ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 1990, p. 1278-1280
Vol. 34, No. 6
0066-4804/90/061278-03$02.00/0 Copyright © 1990, American Society for Microbiology
Mobilization of the Gentamicin Resistance Gene in Enterococcus faecalis SUSAN L. HODEL-CHRISTIAN"2 AND BARBARA E. MURRAYl 3* Program in Infectious Diseases and Clinical Microbiology,' Department of Microbiology,2 and Department of Medicine,3 University of Texas Medical School, Houston, Texas 77030 Received 17 October 1989/Accepted 18 March 1990
Enterococcus faecalis plasmid pBEM10 (a conjugative plasmid encoding j8-lactamase production and gentamicin resistance [Gmr]) was made transfer deficient by using Tn917. Relocation of the Gm' determinant into two sites on pCF10 was observed. Restriction analysis revealed insertion of a common 2.5-kilobase-pair HindIII and a 3.9-kilobase-pair HaeM fragment encoding Gmr, suggesting that this determinant resides on a transposon similar to Tn4001.
High-level gentamicin resistance (Gmr) (MIC, >2,000 ,ug/ ml) in Enterococcus faecalis was first reported in 1979 in France (12). In 1983, high-level resistance to gentamicin and all other commercially available aminoglycosides was reported in nine clinical isolates of E. faecalis in Houston, Tex. (19). In these isolates, the genetic determinants for Gmr were carried on conjugative plasmids that transferred at a high frequency. The conjugative plasmid in one of these isolates, HH22, also carried the ,-lactamase gene (20, 21). Resistance to gentamicin in both Staphylococcus aureus and E. faecalis is due to a bifunctional enzyme with both 6'-acetyltransferase [AAC(6')] and 2"-phosphotransferase [APH(2")] activities; in addition to conferring gentamicin resistance, this enzyme confers resistance to tobramycin, kanamycin, amikacin, netilmicin, and sisomicin (3, 4, 18, 24). Identical nucleotide sequences for the gene encoding this enzyme have been obtained with Tn4001 from staphylococci from Australia and with E. faecalis plasmid pIP800 (10, 22). Tn4001 is a composite transposon that has a 2.0kilobase-pair (kb) region encoding aacA-aphD bounded by 1.35-kb terminal inverted repeats (IS256), for a total length of 4.7 kb (14, 15). Symmetrically located HindIII (2.5-kb) and HaeIII (3.9-kb) fragments are found in Tn4001 (11, 16). The 2.5-kb HindIIl fragment has been shown to hybridize with a 2.5-kb HindIII fragment in plasmids found in North American isolates of Gmr S. aureus (14). However, the North American isolates lack the 3.9-kb HaeIII fragment and have a symmetrically located 3.15-kb BglII fragment (14, 17). The terminal regions surrounding the genetic determinants for Gmr in the North American S. aureus isolates share homology with the IS256 elements of Tn4001; however, stem-loops formed during homoduplex analysis indicate that the terminal inverted repeats are shorter (14), and recent evidence has shown that these determinants apparently are not mobile (23). Since the Gmr (and Bla) genes are known to be present on transposons in some staphylococci, this study was undertaken to investigate the possibility that enterococci also have Gmr genes on a transposon. The first step was to generate Tra- mutants of pBEM10, a conjugative plasmid encoding Gmr and ,-lactamase production. pBEM10 is known to confer a response to the sex pheromone cAD1 (20). Originally found in strain HH22, pBEM10 has been introduced into E. faecalis 67, resulting in *
Corresponding author.
strain 67x22, and is the only plasmid in that strain (Table 1) (20). To generate Tra- mutants, pBEM10 was transferred into OGlX(pTV1-ts) by conjugation (using filter matings), as previously described (2); pTV1-ts is temperature sensitive for replication and contains a chloramphenicol resistance gene and the erythromycin resistance (Emr) transposon, Tn9J7 (9, 25). Transconjugants were selected that were resistant to gentamicin, chloramphenicol, and erythromycin and were Bla+. Transconjugants were grown overnight at 30°C to allow replication of pTV1-ts and then switched to 42°C, which prevented replication of pTV1-ts. Clones that were Gmr, Emr, Bla+, and chloramphenicol susceptible (Cm') were tested for their ability to transfer the gene encoding Gmr to E. faecalis JH2-2. Tra- mutants were selected and used in the mobilization studies. In order to detect mobilization of the determinant for Gmr, the conjugative tetracycline resistance (Tet') plasmid pCF10 was transferred into the Tra- mutant strains by using E. faecalis OGlSSp(pCF10) (a gift from Gary Dunny) (6, 8). Transconjugants that were Gmr, Emr, Bla+, and Tetr were selected. The transconjugants were tested for frequency of comobilization of only Gmr with pCF10 by cross-streak mating with E. faecalis JH2-2 (1). Following mating with JH2-2, two transconjugants (A and B) that were Tetr, Gmr, Ems, and Bla- were found. This resistance pattern suggested that the Gmr gene from the bla Gmr plasmid inserted into the conjugative plasmid pCF10. Resistance from A and B was transferred to OGlSSp to confirm the insertion of the Gmr gene into pCF10; all transconjugants acquired Tetr, Gmr, Ems, and Bla-. Plasmid DNA was collected from 67x22, OGlSSp (pCF10), A, and B by using a modification, for gram-positive bacteria, of the Currier and Nester (5) cell lysis protocol and cesium chloride-ethidium bromide density gradient centrifugation. Comparison of plasmid DNA from these strains was done with restriction endonuclease digestions and agarose gel electrophoresis. Restriction endonucleases were utilized according to the recommendations of the manufacturer (Bethesda Research Laboratories, Gaithersburg, Md.). Patterns generated by digesting plasmid DNA from pCF10, A, and B with EcoRI and BamHI differed from each other only in the size of one fragment (Fig. 1A). When plasmid DNA from A was digested with EcoRI, an increase in size of the largest pCF10 fragment was seen, while in plasmid DNA from B digested with EcoRI, the 7-kb fragment of pCF10 was 1278
VOL. 1990~~~~~~~~~~~NOTES VOL. 1990 ~~~~~~ 1279 34,34,
TABLE 1. E. faecalis strains utilized Strain
Relevant plasmid markers'
Host genotypea
67 x22(pBEM10)
OGlX(pTV1-ts) OGlSSp(pCF10) OG1X(pBEM10, pTV1-ts) OGlX(pBEM10::Tn9J7) OGlX(pBEM10::Tn9J7, pCF10) JH2-2
Gm' Th' Kan' Bla' Emr Cmr Tetr Tra' Gm' Tmr Kan' Bla' GmrTmr Kanr Bla' Gmr mr Kanr Bla'
riffus gel sir SP sir SP str gel str gel riffus str
Tra' Tra' EMr CMr EMr TraEmr Tetr
Reference(s) 20, 21 9, 25 6 This study This study This study 13 7 This study This study
str SP OGlSSp A [JH2-2(pCF10::Gmr9] Tetr Gm' Ems Blastr sp B [JH2-2(pCF10: :Gm9] str sp Tetr GMr Ems Blaa Resistance to rifampin fusidic acid (fus), streptomycin (str), gelatinase (gel), spectinomycin (sp). b Gm, Gentamicin; Th, tobramycin; Kan, kanamycin; Bla, P-lactamase; Em, erythromycin; Cm, chloramphenicol; Tra, transfer function; Tet, tetracycline.
(rip),
replaced by a 12-kb fragment. BamHl digestion of A resulted in the disappearance of the 5-kb pCF10 fragment and the appearance of a 10-kb fragment, while BamHI digestion of B resulted in an increase in size of the largest pCF1O fragment. These results suggest that the GMr gene resides on a transposon and has inserted into two different sites in pCF10. Digestion of plasmid DNA from 67x22, pCF10, A, and B with HindII revealed that 67X 22, A, and B share a 2.5-kb fragment, while most of the remaining HindIII fragments of A and B are identical to those seen in pCF10. The DNA was transferred from the agarose gels to Hybond-N membrane filters (Amersham Corp., Arlington Heights, Ill.), according to the recommendations of the manufacturer. A probe specific for the gene encoding the bifunctional GMr enzyme was prepared by double-digesting pSF815A with EcoRI and HindIll (both sites present in the pUC8
multiple cloning region) (10). This plasmid was originally constructed by Ferretti and co-workers (10) by cloning the 1.5-kb AluI fragment containing the coding region for the bifunctional enzyme encoding Gm' from E. faecalis into the multiple cloning site of pUC8. The 1.5-kb EcoRI-HindIII fragment was labeled with the Random Primed DNA labeling kit (Boehringer Mannheim Biochemicals, Indianapolis, Ind.) and [a_32P]dCTP (Amersham). Hybridization was carried out at 420C under highly stringent conditions. Prehybridization and hybridization solutions were as recommended by Amersham (5 x SSPE [l x SSPE is 0. 18 M NaCl-1 mM EDTA-10 mM NaPO4], 5 x Denhardt solution, 0. 5% sodium dodecyl sulfate, 50% formamide, and 100 ~.g of calf thymus DNA per ml). Hybridization between the probe and one fragment from 67 x22, A, and B plasmid DNA generated by digestion with each restriction endonuclease utilized was
A 1 2 3 45878910191012213 14 15
B 1'2! 3'4' 5'6' 7, 891110'I 12 13'14'15'
FIG. 1. (A) Agarose gel (0.7%) electrophoresis of restriction enidonuclease digestions with EcoRI (lanes 2 through 5), BamHl (lanes 7 through 10), and HindIII (lanes 12 through 15). Plasmid DNA was from 67x22 (lanes 2, 7, and 12), pCF1O (lanes 3, 8, and 13), A (lanes 4, 9, and 14), and B (lanes 5, 10, and 15). Molecular weight standards were lambda digested with HindIII (lanes 1 and 11) and a 1-kb ladder (Bethesda Research Laboratories) (lane 6). (B) Autoradiograph of filter of agarose gel in panel A following hybridization with the 32P-labeled 1.5-kb EcoRI-HindIII fragment from pSF815A. The DNA in the center of the gel (BamHI digests) apparently did not transfer as well as that on the sides of the gel.
1280
NOTES
observed; there was no hybridization to pCF10. The fragments in A and 1B that differed from pCF10 were the ones which showed homology with the probe (Figure 1B), confirming that the Gmr gene had relocated into those fragments. The common 2.5-kb HindlIl fragment seen in 67x22, A, and B plasmid DNA also hybridized with the Gmr probe. Since a 2.5-kb fragment carrying Gmr is also seen in both S. aureus Tn4001 from Australia and the North American S. aureus Gmr isolates (14), the region encoding Gmr from E. faecalis must have at least some similarities to the known Gmr transposon, Tn4001. Digestion of 67x22, A, and B with HaeIII revealed the presence of a common 3.9-kb fragment that also hybridized with the probe (data not shown). This is further evidence that the region surrounding the Gmr gene in E. faecalis 67 x 22 is similar to that found on the Australian S. aureus transposon, Tn4001. Digestion with BglII showed that 67x22, A, and B do not have the 3.15-kb BglII fragment that is present in the North American S. aureus isolates with Gmr (data not shown). In conclusion, the genetic determinant for Gmr in E. faecalis 67x22 may be located on a transposon. The ability of this determinant to be mobilized is shown by the insertion of the Gmr gene into two different sites in the conjugative Tetr plasmid pCF10. The Gmr_carrying element in E. faecalis has a restriction endonuclease pattern similar to that seen with Tn4001. Our data are the first to indicate that genetic determinants for Gmr in E. faecalis may reside on a transposable genetic element. Further studies incorporating the use of a recombination-deficient strain of E. faecalis are needed to determine whether or not mobilization occurs in Rec- cells. This work was supported in part by an American Heart Association Texas Affiliate grant-in-aid and in part by a grant from Pfizer Pharmaceuticals, Roerig Division. LITERATURE CITED 1. Bennett, P. M. 1984. Detection of transposable elements on plasmids, p. 227-231. In P. M. Bennett and J. Grinsted (ed.), Methods in microbiology. Academic Press, Inc., New York. 2. Clewell, D. B., F. Y. An, B. A. White, and C. Gawron-Burke. 1985. Streptococcus faecalis sex pheromone (cAM373) also produced by Staphylococcus aureus and identification of a conjugative transposon (Tn916). J. Bacteriol. 162:1212-1220. 3. Combes, T., C. Carlier, and P. Courvalin. 1983. Aminoglycoside-modifying enzyme content of a multiply resistant strain of Streptococcusfaecalis. J. Antimicrob. Chemother. 11:41-47. 4. Courvalin, P., C. Carlier, and E. Collatz. 1980. Plasmid-mediated resistance to aminocyclitol antibiotics in group D streptococci. J. Bacteriol. 143:541-551. 5. Currier, T. C., and E. W. Nester. 1976. Isolation of covalently closed circular DNA of high molecular weight from bacteria. Anal. Biochem. 76:431-441. 6. Dunny, G. M., J. C. Adsit, and C. Funk. 1981. Direct stimulation of the transfer of antibiotic resistance by sex pheromones in Streptococcus faecalis. Plasmid 6:270-278. 7. Dunny, G. M., B. L. Brown, and D. B. Clewell. 1978. Induced cell aggregation and mating in Streptococcusfaecalis: evidence for a bacterial sex pheromone. Proc. Natl. Acad. Sci. USA 75:3479-3483. 8. Dunny, G. M., M. Yuhasz, and E. Ehrenfeld. 1982. Genetic and physiological analysis of conjugation in Streptococcusfaecalis. J. Bacteriol. 151:855-859.
ANTIMICROB. AGENTS CHEMOTHER. 9. Ehrenfeld, E. E., and D. B. Clewell. 1987. Transfer functions of the Streptococcus faecalis plasmid pADl: organization of plasmid DNA encoding response to sex pheromone. J. Bacteriol. 169:3473-3481. 10. Ferretti, J. J., K. S. Gilmore, and P. Courvalin. 1986. Nucleotide sequence analysis of the gene specifying the bifunctional 6'-aminoglycoside acetyltransferase 2"-aminoglycoside phosphotransferase enzyme in Streptococcus faecalis and identification and cloning of gene regions specifying the two activities. J. Bacteriol. 167:631-638. 11. Gillespie, M. T., B. R. Lyon, L. J. Messerotti, and R. A. Skurray. 1987. Chromosome- and plasmid-mediated gentamicin resistance in Staphylococcus aureus encoded by Tn4001. J. Med. Microbiol. 24:139-144. 12. Horodniceanu, T., L. Bougueleret, N. El-Solh, G. Bieth, and F. Delbos. 1979. High-level, plasmid-borne resistance to gentamicin in Streptococcus faecalis subsp. zymogenes. Antimicrob. Agents Chemother. 16:686-689. 13. Jacob, A. E., and S. J. Hobbs. 1974. Conjugal transfer of plasmid-borne multiple antibiotic resistance in Streptococcus faecalis var. zymogenes. J. Bacteriol. 117:360-372. 14. Lyon, B. R., M. T. Gillespie, M. E. Byrne, J. W. May, and R. A. Skurray. 1987. Plasmid-mediated resistance to gentamicin in Staphylococcus aureus: the involvement of a transposon. J. Med. Microbiol. 23:101-110. 15. Lyon, B. R., M. T. Gillespie, and R. A. Skurray. 1987. Detection and characterization of IS256, an insertion sequence in Staphylococcus aureus. J. Gen. Microbiol. 133:3031-3038. 16. Lyon, B. R., J. W. May, and R. A. Skurray. 1984. Tn4001: a gentamicin and kanamycin resistance transposon in Staphylococcus aureus. Mol. Gen. Genet. 193:554-556. 17. Lyon, B. R., and R. Skurray. 1987. Antimicrobial resistance of Staphylococcus aureus: genetic basis. Microbiol. Rev. 51:88134. 18. Martel, A., M. Masson, N. Moreau, and F. LeGoffic. 1983. Kinetic studies of aminoglycoside acetyltransferase and phosphotransferase from Staphylococcus aureus RPAL. Relationships between the two activities. Eur. J. Biochem. 133:515-521. 19. Mederski-Samoraj, B. D., and B. E. Murray. 1983. High-level resistance to gentamicin in clinical isolates of enterococci. J. Infect. Dis. 147:751-757. 20. Murray, B. E., F. Y. An, and D. B. Clewell. 1988. Plasmids and pheromone response of the ,B-lactamase producer Streptococcus (Enterococcus)faecalis HH22. Antimicrob. Agents Chemother. 32:547-551. 21. Murray, B. E., and B. Mederski-Samaroj. 1983. Transferable P-lactamase: a new mechanism for in vitro resistance in Streptococcus faecalis. J. Clin. Invest. 72:1168-1171. 22. Rouch, D. A., M. E. Byrne, Y. C. Kong, and R. A. Skurray. 1987. The aacA-aphD gentamicin and kanamycin resistance determinant of Tn4001 from Staphylococcus aureus: expression and nucleotide sequence analysis. J. Gen. Microbiol. 133: 3039-3052. 23. Thomas, W. D., Jr., and G. L. Archer. 1989. Mobility of gentamicin resistance genes from staphylococci isolated in the United States: identification of Tn4031, a gentamicin resistance transposon from Staphylococcus epidermidis. Antimicrob. Agents Chemother. 33:1335-1341. 24. Ubukata, K., N. Yamashita, A. Gotob, and M. Konno. 1984. Purification and characterization of aminoglycoside-modifying enzymes from Staphylococcus aureus and Staphylococcus epidermidis. Antimicrob. Agents Chemother. 25:754-759. 25. Youngman, P. 1987. Plasmid vectors for recovering and exploiting Tn917 transpositions in Bacillus and other gram-positive bacteria, p. 79-103. In K. Hardy (ed.), Plasmids, a practical approach. IRL Press, Oxford.
Vol. 34, No. 6
0066-4804/90/061278-03$02.00/0 Copyright © 1990, American Society for Microbiology
Mobilization of the Gentamicin Resistance Gene in Enterococcus faecalis SUSAN L. HODEL-CHRISTIAN"2 AND BARBARA E. MURRAYl 3* Program in Infectious Diseases and Clinical Microbiology,' Department of Microbiology,2 and Department of Medicine,3 University of Texas Medical School, Houston, Texas 77030 Received 17 October 1989/Accepted 18 March 1990
Enterococcus faecalis plasmid pBEM10 (a conjugative plasmid encoding j8-lactamase production and gentamicin resistance [Gmr]) was made transfer deficient by using Tn917. Relocation of the Gm' determinant into two sites on pCF10 was observed. Restriction analysis revealed insertion of a common 2.5-kilobase-pair HindIII and a 3.9-kilobase-pair HaeM fragment encoding Gmr, suggesting that this determinant resides on a transposon similar to Tn4001.
High-level gentamicin resistance (Gmr) (MIC, >2,000 ,ug/ ml) in Enterococcus faecalis was first reported in 1979 in France (12). In 1983, high-level resistance to gentamicin and all other commercially available aminoglycosides was reported in nine clinical isolates of E. faecalis in Houston, Tex. (19). In these isolates, the genetic determinants for Gmr were carried on conjugative plasmids that transferred at a high frequency. The conjugative plasmid in one of these isolates, HH22, also carried the ,-lactamase gene (20, 21). Resistance to gentamicin in both Staphylococcus aureus and E. faecalis is due to a bifunctional enzyme with both 6'-acetyltransferase [AAC(6')] and 2"-phosphotransferase [APH(2")] activities; in addition to conferring gentamicin resistance, this enzyme confers resistance to tobramycin, kanamycin, amikacin, netilmicin, and sisomicin (3, 4, 18, 24). Identical nucleotide sequences for the gene encoding this enzyme have been obtained with Tn4001 from staphylococci from Australia and with E. faecalis plasmid pIP800 (10, 22). Tn4001 is a composite transposon that has a 2.0kilobase-pair (kb) region encoding aacA-aphD bounded by 1.35-kb terminal inverted repeats (IS256), for a total length of 4.7 kb (14, 15). Symmetrically located HindIII (2.5-kb) and HaeIII (3.9-kb) fragments are found in Tn4001 (11, 16). The 2.5-kb HindIIl fragment has been shown to hybridize with a 2.5-kb HindIII fragment in plasmids found in North American isolates of Gmr S. aureus (14). However, the North American isolates lack the 3.9-kb HaeIII fragment and have a symmetrically located 3.15-kb BglII fragment (14, 17). The terminal regions surrounding the genetic determinants for Gmr in the North American S. aureus isolates share homology with the IS256 elements of Tn4001; however, stem-loops formed during homoduplex analysis indicate that the terminal inverted repeats are shorter (14), and recent evidence has shown that these determinants apparently are not mobile (23). Since the Gmr (and Bla) genes are known to be present on transposons in some staphylococci, this study was undertaken to investigate the possibility that enterococci also have Gmr genes on a transposon. The first step was to generate Tra- mutants of pBEM10, a conjugative plasmid encoding Gmr and ,-lactamase production. pBEM10 is known to confer a response to the sex pheromone cAD1 (20). Originally found in strain HH22, pBEM10 has been introduced into E. faecalis 67, resulting in *
Corresponding author.
strain 67x22, and is the only plasmid in that strain (Table 1) (20). To generate Tra- mutants, pBEM10 was transferred into OGlX(pTV1-ts) by conjugation (using filter matings), as previously described (2); pTV1-ts is temperature sensitive for replication and contains a chloramphenicol resistance gene and the erythromycin resistance (Emr) transposon, Tn9J7 (9, 25). Transconjugants were selected that were resistant to gentamicin, chloramphenicol, and erythromycin and were Bla+. Transconjugants were grown overnight at 30°C to allow replication of pTV1-ts and then switched to 42°C, which prevented replication of pTV1-ts. Clones that were Gmr, Emr, Bla+, and chloramphenicol susceptible (Cm') were tested for their ability to transfer the gene encoding Gmr to E. faecalis JH2-2. Tra- mutants were selected and used in the mobilization studies. In order to detect mobilization of the determinant for Gmr, the conjugative tetracycline resistance (Tet') plasmid pCF10 was transferred into the Tra- mutant strains by using E. faecalis OGlSSp(pCF10) (a gift from Gary Dunny) (6, 8). Transconjugants that were Gmr, Emr, Bla+, and Tetr were selected. The transconjugants were tested for frequency of comobilization of only Gmr with pCF10 by cross-streak mating with E. faecalis JH2-2 (1). Following mating with JH2-2, two transconjugants (A and B) that were Tetr, Gmr, Ems, and Bla- were found. This resistance pattern suggested that the Gmr gene from the bla Gmr plasmid inserted into the conjugative plasmid pCF10. Resistance from A and B was transferred to OGlSSp to confirm the insertion of the Gmr gene into pCF10; all transconjugants acquired Tetr, Gmr, Ems, and Bla-. Plasmid DNA was collected from 67x22, OGlSSp (pCF10), A, and B by using a modification, for gram-positive bacteria, of the Currier and Nester (5) cell lysis protocol and cesium chloride-ethidium bromide density gradient centrifugation. Comparison of plasmid DNA from these strains was done with restriction endonuclease digestions and agarose gel electrophoresis. Restriction endonucleases were utilized according to the recommendations of the manufacturer (Bethesda Research Laboratories, Gaithersburg, Md.). Patterns generated by digesting plasmid DNA from pCF10, A, and B with EcoRI and BamHI differed from each other only in the size of one fragment (Fig. 1A). When plasmid DNA from A was digested with EcoRI, an increase in size of the largest pCF10 fragment was seen, while in plasmid DNA from B digested with EcoRI, the 7-kb fragment of pCF10 was 1278
VOL. 1990~~~~~~~~~~~NOTES VOL. 1990 ~~~~~~ 1279 34,34,
TABLE 1. E. faecalis strains utilized Strain
Relevant plasmid markers'
Host genotypea
67 x22(pBEM10)
OGlX(pTV1-ts) OGlSSp(pCF10) OG1X(pBEM10, pTV1-ts) OGlX(pBEM10::Tn9J7) OGlX(pBEM10::Tn9J7, pCF10) JH2-2
Gm' Th' Kan' Bla' Emr Cmr Tetr Tra' Gm' Tmr Kan' Bla' GmrTmr Kanr Bla' Gmr mr Kanr Bla'
riffus gel sir SP sir SP str gel str gel riffus str
Tra' Tra' EMr CMr EMr TraEmr Tetr
Reference(s) 20, 21 9, 25 6 This study This study This study 13 7 This study This study
str SP OGlSSp A [JH2-2(pCF10::Gmr9] Tetr Gm' Ems Blastr sp B [JH2-2(pCF10: :Gm9] str sp Tetr GMr Ems Blaa Resistance to rifampin fusidic acid (fus), streptomycin (str), gelatinase (gel), spectinomycin (sp). b Gm, Gentamicin; Th, tobramycin; Kan, kanamycin; Bla, P-lactamase; Em, erythromycin; Cm, chloramphenicol; Tra, transfer function; Tet, tetracycline.
(rip),
replaced by a 12-kb fragment. BamHl digestion of A resulted in the disappearance of the 5-kb pCF10 fragment and the appearance of a 10-kb fragment, while BamHI digestion of B resulted in an increase in size of the largest pCF1O fragment. These results suggest that the GMr gene resides on a transposon and has inserted into two different sites in pCF10. Digestion of plasmid DNA from 67x22, pCF10, A, and B with HindII revealed that 67X 22, A, and B share a 2.5-kb fragment, while most of the remaining HindIII fragments of A and B are identical to those seen in pCF10. The DNA was transferred from the agarose gels to Hybond-N membrane filters (Amersham Corp., Arlington Heights, Ill.), according to the recommendations of the manufacturer. A probe specific for the gene encoding the bifunctional GMr enzyme was prepared by double-digesting pSF815A with EcoRI and HindIll (both sites present in the pUC8
multiple cloning region) (10). This plasmid was originally constructed by Ferretti and co-workers (10) by cloning the 1.5-kb AluI fragment containing the coding region for the bifunctional enzyme encoding Gm' from E. faecalis into the multiple cloning site of pUC8. The 1.5-kb EcoRI-HindIII fragment was labeled with the Random Primed DNA labeling kit (Boehringer Mannheim Biochemicals, Indianapolis, Ind.) and [a_32P]dCTP (Amersham). Hybridization was carried out at 420C under highly stringent conditions. Prehybridization and hybridization solutions were as recommended by Amersham (5 x SSPE [l x SSPE is 0. 18 M NaCl-1 mM EDTA-10 mM NaPO4], 5 x Denhardt solution, 0. 5% sodium dodecyl sulfate, 50% formamide, and 100 ~.g of calf thymus DNA per ml). Hybridization between the probe and one fragment from 67 x22, A, and B plasmid DNA generated by digestion with each restriction endonuclease utilized was
A 1 2 3 45878910191012213 14 15
B 1'2! 3'4' 5'6' 7, 891110'I 12 13'14'15'
FIG. 1. (A) Agarose gel (0.7%) electrophoresis of restriction enidonuclease digestions with EcoRI (lanes 2 through 5), BamHl (lanes 7 through 10), and HindIII (lanes 12 through 15). Plasmid DNA was from 67x22 (lanes 2, 7, and 12), pCF1O (lanes 3, 8, and 13), A (lanes 4, 9, and 14), and B (lanes 5, 10, and 15). Molecular weight standards were lambda digested with HindIII (lanes 1 and 11) and a 1-kb ladder (Bethesda Research Laboratories) (lane 6). (B) Autoradiograph of filter of agarose gel in panel A following hybridization with the 32P-labeled 1.5-kb EcoRI-HindIII fragment from pSF815A. The DNA in the center of the gel (BamHI digests) apparently did not transfer as well as that on the sides of the gel.
1280
NOTES
observed; there was no hybridization to pCF10. The fragments in A and 1B that differed from pCF10 were the ones which showed homology with the probe (Figure 1B), confirming that the Gmr gene had relocated into those fragments. The common 2.5-kb HindlIl fragment seen in 67x22, A, and B plasmid DNA also hybridized with the Gmr probe. Since a 2.5-kb fragment carrying Gmr is also seen in both S. aureus Tn4001 from Australia and the North American S. aureus Gmr isolates (14), the region encoding Gmr from E. faecalis must have at least some similarities to the known Gmr transposon, Tn4001. Digestion of 67x22, A, and B with HaeIII revealed the presence of a common 3.9-kb fragment that also hybridized with the probe (data not shown). This is further evidence that the region surrounding the Gmr gene in E. faecalis 67 x 22 is similar to that found on the Australian S. aureus transposon, Tn4001. Digestion with BglII showed that 67x22, A, and B do not have the 3.15-kb BglII fragment that is present in the North American S. aureus isolates with Gmr (data not shown). In conclusion, the genetic determinant for Gmr in E. faecalis 67x22 may be located on a transposon. The ability of this determinant to be mobilized is shown by the insertion of the Gmr gene into two different sites in the conjugative Tetr plasmid pCF10. The Gmr_carrying element in E. faecalis has a restriction endonuclease pattern similar to that seen with Tn4001. Our data are the first to indicate that genetic determinants for Gmr in E. faecalis may reside on a transposable genetic element. Further studies incorporating the use of a recombination-deficient strain of E. faecalis are needed to determine whether or not mobilization occurs in Rec- cells. This work was supported in part by an American Heart Association Texas Affiliate grant-in-aid and in part by a grant from Pfizer Pharmaceuticals, Roerig Division. LITERATURE CITED 1. Bennett, P. M. 1984. Detection of transposable elements on plasmids, p. 227-231. In P. M. Bennett and J. Grinsted (ed.), Methods in microbiology. Academic Press, Inc., New York. 2. Clewell, D. B., F. Y. An, B. A. White, and C. Gawron-Burke. 1985. Streptococcus faecalis sex pheromone (cAM373) also produced by Staphylococcus aureus and identification of a conjugative transposon (Tn916). J. Bacteriol. 162:1212-1220. 3. Combes, T., C. Carlier, and P. Courvalin. 1983. Aminoglycoside-modifying enzyme content of a multiply resistant strain of Streptococcusfaecalis. J. Antimicrob. Chemother. 11:41-47. 4. Courvalin, P., C. Carlier, and E. Collatz. 1980. Plasmid-mediated resistance to aminocyclitol antibiotics in group D streptococci. J. Bacteriol. 143:541-551. 5. Currier, T. C., and E. W. Nester. 1976. Isolation of covalently closed circular DNA of high molecular weight from bacteria. Anal. Biochem. 76:431-441. 6. Dunny, G. M., J. C. Adsit, and C. Funk. 1981. Direct stimulation of the transfer of antibiotic resistance by sex pheromones in Streptococcus faecalis. Plasmid 6:270-278. 7. Dunny, G. M., B. L. Brown, and D. B. Clewell. 1978. Induced cell aggregation and mating in Streptococcusfaecalis: evidence for a bacterial sex pheromone. Proc. Natl. Acad. Sci. USA 75:3479-3483. 8. Dunny, G. M., M. Yuhasz, and E. Ehrenfeld. 1982. Genetic and physiological analysis of conjugation in Streptococcusfaecalis. J. Bacteriol. 151:855-859.
ANTIMICROB. AGENTS CHEMOTHER. 9. Ehrenfeld, E. E., and D. B. Clewell. 1987. Transfer functions of the Streptococcus faecalis plasmid pADl: organization of plasmid DNA encoding response to sex pheromone. J. Bacteriol. 169:3473-3481. 10. Ferretti, J. J., K. S. Gilmore, and P. Courvalin. 1986. Nucleotide sequence analysis of the gene specifying the bifunctional 6'-aminoglycoside acetyltransferase 2"-aminoglycoside phosphotransferase enzyme in Streptococcus faecalis and identification and cloning of gene regions specifying the two activities. J. Bacteriol. 167:631-638. 11. Gillespie, M. T., B. R. Lyon, L. J. Messerotti, and R. A. Skurray. 1987. Chromosome- and plasmid-mediated gentamicin resistance in Staphylococcus aureus encoded by Tn4001. J. Med. Microbiol. 24:139-144. 12. Horodniceanu, T., L. Bougueleret, N. El-Solh, G. Bieth, and F. Delbos. 1979. High-level, plasmid-borne resistance to gentamicin in Streptococcus faecalis subsp. zymogenes. Antimicrob. Agents Chemother. 16:686-689. 13. Jacob, A. E., and S. J. Hobbs. 1974. Conjugal transfer of plasmid-borne multiple antibiotic resistance in Streptococcus faecalis var. zymogenes. J. Bacteriol. 117:360-372. 14. Lyon, B. R., M. T. Gillespie, M. E. Byrne, J. W. May, and R. A. Skurray. 1987. Plasmid-mediated resistance to gentamicin in Staphylococcus aureus: the involvement of a transposon. J. Med. Microbiol. 23:101-110. 15. Lyon, B. R., M. T. Gillespie, and R. A. Skurray. 1987. Detection and characterization of IS256, an insertion sequence in Staphylococcus aureus. J. Gen. Microbiol. 133:3031-3038. 16. Lyon, B. R., J. W. May, and R. A. Skurray. 1984. Tn4001: a gentamicin and kanamycin resistance transposon in Staphylococcus aureus. Mol. Gen. Genet. 193:554-556. 17. Lyon, B. R., and R. Skurray. 1987. Antimicrobial resistance of Staphylococcus aureus: genetic basis. Microbiol. Rev. 51:88134. 18. Martel, A., M. Masson, N. Moreau, and F. LeGoffic. 1983. Kinetic studies of aminoglycoside acetyltransferase and phosphotransferase from Staphylococcus aureus RPAL. Relationships between the two activities. Eur. J. Biochem. 133:515-521. 19. Mederski-Samoraj, B. D., and B. E. Murray. 1983. High-level resistance to gentamicin in clinical isolates of enterococci. J. Infect. Dis. 147:751-757. 20. Murray, B. E., F. Y. An, and D. B. Clewell. 1988. Plasmids and pheromone response of the ,B-lactamase producer Streptococcus (Enterococcus)faecalis HH22. Antimicrob. Agents Chemother. 32:547-551. 21. Murray, B. E., and B. Mederski-Samaroj. 1983. Transferable P-lactamase: a new mechanism for in vitro resistance in Streptococcus faecalis. J. Clin. Invest. 72:1168-1171. 22. Rouch, D. A., M. E. Byrne, Y. C. Kong, and R. A. Skurray. 1987. The aacA-aphD gentamicin and kanamycin resistance determinant of Tn4001 from Staphylococcus aureus: expression and nucleotide sequence analysis. J. Gen. Microbiol. 133: 3039-3052. 23. Thomas, W. D., Jr., and G. L. Archer. 1989. Mobility of gentamicin resistance genes from staphylococci isolated in the United States: identification of Tn4031, a gentamicin resistance transposon from Staphylococcus epidermidis. Antimicrob. Agents Chemother. 33:1335-1341. 24. Ubukata, K., N. Yamashita, A. Gotob, and M. Konno. 1984. Purification and characterization of aminoglycoside-modifying enzymes from Staphylococcus aureus and Staphylococcus epidermidis. Antimicrob. Agents Chemother. 25:754-759. 25. Youngman, P. 1987. Plasmid vectors for recovering and exploiting Tn917 transpositions in Bacillus and other gram-positive bacteria, p. 79-103. In K. Hardy (ed.), Plasmids, a practical approach. IRL Press, Oxford.
