title: Regulation of expression of the ethanol dehydrogenase gene (adhE) in Escherichia coli by catabolite repressor activator protein Cra.
abstract: The adhE gene encodes ethanol dehydrogenase and is located at min 27.9 of the Escherichia coli chromosome. Expression of adhE is about 10-fold higher in cells grown anaerobically than in cells grown aerobically and is dependent on both transcriptional and posttranscriptional factors. In this study, a trans-regulatory element repressing adhE expression was characterized by genetic and biochemical approaches. A mutation downregulating adhE expression was mapped at min 2 of the chromosome. DNA sequence analysis revealed a missense mutation in the cra gene, formerly known as fruR. The cra gene encodes a catabolite repressor-activator protein (Cra) involved in the modulation of carbon flow in E. coli. The mutant protein (Cra*) sustained an Arg148-->His substitution causing 1.5- and 3-fold stronger repression of adhE transcription under anaerobic and aerobic conditions, respectively. By contrast, cra null mutants displayed 1.5- and 4-fold increased adhE transcription under those conditions. Disruption of the cra gene did not abolish the anaerobic activation of the adhE gene but diminished it twofold. Cra and Cra* were purified as fusion proteins tagged with an N-terminal 6xHis element. In vitro, both fusion proteins showed binding to the adhE promoter region and to the control fruB promoter region, which is a known Cra target. However, only 6xHis-tagged Cra, and not 6xHis-Cra*, was displaced from the DNA target by the effector, fructose-1-phosphate (F1P), suggesting that the mutant protein is locked in a promoter-binding conformation and is no longer responsive to F1P. We suggest that Cra helps to tighten the control of adhE transcription under aerobic conditions by its repression.
authors.affiliation: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: A
authors.initials: A
authors.lastname: Mikulskis
authors.forename: A
authors.initials: A
authors.lastname: Aristarkhov
authors.forename: E C
authors.initials: EC
authors.lastname: Lin
mesh_terms: D000332:Aerobiosis; D000426:Alcohol Dehydrogenase / Q000235:genetics* / Q000378:metabolism*; D000445:Aldehyde Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D019943:Amino Acid Substitution; D000693:Anaerobiosis; D001120:Arginine / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002874:Chromosome Mapping; D002876:Chromosomes, Bacterial; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D005636:Fructosephosphates / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006639:Histidine / Q000235:genetics; D009097:Multienzyme Complexes / Q000235:genetics* / Q000378:metabolism*; D016254:Mutagenesis, Insertional; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic; D056968:Lac Repressors
chemical_list: D000426:Alcohol Dehydrogenase; D000445:Aldehyde Oxidoreductases; D001120:Arginine; D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D005636:Fructosephosphates; D006639:Histidine; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C489416:FruR protein, E coli; C068837:FruR protein, Bacteria; D056968:Lac Repressors; C032284:fructose-1-phosphate; C068383:adhE protein, E coli