101 Gene, 148 (1994) 101~105

0 1994 Elsevier Science B.V. All rights reserved. 0378-l 119/94/$07.00

GENE 08151

Construction of isogenic mutants of Pasteurella haemolytica by allelic replacement

(Electroporation; homologous recombination; membrane lipoproteins; mutation; pasteurellosis)

George L. Murphy and Lisa C. Whitworth

Department of Veterinary Pathology, Oklahoma State Uniwrsity, Stillwater, OK 74078, USA

Received by R.E. Yasbin: 8 April 1994; Revised/Accepted: 19 May/20 May 1994; Received at publishers: 6 June 1994

SUMMARY

We describe methods for the mutagenesis of cloned Pasteurella haemolytica (Gram-) genes and for the construction

of P. haemolytica mutants by allelic exchange. We used these methods to construct isogenic mutants of P. haemolytica which no longer synthesize three membrane lipoproteins (Lpp). A single genetic locus, consisting of three tandemly

arranged genes encoding 28-30-kDa membrane Lpp, was replaced with a mutated locus which carries the (3-lactamase-

encoding ApR gene from a 4.2-kb l? haemolytica plasmid. The inactivated locus was introduced into P. haemolytica by

electroporation of a plasmid which carries the mutated locus, but is incapable of replicating in P. haemolytica. Southern

and Western blot analyses indicate that the wild-type locus was replaced by the mutated locus through a double-

crossover recombination event and that the membrane Lpp were no longer produced by the mutant strain. These

methods should be useful in constructing mutant loci which can be used to analyze the roles for various P. haemolytica proteins in the pathogenesis of bovine pneumonic pasteurellosis.

INTRODUCTION

Pasteurella haemolytica serotype Al is the most impor-

tant bacterial pathogen in the development of the often

fatal fibrinous pleuropneumonia in beef cattle known as

pneumonic pasteurellosis (shipping fever pneumonia)

(Frank, 1986; reviewed in Frank, 1989). While there has

been a substantial effort in developing vaccines for the

prevention of bovine pneumonic pasteurellosis, field trials

of these vaccines have met with limited success (Mosier

Correspondence to: Dr. G.L. Murphy, Department of Veterinary

Pathology, College of Veterinary Medicine, Oklahoma State University,

Stillwater, OK 74078, USA. Tel. (l-405) 744-4518; Fax (l-405) 744-5275;

e-mail: gmurphy@vms.ucc.okstate.edu

Abbreviations: Ap, ampicillin: BHI, brain heart infusion; bla, B-lactamase-encoding gene; bp, base pair(s); Cm, chloramphenicol; kb,

kilobase or 1000 bp; Lpp, lipoprotein(s); mAb, monoclonal antibody-

(ies); NaPP,, sodium pyrophosphate; OMP, outer membrane protein:

PAGE, polyacrylamide-gel electrophoresis; e, resistance/resistant; SDS,

sodium dodecyl sulfate; SSPE, 20 mM NaH,PO,.H,0/180 mM

NaCl/8 mM NaOH/l mM EDTA pH 7.0; Tc, tetracycline; wt, wild type.

SSDl 0378-I 119(94)00362-V

et al., 1989; Confer, 1993). The development of a more

effective vaccine will require a thorough understanding

of P. haemolytica virulence factors.

Progress in the development of genetic systems for the

analysis of virulence factors in l? haemolytica has been

slow, impeding the elucidation of their roles in the patho-

genesis of bovine pneumonic pasteurellosis. However,

methods for the introduction of plasmid DNA into P.

haemolytica by electroporation and conjugation have

been reported (Craig et al., 1989). The development of a

broad-host-range shuttle vector for use in P. haemolytica was also recently reported (Frey, 1992). Here, we describe

the first use of electroporation to create, by allelic replace-

ment, isogenic mutants,of P. haemolytica Al.

EXPERIMENTAL AND DISCUSSION

(a) Construction of a mutated Zpp locus The purpose of this study was to develop methods for

the mutagenesis of cloned P. haemolytica genes and for

allelic replacement of a wt P. h~~~d~~li~~t~ genetic locus

with a mutated locus. We chose to mutate a IOCLIS con-

sisting of three tandemly arranged genes (Ippl. 1/~(>-3. ip173)

encoding 2%30-kDa Lpp and under the control of a

single promoter (Fig. 1; Murphy and Whitworth. 1993 ).

As a selectable marker for insertional inactivation of the

genes, we utilized the hllr gene from the 4.2-kb plasmid

of P. harn~o/~~icu Al (strain OK: Newman et al.. 1982).

The mutated /pp locus on the recombinant plasmid

pACAMP was introduced into P. Iw~~wlyticu by

electroporation.

l? Imv~w1~~tic.a mutants in which the wt /P/I locus had

been exchanged with the mutated locus would be Lpp-.

elcctroporation of pAC’AM P.301 into 1). /~~cc,~~~c~/~./;c~cc. nu-

mcrous ApK transformants wcrc isolated. WC c~amincci

these transformants for the presence of /!‘(I_?. /I/U and

pACYC184 by colony blot hybridization. Twenty-three

transformants hybrid&d to all three probes. two hybrid-

ized both with pACYC 184 and the klrt gene probe (X-I?.

8-l?), and me hybridized only with the hlir gene prohc

f 1 I-68).

(b) Western blot analyses

To determine the status of Lpp synthesis in the Lpp

transformants (X- 12. X- I3, II-68 1, we examined whole cell

lysates by Wcstcrn immunoblot assay with murine sera

ApR, and lack vector (pACYC184) sequences. Upon directed against each of the three proteins encoded by

Plasmid Reference or Source Restriction map 1 kb

pRC9

BH sux ATG P Xm B

Craven et al. (1991) I Murphy and Whitworth (1993) pAT153

pACAMP This study

Pig. I

kDa ~12345M

106 - 60 -

49.5 -

32.5 - 27.5 -

M 6 7 6 9 10 M

106 - 60 -

49.5 - V ww

32.5 -

27.5 -

Fig. 2.

Fig. 1. Restriction maps of recombinant plasmIds carrying wt and mutant Ipp locus. The recombinant plasmid pRC’9 carrjcs the wt I? hrcww/~~ric~~~

Al Ipp locus. The plasmid pACAMP carries the mutated locus in the pACYCIX4 vecror(Chang and Cohen, 1978: Rose, 1988). Thin lines rcprcscnt

vector sequences, boxed areas represent cloned DNA fragments. and stippled boxes represent gene sequences. ATG. lppl start codon: W, B~I~~IHI: C‘.

ClaI; El, EcoRI; E5, EcoRV; H, HindIll; P, Psfl: S. SalI: Su. Sau3AI: X, Xh& Xm, X,nnI. Methods: Purified pACAMP (100 ng) was ubed for the

electroporation of P. karmol~ticu (89010807N) a nalidixic acid resistant variant of 89010807 (kindly supplied by Dr. Ron Welsh. Oklahoma Animal

Disease Diagnostic Laboratory) which lacks the /3-Iactamase-encoding plasmid. For electroporation. mid-log phase cells, from tryptose broth cultures

(Difco Laboratories, Detroit. MI, USA), were harvested at 6000 xx for 15 min al 4 C, washed once in 1 vol. of 272 mM sucrose, once in 0.1 LOI. of

372 mM sucrose and once in 0.1 vol. of 15% glycerol, and resuspended in 0.01 vol. of 15’%, glycerol. Cells were electroporated in 0. I-mm gap cuvcttcs

using an Electra Cell Manipulator 600 (Biotechnologies and Experimental Research, San Diego. CA. USAl. Instrument settings were I .F kV and RIO

(720 ohm). After pulsing, tryptose broth (0.9 ml) was added, and cells were incubated at 37 C on a rotary shaker ( 150 rpm) for I 11 and with 7 k’g

Apiml for an additional hour before plating on selective media.

Fig. 2. Western immunoblot of P. huerno/~fictr wt and mutant strains and E. c,oli recombinants. Lanes I and 6. H9OlOX07N /p/l (X-13): 2 and 7. !:.

c,oli DH5a (pACAMP301): 3 and 8. 89010807N lpp (I I-68); 4 and 9, wt 89010807N; 5 and IO. E. i,o/i DH5g (pRC9): M. molecular weight marker\

Lanes I-5 were probed with murine polyclonal serum directed against Lppl. Lpp2 and Lpp3. Lanes 6 10 were probed with mAb 6A6C’l I which

recognizes a 42-kDa OMP of P. huemol~tic~n. Methods: P. huemo/ytiu was grown in BHI broth at 37 C on a rotary shaker at 125 rpm. I:. c,r,/i was

grown in Luria-Broth base (Life Technologies, Gaithersburg, MD, USA) at 37 C on a rotary shaker at 250 rpm. Total cellular proteins wcrc scparatcd

by 0.1%) SDS-IO%PAGE, using a discontinuous buffer system (Laemmli, 1970). For immunoassays, proteins wcrc transferred to nitrocellulos: ( Bio-

Rad Laboratories, Richmond, CA, USA) using the Transblot apparatus ( Bio-Rad) according to the manufacturer’s Instructions. After blocking with

1% gelatm (Bio-Rad), blots were incubated wrth murine anti-Lppl, -Lpp2 and -Lpp3 polyclonal serum or with mAb 6A6Ct I. Antibody-antigen

interactions were detected with af?inity purified, alkaline phosphatase-conjugated. goat anti-mouse IgG (Sigma, St. LouiA, MO, USA). The anti-Lpp

polyclonal serum was prepared as previously described (Sherwood et al.. 1987), by injecting mice with purified recombinant forms of Lppl. Lpp7 01

Lpp3 (SM. Dabo, A.W. Confer, D. Styre and G.L.M., data not shown). MAb 6A6Cl I was prepared as previously described (Sherwood et al.. 1987:

Bandla et al., 1993) by injection of mice with gel-purified 42-kDa OMP from f. h<lrnz,,/~ti~~rr Al (OK). Both immunological probes were prepared b\

the Oklahoma State University Hybridoma Center for Agricultural and Biological Sciences (Stillwater, OK. USA).

103

this locus. The sera recognized proteins of 28%30-kDa in

wt P. haemolytica and in an E. coli recombinant carrying

the cloned lpp locus (Fig. 2). However, in transformants

8-12 (data not shown), 8-13 and 11-68, and in recombi-

nant E. coli carrying the mutated locus, no antigens were

detected.

(c) Southern blot analyses

To determine the structure of the mutated lpp locus in

these transformants, we examined chromosomal DNA by

Southern blot hybridization with several DNA probes

(Fig. 3). A 5.6-kb XbaI fragment and a 3.8-kb Xbal- EcoRI fragment from transformant 11-68 hybridized to

the bla probe (Fig. 3A). XhaI fragments of 8.5 and 5.6 kb

from transformant 8-13 (Fig. 3A) hybridized to the hla probe. None of these transformants hybridized with the

Ipp2-specific probe (Fig. 3C), but 8-13 hybridized with

pACYC184 (Fig. 3B). A 5.6-kb XbaI fragment and a

1.6-kb XhaI-EcoRI fragment from 11-68 hybridized to

the Ipp3-specific fragment (Fig. 3D). The 5.6-kb XhaI

fragment is slightly larger than that seen with the wt

parent (5.5 kb), due to the differences between the mu-

tated and wt loci. Transformant 8-12 exhibited the same

pattern of hybridization as did 8-13 (data not shown).

(d) Double-crossover recombination event

These data suggest that, in transformant 1 l-68, the mu-

tated lpp locus from pACAMP replaced the wt locus,

through a double-crossover recombination event

(Fig. 3E). Additional Southern blot hybridization data

indicated that the BamHI site 3’ of the mutated locus, in

pACAMP301, is not present in 11-68, suggesting that

recombination occurred 5’ of that site (data not shown).

Further restriction mapping of transformants 8-12 and

8-13 (data not shown) suggested that two copies of the

mutated locus, flanking a single copy of the vector se-

quences from pACAMP301, are present in each of those

strains (Fig. 3E).

We also examined the lpp locus and Lpp production

in the twenty three transformants whose DNA hybridized

to all three probes (data not shown). We observed no

plasmid DNA in these transformants. Southern blots of

chromosomal DNA indicated the presence of an intact

lpp locus, and Western immunoblot assays revealed an

immunoreactive band identical in size to that observed

in wt P. haemolytica. These data suggest that a single

crossover event, which did not eliminate the lpp locus, had occurred between pACAMP and the P. haemolyt- ica chromosome in these transformants.

All ApR transformants were susceptible to Cm, suggest-

ing the absence of pACYC184 in those organisms.

However, as mentioned above, restriction mapping of

two transformants (8-12, 8-13), indicated the presence of

a complete copy of pACAMP301, which carries a func-

tional cat gene. These data suggest that this cat gene or

its product is not functional in P. haemolytica. The low number of mutants obtained by allelic replace-

ment in our study is comparable to those reported by

others working with different organisms (Labigne-

Roussel et al., 1988; McDevitt et al., 1992).

Electroporation with larger quantities of plasmid DNA

did not increase the number of mutants obtained, nor

did linearization of DNA (our unpublished observations).

It is possible that P. haemolytica Al may restrict foreign

DNA, thus reducing the number of mutant genetic loci

available for allelic exchange.

(e) A single chromosomal lpp locus

Southern blot analyses in this study and others

(Murphy et al., 1991; 1992; Cooney and Lo, 1993) have

suggested that two lpp loci, with multiple genes, are pre-

sent in P. haemolytica. In PstI + XhaI restriction digests

of wt l? haemolytica chromosomal DNA, two fragments

(3.2 kb and 5.5 kb) hybridize to the lpp2-specific probe

(89010807N, Fig. 3C, lane 6). The lpp locus of wt P. haemolytica contains a PstI restriction site within the

coding sequence of Lppl (Fig. 1). These hybridization

results are consistent with the presence of two lpp loci,

one of which lacks a PstI site within lppl. Others have

seen similar results using a probe from within Lpp-

encoding sequences and ClaI-digested P. haemolytica chromosomal DNA (Cooney and Lo, 1993), although no

ClaI sites are present within the lpp locus.

Our results here indicate that a single lpp locus is pre-

sent in wt P. haemolytica. Mutant strains failed to produce

Lpp, and hybridization data indicated the presence of

only a single mutated genetic locus in these strains. Our

unpublished experiments, utilizing the polymerase chain

reaction, also suggest the presence of a single lpp locus

in P. haemolytica, and that the two fragments which hy-

bridize with lpp2 in PstI + XbaI restriction digests are the

result of incomplete digestion of that site by PstI.

The ability to produce isogenic mutants of P. haemolyt- ica should be a valuable addition to the limited number

of genetic techniques currently available for use with this

organism. This will allow for expanded analyses of viru-

lence factors important in the pathogenesis of bovine

pneumonic pasteurellosis.

(f ) Conclusions

(I) Isogenic mutants of Pusteurella haemolytica were

constructed using the technique of allelic replacement.

(2) The mutants lack the ability to produce three

28-30-kDa membrane lipoproteins.

(3) Southern blot analyses indicate that only one chro-

104

A. kb

;=

7- 6-

5--

4-

3-

2-

c. kb 12 3 4

1 2 3 4 5 kb

-2

.,.

2-

6.

kb

‘90 -

8- 7- 6-

5-

4-

3-

2- +

5 6 7 8 D. 12 3 4

3-

kb

6-

5-

3-

2- -i:

1.6 -

1 kb

H,CS 8-12 8-13

Fig. 3. Southern blot analysis of chromosomal DNA from wt and mutant ( I l-68) and (X-13) P. huemo/y/ictr strains. (A) The P. htremolytic~tr hh gent

(Fig. I ) was used as probe. Lanes: 1. wt. Xhul: 2. wt. Xhul+ EcoRI: 3, I l-68, XhuI; 4. I l-68, XbuI+ EcoRI; 5. 8-13, Xhrtl. (B) The vector pACYClX4

was used as probe. All lanes are Xhul digests: I. wt: 2. I I-68; 3. 8-13; 4. pACYCl84. (C) An Ipp2-specific fragment was used as probe. Lanes: I. wt.

Xhtrl; 2. wt. XbuI+ E(,oRI: 3, I l-68, XbuI: 4, 1 l-68. XbtrI + Ec,oRI; 5. wt. Xbal; 6, wt, Xhal f PstI; 7. 8-I 3. Xbul; 8. 8-l 3. Xbtrl + EcoRI. (0) An /pp$

specific fragment was used as probe. Lanes: I. wt. Xhtrl: 2, wt, Xbtrl + EwRI; 3. I l-68, Xhtrl; 4. I l-68. XbrrI + EcoRI. (E) Restriction endonuclcasc

maps of mutant Ipp loci in transformants 11-6X, X-l:! ‘tnd 8-13. ATG, lppl start codon; B. BtrrnHI; C. ClnI: El, EwRI: H. HindIll: P. Psrl: S, G/I: X. Xbal. Methods: Total DNA purification and Southern blot hybridization analyses were performed as previously described (Murphy et al., IOO?).

DNA probes (25 ng) were labeled with [c(-3ZP]dCTP using a random primer labeling kit (Prime-It II. Stratagene. La Jolla, CA. USA). The Ipp-7.

and Ipp3-specific DNA probes have been described (Murphy and Whitworth, 1993). High-stringency washes were in 3 x SSPE ‘2 x Denhardt’\

(Sambrook et al., 198Y)/O.l’% NaPP,:O.I% SDS. 3 x 15 min at 65 C; I x SSPE./O.l% NaPP,!O.I%, SDS. 2 x 60 min at 65 C: 0.1 x SSPE,O.I”,,

NaPP,Ql”/I~ SDS, 2 x I5 mm at 65 C.

105

mosomal locus harboring the three genes (Ippl, lpp2 and

lpp3) encoding Lpps is present in P. haemolytica.

ACKNOWLEDGEMENTS

We thank Melanie J. Palmer and S. Mady Dabo for

critical review of the manuscript. This work was sup-

ported by grant 92-37204-7773 from the National

Research Initiative Competitive Grants Office of the

USDA, and by project OKL02179 of the Oklahoma

Agricultural Experiment Station. This is manuscript

594-003 of the College of Veterinary Medicine and the

Oklahoma Agricultural Experiment Station.

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