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Cloning of mercury-resistance gene from R-plasmid in Escherichia coli isolated from dental hospital sewage
Gen. Pharmac. Vol. 20, No. 5, pp. 609-614, 1989 Printed in Great Britain.All rightsreserved
0306-3623/89$3.00+ 0.00 Copyright © 1989PergamonPress pk
CLONING OF MERCURY-RESISTANCE GENE FROM R-PLASMID IN ESCHERICHIA COLI ISOLATED FROM DENTAL HOSPITAL SEWAGE HIROSHI ARII and YOSHIMITSUABIKO Department of Biochemistry, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba 271, Japan (Tel. 0473 (68) 6111) (Received 7 December 1988)
Abstract--I. Mercury-resistance gene was cloned from a drug-resistance plasmid in Escherichia coil isolated from dental hospital sewage. 2. A 7.7 kb mercury-resistance genc was successfullycloned and expressed in pJC74 cosmid and pBR 322 plasmid vectors. 3. High activities of mercury reductase and 2o~Hg vaporization were demonstrated in cell extracts of mercury-resistant clone. 4. Mercury reductase activity was localized in the cytoplasm. 5. By colony DNA-DNA hybridization, 32P-labelled7.7-kb DNA hybridized with mercury-resistant E. coli isolates from dental hospital sewage.
INTRODUCTION
Elevated concentrations of mercury have been detected in the sediment of several estuaries (Nelson and Colwell, 1975; Craig and Morton, 1976). Mercuryresistant bacteria have been isolated from these environments, and the most completely documented pathway of detoxication of mercury is reduction of the inorganic mercury (Hg 2+ ) form to metallic mercury (Hg°) (Komura and Izaki, 1971). This mercury volatilization in bacteria has been shown to be plasmid mediated (Novick and Roth, 1968; Summers and Silver, 1972), and the plasmid-borne gene encodes a NADPH-dependent mercury reductase. It has been demonstrated that the urine of dental hospital workers contain a higher concentration of mercury than those of the general population (Gronka et al., 1970). The mean concentration of mercury in the waste water discharged from dental offices in Japan was found to be 11.3 ppb (Nishimura and Yamanaka, 1981), a value higher than the maximum permissible level of 5 ppb in discharged water
as specified by the prevention of Water Pollution Law of Japan. Recently, Nagahama (1987) reported that mercuryresistant Escherichia coli ( E. coil) strains were isolated more frequently in dental hospital sewage than in general building sewage, and antibiotic drug-resistant plasmids (R-plasmid) which carried the mercury rcductase gene were successfully isolated from these E. coli strains. In this study, we cloned the mercury-resistance gcne from R-plasmid in E. coli isolated from dental hospital sewage and assessed the prevalence and distribution of this gene in E. coli populations in polluted waters. MATERIALS AND METHODS
Bacteria and media The bacterial strains used are listed in Table 1. Mercury resistant E. coil strains were kindly provided by Dr F. Nagahama of the Department of Conservative Dentistry, Nihon University School of Dentistry at Matsudo. E. coli cells were grown in L agar or broth, and bacterial resistance
Table 1. Bacterial strains
Strain E. coil HBI01
C600 x 1628
;~ 1782 x 1792 ;c 2690 HNI HN2 HN3 HN4
Source or
Description
references
F - , hsdRSI4(rk--, ink+-), fecal3, ara-14 proA2, lacY~, galK2, rpsL20(Smr), xyl-5, mtl-I, supE44, ,~F-, thi lo thr l, leuB6, lac Y lo tonA21, supE44, ).-
Contains plasmidRP4 Contains plasmidR6K Contains plasmid Rldrdl9 Containsplasmidp.lC74 Contains mercury-resistanceplasmidpHNI Containsmercury-resistanceplasmidpHN2 Contains raercury-resistanceplasmidpHN3 Containsmercury-resistanceplumid pHN4
Boyer and RoullandDussoix, 1969 Applcyard, 1954 R. Curtin lip R. Curtiss III R. Curtiss III R. Curtiss III F. Nagahama F. Nagahama
F. Naphama F. Nagahama
I~partment of Biology. Washington University, St Louis, MO 63130, U.S.A.
609
610
HIROSHI ARII and YOSHIMI'rSUABIKO
to mercury was tested on L-agar plates containing 25 #g/ml of HgCI 2.
measured at 2 5 C by the initial decrease in absorbance at 340 nm.
Isolation of plasmid DNA For linear DNA size standards in agarose electrophoresis, 2c1857 was isolated by the method of Thomas and Davis (1975). The R plasmids (Rldrdl9, R6K and RP4) and mercury-resistance plasmids (pHNI, PHN2, pHNS, and pHN4) were isolated by the method of Hansen and Olsen (1978). The cosmid pJC74 used as the vector carries a 2-derived cos site, a bla gene which confers ampicillin resistance, and plasmid ColE! replicator and single Sail endonuclease site (Collins, 1979). Cloning vectors pBR322 and pJC74 were isolated by the method of Gurrey et al. (1973). and were further purified by CsCl-ethidium bromide equilibrium density gradient centrifugation. Plasmid screening of transformants was carried out by the method of Birnboim and Doly (1979). To purify plasmid DNA from transformants, E. coli cells were grown in L-broth at 3 7 C to late logarithmic phase and harvested. The cells were then washed with ET buffer (10mM Tris-HCl, I mM EDTA, pH 8.0) and used for preparation of clear lysates. The clear lysate were extracted twice with phenol saturated with 10 mM Tris-HC1, I mM EDTA (pH 7.6) and the DNA was precipitated with ethanol and then dissolved in ET buffer. When necessary RNA was removed from the preparations by treatment with RNase (I0 u/ml) for 30 min at 37'C and DNA was extracted again by phenol.
Assay ./or '°3Hg vaporization
Cosmid cloning Restriction endonuclease and T4 DNA ligase were purchased from Takara Co. Digestion with restriction endonuclease was carried out by the method described by manufacture. The mercury-resistance plasmid was partially digested with Sail, and cosmid pJC74 was completely digested with Sail. These were ligated in ligation buffer (Maniatis et al., 1980a) with T4 ligase at 12°C for 16 hr. This ligated DNA was packaged into A phage heads in vitro by the method of Hobn and Hohn (1974). Agarose gel electrophoresis DNA was electrophoresed in 0.7% agarose slab gels. The running buffer was 89 mM Tris-borate buffer (pH 8.0) containing 2 m M EDTA. After electrophoresis, the gels were stained with a solution of ethidium bromide (2 tag/ml) and photographed under ultraviolet light. DNA fragment for colony hybridization was isolated from electrophoresis gel by the method of Smith (1980). Transformation Maltose-grown E. coli HBI01 cells were infected with phages from the in vitro packaging mixture, and then the infected cells were incubated for 1 hr at 30°C for expression ofantibiotic-resistance gene and were plated on L-agar plate containing 50,ug/ml of ampicillin and 25 #g/ml of HgCI,. Host strain E. coli HBI01 was transformed with chimeric DNA molecules derived from plasmid vector pBR322 by the method described by Maniatis et al. (1980b). Transformants were selected on L-agar containing 25 ~g/ml of ampicillin and HgCI:. Spectrophotometric assay of mercury reductase Harvested cells were washed with 20mM phosphate buffer, pH 7.0, and were suspended in the same buffer containing I mM 2-mercaptoethanol and disrupted by a sonic oscillator. The supernatant fraction obtained by centrifugation at 28,000g for 20 rain was used for the assay. The reaction mixture contained 20 mM phosphate buffer (pH 7.0), 0.134raM NADPH, 0.02mM HgC12, 0.1 mM MgCI2, and an appropriate amount of cell extract in a total volume of 1.0 ml. The reaction was started by the addition of the cell extract after a 5 min preincubation of the other components. HgCl~-dependent oxidation of NADPH was
Cell extract was incubated with 20 mM phosphate buffer (pH7.0), 0.02mM '°SHgCl2 (0.05/~Ci/mmol), 0.1mM MgCI2, and I mM NADPH in a total volume of 2 ml. After incubation for the appropriate time, 0.1 ml portion of the reaction mixture was removed and mixed with scintillation liquid (PCS, Amersham) and radioactivity was measured in a liquid scintillation counter (Aloka LSC-673). The vaporization of 2°SHg was estimated from the disappearance of radioactivity from the reaction mixture. Isolation of periplasmic protein To determine the localization of mercury reductase, the periplasmic space fraction was prepared by the small-scale cold osmotic shock method described by Hazelbauer and Harayma (1979). To obtain the cytoplasmic protein fraction, the osmotically shocked cells were disrupted by sonication and the supernatant fraction was obtained using a bench top centrifuge (Eppendorf 5412). //-Lactamase activity as a marker enzyme of periplasm was measured by the method of O'Callaghan et al. (1972), and the assay of //-galactosidase as a cytoplasmic marker enzyme was carried out by the method of Miller (1977). Colon)' hybridization E. coli strains from dental hospital sewage were screened for the presence of mercury-resistance gene DNA by a method based on the colony hybridization technique of Grunstein and Hogness (1975). E. coli strains were grown on nitrocellulose filter (Millipore, 0.45 lam pore size) laid on a L-agar plate containing 25 ,ug/ml ampicillin and HgCI 2. The filters were placed in 0.5 M NaOH and 1.5 M NaCI to denature double-stranded DNA, then neutralized with 1M Tris-HC1 (pH 7.6). The filters were washed with 2X SSC (SSC; 0.15 M NaCI, 0.015 M sodium citrate, pH 7.0) and baked for 3 h at 80"C to fix single-stranded DNA onto the filter, then incubated for 3 h in Denhardt solution (0.02% ficoll, 0.02% polyvinylpyrrolidone and 0.02% bovine serum albumin) at 65 C. The filters were hybridized with cloned mercury-resistance DNA fragment labeled by nick translation with [:~-3-'P]-dATP as described by Rigby et al. (1977). Filters were then washed with 2x SSC containing 0.1% sodium dodecyl sulfate. After drying, filters were autoradiographed at - 2 0 C for 24 h. RF.SULTS Restriction endonuclease analysis N a g a h a m a (1987) screened plasmids in mercuryresistant E. coli strains isolated from dental hospital sewage and found that most of the strains carried a single plasmid, t h o u g h some carried multiple plasmids. The investigator succeeded in isolating a single plasmid encoding mercury-resistance gene from multiple plasmids in these strains by t r a n s f o r m a t i o n experiments, a n d designated these plasmids confering mercury resistance as p H N I , p H N 2 , a n d p H N 3 . He also showed these plasmids to have different restriction p a t t e r n s by Eco RI digestion. We analyzed plasmid D N A which was isolated as a single plasmid using E c o R l digestion, and found the most pred o m i n a n t plasmid ( p H N 4 ) type h a d the same pattern as p H N 3 . W e analysed these h o m o l o g o u s plasmid D N A s using B a m H l a n d S a i l , as s h o w n in Fig. 1. p H N 4 also showed the same digestion p a t t e r n s as p H N 3 with these restriction endonucleases. These data suggest that p H N 4 a n d p H N 3 are the same
Cloning of Hg-resistance gene 1
2
3
4
5
6
7
8
9
10 I1
12
13 14 15
611 2
1
3
4
6
5
Fig. 1. Restriction endonuelease analysis of mercuryresistance plasmid DNAs with different endonucleases: 1, pHNi/BamHI; 2, pHN2/BamHI; 3, pHN3/BamHI; 4, pHN4/BamHI; 6, pHNl/SalI; 7, pHN2/SalI; 8, pHN3/ Sail; 9, pHN4/Sall; 11, pHNI/EcoRI; 12, pHN2/EcoRI; 13, pHN3/EcoRI; 14, pHN4/EcoRI; "5, 10, 15, 2ci857 Hindlll cut as size standard. plasmid and that this plasmid may be predominantly carried by mercury-resistant E. coil cells in dental hospital sewage. Therefore we decided to clone this mercury-resistance gene from pHN4, which should be widely prevalent in dental hospital sewage. Cloning of mercury-resistance gene We carried out gene cloning using cosmid vector pJC74 first and then subcloned with plasmid vector pBR322. Cleavage of pHN4 by Sail yielded at least 9 detectable DNA fragments (Fig. 1). We partially digested pHN4 with Sail and iigated the products with completely digested cosmid pJC74, as shown in Fig. 2, to produce high molecular weight chimeric DNAs. These chimeric DNAs were in vitro packaged in k phage heads for infection of E. coli C600 cells, then ampicillin-resistant clones were selected. The packaging efficiency was 3 x 104 PFU//~g of recombinant DNA. Mercury-resistant clones were selected on L-agar plates containing 25/~g/ml of HgCI 2. The plasmid DNA from mercury resistant transformants was isolated and analysed with restriction endonuclease Sail. As shown in Fig. 3, mercury-resistance plasmids yielded different digestion patterns comprising several DNA fragments. A mercury resistant clone which had a small number of DNA fragments was designated as pHN4-I. Then SalI-cut pHN4-I DNA fragments were ligated with Sail-cut pBR322 and transformed into HBI01 cells. The transformants of ampicillin- and HgCI2_ resistant clones were screened for plasmids, and a transformant which carried the smallest size of plasmid was selected and designated as pH4-II. Since pHN4 carries the streptomycin (Sm)-resistance gene (Nagahama, 1987), we also examined the transformants for phenotypic expression of Sm However, HN4-II did not grow on L-agar plate resistance containing 10 pg/ml of Sm. Characterization of cloned DNA fragments The molecular sizes of pHN4-I and pHN4-II
Fig. 2. Ligation of SalI fragments of pHN4 and pJC74 vector. 1, pJC74; 2, pHN4; 3, 2ci857 Hindlll cut as size standards; 4, pHN4/SalI partially cut; 5, pJC74/Sa/l; 6, ligated chimeric DNA of pHN4 and pJC74. supercoiled DNA were estimated to be about 24 and 12 kb, respectively, by comparison of their mobilities during agarose gel electrophoresis with plasmid size standards (Fig. 4). The linear DNA fragments observed after electrophoresis of pHN4-I and pHN4-II on agarose gels after SalI digestion are shown in Fig. 5. The DNA from both plasmids contained the identical 7.7 kb DNA fragment. Thus the mercuryresistance gene must reside on the Sail 7.7 kb DNA fragment. HgCl2-dependent NADPH oxidation Since it is believed that the mechanism of mercury resistance is mercury vaporization by HgCI:1
2
3
4
5
Fig. 3. Sail digestion of mercury-resistance eosmid clones. 1, pHN4/SalI; 2, to 5, mercury-resistance cosmids/SalI. The plasmid of lane 2 has 7.7 and 16 kb DNA fragments, and this plasmid was designated as pHN4-I.
612
HIROSHI ARII a n d YOSHIMITSU ABIKO l
2
3
4
5
6
7
fl
Table 2. Mercury reductase activity in E. coli clones Relative activity (%)
Mercury reductase activity ( N A D P nmol.,min/mg protein)
Clones HBI01 HBI01 (pHN4) HBI01 (pHN4-I) HBI01 (pHN4-1I)
0 28.0 46.5 118.9
-100 166
424
Mercury reductase activity was expressed as N A D P formed from N A D P H oxidation. The assay procedure was described in Materials and Methods. Table 3. Localization of mercury reductase in E. coli _Enzymes
Fig. 4. Size determination of mercury-resistance cloned plasmids. 1, Rldrdl9 (94kb), lower band should be chromosomal DNA, 2, R6K (40 kb); 3, pYA724 (24.5 kb); 4, pBTI-10 (7.7 kb); 5, pJC74 (16 kb); 6, pBR322 (4.3 kb); 7, pHN4-I; 8, pHN4-1I, pHN4-I and pHN4-II were determined to be 24 and 12 kb, respectively. dependent NADPH oxidation, we measured mercury reductase activity in cell extracts of mercury-resistant clones. When the cell extracts of pHN4, pHN4-I, and pHN4-II transformed HBI01 cells were added to the reaction mixture containing NADPH and HgCI2, rapid oxidation of NADPH was observed. However, an extract of a mercury-sensitive strain, host cell HBI01 did not. Mercury reductase activities of pHN4-I, and pHN4-II-bearing cells were 1.7 and 4.2-fold higher respectively than that of pHN4 infected one (Table 2).
Vaporization of 2°3Hg Since high activity of HgCl2-dependent NADPH oxidation was recognized in the cell extract of HN4II, 2°3Hg vaporization was examined. As shown in Fig. 6, the cell extract of HN4-11 showed a significant activity to vaporize 2°3Hg. This activity was greatly diminished when the cell extract was heated at 80°C for 5 rain. On the other hand, no 203Hg vaporization
1
2
3
Relative activity (%) Periplasm Cytoplasm
Mercury reductase fl-galactosidase fl-lactamase
2 4 91
98 96 9
Total enzyme activities of periplasm and cytoplasm were calculated to be 100%. The enzymes assay were described in Materials and Methods.
activity could be detected in the cell extract of host strain HB101.
Localization of mercury reductase In control experiments for marker enzymes of periplasmic and cytoplasmic fractions, we found that 1% of the total fl-lactamase activity was in the periplasmic extract and 96% of the total fl-galactosidase was in the cytoplasmic extract. When mercury reductase activity was measured in periplasmic and cytoplasmic extracts, it was predominantly located in the cytoplasm (Table 3).
Cohmy hybridization with cloned mercury-resistance gene Mercury-resistant E. coli cells were isolated from dental hospital sewage and grown on nitrocellulose
4
g
50 -
".._
ff-
I 0
30 Incubation time (rain)
I 60
Fig. 6. Vaporization of m3Hg by the cell extract of HN4-[I.
Fig. 5. Endonuclease analysis of pHN4-I and pHN4-11. I.
pHN4-1/SalI; 2, pHN4-II/Sall; 3, pJC74/SalI; 4, pBR322/ Sail.
Cell extracts were obtained from HN4-II by sonication followed by centrifugation at 28,0008. The reaction mixture and measurement of radioactivity were described in Materials and Methods. (O), Cell extract of HN4-II; (C)), heated cell extract of HN4-II; (11), cell extract of HBI01.
613
Cloning of Hg-resistance gene
Fig. 7. Colony hybridization of DNA from mercuryresistance E. coli isolated from dental hospital sewage with 32P-labeled 7.7kb DNA encoding mercury resistance. Arrow indicates a colony of HN4 as positive control.
filters. Fifty colonies were tested for hybridization with radioactivity labeled 7.7 kb D N A fragment encoding mercury resistance, and 13 colonies hybridized with the probe, as shown in Fig. 7. DISCUSSION Numerous studies of antibiotic drug-resistant bacteria have clarified the close relationship between the appearance of drug-resistant bacteria and usage of the drug. However, the nature of the selection factor for heavy metal-resistant bacteria has not yet been fully explained from epidemiological and genetic viewpoints. Timoney et al. (1978) surveyed mercury resistance of aerobic bacterial flora in sewage sludge dump areas and unpolluted areas of New York Bight. Mercuryresistant Bacillus populations were found with much greater frequency in sediments containing high concentrations of mercury than in sediments from areas further offshore where dumping has never been practiced and where heavy-metal concentrations were found to be low. Nagahama (1987) also reported that mercury resistant E. coil populations were more numerous in sediments of dental hospital sewage than in sediments from other general building. Therefore, it is suggested that mercury contamination of the sediment was the selective pressure giving rise to the plasmids encoding the mercury resistance. One of our aims for cloning the mercury-resistance gene from an R-plasmid was to assess the prevalence and distribution of such gene in mercury-resistant E. coli populations in polluted sewage. According to our data, the cell extract of HN4-II had high activity of 2°4Hg vaporization, and the heat-treated extract lost this activity. Mercury reductase activities of HN4-II and HN4-I were 4.2- and 1.7-foid higher, respectively, than that of HN4. In general, high molecular size plasmids exist in host cells as a small number of copies, whereas small plasmids are carried as a large number of copies. Therefore, the differences in the
enzyme activity in these mercury-resistant transformant clones are probably dependent on plasmid copy number, for pBR322 (vector for pHN4-II) and pJC74 (vector for pHN4-I) are maintained at a relatively high.copy number of 40 and 15 per cell, respectively (Karn et al., 1979; Collins, 1979). In addition, pHN4-I and pHN4-II represent insertion of the 7.7 kb fragment in vector plasmids. These data suggest that the mercury-resistance gene, encoding mercury reductase, was successfully cloned as a 7.7 k b - S a l I DNA fragment. Since HN4 carries Sm resistance and the HN4-II beating cell is sensitive to Sm, the 7.7 kb mercury-resistance gene was separated from the Sm resistance gene. It has been found that plasmid-determined genes for drug resistance are capable of moving from one replicon to another in the absence of host cell-mediated recombination function, and such genetic elements are called to transposon (Kleckner, 1977). The mercury-resistance gene carried in plasmids often occurs on a transposon (Stanisich et al., 1977) and associated sex factors (Summers and Silver, 1972). From the view points of the origin of mercuryresistant bacteria and their selection, spreading and prevalence, transposon elements may exist in near the mercury-resistance gene of plasmid. The colonies of mercury-resistant E. coli strains isolated from sewage were tested with 32P-labeled S a l I 7.7kb D N A by colony hybridization. Of 50 colonies examined, 13 (26%) reacted with the probe. Thus, this cloned mercury-resistance gene can be detected among diverse mercury-resistance E. coli isolates. These data suggest that the pHN4-II clone is a useful tool for survey of mercury-resistance determinants and ecological changes of mercury pollution in sewage. Furthermore, the cell extract of HN4-II provide a larger quantity of mercury reductase to study protein and enzymological studies. It is likely that mercury concentrations in sediments become less after dumping of mercury compounds because of dilution effects during passage through the water, because of mixing with other sediments on the bottom, and finally because of dissipation of mercury into the water by resistant mercury reducing bacteria. Therefore, HN4-II, which expressed high Hg2+-reductase activity, may also be useful for reduction of the mercury concentration in polluted waters. REFERENCES
Appleyard R. K. (1954) Segregation of new lysogenic types during growth of a doubly lysogenic strain derived from Escherichia coil Ki2. Genetics 39, 440-452. Birnboim H. C. and Doly J. (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acid Res. 7, 1513-1523. Boyer H. W. and Roulland-Dessoix (1969) A complemenration analysis of the restriction and modification of DNA in gscherichia coll. J. molec. Biol. 41, 459-472. Collins J. (1979) Escherichia coil plasmids packageable /n vitro in 2 bacteriophage particles. Methods Enzymol. 68, 309-326. Craig P. J. and Morton S. F. (1976) Mercury in Mersey estuary sediments, and the analytical procedure for total mercury. Nature 261, 125-126. Gronka P. A., Bobkoskie R. L., Tomchick G. J., Bach F. and Rakow A. B. (1970) Mercury vapor exposure in dental offices. J. Amer. Dent. Assoc. gl, 923-925.
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HIROSHI ARI1 and YOSHIMI'rsu AB1KO
Grunstein M. and Hogness (1975) Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc. ham. Aead. Sci. U.S.A. 72, 3961-3965. Gurrey P., LeBlanc D. J. and Falkow S. (1973) General method for the isolation of plasmid deoxyribonucleic acid. J. Bacteriol. 116, 1064-1066. Hansen J. B. and OIsen R, H. (1978) Isolation of large bacterial plasmids and characterization of the P2 incompatibility group plasmids pMGI and pMG5. J. Bacteriol. 135, 227- 238. Hazelbauer G. and Harayama S. (1979) Mutants in transmission of chemotactic signals from two independent receptors of E. coll. Cell 16, 617-625. Hohn B. and Hohn T. (1974) Activity of empty, headlike particles for packaging of DNA of bacteriophage ). in vitro. Proc. natn. Acad. Sci. U.S.A. 71, 2372-2376. Karn M., Kolter R., Thomas C., Figurski D., Meyer R., Remut E. and Helinski D. R. (1979) Plasmid cloning vehicles derived from plasmids ColEI, F, R6K and RK2. Methods Enzymol. 68, 268-280. Kleckner N. (1977) Translocatable elements in prokaryotes. Cell !1, 11-23. Komura I. and lzaki K. (1971) Mechanism of mercuric chloride resistance in microorganism. I. Vaporization of a mercury compound from mercuric chloride by multiple drug resistant strains of Escherichia coli. J. Biochem. 70, 885 893. Maniatis T., Fritsch E. F. and Sambrook J. (1980a) Procedure for cDNA cloning. In Molecular Cloning, pp. 229-246. Cold Spring Harbor, New York. Maniatis T., Fritsch E. F. and Sambrook J. (1980b) Introduction of plasmid and bacteriophage J. DNA into Escherichia coli. In Molecular Cloning, pp. 247-268. Cold Spring Harbor, New York. Miller J. H. (1977) Assay of fl-galactosidase. In Experiments in Molecular Genetics, pp. 352-355. Cold Spring Harbor, New York.
Nagahama F. (1987) Mercury resistant Escherichia coli in the sediments of a dental hospital. Nihon Univ. J. oral Sci. 13, I-9. Nelson J. D. and Colwell R. R. (1975) The ecology of mercury-resistant bacteria in the Chesapeake Bay. Microbiol. Ecol. 1, 191-218. Nishimura M. and Yamanaka S. (1981) Mercury pollution in dental practice. In International Conference on Mercury Hazards in Dental Practice; No. 28. Novick R. P. and Roth C. (1968) Plasmid-linked resistance to inorganic salts in Staphyloccus aureus. J. Bacteriol. 95, 1335-1342. O'Callaghan C. H., Morris A., Kirby S. M. and Shingler (1972) Novel method for detection of fl-lactamases by using a chromogenic cephalosporin substrate. Antimicrobiol. Agents Chemother. 1, 283-288. Rigby P. W. J., Dieckmann M., Rhodes C. and Berg P. (1977) Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase. J. molec. Biol. 113, 237 251. Smith H. O. (1980) Recovery of DNA from gels. Methods En:ymol. 65, 371-380. Stanisich V. A., Bennett P. M. and Richmond M. H. (1977) Characterization of a translocation unit encoding resistance to mercuric ions that occurs on a nonconjugative plasmid in Pseudomonas aeruginosa. J. Bacteriol. 129, 1227- 1233. Summers A. O. and Silver S. (1972) Mercury resistance in a plasmid-bearing strain of Escherichia eoli. J. Bacteriol. 112, 1228 1236. Thomas M. and Davis R. W. (1975) Studies on the cleavage of bacteriophage lambda DNA with EcoRl restriction endonuclease. J. molec. Biol. 91, 315-328. Timoney J. F., Port J., Giles and Spanier J. (1978) Heavymetal and antibiotic resistance in the bacterial flora of sediments of New York Bight. Appl. Environ. Microbiol. 36, 465 472.
0306-3623/89$3.00+ 0.00 Copyright © 1989PergamonPress pk
CLONING OF MERCURY-RESISTANCE GENE FROM R-PLASMID IN ESCHERICHIA COLI ISOLATED FROM DENTAL HOSPITAL SEWAGE HIROSHI ARII and YOSHIMITSUABIKO Department of Biochemistry, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba 271, Japan (Tel. 0473 (68) 6111) (Received 7 December 1988)
Abstract--I. Mercury-resistance gene was cloned from a drug-resistance plasmid in Escherichia coil isolated from dental hospital sewage. 2. A 7.7 kb mercury-resistance genc was successfullycloned and expressed in pJC74 cosmid and pBR 322 plasmid vectors. 3. High activities of mercury reductase and 2o~Hg vaporization were demonstrated in cell extracts of mercury-resistant clone. 4. Mercury reductase activity was localized in the cytoplasm. 5. By colony DNA-DNA hybridization, 32P-labelled7.7-kb DNA hybridized with mercury-resistant E. coli isolates from dental hospital sewage.
INTRODUCTION
Elevated concentrations of mercury have been detected in the sediment of several estuaries (Nelson and Colwell, 1975; Craig and Morton, 1976). Mercuryresistant bacteria have been isolated from these environments, and the most completely documented pathway of detoxication of mercury is reduction of the inorganic mercury (Hg 2+ ) form to metallic mercury (Hg°) (Komura and Izaki, 1971). This mercury volatilization in bacteria has been shown to be plasmid mediated (Novick and Roth, 1968; Summers and Silver, 1972), and the plasmid-borne gene encodes a NADPH-dependent mercury reductase. It has been demonstrated that the urine of dental hospital workers contain a higher concentration of mercury than those of the general population (Gronka et al., 1970). The mean concentration of mercury in the waste water discharged from dental offices in Japan was found to be 11.3 ppb (Nishimura and Yamanaka, 1981), a value higher than the maximum permissible level of 5 ppb in discharged water
as specified by the prevention of Water Pollution Law of Japan. Recently, Nagahama (1987) reported that mercuryresistant Escherichia coli ( E. coil) strains were isolated more frequently in dental hospital sewage than in general building sewage, and antibiotic drug-resistant plasmids (R-plasmid) which carried the mercury rcductase gene were successfully isolated from these E. coli strains. In this study, we cloned the mercury-resistance gcne from R-plasmid in E. coli isolated from dental hospital sewage and assessed the prevalence and distribution of this gene in E. coli populations in polluted waters. MATERIALS AND METHODS
Bacteria and media The bacterial strains used are listed in Table 1. Mercury resistant E. coil strains were kindly provided by Dr F. Nagahama of the Department of Conservative Dentistry, Nihon University School of Dentistry at Matsudo. E. coli cells were grown in L agar or broth, and bacterial resistance
Table 1. Bacterial strains
Strain E. coil HBI01
C600 x 1628
;~ 1782 x 1792 ;c 2690 HNI HN2 HN3 HN4
Source or
Description
references
F - , hsdRSI4(rk--, ink+-), fecal3, ara-14 proA2, lacY~, galK2, rpsL20(Smr), xyl-5, mtl-I, supE44, ,~F-, thi lo thr l, leuB6, lac Y lo tonA21, supE44, ).-
Contains plasmidRP4 Contains plasmidR6K Contains plasmid Rldrdl9 Containsplasmidp.lC74 Contains mercury-resistanceplasmidpHNI Containsmercury-resistanceplasmidpHN2 Contains raercury-resistanceplasmidpHN3 Containsmercury-resistanceplumid pHN4
Boyer and RoullandDussoix, 1969 Applcyard, 1954 R. Curtin lip R. Curtiss III R. Curtiss III R. Curtiss III F. Nagahama F. Nagahama
F. Naphama F. Nagahama
I~partment of Biology. Washington University, St Louis, MO 63130, U.S.A.
609
610
HIROSHI ARII and YOSHIMI'rSUABIKO
to mercury was tested on L-agar plates containing 25 #g/ml of HgCI 2.
measured at 2 5 C by the initial decrease in absorbance at 340 nm.
Isolation of plasmid DNA For linear DNA size standards in agarose electrophoresis, 2c1857 was isolated by the method of Thomas and Davis (1975). The R plasmids (Rldrdl9, R6K and RP4) and mercury-resistance plasmids (pHNI, PHN2, pHNS, and pHN4) were isolated by the method of Hansen and Olsen (1978). The cosmid pJC74 used as the vector carries a 2-derived cos site, a bla gene which confers ampicillin resistance, and plasmid ColE! replicator and single Sail endonuclease site (Collins, 1979). Cloning vectors pBR322 and pJC74 were isolated by the method of Gurrey et al. (1973). and were further purified by CsCl-ethidium bromide equilibrium density gradient centrifugation. Plasmid screening of transformants was carried out by the method of Birnboim and Doly (1979). To purify plasmid DNA from transformants, E. coli cells were grown in L-broth at 3 7 C to late logarithmic phase and harvested. The cells were then washed with ET buffer (10mM Tris-HCl, I mM EDTA, pH 8.0) and used for preparation of clear lysates. The clear lysate were extracted twice with phenol saturated with 10 mM Tris-HC1, I mM EDTA (pH 7.6) and the DNA was precipitated with ethanol and then dissolved in ET buffer. When necessary RNA was removed from the preparations by treatment with RNase (I0 u/ml) for 30 min at 37'C and DNA was extracted again by phenol.
Assay ./or '°3Hg vaporization
Cosmid cloning Restriction endonuclease and T4 DNA ligase were purchased from Takara Co. Digestion with restriction endonuclease was carried out by the method described by manufacture. The mercury-resistance plasmid was partially digested with Sail, and cosmid pJC74 was completely digested with Sail. These were ligated in ligation buffer (Maniatis et al., 1980a) with T4 ligase at 12°C for 16 hr. This ligated DNA was packaged into A phage heads in vitro by the method of Hobn and Hohn (1974). Agarose gel electrophoresis DNA was electrophoresed in 0.7% agarose slab gels. The running buffer was 89 mM Tris-borate buffer (pH 8.0) containing 2 m M EDTA. After electrophoresis, the gels were stained with a solution of ethidium bromide (2 tag/ml) and photographed under ultraviolet light. DNA fragment for colony hybridization was isolated from electrophoresis gel by the method of Smith (1980). Transformation Maltose-grown E. coli HBI01 cells were infected with phages from the in vitro packaging mixture, and then the infected cells were incubated for 1 hr at 30°C for expression ofantibiotic-resistance gene and were plated on L-agar plate containing 50,ug/ml of ampicillin and 25 #g/ml of HgCI,. Host strain E. coli HBI01 was transformed with chimeric DNA molecules derived from plasmid vector pBR322 by the method described by Maniatis et al. (1980b). Transformants were selected on L-agar containing 25 ~g/ml of ampicillin and HgCI:. Spectrophotometric assay of mercury reductase Harvested cells were washed with 20mM phosphate buffer, pH 7.0, and were suspended in the same buffer containing I mM 2-mercaptoethanol and disrupted by a sonic oscillator. The supernatant fraction obtained by centrifugation at 28,000g for 20 rain was used for the assay. The reaction mixture contained 20 mM phosphate buffer (pH 7.0), 0.134raM NADPH, 0.02mM HgC12, 0.1 mM MgCI2, and an appropriate amount of cell extract in a total volume of 1.0 ml. The reaction was started by the addition of the cell extract after a 5 min preincubation of the other components. HgCl~-dependent oxidation of NADPH was
Cell extract was incubated with 20 mM phosphate buffer (pH7.0), 0.02mM '°SHgCl2 (0.05/~Ci/mmol), 0.1mM MgCI2, and I mM NADPH in a total volume of 2 ml. After incubation for the appropriate time, 0.1 ml portion of the reaction mixture was removed and mixed with scintillation liquid (PCS, Amersham) and radioactivity was measured in a liquid scintillation counter (Aloka LSC-673). The vaporization of 2°SHg was estimated from the disappearance of radioactivity from the reaction mixture. Isolation of periplasmic protein To determine the localization of mercury reductase, the periplasmic space fraction was prepared by the small-scale cold osmotic shock method described by Hazelbauer and Harayma (1979). To obtain the cytoplasmic protein fraction, the osmotically shocked cells were disrupted by sonication and the supernatant fraction was obtained using a bench top centrifuge (Eppendorf 5412). //-Lactamase activity as a marker enzyme of periplasm was measured by the method of O'Callaghan et al. (1972), and the assay of //-galactosidase as a cytoplasmic marker enzyme was carried out by the method of Miller (1977). Colon)' hybridization E. coli strains from dental hospital sewage were screened for the presence of mercury-resistance gene DNA by a method based on the colony hybridization technique of Grunstein and Hogness (1975). E. coli strains were grown on nitrocellulose filter (Millipore, 0.45 lam pore size) laid on a L-agar plate containing 25 ,ug/ml ampicillin and HgCI 2. The filters were placed in 0.5 M NaOH and 1.5 M NaCI to denature double-stranded DNA, then neutralized with 1M Tris-HC1 (pH 7.6). The filters were washed with 2X SSC (SSC; 0.15 M NaCI, 0.015 M sodium citrate, pH 7.0) and baked for 3 h at 80"C to fix single-stranded DNA onto the filter, then incubated for 3 h in Denhardt solution (0.02% ficoll, 0.02% polyvinylpyrrolidone and 0.02% bovine serum albumin) at 65 C. The filters were hybridized with cloned mercury-resistance DNA fragment labeled by nick translation with [:~-3-'P]-dATP as described by Rigby et al. (1977). Filters were then washed with 2x SSC containing 0.1% sodium dodecyl sulfate. After drying, filters were autoradiographed at - 2 0 C for 24 h. RF.SULTS Restriction endonuclease analysis N a g a h a m a (1987) screened plasmids in mercuryresistant E. coli strains isolated from dental hospital sewage and found that most of the strains carried a single plasmid, t h o u g h some carried multiple plasmids. The investigator succeeded in isolating a single plasmid encoding mercury-resistance gene from multiple plasmids in these strains by t r a n s f o r m a t i o n experiments, a n d designated these plasmids confering mercury resistance as p H N I , p H N 2 , a n d p H N 3 . He also showed these plasmids to have different restriction p a t t e r n s by Eco RI digestion. We analyzed plasmid D N A which was isolated as a single plasmid using E c o R l digestion, and found the most pred o m i n a n t plasmid ( p H N 4 ) type h a d the same pattern as p H N 3 . W e analysed these h o m o l o g o u s plasmid D N A s using B a m H l a n d S a i l , as s h o w n in Fig. 1. p H N 4 also showed the same digestion p a t t e r n s as p H N 3 with these restriction endonucleases. These data suggest that p H N 4 a n d p H N 3 are the same
Cloning of Hg-resistance gene 1
2
3
4
5
6
7
8
9
10 I1
12
13 14 15
611 2
1
3
4
6
5
Fig. 1. Restriction endonuelease analysis of mercuryresistance plasmid DNAs with different endonucleases: 1, pHNi/BamHI; 2, pHN2/BamHI; 3, pHN3/BamHI; 4, pHN4/BamHI; 6, pHNl/SalI; 7, pHN2/SalI; 8, pHN3/ Sail; 9, pHN4/Sall; 11, pHNI/EcoRI; 12, pHN2/EcoRI; 13, pHN3/EcoRI; 14, pHN4/EcoRI; "5, 10, 15, 2ci857 Hindlll cut as size standard. plasmid and that this plasmid may be predominantly carried by mercury-resistant E. coil cells in dental hospital sewage. Therefore we decided to clone this mercury-resistance gene from pHN4, which should be widely prevalent in dental hospital sewage. Cloning of mercury-resistance gene We carried out gene cloning using cosmid vector pJC74 first and then subcloned with plasmid vector pBR322. Cleavage of pHN4 by Sail yielded at least 9 detectable DNA fragments (Fig. 1). We partially digested pHN4 with Sail and iigated the products with completely digested cosmid pJC74, as shown in Fig. 2, to produce high molecular weight chimeric DNAs. These chimeric DNAs were in vitro packaged in k phage heads for infection of E. coli C600 cells, then ampicillin-resistant clones were selected. The packaging efficiency was 3 x 104 PFU//~g of recombinant DNA. Mercury-resistant clones were selected on L-agar plates containing 25/~g/ml of HgCI 2. The plasmid DNA from mercury resistant transformants was isolated and analysed with restriction endonuclease Sail. As shown in Fig. 3, mercury-resistance plasmids yielded different digestion patterns comprising several DNA fragments. A mercury resistant clone which had a small number of DNA fragments was designated as pHN4-I. Then SalI-cut pHN4-I DNA fragments were ligated with Sail-cut pBR322 and transformed into HBI01 cells. The transformants of ampicillin- and HgCI2_ resistant clones were screened for plasmids, and a transformant which carried the smallest size of plasmid was selected and designated as pH4-II. Since pHN4 carries the streptomycin (Sm)-resistance gene (Nagahama, 1987), we also examined the transformants for phenotypic expression of Sm However, HN4-II did not grow on L-agar plate resistance containing 10 pg/ml of Sm. Characterization of cloned DNA fragments The molecular sizes of pHN4-I and pHN4-II
Fig. 2. Ligation of SalI fragments of pHN4 and pJC74 vector. 1, pJC74; 2, pHN4; 3, 2ci857 Hindlll cut as size standards; 4, pHN4/SalI partially cut; 5, pJC74/Sa/l; 6, ligated chimeric DNA of pHN4 and pJC74. supercoiled DNA were estimated to be about 24 and 12 kb, respectively, by comparison of their mobilities during agarose gel electrophoresis with plasmid size standards (Fig. 4). The linear DNA fragments observed after electrophoresis of pHN4-I and pHN4-II on agarose gels after SalI digestion are shown in Fig. 5. The DNA from both plasmids contained the identical 7.7 kb DNA fragment. Thus the mercuryresistance gene must reside on the Sail 7.7 kb DNA fragment. HgCl2-dependent NADPH oxidation Since it is believed that the mechanism of mercury resistance is mercury vaporization by HgCI:1
2
3
4
5
Fig. 3. Sail digestion of mercury-resistance eosmid clones. 1, pHN4/SalI; 2, to 5, mercury-resistance cosmids/SalI. The plasmid of lane 2 has 7.7 and 16 kb DNA fragments, and this plasmid was designated as pHN4-I.
612
HIROSHI ARII a n d YOSHIMITSU ABIKO l
2
3
4
5
6
7
fl
Table 2. Mercury reductase activity in E. coli clones Relative activity (%)
Mercury reductase activity ( N A D P nmol.,min/mg protein)
Clones HBI01 HBI01 (pHN4) HBI01 (pHN4-I) HBI01 (pHN4-1I)
0 28.0 46.5 118.9
-100 166
424
Mercury reductase activity was expressed as N A D P formed from N A D P H oxidation. The assay procedure was described in Materials and Methods. Table 3. Localization of mercury reductase in E. coli _Enzymes
Fig. 4. Size determination of mercury-resistance cloned plasmids. 1, Rldrdl9 (94kb), lower band should be chromosomal DNA, 2, R6K (40 kb); 3, pYA724 (24.5 kb); 4, pBTI-10 (7.7 kb); 5, pJC74 (16 kb); 6, pBR322 (4.3 kb); 7, pHN4-I; 8, pHN4-1I, pHN4-I and pHN4-II were determined to be 24 and 12 kb, respectively. dependent NADPH oxidation, we measured mercury reductase activity in cell extracts of mercury-resistant clones. When the cell extracts of pHN4, pHN4-I, and pHN4-II transformed HBI01 cells were added to the reaction mixture containing NADPH and HgCI2, rapid oxidation of NADPH was observed. However, an extract of a mercury-sensitive strain, host cell HBI01 did not. Mercury reductase activities of pHN4-I, and pHN4-II-bearing cells were 1.7 and 4.2-fold higher respectively than that of pHN4 infected one (Table 2).
Vaporization of 2°3Hg Since high activity of HgCl2-dependent NADPH oxidation was recognized in the cell extract of HN4II, 2°3Hg vaporization was examined. As shown in Fig. 6, the cell extract of HN4-11 showed a significant activity to vaporize 2°3Hg. This activity was greatly diminished when the cell extract was heated at 80°C for 5 rain. On the other hand, no 203Hg vaporization
1
2
3
Relative activity (%) Periplasm Cytoplasm
Mercury reductase fl-galactosidase fl-lactamase
2 4 91
98 96 9
Total enzyme activities of periplasm and cytoplasm were calculated to be 100%. The enzymes assay were described in Materials and Methods.
activity could be detected in the cell extract of host strain HB101.
Localization of mercury reductase In control experiments for marker enzymes of periplasmic and cytoplasmic fractions, we found that 1% of the total fl-lactamase activity was in the periplasmic extract and 96% of the total fl-galactosidase was in the cytoplasmic extract. When mercury reductase activity was measured in periplasmic and cytoplasmic extracts, it was predominantly located in the cytoplasm (Table 3).
Cohmy hybridization with cloned mercury-resistance gene Mercury-resistant E. coli cells were isolated from dental hospital sewage and grown on nitrocellulose
4
g
50 -
".._
ff-
I 0
30 Incubation time (rain)
I 60
Fig. 6. Vaporization of m3Hg by the cell extract of HN4-[I.
Fig. 5. Endonuclease analysis of pHN4-I and pHN4-11. I.
pHN4-1/SalI; 2, pHN4-II/Sall; 3, pJC74/SalI; 4, pBR322/ Sail.
Cell extracts were obtained from HN4-II by sonication followed by centrifugation at 28,0008. The reaction mixture and measurement of radioactivity were described in Materials and Methods. (O), Cell extract of HN4-II; (C)), heated cell extract of HN4-II; (11), cell extract of HBI01.
613
Cloning of Hg-resistance gene
Fig. 7. Colony hybridization of DNA from mercuryresistance E. coli isolated from dental hospital sewage with 32P-labeled 7.7kb DNA encoding mercury resistance. Arrow indicates a colony of HN4 as positive control.
filters. Fifty colonies were tested for hybridization with radioactivity labeled 7.7 kb D N A fragment encoding mercury resistance, and 13 colonies hybridized with the probe, as shown in Fig. 7. DISCUSSION Numerous studies of antibiotic drug-resistant bacteria have clarified the close relationship between the appearance of drug-resistant bacteria and usage of the drug. However, the nature of the selection factor for heavy metal-resistant bacteria has not yet been fully explained from epidemiological and genetic viewpoints. Timoney et al. (1978) surveyed mercury resistance of aerobic bacterial flora in sewage sludge dump areas and unpolluted areas of New York Bight. Mercuryresistant Bacillus populations were found with much greater frequency in sediments containing high concentrations of mercury than in sediments from areas further offshore where dumping has never been practiced and where heavy-metal concentrations were found to be low. Nagahama (1987) also reported that mercury resistant E. coil populations were more numerous in sediments of dental hospital sewage than in sediments from other general building. Therefore, it is suggested that mercury contamination of the sediment was the selective pressure giving rise to the plasmids encoding the mercury resistance. One of our aims for cloning the mercury-resistance gene from an R-plasmid was to assess the prevalence and distribution of such gene in mercury-resistant E. coli populations in polluted sewage. According to our data, the cell extract of HN4-II had high activity of 2°4Hg vaporization, and the heat-treated extract lost this activity. Mercury reductase activities of HN4-II and HN4-I were 4.2- and 1.7-foid higher, respectively, than that of HN4. In general, high molecular size plasmids exist in host cells as a small number of copies, whereas small plasmids are carried as a large number of copies. Therefore, the differences in the
enzyme activity in these mercury-resistant transformant clones are probably dependent on plasmid copy number, for pBR322 (vector for pHN4-II) and pJC74 (vector for pHN4-I) are maintained at a relatively high.copy number of 40 and 15 per cell, respectively (Karn et al., 1979; Collins, 1979). In addition, pHN4-I and pHN4-II represent insertion of the 7.7 kb fragment in vector plasmids. These data suggest that the mercury-resistance gene, encoding mercury reductase, was successfully cloned as a 7.7 k b - S a l I DNA fragment. Since HN4 carries Sm resistance and the HN4-II beating cell is sensitive to Sm, the 7.7 kb mercury-resistance gene was separated from the Sm resistance gene. It has been found that plasmid-determined genes for drug resistance are capable of moving from one replicon to another in the absence of host cell-mediated recombination function, and such genetic elements are called to transposon (Kleckner, 1977). The mercury-resistance gene carried in plasmids often occurs on a transposon (Stanisich et al., 1977) and associated sex factors (Summers and Silver, 1972). From the view points of the origin of mercuryresistant bacteria and their selection, spreading and prevalence, transposon elements may exist in near the mercury-resistance gene of plasmid. The colonies of mercury-resistant E. coli strains isolated from sewage were tested with 32P-labeled S a l I 7.7kb D N A by colony hybridization. Of 50 colonies examined, 13 (26%) reacted with the probe. Thus, this cloned mercury-resistance gene can be detected among diverse mercury-resistance E. coli isolates. These data suggest that the pHN4-II clone is a useful tool for survey of mercury-resistance determinants and ecological changes of mercury pollution in sewage. Furthermore, the cell extract of HN4-II provide a larger quantity of mercury reductase to study protein and enzymological studies. It is likely that mercury concentrations in sediments become less after dumping of mercury compounds because of dilution effects during passage through the water, because of mixing with other sediments on the bottom, and finally because of dissipation of mercury into the water by resistant mercury reducing bacteria. Therefore, HN4-II, which expressed high Hg2+-reductase activity, may also be useful for reduction of the mercury concentration in polluted waters. REFERENCES
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HIROSHI ARI1 and YOSHIMI'rsu AB1KO
Grunstein M. and Hogness (1975) Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc. ham. Aead. Sci. U.S.A. 72, 3961-3965. Gurrey P., LeBlanc D. J. and Falkow S. (1973) General method for the isolation of plasmid deoxyribonucleic acid. J. Bacteriol. 116, 1064-1066. Hansen J. B. and OIsen R, H. (1978) Isolation of large bacterial plasmids and characterization of the P2 incompatibility group plasmids pMGI and pMG5. J. Bacteriol. 135, 227- 238. Hazelbauer G. and Harayama S. (1979) Mutants in transmission of chemotactic signals from two independent receptors of E. coll. Cell 16, 617-625. Hohn B. and Hohn T. (1974) Activity of empty, headlike particles for packaging of DNA of bacteriophage ). in vitro. Proc. natn. Acad. Sci. U.S.A. 71, 2372-2376. Karn M., Kolter R., Thomas C., Figurski D., Meyer R., Remut E. and Helinski D. R. (1979) Plasmid cloning vehicles derived from plasmids ColEI, F, R6K and RK2. Methods Enzymol. 68, 268-280. Kleckner N. (1977) Translocatable elements in prokaryotes. Cell !1, 11-23. Komura I. and lzaki K. (1971) Mechanism of mercuric chloride resistance in microorganism. I. Vaporization of a mercury compound from mercuric chloride by multiple drug resistant strains of Escherichia coli. J. Biochem. 70, 885 893. Maniatis T., Fritsch E. F. and Sambrook J. (1980a) Procedure for cDNA cloning. In Molecular Cloning, pp. 229-246. Cold Spring Harbor, New York. Maniatis T., Fritsch E. F. and Sambrook J. (1980b) Introduction of plasmid and bacteriophage J. DNA into Escherichia coli. In Molecular Cloning, pp. 247-268. Cold Spring Harbor, New York. Miller J. H. (1977) Assay of fl-galactosidase. In Experiments in Molecular Genetics, pp. 352-355. Cold Spring Harbor, New York.
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