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Journal of Bacteriology, November 2000, p. 6049-6054, Vol. 182, No. 21
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Aerobic Activity of Escherichia coli Alcohol Dehydrogenase Is Determined by a Single Amino Acid

Carol A. Holland-Staley,1 KangSeok Lee,2 David P. Clark,3 and Philip R. Cunningham4,*

Infectious Disease Research, Henry Ford Hospital,1 and Department of Biological Sciences, Wayne State University,4 Detroit, Michigan 48202; Department of Genetics, Stanford University School of Medicine, Stanford, California 943052; and Department of Microbiology, Southern Illinois University, Carbondale, Illinois 629013

Received 3 April 2000/Accepted 14 August 2000

Expression of the alcohol dehydrogenase gene, adhE, in Escherichia coli is anaerobically regulated at both the transcriptional and the translational levels. To study the AdhE protein, the adhE+ structural gene was cloned into expression vectors under the control of the lacZ and trpc promoters. Wild-type AdhE protein produced under aerobic conditions from these constructs was inactive. Constitutive mutants (adhC) that produced high levels of AdhE under both aerobic and anaerobic conditions were previously isolated. When only the adhE structural gene from one of the adhC mutants was cloned into expression vectors, highly functional AdhE protein was isolated under both aerobic and anaerobic conditions. Sequence analysis revealed that the adhE gene from the adhC mutant contained two mutations resulting in two amino acid substitutions, Ala267Thr and Glu568Lys. Thus, adhC strains contain a promoter mutation and two mutations in the structural gene. The mutant structural gene from adhC strains was designated adhE*. Fragment exchange experiments revealed that the substitution responsible for aerobic expression in the adhE* clones is Glu568Lys. Genetic selection and site-directed mutagenesis experiments showed that virtually any amino acid substitution for Glu568 produced AdhE that was active under both aerobic and anaerobic conditions. These findings suggest that adhE expression is also regulated posttranslationally and that strict regulation of alcohol dehydrogenase activity in E. coli is physiologically significant.


* Corresponding author. Mailing address: Department of Biological Sciences, Wayne State University, Detroit, MI 48202. Phone: (313) 577-5029. Fax: (313) 577-6891. E-mail: philip.cunningham{at}wayne.edu.


Journal of Bacteriology, November 2000, p. 6049-6054, Vol. 182, No. 21
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.



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