Cloning and expression of the Lactococcus lactis purDEK genes, required for growth in milk

An operon containing the genes purD and purE and part of the purK gene was cloned from the facultative anaerobic gram-positive bacterium Lactococcus lactis by complementation of the purD mutation in Escherichia coli SO609. The genes encode enzymes in the de novo pathway of purine nucleotides. The expression of the genes was regulated approximately 35-fold at the transcription level by the availability of purines in the growth medium. Deletion analysis of the nucleotide region upstream of purD indicated that a region of 145 bp is enough to give regulated expression of the reporter lacLM genes, which encode beta-galactosidase. Deletion of a region 79 bp upstream of the transcription start point reduced the promoter activity 33-fold when incubated in a purine-free medium and to values below the detection limit when incubated in a purine-containing medium. No secondary transcription start points were mapped in or close to this region, indicating that a putative activator site and not a promoter was deleted or partly destroyed.