Regulation of expression of the ethanol dehydrogenase gene (adhE) in Escherichia coli by catabolite repressor activator protein Cra.
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.
Mikulskis
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Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
Aristarkhov
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Lin
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EC
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
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