Nancy Simonson and Paul Lipson for assistance with the experiments, and Janice ... man, D. J. Russel, R. Tait, and H. W. Boyer. 1978. A general method for the ...
JOURNAL OF BACTERIOLOGY, June 1980, p. 992-1003 0021-9193/80/06-0992/12$02.00/0
Vol. 142, No. 3
Recombination Between Bacteriophage Lambda and Plasmid pBR322 in Escherichia coli KAY L. POGUE-GEILE, SHILADITYA DASSARMA,t STEVEN R. KING, AND S. RICHARD JASKUNAS* Department of Chemistry and the Program in Molecular, Cellular and Developmental Biology, Indiana
University, Bloomington, Indiana 47405
Recombinant A phages were isolated that resulted from recombination between the A genome and plasmid pBR322 in Escherichia coli, even though these deoxyribonucleic acids (DNAs) did not share extensive regions of homology. The characterization of these recombinant DNAs by heteroduplex analysis and restriction endonucleases is described. All but one of the recombinants appeared to have resulted from reciprocal recombination between a site on A DNA and a site on the plasmid. In general, there were two classes of recombinants. One class appeared to have resulted from recombination at the phage attachment site that probably resulted from A integration into secondary attachment sites on the plasmid. Seven different secondary attachment sites on pBR322 were found. The other class resulted from plasmid integration at other sites that were widely scattered on the A genome. For this second class of recombinants, more than one site on the plasmid could recombine with A DNA. Thus, the recombination did not appear to be site specific with respect to A or the plasmid. Possible mechanisms for generating these recombinants are discussed.
There are two general classes of genetic recombination. One class, which involves recombination between homologous DNA molecules, is called general recombination (24). The other class, which involves recombination between DNA molecules or sites on DNA molecules that are not homologous, is called illegitimate recombination (31, 34). This second class is responsible for DNA rearrangements such as deletions, duplications, insertions, transpositions, and inversions. This report concerns the illegitimate recombination that occurs between the bacteriophage A genome and the plasmid pBR322 in Escherichia coli. These DNA molecules do not share extensive regions of homology. However, it is possible to isolate recombinant A phages after infection of strains containing pBR322 that transduce the drug resistance genes encoded by this plasmid, tetracycline resistance and ampicillin resistance. The characterization of these recombinants by heteroduplex analysis and restriction endonucleases is described here. One class of recombinant phages appears to have resulted from A integration into secondary attachment (att) sites on the plasmid by recombination with the phage att site. The other class has resulted from plasmid integration at other regions of the A genome. Three or more sites on
the plasmid can undergo this type of recombination. Thus, the mechanisms responsible for generating these recombinants may not be site specific. One possible mechanism for generating these recombinants is reciprocal recombination between short regions of homology. MATERIALS AND METHODS Bacteriophages, plasmids, and bacterial strains. Two derivatives of phage A were used; Ab5l9b515cI857S7 (called Abb) and AcI857S7 (caUed A). Plasmid pBR322 encodes resistance to ampicillin (Ampr) and tetracycline (Tetr) and is derived from pMB1, a colicin El-like plasmid (3). Plasmid pKPG10 was isolated by cloning a HindIII restriction endonuclease fragment from AKan2 (1) that encoded kanamycin resistance (Kanr), using pBR322 as the vector.
All bacterial strains were derivatives of E. coli K12. MO is an F- rpsL strain that is isogenic with HfrHayes (20). Strains N747 and N761 are MO(pKPG10) and MO(pBR322), respectively. E. coli C600 was used as the host for A lysogens. Isolation of recombinant phages. Recombinant phages were isolated essentially as described by Berg et al. (1). Fresh overnight cultures of N747 and N761 grown in LB broth (LB) plus 0.2% maltose plus 0.01 M MgSO4 were infected with Abb at a multiplicity of 10, incubated at 30°C for 30 min, diluted 1:10 with LB, shaken at 30°C for 16 h, diluted 1:10, shaken at 300C for 1 h, and induced thermally. Phages that transduced antibiotic resistance genes were isolated by infecting C600 and selecting resistant derivatives on LB plus
t Present address: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139. 992
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VOL. 142, 1980 antibiotic agar plates. The drug concentrations were: kanamycin, 20 pg/mnl; tetracycline, 25 ,ug/ml; and ampicillin, 100 ,ug/ml. All of the resistant derivatives of C600 were found to be lysogens. LB medium and the techniques for physically purifying A phages from the C600 lysogens are described by Miller (21). The recombinant phages we have characterized are listed in Table 1. The K (kanamycin), A (ampicillin), or T (tetracycline) in the name refers to the antibiotic resistance genes transduced by each phage, and the number refers to an isolate number. The CsCl gradient of the phage prepared from each of the lysogens contained two bands, the recombinant phage and another phage that appeared to be identical to the parental Abb phage. However, only AKA1, AKA2, AKA3, AKA8, AKAll, and AK36 were found to be defective phages. Nevertheless, we used the original lysogens as our source of the recombinant phages because we found that the yield of phage for some of the recombinants was greater from these lysogens than from single lysogens. The original lysogens were probably double lysogens of the recombinant phage and the parental phage that occurred because we used a high multiplicity of infection (10 to 20) in isolating the drug-resistant lysogens. However, we cannot exclude the possibility that the Abb-like phage in these lysogens arose by excision of the plasmid from the recombinant phage after induction. We also do not know whether the recombinant phage in these lysogens are integrated into the chromosome at the attA site, or whether they are replicating as plasmid by using the pBR322 origin of replication. Recovery of plasmids from recombinant phages. Revertants that had recovered the inactivated gene were obtained by plating the original C600
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lysogens on LB plus antibiotic plates at 30°C: LB plus kanamycin for AA6, LB plus tetracycline for AA4, and LB plus ampicillin for AT2, AK6, AK21, and AK47. Plasmids were purified from these revertants by the cleared-lysate technique described by Post et al. (23). Plasmids were recovered from C600 lysogens of AKA2, AKA3, AKA8, AKAll, and ATA6 by isolating plasmid DNA from 10-ml cultures as described by Post et al. (23), transforming C600 with the plasmid preparation (19), and selecting transformants on LB plus kanamycin or LB plus tetracycline plates. The frequency of formation of these revertant plasmids was estimated by comparing the number of transformants obtained with the plasmid preparations from the lysogens and from C600(pKPG10). Other techniques. DNA was extracted from the physically purified phage as described by Miller (21), digested with restriction endonucleases as described by Greene et al. (15), and electrophoresed on agarose gels as described by Shinnick et al. (30). Sizes of restriction fragments were determined by using the EcoRI, HindIII, and BamHI restriction endonuclease fragments of A DNA as standards (13; also see Fig. 1). The sizes determined in this way are expressed in percent A units (100% A units = length of wild-type A DNA). In some cases these lengths have been converted to base pairs (bp), using the factor 1% A unit = 490 bp (2). The sizes of restriction fragments are expressed in either percent A units or kilobase (kb) units (1 kb = 1,000 bp). Heteroduplex molecules were prepared and analyzed by electron microscopy as described by Davis et al. (7). The double-strand-length standard was nicked circular pBR322 (4.36 kb), and the single-strandlength standard was the Ab519 deletion loop (3.0 kb)
TABLE 1. Summary of recombinant phagesa Class
Mutant phage
Resistance en- Resistance coded
inactivated
Site of insertion (kb) (%) P
Orientation of t plasmid
II 57.3 ± 0.5 6.8 ± 0.3 Kan Amp AA6 Abb::pKPG10 I 57.2 ± 0.3 0.8 ± 0.1 AKA5 -Abb::pKPG10 Kan, Amp I 57.4 ± 0.2 0.7 ± 0.1 AKA4 -Abb::pKPG10 Kan, Amp I 57.7 ± 0.2 (1.5) Tet AA4 Amp Abb::pBR322 II 57.5 ± 0.2 (3.4) Tet Amp AT2 =Abb::pBR322 II 57.3 ± 0.2 3.6 ± 0.3 Kan Amp AK6 -Abb::pKPG10 I 57.2 ± 0.2 4.0 ± 0.2 Kan Amp AK21 Abb::pKPG10 I 57.3 ± 0.4 3.7 ± 0.1 Kan Amp AK47 Abb::pKPG10 II 36.8 ± 0.3 5.7 ± 0.1 Kan, Amp II AKA1 Abb::pKPG10 I 1.4 ± 0.2 99.1 ± 0.1 Kan, Amp AKA2 =Abb::pKPG10 I 23.1 ± 0.7 0.9 ± 0.2 AKA3 = Abb::pKPG10 Kan, Amp II 7.0 ± 0.3 0.3 ± 0.05 XKA8 = Abb::pKPG10 Kan, Amp I 14.0 ± 0.2 1.6 ± 0.2 AKAll -Abb::pKPG10 Kan, Amp I 86.2 ± 0.7 (2.5) ATA6 Tet, Amp Abb::pBR322 I 8.1 ± 0.2 (1.0) Amp AK36 Abb::pKPG10-A36 Kan A a Sites of insertion on were determined from the A or B lengths given in Table 3. Results are given as percentage of the XPAPA genome. Sites of insertion on the plasmids were determined from the S lengths in Table 3 except for the values in parentheses, which were determined from sizes of restriction fragments in Table 2. Results are given in kilobases on the pBR322 or pKPG10 map in Fig. 1. Orientation I is defined as the one in which the plasmid sequences reading left to right with respect to the normal A genetic map is clockwise with respect to the plasmid map in Fig. 1. Orientation II has the opposite order. Class I phages resulted from recombination near the phage att site, 57.4%. Class II phages resulted from recombination at other sites on the A genome. I
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J. BACTERIOL.
(13). Contour lengths were determined as described by Fiandt et al. (10). Measurements in each case were A based on 8 to 15 independent heteroduplex molecules.
RESULTS Nature of recombinant phages. Most of the experiments described here were done with a derivative of pBR322, called pKPG10, that contains a 3.4-kb HindIII fragment from the transposon Tn5, which encodes resistance to kanamycin. The HindIlI sites that generate this fragment are in the 1.45-kb inverted repeats that flank Tn5 (1). Thus, pKPG10 has part of the inverted repeat of Tn5 (about 500 bp) in addition to the loop region of Tn5. The EcoRI, HindIII, BamHI, and PstI restriction endonuclease maps of pKPG10 are given in Fig. 1. pKPG10 encodes resistance to ampicillin and kanamycin, but not tetracycline. The Tetr genes of pBR322 were inactivated by the insertion of the Tn5 fragment at the HindIII site, which has been found previously (3). We were originally trying to determine whether the Tn5 HindIII fragment could transpose with its truncated inverted repeat. After infection of a strain containing pKPG10 (N747) with Abb, we found that it was possible to isolate recombinant X phages that transduced all possible combinations of Kanr and Ampr. These phages occurred at a frequency of about 10-7 compared with the plaque-forming phages in the lysate. The approximate proportions ofthe three types of phages were as follows: AKanr Ampr, 80%; AKanr Amp8, 10%; AKan8 Ampr, 10%. The HindIII Tn5 fragment was not required for these recombinations because analogous recombinant phages could be isolated after infection of a strain that contained pBR322 (N761), i.e., AAmpr Tetr, AAmpr Tet8, AAmp8 Tetr. The frequency and relative occurrence of the three types of phages were about the same as for pKPG10. About 80% of the recombinant phages transduced Ampr and Tetr, and 20% transduced only Ampr or Tetr. This report describes the characterization of 15 of the recombinant phages listed in Table 1 by restriction endonucleases and heteroduplex analysis. AT2, AA4, and ATA6 were obtained from a lysate of Xbb grown on N761 and selecting one phage of each type; XTetr Amp8 (AT2), ATete Ampr (AA4), or XTetr Ampr (XTA6). Similarly, a AAmpr Kan8 phage (AA6) and a AAmp8 Kanr phage (AK6) were obtained from lysates of Abb grown on N747. AKA1, AKA2, AKA3, XKA4, AKA5, XKA6, XKA8, and AKAll were obtained by selecting one Kanr Ampr Abb::pKPG10 phage from each of eight independent lysates. XK21, AK36, and XK47 were obtained by screening 23 lysogens of Kanr Amp8 Xbb::pKPG10 phages
B
2
FIG. 1. Restriction maps of A and pKPG10. (A) A. Restriction fragments are for Ab515b519 (10, 13). The sizes are in percent A units. The approxirnate locations of the A, J, attP, cI, and S genes are indicated. (B) pKPG10. The coordinate system begins 0.03 kb counterclockwise from the right HindIII site so that the coordinates of pKPG10 are the same as pBR322 from 0.03 to 4.36 kb (32). The sizes of the fragments are given in kilobase units and percent A units. The rectangles on the inner circle represent the truncated inverted repeats from Tn5. The locations of the Amp', Kan', and Tetrgenes are indicated (32). The location of the Kanr gene was established from the remainder ofpKPG10 that is present in AK36 and other mutants ofpKPG10 (Fig. 3 and our unpublished observations).
from a single lysate for their frequency of reversion to Ampr at 300C. Eighteen of these lysogens reverted to Ampr at a frequency of about l0', as did the lysogen of AK6. 'Three reverted at a
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Heteroduplex molecules of each of the recom-
frequency of 106. Phages XK21 and AK47 were obtained from two of these latter lysogens. Two lysogens did not revert to Ampr at a measurable frequency (
