Cloning and characterization of the thymidylate synthase gene from Lactococcus lactis subsp. lactis

Abstract

The thymidylate synthase (thyA) gene has been isolated from Lactococcus lactis subsp. lactis. The cloned gene was strongly expressed in Escherichia coli both in vivo and in vitro (maxicells and cell-free transcription and translation systems) and complemented E. coli thyA mutants. DNA-DNA hybridizations demonstrated that the thyA gene is encoded by the chromosome of L. lactis subsp. lactis. By sequential deletion of DNA outside the complementing region, the thyA gene was localized to a 1.1-kilobase DNA fragment. The nucleotide sequence of the lactococcal thyA gene was determined by the dideoxy-chain termination technique. The derived amino acid sequence indicated a protein size of 32,580 daltons, which is in good agreement with results obtained from maxicell and in vitro transcription and translation experiments. The primary sequence is homologous to 12 other thyA proteins from a variety of other organisms. Upstream from the structural gene, -10 and -35 promoter sequences which were almost canonical sigma-70 promoter sequences were identified, which may explain the strong expression of the thyA gene observed in E. coli. An A-T-rich sequence characteristic of gram-positive promoters was also noted adjacent to the -35 region. The thyA gene has potential as a marker for plasmid maintenance and selection in food systems.