Recombination Between Bacteriophage Lambda and Plasmid Pbr322 in Escherichia Coli KAY L

JOURNAL OF BACTERIOLOGY, June 1980, p. 992-1003 Vol. 142, No. 3 0021-9193/80/06-0992/12$02.00/0 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 re- the plasmid can undergo this type of recombi- combination. One class, which involves recom- nation. Thus, the mechanisms responsible for bination between homologous DNA molecules, generating these recombinants may not be site is called general recombination (24). The other specific. One possible mechanism for generating class, which involves recombination between these recombinants is reciprocal recombination DNA molecules or sites on DNA molecules that between short regions of homology. are not homologous, is called illegitimate recombination (31, 34). This second class is responsible MATERIALS AND METHODS for DNA rearrangements such as deletions, du- Bacteriophages, plasmids, and bacterial plications, insertions, transpositions, and inver- strains. Two derivatives of phage A were used; sions. Ab5l9b515cI857S7 (called Abb) and AcI857S7 (caUed This report concerns the illegitimate recom- A). bination that occurs between the bacteriophage Plasmid pBR322 encodes resistance to ampicillin A genome and the plasmid pBR322 in Esche- (Ampr) and tetracycline (Tetr) and is derived from richia coli. These DNA molecules do not share pMB1, a colicin El-like plasmid (3). Plasmid pKPG10 extensive regions of homology. However, it is was isolated by cloning a HindIII restriction endonu- possible to isolate A clease fragment from AKan2 (1) that encoded kana- recombinant phages after mycin resistance (Kanr), using pBR322 as the vector. infection of strains containing pBR322 that All bacterial strains were derivatives of E. coli K- transduce the drug resistance genes encoded by 12. MO is an F- rpsL strain that is isogenic with this plasmid, tetracycline resistance and ampi- HfrHayes (20). Strains N747 and N761 are cillin resistance. The characterization of these MO(pKPG10) and MO(pBR322), respectively. E. coli recombinants by heteroduplex analysis and re- C600 was used as the host for A lysogens. striction endonucleases is described here. One Isolation of recombinant phages. Recombinant class of recombinant phages appears to have phages were isolated essentially as described by Berg resulted from A integration into secondary at- et al. (1). Fresh overnight cultures of N747 and N761 tachment (att) sites on the plasmid by recom- grown in LB broth (LB) plus 0.2% maltose plus 0.01 M bination MgSO4 were infected with Abb at a multiplicity of 10, with the phage att site. The other class incubated at 30°C for 30 min, diluted 1:10 with LB, has resulted from plasmid integration at other shaken at 30°C for 16 h, diluted 1:10, shaken at 300C regions of the A genome. Three or more sites on for 1 h, and induced thermally. Phages that transduced t Present address: Department of Biology, Massachusetts antibiotic resistance genes were isolated by infecting Institute of Technology, Cambridge, MA 02139. C600 and selecting resistant derivatives on LB plus 992 VOL. 142, 1980 A-pBR322 RECOMBINANTS 993 antibiotic agar plates. The drug concentrations were: lysogens on LB plus antibiotic plates at 30°C: LB plus kanamycin, 20 pg/mnl; tetracycline, 25 ,ug/ml; and am- kanamycin for AA6, LB plus tetracycline for AA4, and picillin, 100 ,ug/ml. All of the resistant derivatives of LB plus ampicillin for AT2, AK6, AK21, and AK47. C600 were found to be lysogens. LB medium and the Plasmids were purified from these revertants by the techniques for physically purifying A phages from the cleared-lysate technique described by Post et al. (23). C600 lysogens are described by Miller (21). The recom- Plasmids were recovered from C600 lysogens of binant phages we have characterized are listed in AKA2, AKA3, AKA8, AKAll, and ATA6 by isolating Table 1. The K (kanamycin), A (ampicillin), or T plasmid DNA from 10-ml cultures as described by (tetracycline) in the name refers to the antibiotic Post et al. (23), transforming C600 with the plasmid resistance genes transduced by each phage, and the preparation (19), and selecting transformants on LB number refers to an isolate number. The CsCl gradient plus kanamycin or LB plus tetracycline plates. The of the phage prepared from each of the lysogens con- frequency of formation of these revertant plasmids tained two bands, the recombinant phage and another was estimated by comparing the number of transform- phage that appeared to be identical to the parental ants obtained with the plasmid preparations from the Abb phage. However, only AKA1, AKA2, AKA3, AKA8, lysogens and from C600(pKPG10). AKAll, and AK36 were found to be defective phages. Other techniques. DNA was extracted from the Nevertheless, we used the original lysogens as our physically purified phage as described by Miller (21), source of the recombinant phages because we found digested with restriction endonucleases as described that the yield of phage for some of the recombinants by Greene et al. (15), and electrophoresed on agarose was greater from these lysogens than from single ly- gels as described by Shinnick et al. (30). Sizes of sogens. The original lysogens were probably double restriction fragments were determined by using the lysogens of the recombinant phage and the parental EcoRI, HindIII, and BamHI restriction endonuclease phage that occurred because we used a high multiplic- fragments of A DNA as standards (13; also see Fig. 1). ity of infection (10 to 20) in isolating the drug-resistant The sizes determined in this way are expressed in lysogens. However, we cannot exclude the possibility percent A units (100% A units = length of wild-type A that the Abb-like phage in these lysogens arose by DNA). In some cases these lengths have been con- excision of the plasmid from the recombinant phage verted to base pairs (bp), using the factor 1% A unit after induction. We also do not know whether the = 490 bp (2). The sizes of restriction fragments are recombinant phage in these lysogens are integrated expressed in either percent A units or kilobase (kb) into the chromosome at the attA site, or whether they units (1 kb = 1,000 bp). are replicating as plasmid by using the pBR322 origin Heteroduplex molecules were prepared and ana- of replication. lyzed by electron microscopy as described by Davis et Recovery of plasmids from recombinant al. (7). The double-strand-length standard was nicked phages. Revertants that had recovered the inacti- circular pBR322 (4.36 kb), and the single-strand- vated gene were obtained by plating the original C600 length standard was the Ab519 deletion loop (3.0 kb) TABLE 1. Summary ofrecombinant phagesa Site of insertion Orienta- Resistance en- Resistance tion of Class Mutant phage coded inactivated (%) P (kb) plasmidt I AA6 Abb::pKPG10 Amp Kan 57.3 ± 0.5 6.8 ± 0.3 II AKA5 -Abb::pKPG10 Kan, Amp 57.2 ± 0.3 0.8 ± 0.1 I AKA4 -Abb::pKPG10 Kan, Amp 57.4 ± 0.2 0.7 ± 0.1 I AA4 Abb::pBR322 Amp Tet 57.7 ± 0.2 (1.5) I AT2 =Abb::pBR322 Tet Amp 57.5 ± 0.2 (3.4) II AK6 -Abb::pKPG10 Kan Amp 57.3 ± 0.2 3.6 ± 0.3 II AK21 Abb::pKPG10 Kan Amp 57.2 ± 0.2 4.0 ± 0.2 I AK47 Abb::pKPG10 Kan Amp 57.3 ± 0.4 3.7 ± 0.1 I II AKA1 Abb::pKPG10 Kan, Amp 36.8 ± 0.3 5.7 ± 0.1 II AKA2 =Abb::pKPG10 Kan, Amp 99.1 ± 0.1 1.4 ± 0.2 I AKA3 = Abb::pKPG10 Kan, Amp 23.1 ± 0.7 0.9 ± 0.2 I XKA8 = Abb::pKPG10 Kan, Amp 7.0 ± 0.3 0.3 ± 0.05 II AKAll -Abb::pKPG10 Kan, Amp 14.0 ± 0.2 1.6 ± 0.2 I ATA6 Abb::pBR322 Tet, Amp 86.2 ± 0.7 (2.5) I AK36 Abb::pKPG10-A36 Kan Amp 8.1 ± 0.2 (1.0) I a Sites of insertion on A 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.

Load more