Bacterial Replicon: Construction of a Mini-F'km Plasmid

Transcription

1 JOURNAL OF BACTERIOLOGY, Aug. 1976, p Copyright 1976 American Society for Microbiology Vol. 127, No. 2 Printed in U.S.A. Method for the Isolation of the Replication Region of a Bacterial Replicon: Construction of a Mini-F'km Plasmid MICHAEL A. LOVETT AND DONALD R. HELINSKI* Department of Biology, University of California, San Diego, La Jolla, California Received for publication 3 February 1976 A purified fragment of deoxyribonucleic acid (DNA) that determines resistance to kanamycin and is incapable of self-replication was used to select a selfreplicating fragment from an EcoRI endonuclease digest of the sex factor F'lac. This F'lac fragment, exhibiting a molecular weight of 6 x 106, carries the genes essential for maintenance of the F replicon in Escherichia coli cells. The constructed mini-f'km plasmid also retains the incompatibility properties of the parent F'lac plasmid. Large amounts of the kanamycin resistance fragment can be obtained as a selective agent for this procedure from a hybrid plasmid that was constructed by combining the EcoRI-generated kanamycin resistance fragment of a molecular weight of 4.5 x 106 with an EcoRI-cleaved, self-replicating derivative of colicinogenic plasmid El that has a molecular weight of 2.2 x 10". The recombinant plasmid is able to replicate extensively in E. coli in medium containing chloramphenicol, and, therefore, large quantities of this plasmid DNA can be obtained. The substantial difference in size between the two fragments in the recombinant plasmid greatly facilitates their separation by preparative agarose gel electrophoresis. Recently, the finding that cleavage of deoxyribonucleic acid (DNA) by restriction endonuclease EcoRI generates cohesive termini (7, 13) has greatly facilitated the formation in vitro of recombinant plasmid DNA molecules. These recombinant plasmids have been established (1, 3, 9, 14) in Escherichia coli cells by transformation (4). This report describes the construction of two new plasmids. One plasmid, designated pml21, consists of the 4.5 x 106-dalton fragment of the plasmid psc105 (3), which determines resistance to kanamycin (Km) and is incapable of self-replication, and a mini-colel, a self-replicating derivative of colicinogenic plasmid El (ColEl) that exhibits a molecular weight of 2.2 x 106 (8). The recombinant plasmid possesses the property of the ColEl plasmid of replicating extensively in a medium containing chloramphenicol, making possible the isolation of large amounts of this plasmid DNA. The kanamycin resistance fragment of pml21 was separated from the ColEl fragment by preparative agarose gel electrophoresis and used to select a single EcoRI fragment containing a self-replicating region of the sex factor F'lac. The use of the kanamycin resistance fragment of pml21 to select the replication segment of a plasmid element found in a gram-negative bacterial species has two advantages over the use by the same procedure of an antibiotic resistance fragment from a Staphylococcus aureus plasmid that has been reported previously (17). It avoids extending the range of antibiotic resistance genes in gram-positive bacteria to a gram-negative species, and the kanamycin resistance fragment can be obtained pure and in very high amounts from the amplifiable mini- ColE1 hybrid plasmid. The mini-f'km plasmid, designated pml31, constructed by this method is used in an accompanying paper (6) to localize the F replication region on an F'gal sex factor. MATERIALS AND METHODS Bacterial strains are described in Table 1. ll bacteriophage was provided by M. Guyer. MS2 bacteriophage was provided by V. Hershfield. The F'lac plasmid used in this study was obtained initially from 2310S from the collection of F. Jacob and transferred to the C600 strain by conjugation by J. Collins. Preparation of supercoiled plasmid DNA by dye-buoyant density centrifugation was as described previously (10, 11). A final concentration of Triton of 0.2% was used to prepare the cleared lysate. Transformation ofe. coli cells was performed as described elsewhere (4). Cleavage of DNA with the restriction endonuclease EcoRI was carried out as described previously (9). The reaction was terminated by heat inactivation of the enzyme at 65 C for 10 min. DNA ligase from T4 bacteriophage-infected E. coli cells was purchased from Miles Laboratories, Elkhart, Ind. The buffer used for ligation contained 66 mm tris(hydroxymethyl)aminomethane(tris), ph 7.5,

2 VOL. 127, 1976 TABLE 1. Bacterial strains Strain Propertiesa HfrH Hfr (Hayes) thi-1 C600 thr leu Thi- Lac- C600 reca trpr - thr leu Thi- Lac- trpr- trpe trpe reca CR34 Nalr thr leu Thi- Lac- Thy- Nalr HB 101 thi- leu pro lac gal his hsr hsm reca Strr JC 411 argg his leu metb lacy mala xyl stra a Genetic symbols are as given by Taylor and Trotter (16). mm NaCl, 10 mm MgCl2, 10 mm dithiothreitol, and 6 mm adenosine 5'-triphosphate. The ligation of the EcoRI digest of the F'lac plasmid and the purified Km fragment was carried out at 15 C for 3 h in a total volume of 2 ml, containing equal amounts (8 gg of each) of F'lac and Km DNA and 0.5 units of T4 DNA ligase. The ligation was terminated by the addition of ethylenediaminetetraacetic acid (EDTA) to 25 mm. An appropriate amount of 0.1 M CaCl2 was added to adjust the concentration of CaCl2 to 0.03 M prior to transformation with this DNA sample Ȧgarose slab gel electrophoresis was performed basically as described elsewhere (P. J. Greene, M. C. Betlach, H. M. Goodman, and H. W. Boyer, In R. B. Wickner [ed. ], Methods in Molecular Biology, vol. 9). The Km fragment was separated from the mini- ColEl fragment by preparative agarose gel electrophoresis. The electrophoresis apparatus consisted of a plexiglass cylinder with an internal diameter of 10 cm and a height of 15 cm. The cylinder contained a column of 0.8% agarose gel that was 8 cm in height and was held in the apparatus by placement of dialysis tubing across the bottom of the cylinder. A sample well of 8 cm in diameter was formed in the gel by positioning a plastic beaker approximately 1.5 cm into the molten gel. The top 7 cm of the apparatus was covered with 500 ml of Tris-borate buffer, and the apparatus was positioned on a sponge in a lower buffer chamber that also contained 500 ml of Tris-borate buffer. The DNA sample, containing up to 4.5 mg ofecori-cleaved pml21 DNA with 6% glycerol and % bromophenyl blue, was underlayered into the sample well, and a circular electrode was placed in the buffer above the sample. Electrophoresis was carried out at room temperature at 100 ma until the bromophenyl blue ran off the gel (about 10 h). The gel was then stained for 5 min with ethidium bromide (0.5 gg/ml), and the separated bands of DNA were sliced out of the gel. To recover the DNA, the gel slice was finely minced with a scalpel and covered with 40 to 50 ml of a buffer containing 100 mm Tris, ph 7.5, 50 mm NaCl, and 25 mm EDTA in a 250-ml plastic bottle. A magnetic stirring bar was added, and the DNA was eluted from the gel by mixing at 37 C overnight. The agarose was then pelleted by centrifugation for 5 min at 5,000 rpm at 10 C in a Sorvall GSA rotor. The CONSTRUCTION OF A MINI-F'Km PLASMID 983 recovery of the Km fragment from the gel was approximately 40%. Solid cesium chloride (3.6 g) and 0.3 ml of a 5-mg/ml solution of ethidium bromide was added to each 3.8 ml of the supernatant, and the DNA solution was centrifuged to equilibrium in a Spinco Ti6O rotor at 38,000 rpm for 36 h at 15 C. This equilibrium centrifugation concentrated the Km DNA and removed small pieces of agarose remaining in the supernatant after the low-speed centrifugation step. The presence of ethidium bromide in the gradient allowed the visualization of the DNA band with shortwave ultraviolet light. RESULTS Construction of a mini-cole1-km recombinant plasmid. Supercoiled DNA of plasmid psc105, consisting of the 5.8 x 106-dalton plasmid psc101 and a 4.5 x 106;-dalton Km fragment derived from EcoRI-cleaved R6-5, was digested with EcoRI endonuclease and centrifuged on a preparative, neutral 5 to 20% sucrose density gradient. The slowest sedimenting, one-third portion of the broad DNA peak obtained was precipitated with ethanol. After resuspension in a buffer containing 20 mm Tris, ph 7.5, 20 mm NaCl, and 1 mm EDTA (TEN), an equal amount of EcoRI endonucleasetreated mini-colel DNA in TEN buffer was added. About 2 ug of this mixture was used to transform strain C600 trpr - trpe reca for kanamycin resistance. Kanamycin-resistant colonies obtained from this transformation were tested for immunity to colicin El. Several of the kanamycin-resistant and colicin El-transformed clones were examined further and found to possess supercoiled DNA that sedimented as a 30S molecule. This supercoiled DNA yielded, upon treatment with EcoRI endonuclease, the 4.5 x 106-dalton Km fragment and the 2.2 x 106- dalton mini-colel DNA (Fig. 10). This recombinant plasmid was designated pml21. At least several hundred copies of this plasmid accumulate per chromosome per cell after incubation of the cells in the presence of chloramphenicol (8), as has been described for the ColE1-containing recombinant plasmids PVH5 and pml2 (9). Construction of a recombinant plasmid consisting of the replication region of F'lac and the Km fragment. Under no condition, including attempts to transform several E. coli strains to kanamycin resistance with the purified Km fragment, has the Km fragment been shown to be capable of self-replication. Therefore, this fragment, purified from pml21 as described in Materials and Methods, was used to select for the plasmid replication region present on one or more EcoRI-generated fragments derived from a plasmid element. The procedure used for the purification of the Km fragment

3 A D Li _-a FIG. 1. Agarose slab gel electrophoresis ofecori endonuclease-digested DNA samples. DNA samples (0.1 to 1.0 Ag) contained 6% glycerol and % bromophenyl blue in a volume of100 X. Electrophoresis was carried out at room temperature for 100 min at 160 V. The agarose concentration was 0.8% and the Trisborate buffer contained 10.8 g oftris, 0.93 g ofedta, and 5.5 g ofboric acid per liter. The gel was stained in Tris-borate buffer with 1.0 pg of ethidium bromide per ml and photographed with a shortwave ultraviolet light source. (A) Fragments of F'lac; molecular weights are approximately 13 (doublet), 7.7, 6.8, 6.2 (doublet), 6.0, 5.4, 5.0, 3.4, 3.2 (doublet), 2.9, 1.7, 1.4, and 1.3 x 106, and 4.1, 3.1, and 2.9 x 105. (B) Fragments ofpml31; 6.0 x 106 and 4.5 x 106 molecular weight. (C) Fragments ofpml21; 4.5 x 106 and 2.2 X 106 molecular weight. (D) Purified kanamycin fragment; 4.5 x 106 molecular weight. (E) X Fragments: molecular weights of 13.70, 4.68, 3.70, 3.56, 3.03, and 2.09 x 106 (9). The 3.70 X 106 and 3.56 x 106 fragments appear as a single band under these conditions. 984

4 VOL. 127, 1976 gives little or no detectable contamination with the DNA of the EcoRI-derived mini-colel fragment of pml21 (Fig. 1D). It was expected, therefore, that any kanamycin-resistant clones obtained after transformation with the ligated mixture of the Km fragment and EcoRI-generated fragments derived from a replicon should have resulted from the selection of an EcoRI fragment(s) possessing the essential properties of the replicon. Approximately 5,ug of the ligated mixture of the EcoRI endonuclease-generated fragments of the DNA of the sex factor F'lac, isolated from C600 reca trpr - trpe (F'lac), and the purified Km fragment of pml21 was used to transform E. coli C600. Supercoiled plasmid DNA isolated from three kanamycin-resistant clones was found in each case to sediment as a 36S molecule in a neutral sucrose gradient and yield on EcoRI cleavage the Km fragment and a 6 x 106-dalton fragment that is also generated by EcoRI digestion of the F'lac (Fig. lb). This recombinant plasmid, containing the Km fragment and the segment of the F'lac plasmid responsible for its replication, was designated pml31 and is also referred to as mini-f'km. Properties of pml31. The mini-f'km plasmid does not possess properties associated with the fertility of the sex factor F'lac. The conjugal mating of C600 (pml31) with JC411 did not result in the transfer of pml31 under conditions where a frequency of transfer of 10-9 or greater would have been detected, indicating that pml31 is non-self-transmissible. C600 (pml31) also was found to be resistant to the male-specific MS2 phage and sensitive to the female-specific k11 phage (Table 2). The antibiotic resistance plasmid R100-1 was able to promote the transfer of pml31 at low frequency. Under conjugal mating conditions where the R100-1 plasmid was transferred from C600 (R100-1) (pml31) to a CR34 Nalr recipient at a frequency of 5 x 10-4, the pml31 plasmid was transferred at a frequency of 1.5 x 10-l. The mini-f'km plasmid does retain the property of incompatibility with F-prime plasmids exhibited by the parent F'lac plasmid. When C600 reca trpr - trpe (F'lac) was mated with both HB101 (pml31) and HB101, the number of Lac+ colonies appearing on MacConkey lactose plates containing streptomycin as the counterselective agent was approximately 110 of 210 total colonies and 93 of 155 colonies, respectively, indicating the absence of surface exclusion of the parent F'lac in pml31. However, when the recipient clones of HB101 (pml31) on MacConkey lactose plates containing streptomycin were replica plated to MacConkey lac- CONSTRUCTION OF A MINI-F'Km PLASMID 985 TABLE 2. Bacteriophage typing ofpml31l No. of plaques Strain o11 MS2 5 x lo-5 b x HfrH > 1,000 C600 >1,000 > C600 (pml31) >1,000 > C600 reca trpr >1,000 trpe (Flac) C600 reca trpr- > 1, trpe a A 0.1-ml amount of an overnight L-broth culture of each strain tested was added to 2.5 ml of melted 0.7% L agar containing 2 x 10-3 M CaCl2. A 0.1-ml portion of each dilution of the bacteriophage preparation was added, and the mixtures were poured onto L-agar plates containing 2 x 10-3 M CaCl2, 0.1% glucose, and 0.001% thymidine. Plaques were counted after overnight incubation at 37 C. b Dilution. tose plates containing kanamycin and streptomycin, 58 of 61 Lac- colonies were replicated, but only 39 of 93 original Lac+ colonies were replicated and each of these colonies was Lacupon replica plating to MacConkey plates containing kanamycin. Fifteen Lac+ colonies from the mating were also transferred to MacConkey lactose-streptomycin plates and then replica plated to minimal lactose plates and to Mac- Conkey lactose plates containing streptomycin and kanamycin. Fourteen of the 15 Lac+ colonies replicated to minimal lactose plates, and 8 of the 15 replicated to the kanamycin plates, but each of these 8 colonies was found to be Lac-. These data indicate that the mini-f'km plasmid cannot stably coexist with F'lac. pml31 also retained the stringent control of replication of F plasmids that limits their copy number to one to two per bacterial chromosome. The data presented in Table 3 show that approximately 0.59% of the DNA in C600 (pml31) is in the form of 36S plasmid DNA. Assuming that the size of the E. coli chromosome is 2.5 x 109 daltons, there are approximately 1.4 copies of the 10.5 x 106-dalton pml31 per chromosome. DISCUSSION The selection of a single EcoRI-generated fragment from F'lac that serves as a molecular vehicle for the stable maintenance of the Km fragment derived from pml21 indicates that the purified Km fragment can be used to select EcoRI-generated fragments containing the essential components of a replicon. pml21 offers

5 986 LOVETT AND HELINSKI TABLE 3. Number of copies ofpml31 DNA per bacterial chromosomea % DNA sedimenting No. of cop- Cleared ly- ~ies of Expt sate (cpm); 36S DNA 36S DNA pml31/bactotal lysate cleared ly-(cpm)/total terial (cpm) sate (cpm) lysate (cpm) chromosome a C600 (pml31) spheroplasts labeled with [3H]thymine were lysed with Triton X-100 as described elsewhere (10, 11). The crude lysate was centrifuged at 48,000 x g for 20 min at 0 C. The cleared lysate was analyzed by centrifugation on 5 to 20% neutral sucrose density gradients containing 50 mm Tris, ph 7.5, 0.5 M NaCl, and 5 mm EDTA at 50,000 rpm for 100 min at 15 C in a Spinco SW50.1 rotor. Differentially labeled supercoiled ColEl DNA (23S) was included as a sedimentation reference. The percentage of labeled DNA in the gradient sedimenting at the 36S position was calculated, and the number of copies of pml31 per bacterial chromosome was determined as described in the text. two advantages in the preparation of the Km fragment; the 2.2 x 106-dalton mini-colel vehicle is easily separated from the 4.5 x 106 Km fragment by preparative agarose gel electrophoresis, and the ability of the plasmid to replicate extensively after the addition of chloramphenicol permits the isolation of large quantities of the supercoiled plasmid DNA. Recently, an antibiotic-resistant fragment from an S. aureus plasmid has been used to clone the replication segments of plasmid R6-5 and F'lac (17). The properties of the replication segment of F'lac isolated by this procedure are similar to those reported here. In an accompanying paper (6) the analysis of heteroduplexes between pml31 and two F- prime plasmids by electron microscopy has localized the replication fragment of pml31 to the region of the F-prime plasmid deduced to contain the genes involved in replication of the plasmid. The fact that pml31 retains the stringent control of replication of F elements probably means that the 6 x 106-dalton fragment contains those genes of F'lac responsible for stringent replication control. The retention of the incompatibility property of F'lac by pml31 does not distinguish whether this phenomenon is due to competition of a similar or identical replicon for the same essential cellular site, or if some other gene product or DNA site on the 6.0 x 106 fragment is responsible for this property. It is clear, however, that both replication and incompatibility functions of F'lac are present on this 6.0 x 106 segment of F'lac plasmid. A similar observation has been made for the J. BACTERIOL. isolated replication regions of R6-5 and F'lac described by Timmis et al. (17). The high frequency with which F'lac entered C600 (pml31) suggests that pml31 does not carry the gene for surface exclusion. This function has been found to map within a region of F containing the genes required for the expression of fertility (18). The 6.0 x 106 fragment represents 6.3% ofthe size of F'lac (94.9 ± 2.1 x 106 daltons [15]). Apparent clustering of genes required for DNA replication and existence as a plasmid has also been noted in the cases ofpsc101, which represents less than 10% of the size of the R6-5 plasmid from which it was derived (2), X dv, which represents less than 15% of the size of bacteriophage X (12), the mini-colel plasmid (8), and, recently, for R6-5 and F'lac (17), where a conceptually similar procedure was used for the isolation of the replication region. The reason for this clustering of replication genes and its implications with respect to the control of plasmid replication and the evolution of plasmid elements remain to be determined. ACKNOWLEDGMENTS We thank William A. Moller for his excellent technical assistance, and we thank Herbert Boyer and John Collins for helpful discussions. This investigation was supported by U.S. Public Health Service research grant AI from the National Institute of Allergy and Infectious Diseases and National Science Foundation grant GB LITERATURE CITED 1. Chang, A. C. Y., and S. N. Cohen Genome construction between bacterial species in vitro: replication and expression of Staphylococcus plasmid genes in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 71: Cohen, S. N., and A. C. Y. Chang Recircularization and autonomous replication of a sheared R-factor DNA segment in Escherichia coli transformants. Proc. Natl. Acad. Sci. U.S.A. 70: Cohen, S. N., A. C. Y. Chang, H. W. Boyer, and R. B. Helling Construction of biologically functional bacterial plasmids in vitro. Proc. Natl. Acad. Sci. U.S.A. 70: Cohen, S. N., A. C. Y. Chang, and C. L. Hsu Nonchromosomal antibiotic resistance in bacteria: genetic transformation ofescherichia coli by R-factor DNA. Proc. Natl. Acad. Sci. U.S.A. 69: Davidson, N., R. C. Deonier, S. Hu, and E. Ohtsubo Electron microscope heteroduplex relations among plasmids of Escherichia coli. X. Deoxyribonucleotide acid sequence organization of F and F-primes, and the sequences involved in Hfr formation, p In D. Schlessinger (ed.), Microbiology American Society for Microbiology, Washington, D.C. 6. Guyer, M. S., D. Figurski, and N. Davidson Electron microscope study of a plasmid chimera containing the replication region of the Escherichia coli F plasmid. J. Bacteriol. 127: Hedgpeth, J., H. M. Goodman, and H. W. Boyer DNA nucleotide sequence restricted by the RI endo-

6 VOL. 127, 1976 nuclease. Proc. Natl. Acad. Sci. U.S.A. 69: Hershfield, V., H. W. Boyer, L. Chow, and D. R. Helinski Characterization of a mini-colel plasmid. J. Bacteriol. 126: Hershfield, V., H. W. Boyer, C. Yanofsky, M. A. Lovett, and D. R. Helinski Plasmid ColEl as a molecular vehicle for cloning and amplication of DNA. Proc. Natl. Acad. Sci. U.S.A. 71: Katz, L., D. T. Kingsbury, and D. R. Helinski Stimulation by cyclic adenosine monophosphate of plasmid deoxyribonucleic acid replication and catabolite repression of the plasmid deoxyribonucleic acidprotein relaxation complex. J. Bacteriol. 114: Lovett, M. A., D. G. Guiney, and D. R. Helinski Relaxation complexes of plasmids ColEl and ColE2: unique site of the nick in the open circular DNA of the relaxed complexes. Proc. Natl. Acad. Sci. U.S.A. 71: Matsubara, K., and A. D. Kaiser dv: an autonomously replicating DNA fragment. Cold Spring Harbor Symp. Quant. Biol. 33: CONSTRUCTION OF A MINI-F'Km PLASMID Mertz, J. E., and R. W. Davis Cleavage of DNA by Rl restriction endonuclease generates cohesive ends. Proc. Natl. Acad. Sci. U.S.A. 69: Morrow, J. F., S. N. Cohen, A. C. Chang, H. W. Boyer, H. M. Goodman, and R. Helling Replication and transcription of eukaryotic DNA in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 71: Sharp, P. A., M. Hsu, E. Ohtsubo, and N. Davidson Electron microscope heteroduplex studies of sequence relations among plasmids ofescherichia coli. I. Structure of F-prime factors. J. Mol. Biol. 71: Taylor, A. L., and C. D. Trotter Revised linkage map ofescherichia coli. Bacteriol. Rev. 31: Timmis, K., F. Cabello, and S. N. Cohen Cloning, isolation and characterization of replication regions of complex plasnid genomes. Proc. Natl. Acad. Sci. U.S.A. 72: Willetts, N Mapping loci for surface exclusion and incompatibility on the F factor ofescherichia coli K-12. J. Bacteriol. 118: