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Physical and functional map of the hemolytic plasmid pSU316
PLASMID
11,96-98 (1984)
Physical and Functional Map of the Hemolytic Plasmid pSU316 ISABEL ANDR~S,' Jo& Departamento
de Bioquimica,
~.RODRIGUEZ,
Facultad de Medic&a,
ANDJO&
M. ORTIZ
Poligono de Cazoiia s/n, Santander, Spain
Received June 1, 1983 A restriction endonucleaseanalysis of the hemolytic plasmid pSU3 16 has allowed location of the cleavage sites for the endonucleasesBamHI, XbaI, KpnI, BgllI, SaZGI, EcoRI, and HindIII. Hybridization experiments between pSU316 and pEDlO0 have shown that the tra region of pSU3 I6 lies in a segmentcomprising part of SaK31fragments S-I and S-3 and the entire fragment S-4. The positions of other plasmid coded functions, namely the replication functions and 01hemolysin production, have been determined in the physical map.
The hemolytic plasmid pSU3 16 is a conjugative plasmid with a molecular weight of 76.8 kb, isolated from a human Escherichia coli strain (3). The genetic information required for the production of the a-hemolysin is located in a 6.0-kb DNA sequence shared by several other plasmids belonging to different incompatibility groups (4), as well as by the bacterial chromosome of E. coli hemolytic strains (5,7). Cloning experiments have indicated that an adjacent segment of 6.2 kb carries all the functions required for the autonomous replication of this plasmid (II). Two transposition derivatives of pSU3 16, namely pSU306 (pSU3 16: :Tn802; Hly- Carbenicilin (Cb) resistant) and pSU307 (pSU3 16: :Tn5, Hly- Kanamycin (Km) resistant), have allowed assignment of the plasmid pSU3 16 to the incompatibility complex In&III-FIV (3) and a study of its incompatibility properties (9). Furthermore, cloning experiments have shown that the incompatibilities In&III and FIV are physically separated and that the DNA sequenceinvolved in the IncFIII incompatibility determinant is located in a 0.5-kb PstI fragment within the replication region of pSU3 16 (I I). We present here a restriction map of pSU3 16 for the endonucleasesBumHI, XbaI, KpnI, Bg/II, SulGI, EcoRI, and HindIII. The position of the transposons Tn802 and Tn5 ’ To whom correspondence should be sent. 0147-619X/84 $3.00 Copyright 8 1984 by Academic Pi-a, Inc. All rigJtts of reproduction in any form reserved.
96
in the transposition derivatives pSU306 and pSU307 as well as the hemolytic and tra regions and the region containing the replication functions of pSU3 16 have been located on this physical map. Hybrid plasmid DNA, amplified in the presence of cloramphenicol (Cm) was prepared as described by Timmis et al. (15). Plasmid DNA that does not amplify during Cm treatment was obtained according to Humphreys et al. (6). Restriction enzymes and T4 DNA ligase were obtained from New England Bio-Labs, and used according to the supplier’s recommendations. DNA polymerase I was purchased from Boehringer. For hybridization experiments, DNA fragments generated by restriction endonucleases were separated by agarosegel electrophoresis and transferred to nitrocellulose filters by the Southern blotting technique (13). pED 100 plasmid DNA was labeled with [a-32P]dGTP by nick translation with DNA polymerase I. The 32P-labeledprobe was hybridized against the DNA samples blotted onto nitrocellulose filters. Hybridization was carried out at 68°C in 5-10 ml of hybridization buffer (4) containing about 5 X 1O6cpm of the labeled probe, for about 20 h. pSU3 16 BglII- or KpnI-generated fragments were cloned into the unique BgflI or KpnI cleavage site of pKTO42 and pKTO68, respectively. Both cloning vectors are pBR322/
SHORT COMMUNICATIONS
pSC102 hybrid plasmids containing a unique restriction site for either BgAI or KpnI in the pSC102 PstI fragment inserted into pBR322 (1,12). pSU3 16 SulGI-generated fragments were cloned into the unique SalGI site of pBR322 (2). Agarose gel electrophoresis was performed in a vertical slab gel at agaroseconcentrations between 0.7 and 1.2% depending on the size of the fragment being studied. Electrophoresis was carried out in either TB (90 mM Tris, 2.5 mM EDTA, 90 mM boric acid, pH 8.3) or TP buffer (36 mM Tris, 30 mM NaH2P04, 10 mM EDTA, pH 7.5). Molecular weight standards were the bacteriophage X DNA fragments generated by digestion with Hind111 and the R6-5 DNA fragments generated by digestion with either EcoRI or HindIII. The molecular weights of X/Hind111and R6-S/EcoRI or R6S/Hind111were taken from Murray and Murray (8) and from Timmis et al. (25), respectively. The size of the largest fragment in each single digestion was taken to be the difference between the calculated size of the whole plasmid (76.8 kb, Ref. (3)) and the added sizesof the other restriction fragments. The restriction map has been constructed by comparison of double digest of pSU3 16 DNA with the digestion patterns produced by each of the restriction enzymes alone. When this approach proved insufficient hybrid plasmids containing specific fragments of pSU3 16 were digested with the appropriate restriction enzymes. The map has been orientated using the unique BarnHI site as reference point. The restriction sites produced by the endonucleasesBamHI and XbaI have been used in order to locate fragments generatedby other endonucleases unambiguously, namely BglII fragments B-3 and B-4, SulGI fragment S-2, KpnI fragment K-l, and EcoRI fragment E1. Double digestion of pSU3 16 DNA with both BglII and KpnI allowed assignment of all of the KpnI fragments and all but the smallest BgfiI fragments. This fragment (B-5) was located after a BgllI/SalGI digestion that also produced a SalGI map consistent with those for Bg/II and KpnI. This strategy allowed also construction of the Hind111map. The data
97
on sequencehomology in the hemolytic region between plasmid pHlyl52 and pSU3 16 (4) and on insertion of Tn802 in pSU3 16 (3) have been used to determine the position of some of the EcoRI pSU3 16 generated fragments, namely E-l, E-4, E-5, and E-9. The position of other EcoRI fragments was determined by using hybrid plasmids containing specific pSU3 16 restriction fragments. The location of the smallest EcoRI fragment (E-10, 0.3 kb) has not been determined. Hybridization studies between the hemolytic plasmids pHly152 and pSU3 16 (4) have demonstrated that the extent of their DNA sequence homology is restricted to a 6.0-kb fragment carrying the Hly determinant. The stretch of DNA, flanked at one end by a BumHI site, comprises 1.9 kb of the largest pSU3 16 EcoRI fragment, part of fragment E4 and the whole of EcoRI fragments E-5 and E-9. In the physical map of pSU3 16 (Fig. 1) the hemolytic determinant runs anticlockwise from the BumHI site overlapping 2.9 kb of
FIG. 1. Physicaland functional map of plasmid pSU3 16. @aI, KpnI (K), BgfiI (B), SufCiI (S), EcoRI (E), and Hi&II restriction maps of plasmid pSU3 16 are orientated with respect to the unique BarnHI site. Map coordinates are given in kilobases. Dashed lines in the functional map indicate that the end of the region has not been determined exactly.
98
SHORT COMMUNICATIONS
BgflI fragment B-4 and 3.0 kb of fragment B-3. Two Hly- derivatives of pSU3 16, showing resistance either to Cb (pSU306) or Km (pSU307) were obtained by transposition to pSU3 16 of Tn802 and Tn5, respectively. It has been shown that in pSU306, Tn802 insertion occurs in EC&I fragment E-9, whereas in pSU307 Tn5 is inserted in IIindIII fragment H-6 (3,9). Restriction analysis of the parental plasmid and its two transposition derivatives after Bg& digestion revealed that both insertions lie on fragment B-4, which is in agreement with the location of the hemolytic region. According to the results of Nogel et al. (10) the insertions of Tn802 in pSU306 and of Tn5 in pSU307 lie on cistrons hlyA and h&Z, respectively, responsible for the synthesis of a protein precursor of cu-hemolysin. By means of cloning experiments a Cb’ miniplasmid (pSU3025) derived of pSU306 (pSU3 16: :Tn802) has been constructed (1 I). The miniplasmid contains all the functions required for the autonomous replication of pSU306, and therefore of pSU3 16, in a 6.2kb DNA segmentcontiguous to the hly region, flanked at the ends by the BgfiI site of the Bl-B-4 junction and the unique BarnHI restriction site of pSU316 (see Fig. I). We have studied the sequence homology between pSU316 and pEDlO0 (a mini-F derivative containing the replication and transfer regions of F, Ref. (16)) in order to localize the tra region of pSU3 16. pSU3 16 plasmid DNA digested with Hind111and SalGI endonucleaseswas run on agarosegels, blotted directly on nitrocellulose filters and hybridized against 32P-labeledDNA prepared by nick translation of pED 100 plasmid DNA. An EcoRI digestion of pEDlO0 was also included in the gels as hybridization control. Hybridization was detected in the three largest generated bands, which correspond to S&G1 fragments S-l, S-3, and S-4. BecauseS-4 is the SulGI fragment located between S-l and S-3, this result indicates that the tra region of plasmid pSU3 16 comprises
the whole fragment S-4 and part of the fragments S-l and S-3. ACKNOWLEDGMENTS We thank Dr. F. de la Cruz for helpful discussion and Mrs. Marta Garcia for her excellent assistance.J.C.R. was a recipient of a grant (Institute National de Asistencia y Promotion de1Estudiante) from the Spanish Ministry of Education. This work was supported by a grant from the Comisibn Asesora Cientifica y Tecnica.
REFERENCES 1. ANDRE& I., SLOCOMBE,P. M., CABELLO, F., TIMMIS, J. K., LUFU, R., BURKARDT, H. J., AND TIMMIS, K. N., Molec. Gen. Genef. 168, l-25 (1979). 2. BOLIVAR, F., RODRIGUEZ, R. L., GREENE, P. J., BETLACH, M. C., HEYNEKER, H. L., BOYER, H. W., CROSA, J., AND FALKOW, S., Gene 2,95-
113 (1977). 3. DE LA CRUZ, F., ZABALA, J. C., AND ORTIZ, J. M., Plasmid 2, 507-519 (1979). 4. DE LA CRUZ, F., MOLLER, D., ORTIZ, J. M., AND GOEBEL, W., J. Bacferiol. 143, 825-833 (1980). 5. DE LA CRUZ, F., ZABALA, J. C., AND ORTIZ, J. M. Infect. Immun. 41, 881-887 (1983). 6. HUMPHREYS, G. O., WILLSHAW, G. A., AND ANDERSON, E. S., Biochim. Biophys. Acta 383,457-
463 (1975). 7. MULLER, D., HUGHES, C., AND GOEBEL, W., J. Bacteriol. 153, 846-851 (1983). 8. MURRAY, K., AND MURRAY, N. E., J. Mol. Biol. 98, 551-564 (1975). 9. NAVAS, J., DE LA CRUZ, F., RODRIGUEZ, J. C., ANDRES, I., VULGAR, G., AND ORTIZ, J. M., J. Gen. Microbial. 129, 2277-2283 (1983). 10. NOEGEL, A., RDEST, U., SPRINGER,W., AM) G~EBEL, W., Mol. Gen. Genet. 175, 343-350 (1979). II. RODRIGUEZ, J. C., ANDRES, I., DE LA CRUZ, F., NAVAS, J., I%JLGAR, G., AND ORTIZ, J. M., Plasmid
10, 175-183 (1983). 12. SLOCOMBE, P. M., ANDRES, I., AND TIMMIS, K. N.,
In “Microbial Drug Resistance and Related Plasmid? (S. Mitsuhashi and H. Hashimoto, eds.),pp. 83-87. Tokyo Univ. Press,Tokyo, 1978. 13. SOUTHERN, E. M., J. Mol. Biol. 98,503-517 (1975). 14. TIMMIS, K., CABELLO, F., AND COHEN, S. N., In
“Modem Trends in Bacterial Transformation and Transfection” (A. Portoles, R. L@ez, and H. Espinosa,eds.),pp. 263-276. Elsevier/North-Holland, Amsterdam, 1977 15. TIMMIS, K. N., CABELLO, F., AND COHEN, S. N., Mol. Gen. Genet. 162, 121-137 (1978). 16. WILLETS, N., AND JOHNSON, D., Mol. Gen. Genet.
182, 520-522 (1981).
11,96-98 (1984)
Physical and Functional Map of the Hemolytic Plasmid pSU316 ISABEL ANDR~S,' Jo& Departamento
de Bioquimica,
~.RODRIGUEZ,
Facultad de Medic&a,
ANDJO&
M. ORTIZ
Poligono de Cazoiia s/n, Santander, Spain
Received June 1, 1983 A restriction endonucleaseanalysis of the hemolytic plasmid pSU3 16 has allowed location of the cleavage sites for the endonucleasesBamHI, XbaI, KpnI, BgllI, SaZGI, EcoRI, and HindIII. Hybridization experiments between pSU316 and pEDlO0 have shown that the tra region of pSU3 I6 lies in a segmentcomprising part of SaK31fragments S-I and S-3 and the entire fragment S-4. The positions of other plasmid coded functions, namely the replication functions and 01hemolysin production, have been determined in the physical map.
The hemolytic plasmid pSU3 16 is a conjugative plasmid with a molecular weight of 76.8 kb, isolated from a human Escherichia coli strain (3). The genetic information required for the production of the a-hemolysin is located in a 6.0-kb DNA sequence shared by several other plasmids belonging to different incompatibility groups (4), as well as by the bacterial chromosome of E. coli hemolytic strains (5,7). Cloning experiments have indicated that an adjacent segment of 6.2 kb carries all the functions required for the autonomous replication of this plasmid (II). Two transposition derivatives of pSU3 16, namely pSU306 (pSU3 16: :Tn802; Hly- Carbenicilin (Cb) resistant) and pSU307 (pSU3 16: :Tn5, Hly- Kanamycin (Km) resistant), have allowed assignment of the plasmid pSU3 16 to the incompatibility complex In&III-FIV (3) and a study of its incompatibility properties (9). Furthermore, cloning experiments have shown that the incompatibilities In&III and FIV are physically separated and that the DNA sequenceinvolved in the IncFIII incompatibility determinant is located in a 0.5-kb PstI fragment within the replication region of pSU3 16 (I I). We present here a restriction map of pSU3 16 for the endonucleasesBumHI, XbaI, KpnI, Bg/II, SulGI, EcoRI, and HindIII. The position of the transposons Tn802 and Tn5 ’ To whom correspondence should be sent. 0147-619X/84 $3.00 Copyright 8 1984 by Academic Pi-a, Inc. All rigJtts of reproduction in any form reserved.
96
in the transposition derivatives pSU306 and pSU307 as well as the hemolytic and tra regions and the region containing the replication functions of pSU3 16 have been located on this physical map. Hybrid plasmid DNA, amplified in the presence of cloramphenicol (Cm) was prepared as described by Timmis et al. (15). Plasmid DNA that does not amplify during Cm treatment was obtained according to Humphreys et al. (6). Restriction enzymes and T4 DNA ligase were obtained from New England Bio-Labs, and used according to the supplier’s recommendations. DNA polymerase I was purchased from Boehringer. For hybridization experiments, DNA fragments generated by restriction endonucleases were separated by agarosegel electrophoresis and transferred to nitrocellulose filters by the Southern blotting technique (13). pED 100 plasmid DNA was labeled with [a-32P]dGTP by nick translation with DNA polymerase I. The 32P-labeledprobe was hybridized against the DNA samples blotted onto nitrocellulose filters. Hybridization was carried out at 68°C in 5-10 ml of hybridization buffer (4) containing about 5 X 1O6cpm of the labeled probe, for about 20 h. pSU3 16 BglII- or KpnI-generated fragments were cloned into the unique BgflI or KpnI cleavage site of pKTO42 and pKTO68, respectively. Both cloning vectors are pBR322/
SHORT COMMUNICATIONS
pSC102 hybrid plasmids containing a unique restriction site for either BgAI or KpnI in the pSC102 PstI fragment inserted into pBR322 (1,12). pSU3 16 SulGI-generated fragments were cloned into the unique SalGI site of pBR322 (2). Agarose gel electrophoresis was performed in a vertical slab gel at agaroseconcentrations between 0.7 and 1.2% depending on the size of the fragment being studied. Electrophoresis was carried out in either TB (90 mM Tris, 2.5 mM EDTA, 90 mM boric acid, pH 8.3) or TP buffer (36 mM Tris, 30 mM NaH2P04, 10 mM EDTA, pH 7.5). Molecular weight standards were the bacteriophage X DNA fragments generated by digestion with Hind111 and the R6-5 DNA fragments generated by digestion with either EcoRI or HindIII. The molecular weights of X/Hind111and R6-S/EcoRI or R6S/Hind111were taken from Murray and Murray (8) and from Timmis et al. (25), respectively. The size of the largest fragment in each single digestion was taken to be the difference between the calculated size of the whole plasmid (76.8 kb, Ref. (3)) and the added sizesof the other restriction fragments. The restriction map has been constructed by comparison of double digest of pSU3 16 DNA with the digestion patterns produced by each of the restriction enzymes alone. When this approach proved insufficient hybrid plasmids containing specific fragments of pSU3 16 were digested with the appropriate restriction enzymes. The map has been orientated using the unique BarnHI site as reference point. The restriction sites produced by the endonucleasesBamHI and XbaI have been used in order to locate fragments generatedby other endonucleases unambiguously, namely BglII fragments B-3 and B-4, SulGI fragment S-2, KpnI fragment K-l, and EcoRI fragment E1. Double digestion of pSU3 16 DNA with both BglII and KpnI allowed assignment of all of the KpnI fragments and all but the smallest BgfiI fragments. This fragment (B-5) was located after a BgllI/SalGI digestion that also produced a SalGI map consistent with those for Bg/II and KpnI. This strategy allowed also construction of the Hind111map. The data
97
on sequencehomology in the hemolytic region between plasmid pHlyl52 and pSU3 16 (4) and on insertion of Tn802 in pSU3 16 (3) have been used to determine the position of some of the EcoRI pSU3 16 generated fragments, namely E-l, E-4, E-5, and E-9. The position of other EcoRI fragments was determined by using hybrid plasmids containing specific pSU3 16 restriction fragments. The location of the smallest EcoRI fragment (E-10, 0.3 kb) has not been determined. Hybridization studies between the hemolytic plasmids pHly152 and pSU3 16 (4) have demonstrated that the extent of their DNA sequence homology is restricted to a 6.0-kb fragment carrying the Hly determinant. The stretch of DNA, flanked at one end by a BumHI site, comprises 1.9 kb of the largest pSU3 16 EcoRI fragment, part of fragment E4 and the whole of EcoRI fragments E-5 and E-9. In the physical map of pSU3 16 (Fig. 1) the hemolytic determinant runs anticlockwise from the BumHI site overlapping 2.9 kb of
FIG. 1. Physicaland functional map of plasmid pSU3 16. @aI, KpnI (K), BgfiI (B), SufCiI (S), EcoRI (E), and Hi&II restriction maps of plasmid pSU3 16 are orientated with respect to the unique BarnHI site. Map coordinates are given in kilobases. Dashed lines in the functional map indicate that the end of the region has not been determined exactly.
98
SHORT COMMUNICATIONS
BgflI fragment B-4 and 3.0 kb of fragment B-3. Two Hly- derivatives of pSU3 16, showing resistance either to Cb (pSU306) or Km (pSU307) were obtained by transposition to pSU3 16 of Tn802 and Tn5, respectively. It has been shown that in pSU306, Tn802 insertion occurs in EC&I fragment E-9, whereas in pSU307 Tn5 is inserted in IIindIII fragment H-6 (3,9). Restriction analysis of the parental plasmid and its two transposition derivatives after Bg& digestion revealed that both insertions lie on fragment B-4, which is in agreement with the location of the hemolytic region. According to the results of Nogel et al. (10) the insertions of Tn802 in pSU306 and of Tn5 in pSU307 lie on cistrons hlyA and h&Z, respectively, responsible for the synthesis of a protein precursor of cu-hemolysin. By means of cloning experiments a Cb’ miniplasmid (pSU3025) derived of pSU306 (pSU3 16: :Tn802) has been constructed (1 I). The miniplasmid contains all the functions required for the autonomous replication of pSU306, and therefore of pSU3 16, in a 6.2kb DNA segmentcontiguous to the hly region, flanked at the ends by the BgfiI site of the Bl-B-4 junction and the unique BarnHI restriction site of pSU316 (see Fig. I). We have studied the sequence homology between pSU316 and pEDlO0 (a mini-F derivative containing the replication and transfer regions of F, Ref. (16)) in order to localize the tra region of pSU3 16. pSU3 16 plasmid DNA digested with Hind111and SalGI endonucleaseswas run on agarosegels, blotted directly on nitrocellulose filters and hybridized against 32P-labeledDNA prepared by nick translation of pED 100 plasmid DNA. An EcoRI digestion of pEDlO0 was also included in the gels as hybridization control. Hybridization was detected in the three largest generated bands, which correspond to S&G1 fragments S-l, S-3, and S-4. BecauseS-4 is the SulGI fragment located between S-l and S-3, this result indicates that the tra region of plasmid pSU3 16 comprises
the whole fragment S-4 and part of the fragments S-l and S-3. ACKNOWLEDGMENTS We thank Dr. F. de la Cruz for helpful discussion and Mrs. Marta Garcia for her excellent assistance.J.C.R. was a recipient of a grant (Institute National de Asistencia y Promotion de1Estudiante) from the Spanish Ministry of Education. This work was supported by a grant from the Comisibn Asesora Cientifica y Tecnica.
REFERENCES 1. ANDRE& I., SLOCOMBE,P. M., CABELLO, F., TIMMIS, J. K., LUFU, R., BURKARDT, H. J., AND TIMMIS, K. N., Molec. Gen. Genef. 168, l-25 (1979). 2. BOLIVAR, F., RODRIGUEZ, R. L., GREENE, P. J., BETLACH, M. C., HEYNEKER, H. L., BOYER, H. W., CROSA, J., AND FALKOW, S., Gene 2,95-
113 (1977). 3. DE LA CRUZ, F., ZABALA, J. C., AND ORTIZ, J. M., Plasmid 2, 507-519 (1979). 4. DE LA CRUZ, F., MOLLER, D., ORTIZ, J. M., AND GOEBEL, W., J. Bacferiol. 143, 825-833 (1980). 5. DE LA CRUZ, F., ZABALA, J. C., AND ORTIZ, J. M. Infect. Immun. 41, 881-887 (1983). 6. HUMPHREYS, G. O., WILLSHAW, G. A., AND ANDERSON, E. S., Biochim. Biophys. Acta 383,457-
463 (1975). 7. MULLER, D., HUGHES, C., AND GOEBEL, W., J. Bacteriol. 153, 846-851 (1983). 8. MURRAY, K., AND MURRAY, N. E., J. Mol. Biol. 98, 551-564 (1975). 9. NAVAS, J., DE LA CRUZ, F., RODRIGUEZ, J. C., ANDRES, I., VULGAR, G., AND ORTIZ, J. M., J. Gen. Microbial. 129, 2277-2283 (1983). 10. NOEGEL, A., RDEST, U., SPRINGER,W., AM) G~EBEL, W., Mol. Gen. Genet. 175, 343-350 (1979). II. RODRIGUEZ, J. C., ANDRES, I., DE LA CRUZ, F., NAVAS, J., I%JLGAR, G., AND ORTIZ, J. M., Plasmid
10, 175-183 (1983). 12. SLOCOMBE, P. M., ANDRES, I., AND TIMMIS, K. N.,
In “Microbial Drug Resistance and Related Plasmid? (S. Mitsuhashi and H. Hashimoto, eds.),pp. 83-87. Tokyo Univ. Press,Tokyo, 1978. 13. SOUTHERN, E. M., J. Mol. Biol. 98,503-517 (1975). 14. TIMMIS, K., CABELLO, F., AND COHEN, S. N., In
“Modem Trends in Bacterial Transformation and Transfection” (A. Portoles, R. L@ez, and H. Espinosa,eds.),pp. 263-276. Elsevier/North-Holland, Amsterdam, 1977 15. TIMMIS, K. N., CABELLO, F., AND COHEN, S. N., Mol. Gen. Genet. 162, 121-137 (1978). 16. WILLETS, N., AND JOHNSON, D., Mol. Gen. Genet.
182, 520-522 (1981).