Construction of conjugative shuttle and suicide vectors for Pasteurella haemolytica and P. multocida

Gene, 145 (1994) 81-85 Q 1994 Elsevier Science B.V. All rights reserved. 0378-l 1i9/94~$07.~

81

GENE 08007

Construction of conjugative shuttle and suicide vectors for Pasteurella haemolytica and P. multocida (Cloning vector; ZacZcr;multiple cloning site; mob gene; conjugative transfer; ColEl-type

replicon, oriR6K replicon)

Abul K. Azad, John G. Coote and Roger Parton Department of Microbiology,

University of Glasgow, Glasgow G12 8QQ, UK

Received by J.R. Kinghorn: 3 January 1994; Accepted: 4 February 1994: Received at publishers: 7 April 1994

SUMMARY

A shuttle cloning vector, pAKA16, and suicide derivatives pAKA19 and pAKA22 have been developed for gene transfer to Pasteurella haemolytica and P. multocida. pAKA16 was constructed by insertion of the la&&peptide-encoding region and a multiple cloning site into a plasmid which was originally isolated from P. haemolytica serotype Al. The vector encodes ampicillin resistance, and contains at least 14 unique restriction sites and the property of phenotypic identification of recombinant clones in Escherichia coli by insertional inactivation of P-galactosidase activity. It can be transferred by conjugation to P. haemolytica or l? multocida and is stably maintained in both species. The type-II chloramphenicol acetyltransferase-encoding gene (cat), cloned into pAKAi6, was stably expressed in both P. haemolytica and l? m~ltocida. Plasmids pAKAf9 and pAKA22 were constructed by replacement of the origin of DNA replication (ovi) of pAKA16 with a ColEl-type ori from pBR322 or an ori of piasmid R6K (oriR6K) from pJM703.1, respectively. These derivatives replicate in E. co&, but not in either I? haemolytica or P. multocida, and are suitable for use as suicide vectors for these Paste~re~la species.

INTRODUCTION

Pasteurella haemolytica and P. multocida are economically important bacterial pathogens of domestic ruminants and produce a number of putative virulence factors. Correspondence

to: Dr. J.G. Coote, Department of Microbiology, University of Glasgow, Glasgow G12 8QQ, UK. Tel. (44-41) 339-8855, ext. 5845; Fax (44-41) 330-4600; e-mail: [email protected]

Abbreviations: A. absorbance (1 cm); Ap, ampicillin; j3Gal. h-galactosidase; BHI, brain heart infusion; Bla, S-lactamase; bp, base pair(s); cat, gene encoding Cm acetyltransferase; CIAP, calf intestinal alkaline phosphatase; Cm. chloramphenicol; dNTP, deoxynucleoside triphosphate; kb, kilobase or 1000 bp; Km. kanamycin; LB, LuriaBertani (medium): MCS, multiple cloning site; mob, gene required for plasmid mobilization; nt, nucleotide(s)~ ori, origin of DNA replication; P., Pasreure~lu; PolIk, Klenow (large) fragment of E. coli DNA polymerSm. st~ptomycin: XGal, 5-bromo-4ase I: R, resis~nce/resistant; c~~oro-3-indolyl-~-D-galactopyranoside: [J, denotes plasmid-carrier state; ::, novel junction (insertion or fusion). SSDI 0378-1119(94)00218-H

Elucidation of the role of these factors in disease has been hampered by a lack of genetic analysis, due to the unavailability of shuttle vectors for transfer of cloned genes between E. coli and Pasteurella or for transposon mutagenesis of individual virulence determinants. Previous work in our laboratory showed that broadhost-range plasmids, such as pSa4 and pRK290, could not be propagated in P. haemoiytica, but we identified several small ApR plasmids from serotype Al strains of F. haemolytica that could be transferred between E. coli and P. haemolytica by electroporation or conjugation (Craig et al., 1989; Azad et al., 1992a). These plasmids encoded a ROB-l-type Bla, and one such plasmid, pPH843, was readily amplified in E. coli (Azad et al., 1992a). Here we report the construction of three vectors, derived from the naturally occurring plasmid pPH843, which can be mobilized by conjugation between E. coli and Pasteurella species.

82

/

(5.3 kb)

I

Ndel

OiW-fii with Awl + ApaLl ‘Filling in” (WIIK) \

Diiesfii

Thai

(ApaLl /Oral) .

/

/

pAKAlf3

tiemd&&km ‘Rimming’ (r4 DNA pciymerase)

I

Ndsl ACCI

Ligadonend bansfamation (Aval I Pwll)

Fig. 1. A restriction map of plasmid pPH843 and construction of cloning vectors pAKAl6 and pAKA19. Abbreviations: ori or m-i(P), origin of DNA replication of E. coli (E) or Pasteur& (P) plasmid; mob, mobility region required for conjugative transfer (the regions to which the mob gene extends beyond the PstI and 7’uqI sites are not known and are represented by the hatched lines); la&a, the oc-peptide of ~-galactosidase~ncoding sequence (striped box); iacl’P0, regulatory region ~promotcr-operator and part of the I gene; stippled box) for 1acZ; MCS, multiple cloning site (blackened box) (only shown in detail in the pAKA19 map). Piasmids are not drawn to scale and only relevant restriction sites are shown on the maps. Restriction enzymes shown in bold type in the MCS of pAKA19 have two sites in the vector. Restriction sites displayed in parentheses were destroyed by DNA polymerase. Arrows inside the circular maps indicate the direction of transcription. pPH843 is cleaved by only a few restriction enzymes with a 6-bp recognition sequence, but pAKA16 contains at least 14 unique restriction sites with insertional inactivation of bGa1 activity for recognition of recombinant clones in E. coli. Methods: Plasmid DNA was amplified in E. coli (Azad et al.. 1992a) and extracted by the modified ~kaline-lysis method (Sambrook et al., 1989) or with a QIAGEN plasmid kit (DIAGEN), according to the manufacturer’s instructions. Plasmid DNA was further purified by a modified acid-phenol extraction method (Azad et ai., 1992b). Restriction endonucleases (Gibco-BRL, Paisley, UK; Stratagene, Cambridge, UK) and agarose (type HA, Sigma or low-melting point ultraPureTM; BRL) were used for DNA restriction analysis and a GENECLEAN II@ kit for restriction fragment purification, according to the suppliers’ instructions. (i) Mapping of plasmid pPH843 was done by a combination of single- and double-enzyme digestions, which allowed the relative cleavage positions of the enzymes to be determined from the estimated sizes of different restriction fragments. The underlined and the asterisked (*) Z’ayl sites in the pPH843 map represent those used for enzymatic manipulation (see section a). (ii) Plasmid pPH843 was linearized by ApaL digestion and ligated to a 979-bp ApaLl fragment from pICZOH, which contained lacZa, MCS, Eucl’PO flanked on either side by portions of pIC20H, the larger of which contained a non-functional portion of WI‘(E), to generate pAKA16. The construct was transformed into E. coii DHSa ~~8Od~ucZAMl5 A((lacZYA-argFJU169 endA recA1 hsdl7(r,m:) deoR t&-l supE44 h- gyrA96 relAl), selected as blue clones on LB agar containing XGal and Ap, and confirmed by restriction analysis. A 1.9-kb fragment,

83 EXPERIMENTAL

AND DISCUSSION

(a) Restriction mapping and analysis of plasmid pPH843

Plasmid pPH843 is a small (4.3 kb), ROB-l-type Blaencoding plasmid, derived from a bovine isolate of P. haemolytica serotype Al (Azad et al., 1992a). This plasmid was considered suitable for development of a shuttle vector because of its stability and ready amplification in E. coli, and high mobilization frequency to P. haemolytica (Azad et al., 1992a). A restriction map of pPH843 was constructed (Fig. 1). The plasmid has unique sites for the restriction enzymes &I, AuaI, Bsp12861 and ApaLI. Removal of the small 60-bp ScaI fragment had no effect on plasmid functions, including its transfer to P. haemolytica. The putative regions containing the ApR gene and the ori were located by deletion experiments using DraI and Thai, or by digesting the plasmid at the unique Bsp12861 or ApaLI site followed by religation of the linear plasmid after removal of protruding ends or filling recessed ends, respectively. E. coli transformants were obtained only with plasmid molecules religated at the modified ApaLI site. The location of the ApR gene was further deduced from the similarity to the restriction map of the cloned ROB-l Bla-encoding gene from a similar P. haemolytica ApR plasmid (Livrelli et al., 1991). A plasmid derived from the religated 2.6-kb TaqI fragment (bounded by the underlined and the asterisked 7’aqI sites on the pPH843 map in Fig. I), which contained the ApR and ori regions, could not be mobilized by conjugation from E. coli to P. haemolytica. This indicated that the native mobilization (mob) functions of pPH843 were located within the remaining portion (1.7 kb) of the plasmid. This was supported by observations with two other constructs (pAKA15 and pAKA17), obtained by cloning either a 485-bp Hue11 fragment or a 2032-bp AcyI fragment from pIC20H (Marsh et al., 1984) into the PstI or the asterisked TaqI site, respectively, of pPH843 (Fig. 1). Neither of these constructs was found to be mobilizable to Pasteurella.

(AlS), P. multocida P1552 (Hirsh et al., 1981) and E. coli JC3272 (his, lys, trp) recipients (Table I). Some of the Pasteurella colonies which grew on the initial selection agar (BHI agar + Ap + Sm) proved not to be true transconjugants and failed to grow on further subculture on the selective medium. This was probably due to localized inactivation of Ap in the medium as a result of the potent action of the ROB-l type Bla (Azad et al., 1992a) elaborated by the Pasteurella plasmid-derived vectors. Initially, vector pAKA16 was mobilized to P. haemolytica AlS at a low frequency (approx. low4 transconjugants per donor), but when the pAKA16 DNA, prepared from P. haemolytica, was transformed back into E. coli SMlO, the frequency of retransfer to P. haemolytica AlS was signifiTABLE

I

Mobilization

of vectors

pAKA16 (Fig. 1) could be mobilized by conjugation from E. coli donor strain SMlO (RP4-2-TcR::Mu, KmR) (Simon et al., 1983) to SmR strains of l? haemolytica Al

and pAKA19

from E. coli SmlO by

Donorb

Recipient’

SMlO[pAKA16]

P. haemolytica AlS

lx

SMlO[pAKA16]

P. multocida

1.2 x 1o-4

SMlO[pAKA16] SMlO[pAKA19]

E. cob JC3272 P. haemolyrica AlS

0.25 0

SMlO[pAKA19]

P. multocida

0

SMlO[pAKA19]

E. coli JC3272

“Conjugation

was

performed

Mobilization

P1552

P1552

by

frequencyd

1o-3

0.28 plate-mating

of late

exponential

growth-phase cultures as described by Bradley et al. (1980). Overnight cultures of donor and recipient strains were diluted 20 times into fresh (E. cob) or brain-heart

LB broth strains)

and grown

infusion

at 37°C with shaking

growth phase (approx. of donor and recipient

(BHI)

(Pasteurella

broth

until the late exponential

3-4 h; A,,, nm 1.00). Equal volumes (0.5 ml each) were mixed and a portion (0.3 ml) of the mixture

spread onto prewarmed

BHI agar, which was incubated

surface upper-

most at 37°C. Mating proceeded for at least 2 h and then was stopped by centrifugation and resuspension of cells in saline (3 ml). A O.l-ml aliquot

of the suspension

agar containing transconjugants.

Ap

or a suitable

and

Sm

(each

bE. cob SMlO was used as the mobilizing transfer

functions

incorporated transformed

(Tra)

dilution

was spread

100 pg/ml) donor

of the broad-host-range

for

strain. IncP

onto BHI

selection It contains plasmid

of the RP4

into the chromosome (Simon et al., 1983). Vectors were into SMlO by the rapid protocol of Chung et al. (1989).

‘P. haemolytica and

(b) Construction of pAKA16 and its conjugative properties

pAKA16

conjugation”

P. multocida recipients

mutants isolated in our laboratory. dFrequency of mobilization is expressed

were spontaneous

as the number

SmR

of transconju-

gants per donor cell. Each value shown is the mean from two or three independent experiments. Vector pAKA19 did not yield any viable transconjugants with Ap-selective agar.

either

P. haemolytica or P. mukocida on the

containing ori and lacZa-MCS, was then deleted from pAKA16 (see also section c) by digestion with ha1 + ApaLI. The ends of the remaining 3.4-kb fragment of pAKA16 were filled-in with PolIk in the presence of dNTPs and ligated to a 1166-bp PuuII-DraI fragment (cross-hatched box), containing the ColEl-type ori from pBR322 (Gibco-BRL), to produce pAKA18. A 485-bp fragment, containing IacZa-MCS, was isolated from pAKA16 by digestion with HaeII and the fragment was blunted by trimming with T4 DNA polymerase in the presence of dNTPs. pAKA18 was digested with ScaI and dephosphorylated with CIAP and then subjected to blunt-end ligation with the above 485-bp fragment, which resulted in the construction of pAKA19. All ligations were performed according to standard procedures (Sambrook et al., 1989). In each case, the ligation mix was transformed into DH5aTM Library Efficiency competent cells (Gibco-BRL), according to the supplier’s instructions. Transformant clones were selected on LB agar containing XGal and Ap (each 100 m/ml) and identified by direct colour (blue/white) screening of colonies.

84 cantly increased (Table I}. However, this was not true if P. ~~f~oc~~u P1552 was used as second recipient. The reason for the increased frequency in the former case remains unclear, but pAKA16, passaged through P. haemolytica, was used in all subsequent experiments. The shuttle vector was found to be stably inherited for at least 60 generations in both Pasteurella species in the absence of Ap selection. Restriction analysis of pAKA16 prepared from Pasteurella after serial passage, when compared with the original construct prepared from E. coli, showed no evidence of DNA deletion or rearrangement. Frey (1992) described a vector (pJFF2241, derived from the RSFlOlO replicon, which could be introduced by electroporation into Actinobacillus pteuropneumoniae and P. haemolytica. The vector contained a type-11 cut gene as a selectable marker. Here, the corresponding gene from plasmid pSa152 (Tait et al., 1983) was removed as a 1.5kb BamHI fragment and cloned into the MCS of pAKA16; the latter was also digested with BamHI and with CIAP before ligation. dephosphorylated Recombinants selected as white clones of E. coli DHSa on LB-XGal indicator medium containing Ap were then subcuitured on selective agar containing 20 ug Cm/ml. With E. coli SMlO as donor, the recombinant construct (pAKA16:CmR) produced CmR transconjugants at a low frequency with both P. haemolytica and P. multocida (1.2 x lo-’ and 2 x 10T7 transconjugants per donor, respectively) and only when selected at a Cm concentration not exceeding 5 ug/ml. The transconjugants were slow-growing, but stable and did not lose the CmR phenotype after ten subcultures in the absence of antibiotic selection. However, the low-transfer frequency of recombinant DNA indicates the presence of a restriction system operating in Pusteurellu spp. (c) Construction of pAKA19 and pAKA22

We have previously shown that ColEl-based plasmids did not replicate in P. haemolytica (Craig et al., 1989; Azad et al., 1992a) and this has also been reported for P. multocida (Nnalue and Stocker, 1989). Repeated attempts to directly modify pAKA16 into a suicide derivative, by deletion of the 0.9-kb AvaI-NdeI fragment (containing ori of the Pasteurella plasmid; Fig. 1) and replacement with a 2.1-kb Avnl-AseI fragment (containing pMB1 or ColEl-type ori) from plasmid pBR322 (Bolivar et al., 1977), were unsuccessful. The reasons for this are not clear, but may have been due to the presence of a truncated portion of the ColEl-type ori region of pIC20H. included in the 979-bp ApaLI fragment which had been cloned into pPH843 to create pAKA16 (see also section ii of the legend to Fig. 1). The 1.9-kb AuaI-ApaLI fragment spanning the ori and EffcZa-MCS of pAKA16 was, therefore, deleted and the remaining 3.4-kb Avai-ApaLI

fragment blunt-ended by the ‘filling-in’ reaction of PolIk and ligated to a 1166-bp PnuII-DraI fragment containing the ColEl-type ori from pBR322 to produce pAKA18 (Fig. 1). The 485bp HaeII fragment from pAKA16 containing lacZa-MCS was isolated, blunt-ended by the ‘trimming’ reaction of T4 DNA polymerase and then cloned into the ScnI sites (dephosphorylated with CIAP) of pAKAl8 to construct pAKA19 (Fig. 1). Conjugative transfer from E. coli SMlO to P. haemolytica AlS, P. multocida PI552 and E. coli JC3272 showed that pAKA19 was only able to produce transconjugants with E. coli (Table I), although several Pasteurella colonies appeared on the initial selection agar which were not viable upon further subculture on the same medium (see section b). In order to confirm that pAKA19 had been mobilized into Pasteurella in a non-replicative form, the native ori region of the Pasteurella plasmid, contained in a 0.9-kb AvaI-ApaLI fragment of pAKA16, was cloned into the MCS of pAKA19. The resulting construct was mobilized at a low frequency from E. coli SMlO into both P. haemolytica and P. m~ltoeida (data not shown). Finally, another suicide derivative, pAKA22 (4.2 kb), was constructed by a procedure similar to that used for pAKA19 except that, in place of the 1166-bp Pt~uII-DraI fragment from pBR322, an approx. 0.5-kb BnmHI fragment from pJM703.1 (Miller and Mekalanos, 1988) containing oriR6K was ligated, after a filling-in reaction with PolIk, to the 3.4-kb AvaI-ApaLI fragment of pAKA16. The R6K ori is dependent on the activity of the pir gene product (7~ protein) and will only replicate in E. coli strains expressing this protein, such as the h pir lysogens SY327 or SMlO (Miller and Mekalanos, 1988).

(d) Conclusions (1) We have

described the construction of two Pasteurella cloning vectors, pAKAl6 and pAKA 19, which possess the property of blue/white-colony screening for recombinant selection in E. coli. They are mobilizable to P. haemolytica and P multocida by conjugation which provides a more convenient means of gene transfer than electroporation. One vector (pAKA16) contains the native ori from a Paste~rellu plasmid and is suitable for expression of cloned genes in Pasteurella and the other (pAKA19) contains the ColEl-type ori and is suitable as a suicide vector for allelic exchange of cloned Pasteurella DNA or for transposon mutagenesis. (2) A further suicide vector, pAKA22, was constructed which contains oriR6K and will not replicate in strains lacking the pir gene. Although suitable E. coli strains for blue/white-colony screening are not available for this derivative, it offers an alternative counterselection against plasmid replication.

85 plasmid

ACKNOWLEDGEMENTS

which

can

be transferred

Pasteurella haemolytica

We thank R. Aitken for help with computer-drawn plasmid maps. A.K.A. is a Research Fellow of the Wellcome Trust, UK.

Microbial.

and conjugation.

of a broad

host range

D.C., Martin,

L.D. and Rhoades,

in Pasteurella multocida.

ization of the ROB-l Antimicrob.

ROB-l

8-lactamase.

J.

Gen.

Microbial.

138

(1992a)

1185-1196. Azad, A.K., Coote, J.G. and Parton, purification

of covalently

R.: An improved

closed circular

plasmid

method

for rapid

DNA over a wide

H.L.. Boyer,

H.W., Crosa,

characterization

J.H. and Falkow,

of new cloning

vehicles. II. A multipurpose

system. Gene 2 (1977) 95-l 13. Bradley, D.E., Taylor, D.E. and Cohen, mating

systems

Escherichia Chung,

among

and cloning

D.R.: Specification drug-resistance 143

of surface plasmids

S.L. and Miller, R.H.: One-step

preparation

brane

proteins N.A. and

B.A.D.:

Transfer

and properties

J., Fritsch, Manual,

Cold Spring

Harbor,

E.F. and Maniatis,

T.: Molecular

2nd ed. Cold Spring Harbor

of some J. Gen.

Cloning.

Laboratory

A

Press,

NY, 1989.

of

N.J.L.: A

in Vibrio cholerae re-

determinants

natural and suicide replicons in Pasteurella multocida. Microbial. 135 (1989) 3345-3352. Sambrook.

by in-

170 (1988) 2575-2583.

system for in vivo genetic engineering: Gram-negative bacteria. Bio/Technology

217222175. R., Freer, J.H. and Gilmour,

Stocker,

and phage

selection

J.J.: A novel suicide vector and its use in mutations: osmoregulation of outer mem-

and virulence

quires toxR. J. Bacterial.

character-

35 (1991) 242-251.

cloning sites for recombinant Gene 32 (1984) 481-485.

Miller. V.L. and Mekalanos, construction of insertion

of an

haemolytica.

E.J.: The pIC plasmid

Simon, R.. Priefer, U. and Ptihler, A.: A broad

competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc. Natl. Acad. Sci. USA 86 (1989) Craig, F.F., Coote, J.G.. Parton.

Agents Chemother.

vectors with versatile sertional inactivation.

transfer

and molecular

in

( 1980) 1466-1470.

for gene

Agents Chemother.

gene from Pasteur&

8-lactamase

J.L., Erfle. M. and Wykes.

Laboratory

conjugative

coli K-12. J. Bacterial.

C.T., Niemela,

S.: Construction

Marsh,

Nnalue,

size range. Lett. Appl. Microbial. 14 (1992b) 250-254. Bolivar, F., Rodriguez, R.L., Greene, P.J.. Betlach, M.C., Heyneker.

vector

K.R.: Conjugal Antimicrob.

20(1981)415-417. Livrelli, V., Peduzzi. J. and Joly, B.: Sequence

coding

shuttle

cloning and expression in Actinobacillus pleuropneumoniae and other Pasteurellaceae. Res. Microbial. 143 (1992) 263-269. R-plasmid

Azad, A.K., Coote, J.G. and Parton, R.: Distinct plasmid profiles of Pasteurella haemolytica serotypes and the characterization and amplification in Escherichia coli of ampicillin-resistance plasmids en-

J. Gen.

135 (1989) 288552890.

Frey, J.: Construction

Hirsh,

REFERENCES

Escherichia coli and

between

by electroporation

Tait, R.C.. Close, T.J., Lundquist,

host range mobilization

transposon mutagenesis 1 (1983) 7844791.

R.C.. Hagiya,

M., Rodriguez,

in R.L.

and Kado, C.I.: Construction and characterization of a versatile broad host range DNA cloning system for Gram-negative bacteria. Bio/Technology

1 (1983) 269-275.