CHARACTERIZATION AND EXPRESSION OF THE LACTOBACILLUS-HELVETICUS PEPC GENE ENCODING A GENERAL AMINOPEPTIDASE

An aminopeptidase C gene (pepC) was detected by nucleic acid hybridiza tion from an industrially important Lactobacillus helveticus strain. T hree hybridization positive clones were isolated from a gene library o f this L. helveticus strain, and one of them was characterized in more detail. Deletion mapping localized the hybridization positivity into a 2.8-kb fragment, which also encoded aminopeptidase activity. This fr agment was sequenced and two open reading frames (ORF1 and 2) of 1347 and 840 base pairs were identified. The ORF1 was preceded by a typical prokaryotic promoter region, and an inverted repeat structure with De lta G of -49.0 kJ mol(-1) was found downstream of the coding region. T he deduced amino acid sequence of ORF1, with an encoding capacity for a 51.4-kDa protein, was shown to share 48.3% and 98.0% identities with the PepC proteins from Lactococcus lactis and L. helveticus CNRZ32, r espectively, thus confirming that ORF1 codes for an aminopeptidase C. mRNA size analyses revealed 1.7-kb and 2.7-kb transcripts in Northern blot with the pepC-specific probe. A further analysis with the pepC- a nd ORF2-specific probes showed that downstream ORF2 is co-transcribed with the pepC gene at the exponential phase of growth whereas, at the stationary growth phase, transcripts derived from the pepC promoter we re below the detection limit, and the ORF2 was expressed by its own pr omoter. The 5' end mapping of the pepC transcripts with primer extensi on revealed one transcription start site suggesting a new position for the pepC promoter region when compared to that predicted for the L. h elveticus CNRZ32 pepC gene. Expression of pepC was also studied in L. helveticus as the function of growth in a bioreactor study. Transcript ion of pepC was typical to exponential growth phase expression. The le vel of total thiol-aminopeptidase activity, however, remained nearly c onstant throughout the stationary growth phase.