Directed evolution of cellobiose utilization in Escherichia coli K12

The cellobiose catabolic system of Escherichia coli K12 is being used tostudy the role of cryptic genes in evolution of new functions. Escherichiacoli does not use beta-glucoside sugars; however, mutations in several locican activate the cryptic bgl operon and permit growth on the beta-glucosidesugars arbutin and salicin. Such Bgl+ mutants do not use cellobiose, whichis the most common beta-glucoside in nature. We have isolated a Cel+(cellobiose-utilizing) mutant from a Bgl+ mutant of E. coli K12. The Cel+mutant grows well on cellobiose, arbutin, and salicin. Genes forutilization of these beta-glucosides are located at 37.8 min on the E. colimap. The genes of the bgl operon are not involved in cellobioseutilization. Introduction of a deletion covering bgl does not affect theability to utilize cellobiose, arbutin, or salicin, indicating that the newCel+ genes provide all three functions. Spontaneous cellobiose negativemutants also become arbutin and salicin negative. Analysis ofbeta-glucoside positive revertants of these mutants indicates that thereare separate loci for utilization of each of the beta-glucoside sugars. Thegenes are closely linked and may be activated from a single locus. A fourthgene at an unknown location increases the growth rate on cellobiose. Thecel genes constitute a second cryptic system for beta-glucoside utilizationin E. coli K12.