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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM039693-05
Application #
3296798
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1988-04-01
Project End
1993-03-31
Budget Start
1992-04-01
Budget End
1993-03-31
Support Year
5
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Harvard University
Department
Type
Schools of Medicine
DUNS #
082359691
City
Boston
State
MA
Country
United States
Zip Code
02115
De Wulf, P; Lin, E C (2000) Cpx two-component signal transduction in Escherichia coli: excessive CpxR-P levels underlie CpxA* phenotypes. J Bacteriol 182:1423-6
Pellicer, M T; Fernandez, C; Badia, J et al. (1999) Cross-induction of glc and ace operons of Escherichia coli attributable to pathway intersection. Characterization of the glc promoter. J Biol Chem 274:1745-52
Membrillo-Hernandez, J; Lin, E C (1999) Regulation of expression of the adhE gene, encoding ethanol oxidoreductase in Escherichia coli: transcription from a downstream promoter and regulation by fnr and RpoS. J Bacteriol 181:7571-9
De Wulf, P; Kwon, O; Lin, E C (1999) The CpxRA signal transduction system of Escherichia coli: growth-related autoactivation and control of unanticipated target operons. J Bacteriol 181:6772-8
McGuire, A M; De Wulf, P; Church, G M et al. (1999) A weight matrix for binding recognition by the redox-response regulator ArcA-P of Escherichia coli. Mol Microbiol 32:219-21
Pogliano, J; Lynch, A S; Belin, D et al. (1997) Regulation of Escherichia coli cell envelope proteins involved in protein folding and degradation by the Cpx two-component system. Genes Dev 11:1169-82
Aristarkhov, A; Mikulskis, A; Belasco, J G et al. (1996) Translation of the adhE transcript to produce ethanol dehydrogenase requires RNase III cleavage in Escherichia coli. J Bacteriol 178:4327-32
Lynch, A S; Lin, E C (1996) Transcriptional control mediated by the ArcA two-component response regulator protein of Escherichia coli: characterization of DNA binding at target promoters. J Bacteriol 178:6238-49
Chen, Y M; Lin, E C (1991) Regulation of the adhE gene, which encodes ethanol dehydrogenase in Escherichia coli. J Bacteriol 173:8009-13
Hidalgo, E; Chen, Y M; Lin, E C et al. (1991) Molecular cloning and DNA sequencing of the Escherichia coli K-12 ald gene encoding aldehyde dehydrogenase. J Bacteriol 173:6118-23

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