Fitness effects of amino acid replacements in the beta-galactosidase of Escherichia coli

Two genetic procedures were used to obtain amino acid replacements in thelacZ-encoded beta-galactosidase in Escherichia coli. Amino acidreplacements could be obtained without regard to their effects on lactaseactivity by selecting spontaneous mutations that relieved the strongpolarity of six nonsense mutations. When streaked on MacConkey- lactoseindicator plates, approximately 75% of these mutants gave strong redlactose-fermenting colonies, and 25% gave white nonfermenting colonies.Mutants from 11 other nonsense codons were isolated directly usingMacConkey-lactose indicator plates, on which positive color indicationrequires only 0.5% of the wildtype lactase activity. Among the total of 17codons, 25 variant beta-galactosidases were identified usingelectrophoresis and thermal denaturation studies. The fitness effects ofthese variant beta-galactosidases were determined using competitionexperiments conducted with lactose as the sole nutrient limiting the growthrate in chemostat cultures. Three of the replacements were deleterious, onewas selectively advantageous, and the selective effects of the remaining 21were undetectable under conditions in which the smallest detectableselection coefficient was approximately 0.4%/generation.