Development of M13 as a Single-stranded Cloning Vector: Insertion of the Tn3 Transposon into the Genome of M13

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1 Development of M13 as a Single-stranded Cloning Vector: Insertion of the Tn3 Transposon into the Genome of M13 Dan S. Ray and Karin Kook Molecular Biology Institute and Department of Biology University of California Los Angeles, California As a first step towards developing phage Ml3 as a cloning vector we have investigated the in vivo insertion of a transposable ampicillin-resistance element (Hedges and Jacob 194; Heffron et al. 195a,b, 19) into its genome. Passage of M13 through Escherichia coli RSF2124, a strain carrying the element To? inserted into the plasmid ColEl (So et al. 195), yielded phage preparations capable of transducing sensitive E. coli strains to ampicillin resistance. By repeated colony purification of transduced cells on ampicillin plates we have isolated clones that produce homogeneous transducing phage capable of transducing ampicillin resistance with high efficiency. We describe here the properties of one of these isolates, which we have termed Ml3 Tn?-15. To establish that the transducing activity is associated with the M13 phage produced by resistant cells, we have investigated the ability of antibodies against the M13 coat protein to inactivate both the plaque-forming ability and the transducing activity of the M13 TnJ-15 phage. Table 1 shows that both plaque formation and transduction to drug resistance are inactivated by purified antibodies to the viral coat protein. Gamma globulins from rabbits that had not been injected with coat protein had no effect on either plaque formation or transduction. Electron microscopic observation of the M13 Ta?-15 phages shows them to have a mean length approximately 1. times that of the Ml 3 unit length. Figure 1 shows Ml3 and Ml3 TnJ-15 in a mixture of the two phages. Replicative forms (RF) of the Ml 3 TnJ-15 DNA have been isolated from infected cells and also examined by electron microscopy. Figure 2 shows electron micrographs of both Ml 3 and Ml 3 TnJ-15 RF molecules prepared by the aqueous Kleinschmidt procedure. The length of M13 TnJ-15 RF DNA is 1.8 times that of Ml 3 RF DNA. The increased lengths of both the M13 TnJ-15 phage DNA and its duplex RF DNA agree (within experimental error) with the length predicted from the insertion of the 4.8-kb TnJ 455 The Single-Stranded DNA Phages 198 Cold Spring Harbor Laboratory Press

2 456 D. S. Ray and K. Kook Table 1 Inactivation of M13 Tn3-15 by antibodies against M13 coat protein Gamma globulin (/ig/ml) anti-m13 control Plaque-forming units/ml 0 2. xlo X x10* xlo 2 5.1x10' xl0 Ap' colony-forming units/ml 6 2. XlO 6 2. XlO 1.2 xlo XlO 2.4X10 5.2X10 2.3X10 1.X10 M13 Tni-15 plaque-forming ability and ampicillin-resistance transforming activity were assayed after incubation of M13 TnJ-15 with purified M13 anti-coat protein gamma globulins or gamma globulins from rabbits that had not been exposed to Ml3 coat protein. Incubations were for 15 min at 3 C in 0.01 M Tris (ph 8.0), 1 nw EDTA. One aliquot was diluted and assayed for infectivity by the plaque assay and a second aliquot was incubated with E. coli K3 for 10 min at 3 C and then spread on nutrient plates containing ampicillin at 0 /*g/ml. 4 into the 6.4-kb M13 genome to yield an M13 Tn3-15 RF DNA of 11.2 kb, or 1.5 times the size of Ml 3 RF DNA. To determine both the site and polarity of insertion of the transposable element into the Ml3 genome, we have analyzed various restriction fragments of M13 Tn?-15 RF DNA. Restriction analyses with the enzymes Haell, Haelll, and Hpall indicate that the site of insertion must be just clockwise from the single Haell site in the intergenic space (data not shown). Figure 1 Electron micrograph of Ml3 and Ml3 Tni-15 phage prepared by the aqueous DNA spreading technique. (Bar = 1 fim). The Single-Stranded DNA Phages 198 Cold Spring Harbor Laboratory Press

3 M13 as Cloning Vector 45 Figure 2 Electron micrographs of M13 RF DNA and M13 Tn3-15 RF DNA prepared by the aqueous DNA spreading technique. (Bar = 1 /im). In each case all but one of the usual Ml3 fragments were observed in the digest of M13 Tn5-15 RF DNA. The missing fragments were HaeW B and one member of the Haelll E doublet. The Hpall A fragment appeared to be slightly increased in size. Additional digestions with Baml, Hindi, HaeW, and combinations of these enzymes (Fig. 3) confirm the location indicated above and establish a unique orientation of the transposon in the viral genome (Fig. 4). Baml, Hindi, and HaeW cleave M13Tn3-15 RFDNA into Figure 3 Gel electrophoresis of restriction fragments of M13 Tn3-15 RF DNA produced by treatment with (b) HaeW, (c) HaeW + Hindi, (d) HaeW + Baml, (f) Hindi, (g) Baml, and (h) Hindi + Baml. Lanes a, e, and /' contain markers of known molecular weight: M13 RFIII (6400 bp), M13 Bam + Hin A (420 bp), M13 Bam + Hin B (2130 bp), M13 Haelll A (2500 bp), Ml3 Haelll B + D (1940 bp), Ml3 Haelll B (1630 bp), M13 Haelll C (820 bp), and M13 Haelll D (310 bp). The Single-Stranded DNA Phages 198 Cold Spring Harbor Laboratory Press

4 L V T T 458 D. S. Ray and K. Kook BO0 Figure 4 Restriction map of the M13 Tn?-15 phage genome. Numbered sectors indicate the locations of Ml 3 genes and the intergenic region (IR) containing both the viral- and complementary-strand origins (ori v and ori c ). The inverted repeat sequences of the Tni element are indicated by black rectangles. The ^-lactamase gene confers resistance to ampicillin and is indicated by Ap'. The relative sizes of the Ml 3 and TnJ portions of the Ml 3 Tn?-15 symbols M13 TnJ-15 genome are 6.4 kb and 4.8 kb, respectively. HoeU A Hae HI o HpoU two, three, and seven fragments, respectively (Table 2). In each case the number of fragments is equal to the sum of the number of restriction sites for that particular enzyme in the transposable element and in Ml 3 RF DNA. The sum of the molecular weights of the fragments produced by each enzyme is equivalent to 11.2 kb, again in agreement with the formation of the M13 TnJ-15 RF DNA by insertion of TnJ into M13 RF DNA. On the basis of the observations presented here and others to be described elsewhere (D. S. Ray and K. Kook, in prep.), we conclude that the ability of the Ml3 Tn?-15 phage to transduce ampicillin resistance is the result of an Table 2 Hindi, Baml, and Haell fragments of M13 Tn3-15 RF DNA Size (bp) Fragment flmcll Baml Haell A * B C D 525* E ,230 11,300 11,250 Molecular weights of M13 TnJ-15 restriction fragments were determined by agarose gel electrophoresis. Asterisks indicate fragments tentatively identified as doublets based on staining intensity. The Haell A band has been shown to be a doublet by restriction with either Hindi or Baml. The Single-Stranded DNA Phages 198 Cold Spring Harbor Laboratory Press

5 M13 as Cloning Vector 459 insertion of the Tn5 transposon into the intergenic region of Ml3 close to the terminus of gene IV. The insertion of the ampicillin-resistance gene into the viral genome provides a direct selection for infected cells. Future efforts to develop this phage as a cloning vector will include the deletion of a large fraction of the transposon so as to reduce the size of the vector and to prevent further transposition. It may be possible to use M13 as an EK2 vector system if we can obtain mutants with small deletions within viral genes corresponding to the Ml3 DNA fragments that have been cloned by Kaplan et al. (this volume). Such deletion mutants would be dependent on the host strain carrying the appropriate cloned gene of Ml3 and would therefore have a greatly reduced ability to propagate in the absence of this specially constructed host. ACKNOWLEDGMENTS The authors are grateful to Dr. William Wickner for providing us with purified antibodies against the M13 coat protein and to Drs. Ron Gill and Stanley Falkow for providing us with E. coli RSF2124. This work was supported by National Institutes of Health contract NOI Al and grant Al 1052, and by a grant from the National Science Foundation (PCM ). REFERENCES Hedges, R. W. and A. Jacob Transposition of ampicillin resistance from RP4 to other replicons. Mol. Gen. Genet. 132: 31. Heffron, F., C. Rubens, and S. Falkow. 195a. Translocation of a plasmid DNA sequence which mediates ampicillin resistance: Molecular nature and specificity of insertion. Proc. Natl. Acad. Sci. 2: Heffron, F., P. Bedinger, J. J. Champoux, and S. Falkow. 19. Deletions affecting the transposition of an antibiotic resistance gene. Proc. Natl. Acad. Sci. 4: 02. Heffron, F., R. Sublett, R. Hedges, A. Jacob, and S. Falkow. 195b. Origin of the TEM beta-lactamase gene found on plasmids. J. Bacteriol. 122: 250. So, M., R. Gill, and S. Falkow The generation of a ColEl-Ap r cloning vehicle which allows detection of inserted DNA. Mol. Gen. Genet. 142: 239. The Single-Stranded DNA Phages 198 Cold Spring Harbor Laboratory Press