Anaerobic regulation of the adhE gene, encoding the fermentative alcohol dehydrogenase of Escherichia coli.
The regulation of the adhE gene, which encodes the trifunctional fermentative acetaldehyde-alcohol dehydrogenase of Escherichia coli, was investigated by the construction of gene fusions and by two-dimensional protein gel electrophoresis. Both operon and protein fusions of adhE to lacZ were induced 10- to 20-fold by anaerobic conditions, and both fusions were repressed by nitrate, demonstrating that regulation is at the level of transcription. Nitrate repression of phi (adhE-lacZ) expression, as well as of alcohol dehydrogenase enzyme activity, was partly relieved by a mutation in narL. Mutations in rpoN or fnr had no effect on the expression of adhE. Two-dimensional protein gels demonstrated that increases in the amount of adhE protein correlated with increases in enzyme activity, demonstrating that induction was due to synthesis of new protein, not to activation of preexisting protein. When oxidized sugar derivatives such as gluconate or glucuronate were used as carbon sources, the anaerobic expression of phi (adhE-lacZ) was greatly reduced, whereas when sugar alcohols such as sorbitol were used, the expression was increased compared with expression when glucose was the carbon source. This observation suggested that induction of phi (adhE-lacZ) might depend on the level of reduced NADH, which should be highest with sorbitol-grown cells and lowest with glucuronate-grown cells. When phi (adhE-lacZ) was present in a strain deleted for the adhE structural gene, anaerobic expression of phi (adhE-lacZ) was approximately 10-fold higher than in an adhE+ strain. Since the presence of alcohol dehydrogenase would serve to decrease NADH levels, this finding again implies that the adhE gene is regulated by the concentration of reduced NAD. Introduction of a pgi (phosphoglucose isomerase) mutation reduced the anaerobic induction of phi(adhE-lacZ) when the cells were grown on glucose, but had little effect on fructose-grown cells. Pyruvate did not overcome the pgi effect, but glycerol 3-phosphate did, which is again consistent with the possibility that adhE expression responds to the level of reduced NAD rather than to a glycolytic intermediate.
Leonardo
M R
MR
Department of Microbiology, Southern Illinois University, Carbondale, 62901.
Cunningham
P R
PR
Clark
D P
DP
D000426:Alcohol Dehydrogenase / Q000235:genetics*; D000693:Anaerobiosis; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D015180:Electrophoresis, Gel, Two-Dimensional; D004790:Enzyme Induction; D004926:Escherichia coli / Q000235:genetics*; D005285:Fermentation; D015964:Gene Expression Regulation, Bacterial; D006019:Glycolysis; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D009243:NAD; D000426:Alcohol Dehydrogenase