1887

Abstract

Under anaerobic conditions, especially at low pH, 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 gene, encoding the fermentative lactate dehydrogenase of , was cloned using λ10E6 of the Kohara collection as the source of DNA. The gene was subcloned on a 2.8 kb I fragment into a multicopy vector and the region encompassing the gene was sequenced. The gene of was highly homologous to genes for other -lactate-specific dehydrogenases but unrelated to those for the -lactate-specific enzymes. We constructed a disrupted derivative of the gene by inserting a kanamycin resistance cassette into the unique I site within the coding region. When transferred to the chromosome, the :Kan construct abolished the synthesis of the D-LDH completely. When present in high copy number, the 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 gene fusion construct we isolated mutants which no longer showed pH-dependent regulation of the gene. Some of these appeared to be in the gene, which encodes phosphotransacetylase, suggesting the possible involvement of acetyl phosphate in regulation.

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1997-01-01
2021-07-27
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