D N A Sequence--/.DNA Sequencing andMapping, Vol. 3 , pp. 89-97 Reprints available directly from the publisher Photocopying permitted by license only
0 1992 Harwood Academic Publisher5 GmbH Printed in the United Kingdom
Mitochondrial DNA Downloaded from informahealthcare.com by McMaster University on 01/09/15 For personal use only.
Characterization of plasmids with antimicrobial resistant genes in Pasfewella haemolytica A1 Y.
F. CHANG’, D. P.MA2, H. Q. BAI’, R.
YOUNG’, D. K. STRUCK‘, S. j. SHIN’ and D. H . LEI”
‘Diagnostic Laboratory, College of Veterinary Medicine, Cornell University, Ithaca, NY 1485 I , USA ’Department of Biochemistry and Molecular Biology, College of Agriculture, Mississippi State University, Mississippi State, MS 39762, USA ‘Department of Biochemistry and Biophysics, College of Agriculture and Life Sciences, Texas A&M University, College Station, TX 77843, USA ‘Department of Medical Biochemistry and Genetics, College o f Medicine, Texas A&M University, College Station, TX 77843, USA
Two R plasmids, pYFCl and pYFC2, from PasteureNa haemolytica A1 encoding sulfonamide, streptomycin (pYFC1), and ampicillin (pYFC2) resistances have been characterized by restriction endonuclease digestions, subcloning or DNA sequencing. pYFCl consists of 4225 bp and is 51.9% in AT content. Physical mapping indicated a highly conserved region of restriction sites among pYFC1, R S F l O l 0, pGS05, pFM739, pHD148 and pGSO3B. pYFCl encoded a dihydropteroate synthase (29.8 kDa), and streptomycin kinase (29.6 kDa) which i s homologous in nucleotide sequences or deduced amino acid sequence to that encoded by a broad-host range lncQ plasmid RSFl 010. Based on the primary structure of pYFC1, the sulfonamide and streptomycin genes are derived from the same ancestor of RSF1010. pYFC2 i s similar to the plasmid from P. haemofytica LNPBSl isolated i n France by partial restriction enzyme mapping. pYFCl and pYFC2 have the same size of 4.2 kbp.
of animals to reduce economic losses (Schwarz et a/., 1989). We have reported on the P. haemolytica strains which were isolated from cattle that died of pneumonic pasteurellosis and defined their leukotoxin production, plasmid content, and antimicrobial susceptibility (Chang et a/., 1987). The wide and rapid spread of antibiotic resistance among different pathogenic organisms is probably due to recombinational incorporation of resistance genes into the P. haemolytica plasmids. Sulfonamides are one of the common group of antibacterial agents used in chemotherapy of animals. Plasmid-mediated sulfonamide resistance is widely distributed in Gram-negative bacteria. There are two types of plasmids (type 1 and type 2) that express dihydropteroate synthase (DHPS). Type 1 sulfonamide resistance DHPS, sull, is usually found i n the large self-transmissible resistance plasmids and on a transposon (Sundstrom et a/., 1988). Type II DPHS i s usually found in small nonconjugative plasmids (Swedberg, 1987). Streptomycin resistance has also been found to be very common in Gram-negative organisms. The resistance is due to the expression of a gene encoding ki nase (ami noglycoside 3‘streptomycin phosphotransferases) located on the plasmid. It has been reported that the streptomycin resistance gene i s located downstream from the sulfonamide gene in several isolated plasmids (Radstrom et a/., 1988; Rotger et a/., 1986; Scholz et a/., 1989).
KEY WORDS: P. haemolytica, bla, sulll, and aph(strA)
INTRODUCTION Pasteurella haemolytica biotype A, serotype 1 (A1) is one of the major etiologic agents of pneumonic pasteurellosis (Yates, 1982). This disease i s responsible for substantial economic losses to the cattle and sheep industries (Yates, 1982). Recently, antimicrobial resistant strains of P. haemolytica have been isolated from sheep and cattle, probably due to the increased use of antibiotics in the treatment Address Correspondence to: Dr Y. F. Chang.
89
')O
Y. F. CHANC ET AL
1hc K O U - 1 p-lactamase has been described in
Mitochondrial DNA Downloaded from informahealthcare.com by McMaster University on 01/09/15 For personal use only.
wJver31 org'ini5ms inc luding Haemophilus influenza~~ Ac, tinohac.i//iir pleuropneumoniae (Juteau et J / , 1990, ( hang et a / , 1992), P muftocida, P. aerogei~c,, dnd P haemolytica (Liverlli et a/., 1991). Tho R O B - I -en( oding plasmid from P. haemolytica 5tr'tins has been Isol,ited in the U.S A and other
countries (C hang ef < I / , 1987, Liverlli ef a/., 1991). Ihe R O B - 1 p-lactamase gene from Haemophilus has been sequenced intluenrae and P hc~enio/ytica and the gene product belongs to a class A p-lactam a w (JutCCkie i t a / , 1990, Liverlli e t a / , 1991). 111 order t o understand the relationship of the tl i hytf ropt er oate 5 y nt ha se and s t rept o m y c i n k i na s e genes from the P hawrmlytica plasmid to other pathogenic organisms and to facilitate its use as a \tiuttle vector for P h;lemo/ytica and E. coli. We se(4uence.d the pla5mid D N A of the P. haemolytica c o n t a i ti Irig the su I fonamide and streptomycin re5istancc' genes In this paper, we describe the typ" 11 5ulfonamide qrne and streptomycin resist~ n c t ,gcrie trom 1' haemo/yt/ta which are conL C ~ among the broad-host-range plasmid KSO 1 0 1 0 ('x holz et , I / , 1989) pGS05 (Radstrom
1
23.1 30-9,416--
6,682-
4 , 7 7 3--
2.322-Z,C)?7-
2
3
4
5
6 7 8
9
et a/., 1988), pCS03B (Radstrom et ' I / . , 1988), and pFM739 (Rotger et a/., 1986). We also report that P. haemolytica contains a KOB-1 p-l,ictcimaseencoding plasmid.
RESULTS Isolation of plasmid and restriction enzyme digestion We have screened 15 P. h,iemo/ytic,i A1 stidins for the presence of plasmids. Nine strains of P. haemolytica were found to contain plasmids with a size of 4.2 kbp (Fig. 1). The other 6 P. hat~molytica strains contained no plasmids. We discovered that direct analysis of the crude plasmid preparations in agarose gels revealed a number of bands (Fig. 1, lanes 18-21) most of which disappeared upon purification in isopycnic Cs(:L or CsTFA gradients (data not shown). Our attempts to characterize the pl,ismid by digestion with EcoRl resulted in apparent pirtial digestion, in which a signific-ant portion or the D N A remained in supercoiled or relaxe5 c-ir( u l a r
10 I 1
12 13 14 15 16 17 18 19 20 21 2 2
ANTIMICROBIAL RESISTANT GENES
Mitochondrial DNA Downloaded from informahealthcare.com by McMaster University on 01/09/15 For personal use only.
forms (Fig. 2, lane 9) even in enzyme excess. We digested the DNA with Scal which showed that there are actually two plasmid species, indistinguishable by size with distinctly different restriction maps (Fig. 3). One plasmid, designated pYFCl was present in all but one strain, and contained Pstl, Hindlll and Sac1 sites; the other plasmid, designated pYFC2 was present in 10 strains and contained Scal, Aval, and HgiAl sites (Fig. 3).
Leukotoxin production Regardless of plasmid content, all 15 strains were leukotoxin positive when culture supernatants were checked for BL-3 (bovine leukemia cells) cell cytotoxicity. Transformation of pYFCl and pYFC2 and subclones into E. coli and P. haemolytica Plasmids, pYFCl, pYFC2, pYFC4, pYFC5, pYFC6, and pYFC70 can be transformed into E. coli by the CaCI, method or that of Hanahan (Hanahan, 1983) and the DNA can be reisolated (data not shown). Attempts to transform pYFC4, pYFC5, pYFC6 and pYFC70 into P. haemolytica 5 (this strain does not have any plasmid) by either the procedure of Hanahan (Hanahan, 1983) or an electroporation technique (Craig et a/., 1989)
91
were not successful. However, pYFCl and pYFC2 can be transformed into P. haemolytica by these two latter techniques (data not shown). DNA sequence of the pYFCl The small plasmid, pYFCl was subjected to DNA sequence analysis and three open reading frames (ORFs) were found (Fig. 4). The first ORF encodes a sulfonamide resistant dihydropteroate synthase, the second reading frame encodes an aminoglycoside 3'-phosphotransferase, and the third encodes a partial MbeA-like protein. This is similar to the R S F l O l O which was originally described by Falkow's group, in which the sulfonamide and streptomycin resistance genes are closely linked and transcribed from the same promoter as the sulfonamide resistance gene (Guerry et a/., 1974; Scholz et a/., 1989). The D N A sequence was screened for E. coli promoter-like sequences using the homology score method (Mulligan et a/., 1984). There were t w o sequences similar to the TATAAT consensus promoter sequence (-1 0 region) and two sequences similar to the RNA polymerase-binding site, TTGACA (19) proximal to the dihydropteroate synthase gene (Fig. 4). The sulfonamide resistance gene has a methionine start codon with a reasonable Shine-Dalgarno
1 2 3 4 5 6 7 8 9 10 11 12 1314 151617 18 19 20 23,130 99416 6,6824,3 73 2,3222,027-
Figure 2 Comparative restriction endonuclease digestions. Plasmid D N A from P. haemolytica strains 43 (lanes 2-7) and 630 (lanes 8-13, 15-20) are digested with EcoRl (lanes 2 and 8), Scal (lanes 3 and 9), or EcoRl and Scal (lanes 4 and 10). Lanes 5-7, 1 1 - 1 1 are mock-digested (no enzyme) controls for lanes 2-4 and 8-1 0 respectively. Lanes 15, 16, 17, 18 are digestions with BamHI, EcoRI, Hindlll, or Hindlll plus EcoKI. Lanes 19, 20 are mock-digested controls for lanes 15 and 16. Lanes 1,4 are Hindlll-digested lambda DNA.
02
Y. F CHANC ET AL
-
’0
c
(I)
a
-7
i
I
I
l
l
I
STR
Mitochondrial DNA Downloaded from informahealthcare.com by McMaster University on 01/09/15 For personal use only.
i
*
S U L I1
-
, pxr-b
AMP
1 Kb
c’ Ioc ated up5treani (Fig. 4) The streptoiiiyc i n kinaw gene’ h‘i5 a niethionine start codon /lI L ’(,I w i t h ‘1 Shine-[lalgarno sequence (AGGA; kig 1) The st,irt codon and Shine-Dalgarno w ~ i i c ~ i i corc ~the P. haemolytica streptomycin kin‘i-+ gene’ n a s icfentiml t o that of the strA of the Inc (2 RSF I010 (ScholJ et < I / . , 1989). Immediately downstrean1 of the strA, thew was a “pseudogt.tit3” ot strH characterized by only the N-terniindl 1 d c c i u c d cirnino acids showing signific ,int \iniil,irity t o the Srr5 of R S F l 01 0 (Fig. 4). 5 e q u t ~ n c(25 t)eyond this point immediately cli\ttrged tram 5trH with multiple stop codons ,tppcwrrng in a11 t h r w redding frames. The third it1,itling trarrie contained 137 deduced amino < I ( it15 ,>nd showed 70 6”% homology to MbeA of ( oII 1 it3oycl el , I / , 1989) i n the N-terminal overI‘ip and i t c o t r l t l also h e a “pseudogene” since the MhcA in the C o l E l (Boyd et a/., 1989) contains 7 I 7 ,timino d ( i d s (Fig 4 ) Homology comparison o t t h c clrdut c d amino acid sequence from P. haemo/vri( 5 u I I I indicated that it has 98.1, 100 and 70”,8\iniil,iritv to that o t l n t Q R S F l O l 0 (Scholz et , I / , 1 O t ~ O j , p(JS05 (R,icJstrom et a / , 1988), and plb.1OLO iSl)r,itt rt
0 1992 Harwood Academic Publisher5 GmbH Printed in the United Kingdom
Mitochondrial DNA Downloaded from informahealthcare.com by McMaster University on 01/09/15 For personal use only.
Characterization of plasmids with antimicrobial resistant genes in Pasfewella haemolytica A1 Y.
F. CHANG’, D. P.MA2, H. Q. BAI’, R.
YOUNG’, D. K. STRUCK‘, S. j. SHIN’ and D. H . LEI”
‘Diagnostic Laboratory, College of Veterinary Medicine, Cornell University, Ithaca, NY 1485 I , USA ’Department of Biochemistry and Molecular Biology, College of Agriculture, Mississippi State University, Mississippi State, MS 39762, USA ‘Department of Biochemistry and Biophysics, College of Agriculture and Life Sciences, Texas A&M University, College Station, TX 77843, USA ‘Department of Medical Biochemistry and Genetics, College o f Medicine, Texas A&M University, College Station, TX 77843, USA
Two R plasmids, pYFCl and pYFC2, from PasteureNa haemolytica A1 encoding sulfonamide, streptomycin (pYFC1), and ampicillin (pYFC2) resistances have been characterized by restriction endonuclease digestions, subcloning or DNA sequencing. pYFCl consists of 4225 bp and is 51.9% in AT content. Physical mapping indicated a highly conserved region of restriction sites among pYFC1, R S F l O l 0, pGS05, pFM739, pHD148 and pGSO3B. pYFCl encoded a dihydropteroate synthase (29.8 kDa), and streptomycin kinase (29.6 kDa) which i s homologous in nucleotide sequences or deduced amino acid sequence to that encoded by a broad-host range lncQ plasmid RSFl 010. Based on the primary structure of pYFC1, the sulfonamide and streptomycin genes are derived from the same ancestor of RSF1010. pYFC2 i s similar to the plasmid from P. haemofytica LNPBSl isolated i n France by partial restriction enzyme mapping. pYFCl and pYFC2 have the same size of 4.2 kbp.
of animals to reduce economic losses (Schwarz et a/., 1989). We have reported on the P. haemolytica strains which were isolated from cattle that died of pneumonic pasteurellosis and defined their leukotoxin production, plasmid content, and antimicrobial susceptibility (Chang et a/., 1987). The wide and rapid spread of antibiotic resistance among different pathogenic organisms is probably due to recombinational incorporation of resistance genes into the P. haemolytica plasmids. Sulfonamides are one of the common group of antibacterial agents used in chemotherapy of animals. Plasmid-mediated sulfonamide resistance is widely distributed in Gram-negative bacteria. There are two types of plasmids (type 1 and type 2) that express dihydropteroate synthase (DHPS). Type 1 sulfonamide resistance DHPS, sull, is usually found i n the large self-transmissible resistance plasmids and on a transposon (Sundstrom et a/., 1988). Type II DPHS i s usually found in small nonconjugative plasmids (Swedberg, 1987). Streptomycin resistance has also been found to be very common in Gram-negative organisms. The resistance is due to the expression of a gene encoding ki nase (ami noglycoside 3‘streptomycin phosphotransferases) located on the plasmid. It has been reported that the streptomycin resistance gene i s located downstream from the sulfonamide gene in several isolated plasmids (Radstrom et a/., 1988; Rotger et a/., 1986; Scholz et a/., 1989).
KEY WORDS: P. haemolytica, bla, sulll, and aph(strA)
INTRODUCTION Pasteurella haemolytica biotype A, serotype 1 (A1) is one of the major etiologic agents of pneumonic pasteurellosis (Yates, 1982). This disease i s responsible for substantial economic losses to the cattle and sheep industries (Yates, 1982). Recently, antimicrobial resistant strains of P. haemolytica have been isolated from sheep and cattle, probably due to the increased use of antibiotics in the treatment Address Correspondence to: Dr Y. F. Chang.
89
')O
Y. F. CHANC ET AL
1hc K O U - 1 p-lactamase has been described in
Mitochondrial DNA Downloaded from informahealthcare.com by McMaster University on 01/09/15 For personal use only.
wJver31 org'ini5ms inc luding Haemophilus influenza~~ Ac, tinohac.i//iir pleuropneumoniae (Juteau et J / , 1990, ( hang et a / , 1992), P muftocida, P. aerogei~c,, dnd P haemolytica (Liverlli et a/., 1991). Tho R O B - I -en( oding plasmid from P. haemolytica 5tr'tins has been Isol,ited in the U.S A and other
countries (C hang ef < I / , 1987, Liverlli ef a/., 1991). Ihe R O B - 1 p-lactamase gene from Haemophilus has been sequenced intluenrae and P hc~enio/ytica and the gene product belongs to a class A p-lactam a w (JutCCkie i t a / , 1990, Liverlli e t a / , 1991). 111 order t o understand the relationship of the tl i hytf ropt er oate 5 y nt ha se and s t rept o m y c i n k i na s e genes from the P hawrmlytica plasmid to other pathogenic organisms and to facilitate its use as a \tiuttle vector for P h;lemo/ytica and E. coli. We se(4uence.d the pla5mid D N A of the P. haemolytica c o n t a i ti Irig the su I fonamide and streptomycin re5istancc' genes In this paper, we describe the typ" 11 5ulfonamide qrne and streptomycin resist~ n c t ,gcrie trom 1' haemo/yt/ta which are conL C ~ among the broad-host-range plasmid KSO 1 0 1 0 ('x holz et , I / , 1989) pGS05 (Radstrom
1
23.1 30-9,416--
6,682-
4 , 7 7 3--
2.322-Z,C)?7-
2
3
4
5
6 7 8
9
et a/., 1988), pCS03B (Radstrom et ' I / . , 1988), and pFM739 (Rotger et a/., 1986). We also report that P. haemolytica contains a KOB-1 p-l,ictcimaseencoding plasmid.
RESULTS Isolation of plasmid and restriction enzyme digestion We have screened 15 P. h,iemo/ytic,i A1 stidins for the presence of plasmids. Nine strains of P. haemolytica were found to contain plasmids with a size of 4.2 kbp (Fig. 1). The other 6 P. hat~molytica strains contained no plasmids. We discovered that direct analysis of the crude plasmid preparations in agarose gels revealed a number of bands (Fig. 1, lanes 18-21) most of which disappeared upon purification in isopycnic Cs(:L or CsTFA gradients (data not shown). Our attempts to characterize the pl,ismid by digestion with EcoRl resulted in apparent pirtial digestion, in which a signific-ant portion or the D N A remained in supercoiled or relaxe5 c-ir( u l a r
10 I 1
12 13 14 15 16 17 18 19 20 21 2 2
ANTIMICROBIAL RESISTANT GENES
Mitochondrial DNA Downloaded from informahealthcare.com by McMaster University on 01/09/15 For personal use only.
forms (Fig. 2, lane 9) even in enzyme excess. We digested the DNA with Scal which showed that there are actually two plasmid species, indistinguishable by size with distinctly different restriction maps (Fig. 3). One plasmid, designated pYFCl was present in all but one strain, and contained Pstl, Hindlll and Sac1 sites; the other plasmid, designated pYFC2 was present in 10 strains and contained Scal, Aval, and HgiAl sites (Fig. 3).
Leukotoxin production Regardless of plasmid content, all 15 strains were leukotoxin positive when culture supernatants were checked for BL-3 (bovine leukemia cells) cell cytotoxicity. Transformation of pYFCl and pYFC2 and subclones into E. coli and P. haemolytica Plasmids, pYFCl, pYFC2, pYFC4, pYFC5, pYFC6, and pYFC70 can be transformed into E. coli by the CaCI, method or that of Hanahan (Hanahan, 1983) and the DNA can be reisolated (data not shown). Attempts to transform pYFC4, pYFC5, pYFC6 and pYFC70 into P. haemolytica 5 (this strain does not have any plasmid) by either the procedure of Hanahan (Hanahan, 1983) or an electroporation technique (Craig et a/., 1989)
91
were not successful. However, pYFCl and pYFC2 can be transformed into P. haemolytica by these two latter techniques (data not shown). DNA sequence of the pYFCl The small plasmid, pYFCl was subjected to DNA sequence analysis and three open reading frames (ORFs) were found (Fig. 4). The first ORF encodes a sulfonamide resistant dihydropteroate synthase, the second reading frame encodes an aminoglycoside 3'-phosphotransferase, and the third encodes a partial MbeA-like protein. This is similar to the R S F l O l O which was originally described by Falkow's group, in which the sulfonamide and streptomycin resistance genes are closely linked and transcribed from the same promoter as the sulfonamide resistance gene (Guerry et a/., 1974; Scholz et a/., 1989). The D N A sequence was screened for E. coli promoter-like sequences using the homology score method (Mulligan et a/., 1984). There were t w o sequences similar to the TATAAT consensus promoter sequence (-1 0 region) and two sequences similar to the RNA polymerase-binding site, TTGACA (19) proximal to the dihydropteroate synthase gene (Fig. 4). The sulfonamide resistance gene has a methionine start codon with a reasonable Shine-Dalgarno
1 2 3 4 5 6 7 8 9 10 11 12 1314 151617 18 19 20 23,130 99416 6,6824,3 73 2,3222,027-
Figure 2 Comparative restriction endonuclease digestions. Plasmid D N A from P. haemolytica strains 43 (lanes 2-7) and 630 (lanes 8-13, 15-20) are digested with EcoRl (lanes 2 and 8), Scal (lanes 3 and 9), or EcoRl and Scal (lanes 4 and 10). Lanes 5-7, 1 1 - 1 1 are mock-digested (no enzyme) controls for lanes 2-4 and 8-1 0 respectively. Lanes 15, 16, 17, 18 are digestions with BamHI, EcoRI, Hindlll, or Hindlll plus EcoKI. Lanes 19, 20 are mock-digested controls for lanes 15 and 16. Lanes 1,4 are Hindlll-digested lambda DNA.
02
Y. F CHANC ET AL
-
’0
c
(I)
a
-7
i
I
I
l
l
I
STR
Mitochondrial DNA Downloaded from informahealthcare.com by McMaster University on 01/09/15 For personal use only.
i
*
S U L I1
-
, pxr-b
AMP
1 Kb
c’ Ioc ated up5treani (Fig. 4) The streptoiiiyc i n kinaw gene’ h‘i5 a niethionine start codon /lI L ’(,I w i t h ‘1 Shine-[lalgarno sequence (AGGA; kig 1) The st,irt codon and Shine-Dalgarno w ~ i i c ~ i i corc ~the P. haemolytica streptomycin kin‘i-+ gene’ n a s icfentiml t o that of the strA of the Inc (2 RSF I010 (ScholJ et < I / . , 1989). Immediately downstrean1 of the strA, thew was a “pseudogt.tit3” ot strH characterized by only the N-terniindl 1 d c c i u c d cirnino acids showing signific ,int \iniil,irity t o the Srr5 of R S F l 01 0 (Fig. 4). 5 e q u t ~ n c(25 t)eyond this point immediately cli\ttrged tram 5trH with multiple stop codons ,tppcwrrng in a11 t h r w redding frames. The third it1,itling trarrie contained 137 deduced amino < I ( it15 ,>nd showed 70 6”% homology to MbeA of ( oII 1 it3oycl el , I / , 1989) i n the N-terminal overI‘ip and i t c o t r l t l also h e a “pseudogene” since the MhcA in the C o l E l (Boyd et a/., 1989) contains 7 I 7 ,timino d ( i d s (Fig 4 ) Homology comparison o t t h c clrdut c d amino acid sequence from P. haemo/vri( 5 u I I I indicated that it has 98.1, 100 and 70”,8\iniil,iritv to that o t l n t Q R S F l O l 0 (Scholz et , I / , 1 O t ~ O j , p(JS05 (R,icJstrom et a / , 1988), and plb.1OLO iSl)r,itt rt
