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Characterization of sulfonamide resistance determinants and relatedness of Bordetella avium R plasmids
PLASMID
26, 136-140
(1991)
SHORT COMMUNICATIONS Characterization of Sulfonamide Resistance Determinants and Relatedness of Bordetella avium R Plasmids DEBORAH L. CUTTERANDGERALDINE
H. LUGINBUHL
Department of Microbiology, North Carolina State University, Raleigh, North Carolina 27695-7615 Received Five
plasmids,
December
11, 1990; revised
April
22, 199 1
varying
in size from 16 to 5 1.5 kb, were isolated from virulent strains of by restriction endonuclease digestion and DNA-DNA hybridization. These plasmids confer resistance to streptomycin and sulfonamides, and three of the five also confer resistance to tetracycline, but they are not closely related. Four of the plasmids, pRLlO0, p4093, pCW, and pWAM, carried determinants related to the heat-labile type I plasmid-mediated dihydropteroate synthase of the plasmid R388, while one plasmid, p4 168, carried a determinant related to the heat-stable type II dihydropteroate synthase of pGSO5. o 1991
Bordetella avium and compared
Academic
Press, Inc.
Bordetella avium, formerly identified as mids to one another through hybridization Alcaligenes faecalis, causescoryza, a severe analyses. respiratory diseasein turkeys and a mild to inapparent infection in other avian species (Simmons, 1984; Kersters et al., 1984). Plasmids have been found in 20-30% of field isolates of B. avium and so far all such plasmids encode resistance to sulfonamides and streptomycin or sulfonamides, streptomycin, and tetracycline (Luginbuhl et al., 1986; Jackwood et al., 1987). B. avium virulence is not affected by plasmid lossor gain (Simmons et al., 1986). Antibiotic resistance plasmids recovered from field isolates of B. bronchiseptica have been shown to confer sulfonamide resistance and are markedly similar to one another basedon restriction endonucleasedigestion patterns (Hedges et al., 1974; Graham and Abruzzo, 1982; Lax and Walker, 1986; Jackwood et al., 1987). On the other hand, the B. avium plasmids show little evidence of relatednessbased on restriction endonuclease digestion patterns (Luginbuhl et al., 1986; Jackwood et al., 1987). One of the purposes of the present study was to further examine the relatednessof the B. avium plas0147-619X/91
$3.00
Copyright 0 1991 by Academic Press, Inc. All rights of reproduction m any form reserved.
136
Plasmid-mediated sulfonamide resistance is due to the duplication of the target enzyme, dihydropteroate synthase. (DHPS)’ (Wise and Abou-Donia, 1975; Skold, 1976). Sulfonamides exert a bacteriostatic effect by interfering with folic acid synthesis at the step in which dihydropteridine is condensed with p-aminobenzoic acid (PABA) to form dihydropteroate; sulfonamides are structural analogs of PABA and cause competitive inhibition of the chromosome-encoded DHPS (Foster, 1983). The plasmid-specified DHPS is extremely resistant to sulfonamides but maintains the same affinity for PABA as the chromosomally encoded enzyme (Wise and Abou-Donia, 1975; Swedberg and Skold, 1980). Swedborg and Skold ( 1980, 1983) have identified two different types of plasmid-encoded DHPS. They characterized the type I DHPS as very labile; it is encoded by
’ Abbreviations used: DHPS, dihydropteroate thase; PABA, p-aminobcnzoic acid.
syn-
SHORT
COMMUNICATIONS
137
the sul1 gene and found on large conjugative into pBR322 and the recombinant plasmids plasmids such as RlOO. The type II DHPS is were introduced into Escherichia coli strain encoded by &II and is found primarily on DS410 by transformation (Hanahan, 1983). the small broad-host-range IncQ plasmids The plasmid pGS03B was restricted with such as RSFlOlO (Barth and Grinter, 1974; AvaI and ligated into pBR322, resulting in Smith et al., 1974). pB1; plasmid pB 1 contained a 1. I-kb fragThe five B. avium strains and plasmids ment of pGS03B and conferred resistance to used in this study have been described previsulfonamides at a concentration of 1200 pg/ ml when transferred into E. coli strain ously (Luginbuhl et al., 1986). DNA-DNA hybridization and restriction digest patterns DS410. Plasmid pDCl0 contained a 1. I-kb were used as indicators of the relatedness of EcoRI fragment of pG505 cloned into these plasmids. The plasmids were isolated pBR322; plasmid pDC 10 also conferred resisby standard methods (Maniatis et al., 1982) tance to sulfonamides when transferred into and digested with the enzyme AvaI or HincII E. coli strain DS4 10. Plasmid pDC 10 agreed according to the supplier’s recommendations fairly closely in size to plasmid pGS51 de(International Biotechnologies, Inc.). Plas- scribed by Swedberg and Skold ( 1983), which mids pRL100, p4 168, and p4093 were nick also consists of pBR322 plus an EcoRI fragtranslated (Maniatis et al., 1982) and used to ment of pGSO5. Plasmids pB 1 and pDCl0 probe nitrocellulose lilters to which the AvaI represented &II probes. The plasmid or HincII digests of pRL100, ~4168, p4093, pDC20 consisted of pBR322 plus the 1.8-kb pCW, and pWAM had been transferred after BamHI fragment of R388 specifying sulfonaelectrophoresis. We had previously shown mide resistance (Ward and Grinsted, 1978); (Luginbuhl et al., 1986) that there were frag- pDC20 was the source of the sul1 probe and ments of approximately 1.5 and 0.9 kb appar- conferred sulfonamide resistance when transently common to the AvaI digests of these ferred into E. coli strain DS410. Plasmids plasmids and a 2.2-kb fragment that also ap- pB1, pDCl0, and pDC20 were nick transpeared in HincII digests of the plasmids. Hy- lated and used to probe the restriction digests bridization data supported the relatedness of of the B. avium plasmids. Controls included the 2.2-kb HincII fragments from all of the hybridization of the probes to restriction diplasmids with the exception of p4093 (Fig. 1). gests of the plasmids pGSO1, pGS03B, The heterogeneity of the plasmids that was pGSO5, and R388. revealed by the restriction endonuclease The sul1 gene probe, pDC20, hybridized digestion patterns was substantiated by the with the 5.8-kb pRLlO0 fragment and with a hybridization data. There were clearly, how- 4.2- to 4.6-kb HincII fragment from plasmids ever, regions of homology among these plas- p4093, pCW, and pWAM (Fig. 2). The same mids; in particular, the two smallest plas- fragments had hybridized to the pRL 100 mids, pRL 100 and p4 168, shared the greatest probe and not to the ~4168 probe. The plasdegree of homology. mid pDC20 did not hybridize with any of the The second objective of this study was to p4 168 fragments. The faint 1.3-kb p4093 and localize the sulfonamide resistance genes on 1.2-kb pWAM bands in the autoradiogram in the B. avium plasmids and determine if they Fig. 2A were due to hybridization with the were homologous to the previously described vector pBR322. pDC20 did not show homolsul1 or sulI1 genes. Plasmids pGSO1, pGS03B, ogy to pGSO5 or to any of the cloned fragand pGSO5 were kindly provided by G. ments of the &II probes (not shown). Swedberg, University of Uppsala, BiomediThe &II gene probes, pB 1 and pDC 10, cum, Uppsala, Sweden. Plasmid R388 was gave identical results when hybridized to the provided by L. Elwell, Wellcome Research B. avium plasmids. The results of hybridizaLaboratories, Research Triangle Park, North tion of labeled pDC 10 to the H&II fragCarolina. The sulfonamide resistance deter- ments of the B. avium plasmids are shown in minants from these plasmids were subcloned Fig. 2B. The sulI1 gene probe hybridized only
138
SHORT COMMUNICATIONS
6-
4-
l-
FIG. 1. Hybridization of Bordetellu avium plasmids pRL 100 (A) and p4 168 (B) to HincII digests of the B. avium plasmids. Probe DNA was isolated from B. avium strains NCD and P4168, respectively, and “P-labeled by nick translation. Plasmid DNA to be restricted was isolated from E. coli strain DS410, except for pWAM, which was isolated from B. avium strain Wampler. Restriction fragments were separated on 1.2% agarose gels and transferred to nitrocellulose paper. The labeled probe was added and allowed to hybridize overnight at 37°C. The washed filter was exposed to X-ray film at -70°C overnight. Lanes: (a) pRL100, (b) p4 168, (c) ~4093, (d) pCW, (e) pWAM. Numbers on the left refer to fragment sizes in kilobases.
with the 0.9-kb ~4168 fragment in lane b. The other bands in Fig. 2B are the same size as the faint bands seen when the sul1 gene probe was used (Fig. 2A) and are most likely
Aa
b
c
d
e
f
due to hybridization with the pBR322 sequences in the probe. We therefore found that the two plasmids that shared the most sequence homology
Ba
b
c
d
e
f
FIG. 2. (A) Hybridization ofthe sul1 probe pDC20 to HincII digests ofthe Bordetella avium plasmids. (B) Hybridization of the sulII probe pDC 10 to HincII digests ofthe B. avium plasmids. In both panels lanes a-e contain HincII digests of the B. avium plasmids: (a) pRL100, (b) p4168, (c) p4093, (d) pCW, (e) pWAM; lane f contains pDC20 digested with BarnHI. Numbers on the left refer to fragment sizes in kilobases.
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SHORT COMMUNICATIONS
based on DNA-DNA hybridization, pRL100 and p4 168, did not hybridize to the same sulfonamide resistance determinant, while pRLlO0 and p4093 appeared to have only this determinant in common. There was very little similarity in the restriction endonuclease digestion patterns of these plasmids, and thus the regions surrounding the resistance determinants may differ in the different plasmids. Restriction analysis and hybridization studies of cloned sulfonamide resistance genes from a wide variety of R factors support the classification of plasmid-specified enzymes into two groups. One group, consisting of plasmids such as the IncFII R plasmids R 1, RlOO, and R6, the IncW plasmid R388, R22259, and plasmids pGSO1 and pGSO2 from more recent E. coli clinical isolates, share a common 1.3-kb HindIII-BumHI fragment carrying the gene sul1 that specifies the heat-labile type I enzyme (Ward and Grinsted, 1978). The other group, consisting of plasmids pGS03B, pGS04, and pGSO5, share a 1-kb EcoRI fragment containing the gene sulI1 that encodes the stable type II DHPS (Swedberg and Skold, 1983). The small broad-host-range RSF 10 10 family of SuSm resistant plasmids also carry sulI1, even though most of these have only one EcoRI site (Grinter and Barth, 1976). DNA hybridization studies show no homology between DNA fragments carrying the sul1 and ~~111 genes (Foster, 1983; Swedberg and Skold, 1983). Nucleotide sequence analyses have revealed 57% similarity between sul1 and suZI1 (Radstrom and Swedberg, 1988). Both types of sulfonamide resistance are very widespread in nature and appear in a variety of plasmid backgrounds (Akari et al., 1987; Swedberg, 1987; Willson et al., 1989). The B. avium plasmids examined encoded determinants representing both the type I and type II enzymes. ACKNOWLEDGMENTS This work was supported by U.S. Department of Agriculture Grant 59-2371-O-2-120-0 and by the North Carolina Agricultural Research Service. D. L. Cutter was
supported by a National Science Foundation predoctoral
fellowship. REFERENCES ARAKI, Y., INOUE, M., AND HASHIMOTO, H. (1987). Characterization and mobilization of nonconjugative plasmids encoding resistance to streptomycin and sulfanilamide. Microbial. Immunol. 31, 1107-l 111. BARTH, P. T., AND GRINTER, N. J. (1974). Comparison of the deoxyribonucleic acid molecular weights and homologies of plasmids conferring linked resistance to streptomycin and sulfonamides. J. Bacterial. 120, 618-630. BOLIVAR, F., RODRIGUEZ, R. L., GREENE, P. J., BETLACH, M. C., HEYNEKER, H. L., AND BOYER, H. W. (1977). Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2,95-l 13. FOSTER, T. J. (1983). Plasmid-determined resistance to antimicrobial drugs and toxic metal ions in bacteria. Microbial. Rev. 47, 361-409. GRAHAM, A. C., AND ABRUZZO, G. K. (1982). Occurrence and characterization of plasmids in field isolates of Bordetella bronchiseptica. Am. J. Vet. Rex 43, 1852-1855. GRINTER, N. J., AND BARTH, P. T. (1976). Characterization of SmSu plasmids by restriction endonuclease cleavage and compatibility testing. J. Bacterial. 128, 394-400. HANAHAN, D. (1983). Studies on transformation ofE.rcherichia coli with plasmids. J. Mol. Biol. 166,557-580. HEDGES, R. W., JACOB, A. E., AND SMITH, J. T. (1974). Properties of an R. factor from Bordetella bronchiseptica. J. Gen. Microbial. 84, 199-204. JACKWOOD, M. W., SAIF, Y. M., AND COPLIN, D. L. (1987). Isolation and characterization of Bordetella avium plasmids. Avian Dis. 31, 782-786. KERSTERS, K., HINZ, K.-H., HERTLE, A., SEGERS,P., LIEVENS, A., SIEGMANN, O., AND DE LEY, J. (1984). Bordetella avium sp. nov., isolated from the respiratory tracts of turkeys and other birds. ht. J. Syst. Bacteriol. 34, 56-70. LAX, A. J., AND WALKER, C. A. (1986). Plasmids related to RSFlOlO from Bordetella bronchiseptica. Plasmid 15,210-216. LUGINBUHL, G. H., CUTTER, D., CAMPODONICO, G., PEACE,J., AND SIMMONS, D. G. (1986). Plasmid DNA of virulent Alcaligenes faecalis. Am. J. Vet. Res. 47, 619-621. MANIATIS, T., FRITSCH, E. F., AND SAMBROOK, J. (1982). “Molecular Cloning: A Laboratory Manual.” Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. RADSTROM, P., AND SWEDBERG,G. (1988). RSFlOlO and a conjugative piasmid contain sulI1, one of two known genes for plasmid-borne sulfonamide resistance dihydropteroate synthase. Antimicrob. Agents Chemother. 32, 1684- 1692. SIMMONS, D. G. (1984). Turkey coryza. In “Diseases of
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Poultry” (M. S. Hofstad, Ed.), Iowa State Univ. Press, Ames. SIMMONS, D. G., ROSE, L. P., FULLER, F. J., MAURER, L. C., AND LUGINBUHL, G. H. (1986). Turkey coryza: Lack of correlation between plasmids and pathogenicity of Bordeiellu avium. Avian Es. 30, 593-597. SKOLD, 0. (1976). R-factor-mediated resistance to sulfonamides by a plasmid-borne, drug-resistant dihydropteroate synthase. Antimicrob. Agents Chemother. 9,49-54.
SMITH, H. R., HUMPHREYS, G. O., AND ANDERSON, E. S. (1974). Genetic and molecular characterisation of some non-transferring plasmids. Mol. Gen. Genet. 129,229-242.
SWEDBERG,G., AND SKOLD, 0. (1980). Characterization of different plasmid-borne dihydropteroate synthases mediating bacterial resistance to sulfonamides. J. Bacteriol. 142, 1-7. SWEDBERG, G., AND SKOLD, 0. (1983). Plasmid-borne
sulfonamide resistance determinants studied by restriction enzyme analysis. J. Bacterial. 153, 1228-1237. SWEDBERG,G. (1987). Organization of two sulfonamide resistance genes on plasmids of gram-negative bacteria. Antimicrob. Agents Chemother. 31, 306-3 11. WARD, J. M., AND GRINSTED, J. (1978). Mapping of functions in the R-plasmid R388 by examination of deletion mutants generated in vitro. Gene 3, 87-95. WILLSON, P. J., DENEER, H. G., POTTER, A., AND ALBRI~TON, W. (1989). Characterization of a streptomycin-sulfonamide resistance plasmid from Actinobacillus pleuropneumoniae Antimicrob. Agents Chemother. 33,235-238. WISE, E. M.,
AND ABOU-DONIA, M. M. (1975). Sulfonamide resistance mechanism in Escherichia coli: R plasmids can determine sulfonamide-resistant dihydropteroate synthases. Proc. Nat/. Acad. Sci. USA 72, 262 I-2625.
Communicated by Stewart B. Levy
26, 136-140
(1991)
SHORT COMMUNICATIONS Characterization of Sulfonamide Resistance Determinants and Relatedness of Bordetella avium R Plasmids DEBORAH L. CUTTERANDGERALDINE
H. LUGINBUHL
Department of Microbiology, North Carolina State University, Raleigh, North Carolina 27695-7615 Received Five
plasmids,
December
11, 1990; revised
April
22, 199 1
varying
in size from 16 to 5 1.5 kb, were isolated from virulent strains of by restriction endonuclease digestion and DNA-DNA hybridization. These plasmids confer resistance to streptomycin and sulfonamides, and three of the five also confer resistance to tetracycline, but they are not closely related. Four of the plasmids, pRLlO0, p4093, pCW, and pWAM, carried determinants related to the heat-labile type I plasmid-mediated dihydropteroate synthase of the plasmid R388, while one plasmid, p4 168, carried a determinant related to the heat-stable type II dihydropteroate synthase of pGSO5. o 1991
Bordetella avium and compared
Academic
Press, Inc.
Bordetella avium, formerly identified as mids to one another through hybridization Alcaligenes faecalis, causescoryza, a severe analyses. respiratory diseasein turkeys and a mild to inapparent infection in other avian species (Simmons, 1984; Kersters et al., 1984). Plasmids have been found in 20-30% of field isolates of B. avium and so far all such plasmids encode resistance to sulfonamides and streptomycin or sulfonamides, streptomycin, and tetracycline (Luginbuhl et al., 1986; Jackwood et al., 1987). B. avium virulence is not affected by plasmid lossor gain (Simmons et al., 1986). Antibiotic resistance plasmids recovered from field isolates of B. bronchiseptica have been shown to confer sulfonamide resistance and are markedly similar to one another basedon restriction endonucleasedigestion patterns (Hedges et al., 1974; Graham and Abruzzo, 1982; Lax and Walker, 1986; Jackwood et al., 1987). On the other hand, the B. avium plasmids show little evidence of relatednessbased on restriction endonuclease digestion patterns (Luginbuhl et al., 1986; Jackwood et al., 1987). One of the purposes of the present study was to further examine the relatednessof the B. avium plas0147-619X/91
$3.00
Copyright 0 1991 by Academic Press, Inc. All rights of reproduction m any form reserved.
136
Plasmid-mediated sulfonamide resistance is due to the duplication of the target enzyme, dihydropteroate synthase. (DHPS)’ (Wise and Abou-Donia, 1975; Skold, 1976). Sulfonamides exert a bacteriostatic effect by interfering with folic acid synthesis at the step in which dihydropteridine is condensed with p-aminobenzoic acid (PABA) to form dihydropteroate; sulfonamides are structural analogs of PABA and cause competitive inhibition of the chromosome-encoded DHPS (Foster, 1983). The plasmid-specified DHPS is extremely resistant to sulfonamides but maintains the same affinity for PABA as the chromosomally encoded enzyme (Wise and Abou-Donia, 1975; Swedberg and Skold, 1980). Swedborg and Skold ( 1980, 1983) have identified two different types of plasmid-encoded DHPS. They characterized the type I DHPS as very labile; it is encoded by
’ Abbreviations used: DHPS, dihydropteroate thase; PABA, p-aminobcnzoic acid.
syn-
SHORT
COMMUNICATIONS
137
the sul1 gene and found on large conjugative into pBR322 and the recombinant plasmids plasmids such as RlOO. The type II DHPS is were introduced into Escherichia coli strain encoded by &II and is found primarily on DS410 by transformation (Hanahan, 1983). the small broad-host-range IncQ plasmids The plasmid pGS03B was restricted with such as RSFlOlO (Barth and Grinter, 1974; AvaI and ligated into pBR322, resulting in Smith et al., 1974). pB1; plasmid pB 1 contained a 1. I-kb fragThe five B. avium strains and plasmids ment of pGS03B and conferred resistance to used in this study have been described previsulfonamides at a concentration of 1200 pg/ ml when transferred into E. coli strain ously (Luginbuhl et al., 1986). DNA-DNA hybridization and restriction digest patterns DS410. Plasmid pDCl0 contained a 1. I-kb were used as indicators of the relatedness of EcoRI fragment of pG505 cloned into these plasmids. The plasmids were isolated pBR322; plasmid pDC 10 also conferred resisby standard methods (Maniatis et al., 1982) tance to sulfonamides when transferred into and digested with the enzyme AvaI or HincII E. coli strain DS4 10. Plasmid pDC 10 agreed according to the supplier’s recommendations fairly closely in size to plasmid pGS51 de(International Biotechnologies, Inc.). Plas- scribed by Swedberg and Skold ( 1983), which mids pRL100, p4 168, and p4093 were nick also consists of pBR322 plus an EcoRI fragtranslated (Maniatis et al., 1982) and used to ment of pGSO5. Plasmids pB 1 and pDCl0 probe nitrocellulose lilters to which the AvaI represented &II probes. The plasmid or HincII digests of pRL100, ~4168, p4093, pDC20 consisted of pBR322 plus the 1.8-kb pCW, and pWAM had been transferred after BamHI fragment of R388 specifying sulfonaelectrophoresis. We had previously shown mide resistance (Ward and Grinsted, 1978); (Luginbuhl et al., 1986) that there were frag- pDC20 was the source of the sul1 probe and ments of approximately 1.5 and 0.9 kb appar- conferred sulfonamide resistance when transently common to the AvaI digests of these ferred into E. coli strain DS410. Plasmids plasmids and a 2.2-kb fragment that also ap- pB1, pDCl0, and pDC20 were nick transpeared in HincII digests of the plasmids. Hy- lated and used to probe the restriction digests bridization data supported the relatedness of of the B. avium plasmids. Controls included the 2.2-kb HincII fragments from all of the hybridization of the probes to restriction diplasmids with the exception of p4093 (Fig. 1). gests of the plasmids pGSO1, pGS03B, The heterogeneity of the plasmids that was pGSO5, and R388. revealed by the restriction endonuclease The sul1 gene probe, pDC20, hybridized digestion patterns was substantiated by the with the 5.8-kb pRLlO0 fragment and with a hybridization data. There were clearly, how- 4.2- to 4.6-kb HincII fragment from plasmids ever, regions of homology among these plas- p4093, pCW, and pWAM (Fig. 2). The same mids; in particular, the two smallest plas- fragments had hybridized to the pRL 100 mids, pRL 100 and p4 168, shared the greatest probe and not to the ~4168 probe. The plasdegree of homology. mid pDC20 did not hybridize with any of the The second objective of this study was to p4 168 fragments. The faint 1.3-kb p4093 and localize the sulfonamide resistance genes on 1.2-kb pWAM bands in the autoradiogram in the B. avium plasmids and determine if they Fig. 2A were due to hybridization with the were homologous to the previously described vector pBR322. pDC20 did not show homolsul1 or sulI1 genes. Plasmids pGSO1, pGS03B, ogy to pGSO5 or to any of the cloned fragand pGSO5 were kindly provided by G. ments of the &II probes (not shown). Swedberg, University of Uppsala, BiomediThe &II gene probes, pB 1 and pDC 10, cum, Uppsala, Sweden. Plasmid R388 was gave identical results when hybridized to the provided by L. Elwell, Wellcome Research B. avium plasmids. The results of hybridizaLaboratories, Research Triangle Park, North tion of labeled pDC 10 to the H&II fragCarolina. The sulfonamide resistance deter- ments of the B. avium plasmids are shown in minants from these plasmids were subcloned Fig. 2B. The sulI1 gene probe hybridized only
138
SHORT COMMUNICATIONS
6-
4-
l-
FIG. 1. Hybridization of Bordetellu avium plasmids pRL 100 (A) and p4 168 (B) to HincII digests of the B. avium plasmids. Probe DNA was isolated from B. avium strains NCD and P4168, respectively, and “P-labeled by nick translation. Plasmid DNA to be restricted was isolated from E. coli strain DS410, except for pWAM, which was isolated from B. avium strain Wampler. Restriction fragments were separated on 1.2% agarose gels and transferred to nitrocellulose paper. The labeled probe was added and allowed to hybridize overnight at 37°C. The washed filter was exposed to X-ray film at -70°C overnight. Lanes: (a) pRL100, (b) p4 168, (c) ~4093, (d) pCW, (e) pWAM. Numbers on the left refer to fragment sizes in kilobases.
with the 0.9-kb ~4168 fragment in lane b. The other bands in Fig. 2B are the same size as the faint bands seen when the sul1 gene probe was used (Fig. 2A) and are most likely
Aa
b
c
d
e
f
due to hybridization with the pBR322 sequences in the probe. We therefore found that the two plasmids that shared the most sequence homology
Ba
b
c
d
e
f
FIG. 2. (A) Hybridization ofthe sul1 probe pDC20 to HincII digests ofthe Bordetella avium plasmids. (B) Hybridization of the sulII probe pDC 10 to HincII digests ofthe B. avium plasmids. In both panels lanes a-e contain HincII digests of the B. avium plasmids: (a) pRL100, (b) p4168, (c) p4093, (d) pCW, (e) pWAM; lane f contains pDC20 digested with BarnHI. Numbers on the left refer to fragment sizes in kilobases.
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based on DNA-DNA hybridization, pRL100 and p4 168, did not hybridize to the same sulfonamide resistance determinant, while pRLlO0 and p4093 appeared to have only this determinant in common. There was very little similarity in the restriction endonuclease digestion patterns of these plasmids, and thus the regions surrounding the resistance determinants may differ in the different plasmids. Restriction analysis and hybridization studies of cloned sulfonamide resistance genes from a wide variety of R factors support the classification of plasmid-specified enzymes into two groups. One group, consisting of plasmids such as the IncFII R plasmids R 1, RlOO, and R6, the IncW plasmid R388, R22259, and plasmids pGSO1 and pGSO2 from more recent E. coli clinical isolates, share a common 1.3-kb HindIII-BumHI fragment carrying the gene sul1 that specifies the heat-labile type I enzyme (Ward and Grinsted, 1978). The other group, consisting of plasmids pGS03B, pGS04, and pGSO5, share a 1-kb EcoRI fragment containing the gene sulI1 that encodes the stable type II DHPS (Swedberg and Skold, 1983). The small broad-host-range RSF 10 10 family of SuSm resistant plasmids also carry sulI1, even though most of these have only one EcoRI site (Grinter and Barth, 1976). DNA hybridization studies show no homology between DNA fragments carrying the sul1 and ~~111 genes (Foster, 1983; Swedberg and Skold, 1983). Nucleotide sequence analyses have revealed 57% similarity between sul1 and suZI1 (Radstrom and Swedberg, 1988). Both types of sulfonamide resistance are very widespread in nature and appear in a variety of plasmid backgrounds (Akari et al., 1987; Swedberg, 1987; Willson et al., 1989). The B. avium plasmids examined encoded determinants representing both the type I and type II enzymes. ACKNOWLEDGMENTS This work was supported by U.S. Department of Agriculture Grant 59-2371-O-2-120-0 and by the North Carolina Agricultural Research Service. D. L. Cutter was
supported by a National Science Foundation predoctoral
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Communicated by Stewart B. Levy