The L-fucose (fuc) and L-rhamnose (rha) systems of Escherichia coli will be studied as models for experimental evolution of novel functions. The fuc structural genes are partitioned in 3 contiguous operons under complex positive control: fucO (encoding propanediol oxidoreductase), fucA (encoding fuculose 1- phosphate aldolase), fucPIK (respectively encoding fucose permease, fucose isomerase, and fuculose kinase). Selection for aerobic constitutive activity of the oxidoreductase by growth on propanediol eventually causes co-constitutivity of fucO and fucA and simultaneous non-inducibility of fucPIK. For testing models of regulation that might account for this mutant phenotype, the regulator fucR locus and the target promoter regions from wild- type and mutant strains will be cloned for functional analysis, DNA sequencing, and in vitro binding to regulatory proteins. Anaerobic growth on rhamnose, involving a different permease, isomerase, kinase, and aldolase, depends on the oxidoreductase encoded by fucO. To explain how rhamnose cross induces the fuc system, the interaction of the rha and fuc controlling elements will be explored by mutant analysis. In wild-type cells the fucO protein appears in a catalytically active form anaerobically but not aerobically. Genetic and biochemical studies will be carried out to characterize this post-transcriptional control and to discover the nature of mutations that allow the synthesis of an enzymically active protein aerobically. Additional mutants will be selected to explore the adaptive potential of E. coli. Mutants that can grow on several analogs of fucose or rhamnose will be selected to see if similar genetic systems follow parallel mechanistic pathways in evolution. Attempts will be made to convert the propanediol oxidoreductase to an aldehyde dehydrogenase and vice versa by using appropriate strains for selection. Attempts will also be made to interconvert the function of the regulatory proteins of the fuc and rha systems by selection of mutants with appropriate deletions. Possible influences on the routes of adaptation by a transposable element near the evolving locus will be probed.
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