Cloning, mapping, and expression of genes involved in the fatty acid-degradative multienzyme complex of Escherichia coli

S. K. Spratt, P. N. Black, M. M. Ragozzino, W. D. Nunn

Research output: Contribution to journalArticle

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Abstract

Two protein subunits (42,000 and 78,000 daltons) encoded by the fadAB genes form a multifunctional enzyme complex containing thiolase, 3-hydroxyacyl-coenzyme A dehydrogenase, crotonase, epimerase, and isomerase activities (S. Pawar and H. Schulz, J. Biol. Chem. 256:3894-3899, 1981). In an attempt to characterize the structural organization and regulatory properties of these genes, a 5.2-kilobase pair fragment containing the fadAB genes has been isolated. Plasmids containing this fragment (i) complement mutations in the fadAB genes; (ii) overproduce by 10- to 50-fold thiolase, 3-hydroxyacyl-coenzyme A dehydrogenase and crotonase; and (iii) specify a 42,000- and a 78,000-dalton protein. The fadA gene, which encodes the 42,000-dalton protein, has been localized within the original clone to a 3.3-kilobase pair fragment. Thiolase activity, which is encoded by the 42,000-dalton protein, was not observed in the absence of the 78,000-dalton protein, suggesting that an intact complex is required for function. Transposon Tn5 insertional mutagenesis of the cloned fadAB genes has demonstrated that both fadA and fadB are transcribed as a single transcriptional unit with the direction of transcription from fadA to fadB. The molecular cloning and characterization of the fadAB region confirm the original genetic contention that the genes encoding the proteins for the multifunctional complex form an operon.

Original languageEnglish (US)
Pages (from-to)535-542
Number of pages8
JournalJournal of bacteriology
Volume158
Issue number2
StatePublished - Jan 1 1984

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Multienzyme Complexes
Chromosome Mapping
Organism Cloning
Fatty Acids
Escherichia coli
Gene Expression
Enoyl-CoA Hydratase
Genes
Coenzyme A
Proteins
Oxidoreductases
Multifunctional Enzymes
Racemases and Epimerases
Isomerases
Insertional Mutagenesis
Protein Subunits
Molecular Cloning
Regulator Genes
Operon
Plasmids

ASJC Scopus subject areas

  • Microbiology
  • Molecular Biology

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Cloning, mapping, and expression of genes involved in the fatty acid-degradative multienzyme complex of Escherichia coli. / Spratt, S. K.; Black, P. N.; Ragozzino, M. M.; Nunn, W. D.

In: Journal of bacteriology, Vol. 158, No. 2, 01.01.1984, p. 535-542.

Research output: Contribution to journalArticle

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