The ldhA gene encoding the fermentative lactate dehydrogenase of Escherichia coli.
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 ldhA gene, encoding the fermentative lactate dehydrogenase of E. coli, was cloned using lambda 10E6 of the Kohara collection as the source of DNA. The IdhA gene was subcloned on a 2.8 kb MluI-MluI 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 ldhA gene by inserting a kanamycin resistance cassette into the unique KpnI site within the coding region. When transferred to the chromosome, the ldhA::Kan construct abolished the synthesis of the D-LDH completely. When present in high copy number, the ldhA 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 ldhA-cat gene fusion construct we isolated mutants which no longer showed pH-dependent regulation of the ldhA gene. Some of these appeared to be in the pta gene, which encodes phosphotransacetylase, suggesting the possible involvement of acetyl phosphate in ldhA regulation.
Bunch
Pamela K
PK
Department of Microbiology, Southern Illinois University,Carbondale, IL 62901,USA.
Mat-Jan
Fairoz
F
Department of Microbiology, Southern Illinois University,Carbondale, IL 62901,USA.
Lee
Norizan
N
Department of Microbiology, Southern Illinois University,Carbondale, IL 62901,USA.
Clark
David P
DP
Department of Microbiology, Southern Illinois University,Carbondale, IL 62901,USA.
D000595:Amino Acid Sequence; D000693:Anaerobiosis; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005285:Fermentation / Q000235:genetics; D018628:Gene Dosage; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D007770:L-Lactate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010755:Organophosphates / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D010755:Organophosphates; D011994:Recombinant Proteins; C011632:acetyl phosphate; D007770:L-Lactate Dehydrogenase