Sep 19, 1977 - unit of HapI for 2 h, five different bands were visualized (Fig. 2, lane 4). The upper band is the linear pSN1; below it is the HpaI-A fragment.
JOURNAL
OF
Vol. 134, No. 1
BACTERIOLOGY, Apr. 1978, p. 345-348
0021-9193/78/0134-0345$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
NOTES Cleavage Maps of a Tetracycline Plasmid from Staphylococcus aureus AVIGDOR SHAFFERMAN,* ZAMIR SHALITA, AND ISRAEL HERTMAN Israel Institute for Biological Research, Ness-Ziona, Israel Received for publication 19 September 1977
The cleavage maps of a Staphylococcus aureus plasmid, pSN1 (2.75 megadaltons), conferring tetracycline resistance, were determined. Cleavage maps are given for HpaI and HindIII restriction endonucleases by using the single HpaII site as a reference point. Nucleases EcoRI, BamHI, Sall, and HaeIII have no sites on this plasmid.
Recently a 2.8-megadalton (Mdal) tetracycline plasmid was isolated from Staphylococcus aureus SN109 (Pen" Mecr Tetr EntB+ 200) and characterized (5). Strain SN109 harbors another miniplasmid, pEnt, involved with enterotoxin production. The tetracycline plasmid is now designated pSN1. Another tetracycline plasmid, pT127, of a molecular weight similar to pSNl, was isolated from a different S. aureus strain (4). The pT127 was used successfully to transform B. subtilis bacteria to Tetr (1). To find out whether there is a relationship between pSN1 and pT127 and because of its potential use in cloning experiments, restriction mapping of pSN1 was carried out. The tetracycline plasmid pSN1 was isolated from strain SN109 by the clear lysate procedure (5) followed by dye-bouyant density equilibrium centrifugation. The two different supercoiled DNA molecules harbored in SN109 were then separated on a dye sucrose gradient (5). Alternatively, the strain SN576 (5), harboring only the tetracycline plasmid, was used to isolate pSN1; in this case, the sucrose gradient separation was omitted. Restriction endonucleases HpaI, HpaII, SalI, HindIII, and HaeIII were from Biolabs, BamHI was prepared by the method of Wilson and Young (7), and EcoRI was prepared from Escherichia coli RY13 by the method of Tanaka and Weisblum (6). Activity of enzymes was determined by the use of known substrates, e.g., A DNA, simian virus 40 DNA, and pColEl DNA. Restriction fragments were resolved by electrophoresis on 1% agarose gel (2). We used phage A DNA, which produces a well-defined (3) cleavage pattern by HindIII and EcoRI, as a molecular weight marker.
The four restriction endonucleases, EcoRI, BamHI, SalI, and HaeIII, have no cleavage sites on pSN1. When pSN1 DNA was subjected to complete digestion with either HindIII or HpaI, three specific fragments were visualized; HpaII has only one cleavage site on pSN1, converting the supercoiled molecule to the linear form. A molecular weight of 2.8 x 106 was previously estimated for pSN1 by relative sedimentation in a sucrose gradient and by electron microscopy (5). We determined the molecular weight of pSN1 by using the relative mobilities in agarose gels of the linear form of the plasmid (produced by HpaII) together with data obtained from the sum of the molecular weights produced by the action of HindIII and HpaI and pSN1. The value obtained is 2.75 + 0.02 Mdal. The molecular weights of each of the fragments produced by either HindlIl or HpaI were then recalculated to best fit the mean value of 2.75 x 106. This was done by subjecting pSN1 DNA to various combinations of endonucleases as follows (i) HindIII partial digest; (ii) HpaI partial digest; (iii) HindIII + HpaII; (iv) HindIII + HpaI; (v) HpaII + HindIII partial digest; (vi) HindIII partial digest + HpaII partial digest; (vii) HpaI partial digest + HindIll. The calculations were actually carried out after we determined the relative positions of the various endonuclease sites on pSN1. To calculate averages and fittings, we used those fragments that were observed in the linear range of migration, namely, fragments ranging from 0.3 to 1.9 Mdal. In Table 1, a comparison of calculated and experimental (from relative mobilities) values in different experiments is presented. The various fragments were then oriented relative to one another by using the single HpaII
345
346
NOTES
J. BACTERIOL.
site as a reference point. First, we oriented the three HindIII DNA fragments. HpaII cleaves the HindIII-B fragment and produces two uneTABLE 1. Calculated and experimental values (in Mdal) of DNA fragments produced when subjecting pSNI DNA to various restriction endonucleases Restriction endonucle-
Calculated
Experimental
ase
HpaII
2.75
2.70 2.75 2.75 2.65
HpaI
1.52
1.55 1.50 1.55
HindIII
0.8
0.8 0.8 0.8
0.43
0.4 0.42 0.42
1.47
1.45 1.50 1.45
1.00
1.00 0.95
1.00
HpaI + HindIII
0.28
0.3 0.3 0.3
1.09
1.15
1.10 1.10 0.43a 0.42a
0.38a
HpaII + HindIII
0.4a 0.4a 0.4a
0.28
0.3 0.3 0.3
0.15
0.20 0.13 0.17
1.47
1.45 1.45 1.50
0.58
0.55 0.52 0.55
0.42
0.42 0.42 0.40
TABLE 1. Continued Restriction endonuclease
Calculated
Experimental
0.28
0.3 0.3 0.3
aThese fragments were not resolved experimentally, and the bands related to them appeared as a single large band (Fig. 2, lane 3).
qual fragments, B1 (0.58 Mdal) and B2 (0.42 Mdal) (Fig. 1, lane 4). To determine the correct orientation, it was necessary to find out which fragment is adjacent to the HindIII-C or A fragment. For this purpose different conditions for partial digestion of pSNl DNA by HindIII were tried. When pSN1 was subjected to an almost complete digestion by HpaII and then treated with HindIII for 20 min (2 units of enzyme and 1.5 ,tg of DNA), 12 different migration bands were observed (Fig. 1, lane 5). These fragments correspond to all the expected possibilities from partial and full digestion of pSN1 DNA together with the supercoiled and the open circular conformations. Because of the limits of precision, most of the bands would be expected to appear in either of the two possible orientations in similar positions. However, there is a band (fourth from the bottom in Fig. 1, lane 5) with a mobility of 5.3 cm, corresponding to a molecular weight of 0.86 x 106. This is the value expected to be found from the partially fragmented BIC DNA piece. In the second possible orientation, we would have expected to find the B2C fragment, which would have had a different and clearly separable band with a mobility of 5.7 cm. To conclude, the HpaII site in the HindIII-B fragment is oriented relative to A and C in such a way that the larger B1 fragment produced by HpaII is closer to C, and the smaller B2 fragment is closer to the HindHI-A fragment. Given the relative orientations of the HindIlI and the HpaII sites on pSN1, we tried to orient them relative to the HpaI sites. A comparison of lanes 1 and 2 in Fig. 2 suggests that the single HpaII site is very close to one of the HpaI sites-so close that under our conditions of separation it looks as if they overlap. To determine the exact orientation, conditions for partial digestion of pSN1 DNA by HpaI were sought. When 1.5 ,ug of pSN1 DNA was treated by 0.5 unit of HapI for 2 h, five different bands were visualized (Fig. 2, lane 4). The upper band is the linear pSN1; below it is the HpaI-A fragment (1.52 Mdal); beneath that, a 1.2-Mdal DNA piece, probably related to the HpaI-B, C fragment; and at the bottom appear two very faint bands corresponding to the HpaI-B (0.8 Mdal)
VOL. 134, 1978
347
NOTES
where the HpaI cleavage map is rotated (1800) around the HpaII axis (see Fig. 3). In the latter orientation, no HpaI sites are expected to lie within the HindIII-A fragment, and this seems not to be the case (cf. Fig. 2, lanes 3 and 7). Even when considering the largest experimental errors in placing restriction sites (0.06 Mdal, Table 1), the rotated orientation cannot account for the observed sizes of fragments obtained after digestion of pSN1 by HpaI + HindIII (Fig. 2, lanes 3, 4, and 5; Table 1). It follows, then, that the position of HindIIl sites in the HpaI cleavage map should be as depicted in Fig. 3. We can summarize all the foregoing data in a single cleavage map incorporating the various sites and molecular-weight estimates of the fragments (Fig. 3). The 2.8-Mdal tetracycline plasmid pT127, which was isolated from a different S. aureus strain (4), has three cleavage HindIII sites (1), md the three fragments produced have sizes
12
34
_
5 6 7
FIG. 1. Gel electrophoretic patterns of pSNI DNA and its fragments. (1) Untreated supercoiled pSN1; (2) digested with HpaII; (3) digested with HindIII; (4) digested with HindIII and then with HpaII; (5) digested with HpaII and then partially digested with HindIII; (6) phage A DNA digested with HindIII; (7) phage A DNA digested with HindIII and EcoRI. Agarose gels (1%) were in 40 mM tris(hydroxymethyl)aminomethane buffer-20 mM sodium acetate-1 mM EDTA; electrophoresis was for 2.5 h at ca. 4.5 V/cm.
and C (0.43 Mdal) fragments. When the pSN1 DNA is partially digested with HpaI as described above (during inactivation of HpaI the linear pSN1 is further degraded) and then treated with HpaII, the 1.2-Mdal DNA piece disappears and the two foremost lower bands
corresponding to HpaI-B and C fragments become clearly visible (cf. Fig. 2, lane 6, to lanes 2 and 5). It follows then that the HpaII site is very close to the HpaI site located on the HpaI-B, C partially digested fragment. This result allows us to define all the HpaI sites on pSN1 relative to HpaII. With this to possible to it was possible at hand hand it result at With this result orient the HpaI sites relative to HindIII. There are only two possible orientations to be considered: one is that shown in Fig. 3 and the other is was
I
2
3
4
5
6
7
8
9
FIG. 2. Gel electrophoretic patterns of pSN1 DNA fragments. (1) Digested with HpaI; (2) digested with
HpaI and then with HapII; (3) digested with HpaI and then with HindIII; (4) partially digested with digested with HpaI and then fully HpaI; (5) partially with with
HindIII; (6) partially digested digested HpaI and then fully digested with HpaII; (7) digested with HindIII; (8) phage A DNA digested with HindIII; (9) phage A DNA digested with HindIII and
EcoRI.
348
NOTES
J. BACTERIOL.
HPA ]
similar to those determined for pSNl DNA. As with pSN1, plasmid pT127 also has no EcoRI sites. It seems probable that the two S. aureus Tetr strains harbor similar or identical plasmids that carry the antibiotic resistance determinant. A4
HIN
IE
O 6
\ \
0 o.55
< \ \ \ \ \ I
| // 0
HIND1ll Pk
/
/
/
',V/
FIG. 3. Cleavage meap of pSNI. The inner and outer rings show HindMIII and HpaI fragments, reL rr-Trr M:. I he spectivety. Hpall cleavage site was designated zero point. The map distance from the zero point is shown in Mdal.
LITERATURE CITED
1. Ehrlich, S. D. 1977. Replication and expression of plasmids from Staphylococcus aureus in Bacillus subtilis. Proc. Natl. Acad. Sci. U.S.A. 74:1680-1682. 2. Green, P. J., M. C. Betlach, and H. W. Boyer. 1974. EcoRI restriction endonuclease. Methods Mol. Biol. 7:87-111. 3. Murray, K., and N. E. Murray. 1975. Phage lambda receptor chromosomes for DNA fragments made with restriction endonuclease III of Haenmphilus influenzae and restriction endonuclease I of Escherichia coli. J. Mol. Biol. 98:551-564. 4. Novick, R. 1976. Plasmid-protein relaxation complexes in Staphylococcus aureus. J. Bacteriol. 127:1177-1187. 5. Shalita, Z., L Hertman, and S. Sarid. 1977. Isolation and characterization of a plasmid involved with enterotoxin B production in Staphylococcus aureus. J. Bacteriol. 129:317-325. 6. Tanaka, T., and B. Weisblum. 1975. Construction of colicin E1-R factor composited plasmid in vitro: means for amplification of deoxyribonucleic acid. J. Bacteriol.
121:354-362. 7. Wilson, G. A., and F. E. Young. 1975. Isolation of a
sequence-specific endonuclease (BamI) from Bacillus
amyloliquefaciens H. J. Mol. Biol. 97:123-125.
