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Physical and genetic structure of the IncN plasmid R15
PLASMID
14,99-105 (1985)
Physical and Genetic Structure of the IncN Plasmid R15 A. P. DOBRITSA, TATYANA G. MIKHAILOVA,
AND VERA I. DUBOVAYA
Institute of Applied Microbiology, Serpukhov. Moscow Region, 142200. USSR Received August 7, 1984; revised June 12, 1985 Restriction sites for seven hexanucleotide-specific endonucleaseswere located on the map of the conjugative IncN plasmid RI5 (Sm?WI-Ig, 62.3 kb). The distribution of the cleavage sites is strongly asymmetric. Twenty-eight of thirty-four sites for BarnHI, EcoRI, Hi&II, WI, SmaI, and PstI were located close to or within the sequencesof an 1%like element and the transposons Tn2353 and Tn2354. By analysis of R 15::Tnl756 deletion derivatives and recombinant plasmids harboring R 15 fragments,the genetic determinants for the streptomycin, sulfonamide, and mercury resistanceswere mapped, as well as the regions necessaryfor EcoRII restriction-modification and for plasmid replication and conjugation. The features of physical and genetic structures of the plasmid R15 and other IncN plasmids are discussed. o 1985Academic p, I~C.
R-plasmids of the incompatibility group N (IncN)’ have been detected in strains of many bacterial speciesisolated in different parts of the world. The host range of these plasmids includes many genera of the Enterobacteriaceae,and some IncN plasmids are also transmissible to Pseudomonas. In addition to antibiotic resistance determinants, IncN plasmids often harbor genes encoding DNA restriction and modification activities, and for a system for DNA repair and a specific DNA polymerase. In recent years, the genetic and physical organization of some plasmids determining these functions have been studied (Brown and Willetts, 1981; Ando and Arai, 1981; Langer and Walker, 198I ; Langer et al., 1981; Konarska-Kozlowska and Iyer, 198I ; Thatte and Iyer, 1983). This paper presents a physical and genetic map of the IncN plasmid R 15. This plasmid, isolated from a Proteus vulgaris strain (Watanabe et al., 1964), is conjugative, has a size of about 60 kb, and confers resistance to streptomycin, sulfonamides, and mercury ions.
MATERIALS
AND
METHODS
Bacterial strains andplasmids. Escherichia coli K12 strains used were 553 (pro-met-; Clowes and Rowley, 1954), 802 (met-gatWood, 1966), and C600 (thfleu-rk ml; thi-lac-tonA supE; Appleyard, 1954). The plasmids were R 15 (SuSm’HgVra+; Watanabe et al., 1964), pBR322 (Ap’Tc’; Bolivar et al., 1977), pBR328 (Ap’Tc’Cm’; Soberon et al., 1980), pAD838 (Ap’Km’Tc’Sm’Su’Hg’; Dobritsa, 1984), pAD8 16 and pAD822. The latter two plasmids are R 15 derivatives which haveacquired the transposon Tn17.56(AprTc3 from the plasmid RP4: :Tnl. Tn17.56 is a composite transposon which is flanked by two inverted copies of a Tnl element (Dobritsa et al., 1983). Media. Cultures were grown in L-broth or in A-medium (Miller, 1972) supplemented, if required, with L-amino acids (20 pg/ml) and thiamine (1 pg/ml). To determine bacterial resistance to drugs and mercury, ampicillin (Ap), chloramphenicol (Cm), streptomycin (Sm), sulfanilamide (Su), and HgC12 were added to media at 200, 25, 50, 20, 100, and ’ Abbreviations use& Ap, ampicillin; Cm, chloram- 20 /*g/ml, respectively. Plasmid transfer. Conjugal crosses were phenicol; IncN, incompatibility group N, R-M, restriction and modification; Sm, streptomycin; Su, sulfanilamide. performed as described previously (Dobritsa 99
0147-619X/85 $3.00 Copyright 0 1985 by Academic Press Inc. All rights of reproducnon in any form reserved.
100
DOBRITSA, MIKHAILOVA,
et al., 1981). Bacterial transformation was carried out under conditions recommended by Weston et al. (198 1). Isolation of plasmids. Screening of clones for the presenceof plasmids was accomplished by the procedure of Eckhardt (1978). Small amounts of plasmid DNAs were obtained according to Holmes and Quigley (1981) and Birnboim and Doly ( 1979). More purified and large stocks of plasmid DNAs were prepared by centrifugation in cesium chloride-ethidium bromide gradients. Before equilibrium density-gradient centrifugation, the DNAs from bacterial cells lysed with Triton X-100 (Meagher et al., 1977) were concentrated by precipitation with polyethylene glycol 6000 (Humphreys et al., 1975). Restriction analysis of plasmids. Restriction-enzyme digestsand gel electrophoresis of DNAs were performed as described previously (Dobritsa et al., 1978, 1980). EcoRI, HindUI, and EcoRI-Hind111 fragments of phage X DNA were usedasmolecular-weight standards (Sanger et al., 1982). Construction of recombinant plasmids and plasmid derivatives. The DNA of R 15 and of a vector plasmid, either pBR322 or pBR328, were cleaved by the appropriate restriction enzymes. The digested DNAs were mixed in equimolar proportions and ligated. The ligation mixture contained 50 mM Tris-HCl (pH 7.5) 10 mM MgC12, 10 mM dithiothreitol, 0.5 mM ATP, DNA (20 to 40 pg/ml), and T4 DNA ligase (0.5 to 1 units/ml). Ligation was carried out at 4°C for 4 to 6 h, after which the mixture was diluted by a factor of 5 to 10 and the reaction continued for 12 to 18 h. When constructing the deletion derivatives of R 15, pAD8 16, and pAD822, we used the sameprocedure to ligate restriction fragments of these plasmids. The ligated DNAs were used to transform Escherichia coli 802 cells. The transformants carrying hybrid plasmids were detected by their Ap’T? or Ap’Tc%mS phenotypes using pBR322 or pBR328, respectively, as vectors. The bacterial cells harboring deletion plasmids were selected on plates containing Ap, Sm, Su, or HgCl* .
AND DUBOVAYA
Determination of modification and restriction phenotype of transformants. Restriction-
modification genesin plasmids were detected by determining the efficiency of modified and unmodified phage X plating on plasmidless and plasmid-containing E. coli 802 cells. The details of the procedure were as given by Clowes and Hayes (1968). RESULTS
R15 Restriction Map
R15 DNA is cleaved with Bg/II, HindIII, EcoRI, BamHI, SalI, SmaI, and PstI into 9, 11, 6, 5, 4, 4, and 4 fragments, respectively. The cleavagesitesfor theseendonucleaseswere mapped by single, double, triple, and partial digests of RI 5 DNA. For some BgfiI and Hind111sites,further information was deduced from the restriction analysis of recombinant plasmids containing R 15 DNA fragments. The physical map of the RI 5 plasmid is shown in Fig. 1. The plasmid size calculated as the sum of the sizesof restriction fragments is 62.3 kb. Localization of the Drug-Resistance Determinants
When constructing recombinant plasmids by ligation of BamHI fragments of RI 5 to
50
FIG. 1. Restriction map of the plasmid R 15. The map is calibrated in kb, from one arbitrarily chosen EcoRI site as zero coordinate.
IncN PLASMID R15 STRUCTURE
BarnHI-cleaved pBR322, the fragment extending from the R15 coordinate 29.65 to 33.3 kb was found to carry genes for resistance to Sm and Su. The hybrid plasmids carrying the BamHI-EcoRI segments29.65 to 3 1.75 kb or 31.75 to 33.3 kb and plasmids harboring EcoRI fragments 29.65 to 31.75 kb or 31.75 to 38.7 kb did not confer resistance to Sm. However, E. coli cells carrying recombinant plasmids incorporating the 26.25 to 3 1.75 kb or 29.65 to 31.75 kb regions of R15 were resistant to Su. Thus, the Sm’ gene sequenceis cleaved by EcoRI at coordinate 3 1.75 kb and the sulfonamide resistance determinant is located within the BumHI-EcoRI fragment 29.65 to 3 1.75 kb adjoining this site. Localization of the Hg’ Gene Among hybrid plasmids incorporating SalI fragments of R 15, only those harboring a fragment with coordinates 11.7 to 28.8 kb determine resistance to HgC&. Plasmids carrying EcoRI fragments 23.6 to 26.25 kb or 26.25 to 3 1.75 kb inserted into EcoRI-cleaved pBR328 lack the active Hg’ gene. Mercury resistance is not determined by recombinant plasmids carrying the pAD8 16 BamHI-EcoRI fragments 18.2 to 23.6 kb and 26.25 to 29.65 kb either (seeFig. 2). However, E. coli cells harboring a plasmid carrying the EcoRI-BamHI fragment 23.6 to 29.65 kb inserted into pBR322 were resistant to Hg. Thus, the Hg’ gene sequence appears to include the EcoRI site at 26.25 kb. Mapping of the Determinants for EcoRII Restriction-Modification The plasmid R 15 and other IncN plasmids determine HspII restriction and modification (R-M) specificity (Hedges, 1972). Our data on the efficiency of X phage plating and the analysis of digestion of unmodified pBR322 DNA with a restriction endonuclease isolated from E. coli 553 (R 15) cells confirmed that R 15 also
101
FIG. 2. Linear maps of R 15: :Tnl756 plasmids, pAD8 16 and pAD822, and their deletion derivatives. Coordinates in pAD816 and pAD822 correspond to those of R15 indicated in Fig. 1. In these maps, the Tn1756 region is drawn above the lines representing the R15 sequencesand is calibrated independently. The segment of Tnl756 retained in deletion derivatives is delineated by a thick line; the thin lines representR 15regions.Note that the pAD8225 region from 6.9 to 10.55 kb and the pAD822-2 region from 0 to 3.2 kb are inverted and correspond to the R15 sequencesfrom 33.3 to 29.65 kb and from 33.3 to 30.1 kb, respectively. Discontinuities indicated by broken lines, and as unfilled gapsrepresentdeletions. Restriction enzyme symbols shown at deletion sites and at sites of plasmid linearization represent “half sites.” Abbreviations: B, BumHI; Bg, &/II; E, EcoRI; Sa, S&I.
encoded EcoRII restriction-modification. The unmodified infecting phage was also restricted in E. coli 802 cells carrying a hybrid plasmid incorporating the Hg’ &z/I fragment of RI 5 with coordinates 11.7 to 28.8 kb. The plasmid pAD8 16 also determines the R-M activity, but restriction and modification of the bacteriophage did not occur in an E. coli strain carrying a plasmid constructed by cyclization of a BumHI fragment of pAD8 16 with the coordinates 44.45 to 18.2 kb, nor in an E. coli strain harboring pAD838 (RP4::Tnl::Tn2353, Dobritsa, 1984).In R15, Tn2353 extends from coordinates 23.55 to 36.6 kb. These results demonstrate that the genes of the R-M system map between coordinates 18.2 and 23.55 kb. This conclusion was confirmed by data on phage restriction and modification in E. coli cells harboring a 6.8-kb BamHI-EcoRI fragment of pAD816 of coordinates 18.2 to 23.6 kb cloned into pBR322.
102
DDBRITSA, MIKHAILOVA,
Determination of the Position of the Replication Genes
AND DUBOVAYA
sulted in clones carrying plasmids which composed of two BgfiI fragments. One of these plasmids is named pAD822-4 (Fig. 2). For the The 23.55 to 44.5 kb region of R15 carries same purpose, pAD822-5 was cleaved with the transposons Tn2353 23.55 to 36.6 kb BgfiI and BarnHI, and then ligated and trans(Dobritsa, 1984) and Tn2354 36.7 to 44.5 kb formed into E. coli 802. [Both Sg/II and (Dobritsa and Dergunova, 1984). The se- BamHI fragments have identical cohesiveends quence between the transposons is overlapped (Roberts, 1983).] The transformants were seby the Sm’Su’ SalI fragment at 29.45 to 42.55 lected on media containing either Ap or Sm. kb which fails to self-replicate. Thus, the rep The structure of plasmids pAD822-3 and genes appear to be located in the other RI 5 pAD822-2 isolated from Ap’ and Sm’ transregion which unfortunately, lacks determi- formants, respectively, is shown in Fig. 2. nants that can be used as markers. Therefore, Comparison of the structures of pAD822-2, the RI 5 derivatives, pAD8 16 and pAD822, pAD822-3, pAD822-4, and other R 15 derivcarrying the transposon Tnl756 integrated atives indicates that the R 15 BgfiI fragment into sites at the coordinates 18.2 and 5.5 kb, extending from a coordinate 46.0 to 53.7 kb respectively (Fig. 2), were chosen as a source carries genetic elements necessary for R15 of R 15 deletions. Due to the locations of the replication. BamHI sites and the Apr genes,TnZ 756 may be used to construct Ap’ minireplicons incor- Mapping of the R15 Conjugation Genes porating BamHI fragments located either side When studying the transfer ability of the of the transposon. pAD8 16 and pAD822 deletion derivatives deThe Ap’ clones of E. coli 802 cells transscribed above, only pAD816-7 was shown to formed with pAD8 16 or pAD822 DNAs be transferred from E. coli 802 to E. coli C600 treated with BamHI and T4 DNA ligase were by conjugation. Thus, the tra genesare located found to harbor only plasmids generated by between coordinates 44.45 and 18.2 kb. The cyclization of the fragments containing the positions of theseand other R 15genesmapped R15 sequencesfrom coordinates 44.45 to 18.2 by us are shown in Fig. 3. kb and 44.45 to 5.5 kb, respectively. The plasmids were designatedpAD8 16-7and pAD8226. Plasmids consisting of two BamHI fragments, 44.45 to 5.5 kb and 29.65 to 33.3 kb, were isolated from Sm’ clones selected after transformation of E. coli 802 with ligated BamHI fragments of pAD822. The structure of one of these plasmids designated pAD8225 is shown in Fig. 2. Thus, the plasmid pAD822-6 is the minimal replicon obtained by ligation of the BamHIcleaved pAD816 or pAD822 DNAs, and its R 15 sequence is common to all the deletion derivatives constructed. Hence, the R 15 rep genesappear to be located only in this region. Since it has several targets for BgfiI, pAD8223. Genetic and physical map of the plasmid R15. 6 DNA was digested with Bg/II, then ligated TheFIG. sequencesof Tn2353, Tn2354, and the IS element and transformed into E. coli 802 cells to map (presumably IS5) are shown by heavy lines. Abbreviations: the determinants of R15 replication more B, BumHI; Bg, &$I; E, EC&I; H, HirzdIII; S, SmuI; Sa, precisely. Ap’ selection of transformants re- SaA; P, PstI; X, XhoI.
IncN PLASMID R15 STRUCTURE
DISCUSSION
In this paper, we report the results of restriction enzyme and genetic mapping of the IncN plasmid R15 (Fig. 3). Comparisons of the R 15 map with those of other IncN plasmids illustrate the evolution of this plasmid group. Maps of N3, pCU1, R46, and the R46 derivative, pKM 101, have previously been reported (Ando and Arai, 1981; Konarska-Kozlowska and Iyer, 1981; Thatte and Iyer, 1983; Brown and Willetts, 1981; Brown et al., 1984; Ianger and Walker, 1981; Langer et al., 1981). The sulfonamide resistancedeterminant of R 15 maps on a BamHI-EcoRI fragment that carries &$I, PstI, and Hind111sites. A similar restriction pattern of BarnHI, Bg/II, PstI, and Hind111 sites was observed in the Su’ regions of R46 and N3 (Brown and Willetts, 1981; Ando and Arai, 1981) and of the IncW plasmids R388 and Sa (Ward and Grinsted, 1978; Brown and Willetts, 1981). The analogous distribution of BglII, PstI, and Hind111 sites has also been found in Su’ genesof TnZZ-like elements (Kratz et al., 1983). All these Su’ determinants are thus likely to be homologous. Streptomycin resistance determinants in R 15 and N3 may also be related or identical. Both include an EcoRI site and are located close to the Su’ gene. In R 15, the Sm’ and Su’ determinants are part of the transposon Tn2353 (Dobritsa, 1984) which, in addition, harbors the Hg’ gene.The structure of one of Tn2353 terminal regions is indistinguishable from that of the left-end sequencesof Hg’ transposons Tn2608 and Tn2613 (Tanaka et al., 1983). These regions carry a mercury-resistance gene, one Hind111 and two EcoRI sites, and one of the EcoRI sites is near or in the terminal repeat of the elements Tn2608 and Tn2613. This similarity in Tn2353, Tn2608, and Tn2613 may have resulted from the insertion (transposition) of identical or similar sequencescarrying the Hg’ gene into ancestral transposons. The formation of a Hg’ transposon by the translocation of a Tndl-like structure has been demonstrated by Grinsted and Brown ( 1984).
103
An alternative explanation is that Tn2353, Tn2608, and Tn2613 are related mobile elements, which have evolved from a common ancestral transposon. The Tn2353 sequence borders on a region of R 15 which carries EcoRII restriction-modification activity corresponding to that of N3. However, the N3 genes carry BamHI, PstI, and Sall sites (Ando and Arai, 1981; Brown et al., 1984). The rep genesof R 15 were mapped to a 7.7kb BglII segment which carries only two Hind111 sites, whereas N3 carries SalI sites (Ando and Arai, 1981). In R46 and pCU 1, the rep genesalso have no SalI, BamHI, and PstI sites (Brown and Willetts, 1981; Thatte and Iyer, 1983). R46 and pCU 1 carry tra regions which have close similarities in the location of restriction sites (Langer et al., 1981; Brown and Willetts, 1981; Konarska-Kozlowska et al., 1983), but those of the tra sequence on N3 differs from those of R46 and pCU1 or R15 (Ando and Arai, 1981; Brown et al., 1984). In R46 and pCU1, the tra and rep clusters are separated by relatively long DNA sequencesencoding other functions, whereasin R 15 and N3, these determinants are located close to one another to form a “core” structure. The IncN plasmids N3, R46, pCU1 and RI 5 differ in molecular weight, the number and locations of restriction sites, and phenotype aswell asin the arrangement of the genes, but sharecommon features in the nonrandom distribution of restriction sites. Those regions carrying replication and conjugation genesessential for plasmid maintenance and transfer carry relatively few restriction sites, whereas the overwhelming majority of these sites are located close to, or within, the regions containing the antibiotic-resistance determinants. In particular, 28 of 34 sites for BamHI, EcoRI, HindIII, SalI, SmaI, and PstI cleavageare localized in a Tn2353- and Tn2354- containing 23-kb region of the 62.3-kb plasmid R 15. In this region, we have recently found an insertion sequence, which is similar or identical to IS5 by its size and the distribution of
104
DOBRITSA, MIICHAILOVA,
AND DUBOVAYA
T. G. (1984). Detection and characterization of an IS EcoRI, PvuII, and Bg/II sites (Dobritsa et al., element in the structure of the plasmid R 15.In “Hybrid 1984). It is located between coordinates 45.0 plasmids and geneexpression” (I. V. Domaradskii, ed.), and 46.2 kb (Fig. 3). pp. 8 1-83. NCBI AN SSSR,Pushchino. [in Russian] These data support the hypothesis that one DOBRIA, A. P., D~BRI’ISA, S. V., POPOV,E. I., AND of the consequencesof evolution of wide-hostFEDOSEEVA,V. B. (1981). Transposition of a DNA fragment flanked by two inverted Tnl sequences.Gene range plasmids is a decreasing number of tar14,2 17-225. getsfor restriction endonucleases,whereasthe A. P., DOBRITSA,S. V., AND TANYASHIN, great number of cleavage sites in plasmid re- DOBRITSA, V. I. (I 978). Isolation and characterization of plasmid gions carrying resistancedeterminants may be from the Bacillus brevis var.G.-B. c&s. Mol. Gen. Genet. due to relatively recent events in which acquis164, 195-204. ition of these genes has occurred by transpo- D~BRITSA, A. P., IVANOVA, Z. A., AND FEWGEEVA, V. B. (1983). Transposition of DNA fragments flanked sition or otherwise.
ACKNOWLEDGMENTS The authors are grateful to Irina Filonova for her help in performing some stagesof the work and to Dr. V. M. Kramarov for determination of the restriction enzyme activity in the extracts of the E. coli cells.
REFERENCES ANDO, T., AND ARAI, T. (1981). Genetic structure of the IncN plasmid N3. Plasmid 6,293-30 1. APPLEYARD,R. K. (1954). Segregation of new lysogenic types during growth of a doubly lysogenic strain from E. coli K-12. Genetics 39,440-452. BIRNBOIM,H. C., AND DOLY, J. (1979). A rapid alkaline extraction procedure for screeningrecombinant plasmid DNA. Nucleic Acids Res. 7, I5 13-l 523. BOLIVAR,F., RODRIGUEZ,R., GREENE,P. J., BETLACH, M., HEYNEKER,H. L., BOYER,H. W., CROSA,J., AND FALKOW,S. (1977). II. Construction and characterization of new cloning vehicles. A multipurpose cloning system. Gene 2,95-l 13. BROWN,A. M., COUPLAND,G. M., ANDWILLE~S, N. S. (1984). Characterization of IS&, an insertion sequence found on two IncN plasmids. J. Bacterial. 159, 472481. BROWN,A. M. C., ANDWILLE-IX, N. S. ( 1981).A physical and genetic map of the IncN plasmids R46. Plasmid 5, 188-201. CLOWES,R. C., AND HAYES, W. (eds.). (1968). “Experiments in Microbial Genetics.” Blackwell, Oxford/Edinburg. CLOWES,R. C., AND ROWLEY,D. (1954). Some observations on linkage effects in genetic recombination in Escherichia coli K12. J. Gen. Microbial. l&250-260. DOBRITSA,A. P. (1984). IS8- and Tn2353-mediated cointegration of the plasmids R 15 and RP4: :Tnl. Mol. Gen. Genet. 194,206-2 10. DOBRITSA,A. P., ANDDERGUNOVA,L. V. (1984). Mobile genetic elements of the plasmid R 15.Dokl. Akad. Nauk SSSR, Ser. Biol. 271,980-983. [in Russian] DOBRITSA,A. P., DERGUNOVA,L. V., ANDMIKHAILOVA,
by two inverted Tnl sequences:translocation of the plasmid RP4::Tnl region harboring the Tc’ marker. Gene 22,237-243. D~BRITSA, A. P., K.SENZENKO, V. N., FEDOSEEVA, V. B., ALEXANDROV,A. A., KAMYNINA, T. P., ANDKHMELNITSKY,M. I. (1980). Isolation oftransposon Tn4 from plasmid RP4 carrying two copies of this element. Gene 8, 153-162. ECKHARDT,T. (1978). A rapid method for the identification of plasmid deoxyribonucleic acid in bacteria. Plasmid 1, 584-588. GRINSTED,J., ANDBROWN,N. L. (1984).A Tn2l terminal sequencewithin Tn501: Complementation of tnpA gene function and transposon evolution. Mol. Gen. Genet. 197,497-502.
HEDGES,R. W. (1972). Phenotypic characterization of fiR factors determining the restriction and modification HsplI specificity. Mol. Gen. Genet. 115, 225-233. HOLMES,D. S., ANDQUIGLEY,M. (198 I). A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. 114, 193-197. HUMPHREY%G. O., WILLSHAW,G. A., AND ANDERSON, E. C. (1975). A simple method for the preparation of large quantities of pure plasmid DNA. B&him. Biophys. Acta 383,457-463. KONARSKA-KOZLOWSKA, M., AND IYER, V. N. (1981). Physical and genetic organization of the IncN group plasmid pCU 1. Gene 14, 195-204. KONARSKA-KOZLOWSKA, M., THA~E, V., AND IYER, V. N. (1983). Inverted repeats visualized in strands of the DNA of plasmid pCU I. J. Bacterial. 153, 15021512. KRATZ, J., SCHMIDT,F., AND WIEDEMANN,B. (1983). Characterization of Tn2411 and Tn2410, two transposons derived from R-plasmid R1767 and related to Tn2603 and Tn21. J. Bacterial. 155, 1333-1342. LANCER, P. J., SHANABRUCH,W. G., AND WALKER, G. C. (1981). Functional organization of plasmid pKMlOI. J. Bacterial. 145, 1310-1316. LANCER, P. J., AND WALKER, G.C. (1981). Restriction endonuclease cleavage map of pKM IO1: Relationship to parental plasmid R46. Mol. Gen. Genet. 182, 268272. MEAGHER,R. B., TAIT, R. C., BETLACH,M., ANDBOYER,
IncN PLASMID Rl5 STRUCTURE H. W. (1977). Protein expression in E. coli minicells by recombinant plasmids. Cell 10, 521-536. MILLER, J. H. (1972). “Experiments in Molecular Genetics.” Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. ROBERTS,R. J. (1983). Restriction and modification enzymes and their recognition sequences.Nucleic Acids Rex 11, rl35-r167. SANGER,F., COULSON,A. R., HONG, G. F., HILL, D. F., AND PETERSON,C. B. (I 982). Nucleotide sequence of bacteriophage X DNA. J. Mol. Biol. 162, 729-773. SOBERON, X., COVARRUBIAS, L., ANDBOLIVAR,F. ( 1980). Construction and characterization of new cloning vehicles. IV. Deletion derivatives of pBR322 and pBR325. Gene 9,287-305. TANAKA, M., YAMAMOTO, T., AND SAVAI, T. (1983). Evolution of complex resistance transposons from an ancestral mercury transposon. J. Bucteriol. 153, 14321438.
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THA’ITE, V., ANDIYER,V. N. ( 1983).Cloning of a plasmid region specifying the system of bacterial conjugation. Gene 21,227-236. WARD, J. M., AND GRINSTED,J. (1978). Mapping of functions in the R-plasmid R388 by examination of deletion mutants generated in vitro. Gene 3, 87-95. WATANABE,T., NISHIDA, H., OGATA,C., ARAI, T., AND SATO,S. ( 1964).Episome-mediated transfer of drug resistance in Enterobacteriaceae. VII. Two types of naturally occurring R factors. J. Bucteriol. 88, 7 16-726. WESTON,A., BROWN,M. G. M., PERKINS,H. R., SAUNDERS,J. R., AND HUMPHREYS,G. 0. (1981). Transformation of Escherichiu coli with plasmid deoxyribonucleic acid: Calcium-induced binding of deoxyribonucleic acid to whole cells and to isolated membrane fractions. J. Bacterial. 145, 780-787. WOOD,W. B. (1966). Host specificity of DNA produced by E. coli: Bacterial mutations affecting the restriction and modification of DNA. J. Mol. Biol. 16. 118-133.
14,99-105 (1985)
Physical and Genetic Structure of the IncN Plasmid R15 A. P. DOBRITSA, TATYANA G. MIKHAILOVA,
AND VERA I. DUBOVAYA
Institute of Applied Microbiology, Serpukhov. Moscow Region, 142200. USSR Received August 7, 1984; revised June 12, 1985 Restriction sites for seven hexanucleotide-specific endonucleaseswere located on the map of the conjugative IncN plasmid RI5 (Sm?WI-Ig, 62.3 kb). The distribution of the cleavage sites is strongly asymmetric. Twenty-eight of thirty-four sites for BarnHI, EcoRI, Hi&II, WI, SmaI, and PstI were located close to or within the sequencesof an 1%like element and the transposons Tn2353 and Tn2354. By analysis of R 15::Tnl756 deletion derivatives and recombinant plasmids harboring R 15 fragments,the genetic determinants for the streptomycin, sulfonamide, and mercury resistanceswere mapped, as well as the regions necessaryfor EcoRII restriction-modification and for plasmid replication and conjugation. The features of physical and genetic structures of the plasmid R15 and other IncN plasmids are discussed. o 1985Academic p, I~C.
R-plasmids of the incompatibility group N (IncN)’ have been detected in strains of many bacterial speciesisolated in different parts of the world. The host range of these plasmids includes many genera of the Enterobacteriaceae,and some IncN plasmids are also transmissible to Pseudomonas. In addition to antibiotic resistance determinants, IncN plasmids often harbor genes encoding DNA restriction and modification activities, and for a system for DNA repair and a specific DNA polymerase. In recent years, the genetic and physical organization of some plasmids determining these functions have been studied (Brown and Willetts, 1981; Ando and Arai, 1981; Langer and Walker, 198I ; Langer et al., 1981; Konarska-Kozlowska and Iyer, 198I ; Thatte and Iyer, 1983). This paper presents a physical and genetic map of the IncN plasmid R 15. This plasmid, isolated from a Proteus vulgaris strain (Watanabe et al., 1964), is conjugative, has a size of about 60 kb, and confers resistance to streptomycin, sulfonamides, and mercury ions.
MATERIALS
AND
METHODS
Bacterial strains andplasmids. Escherichia coli K12 strains used were 553 (pro-met-; Clowes and Rowley, 1954), 802 (met-gatWood, 1966), and C600 (thfleu-rk ml; thi-lac-tonA supE; Appleyard, 1954). The plasmids were R 15 (SuSm’HgVra+; Watanabe et al., 1964), pBR322 (Ap’Tc’; Bolivar et al., 1977), pBR328 (Ap’Tc’Cm’; Soberon et al., 1980), pAD838 (Ap’Km’Tc’Sm’Su’Hg’; Dobritsa, 1984), pAD8 16 and pAD822. The latter two plasmids are R 15 derivatives which haveacquired the transposon Tn17.56(AprTc3 from the plasmid RP4: :Tnl. Tn17.56 is a composite transposon which is flanked by two inverted copies of a Tnl element (Dobritsa et al., 1983). Media. Cultures were grown in L-broth or in A-medium (Miller, 1972) supplemented, if required, with L-amino acids (20 pg/ml) and thiamine (1 pg/ml). To determine bacterial resistance to drugs and mercury, ampicillin (Ap), chloramphenicol (Cm), streptomycin (Sm), sulfanilamide (Su), and HgC12 were added to media at 200, 25, 50, 20, 100, and ’ Abbreviations use& Ap, ampicillin; Cm, chloram- 20 /*g/ml, respectively. Plasmid transfer. Conjugal crosses were phenicol; IncN, incompatibility group N, R-M, restriction and modification; Sm, streptomycin; Su, sulfanilamide. performed as described previously (Dobritsa 99
0147-619X/85 $3.00 Copyright 0 1985 by Academic Press Inc. All rights of reproducnon in any form reserved.
100
DOBRITSA, MIKHAILOVA,
et al., 1981). Bacterial transformation was carried out under conditions recommended by Weston et al. (198 1). Isolation of plasmids. Screening of clones for the presenceof plasmids was accomplished by the procedure of Eckhardt (1978). Small amounts of plasmid DNAs were obtained according to Holmes and Quigley (1981) and Birnboim and Doly ( 1979). More purified and large stocks of plasmid DNAs were prepared by centrifugation in cesium chloride-ethidium bromide gradients. Before equilibrium density-gradient centrifugation, the DNAs from bacterial cells lysed with Triton X-100 (Meagher et al., 1977) were concentrated by precipitation with polyethylene glycol 6000 (Humphreys et al., 1975). Restriction analysis of plasmids. Restriction-enzyme digestsand gel electrophoresis of DNAs were performed as described previously (Dobritsa et al., 1978, 1980). EcoRI, HindUI, and EcoRI-Hind111 fragments of phage X DNA were usedasmolecular-weight standards (Sanger et al., 1982). Construction of recombinant plasmids and plasmid derivatives. The DNA of R 15 and of a vector plasmid, either pBR322 or pBR328, were cleaved by the appropriate restriction enzymes. The digested DNAs were mixed in equimolar proportions and ligated. The ligation mixture contained 50 mM Tris-HCl (pH 7.5) 10 mM MgC12, 10 mM dithiothreitol, 0.5 mM ATP, DNA (20 to 40 pg/ml), and T4 DNA ligase (0.5 to 1 units/ml). Ligation was carried out at 4°C for 4 to 6 h, after which the mixture was diluted by a factor of 5 to 10 and the reaction continued for 12 to 18 h. When constructing the deletion derivatives of R 15, pAD8 16, and pAD822, we used the sameprocedure to ligate restriction fragments of these plasmids. The ligated DNAs were used to transform Escherichia coli 802 cells. The transformants carrying hybrid plasmids were detected by their Ap’T? or Ap’Tc%mS phenotypes using pBR322 or pBR328, respectively, as vectors. The bacterial cells harboring deletion plasmids were selected on plates containing Ap, Sm, Su, or HgCl* .
AND DUBOVAYA
Determination of modification and restriction phenotype of transformants. Restriction-
modification genesin plasmids were detected by determining the efficiency of modified and unmodified phage X plating on plasmidless and plasmid-containing E. coli 802 cells. The details of the procedure were as given by Clowes and Hayes (1968). RESULTS
R15 Restriction Map
R15 DNA is cleaved with Bg/II, HindIII, EcoRI, BamHI, SalI, SmaI, and PstI into 9, 11, 6, 5, 4, 4, and 4 fragments, respectively. The cleavagesitesfor theseendonucleaseswere mapped by single, double, triple, and partial digests of RI 5 DNA. For some BgfiI and Hind111sites,further information was deduced from the restriction analysis of recombinant plasmids containing R 15 DNA fragments. The physical map of the RI 5 plasmid is shown in Fig. 1. The plasmid size calculated as the sum of the sizesof restriction fragments is 62.3 kb. Localization of the Drug-Resistance Determinants
When constructing recombinant plasmids by ligation of BamHI fragments of RI 5 to
50
FIG. 1. Restriction map of the plasmid R 15. The map is calibrated in kb, from one arbitrarily chosen EcoRI site as zero coordinate.
IncN PLASMID R15 STRUCTURE
BarnHI-cleaved pBR322, the fragment extending from the R15 coordinate 29.65 to 33.3 kb was found to carry genes for resistance to Sm and Su. The hybrid plasmids carrying the BamHI-EcoRI segments29.65 to 3 1.75 kb or 31.75 to 33.3 kb and plasmids harboring EcoRI fragments 29.65 to 31.75 kb or 31.75 to 38.7 kb did not confer resistance to Sm. However, E. coli cells carrying recombinant plasmids incorporating the 26.25 to 3 1.75 kb or 29.65 to 31.75 kb regions of R15 were resistant to Su. Thus, the Sm’ gene sequenceis cleaved by EcoRI at coordinate 3 1.75 kb and the sulfonamide resistance determinant is located within the BumHI-EcoRI fragment 29.65 to 3 1.75 kb adjoining this site. Localization of the Hg’ Gene Among hybrid plasmids incorporating SalI fragments of R 15, only those harboring a fragment with coordinates 11.7 to 28.8 kb determine resistance to HgC&. Plasmids carrying EcoRI fragments 23.6 to 26.25 kb or 26.25 to 3 1.75 kb inserted into EcoRI-cleaved pBR328 lack the active Hg’ gene. Mercury resistance is not determined by recombinant plasmids carrying the pAD8 16 BamHI-EcoRI fragments 18.2 to 23.6 kb and 26.25 to 29.65 kb either (seeFig. 2). However, E. coli cells harboring a plasmid carrying the EcoRI-BamHI fragment 23.6 to 29.65 kb inserted into pBR322 were resistant to Hg. Thus, the Hg’ gene sequence appears to include the EcoRI site at 26.25 kb. Mapping of the Determinants for EcoRII Restriction-Modification The plasmid R 15 and other IncN plasmids determine HspII restriction and modification (R-M) specificity (Hedges, 1972). Our data on the efficiency of X phage plating and the analysis of digestion of unmodified pBR322 DNA with a restriction endonuclease isolated from E. coli 553 (R 15) cells confirmed that R 15 also
101
FIG. 2. Linear maps of R 15: :Tnl756 plasmids, pAD8 16 and pAD822, and their deletion derivatives. Coordinates in pAD816 and pAD822 correspond to those of R15 indicated in Fig. 1. In these maps, the Tn1756 region is drawn above the lines representing the R15 sequencesand is calibrated independently. The segment of Tnl756 retained in deletion derivatives is delineated by a thick line; the thin lines representR 15regions.Note that the pAD8225 region from 6.9 to 10.55 kb and the pAD822-2 region from 0 to 3.2 kb are inverted and correspond to the R15 sequencesfrom 33.3 to 29.65 kb and from 33.3 to 30.1 kb, respectively. Discontinuities indicated by broken lines, and as unfilled gapsrepresentdeletions. Restriction enzyme symbols shown at deletion sites and at sites of plasmid linearization represent “half sites.” Abbreviations: B, BumHI; Bg, &/II; E, EcoRI; Sa, S&I.
encoded EcoRII restriction-modification. The unmodified infecting phage was also restricted in E. coli 802 cells carrying a hybrid plasmid incorporating the Hg’ &z/I fragment of RI 5 with coordinates 11.7 to 28.8 kb. The plasmid pAD8 16 also determines the R-M activity, but restriction and modification of the bacteriophage did not occur in an E. coli strain carrying a plasmid constructed by cyclization of a BumHI fragment of pAD8 16 with the coordinates 44.45 to 18.2 kb, nor in an E. coli strain harboring pAD838 (RP4::Tnl::Tn2353, Dobritsa, 1984).In R15, Tn2353 extends from coordinates 23.55 to 36.6 kb. These results demonstrate that the genes of the R-M system map between coordinates 18.2 and 23.55 kb. This conclusion was confirmed by data on phage restriction and modification in E. coli cells harboring a 6.8-kb BamHI-EcoRI fragment of pAD816 of coordinates 18.2 to 23.6 kb cloned into pBR322.
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DDBRITSA, MIKHAILOVA,
Determination of the Position of the Replication Genes
AND DUBOVAYA
sulted in clones carrying plasmids which composed of two BgfiI fragments. One of these plasmids is named pAD822-4 (Fig. 2). For the The 23.55 to 44.5 kb region of R15 carries same purpose, pAD822-5 was cleaved with the transposons Tn2353 23.55 to 36.6 kb BgfiI and BarnHI, and then ligated and trans(Dobritsa, 1984) and Tn2354 36.7 to 44.5 kb formed into E. coli 802. [Both Sg/II and (Dobritsa and Dergunova, 1984). The se- BamHI fragments have identical cohesiveends quence between the transposons is overlapped (Roberts, 1983).] The transformants were seby the Sm’Su’ SalI fragment at 29.45 to 42.55 lected on media containing either Ap or Sm. kb which fails to self-replicate. Thus, the rep The structure of plasmids pAD822-3 and genes appear to be located in the other RI 5 pAD822-2 isolated from Ap’ and Sm’ transregion which unfortunately, lacks determi- formants, respectively, is shown in Fig. 2. nants that can be used as markers. Therefore, Comparison of the structures of pAD822-2, the RI 5 derivatives, pAD8 16 and pAD822, pAD822-3, pAD822-4, and other R 15 derivcarrying the transposon Tnl756 integrated atives indicates that the R 15 BgfiI fragment into sites at the coordinates 18.2 and 5.5 kb, extending from a coordinate 46.0 to 53.7 kb respectively (Fig. 2), were chosen as a source carries genetic elements necessary for R15 of R 15 deletions. Due to the locations of the replication. BamHI sites and the Apr genes,TnZ 756 may be used to construct Ap’ minireplicons incor- Mapping of the R15 Conjugation Genes porating BamHI fragments located either side When studying the transfer ability of the of the transposon. pAD8 16 and pAD822 deletion derivatives deThe Ap’ clones of E. coli 802 cells transscribed above, only pAD816-7 was shown to formed with pAD8 16 or pAD822 DNAs be transferred from E. coli 802 to E. coli C600 treated with BamHI and T4 DNA ligase were by conjugation. Thus, the tra genesare located found to harbor only plasmids generated by between coordinates 44.45 and 18.2 kb. The cyclization of the fragments containing the positions of theseand other R 15genesmapped R15 sequencesfrom coordinates 44.45 to 18.2 by us are shown in Fig. 3. kb and 44.45 to 5.5 kb, respectively. The plasmids were designatedpAD8 16-7and pAD8226. Plasmids consisting of two BamHI fragments, 44.45 to 5.5 kb and 29.65 to 33.3 kb, were isolated from Sm’ clones selected after transformation of E. coli 802 with ligated BamHI fragments of pAD822. The structure of one of these plasmids designated pAD8225 is shown in Fig. 2. Thus, the plasmid pAD822-6 is the minimal replicon obtained by ligation of the BamHIcleaved pAD816 or pAD822 DNAs, and its R 15 sequence is common to all the deletion derivatives constructed. Hence, the R 15 rep genesappear to be located only in this region. Since it has several targets for BgfiI, pAD8223. Genetic and physical map of the plasmid R15. 6 DNA was digested with Bg/II, then ligated TheFIG. sequencesof Tn2353, Tn2354, and the IS element and transformed into E. coli 802 cells to map (presumably IS5) are shown by heavy lines. Abbreviations: the determinants of R15 replication more B, BumHI; Bg, &$I; E, EC&I; H, HirzdIII; S, SmuI; Sa, precisely. Ap’ selection of transformants re- SaA; P, PstI; X, XhoI.
IncN PLASMID R15 STRUCTURE
DISCUSSION
In this paper, we report the results of restriction enzyme and genetic mapping of the IncN plasmid R15 (Fig. 3). Comparisons of the R 15 map with those of other IncN plasmids illustrate the evolution of this plasmid group. Maps of N3, pCU1, R46, and the R46 derivative, pKM 101, have previously been reported (Ando and Arai, 1981; Konarska-Kozlowska and Iyer, 1981; Thatte and Iyer, 1983; Brown and Willetts, 1981; Brown et al., 1984; Ianger and Walker, 1981; Langer et al., 1981). The sulfonamide resistancedeterminant of R 15 maps on a BamHI-EcoRI fragment that carries &$I, PstI, and Hind111sites. A similar restriction pattern of BarnHI, Bg/II, PstI, and Hind111 sites was observed in the Su’ regions of R46 and N3 (Brown and Willetts, 1981; Ando and Arai, 1981) and of the IncW plasmids R388 and Sa (Ward and Grinsted, 1978; Brown and Willetts, 1981). The analogous distribution of BglII, PstI, and Hind111 sites has also been found in Su’ genesof TnZZ-like elements (Kratz et al., 1983). All these Su’ determinants are thus likely to be homologous. Streptomycin resistance determinants in R 15 and N3 may also be related or identical. Both include an EcoRI site and are located close to the Su’ gene. In R 15, the Sm’ and Su’ determinants are part of the transposon Tn2353 (Dobritsa, 1984) which, in addition, harbors the Hg’ gene.The structure of one of Tn2353 terminal regions is indistinguishable from that of the left-end sequencesof Hg’ transposons Tn2608 and Tn2613 (Tanaka et al., 1983). These regions carry a mercury-resistance gene, one Hind111 and two EcoRI sites, and one of the EcoRI sites is near or in the terminal repeat of the elements Tn2608 and Tn2613. This similarity in Tn2353, Tn2608, and Tn2613 may have resulted from the insertion (transposition) of identical or similar sequencescarrying the Hg’ gene into ancestral transposons. The formation of a Hg’ transposon by the translocation of a Tndl-like structure has been demonstrated by Grinsted and Brown ( 1984).
103
An alternative explanation is that Tn2353, Tn2608, and Tn2613 are related mobile elements, which have evolved from a common ancestral transposon. The Tn2353 sequence borders on a region of R 15 which carries EcoRII restriction-modification activity corresponding to that of N3. However, the N3 genes carry BamHI, PstI, and Sall sites (Ando and Arai, 1981; Brown et al., 1984). The rep genesof R 15 were mapped to a 7.7kb BglII segment which carries only two Hind111 sites, whereas N3 carries SalI sites (Ando and Arai, 1981). In R46 and pCU 1, the rep genesalso have no SalI, BamHI, and PstI sites (Brown and Willetts, 1981; Thatte and Iyer, 1983). R46 and pCU 1 carry tra regions which have close similarities in the location of restriction sites (Langer et al., 1981; Brown and Willetts, 1981; Konarska-Kozlowska et al., 1983), but those of the tra sequence on N3 differs from those of R46 and pCU1 or R15 (Ando and Arai, 1981; Brown et al., 1984). In R46 and pCU1, the tra and rep clusters are separated by relatively long DNA sequencesencoding other functions, whereasin R 15 and N3, these determinants are located close to one another to form a “core” structure. The IncN plasmids N3, R46, pCU1 and RI 5 differ in molecular weight, the number and locations of restriction sites, and phenotype aswell asin the arrangement of the genes, but sharecommon features in the nonrandom distribution of restriction sites. Those regions carrying replication and conjugation genesessential for plasmid maintenance and transfer carry relatively few restriction sites, whereas the overwhelming majority of these sites are located close to, or within, the regions containing the antibiotic-resistance determinants. In particular, 28 of 34 sites for BamHI, EcoRI, HindIII, SalI, SmaI, and PstI cleavageare localized in a Tn2353- and Tn2354- containing 23-kb region of the 62.3-kb plasmid R 15. In this region, we have recently found an insertion sequence, which is similar or identical to IS5 by its size and the distribution of
104
DOBRITSA, MIICHAILOVA,
AND DUBOVAYA
T. G. (1984). Detection and characterization of an IS EcoRI, PvuII, and Bg/II sites (Dobritsa et al., element in the structure of the plasmid R 15.In “Hybrid 1984). It is located between coordinates 45.0 plasmids and geneexpression” (I. V. Domaradskii, ed.), and 46.2 kb (Fig. 3). pp. 8 1-83. NCBI AN SSSR,Pushchino. [in Russian] These data support the hypothesis that one DOBRIA, A. P., D~BRI’ISA, S. V., POPOV,E. I., AND of the consequencesof evolution of wide-hostFEDOSEEVA,V. B. (1981). Transposition of a DNA fragment flanked by two inverted Tnl sequences.Gene range plasmids is a decreasing number of tar14,2 17-225. getsfor restriction endonucleases,whereasthe A. P., DOBRITSA,S. V., AND TANYASHIN, great number of cleavage sites in plasmid re- DOBRITSA, V. I. (I 978). Isolation and characterization of plasmid gions carrying resistancedeterminants may be from the Bacillus brevis var.G.-B. c&s. Mol. Gen. Genet. due to relatively recent events in which acquis164, 195-204. ition of these genes has occurred by transpo- D~BRITSA, A. P., IVANOVA, Z. A., AND FEWGEEVA, V. B. (1983). Transposition of DNA fragments flanked sition or otherwise.
ACKNOWLEDGMENTS The authors are grateful to Irina Filonova for her help in performing some stagesof the work and to Dr. V. M. Kramarov for determination of the restriction enzyme activity in the extracts of the E. coli cells.
REFERENCES ANDO, T., AND ARAI, T. (1981). Genetic structure of the IncN plasmid N3. Plasmid 6,293-30 1. APPLEYARD,R. K. (1954). Segregation of new lysogenic types during growth of a doubly lysogenic strain from E. coli K-12. Genetics 39,440-452. BIRNBOIM,H. C., AND DOLY, J. (1979). A rapid alkaline extraction procedure for screeningrecombinant plasmid DNA. Nucleic Acids Res. 7, I5 13-l 523. BOLIVAR,F., RODRIGUEZ,R., GREENE,P. J., BETLACH, M., HEYNEKER,H. L., BOYER,H. W., CROSA,J., AND FALKOW,S. (1977). II. Construction and characterization of new cloning vehicles. A multipurpose cloning system. Gene 2,95-l 13. BROWN,A. M., COUPLAND,G. M., ANDWILLE~S, N. S. (1984). Characterization of IS&, an insertion sequence found on two IncN plasmids. J. Bacterial. 159, 472481. BROWN,A. M. C., ANDWILLE-IX, N. S. ( 1981).A physical and genetic map of the IncN plasmids R46. Plasmid 5, 188-201. CLOWES,R. C., AND HAYES, W. (eds.). (1968). “Experiments in Microbial Genetics.” Blackwell, Oxford/Edinburg. CLOWES,R. C., AND ROWLEY,D. (1954). Some observations on linkage effects in genetic recombination in Escherichia coli K12. J. Gen. Microbial. l&250-260. DOBRITSA,A. P. (1984). IS8- and Tn2353-mediated cointegration of the plasmids R 15 and RP4: :Tnl. Mol. Gen. Genet. 194,206-2 10. DOBRITSA,A. P., ANDDERGUNOVA,L. V. (1984). Mobile genetic elements of the plasmid R 15.Dokl. Akad. Nauk SSSR, Ser. Biol. 271,980-983. [in Russian] DOBRITSA,A. P., DERGUNOVA,L. V., ANDMIKHAILOVA,
by two inverted Tnl sequences:translocation of the plasmid RP4::Tnl region harboring the Tc’ marker. Gene 22,237-243. D~BRITSA, A. P., K.SENZENKO, V. N., FEDOSEEVA, V. B., ALEXANDROV,A. A., KAMYNINA, T. P., ANDKHMELNITSKY,M. I. (1980). Isolation oftransposon Tn4 from plasmid RP4 carrying two copies of this element. Gene 8, 153-162. ECKHARDT,T. (1978). A rapid method for the identification of plasmid deoxyribonucleic acid in bacteria. Plasmid 1, 584-588. GRINSTED,J., ANDBROWN,N. L. (1984).A Tn2l terminal sequencewithin Tn501: Complementation of tnpA gene function and transposon evolution. Mol. Gen. Genet. 197,497-502.
HEDGES,R. W. (1972). Phenotypic characterization of fiR factors determining the restriction and modification HsplI specificity. Mol. Gen. Genet. 115, 225-233. HOLMES,D. S., ANDQUIGLEY,M. (198 I). A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. 114, 193-197. HUMPHREY%G. O., WILLSHAW,G. A., AND ANDERSON, E. C. (1975). A simple method for the preparation of large quantities of pure plasmid DNA. B&him. Biophys. Acta 383,457-463. KONARSKA-KOZLOWSKA, M., AND IYER, V. N. (1981). Physical and genetic organization of the IncN group plasmid pCU 1. Gene 14, 195-204. KONARSKA-KOZLOWSKA, M., THA~E, V., AND IYER, V. N. (1983). Inverted repeats visualized in strands of the DNA of plasmid pCU I. J. Bacterial. 153, 15021512. KRATZ, J., SCHMIDT,F., AND WIEDEMANN,B. (1983). Characterization of Tn2411 and Tn2410, two transposons derived from R-plasmid R1767 and related to Tn2603 and Tn21. J. Bacterial. 155, 1333-1342. LANCER, P. J., SHANABRUCH,W. G., AND WALKER, G. C. (1981). Functional organization of plasmid pKMlOI. J. Bacterial. 145, 1310-1316. LANCER, P. J., AND WALKER, G.C. (1981). Restriction endonuclease cleavage map of pKM IO1: Relationship to parental plasmid R46. Mol. Gen. Genet. 182, 268272. MEAGHER,R. B., TAIT, R. C., BETLACH,M., ANDBOYER,
IncN PLASMID Rl5 STRUCTURE H. W. (1977). Protein expression in E. coli minicells by recombinant plasmids. Cell 10, 521-536. MILLER, J. H. (1972). “Experiments in Molecular Genetics.” Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. ROBERTS,R. J. (1983). Restriction and modification enzymes and their recognition sequences.Nucleic Acids Rex 11, rl35-r167. SANGER,F., COULSON,A. R., HONG, G. F., HILL, D. F., AND PETERSON,C. B. (I 982). Nucleotide sequence of bacteriophage X DNA. J. Mol. Biol. 162, 729-773. SOBERON, X., COVARRUBIAS, L., ANDBOLIVAR,F. ( 1980). Construction and characterization of new cloning vehicles. IV. Deletion derivatives of pBR322 and pBR325. Gene 9,287-305. TANAKA, M., YAMAMOTO, T., AND SAVAI, T. (1983). Evolution of complex resistance transposons from an ancestral mercury transposon. J. Bucteriol. 153, 14321438.
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THA’ITE, V., ANDIYER,V. N. ( 1983).Cloning of a plasmid region specifying the system of bacterial conjugation. Gene 21,227-236. WARD, J. M., AND GRINSTED,J. (1978). Mapping of functions in the R-plasmid R388 by examination of deletion mutants generated in vitro. Gene 3, 87-95. WATANABE,T., NISHIDA, H., OGATA,C., ARAI, T., AND SATO,S. ( 1964).Episome-mediated transfer of drug resistance in Enterobacteriaceae. VII. Two types of naturally occurring R factors. J. Bucteriol. 88, 7 16-726. WESTON,A., BROWN,M. G. M., PERKINS,H. R., SAUNDERS,J. R., AND HUMPHREYS,G. 0. (1981). Transformation of Escherichiu coli with plasmid deoxyribonucleic acid: Calcium-induced binding of deoxyribonucleic acid to whole cells and to isolated membrane fractions. J. Bacterial. 145, 780-787. WOOD,W. B. (1966). Host specificity of DNA produced by E. coli: Bacterial mutations affecting the restriction and modification of DNA. J. Mol. Biol. 16. 118-133.