Proceedings of the National Academy of Sciences Vol. 66, No. 4, pp. 1083-1088, August 1970 Transduction by Bacteriophage Ti * Henry Drexler DEPARTMENT OF MICROBIOLOGY, BOWMAN GRAY SCHOOL OF MEDICINE, WAKE FOREST UNIVERSITY, WINSTON-SALEM, NORTH CAROLINA Communicated by Edward L. Tatum, May 25, 1970 Abstract. Amber mutants of the virulent coliphage T1 are able to transduce a wide variety of genetic characteristics from permissive to nonpermissive K strains of Escherichia coli. The virulent coliphage T1 is not known to be related to any temperate phage. Certain of the characteristics of T1 seem to be incompatible with its potential existence as a temperate phage; for example, the average latent period for T1 is only 13 min' and about 70% of its DNA is derived from the host.2 In order to demonstrate transduction by T1 it is necessray to provide condi- tions in which potential transductants are able to survive; this is accomplished by using amber mutants of T1 to transduce nonpermissive recipients. Experi- ments which show that T1 is a transducing phage are presented and have been designed chiefly to illustrate the following points: (1) Infection by T1 is able to cause heritable changes in recipients; (2) the genotype of the donor host is im- portant in determining what characteristics can be transferred to recipients; (3) the changes in the recipients are not caused by transformation; and (4) there is a similarity between the transducing activity and the plaque-forming ability of T1 with respect to serology, host range, and density. Materials and Methods. Bacterial strains: The abbreviations and symbols of Demerec et al.3 and Taylor and Trotter4 are used to describe all pertinent genotypes. The phenotype of each strain is given below, together with the symbols used to identify them, and are repeated at each point where their inclusion is useful for clarity. All strains used are members of the species Escherichia coli. KB-5 is a streptomycin-resistant (StrR) strain which is a permissive host (Su+) for all the amber mutants of T1 used here. KB-3 is able to ferment lactose (Lac+) and galac- tose (Gal+) and is independent of any requirement for arginine (Arg+), tryptophan (Trp+), serine (Ser+), threonine (Thr+), and biotin (Bio+). A tryptophan-dependent (Trp-) variant of KB-3 which requires tryptophan to satisfy its growth requirement was used in one experiment. CS1006 is a streptomycin-sensitive (StrS) strain which is Su+. A spontaneous StrR variant of CS100 was isolated in this laboratory. The strain W33507 is a nonpermissive host (Su-) for the amber mutants used here; it is StrS and is unable to ferment either galactose (Gal-) or lactose (Lac-). Mutants of W3350 which are de- pendent on either arginine (Arg-), tryptophan (Trp-), or serine and threonine (Ser-Thr-) were obtained by exposing W3350 to nitrosoguanidine. W3350/1 is a spontaneous, T1- resistant mutant isolated in this laboratory. Strain R9018 was derived from W3350 (X); it has a deletion from galE through XQ and also from XB through chlA including one or more genes controlling the biosynthesis of biotin; phenotypically R901 is Su-Gal-Bio-. Strains KB-3, CS100, W3350, and R901 were kindly provided by Drs. W. Michalke, J. R. Christensen, E. Six, and A. Campbell respectively. 1083 Downloaded by guest on September 29, 2021 10841084GENETICS: H. DREXLER PROC. N. A. S. Phage strains: T1 amber mutants (am) are from Michalke.' Efficiencies of plat- ing strains Tlamll and Tlam5amll on W3350 compared to KB-3 were 10-6 and less than 10-8 respectively. T1 plaques were less than 4 mm in diameter on the various selective media. The infected recipient cells were diluted to give less than 40 plaques per plate. Materials: L broth,9 L agar,9 saltless nutrient broth,10 synthetic T medium with glucose and agar (TMGA),"1 and complete eosin methylene blue agar (EMB)12 have been described elsewhere. T1 resuspension medium is a sterile solution of 1.21% tris(hydroxymethyl)aminomethane (Trizma Base, Sigma Chemical Corp.), 0.05% CaCl2, 0.06% MgSO4, and 0.15% gelatin. Streptomycin is an aqueous solution of streptomycin sulfate (Eli Lilly and Co.). Specific amino acids were used to supplement the T medium in 0.002% amounts. Biotin, when needed, was used at concentrations of 5 ,ug/ml. DNase was a solution of a crystalline preparation of Nutritional Biochem. Corp. CsCl solution contained 1.12 g/ml of CsCl dissolved in 1.21% Trizma base. Methods: In experiments, cells were grown to log phase in L broth and harvested at a titer of about 5 X 108/ml. Cells were centrifuged three times and resuspended each time in saltless nutrient broth and finally concentrated to about 1-2 X 109/ml. Cells and phage (multiplicity of infection of about 1) were incubated at 37°C for 7 min, chilled, and a representative sample was centrifuged in order to test the supernatant for un- adsorbed phage. The adsorption mixture was maintained in an ice bath during plating. In experiments where the transfer of the streptomycin-resistant phenotype was being selected, a sample of the adsorption mixture was plated by overlay on a L agar plates; these plates were incubated at 37°C for 3 hr and streptomycin solution was added via an additional overlay and incubation continued.'0 In experiments other than the strepto- mycin experiments, the adsorption mixture was spread on the surface of either EMB plates or TMGA plates. A volume of uninfected recipient cells equal in number to the number of cells exposed to T1 were plated as controls in all experiments. Anti-Ti serum wasl)repared by injecting wild type T1 which had been grown on Shi- gella dysenteriae into a rabbit; before use the complement was inactivated by incubation at 56°C for 30 min. Before use, all stocks of Ti were washed several times in Ti resuspension medium, re- frigerated overnight, and filtered. All stocks were routinely tested for sterility. With CsCl density gradient centrifugation, 2 ml of stock phage (about 1-3 X 1011 plaque-forming units/ml) were mixed with 3 ml of CsCl solution. The mixture was cen- trifuged for 18 hr in a model L, Beckman utracentrifuge in a SW50L rotor at 100,000 X g (36,000 rpm). Results: Transduction of various markers: Table 1 presents data from experiments in which Tlam phage grown on the Su+, Gal+, Arg+, Trp+, and StrR strain KB-3 were used to infect variants of W3350; all W3350 variants are Gal- and StrS; Arg- and Trp- isolates of W3350 were also used. In experiment 3 it can be observed that strain CS100 Su + StrR is also able to serve as a donor for Ti-mediated transduction. The results in Table 1 demonstrate the generalized nature of Tl transduction. Since the amber mutants of T1 which were used here (i.e., Tlamll and Tlam- 5aml 1) kill W3350 even though no phage are produced, transductants must arise from cells which obtained a transducing particle (containing donor DNA but little or no phage DNA") but no phage particle. The efficiency of transduction is given as the number of tranductants per milliliter less control values per singly infected cell as calculated using the multiplicity of infection, the number of cells, and the Poisson distribution. When KB-3Su+ is used as the donor, the number of transductants per singly infected cell varies from about 3 X 10- to 1 X 10-. Importance of the previous host: Tlam5amll was grown on CS100SutStrR Downloaded by guest on September 29, 2021 VOL. 66, 1970 GENETICS: H. DREXLER 1085 TABLE 1. Generalized tranduction by Ti; importance of previous host; host range of trans- ducing particles. * Colonies Transductants/ Phenotype Su + Su - with selected singly infected Expt. selected donor recipient phenotype cells 1 Gal + KB-3Gal + W335OGal- 157 5 X 10-7 Control 0 2 Arg+ KB-3Arg + W3350Arg- 211 3 X 10-7 Control 0 3 StrR CS10OStrR W335OStrS 2155 9 X 10-6 CSLOOStrS W335OStrS 35 7 X 10-8 Control 13 4 Trp + KB-3Trp+ W3350Trp- 129 1 X 10-6 KB-3Trp- W3350Trp- 21 7 X 10-8 Control 1 5 StrR KB-3StrR W335OStrS 248 1 X 10-6 Control 0 W335OStrS/1 0 Control 0 * Tlamll was used in Expts. 1, 2, and 5. Tlam5amll was used in Expts. 3 and 4. and CS100Su+StrS respectively. The third experiment in Table 1 shows that phage grown on CS100Su+StrR can transfer streptomycin resistance to W3350- Su-StrS but that phage grown on CS100Su+StrS cannot. Experiment 4 of Table 1 shows that if a tryptophan-dependent auxotroph (KB-3Su-Trp-), which requires tryptophan to satisfy its requirement, is used as the host for Tlam5amll, the phage is either not able to transfer tryptophan independence to a recipient with a similar requirement (W3350Su-Trp-) or it does so at a very low rate compared to phage grown on Trp+ donors. Since KB-3Trp- donors and W3350Trp- recipients do not necessarily represent iden- tical point mutations, a small amount of transduction may occur in this instance. Host range of transducing activity: Experiment 5 in Table 1 gives the results of a representative experiment in which attempts were made to transduce the Tl-resistant, Su-, StrS strain W3350/1 using Tlaml which had been grown on KB-3Su+StrR. It can be seen that while W335OSu-StrS could be transduced for StrR by the phage, no W3350/1 transductants were observed. Effect of specific anti-Ti serum: Antiserum was used in concentrations which, on the one hand, would inactivate 70-90% of the phage and which, on the other hand, would inactivate less than 10% of the phage after a 1:10 dilution of the antiserum.