Cloning of a recA-like gene of Proteus mirabilis

Ceue. !4 (1981) 301-308 Elsevier/North-HollandBiomedicalPress

301

Cloning o f a recA-fike gene o f P r o t e u s mirabilis •

.

.

.

.

(recAp.m., gene cloning, restriction map, UV resistance)

G. Eitner, A.S. Solonin * and V.l, Tanyashin * Zentralinstitut ffir Genetik und KulturPllanzenforschungof the Academy of Sciences of the GDR, 4325 Gatersleben (GDR}, and Institute of Biochemistry and Physiology of M~eroorganisms,Academy of Sciences of the USSR, 142292, Pushchino (USS.~)

(ReceivedDecember28th, 1980) (Accepted March26th, 1981)

SUMMARY

A gene of Proteus mirabilis that can substitute for functions of the recA gene of Escherichia coil has been cloned into the plasmid pBR322, using shotgun experiments. The recA-fike gene (recAp.m.)has been localized by restriction mapping within a 1.5-Md PstI fragment that is a part of two cloned HindIII fragments of the chromosome ofP. mirabilis. The restriction map of the recAp.m, gene differs from that of the reeA gene of E. coll. Functionally, the recombinant plasmids containing the recAp.re, gene restore a nearly wild-type level of UV.resistance to several poinl and deletion mutants in the recA gene of E. coli.

INTRODUCTION

bilis has been found to express SOSfqnctions in a way similar to that of E. coll. After irradiation with

The recA gene product plays a key role in pro. cesses of general recombination and in induction of SOS-functions. SOS-functions (Radman, 1974)are a response of the cell to the DNA damage caused by UV-irrad.iation and related agents such as mitomycin C and methylmethane sulfonate. They involve the induction of mutations, prophages and repair systems (for review see Witkin, 1976), i n c l u ~ the induction of the reeA protein itself (Emmerson and West, 1977; McEntee, 1977b; Little and Kleid, 1977; Gudas and Mount, 1977; Sedgwick, 1978). P. mira-

UV, repair systems and prophages are induced (Hofemeister, 1977a, b; Hofemeister et al., 1979a, b). Additionally, a recA-like mutant of P. mirabilis has been isolated (Boehme, 1968) and characterized as being of the "reckless" phenotype (Hofemeister, 1977a). These findings indicate the presence of a recA.like gene function in P. mirabilis. But contrary to E. co~~,no induction of SOS.mutagenesis is observable in P. mirabl?is (Mates and Boehme, 1960; Steinborn, 1975; Hofemei~r et el., 1979b). To find out whether or not regulation and functional control of the SOS.processes in P. mirabilis are comparable to those of E. colt" we cloned the proposed recA.like gene of P. m/rabl//s and tested its expression in E. co//. Since the recA gene of E. coli has been cloned

Abbreviations: Amp,ampicHlin;bp, base ~ ; ~ , dithiothteitol; Md, megadaltons;recAp.re., recA gene of Proteus mlrabills; SDS, ~ dodecyl sulfate; Tet, tetracycline; [] brackets indicateplasmklcartier state.

0378-1119/81/0000-0000/$02.50 0 1981 Elsevier/North-HollandBiomedicalPress

302

and mapped for restriction sites (Clarke and Carbon, 1976; Ogawa et al., 1979; Little, 1979; Sanear and Rupp, 1979), sequenced (Horii et al., 1980; Sanear et al., 1980) and the protein electrophoretically identified (Emmerson and West, 1977; Gudas and Mount, 1977; Little and Hanawalt, 1977), a comparison of the fhnctions of the gene products as well as of the structures of the two genes is possible. In this paper we report on cloning and characterization of a ct~omosomal fragment of P. mirabi!is that is able to substitute for the recA gene in recA point and deletion mutants of E. coli with respect to conferring resistance against UV.

nisms, Academy of Sciences USSR, Pushchino), and by Dr. M. Hartmann (Central histRute of Experimental Therapy and bticrobiology, Academy of Sciences of GDR, Jena). [a-32p]dATP was obtained from The Radiochemical Centre, Amersham.

(d) Enzyme digestions Chromosomal and plasmid DNAs were digested with EcoRl and Sail in 50 mM Tris (pH 7.5), 10 mM .dgC12, 50 mM NaC1; with 8amHl, Hindlll, Kpnl, Xhol and BglII in 10 mM Tris (pH 7.5), 10 rnM MgO.2, 10 mM NaCl; with PstI in 90 mM Tris (pH 7A), lO mM MgCI2. In the case of ligation experiments all enzymatic reactions were carded out in 50 mM Tris (pH 7.5), 10 mM MgCl2, 10 mM DTT.

MATERIALS AND METHODS

(e) Transformation {a) Bacterial strains

P. mirabilis PG 1300 (arg, thr, ,redo 199) (i iofemeister et al., 1979) and E. coil ABII57 (thr-I, leu-6, proA2, his-4, argE3, thi.1, ~cY1, galK2, tsx, strA31) were used for isolation of chromosomal DNA. For transformation E. coli ItB101 (pro, ieu, lacY, thi, str r, r~m~, endA-, F-, recAl) was used. The recA deletion mutant KM 2321 (McEntee, 1977a) was kindly provided by R. Devoret. AB2463 is a recA 13 derivative of AB1157 (Howard-Flanders and Theriot, 1966). The plasmid pBR322 was used in all cloning experiments. It h~s been described by Bolivar et al. (1977). (b) Media The cuP.ures were grown in TBY medium (10 g tryptone-Difco, 5 g ,/east extract, 5 g NaCI]liter), with or without adq-: ion of ampiciUin (50/zg]ml) or tetracycline (10 gg:ml) depending on the antibiotic resistance conferred by the plasmids. For plates the medium was solidified with 2% agar.

(c) Enzymes and chemicals

Cells were transformed according to the procedure described by Cohen et al. (1972). (0 UV irradiation The conditions used were described by Eitner et al. (1980). For the ~lection of UVf clones the cells were irradiated on the plate. UV survival curves were obtained by irradiation of appropriate dilutions in 0.9% NaC! of a freshly grown log-culture, and plating of the irradiated cells on agar (Morand et al., 1977a), with or without ampicillin or tetracycline.

(g) Selection For the selection after transformation of phenotype UVrAmpf Tet s the cells were tenfold diluted in TBY and incubated at 370C for 2 h to express the resistance. Subsequently the cells were spread on TBY agar containing 30/zg/m! ampiciUin and directly irradiated with UV on the agar. The plates were incubated overnight at 37°C in the dark. Surviving clones were once again tested for resistance to UV ~ d ampicillin and, additionally, for sensitivity against tetra-

cycline I0 ttg/ml. (h) Plasmid isolation

Restriction enzymes and T4 DNA ligase were kindly provided by Drs. L. Li and K. Kusmin (Institute of Biochemistry and Physiology of Microorga-

Plasmid DNA was prepared from a 100 ml overnight culture either with or without addition of chlor-

303 amphenicol (100 ~g/ml) for plasmid amplification. The cells were centrifuged, frozen at -200C for 20 min and lysed by addition of 5 ml 25% sucrose in 10 mM Tris (pH 8.0) and 1 ml lysozyme (5 mg/ml in 10 mM Tris, pH 8.0). After 5 rain incubation at room temperature 0.5 ml 0.25 M EDTA (pH 8.0) and, after a further 10 rain incubation, 2 rnl 5 M NaCI and 1 rnl 10% SDS in 0.25 M EDTA were added. The lysate was incubated at 0°C for 4 - 6 h. Then it was centrifuged for 90 rain at 16000 rev./min (K24, Janetzkicentrifuge); the supernatant was decanted and the nucleic acids were precipitated by 2 vols. of 96% ethanol at -20°C during 2 - 1 2 h. After centrifugation at 5 000 rev./min (I(23, Janetzki-centrifuge) for 15 rnin the precipitate was dissolved in 14 ml TE-buffer (10 mM Tris, pH 8.0, 1 rnM EDTA). CsCl (1.625 g/era a) and ethidium bromide (400 tag/rnl) were added. Equilibrium centrifugation was done in the Spinco L-5 centrifuge at 40000 rev./min for 36 h. Ethidium bromide was removed with isoamyl alcohol and CsC1 was removed by dialysis against TE-buffer. Then the solution was treated with 0.5 vol. phenol (distilled and saturated with 10 mid Tris, pH 8). Phenol was removed by ether treatment. 5 M NaC1 v-as added to the solution to a f'mal concentration of 0.2 M and the plasmid DNA was precipitated with 2 vols. ethanol at -20°C overnight. After centrifugation the DNA was dissolved in about 0.7 ml TE-buffer. Its final concentration was 80-100 t~g/ml.

(j) Gel electrophor.~si~ Electrophoresis wzs carried out in Tris-acetatebuffer (0.04 M Tris, 0.02 M iqa-acetate, 0.002 M EDTA, pH 7.8), either in 12 cm long 0.8% vertical agarose gels or 6% polyaerylarnide gels. The fragments were separated at 50 V in agarose or at 150 V in acrylamide gels, respectively. As standard for "the determination of the Mr, a HindllI digest of phage ~, DNA was used in agarose gels and a HaelII (BspI) digest of pBR322 in acrylamide gels. DNA probes for hyrbidization were separated in 20 cm long agarose gels at 20 V for 12 h. (k) Hybridization techniques Chromosomal DNA of E. coli and of P. mirabilis were digested by BamHI, EcoRI and PstI, electrophoresed and transferred to nitrocellulose filters according te the method of Southern (1975). The recombinant plasmid pPM1 was labeled by nick translation with [cz32PIdATP (300 Ci/mmol) and hybridized with falter-bound chromosomal digests at 65°C for 12 h in a solution containing 2 × SSC, 10 × Denhardt's solution (Denhardt, 1966), 0.1% SDS, 10% dextran sulfate, 100 tag/ml poly(A), 100 #g/ ml tRNA, 200/ag/ml denatured calf thymus DNA.

(i) Isolation of chromosomal DNA

RESULTS

Ctuomosomal DNA was psepared from 400 mg of wet cells, harvested in the log.phase. Lysis was performed as descr;bed for plasmid isolation, using 5 ml lysis medium, 3 ml H20, 2 ml lysozyme, 0.5 ml EDTA and 1.5 ml SDS. After deproteinization by treatment with l X phenol (buffer saturated) and 3 - 5 X chloroform-isoamyl alcohol (24 : 1), the nucleic acids were precipitated with 2 vols. cold (-20°C) ethanol, spooled out on a glass rod, and dissolved in

(a) Cloning of the recAp.re, gene

4 ml 1 X SSC (0.15 M NaC1, 0.015 sodium citrate, pH 7.6). RNA was digested by incubation with 100/~g/ ml RNase (pretreated at lO0*C for 10 rain) for 30 rain at 3T'C. After repeated deproteinisation with ehloroform-isoamyl alcohol the DNA was ethanol precipitated (on a glass rod) and dissolved in 1.5 nd TE-buffer. The f'mal concentration was about 700 ,ug DNA/ml.

The DNA of/'. mirabilis was digested with H/ndllI and the fragments were inserted by T4-1igase into the HindlIl site of plasmid pBR322. Since with the methods used P. mirabilis proved to be not transformable by plasmid DNA (recently we were successful in transiormation of P. mirabilis with plasrnid DNA using the method described by Law et al., 1980), the recA ÷ clones had to be selected in a recA- background of E. cell, i.e. in strain HB101 recA 1. Therefore, already during the selection step interspecies gene expression had to be presupposed. Increase in UV resistance was employed for selection of those recA- cells that become recA*. A dose of 20 J/m 2 was found to kill E. coli HB101 completely, whereas almost all cells of the isogenic recA ÷ strain RRI sur-

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recAp.re. Fig. 1. Map of the restriction endonucle&qe cleavage sites in recAp.re. (a) Map of two (A and B) Hindlll fragments cloned in pPM1; (b, c) Deletion of fragments and cloning of subfragments of pPMl, respectively, performed in order to localize gene recAp.re.; (b) Hindlll deletion of fragment A resulting in pPM2; (c) EcoRl deletion resulting in pPM3; (d) Recloning of the Pstl fragment resulting in pPM4. The recAp.m, gene (heavy line) has been localized in "he region cut by two neighboufing EcoRl sites m~d probably also by the nearby H/ndlH site.

vive,~ such UV treatment. Previous experience in handling cells carrying the cloned recA* gene orE. coli in plasmid pBR322 (Kuzmin, N.P., Solonin, A.S., Eitaer, G., Glukhov, I.L., Tanya~hin, V.I., Bayev, A.A., in prep.), provided the experimental base for the present selection. After two cycles of selection of UV.resistant transformants on ampiciUinplates (ampicillin allows growth only of tra.,~sformants), two colonies showed the searched for phenotype UVrAmprTets (Tets indicates insertion of DNA in the Hindlll site of pBR322). These two colonies were later shown by restriction analysis to contain the same plasmid, which we called pPMl (plasmid, containing a fragment of the chromosomal DNA of P. mirabilis). The origin of cloned DNA from P. mirabilis was confirmed by DNA hybridization according to Southern (19~75). Hybridization between the recombinant plasmid and the chromosomal DNA of P. mirabilis was highly specific; between plasmid and chromosomal DNA of E. coli hybrid'~ation was only weak (results not shown). (b) Localization of the gene The whole 5A-Md piece of cloned DNA was mapped for HindIlI (with which it has been cloned), BamHi, SalI, PstI, Sinai, BglII, KpnI and Xhol cleavage sites. As demonstrated in Fig. 1 the cloned DNA

consisted of two Hindlll fragments, A and B. It further contained one site for Xhol, two for EcoRI, Pstl and Kpnl, three for Bglll and no sites ior BamHI, Sall and Sinai. The restriction map was obtained by a series of double digestions and electrophoretic analysis in 0.8% agarose gels. The very close proximity of two sites for Bglll and EeoRI cleavage became detectable only in acr~,lamide gels (6%). To localize the gene recAe.m, within the cloned two HindIII fragments parts of the DNA were deleted by HindIII, EcoRI and PstI (Fig. lb, c, d). The intactness of the recAp.re, gene on the fragment was determined by its ability to transform UV-sensitive recA- mutant cells to UV resistance. After separation of the two HindlII fragments, the gene in question seemed to be contained in the larger 3.4.Md (B) fragment, contained in plasmid pPM2 (F~. Ib). However, only partial UV resistance v:ds conferred by pPM2 (see below and Fig. 3). Deletion of the HindIII/EeoRI fragment, i.e. subcloning of the C.fragment (see pPM3; Fig. lc), was found to eliminate the recA gene function completely, indicating that one (or two) EcoRI site is within recA. Since the gene function was retained by the 1.5 Md Pstl.D-fragment (plasmid pPM4; Fig. l d), the gene must be localized in this region. More significant, the rec-gene~ontaining fragment is inserted not only into another restriction site of pPM4, but also with the inverse orientation in relation to pPM1

(Fig. 2).

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4 Fig. 2. Localization of the recAp.m.-Containing fragment in pPMI and pPM4. In pPM1 the gene is contained within a 5o4-Md fragment (heavy lines) that is inserted into the H/ndlll site of pBR322. The HindllI and the EcoRl sites, which cut the recAp.r,. gene, are arranged clockwise. In pPM4, the gene is contained in a 1.5 Md fragment (heavy line), inserted in the Pstl site of pBR322. The orientation of that fragment is counter-clockwise as compared with pPM 1.

(c) UV resistance

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Plasmids pPM 1 and pPM4 confer UV resistance on strain KM 2321, in which A21 deleted theE. colirecA gene (McEntee, 1977a), as well as on strains HB101 and AB2463, which both carry point mutations in the recA gene. Therefore, we conclude that the com. plete structural gene of P. mirabilis has been cloned. Fig. 3 shows UV.survival curves for the rec4 - mutant HB101 containing plasmid pPM1 and its derivatives pPM2 and pPM4. UV resistance becomes almost completely reslored to the wild.type level by pPM1, nearly so by pPM4, but to a significantly lower extent by pPM2. Obviously, the gene is not fully active when the smaller ttindIH.A fragment is deleted. The somewhat irregular shape of the survival curves of HB101 [pPM2] and HB101 [pPM4] are not due to segregat:on of the plasmid; this was e~cluded by testing each of 100 colonies as to their UV resistance.

DISCUSSION

We have cloned a chromosomal fragment of P. mirabilis containing a gene that - when introduced into recA- mutants of E. coli by recombinant plasmids pPMI and pPM4 (Fig. 2) - restores UV survival

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of the recA- mutants to nearly the UV resistance level of the wild type. Consequently, the cloned gene of t". mirabilis called recA~,m, must be expressed in E. coli, ther.eby replacing the E. coli recA gene in repair of UV.~]amaged DNA. The P. mirabil!s origin of the recA gen~ was confirmeJ by DNA-DNA hybridization. The gene was originally cloned in shotgun experiments using partial Hindlll rest.fiction followed by li~tion, and resulting in the recombinant plasmid pPMI, which contains two of the Hindlll fragments (Fig. 1). Since the gene function is impaired by the imernal Hindlll cut, as shown by the lower level of UV resistance conferred by pPM2 (Figs. 1 a~_d 3), both these A and B fragments seem to represent a contiguous piece of the chromosome. The UV inactivation data clearly indicate that the gene straddles the HindlIl and EcoRI sites (Figs. 1-3). As to whether the Hindlll site effects a structural or a regulatory region of the gene could be determined only in more detailed studies. Alternatively one could propose that another gene, located within the HindIIlA-fragment between the Pstl and the second Hindlll site and missing in pPM2, substantially contribute to the UV resistance conferred by pPM1 and pPM4. Although the genetics of the recA gene of E. coli does not provide any hints as to the presence of such a "recA-helper" gene (Clark, 1971; Morand et al., 1977b) we can currently not completely exclude this

possibility for P. mirabilis. The slightly decreased UV resistance conferred by pPM4, as compared with pPMI, appears to be of questionable significance. One may speculate that the Pstl cut slightly affects the regulator region of recAp.m, or the proposed second gene; it seems to be more probable, however, that this small difference in UV resistance is due to the different position and orientation of the cloned fragment in pPM4, ~s compared with pPM1 (Fig. 2). A comparison of the restriction map of the recA gene of L: coli (Sancar and Rupp, 1979; Kuzmin, N.P., Solonin, A.S., Either, G., Glukhov, I.L., Tanyas.hin, V.I., Bayev, A.A., in prep.) with the map ef our cloned P. mirabilis gene (Fig. 4) gives evidence of completely different patterns of restriction sites. If one assumes that the Mr of the recAp.re, protein is similar to that of the recA protein of E. coli, then the protein should be code~ by 1100 to 1200 bp localized mostly rightwards fiom the HindIH site and therefore containing two EcoRI sites. Although the structure of the cloned gene of P. mirabilis differs from that of the recA gene of E. coli, it functionally replaces the recA gene of E. coil in the repair of UV damaged DNA and moreover in general recombination, UV mutagenesis and induction of prophages (Eitner, G., Adler, B., Lanzov, V.I. and Hofemeister, J., in prep.). Therefore, we propose that the cloned gene recAp.re, corresponds to the recA ge~e of E. coll.

307 ACKNOV¢LEDGEMENTS Most o f the experiments were done during a visit o f G. Eitner in the department o f Prof. A. Bayev in Pushchino, USSR. G. Eitner would like to t h a n k Prof. A. Bayev for the s u p p o ~ o f the work. We would further like to t h a n k Drs. L Li, N. Kuzm i n and M. l-Iartmann for the enzymes used, Dr. V. Kryukov for his advice on restriction mapping, Dr. U. Wobus for his guidance and help in performing the DNA hybridization experiments, Dr. B. Adler and Miss J. Dommes, who helped in the UV survival experiments, and Dr. J. Hofemeister and Prof. R. Rieger for critical reading o f the manuscript.

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