Expression of the phage λ recombination genes exo and bet under lacPO control on a multi-copy plasmid

The bacteriophage λ genes exo and bet, whose products (λ exonuclease and β protein, respectively; Red phenotype) mediate homologous recombination of λ phages, have been cloned under lacPOlacI^q control on multi-copy plasmids. Induction of recA3 cells harboring these plasmids with isopropylthiogalactoside (IPTG) resulted in λ exonuclease levels (assayed in vitro) that were proportional to the time of induction (for at least 4 h); recombination of λ Red^? phages in vivo was similarly inducible. Only one out of 25 bet^Δ plasmids (constructed by a variety of in vitro techniques) expressed λ exonuclease, a result consistent with the polarity of several known phage bet mutations. A general method for transferring phage exo and bet mutations to plasmids was devised and plasmids bearing polar (bet3) and nonpolar (bet113) mutations were constructed. Mutant derivatives of the plasmid showed the same complementation pattern as analogous phage red mutants. When λbet3 phages (Exo^?Bet^?) infected IPTG-induced recA3 bacteria containing exo⁺bet⁺ plasmids, recombination frequencies were no more than twice those typical for infection of plasmid-free recA3 cells with exo⁺bet⁺ phages, even in the case of IPTG induction sufficient to elevate the production of λ exonuclease about 100-fold. Even when plasmid induction was delayed till as late as 50 min after infection, recombination was significant. Preliminary experiments suggest that these plasmids encode a polypeptide with Gam activity that corresponds to the 98-amino acid “shorter” open reading frame assigned to gam by Sanger et al.