Specialized transduction of colicin El DNA in Escherichia coli K-12 by phage lambda

Genetic studies were made on E. coli K-12 TM96, which carries recombinant molecules constructed by in vitro combination of colicin El DNA and a DNA fragment of E. coli for guanine synthesis derived from transducing phage. The recombinant molecules existed as stable plasmids within the cell and contained genes for colicin El immunity and the guaA enzyme (xanthosine 5'-monophosphate aminase) together with a part of the X genome, R through J: (R-A-F-J)+. A block of the X genome, int through Q, was not detected in the recombinant molecule. Thus, this recombinant molecule was named ColEI-cosX-guaA, and the specialized transduction of the ColEl-cosX-guaA DNA into various E coli K-12 cells by X phage was described. Lysates prepared by lytic infection of X phage onto TM96 or by induction of TM96(X) lysogens contained transducing particles which could transduce gua-deleted E coli to stable guaA + cells. These transductants were proved to have similar genetic properties as those of TM96. The frequency of transduction was not affected by the presence of an attachment site for A, prophage X, colicin El plasmids, or the recA property within gua-deleted recipient cells. Transducing particles were resistant to EDTA treatment and most of them'had an average density of about 1.472. This value corresponds to that of X phage particles, which contain about 72% of the length of X DNA. Colicinogenic factor El (ColEl) is an Escherichia coli plasmid that directs the production of a specific antibiotic protein. The plasmid DNA has a molecular weight of 4.2 X 106 (1), corresponding to about 14% of that of the well-studied bacteriophage ADNA. The ColEl plasmid exists as multiple copies, in contrast to the F factor or R factor. The number of plasmid DNAs in a cell is carefully controlled, probably, by the function(s) of the host cell together with that of plasmid DNA itself. The mechanism of maintenance of a stable level of plasmid DNA is an interesting subject. It is reported that the replication of ColEl DNA depends on host dnaA (2) and polA (3) functions but not on the dnaE function (4), and that ColEl DNA initiates and completes a round of semiconservative replication in a crude in vitro system (5). However, the role of ColEl genes in controlling the replication of its DNA is poorly understood. The large number of ColEl DNA copies within a cell makes genetic analysis very difficult. This paper describes the specialized transduction of a recombinant molecule, which carries a complete ColEl genome joined in vitro with a part of the X phage genome and a fragment of an E. coli chromosome for guanine synthesis, in E. coli K12 by X phage. This system makes it possible to introduce the established phage genetics into genetic study of ColEl DNA replication. MATERIALS AND METHODS Strains. The bacterial strains used are listed in Table 1. E. coli K-12 TM96, which carries the in vitro ColEl-guaA recombinant, was obtained from Dr. T. Mukai and is described in ref. 6. Most of the phage strains used are listed in (7). XpguaBA504(Xpgal type transducing phage) was isolated from heat-induced lysates of KS504 (XcI857 within guaB lysogens) (10). BF23 phage cannot kill ColEl resistant cells, but its growth is not affected by the presence of ColEl immunity (14). Media. Polypeptone bonito extract broth (PBB) medium (see ref. 15) was used for bacterial growth. PBB agar was used for phage and bacterial assays. Minimal agar was supplemented with 0.1 ,Ag/ml of biotin and thiamine and, where required, 20 /Ag/ml of xanthine. These media have been described previously (7, 15). Suspensions of bacteria and phages were diluted with 0.01 M MgSO4. Lysogens were isolated on EMB-O agar (7). Phage Assays and Phage Stocks. Free phage was assayed as described previously (7). Phage stocks were obtained by the plating method or by induction of X lysogens (see legend to Table 3). Measurement of Transducing Ability. Overnight cultures of various recipients in PBB supplemented with 0.1% maltose, were centrifuged and the cells were resuspended in 0.4 volume of 0.01 M MgSO4 and aerated for 1 hr at 37'. The final transduction mixture in a volume of 0.2 ml had a phage multiplicity of less than 10-2 per cell. After incubation for 30 min at 30°, to allow adsorption of phage, the mixture was plated on minimal agar supplemented with 20 ,ug/ml of xanthine. Plates were examined after incubation for 2 days at 30° or at 37O. CoWEl Immunity Test. Crude ColEl protein was prepared from Kp482 as described in ref. 16. This preparation contained about 1600 colicin units (17) per ml. The presence of ColE1 immunity was determined by spotting full grown cultures onto plates of PBB agar overlayered with 0.2-0.5 ml of crude ColEl preparation. Growth at this spot after 12 hr of incubation indicated that the cell was either immune or resistant to ColE1 protein. BF23 phage was used to distinguish ColEl resistant cells from ColE1 immune cells. Cultures of Kp482 which contains ColEl plasmid, and of KS1616 were always used as controls. Detection of CoIEl Protein Production. Colicin production by single colonies was assayed by stabbing samples of each colony into a fresh plate and incubating the plate at 37° for 12 hr. The surface of this plate was then exposed to chloroform vapor for 10 min to kill the bacteria. The plate was then overlayered with 2.5 ml of soft agar containing 5 X 107 indicator bacteria, either HfrH or HfrH(X+), and incubated overnight at 37°. The position of colicinogenic colonies was seen as a clear zone in the turbid lawn of indicator bacteria. Estimation of Int Function and Measurement of EDTASensitivity of Phage X. The methods used were as described in previous papers (18, 19). Enzyme Assays. Crude enzyme extracts were prepared (20) 3238 Abbreviations: ColEl, colicinogenic factor El; PBB medium, polypeptone bonito extract broth. Proc. Nati. Acad. Sci. USA 73 (1976) 3239 Tabie 1. Bacterial strains Strain Relevant genotype* Source or reference C600 XS supII+ R. Appleyard (see ref. 7, 8) KL16-99 Hfr KL16 recA B. Low (see ref. 7, 8)