PEDIOCOCCUS-ACIDILACTICI IDHD GENE - CLONING, NUCLEOTIDE-SEQUENCE, AND TRANSCRIPTIONAL ANALYSIS

The gene encoding D-lactate dehydrogenase was isolated on a 2.9-kb ins ert from a library of Pediococcus acidilactici DNA by complementation for growth under anaerobiosis of an Escherichia coli lactate dehydroge nase and pyruvate-formate lyase double mutant. The nucleotide sequence of IdhD encodes a protein of 331 amino acids (predicted molecular mas s of 37,210 Da) which shows similarity to the family of D-2-hydroxyaci d dehydrogenases. The enzyme encoded by the cloned fragment is equally active on pyruvate and hydroxypyruvate, indicating that the enzyme ha s both D-lactate and D-glycerate dehydrogenase activities. Three other open reading frames were found in the 2.9-kb insert, one of which (rp sB) is highly similar to bacterial genes coding for ribosomal protein S2. Northern (RNA) blotting analyses indicated the presence of a 2-kb dicistronic transcript of IdhD (a metabolic gene) and rpsB (a putative ribosomal protein gene) together with a l-kb monocistronic rpsB mRNA. These transcripts are abundant in the early phase of exponential grow th but steadily fade away to disappear in the stationary phase. Primer extension analysis identified two distinct promoters driving either c otranscription of ldhD and rpsB or transcription of rpsB alone.