Evolutionary potential of the ebgA gene.

Twenty years ago a study was initiated (Hall and Hart1 1974) whose purposes were to understand the evolutionary potential that lay within the genome of an organism, and to understand the processes and mechanisms by which the organism could exploit that potential to adapt to new selective pressures (Hall 1990). That study exploited the ebg operon of Escherichia coli K 12. The ebg operon encodes a second P-galactosidase ( Hart1 and Hall 1974)) whose existence was detected when the LacZ gene was deleted (Campbell et al. 1973 ) . The ebg operon was used extensively as a model system to study the acquisitive evolution of new enzymatic functions at the genetic level (reviewed in Hall 1984, 1990), at the biochemical level (Hall 198 1; Burton and Sinnott 1983; Hall et al. 1983; Li et al. 1983; Elliott et al. 1992; K et al. 1993), and at the molecular level (Hall et al. 1989). The ebg operon includes the ebgR gene, which specifies a repressor that controls expression of the ebgACB genes (Hall and Hart1 1975; Hall and Clarke 1977; Hall 1978b; Hall et al. 1989). ebgA encodes the 118-kilodalton a subunit, and ebgC encodes the 20-kilodalton p subunit of EBG P-galactosidase enzyme ( Hall et al. 1989 ), while ebgB encodes a product whose function is unknown (Hall and Zuzel 1980a). EBG enzyme is a heterooctamer consisting of four a and four p subunits. The wild-type enzyme, whose function in nature is unknown, is a very feeble P-galactosidase whose activity toward lactose (galactosyl-P1,4-D-glucose) and its analog lactulose (galactosyl-B1,4-D-fructose) is so ineffective that, in AZacZ strains, those sugars cannot be utilized for growth even when the operon is expressed constitutively (ebgR_) at a level such that EBG enzyme constitutes 5% of the cell’s soluble protein (Hall 1982). When a AZacZ ebgR strain is subjected to selection for growth on lactose, only two phenotypic classes of singlestep spontaneous mutants have been obtained among several hundred independent isolates. Class I mutants grow rapidly on lactose, but fail to grow on lactulose, as the result of a mutation that increases the catalytic ef-