Proc. Nat!. Acad. Sci. USA Vol. 76, No. 1, pp. 400-404, January 1979
Genetics
Genetic translocation in Staphylococcus aureus (transposon/erythromycin resistance/insertional inactivation/staphylococcal plasmids)
RICHARD P. NovICK*, IRIT EDELMAN*, MARJORIE D. SCHWESINGER*, ALEXANDRA D. GRUSS*, EVRYLL C. SWANSONt, AND PETER A. PATTEEt *Department of Plasmid Biology, The Public Health Research Institute of The City of New York, Inc., New York, New York 10016; and tDepartment of
Bacteriology, Iowa State University, Ames, Iowa 50011 Communicated by George K. Hirst, October 3,1978
A 5.2-kilobase pair transposon, TnS5l, has been ABSTLAC found in Staphylococcus aureus, a Gram-positive bacterium. Initially detected on plasmid pI258, it undergoes rec-independent transposition to multiple chromosomal and plasmid sites, sometimes causing insertional inactivation. Unlike most other transposons, TnSl undergoes apparently precise excision as a rule. The initial observation of TnSSl transition involved UV inactivation of the carrier plasmid; this would appear to be a general means of detecting transposable elements. In the early to middle 1960s, "illegitimate" recombination involving plasmid-borne antibiotic resistance genes was observed in Staphylococcus aureus as well as in Escherichia colt. In particular, the erythromycin resistance determinant, ermB, of plasmid p1258 was found to insert into the recipient chromosome after transduction with UV-irradiated phage grown on a pI258 donor (1). Further, the inserted ermB gene could be transferred onto other plasmids, also by means of transduction (1). Similarly, Asheshov (2) described a S. aureus strain with a chromosomal fi-lactamase gene that could insert into a plasmid carried by the same strain. In this report, we present definitive genetic and molecular evidence that the ermB gene of pI258 belongs to a typical transposon. MATERIALS AND METHODS Organisms and Culture Procedures. The staphylococcal strains and plasmids used in this study are listed in Table 1. In general, the nomenclature follows the recommendations of Novick et al. (8) and Berg et al. (9). Translocations are indicated by a Greek omega (Q) followed by a serial isolation number and then by information about the insertion in square brackets. Thus, pI6187Q113[EcoA::TnS51] indicates that insertion 113 consists of a transposition of TnS51 into EcoRI fragment A of plasmid p16187. Culture media (CY broth, GL agar, and phage buffer), culture conditions, phages, preparation of phage lysates, and transduction procedures were as previously described (7). Resistance phenotypes were scored by standard methods (6). Genetic mapping by transformation was performed by- the method of Pattee and Neveln (5). Plasmid DNA Isolation. Plasmid DNA was isolated from cleared lysates of acetone-ethanol-treated organisms (7), subjected to two cycles of dye buoyant density centrifugation, dialyzed against 10 mM Tris.HCl/1 mM EDTA (pH 8), and stored at 40C. Restriction Endonuclease Analysis. Restriction endonucleases were obtained from Miles and were used according to the manufacturer's instructions. Electrophoresis was performed on horizontal slab gels with 0.8% Seakem agarose (Marine
Table 1. Staphylococcal strains and plasmids Parental strain or plasmid and relevant genotype Strains RN21 8325-398[Chr::Tn5511 RN43 8325(pRN4001) RN47 8325(pRN4007) RN53 8325(pRN4008) RN981 8325-4 his-7 recAl RN1438 8325-4(pRN3091) ISP7 8325 thy-101 his-116 trp-103 Plasmids p1258 bla cad bis lea asa asi ant mer ermB incl bla cad bis lea asa asi ant mer incl pI524 bla cadA cadB bis mer lea asa inc2 pI1147 pI618,7 bla cad bis lea asa asi ant mer incl pRN4001 pI524[blaI1]:pI258[ermBj
pRN4007 pRN4008 pRN4109 pRN4110 pRN4111 pRN4112 pRN4113 pRN3184 pRN3186 pRN3091
p1524U1O8[EcoA::Tn551J
Derivation
(1) (1) (1) (1) (3) (4) (5)
NO, (6) (6)
(6) (7) Recombinant,
(1)
pRI258A114[ermBJ pI258A116[ermBl
This paper This paper UV (1) UV (1) UV (1) UV (1) UV (1) UV (1) UV (1)
pI258 bLa-401 cadA52 mer-14 repA36
(14)
pII147fl109[EcoA::Tn551] pRN4001A112[ermBJ pRN4001A113[ermBj pRN4001A115[ermBJ pRN4001A117[ermBJ pRN4001A118[ermBJ
NO, naturally occurring.
Colloids, Rockland, ME) in Tris/borate/EDTA buffer, pH 8.3 (10), run at 14 mA overnight at room temperature. Gels were stained with ethidium bromide and photographed with Polaroid film through red and yellow filters (Kodak 23A and 8) and with UV transillumination. Molecular weights of fragments were calculated from relative mobilities in agarose in comparison with linear DNA standards. Electron Microscopy. Heteroduplexes were prepared by the alkali denaturation method of Davis et al. (11) except that the dialysis after renaturation was omitted. Specimens were mounted on parlodion-coated copper grids and were examined and photographed with a Philips EM300 electron microscope. Coliphage OX174 DNA was used as a single-stranded DNA length standard. RESULTS Mapping of Chromosomal Insertions. Some 40 different chromosomal insertions of ermB that had previously been obtained after transduction of p1258 by UV-irradiated phage 80c with selection for erythromycin resistance were mapped by
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Abbreviation: kb, kilobase pair. 400
Proc. Natl. Acad. Sci. USA 76 (1979)
Genetics: Novick et al. Table 2. Transformation of strain ISP7 (8325 thy-101 his-116 trp-103) with DNA from strain RN21 (8325-3Q8[chr::Tn551])
B;JC3c
.cRl
2
Selection for
3
C
9~
li
2 3 4 U5
401
Hoai B3r-4i
1l E8
Thy+ Trp+ Thy+ Trp+ 4.50 68 281 250
Frequency of reversion
Frequency of transformation
Genetics
Genetic translocation in Staphylococcus aureus (transposon/erythromycin resistance/insertional inactivation/staphylococcal plasmids)
RICHARD P. NovICK*, IRIT EDELMAN*, MARJORIE D. SCHWESINGER*, ALEXANDRA D. GRUSS*, EVRYLL C. SWANSONt, AND PETER A. PATTEEt *Department of Plasmid Biology, The Public Health Research Institute of The City of New York, Inc., New York, New York 10016; and tDepartment of
Bacteriology, Iowa State University, Ames, Iowa 50011 Communicated by George K. Hirst, October 3,1978
A 5.2-kilobase pair transposon, TnS5l, has been ABSTLAC found in Staphylococcus aureus, a Gram-positive bacterium. Initially detected on plasmid pI258, it undergoes rec-independent transposition to multiple chromosomal and plasmid sites, sometimes causing insertional inactivation. Unlike most other transposons, TnSl undergoes apparently precise excision as a rule. The initial observation of TnSSl transition involved UV inactivation of the carrier plasmid; this would appear to be a general means of detecting transposable elements. In the early to middle 1960s, "illegitimate" recombination involving plasmid-borne antibiotic resistance genes was observed in Staphylococcus aureus as well as in Escherichia colt. In particular, the erythromycin resistance determinant, ermB, of plasmid p1258 was found to insert into the recipient chromosome after transduction with UV-irradiated phage grown on a pI258 donor (1). Further, the inserted ermB gene could be transferred onto other plasmids, also by means of transduction (1). Similarly, Asheshov (2) described a S. aureus strain with a chromosomal fi-lactamase gene that could insert into a plasmid carried by the same strain. In this report, we present definitive genetic and molecular evidence that the ermB gene of pI258 belongs to a typical transposon. MATERIALS AND METHODS Organisms and Culture Procedures. The staphylococcal strains and plasmids used in this study are listed in Table 1. In general, the nomenclature follows the recommendations of Novick et al. (8) and Berg et al. (9). Translocations are indicated by a Greek omega (Q) followed by a serial isolation number and then by information about the insertion in square brackets. Thus, pI6187Q113[EcoA::TnS51] indicates that insertion 113 consists of a transposition of TnS51 into EcoRI fragment A of plasmid p16187. Culture media (CY broth, GL agar, and phage buffer), culture conditions, phages, preparation of phage lysates, and transduction procedures were as previously described (7). Resistance phenotypes were scored by standard methods (6). Genetic mapping by transformation was performed by- the method of Pattee and Neveln (5). Plasmid DNA Isolation. Plasmid DNA was isolated from cleared lysates of acetone-ethanol-treated organisms (7), subjected to two cycles of dye buoyant density centrifugation, dialyzed against 10 mM Tris.HCl/1 mM EDTA (pH 8), and stored at 40C. Restriction Endonuclease Analysis. Restriction endonucleases were obtained from Miles and were used according to the manufacturer's instructions. Electrophoresis was performed on horizontal slab gels with 0.8% Seakem agarose (Marine
Table 1. Staphylococcal strains and plasmids Parental strain or plasmid and relevant genotype Strains RN21 8325-398[Chr::Tn5511 RN43 8325(pRN4001) RN47 8325(pRN4007) RN53 8325(pRN4008) RN981 8325-4 his-7 recAl RN1438 8325-4(pRN3091) ISP7 8325 thy-101 his-116 trp-103 Plasmids p1258 bla cad bis lea asa asi ant mer ermB incl bla cad bis lea asa asi ant mer incl pI524 bla cadA cadB bis mer lea asa inc2 pI1147 pI618,7 bla cad bis lea asa asi ant mer incl pRN4001 pI524[blaI1]:pI258[ermBj
pRN4007 pRN4008 pRN4109 pRN4110 pRN4111 pRN4112 pRN4113 pRN3184 pRN3186 pRN3091
p1524U1O8[EcoA::Tn551J
Derivation
(1) (1) (1) (1) (3) (4) (5)
NO, (6) (6)
(6) (7) Recombinant,
(1)
pRI258A114[ermBJ pI258A116[ermBl
This paper This paper UV (1) UV (1) UV (1) UV (1) UV (1) UV (1) UV (1)
pI258 bLa-401 cadA52 mer-14 repA36
(14)
pII147fl109[EcoA::Tn551] pRN4001A112[ermBJ pRN4001A113[ermBj pRN4001A115[ermBJ pRN4001A117[ermBJ pRN4001A118[ermBJ
NO, naturally occurring.
Colloids, Rockland, ME) in Tris/borate/EDTA buffer, pH 8.3 (10), run at 14 mA overnight at room temperature. Gels were stained with ethidium bromide and photographed with Polaroid film through red and yellow filters (Kodak 23A and 8) and with UV transillumination. Molecular weights of fragments were calculated from relative mobilities in agarose in comparison with linear DNA standards. Electron Microscopy. Heteroduplexes were prepared by the alkali denaturation method of Davis et al. (11) except that the dialysis after renaturation was omitted. Specimens were mounted on parlodion-coated copper grids and were examined and photographed with a Philips EM300 electron microscope. Coliphage OX174 DNA was used as a single-stranded DNA length standard. RESULTS Mapping of Chromosomal Insertions. Some 40 different chromosomal insertions of ermB that had previously been obtained after transduction of p1258 by UV-irradiated phage 80c with selection for erythromycin resistance were mapped by
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
Abbreviation: kb, kilobase pair. 400
Proc. Natl. Acad. Sci. USA 76 (1979)
Genetics: Novick et al. Table 2. Transformation of strain ISP7 (8325 thy-101 his-116 trp-103) with DNA from strain RN21 (8325-3Q8[chr::Tn551])
B;JC3c
.cRl
2
Selection for
3
C
9~
li
2 3 4 U5
401
Hoai B3r-4i
1l E8
Thy+ Trp+ Thy+ Trp+ 4.50 68 281 250
Frequency of reversion
Frequency of transformation
