f The IdhA Gene Encoding the Fermentative Lactate Dehydrogenase of Escherichia Coli
- Authors: Pamela K. Bunch, Fairoz Mat-Jan†, Norizan Lee, David P. Clark1
- 1Author for correspondence: David P. Clark. Tel: + 1 618 453 3737. Fax: +1 618 453 8036.
- Microbiology, January 1997 143: 187-195, doi: 10.1099/00221287-143-1-187
- Subject: Physiology And Growth
- Published Online:
Under anaerobic conditions, especially at low pH, Escherichia coli converts pyruvate to D-lactate by means of an NADH-linked lactate dehydrogenase (LDH). This LDH is present in substantial basal levels under all conditions but increases approximately 10-fold at low pH. The IdhA gene, encoding the fermentative lactate dehydrogenase of E. coli, was cloned using λ10E6 of the Kohara collection as the source of DNA. The IdhA gene was subcloned on a 2.8 kb MIuI-MIuI fragment into a multicopy vector and the region encompassing the gene was sequenced. The IdhA gene of E. coli was highly homologous to genes for other D-lactate-specific dehydrogenases but unrelated to those for the L-lactate-specific enzymes. We constructed a disrupted derivative of the IdhA gene by inserting a kanamycin resistance cassette into the unique KpnI site within the coding region. When transferred to the chromosome, the IdhA::Kan construct abolished the synthesis of the D-LDH completely. When present in high copy number, the IdhA gene was greatly overexpressed, suggesting escape from negative regulation. Cells expressing high levels of the D-LDH grew very poorly, especially in minimal medium. This poor growth was largely counteracted by supplementation with high alanine or pyruvate concentrations, suggesting that excess LDH converts the pyruvate pool to lactate, thus creating a shortage of 3-carbon metabolic intermediates. Using an IdhA-cat gene fusion construct we isolated mutants which no longer showed pH-dependent regulation of the IdhA gene. Some of these appeared to be in the pta gene, which encodes phosphotransacetylase, suggesting the possible involvement of acetyl phosphate in IdhA regulation.
Present address: Immucor Inc., 3130 Gateway Drive, PO Box 5265, Norcross, GA 30071, USA.
© Society for General Microbiology 1997 | Published by the Microbiology Society
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