The rap gene of bacteriophage was placed in the chromosome of an Escherichia coli K-12 strain in which the recBCD gene cluster had previously been replaced by the red genes and in which the recG gene had been deleted. Recombination between linear double-stranded DNA molecules and the chromosome was tested in variants of the recG⌬ red ؉ rap ؉ strain bearing mutations in genes known to affect recombination in other cellular pathways. The linear DNA was a 4-kb fragment containing the cat gene, with flanking lac sequences, released from an infecting phage chromosome by restriction enzyme cleavage in the cell. Replacement of wild-type lacZ with lacZ::cat was monitored by measuring the production of Lac-deficient chloramphenicolresistant bacterial progeny. The results of these experiments indicated that the rap gene could functionally substitute for the E. coli ruvC gene in Red-mediated recombination.

Replacement of the recBCD genes of Escherichia coli with the red recombination genes of bacteriophage lambda results in a strain in which adaptive mutation occurs at an elevated frequency. Like RecBCDdependent adaptive mutation, Red-mediated adaptive mutation is dependent upon recA and ruvABC functions.Adaptive mutation is a process by which an organism under nonlethal selective pressure produces mutations that relieve the selective pressure (see reference 5 for a review). It is illustrated particularly well by Escherichia coli strain FC40, a strain bearing an FЈ episome carrying a lac allele with a frameshift mutation that makes it phenotypically Lac Ϫ . When FC40 is plated on lactose-minimal medium, Lac ϩ revertants appear at a high rate over several days, largely in the absence of cell growth, death, or turnover (2, 4). The formation of these adaptive mutations, unlike mutations that occur during growth under nonselective conditions, depends on the RecA-RecBCD recombination pathway (2,8).E. coli strains in which the RecBCD function is replaced by the Red recombination system of bacteriophage exhibit a hyperrecombination phenotype (13,14). The possibility that the activity of the cell's recombination system might be a ratelimiting factor in the production of adaptive revertants led us to test whether Red ϩ bacteria might produce such revertants at an elevated rate. We report here that they do.Bacterial strains. P1 transduction was used to replace the recC-ptr-recB-recD gene cluster in FC40 and FC691 with a sequence bearing both P tac -gam-bet-exo and the cat gene. (The P tac -gam-bet-exo sequence is designated red.) Recombinants were selected for chloramphenicol resistance, producing strains TP694 and TP730. Strains TP705 and TP732 were made by transforming TP694 and TP730 with linear DNA resulting from the digestion of plasmid pTP822 (16). Recombinants, in which ⌬recBCD::red-cat was replaced by ⌬recBCD::red-paecI822, were selected for immunity to phage and screened for sensitivity to chloramphenicol and kanamycin. These and other strains employed in this study are described in Table 1.Media. The lysogeny broth (LB) used contained, per liter, 10 g of tryptone, 5 g of yeast extract, 5 g of NaCl, and 1 ml of 1 M NaOH. It was solidified with 15 g of agar per liter and supplemented as necessary with 25 g of tetracycline/ml, 20 g of chloramphenicol/ml, 0.1 mM isopropylthiogalactopyranoside (IPTG), or 80 g of 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside (X-Gal)/ml.M9 salt solution contained, per liter, 6.3 g of Na 2 HPO 4 , 3.0 g of KH 2 PO 4 , 0.5 g of NaCl, and 1.0 g of NH 4 Cl. M9 glycerolminimal medium consisted of M9 salt solution plus 1 mM MgSO 4 , 5 g of thiamine/ml, 0.1 mM CaCl 2 , 10 g of gelatin/ ml, and 1 mg of glycerol/ml. M9 lactose-minimal agar consisted of M9 salt solution plus 1 mM MgSO 4 , 5 g of thiamine/ml, 1 mg of lactose/ml, and 15 mg of agar/ml. Measurement of stationary-phase reversion to Lac؉ . The strains to be tested were scraped from frozen cultures in storage vials and streaked on LB ...