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Possession of identical nonconjugative plasmids by different isolates of pasteurella multocida does not imply clonality
Veterinary Microbiology, 22 (1990) 79-87 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
79
Possession of Identical Nonconjugative Plasmids by Different Isolates of P a s t e u r e U a m u l t o c i d a does not Imply Clonality JACK S. IKEDA and DWIGHT C. HIRSH
Department of Veterinary Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, CA 95616 (U.S.A.) (Accepted 12 September 1989)
ABSTRACT Ikeda, J.S. and Hirsh, D.C., 1990. Possession of identical nonconjugative plasmids by different isolates of Pasteurella multocida does not imply clonality. Vet. Microbiol., 22: 79-87. Experiments were performed to test the hypothesis that possession of the same nonconjugative R plasmid by different isolates of Pasteurella multocida implied that they were of the same clone. Seven isolates ofP. multocida were studied, two possessed an identical nonconjugativeR plasmid (pVM109), four possessed another (pVMll0), and one isolate possessed a nonconjugative R plasmid related to pVM110. Phenotypic and genotypic characteristics of the isolates were determined and compared. Isolates possessing the same nonconjugative R plasmid were shown to be different, and isolates possessing a different nonconjugativeR plasmid were shown to be the same. We conclude that possession of an identical nonconjugative R plasmid by two isolates of P. multocida does not imply clonality.
INTRODUCTION
Pasteurella multocida from turkeys possess nonconjugative (non self-transmissible) R plasmids (Hirsh et al., 1985). Since nonconjugative plasmids, by virtue of their biology, would tend to stay within a particular clone, it has been suggested that characterization of these plasmids (by the use of restriction endonuclease digestion patterns) would be a way of ascertaining the similarity between two isolates of P. multocida, i.e. isolates possessing the same nonconjugative plasmid would most likely belong to the same clone. The purpose of the present study was to test the hypothesis that possession of identical nonconjugative plasmids by isolates of P. multocida implied that they belonged to the same clone. Seven isolates of P. multocida with well-characterized nonconjugative R plasmids were compared with respect to serotype, biotype (subspecies), number and weight of whole-cell proteins and outer0378-1135/90/$03.50
© 1990 Elsevier Science Publishers B.V.
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J.S. IKEDAAND D.C. HIRSH
membrane proteins, restriction endonuclease digestion patterns of chromosomal DNA, and patterns of hybridization with a ribosomal RNA probe. M A T E R I A L S AND M E T H O D S
Bacterial isolates Isolates of P. multocida used in this study were obtained from the tissues of turkeys that had died from fowl cholera throughout the U.S.A. from 1960 to 1975 (Table 1 ). These isolates were previously shown to possess nonconjugative resistance plasmids pVM109, pVMll0, or p V M l l l (Hirsh et al., 1985). Media and growth conditions Bacteria were grown in brain heart infusion (BHI; Difco Laboratories, Detroit, MI) broth or BHI broth containing ~,~'dipyridyl (20 ~g ml-1; Sigma Chemical Co., St. Louis, MO) and incubated overnight at 37 °C with shaking (150 rpm). Capsule typing The possession of the A capsule type was determined nonserologically with hyaluronidase (Sigma Chemical Co.) (Carter and Rundell, 1975). Somatic serotyping Somatic serotyping was performed by the agar gel immunodiffusion method (Heddleston et al., 1972). TABLE1 Source and characteristics of R plasmid carrying isolates of PasteureUa multocida Isolate
Year a
Location b
Plasmid (resistance) c
Somatic serotype d
P1085 Pl167 P1513 P1552 P1845 P1975 P2862
1960 1962 1966 1967 1970 1971 1975
South Carolina Iowa Utah South Carolina California Texas California
pVM109 pVM109 pVMll0 pVMll0 pVMll0 pVMll0 pVMlll
3 3,4 3,4,12 3,4 3 3,4 3
(SmSu) (SmSu) (SmSu) (SmSu) (SmSu) (SmSu) (SmSuTc)
aDenotes year of isolation. bDenotes location in U.S.A. CFrom Hirsh et al. ( 1985 ). Sm = streptomycin, Su = sulfonamides, Tc = tetracycline. dThis work.
IDENTICAL NONCONJUGATIVE PLASMIDS OF P. MULTOCIDA DOES NOT IMPLY CLONALITY
81
Biotyping Biotyping was performed by testing the ability of the isolates to ferment 29 carbohydrates: adonitol, amygdalin, arabinose, cellobiose, dextrin, dulcitol, erythritol, esculin, fructose, galactose, glucose, glycerol, glycogen, inositol, inulin, lactose, maltose, mannitol, mannose, melibiose, melezitose, raffinose, rhamnose, salicin, sorbitol, sorbose, starch, trehalose and xylose (Mutters et al., 1985 ).
Preparation of whole-ceUproteins Whole-cell proteins were prepared from overnight BHI broth cultures as described by Nicolet et al., 1980. The concentration of protein was determined by using a protein assay kit (Bio-Rad Laboratories, Richmond, CA).
Preparation of outer-membrane proteins Outer-membrane proteins were prepared with N-lauroylsarcosine (Sigma Chemical Co.) as described previously (Schnaitman, 1970; Filip et al., 1973; Snipes et al., 1988). Membranes were prepared from BHI broth cultures conraining dipyridyl.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PA GE) Proteins were separated by SDS-PAGE as described by Laemmli (1970). A 3% stacking gel overlaying a 12.5% separating gel was used. The amount of protein per lane was 37.5 ttg of whole-cell proteins and 20/tg of outer-membrane proteins.
Preparation of chromosomal DNA Chromosomal DNA was isolated by a modification of the method described by Hull et al. (1981). Bacteria were grown overnight at 37°C in 100 ml BHI broth with shaking (150 rpm). The culture was centrifuged at 12 000 x g for 20 min at 4°C. The pellet was resuspended in TES (50 m M Tris buffer, pH 8.0 containing 5 m M EDTA and 50 m M NaC1). The suspension was recentrifuged and the pellet suspended in 1.6 ml sucrose solution (50 m M Tris buffer, pH 8.0 containing 25% sucrose, and 1 m M EDTA) and transferred to a 25X89 m m centrifuge tube (Quick-seal, Beckman Instruments, Fullerton, CA). Lysozyme (Sigma Chemical Co.; 0.01 g in 0.4 ml sucrose solution) was added and the cells were placed on ice for 15 rain. Proteinase K (Sigma Chemical Co.; 0.2 mg in 10/~1 water) was added and the suspension was placed on ice for another 15 min. A 0.6 ml solution containing EDTA (333 mM) and N-lauroylsarcosine ( 3.3 % ) in water was added and the mixture incubated overnight at 65 ° C. Thirty ml of TES containing 1 mg phenylmethylsulfonyl fluoride (Sigma Chemical Co.) was combined with 37.83 g cesium chloride and added to the tube. The tube was centrifuged at 123 000 Xg at 15 ° C for 42 h. DNA was collected through the side of the tube with a 16-gauge needle and dialyzed in 10 m M Tris buffer,
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J.S. IKEDA AND D.C. HIRSH
pH 8.0 containing EDTA (1 mM) at 4 °C for 48 h. The concentration of DNA was determined spectrophotometrically at 260 nm (Maniatis et al., 1982). Restriction endonuclease digestion of chromosomal DNA Chromosomal DNA (3 ]~g) was digested with the restriction endonuclease Barn HI (New England Biolabs, Beverly, MA) (Maniatis et al., 1982). Fragments were electrophoretically separated in a 4% polyacrylamide gel with Trisborate-EDTA electrophoresis buffer (89 mM Tris pH 8.0, 89 mM boric acid, 2 mM EDTA) at 30 mA for 2 h. The DNA was stained with ethidium bromide (0.5 zg ml- 1) and visualized with ultraviolet light. Lambda bacteriophage DNA digested with HindIII (New England Biolabs) was used as a molecular weight standard. Hybridization with ribosomal RNA Hybridization with ribosomal RNA (rRNA) was performed by the method of Stull et al. (1988). Escherichia coli rRNA (Sigma Chemical Co.) was dephosphorylated with calf intestinal alkaline phosphatase (Boehringer Mannheim Biochemicals, Indianapolis, IN) and end-labeled with ~2P-ATP (Amersham Corp., Arlington Heights, IL) using T4 polynucleotide kinase (Bethesda Research Laboratories, Gaithersburg, MD) to a specific activity of 107 cpm gg-1 of RNA (Maniatis et al., 1982). Chromosomal DNA (3 #g) was digested with Eco RI (New England Biolabs ) (Maniatis et al., 1982). Fragments were electrophoretically separated in a 0.7% agarose gel with Tris-borate-EDTA buffer at 35 V for 15 h (Meyers et al., 1976). The DNA was stained with ethidium bromide and transferred to a nitrocellulose membrane (0.2 pm pore size, Schleicher and Schuell, Keene, NH) by the method of Southern (1975). The membrane was prehybridized for 1 h at 60 °C in 10 ml prehybridization solution containing 5 × SSPE (1 × SSPE is 10 mM NaH2PQ pH 7.4, 150 mM NaC1, 1 mM EDTA), 0.1% SDS, 5×Denhardt's solution (bovine serum albumin, polyvinylpyrrolidone, and ficoll each at 1 mg ml-1), and 0.1 mg denatured salmon sperm DNA ml-1 (Sigma Chemical Co.). Hybridization occurred at 60°C, gradually decreasing to 40°C overnight, in 10 ml prehybridization solution plus the 32P-labeled rRNA probe (7 × 105 cpm ml-1). The membrane was washed with 0.1 × SSPE, 0.1% SDS at room temperature for 20 min, followed by three more washes at 55 °C for 20 min each. Autoradiography was performed with Kodak X-OMAT AR film (Eastman Kodak, Rochester, NY) and a single intensifying screen at - 70 ° C for 48 h. RESULTS
A previous study had shown that the nonconjugative R plasmids from isolates P1085 and Pl167 were the same (contained pVM109), and the nonconjugative ~ plasmids in isolates P1513, P1552, P1845 and P1975 were the same
IDENTICAL NONCONJUGATIVE PLASMIDS OF P. MULTOCIDA DOES NOT IMPLY CLONALITY
83
AB C DE F G 92.5 6612 45.0 31.0 2t .5 14.0 Fig. 1. Whole-cell proteins separated by SDS-PAGE and stained with Coomassie brilliant blue. Lane A = P1167, Lane B = P1085, Lane C = P2862, Lane D -- P1845, Lane E = P1513, Lane F = 1552, Lane G = P1975. Numbers at the right margin represent molecular mass in kDa.
(pVMl10) (Hirsh et al., 1985). The plasmid ( p V M l l l ) , found in isolate P2862, was thought to be identical to p V M l l 0 except for the insertion of a tetracycline-resistance encoding transposon. All of the isolates possessed the A capsule type. Originally, all were found to be somatic serotype 3 (as reported by the National Animal Disease Laboratory, Ames, IA). However, using reagents made at our institution, we found that three of the isolates were somatic serotype 3, three were 3,4 and one was 3,4,12 (Table 1). All b u t one of the isolates (isolate P1975) were P. multocida subspp, multocida. Isolate P1975 was P. multocida subspp, septica. Isolate Pl167, though P. multocida subspp, multocida, was different from all the other isolates in that it fermented trehalose and raffinose. Inspection of the electropherograms of the whole-cell proteins, outer-membrane proteins, restriction digests of chromosomal DNA, and hybridization with the r R N A probe revealed the following (Figs. 1, 2, 3 and 4). Isolates possessing the same plasmid, P1085 and P l 1 6 7 (pVM109) appear dissimilar, as do isolates P1975 and P1513 ( p V M l l 0 ) . On the other hand, isolates possessing a different plasmid, P1085 (pVM109), P2862 ( p V M l l l ) , P1845 ( p V M l l 0 ) and perhaps P1552 ( p V M l l 0 ) , appear similar. The similarities (dissimilari-
84
A BC
J.S. IKEDA A N D D.C. H I R S H
D E F G - 92,5
- 66.2
-45.0
- 31.0
- 21.5 - 14.0
Fig. 2. Outer-membrane proteins separated by SDS-PAGE and stained with Coomassie brilliant blue. Lane A=Pl167, Lane B=P1085, Lane C-- P2862, Lane D =P1845, Lane E=P1513, Lane F =P1552, Lane G=P1975. Numbers at the right margin represent molecular mass in kDa.
ABCD
E F G
.2322
"2027
-564
Fig. 3. Chromosomal DNA digested with B a m HI and separated by polyacrylamide gel electrophoresis. Lane A=P1167, Lane B=P1085, Lane C--P2862, Lane D=P1845, Lane E=P1513, Lane F = p1552, Lane G = P1975. Numbers at the right margin represent size of fragments in base pairs.
IDENTICAL NONCONJUGATIVE PLASMIDS OF P. MULTOCIDA DOES NOT IMPLY CLONALITY
A
B
C
D
E
F
85
G
Fig. 4. Chromosomal DNA digested with Eco RI, separated by agarose gel electrophoresis, transferred to nitrocellulose, and probed with 32p labeled ribosomal RNA. Lane A=Pl167, Lane B = P1085, lane C = P2862, Lane D =P1845, Lane E=P1513, Lane F=P1552, Lane G= P1975. ties) between isolates are supported by the serotype data, and in the case of P1975, by the biotype results as well. DISCUSSION The experiments were performed to determine whether isolates possessing the same nonconjugative R plasmid belonged to the same clone. Certain phenotypic characteristics (capsule type, somatic serotype, biotype, outer-membrane protein profile and whole-cell protein profile) as well as certain genotypic characteristics (restriction endonuclease digestion patterns, r R N A hybridization) were determined in an attempt to ascertain relativeness between isolates. Those isolates which were similar (P1085, P1845, P2862 and perhaps P1552) were assumed to belong to the same clone, those dissimilar (Pl167, P1513 and P1975) to different clones. It is important to keep in mind that assigning a particular isolate to a particular clone is a bit arbitrary since it is difficult to determine whether differences observed are indications of membership in a different clone, or the result of naturally occurring diversity within a clone. The data do not support the hypothesis that isolates possessing the same nonconjugative R plasmid belong to the same clone. The data show that closely related isolates (P1085, P1845, P2862 and perhaps P1552 ) have different nonconjugative R plasmids, and the more dissimilar isolates (P1513 and P1975) have the same nonconjugative R plasmid. These results might be explained in the following ways.
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J.S. IKEDAANDD.C.HIRSH
After acquiring plasmid DNA, a pasteurella cell, if it continued to evolve, would generate with time, other clones each containing the same nonconjugal plasmid. For example, isolates Pl167 and P1085 could have evolved from a clone that contained pVM109. If, for example, these isolates were obtained shortly after acquisition of the plasmid, it is quite likely that they would be considered members of the same clone. On the other hand, pasteurellae might acquire plasmid DNA at any time. If this were the case, any clone might contain any particular plasmid DNA. But how pasteurellae acquire DNA in nature is not known. The following are ways in which such acquisition might occur. Some avian strains ofP. multocida possess plasmids with sex factor function (Hirsh et al., 1981). These plasmids have been shown to promote the transfer of small nonconjugal R plasmids from pasteurella to pasteurella without promoting their own transfer. However, only one such transfer could occur since the transferred nonconjugal plasmid does not have the ability to promote another round of transfer. Another possibility is that DNA is acquired by different clones by transformation. Since nobody has shown or reported a transformation system in pasteurellae, we feel that this mode of DNA transfer is probably not the manner in which P. multocida acquires DNA. This leaves transduction as the way in which DNA is acquired by pasteurellae, and since avian strains of P. multocida are lysogenized by a variety of bacteriophage, transfer of DNA by this method is an appealing explanation (Kirchner and Eisenstark, 1956). We do not know which of the above is a more accurate description of the acquisition and distribution of nonconjugal plasmid DNA in pasteurellae in nature. It is our guess that both occur.
REFERENCES Carter, G.R. and Rundell, S.W., 1975. Identification of type A strains of P. multocida using staphylococcal hyaluronidase. Vet. Rec., 96: 343. Filip, C., Fletcher, G., Wulff, J.L. and Earhart, C.F., 1973. Solubilization of the cytoplasmic membrane of Escherichia coil by the ionic detergent sodium-lauryl sarcosinate. J. Bacteriol., 115: 717-722. Heddleston, K.L., Gallagher, J.E. and Rebers, P.A,, 1972. Fowl cholera: gel diffusion precipitin test for serotyping PasteureUa multocida from avian species. Avian Dis., 16: 925-936. Hirsh, D.C., Martin, L.D. and Rhoades, K.R., 1981. Conjugal transfer of an R-plasmid in Pasteu rella multocida. Antimicrob. Agents Chemother., 20: 415-417. Hirsh, D.C., Martin, L.D. and Rhoades, K.R., 1985. Resistance plasmids of Pasteurella multocida isolated from turkeys. Am. J. Vet. Res., 46: 1490-1493. Hull, R.A., Gill, R.E., Hsu, P., Minshew, B.H. and Falkow, S., 1981. Construction and expression of recombinant plasmids encoding type 1 or D-mannose-resistant pili from a urinary tract infection Escherichia coli isolate. Infect. Immunol., 33: 933-938. Kirchner, C. and Eisenstark, A., 1956. Lysogeny in PasteureUa multocida. Am. J. Vet. Res., 17: 547-548.
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Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London), 227: 680-685. Maniatis, T., Fritsch, E.F. and Sambrook, J., 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring harbor, NY. Meyers, J.A., Sanchez, D., Elwell, L.P. and Falkow, S., 1976. Simple agarose gel electrophoretic method for the identification and characterization of plasmid deoxyribonucleic acid. J. Bacteriol., 127: 1529-1537. Mutters, R., Ihm, P., Pohl, S., Frederiksen, W. and Mannheim, W., 1985. Reclassification of the genus Pasteurella Trevisan 1887 on the basis of deoxyribonucleic acid homology, with proposals for the new species Pasteurella dagmatis, PasteureUa canis, Pasteurella stomatis, Pasteu reUa anatis, and Pasteurella langaa. Int. J. Syst. Bacteriol., 35: 309-322. Nicolet, J., Paroz, P. and Krawinkler, M., 1980. Polyacrylamide gel electrophoresis of whole-cell proteins of porcine strains of Haemophilus. Int. J. Syst. Bacteriol., 30: 69-76. Schnaitman, C.A., 1970. Examination of the protein composition of the cell envelope of Esche richia coli by polyacrylamide gel electrophoresis. J. Bacteriol., 104: 882-889. Snipes, K.P., Hansen, L.M. and Hirsh, D.C., 1988. Plasma- and iron-regulated expression of high molecular weight outer membrane proteins by PasteureUa multocida. Am. J. Vet. Res., 49: 1336-1338. Southern, E.M., 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol., 98: 503-517. Stull, T.L., LiPuma, J.J. and Edlind, T.D., 1988. A broad-spectrum probe for molecular epidemiology of bacteria: ribosomal RNA. J. Infect. Dis., 157: 280-286.
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Possession of Identical Nonconjugative Plasmids by Different Isolates of P a s t e u r e U a m u l t o c i d a does not Imply Clonality JACK S. IKEDA and DWIGHT C. HIRSH
Department of Veterinary Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, CA 95616 (U.S.A.) (Accepted 12 September 1989)
ABSTRACT Ikeda, J.S. and Hirsh, D.C., 1990. Possession of identical nonconjugative plasmids by different isolates of Pasteurella multocida does not imply clonality. Vet. Microbiol., 22: 79-87. Experiments were performed to test the hypothesis that possession of the same nonconjugative R plasmid by different isolates of Pasteurella multocida implied that they were of the same clone. Seven isolates ofP. multocida were studied, two possessed an identical nonconjugativeR plasmid (pVM109), four possessed another (pVMll0), and one isolate possessed a nonconjugative R plasmid related to pVM110. Phenotypic and genotypic characteristics of the isolates were determined and compared. Isolates possessing the same nonconjugative R plasmid were shown to be different, and isolates possessing a different nonconjugativeR plasmid were shown to be the same. We conclude that possession of an identical nonconjugative R plasmid by two isolates of P. multocida does not imply clonality.
INTRODUCTION
Pasteurella multocida from turkeys possess nonconjugative (non self-transmissible) R plasmids (Hirsh et al., 1985). Since nonconjugative plasmids, by virtue of their biology, would tend to stay within a particular clone, it has been suggested that characterization of these plasmids (by the use of restriction endonuclease digestion patterns) would be a way of ascertaining the similarity between two isolates of P. multocida, i.e. isolates possessing the same nonconjugative plasmid would most likely belong to the same clone. The purpose of the present study was to test the hypothesis that possession of identical nonconjugative plasmids by isolates of P. multocida implied that they belonged to the same clone. Seven isolates of P. multocida with well-characterized nonconjugative R plasmids were compared with respect to serotype, biotype (subspecies), number and weight of whole-cell proteins and outer0378-1135/90/$03.50
© 1990 Elsevier Science Publishers B.V.
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J.S. IKEDAAND D.C. HIRSH
membrane proteins, restriction endonuclease digestion patterns of chromosomal DNA, and patterns of hybridization with a ribosomal RNA probe. M A T E R I A L S AND M E T H O D S
Bacterial isolates Isolates of P. multocida used in this study were obtained from the tissues of turkeys that had died from fowl cholera throughout the U.S.A. from 1960 to 1975 (Table 1 ). These isolates were previously shown to possess nonconjugative resistance plasmids pVM109, pVMll0, or p V M l l l (Hirsh et al., 1985). Media and growth conditions Bacteria were grown in brain heart infusion (BHI; Difco Laboratories, Detroit, MI) broth or BHI broth containing ~,~'dipyridyl (20 ~g ml-1; Sigma Chemical Co., St. Louis, MO) and incubated overnight at 37 °C with shaking (150 rpm). Capsule typing The possession of the A capsule type was determined nonserologically with hyaluronidase (Sigma Chemical Co.) (Carter and Rundell, 1975). Somatic serotyping Somatic serotyping was performed by the agar gel immunodiffusion method (Heddleston et al., 1972). TABLE1 Source and characteristics of R plasmid carrying isolates of PasteureUa multocida Isolate
Year a
Location b
Plasmid (resistance) c
Somatic serotype d
P1085 Pl167 P1513 P1552 P1845 P1975 P2862
1960 1962 1966 1967 1970 1971 1975
South Carolina Iowa Utah South Carolina California Texas California
pVM109 pVM109 pVMll0 pVMll0 pVMll0 pVMll0 pVMlll
3 3,4 3,4,12 3,4 3 3,4 3
(SmSu) (SmSu) (SmSu) (SmSu) (SmSu) (SmSu) (SmSuTc)
aDenotes year of isolation. bDenotes location in U.S.A. CFrom Hirsh et al. ( 1985 ). Sm = streptomycin, Su = sulfonamides, Tc = tetracycline. dThis work.
IDENTICAL NONCONJUGATIVE PLASMIDS OF P. MULTOCIDA DOES NOT IMPLY CLONALITY
81
Biotyping Biotyping was performed by testing the ability of the isolates to ferment 29 carbohydrates: adonitol, amygdalin, arabinose, cellobiose, dextrin, dulcitol, erythritol, esculin, fructose, galactose, glucose, glycerol, glycogen, inositol, inulin, lactose, maltose, mannitol, mannose, melibiose, melezitose, raffinose, rhamnose, salicin, sorbitol, sorbose, starch, trehalose and xylose (Mutters et al., 1985 ).
Preparation of whole-ceUproteins Whole-cell proteins were prepared from overnight BHI broth cultures as described by Nicolet et al., 1980. The concentration of protein was determined by using a protein assay kit (Bio-Rad Laboratories, Richmond, CA).
Preparation of outer-membrane proteins Outer-membrane proteins were prepared with N-lauroylsarcosine (Sigma Chemical Co.) as described previously (Schnaitman, 1970; Filip et al., 1973; Snipes et al., 1988). Membranes were prepared from BHI broth cultures conraining dipyridyl.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PA GE) Proteins were separated by SDS-PAGE as described by Laemmli (1970). A 3% stacking gel overlaying a 12.5% separating gel was used. The amount of protein per lane was 37.5 ttg of whole-cell proteins and 20/tg of outer-membrane proteins.
Preparation of chromosomal DNA Chromosomal DNA was isolated by a modification of the method described by Hull et al. (1981). Bacteria were grown overnight at 37°C in 100 ml BHI broth with shaking (150 rpm). The culture was centrifuged at 12 000 x g for 20 min at 4°C. The pellet was resuspended in TES (50 m M Tris buffer, pH 8.0 containing 5 m M EDTA and 50 m M NaC1). The suspension was recentrifuged and the pellet suspended in 1.6 ml sucrose solution (50 m M Tris buffer, pH 8.0 containing 25% sucrose, and 1 m M EDTA) and transferred to a 25X89 m m centrifuge tube (Quick-seal, Beckman Instruments, Fullerton, CA). Lysozyme (Sigma Chemical Co.; 0.01 g in 0.4 ml sucrose solution) was added and the cells were placed on ice for 15 rain. Proteinase K (Sigma Chemical Co.; 0.2 mg in 10/~1 water) was added and the suspension was placed on ice for another 15 min. A 0.6 ml solution containing EDTA (333 mM) and N-lauroylsarcosine ( 3.3 % ) in water was added and the mixture incubated overnight at 65 ° C. Thirty ml of TES containing 1 mg phenylmethylsulfonyl fluoride (Sigma Chemical Co.) was combined with 37.83 g cesium chloride and added to the tube. The tube was centrifuged at 123 000 Xg at 15 ° C for 42 h. DNA was collected through the side of the tube with a 16-gauge needle and dialyzed in 10 m M Tris buffer,
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J.S. IKEDA AND D.C. HIRSH
pH 8.0 containing EDTA (1 mM) at 4 °C for 48 h. The concentration of DNA was determined spectrophotometrically at 260 nm (Maniatis et al., 1982). Restriction endonuclease digestion of chromosomal DNA Chromosomal DNA (3 ]~g) was digested with the restriction endonuclease Barn HI (New England Biolabs, Beverly, MA) (Maniatis et al., 1982). Fragments were electrophoretically separated in a 4% polyacrylamide gel with Trisborate-EDTA electrophoresis buffer (89 mM Tris pH 8.0, 89 mM boric acid, 2 mM EDTA) at 30 mA for 2 h. The DNA was stained with ethidium bromide (0.5 zg ml- 1) and visualized with ultraviolet light. Lambda bacteriophage DNA digested with HindIII (New England Biolabs) was used as a molecular weight standard. Hybridization with ribosomal RNA Hybridization with ribosomal RNA (rRNA) was performed by the method of Stull et al. (1988). Escherichia coli rRNA (Sigma Chemical Co.) was dephosphorylated with calf intestinal alkaline phosphatase (Boehringer Mannheim Biochemicals, Indianapolis, IN) and end-labeled with ~2P-ATP (Amersham Corp., Arlington Heights, IL) using T4 polynucleotide kinase (Bethesda Research Laboratories, Gaithersburg, MD) to a specific activity of 107 cpm gg-1 of RNA (Maniatis et al., 1982). Chromosomal DNA (3 #g) was digested with Eco RI (New England Biolabs ) (Maniatis et al., 1982). Fragments were electrophoretically separated in a 0.7% agarose gel with Tris-borate-EDTA buffer at 35 V for 15 h (Meyers et al., 1976). The DNA was stained with ethidium bromide and transferred to a nitrocellulose membrane (0.2 pm pore size, Schleicher and Schuell, Keene, NH) by the method of Southern (1975). The membrane was prehybridized for 1 h at 60 °C in 10 ml prehybridization solution containing 5 × SSPE (1 × SSPE is 10 mM NaH2PQ pH 7.4, 150 mM NaC1, 1 mM EDTA), 0.1% SDS, 5×Denhardt's solution (bovine serum albumin, polyvinylpyrrolidone, and ficoll each at 1 mg ml-1), and 0.1 mg denatured salmon sperm DNA ml-1 (Sigma Chemical Co.). Hybridization occurred at 60°C, gradually decreasing to 40°C overnight, in 10 ml prehybridization solution plus the 32P-labeled rRNA probe (7 × 105 cpm ml-1). The membrane was washed with 0.1 × SSPE, 0.1% SDS at room temperature for 20 min, followed by three more washes at 55 °C for 20 min each. Autoradiography was performed with Kodak X-OMAT AR film (Eastman Kodak, Rochester, NY) and a single intensifying screen at - 70 ° C for 48 h. RESULTS
A previous study had shown that the nonconjugative R plasmids from isolates P1085 and Pl167 were the same (contained pVM109), and the nonconjugative ~ plasmids in isolates P1513, P1552, P1845 and P1975 were the same
IDENTICAL NONCONJUGATIVE PLASMIDS OF P. MULTOCIDA DOES NOT IMPLY CLONALITY
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AB C DE F G 92.5 6612 45.0 31.0 2t .5 14.0 Fig. 1. Whole-cell proteins separated by SDS-PAGE and stained with Coomassie brilliant blue. Lane A = P1167, Lane B = P1085, Lane C = P2862, Lane D -- P1845, Lane E = P1513, Lane F = 1552, Lane G = P1975. Numbers at the right margin represent molecular mass in kDa.
(pVMl10) (Hirsh et al., 1985). The plasmid ( p V M l l l ) , found in isolate P2862, was thought to be identical to p V M l l 0 except for the insertion of a tetracycline-resistance encoding transposon. All of the isolates possessed the A capsule type. Originally, all were found to be somatic serotype 3 (as reported by the National Animal Disease Laboratory, Ames, IA). However, using reagents made at our institution, we found that three of the isolates were somatic serotype 3, three were 3,4 and one was 3,4,12 (Table 1). All b u t one of the isolates (isolate P1975) were P. multocida subspp, multocida. Isolate P1975 was P. multocida subspp, septica. Isolate Pl167, though P. multocida subspp, multocida, was different from all the other isolates in that it fermented trehalose and raffinose. Inspection of the electropherograms of the whole-cell proteins, outer-membrane proteins, restriction digests of chromosomal DNA, and hybridization with the r R N A probe revealed the following (Figs. 1, 2, 3 and 4). Isolates possessing the same plasmid, P1085 and P l 1 6 7 (pVM109) appear dissimilar, as do isolates P1975 and P1513 ( p V M l l 0 ) . On the other hand, isolates possessing a different plasmid, P1085 (pVM109), P2862 ( p V M l l l ) , P1845 ( p V M l l 0 ) and perhaps P1552 ( p V M l l 0 ) , appear similar. The similarities (dissimilari-
84
A BC
J.S. IKEDA A N D D.C. H I R S H
D E F G - 92,5
- 66.2
-45.0
- 31.0
- 21.5 - 14.0
Fig. 2. Outer-membrane proteins separated by SDS-PAGE and stained with Coomassie brilliant blue. Lane A=Pl167, Lane B=P1085, Lane C-- P2862, Lane D =P1845, Lane E=P1513, Lane F =P1552, Lane G=P1975. Numbers at the right margin represent molecular mass in kDa.
ABCD
E F G
.2322
"2027
-564
Fig. 3. Chromosomal DNA digested with B a m HI and separated by polyacrylamide gel electrophoresis. Lane A=P1167, Lane B=P1085, Lane C--P2862, Lane D=P1845, Lane E=P1513, Lane F = p1552, Lane G = P1975. Numbers at the right margin represent size of fragments in base pairs.
IDENTICAL NONCONJUGATIVE PLASMIDS OF P. MULTOCIDA DOES NOT IMPLY CLONALITY
A
B
C
D
E
F
85
G
Fig. 4. Chromosomal DNA digested with Eco RI, separated by agarose gel electrophoresis, transferred to nitrocellulose, and probed with 32p labeled ribosomal RNA. Lane A=Pl167, Lane B = P1085, lane C = P2862, Lane D =P1845, Lane E=P1513, Lane F=P1552, Lane G= P1975. ties) between isolates are supported by the serotype data, and in the case of P1975, by the biotype results as well. DISCUSSION The experiments were performed to determine whether isolates possessing the same nonconjugative R plasmid belonged to the same clone. Certain phenotypic characteristics (capsule type, somatic serotype, biotype, outer-membrane protein profile and whole-cell protein profile) as well as certain genotypic characteristics (restriction endonuclease digestion patterns, r R N A hybridization) were determined in an attempt to ascertain relativeness between isolates. Those isolates which were similar (P1085, P1845, P2862 and perhaps P1552) were assumed to belong to the same clone, those dissimilar (Pl167, P1513 and P1975) to different clones. It is important to keep in mind that assigning a particular isolate to a particular clone is a bit arbitrary since it is difficult to determine whether differences observed are indications of membership in a different clone, or the result of naturally occurring diversity within a clone. The data do not support the hypothesis that isolates possessing the same nonconjugative R plasmid belong to the same clone. The data show that closely related isolates (P1085, P1845, P2862 and perhaps P1552 ) have different nonconjugative R plasmids, and the more dissimilar isolates (P1513 and P1975) have the same nonconjugative R plasmid. These results might be explained in the following ways.
86
J.S. IKEDAANDD.C.HIRSH
After acquiring plasmid DNA, a pasteurella cell, if it continued to evolve, would generate with time, other clones each containing the same nonconjugal plasmid. For example, isolates Pl167 and P1085 could have evolved from a clone that contained pVM109. If, for example, these isolates were obtained shortly after acquisition of the plasmid, it is quite likely that they would be considered members of the same clone. On the other hand, pasteurellae might acquire plasmid DNA at any time. If this were the case, any clone might contain any particular plasmid DNA. But how pasteurellae acquire DNA in nature is not known. The following are ways in which such acquisition might occur. Some avian strains ofP. multocida possess plasmids with sex factor function (Hirsh et al., 1981). These plasmids have been shown to promote the transfer of small nonconjugal R plasmids from pasteurella to pasteurella without promoting their own transfer. However, only one such transfer could occur since the transferred nonconjugal plasmid does not have the ability to promote another round of transfer. Another possibility is that DNA is acquired by different clones by transformation. Since nobody has shown or reported a transformation system in pasteurellae, we feel that this mode of DNA transfer is probably not the manner in which P. multocida acquires DNA. This leaves transduction as the way in which DNA is acquired by pasteurellae, and since avian strains of P. multocida are lysogenized by a variety of bacteriophage, transfer of DNA by this method is an appealing explanation (Kirchner and Eisenstark, 1956). We do not know which of the above is a more accurate description of the acquisition and distribution of nonconjugal plasmid DNA in pasteurellae in nature. It is our guess that both occur.
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Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London), 227: 680-685. Maniatis, T., Fritsch, E.F. and Sambrook, J., 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring harbor, NY. Meyers, J.A., Sanchez, D., Elwell, L.P. and Falkow, S., 1976. Simple agarose gel electrophoretic method for the identification and characterization of plasmid deoxyribonucleic acid. J. Bacteriol., 127: 1529-1537. Mutters, R., Ihm, P., Pohl, S., Frederiksen, W. and Mannheim, W., 1985. Reclassification of the genus Pasteurella Trevisan 1887 on the basis of deoxyribonucleic acid homology, with proposals for the new species Pasteurella dagmatis, PasteureUa canis, Pasteurella stomatis, Pasteu reUa anatis, and Pasteurella langaa. Int. J. Syst. Bacteriol., 35: 309-322. Nicolet, J., Paroz, P. and Krawinkler, M., 1980. Polyacrylamide gel electrophoresis of whole-cell proteins of porcine strains of Haemophilus. Int. J. Syst. Bacteriol., 30: 69-76. Schnaitman, C.A., 1970. Examination of the protein composition of the cell envelope of Esche richia coli by polyacrylamide gel electrophoresis. J. Bacteriol., 104: 882-889. Snipes, K.P., Hansen, L.M. and Hirsh, D.C., 1988. Plasma- and iron-regulated expression of high molecular weight outer membrane proteins by PasteureUa multocida. Am. J. Vet. Res., 49: 1336-1338. Southern, E.M., 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol., 98: 503-517. Stull, T.L., LiPuma, J.J. and Edlind, T.D., 1988. A broad-spectrum probe for molecular epidemiology of bacteria: ribosomal RNA. J. Infect. Dis., 157: 280-286.