Gene, 8 (1980) 153--162
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© Elsevier/North-Holland Biomedical Press
ISOLATION OF T R A N S P O S O N TnA F R O M PLASMID RP4 C A R R Y I N G TWO COPIES OF THIS ELEMENT (Restriction endonucleases E c o R I , H i n d I I I , B a m H I ; heteroduplex analysis; endonuclease S1)
A.P. DOBRITSA, V.N. KSENZENKO, V.B. FEDOSEEVA*, A.A. ALEXANDROV*, T.P. KAMYNINA and M.I. KHMELNITSKY Institute of Biochemistry and Physiology of Microorganisms, USSR Academy of Sciences, Pushchmo, Moscow Regmn, 142292, and Institute of Molecular Genetw~ USSR Academy of Sciences, 46, Kurchatov Square, Moscow, 123182 (U.S.S.R.)
(Received May 24th, 1979) (Accepted July 6th, 1979) SUMMARY Employing heteroduplex and restriction analyses, t w o inverted copies of a 3.2 • 106 dalton transposable sequence, TnA, were found in RP4::TnA, a spontaneously arisen derivative o f the plasmid RP4. Integration of the second c o p y of TnA causes loss of the con.iugative properties of RP4. Both TnA sequences in RP4 :: TnA were localized and found to have opposite orientations. The DNA fragment corresponding to the individual transposon TnA was isolated after the endonuclease S1 digestion of RP4:: TnA molecules annealed under conditions favoring intramolecular renaturation. The attempts to transform the cells of Escherichia coil QD5003, HB101 [pCRI] and JC7623 with the isolated transposon were unsuccessful.
INTRODUCTION
The conjugative plasmid RP4 confers resistance to ampicillin, tetracycline and kanamycin (Datta et al., 1971). The gene for ampiciUin resistance resides upon a discrete DNA fragment, transposon TnA, that is capable of recA-independent translocation from replicon to replicon (Hedges and Jacob, 1974; Heffron et al., 1975b; Kopecko and Cohen, 1975; Heffron et al., 1975a; Bennett and Richmond, 1976; Rubens et al., 1976). TnA is 3.2 - 106 daltons in size and is flanked by inverted-repeated sequences of a b o u t 140 base pairs (Heffron et al., 1975a). Probably transposition of TnA does not generate deletions of this sequence in the donor plasmid (Bennett et al., 1977). Insertion of TnA is mutagenic when it occurs within structural genes and polar toward the distal genes of an operon (Rubens et al., 1976).
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Until recently plasmids containing a transposon were thought to be immune to the insertion of its second copy (Robinson et al., 1977). However, they managed to obtain the plasmids carrying two copies of Tn501, encoding resistance to mercuric ions (Bennett et al., 1978a) and the plasmids carrying two copies of TnA (Robinson et al., 1978; Bennett et al., 1978b). But these authors still maintain that insertion of the second copy of a transposon to a plasmid which already carries one is specifically prevented. The formation of plasmids with two transposon copies was conditioned in their opinion by the simultaneous translocation of these DNA sequences. The results presented here show that insertion of the second transposon copy is nevertheless possible. This paper deals with the study of the properties of plasmid RP4 containing the additional TnA sequence and describes the isolation of the individual transposon TnA. MATERIALS AND METHODS
(a ) Bacterial strains E. coli J53 (pro-, met-) cjarrying RP4 was obtained from Dr. T i c h y (Institute of Microbiology, CSSR). A spontaneous derivative of RP4, which carried an additional TnA element, was isolated from this strain. For transformation, E. coli strains HB101 (leu-, pro-, lac-, gal-, thi-, str R, recA56, r-m-) carrying the plasmid pCR1 (Covey et al., 1976), QD5003 ( m e t , supE57, supF58) and JC7623 (leu-, pro-, arg-, his-, thr-, thi-, gal-, met-, xyl-, ara-, str R, recB21, recC22, sbcB15) were used. A streptomycin-resistant mutant of E. coil C600 (thi-, thr-, leu-, lacY-) was used as recipient in the mating procedure. Bacterial cells were grown on LB and M9 media (Miller, 1972).
(b ) Investigation of RP4 :: TnA phenotype Plasmid RP4 carries TnI transposon and symbol TnA is a generic term for several independently isolated transposons, including Tnl, Tn2 and Tn3 (Campbell et al., 1979). Drug resistance markers of RP4 were examined on plates containing ampicillin (100 pg/ml), tetracycline (10 #g/ml) or kanamycin (25 ug/ml). Transferability of the plasmids was determined by a membrane mating procedure (Jacob et al., 1976). Donors were counterselected with streptomycin (200 #g/ml) and transferred plasmids were selected with kanamycin. To test phage sensitivity, about 0.1 ml of an overnight culture was spread onto LB agar plate. When dry, a drop of high titer (approx. 10 ~°plaqueforming units/ml) preparations of phage PRR1 specific for cells with Pl-pili (Olsen and Shipley, 1973) was added to the plate.
(c) Plasmid DNA isolation Plasmid DNAs were isolated according to Guerry et al. (1973). Before the CsC1 banding step, the DNA was concentrated as described by Humphrey et al. (1975).
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(d) Restriction analysis of plasmid DNA Cleavage of plasmid DNA with EcoRI, HindIII and BamHI restriction endonucleases and analysis of DNA fragments b y agarose gel electrophoresis were conducted as described previously (Dobritsa et al., 1978).
(e) Analysis of plasmid DNA by electron microscopy The preparation of DNA, its denaturation, renaturation in formamide and final preparation for electron microscopy were performed using the Westmoreland et al. (1969) formamide technique as modified b y Davis et al. (1971). The formulae Cot = 5 • 10 -2 m o l e - sec/1 and Cot = 5 • 10 -4 m o l e - sec/1 were used to calculate the duration o f inter- and intramolecular renaturation, respectively. Nucleic acids were visualized with an electron microscope JEM-7 (JEOL). The length markers of single- and double-chain DNAs were phage ~X174 and plasmid ColE1 DNAs, respectively. The magnification of the electron microscope was calibrated with a line-grating replica (spacing 1.59 ~m). The contour length of the DNA molecules was measured with a map measurer.
(f) Isolation of nuclease $1 Endonuclease S1 was purified b y the modified technique of Vogt (1973). Sulphosephadex C-50 chromatography and Sephadex G-100 gel-filtration were replaced with Sephadex G-75 gel-filtration (Wiegand et al., 1975).
(g) Isolation of the transposon TnA RP4 :: TnA molecules linearized with HindIII were denatured with alkali for 15 min at room temperature. The denaturation solution contained 100--150 /~g of DNA in 11 ml of 0.1 M NaOH, 20 mM NaC1, 20 mM EDTA. Then 12.2 ml of formamide and 1.2 ml of 2.0 M Tris- HC1 were added to this solution. The renaturation mixture was incubated at 25°C for 4 h. After this the solution was applied to a hydroxylapatite column (Bio-Gel HT, volume 400 p]) equilibrated with 0.05 M potassium phosphate buffer, pH 6.8 (K-P buffer). The column was washed with 10 vol. of this buffer and DNA was eluted with 0.6 ml of 0.5 M K-P buffer. The eluate was dialysed against 0.3 M NaC1, 0.001 M Z n S O 4 , 0 . 0 3 M N a - acetate, 5% glycerol, pH 4.6. After dialysis the solution was supplemented with 60 pl of nuclease SI (specific activity about 400 units/ml according to Vogt, 1973) and allowed to stand at 35°C for 4 h. The products of hydrolysis were separated b y electrophoresis in 0.7% agarose gel (BioRad). A b o u t 0.4 ml of hydrolysate was applied to gel slab (dimensions 13X 11X0.3 cm). Electrophoresis was carried o u t for 12 h at 28 V. After completion of electrophoresis the gel was stained in the ethidium bromide solution (0.5 pg/ml) and examined under a long wavelength UV light. The gel band corresponding to the transposon TnA was cut o u t and dissolved at 37°C in 5 ml of 6 M KI, 0.03 M/3-mercaptoethanol (Smith and Birnstiel, 1976). Further purification and concentration of TnA were achieved b y hydroxylapatite chromatography as above. The obtained preparation of TnA was dialysed
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against 10 mM Tris- HC1, pH 8.0, 20 mM NaC1, 1 mM EDTA and stored at --20°C.
(h ) Transformation Transformation was performed as described by Cohen et al. (1972). The selection of transformants was carried out by their resistance to ampicillin (30 ug/ml). RESULTS AND DISCUSSION
By electron microscope examination a double-strand region was found in the structure of intramolecularly reannealed chains of plasmid molecules isolated from a clone of E. coil J53 [RP4] (Fig. 1). This fragment was flanked by two singie-stranded loops 4.2 + 0.08 pm and 13.5 -+0.15 ~m in length. The molecular weights of double-stranded sequences corresponding to these loops are 8.0 - 106 and 25.7 • 106 . The molecular weight of the double-stranded region (contour length is 1.65 + 0.05 pm) is 3.2 • 106 being in good agreement with that one of the transposon TnA. The molecular size of the plasmid under study exceeds t h a t of the original one by the same value. Therefore it is reasonable to assume that this plasmid is an RP4 derivative formed by insertion of the second copy of TnA in the orientation inverted with respect to the first one. This derivative was named as RP4 :: TnA. The translocation of the transposon to RP4 did not cause marked deletions or substitutions in the
Fig. 1. Electron micrograph of intramolecularly reannealed DNA strand of RP4 :: TnA. SS, single-stranded region; DS, double-stranded region. Bar represents I ~m.
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Fig. 2. Electron micrograph of the RP4/RP4 :: TnA heteroduplex. SS, single-stranded region; DS, double-stranded region. Bar represents 1 urn.
structure of this plasmid. Only one nonhomologous fragment is seen on the electron micrographs of the heteroduplex RP4 :: TnA/RP4 (Fig. 2). The size of the single-stranded loop of this heteroduplex makes up 1.7 -+0.1 pro, t h a t corresponds to the length of TnA. Localization o f the second TnA copy on the physical map o f the plasmid RP4 :: TnA RP4 DNA has a single BamHI cleavage site and this site is localized within TnA (Heffron et al., 1977; Barth and Grinter, 1977). Insertion of the second TnA unit should result in the appearance of the additional BamHI site. RP4 :: TnA contains the single sites of E c o R I and HindIII endonucleases and as would be expected two BamHI sites (Fig. 3). These restriction sites were localized by hydrolysis of RP4 :: TnA with HindIII + EcoRI, BamHI + EcoRI, BamHI + HindIII. The physical map of the plasmid RP4 :: TnA is presented in Fig. 4. The location of EcoRI, HindIII and one of the BamHI sites on RP4 :: TnA is consistent with the restriction map of RP4 constructed by Barth and Grinter (1977). The second BamHI site has the coordinate 16.85 • 106 daltons. The molecular weights of the BamHI fragments of RP4 :: TnA are equal to 12.4 • 106 and 27.85 • 106 . TnA units in the genome of RP4 :: TnA are separated by two segments with molecular weights of 8.0 • 106 and 25.7 106 according to heteroduplex analysis data. These segments, therefore, should be within the first and the second BamHI fragments, respectively. Thus 2.2 • 106 dalton parts of both TnA copies are localized on the smaller BamHI fragment. Endonuclease BamHI is k n o w n to cleave TnA into two fragments which are about one-third and two-thirds of its length. Apr has been mapped
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Fig. 3. Agarose gel electrophoresis of DNA of RP4 :: TnA cleaved with E c o R I (2), E c o R I + HindIII (3), HindIII (4), E c o R I + BamHI (5), HindIII + BamHI (6), Ba m H I (7) and RP4 DNA digested with B a m H I (8). Markers: E c o R I fragments (1) and HindIII fragments (9) of bacteriophage k DNA. within the first fragment mentioned (Heffron et al., 1977). Hence, from the data of electron microscope and restriction analyses, the location and the orientation of both TnA sequences in the RP4 :: TnA genome may be determined (Fig. 4). As shown in Fig. 4, the 2.2- 106 dalton part of the sequence of the inserted TnA element is situated at the boundary of the RP4 :: TnA fragment of 1 6 . 4 . 1 0 6 daltons, which is flanked by B a m H I and E c o R I sites. This indicates t h a t the TnA translocation into RP4 occurred in the point separated by a 14.2 • 106 dalton fragment from the E c o R I site. TnA insertion into the given region of the plasmid RP4 does n o t affect the markers of antibiotic resistance but results in the loss of the plasmid transmissability (see Table I). Cells of E. c o i l J53 a n d E. c o i l HB101 carrying
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Fig. 4. Restriction map of RP4 ::TnA. The sizes of fragments are given in megadaltons. TABLEI FREQUENCY
OF PLASMID TRANSFER
Donor
Recipient
J53 [RP4] J53 [RP4::TnA]
C600 str R C600 str R
Transfer frequency (per donor)
2" 10-' <1 • 10-8
this non-conjugative derivative of t he plasmid RP4 unlike cells of E. coli J53 [RP4] and E. coli HB101 [RP4] are resistant to the Pl-pili-specific phage PRR1. According to Barth and Grinter (1977), the genes responsible for t he Tra + p h e n o t y p e are located near t he c oor di nat e o f 28 - 106 daltons on the RP4 map. Insertion o f TnA which caused the inactivation of t he tra system occurred in the site at 14.2 • 104 daltons. Hence, the tra genes of RP4 are n o t assembled in a cluster but are located in at least two d if f er e nt regions of the plasmid genome. This is confirmed by the results o f Barth et al. (1978). According to these authors, coordinates o f t h e second tra region on the genetic map of RP4 are a b o u t 16.5--17.5 - 104 daltons. Isolation o f the transposon T n A
The presence o f the two inverted copies of TnA in the plasmid RP4 :: TnA enables an isolation of the DNA fragment corresponding to the individual transposon. The steps o f the TnA isolation are given in Fig. 5. DNA of plasmid RP4 :: TnA was converted into the linear form by the cleavage with endonuclease H i n d I I I , t h e n denat ur e d with alkali and annealed under conditions
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0
H i n d II
RFI
/ O'n"ur'1"°nt HY'r'd+""°n OqM NIOH
NUCteSSQ $1
SO% Formlmide
RFIm
Fig. 5. The scheme of TnA isolation.
favourable for intramolecular renaturation (see MATERIALS A N D M E T H O D S ). Under these conditions the inverted T n A units form double-stranded regions in single RP4 ::T n A chains. The non-renatured regions (single~hain regions) of this D N A were removed by hydrolysis with nuclease $1. Further purification of T n A by agarose gel electrophoresis resulted in a D N A fragment of 3.2 • 106 daltons. BamHI cleavage produces two fragments of about 2.3- 10 + and 1.05 - 106 daltons (Fig. 6), which confirms t h a t the isolated DNA fragment corresponds to TnA.
Fig. 6. Agarose gel electrophoresis of the DNA fragment corresponding to the transposon TnA (2) and products of its incomplete hydrolysis with Ba m H I (3). Markers: HindIII fragments of phage T5 DNA (1).
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The attempts to transform the isolated DNA fragment into E. coli QD5003, HB101 [pCRI] and JC7623 failed. Probably this failure results from degradation of the inverted repeats during its isolation or transformation into bacteria. These repeats are known to be essential for the TnA translocation (Heffron et al., 1977). Moreover, TnA-mediated protein synthesis may be required for the transformation of this element. It is also possible that during translocation the TnA element has the structure other than the fragment we isolated. REFERENCES Barth, P.T. and Grinter, N.J., Map of plasmid RP4 derived by insertion of transposon C, J. Mol. Biol., 113 (1977) 455--474. Barth, P.T., Grinter, N.J. and Bradley, D.E., Conjugal transfer system of plasmid RP4: analysis by transposon 7 insertion, J. Bacteriol., 133 (1978) 43--52. Bennett, P.M., Grinsted, J., Choi, C.L. and Richmond, M.H., Characterization of Tn 501, a transposon determining resistance to mercuric ions, Mol. Gen. Genet., 159 (1978a) 101--106. Bennett, P.M., Grinsted, J. and Richmond, M.H., Transposition of TnA does not generate deletions. Mol. Gen. Genet., 154 (1977) 205--211. Bennett, P.M. and Richmond, M.H., Translocation of a discrete piece of deoxyribonucleic acid carrying an amp gene between replicons in Escherichia coli, J. Bacteriol., 126 (1976) 1--6. Bennett, P.M., Robinson, M.K. and Richmond, M.H., Self-limitation of multiple transposition of TnA, Microbiology -- 1978, Am. Soc. Microbiol., (1978b), 16--18. Campbell, A., Berg, D.E., Botstein, D., Lederberg, E.M., Novick, R.P., Starlinger, P. and Szybalski, W., Nomenclature of transposable elements in prokaryotes, Gene 5 (1979) 197--206. Cohen, S.N., Chang, A.C.Y. and Hsu, L., Nonchromosomal antibiotic resistance in bacteria: genetic transformation of E. coil by R-factor DNA, Proc. Natl. Acad. Sci. USA, 69 (1972) 2110--2114. Covey, C., Richardson, D. and Carbon, J., A method for the deletion of restriction sites in bacterial plasmid deoxyribonucleic acid, Mol. Gen. Genet., 145 (1976) 155--158. Datta, N., Hedges, R.W., Shaw, E.J., Sykes, R.B. and Richmond, M.H., Properties of an R factor from Pseudomonas aeruginosa, J. Bacteriol., 108 (1971) 1244--1249. Davis, R.W., Simon, M. and Davidson, N., Electron microscope heteroduplex methods for mapping regions of base sequence homology in nucleic acids, in Grossman, L. and Moldave, K. (Eds.), Methods in Enzymology, Vol. 21, Academic Press, New York, 1971, pp. 413--428. Dobritsa, A.P., Dobritsa, S.V. and Tanyashin, V.I., Isolation and characterization of plasmid from the Bacillus brevis var.G.-B, cells, Mol. Gen. Genet., 164 (1978) 195--204. Guerry, P., Le Blanc, D.J. and Falkow, S., General method for the isolation of plasmid deoxyribonucleic acid, J. Bacteriol., 116 (1973) 1064--1066. Hedges, R.W. and Jacob, A.E., Transposition of ampicillin resistance from RP4 to other replicons, Mol. Gen. Genet., 132 (1974) 31--40. Heffron, F., Bedinger, P., Champoux, J.J. and Falkow, S., Deletions affecting the transposition of an antibiotic resistance gene, Proc. Natl. Acad. Sci. USA, 74 (1977) 702--706. Heffron, F., Rubens, C. and Falkow, S., Translocation of a plasmid DNA sequence which mediates ampicillin resistance: molecular nature and specificity of insertion, Proc. Natl. Acad. Sci.USA, 72 (1975a) 3623--3627. Heffron, F., Sublett, R., Hedges, R., Jacob, A. and Falkow, S., Origin of the TEM betalactamase gene found on plasmids, J. Bacteriol., 122 (1975b) 250--256.
162 Humphreys, G.O., Willshaw, G.A. and Anderson, E.S., A simple method for the preparation of large quantities of pure plasmid DNA, Bioehim. Biophys. Acta, 383 (1975) 457--463. Jacob, A.E., Gresswell, J.M., Hedges, R.W., Coetzee, J.N. and Beringer, J.E., Properties of plasmids constructed by the in vitro insertion of DNA from Rhizobium leguminosarum or Proteus mirabilis into RP4, Mol. Gen. Genet., 147 (1976) 315--323. Kopecko, D.J. and Cohen, S.N., Site-specific recA-independent recombination between bacterial plasmids: involvement of palindromes at the recombinational loci, Proc. Natl. Acad. Sci. USA, 72 (1975) 1373--1377. Miller, J.H., Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor. NY, 1972. Olsen, R.H. and Shipley, P.L., Host range and properties of the Pseudomonas aeruginosa R factor R1822, J. Bacteriol., 113 (1973) 772--780. Robinson, M.K., Bennett, P.M., Grinsted, J. and Richmond, M.H., The stable carriage of two TnA units on a single replicon, Mol. Gen. Genet., 160 (1978) 339--346. Robinson, M.K., Bennett, P.M. and Richmond, M.H., Inhibition of TnA translocation by TnA, J. Baeteriol., 129 (1977) 407--414. Rubens, C., Heffron, F. and Falkow, S., Transposition of a plasmid deoxyribonucleic acid sequence that mediates ampicillin resistance: independence from host rec functions and orientation of insertion, J. Bacteriol., 128 (1976) 425--434. Smith, H.H. and Birnstiel, M.L., A simple method for DNA restriction site mapping, Nucl. Acids Res., 3 (1976) 2387--2398. Vogt, V.H., Purification and further properties of single-strand-specific nuclease from AspergiUus oryzae, Eur. J. Biochem., 33 (1973) 192--200. Westmoreland, B.C., Szybalski, W. and Ris, H., Mapping of deletions and substitutions in heteroduplex DNA molecules of bacteriophage lambda by electron microscopy, Science, 163 (1969) 1343--1348. Wiegand, R.C., Godson, G.N. and Radding, Ch.M., Specificity of the S1 nuclease from Aspergillus oryzae, J. Biol. Chem., 250 (1975)8848--8855. Communicated by A.A. Bayev.