaging of two long vector arms through their BamHI sites will not result in viable E. coli transductants. We and our colleagues have routinely obtained several gene libraries with 1% or less of the clones lacking suitable inserts. The use of insert DNA that has not been size-fractionated has occasionally resulted in some clones with unexpectedly small DNA inserts (approx. lo-15 kb). For this reason, it is probably safer to size-fractionate the

Sazl3A-digested insert DNA on sucrose gradients. As with pLAFR1 and pLAFR3, cosmid clones constructed in pLAFR5 can be transferred to Pseudomonas spp. and other bacteria by conjugational matings (Ditta et al., 1980). We have observed that DNA cloned in these vectors is considerably more stable upon introduction into P. syringae pv. glycineu than DNA maintained in cosmid vectors based cgn RSFlOlO or pRK404 unpublished obse~ations). However, as with pRK4 15 and other relatively small RK2 derivatives, pLAFR5 clones rapidly cure when antibiotic pressure is removed. (e) ~onciusions

The improved vectors described here should facilitate the cloning, sequencing and expression of genes from various Gram-negative bacteria. Selected cosmid clones in pLAFR5 can be subcloned into pUC128 or pUC129 and sequencing may be performed directly on the plasmids following construction of an ordered series of ExoIII/S 1 nuclease deletions. For expression of these genes in various Gram-negative bacteria, the pUC128J129 inserts can readily be transferred to the broad-host-rage vectors we have constructed via approp~ate flanking polylinker restriction sites. Finally, these plasmids can be introduced into the bacterium of choice, via triparental conjugations, for expression studies.

ACKNOWLEDGEMENTS

We thank Dr. B. Hohn for the generous gift of pCos0 DNA, Dr. G. Ditta for pRK404 and Dr. J. Messing for pUC118 and pUC119. Several colleagues, including D. Cooksey and J. Leary, tested some of the new plasmids.

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