Formation of P22 generalized transducing particles in vitro

VIROLOGY

95,

574-576 (197%

SHORT COMMUNICATIONS Formation

of P22 Generalized

DAVID BOTSTEIN’ Department

of Biology,

Transducing

AND

Massachusetts Institute

Particles

in Vitro

ANTHONY

R. POTEETE*

of Technology,

Cambridge,

Accepted February

Massachusetts

02139

26, 1979

Biologically active Salmonella phage P22 generalized transducing particles (i.e., P22 capsids tilled with bacterial DNA) are formed in vitro in extracts in which phage DNA is encapsulated to produce viable phage.

Transfer of bacterial genes from one cell to another by bacteriophage particles is called transduction. When any gene can be transduced (i.e., transfer is not limited to a few specific bacterial genes) the process is called generalized transduction. The phenomenon of generalized transduction was first described in Salmonella typhimurium by Zinder and Lederberg in 1952 (1). The bacteriophage which is responsible, phage P22, is the best understood of the generalized transducing phages (see (2) for a recent review). Generalized transduction by phage P22 (and also coliphage Pl) is carried out by a subclass of particles in lysates which contain primarily bacterial DNA synthesized before infection instead of phage DNA (35). The simplest model for the basis of generalized transduction by P22 is that the phage’s DNA encapsulation mechanism has an imperfect specificity for the intracellular concatemeric DNA which is its normal substrate. As a result, occasionally the bacterial chromosome is mistakenly used to initiate DNA encapsulation. Since encapsulation of phage DNA proceeds sequentially once initiated (6), several particles might be formed from such a mistaken initiation. Supporting this simple view are data sugL To whom reprint requests should be sent. * Present address: Biological Laboratories, Harvard University, 16 Divinity Avenue, Cambridge, Mass. 02138. 0042~6822/79/080574-03$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

gesting specific initiation and sequential encapsulation of bacterial DNA during the formation of transducing particles (7>, the observation that high-transducing mutants occur in P22 gene 3 (8), and the demonstration that these mutants have lost or altered specificity for encapsulation of phage DNA (9) and bacterial DNA (10). An alternative view postulates that recombination between phage and bacterial DNA is involved in the generation of generalized transducing particles (3, 11). In this note, we demonstrate the production of generalized transducing particles in vitro, using an in vitro DNA encapsulation system described in the accompanying papers (12,13). As described in the accompanying papers (12, A?), the P22 in vitro DNA encapsulation system involves the preparation of a head-defective “DNA donor” extract to which purified proheads (or a second extract containing proheads) can be added resulting in the formation (in the presence of ATP) of viable phage particles. Since the DNA donor extract contains bacterial DNA as well as concatemeric phage DNA, production of generalized transducing particles might be expected. In order to detect transducing particles, DNA donor extracts were prepared in the usual way (12) using a wild-type (i.e., his+ pyr+) host. Structure donor extracts were made by infection of either a hisG deletion orpyrA deletion host with P22 3%am phage. Thus the only source of his+ or pyr+ trans-

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SHORT COMMUNICATIONS

To verify the supposition that the transducing activity is due to P22 capsids, porFORMATIONOF TRANSDUCINGPARTICLESIN VITRO” tions of the reaction mixtures described in Table 1 were centrifuged to equilibrium in Head immL DNA CsCl to determine the densities of the parTransductants/ml donor phage/ml donor ticles produced. Fractions of the gradient 3 x 109 1180 (pyr +) + were collected and assayed for transducing PY~A+ hisG2 x 109 160 (his+) activity, titers of in vitro encapsulated
missive strains used for preparation of prohead-donor extracts were NK361 (pyr MOl-deletion supplied by P. Margolin) and TR3068 (his01242 hisG8444-deletion supplied by J. Roth). They were infected with P22 3-am N6 13-am HI01 cl-7 and S-30 extracts prepared with chloroform as before (12). Aliquots (0.25 ml) of extract or equivalent buffer were mixed after addition of 15 mM ATP and incubated for 60 min at 35”. Output P22 (i.e., leakage of the head-donor) was assayed on a permissive strain and found to be about l-5 x log phage/ml; in vitro encapsulated phage (immL) were titered on strain DB7221 [leuA414 supE (P22 int3 sie S)] as before (12). Transduction was assayed on P22 int3 sie6 lysogens of NK361 and TR3068 as described elsewhere (14). '0

ducing activity (assayed on the same deletion strains used to make the structure donor extracts) is encapsulation in vitro of bacterial DNA in the DNA donor extracts. Encapsulation of phage DNA in the extracts was assayed, as before, by measuring formation of phage particles carrying the immunity determinant in the DNA donor phage. The data (Table 1) show that transducing activity for two bacterial genes (h&G and pyrA) was observed in mixtures of extracts which also produced viable phage in vitro. None of the extracts incubated alone showed transducing activity. It should be noted that the pyrA and h&G genes are widely separated on the genetic map ofS. typhimurium (15). Generalized transducing particles thus appear to be made in vitro in this system. The frequency (3 x lo-’ pyr+lviable phage and 1 x 1O-7 /&+/viable phage) is well within the range found in vivo for P22 lysates.

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FIG. 1. Density of transducing particles made in vitro and in vivo. Samples were diluted in 50 mM Tris, pH 7.4, and solid C&l was added to make 5.3 ml of a 45% (w/w) solution. The mixtures were centrifuged at 23,000 rpm at 20” for 30 hr in a Beckman SW 50.1 rotor. Four-drop fractions were collected through holes in the bottom of the tubes. Samples were assayed for P22 transduction and P22 phage titers as described in the legend to Table 1. A was titered on strain C600. (a) Density protile of the reaction mixture described in the fmst line of Table 1. About 2 x lo9 immL PFU and 1 x 103pyr+ transducing particles were applied. (b) Density profile of an in vivo lysate of the DNA donor phage made in a permissive host (DB7002,leuA414 supE). About 1.6 x lOI* PFU and 2.6 x 104pyr+ transducing particles were applied.

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particles, while Fig. lb shows a similar anal- encapsulated in place of the intracellular ysis of a lysate of the DNA donor phage concatemeric phage DNA. grown in vivo on a permissive pyr+ host. It is clear that the in vitro transducing acACKNOWLEDGMENTS tivity has a density similar to that of P22 This research was supported by Grants GM21253 phage and identical to transducing particles made in vivo. Analysis of the density of and GM13973 from the National Institutes of Health. his+ transducing activity gave similar results (not shown). Further evidence that REFERENCES P22 capsids are responsible for the transN. D., and LEDERBERG, J., J. Bacterial. ducing activity in the mixed extracts was 1. ZINDER, 64, 6’79-699 (1952). obtained by showing that the activity is sen- 2. SUSSKIND, M. M., and BOTSTEIN, D., Microbial. sitive to anti-P22 serum (data not shown). Rev. 42, 385-413 (1978). We conclude that generalized transduc3. SCHMIEGER, H., Mol. Gen. Genet. 109, 323-337 ing particles are made in normal numbers (1970). in our in vitro DNA encapsulation system. 4. EBEL-TSIPIS, J., BOTSTEIN, D., and Fox, M. S., J. Mol. Biol. 71, 433-448 (1972). As shown in the accompanying papers (12, 13), the critical action of the product of gene 5. IKEDA, H., and TOMIZAWA, J., J. Mol. Biol. 14, 85- 109 (1965). 3 has not been successfully observed in 6. TYE, B. K., HUBERMAN, J. A., and BOTSTEIN, vitro, and thus it seems likely that the bacD., J. Mol. Biol. 85, 501-532 (1974). terial DNA which is encapsulated in vitro 7. CHELALA, C. A., and MARGOLIN, P., Mol. Gen. has already been acted upon in vivo by the Genet. 131, 97-112 (1974). product of gene 3 before the extracts were 8. RAJ, A. S., RAT, A. Y., and SCHMIEGER, H., Mol. prepared. Thus it seems unlikely that DNA Gen. Genet. 135, 175- 184 (1974). from uninfected cells or other sources can 9. TYE, B. K., J. Mol. Biol. 100, 421-426 (1976). be encapsulated in the system in its present 10. SCHMIEGER, H., and BACKHAUS, H., Mol. Gen. Genet. 143, 307-309 (1976). form. It also seems worth noting that the DNA 11. BACKHAUS, H., and SCHMIEGER, H., Mol. Gen. Genet. 131, 123-135 (1974). encapsulation system is insensitive to novo12. POTEETE, A. R., JARVIK, V., and BOTSTEIN, D., biocin and nalidixic acid, inhibitors of DNA Virology 95, 550-564 (1979). replication, and that recombination among 1s. POTEETE, A. R., and BOTSTEIN, D., Virology 95, phages has not been observed in the system. 565-573 (1979). We suggest that these observations are 14. EBEL-TSIPIS, J., and BOTSTEIN, D., Virology 45, most consistent with a simple model of the 629-637 (1971). origin of generalized transducing particles: 15. SANDERSON, K. E., and HARTMAN, P., Microbial. Rev. 42, 471-519 (1978). bacterial DNA is occasionally mistakenly