A non-essential glutamyl aminopeptidase is required for optimal growth of Lactococcus lactis MG1363 in milk

Institute of Food Research, Norwich Laboratory, Norwich Research Park, Colney, Norwich NR4 7UA, UK Institute of Food Research, Reading Laboratory, Earley Gate, Whiteknights Road, Reading RG6 6BZ, UK Biochemistry and Molecular Biology Department, Leeds University, Leeds LS2 9JT, UK ABSTRACT Summary: Degenerate PCR primers were designed from the N-terminal amino acid sequence of a glutamyl aminopeptidase (PepA) from Lactococcus lactis . These primers were used to screen a lambda library for clones containing the gene ( pepA ) encoding PepA. The DNA sequence of a 2-1 kb fragment containing pepA was determined. The sequence revealed the presence of one complete and two incomplete open reading frames (ORFs). The complete ORF encodes a putative protein of 353 amino acids with a predicted N-terminal sequence identical to that determined for purified PepA. The pepA gene was subcloned on an Escherichia coli plasmid vector and production of active PepA was confirmed by means of a zymogram. Mutants of L. lactis in which the pepA gene was inactivated grew to normal cell densities in milk but exhibited a reduced growth rate during the exponential phase. Thus whilst PepA is required for optimal growth it is not essential. Author for correspondence: K. I'Anson. Tel: + 44 1603 255000. Fax: +44 1603 507723. Keywords: aminopeptidase, PCR screening, Lactococcus lactis subsp. lactis , nucleotide sequence analysis