Phage Red-Mediated Adaptive Mutation

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 F episome carrying a lac allele with a frameshift mutation that makes it phenotypically Lac . When FC40 is plated on lactose-minimal medium, Lac 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 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 Ptac-gam-bet-exo and the cat gene. (The Ptac-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 Na2HPO4, 3.0 g of KH2PO4, 0.5 g of NaCl, and 1.0 g of NH4Cl. M9 glycerolminimal medium consisted of M9 salt solution plus 1 mM MgSO4, 5 g of thiamine/ml, 0.1 mM CaCl2, 10 g of gelatin/ ml, and 1 mg of glycerol/ml. M9 lactose-minimal agar consisted of M9 salt solution plus 1 mM MgSO4, 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 plates supplemented with appropriate antibiotics. A single colony was used to inoculate 5 ml of LB broth, and the culture was grown with aeration at 30 or 37°C to apparent saturation. The LB culture was diluted 10fold with M9 glycerol-minimal medium, with a final 100-fold dilution into three to six tubes containing 1 ml each. These cultures in M9 glycerol-minimal medium were grown with aeration at 30 or 37°C to apparent saturation, and 0.01 to 0.5 ml of each culture was plated on one M9 lactose-minimal plate with approximately 2 10 scavenger cells. Scavenger cells (FC29) were grown to saturation in M9 glycerol-minimal medium and concentrated by centrifugation and resuspension in 0.1 volume of M9 salt solution; thereafter, 0.1 ml was plated. The plates were incubated at 37°C; colony counts were recorded on days 2 through 6 or 7. (Lac cells present in the culture at the time of plating take 2 days to form colonies.) The few plates with large numbers of preexisting revertants were ignored. Titers of the tested cultures were determined by plating on LB agar at 37°C. Red-mediated reversion of episomal lac. A derivative of the F (lacI33-lacZ)-bearing E. coli strain FC40, in which the recBCD gene cluster is replaced by the red system of phage , exhibits an elevated rate of adaptive mutation to Lac relative to its recBCD parent (Fig. 1a). As in recBCD bacteria (7, 9), the rate is significantly lower in the absence of the ruv genes (Fig. 1b) and lower still in the absence of recA (Fig. 1c). Deletion of recG from the red-substituted strain mildly increases the rate of reversion (Fig. 1d). The red recG strain and its recBCD recG counterpart exhibit approximately equal rates of reversion (data not shown). Although Red stimulates the rate of adaptive reversion, it apparently has little effect on the rate of mutation occurring during nonselective growth. Revertants arising during the growth of the cultures form visible colonies in 2 days. The average numbers of colonies appearing on day 2 were 3.5 per 10 cells plated for the wild type (average of 8 experiments) * Corresponding author. Mailing address: Dept. of Molecular Genetics & Microbiology, University of Massachusetts Medical School, 55 Lake Ave. North, Worcester, MA 01655. Phone: (508) 856-3708. Fax: (508) 856-5920. E-mail: [email protected].