The X-prolyl dipeptidyl aminopeptidase gene (pepx) of an industrially used Lactobacillus helveticus strain has been detected by nucleic acid hybridization, cloned, characterized and sequenced. One ORF of 2379 bp with coding capacity for a 906 kDa protein (PepX) was found. The ORF was preceded by a typical prokaryotic promoter region. An inverted repeat structure with AG of -84.1 kJ mol-1 was found downstream of the coding region. The deduced amino acid sequence of the 906 kDa protein showed 49*3,494 and 777% homology with the PepX proteins from Lactococcus lactis subsp. lactis, Lc. lactis subsp. cremoris and Ladobacillus delbnreckii subsp. Iactis, respectively. Northern blotting revealed a 2.6 kb transcript and one transcription start site was identified via primer extension analysis using an A.L.F. sequencer. In a bioreactor study, the expression of pepX in Lb. helveticus was studied as a function of growth. Transcription of pepX was typical of exponential growth phase expression. The pepX gene has been cloned into pKK223-3 and expressed at a high level in Escherichia coli JM105. PepX was purified to homogeneity by ion-exchange and hydrophobic interaction chromatography. Optimum PepX activity was observed at pH 6 5 and 45 OC. According to gel filtration analysis, PepX is a dimer of 165 kDa. The enzyme was inactivated by heavy metal ions such as Cu2+, Cd2+ and Zn2+. EDTA and l,l0-phenanthroline did not decrease PepX activity significantly. It was completely inhibited by p-hydroxymercuribenzoate and reactivated by adding Dll, and strongly inhibited by PMSF. PepX is thus a metal-independent serine peptidase having functional sulfhydryl groups at or near the active site.

An aminopeptidase C gene (pepC) was detected by nucleic acid hybridization from an industrially important Lactobacillus helveticus strain. Three hybridization positive clones were isolated from a gene library of 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 fragment was sequenced and two open reading frames (ORF1 and 2) of 1347 and 840 base pairs were identified. The ORFl was preceded by a typical prokaryotic promoter region, and an inverted repeat structure with AG of -49.0 kJ mol-' was found downstream of the coding region. The 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, respectively, thus confirming that ORFl 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-and 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 were below the detection limit, and the ORF2 was expressed by its own promoter. The 5' end mapping of the pepC transcripts with primer extension revealed one transcription start site suggesting a new position for the pepC promoter region when compared to that predicted for the L. helveticus CNRZ32 pepC gene. Expression of pepC was also studied in L. helveticus as the function of growth in a bioreactor study. Transcription of pepC was typical to exponential growth phase expression. The level of total thiol-aminopeptidase activity, however, remained nearly constant throughout the stationary growth phase.

A dipeptidase gene (pepD) from an industrial Lactobacillus helveticus strain was isolated by colony hybridization. An open reading frame (ORF) of 1422 base pairs (bp) with a coding capacity for a 53.5-kDa protein (PepD) was identified. The ORF was preceded by a typical prokaryotic promoter region, and an inverted repeat structure with delta G of -51.0 kJ mol-1 was found downstream of the coding region. The deduced amino acid sequence of the 53.5-kDa protein revealed no marked homologies when compared to the data bases of EMBL and SWISS-PROT. The 5'end of the 1.6-kb pepD transcript was determined both by a conventional primer extension method and using an automated sequencer. pepD was found to be maximally expressed at late exponential growth. The pepD gene was cloned into an expression vector to over-produce PepD in Escherichia coli JM105. Purification of PepD to homogeneity was achieved using three chromatographic steps. PepD was able to hydrolyze a number of dipeptides with the exception of those containing a proline residue. Optimal PepD activity was observed at pH 6.0 and 55 degrees C. The enzyme was inhibited by p-hydroxymercuribenzoate and reactivated by dithiothreitol whereas ethylenediaminetetraacetate had no inhibitory effect on PepD. The enzymatic properties of PepD suggest that it represents a novel dipeptidase type among lactic acid bacteria.

The operon of the putative lactobacillar oligopeptide transport system (Opp) from Lactobacillus delbrueckii subsp. bulgaricus B14 was cloned and characterized. The opp operon was found to consist of five genes, oppD, oppF, oppB, oppC and oppA (1). In addition, an oppA (1) homolog, oppA (2), was found downstream of the operon. Sequence comparisons of the L. delbrueckii subsp. bulgaricus Opp system with other bacterial transport systems revealed the highest similarity to the oligopeptide transport system of Lactococcus lactis. Northern analyses of oppmRNAs revealed 6.1-kb and 2.1-kb transcripts, confirming that, in addition to the operon structure oppDFBCA (1), the oppA (1) gene was also expressed as a monocistronic transcript. The oppA (2) gene was expressed as a separate 2.1-kb monocistronic transcript with a low expression level. Primer-extension mapping of the 5'end of oppDFBCA (1) mRNA revealed two adjacent transcriptional start sites, and primer extension analyses of oppA (1) and oppA (2) mRNAs confirmed the location of the predicted promoters of these genes. For complementation analysis, oppA (1) alone and the operon constructs oppDFBCA (1) and oppDFBCA (2) were fused with the nisA promoter and expressed in Lactococcus lactisNZ9000Delta oppA strain. Only the L. delbrueckii subsp. bulgaricus oppDFBCA (1)genes were able to complement the L. lactis oppA mutation.

An aminopeptidase N (pepN) gene was detected by DNA hybridization from an industrially important Lactobacillus helveticus strain using part of the L. helveticus CNRZ32 pepN gene as the probe. One of five hybridization positive clones was characterized in more detail. A subcloned 3.7 kb fragment, positive in hybridization and encoding aminopeptidase activity, was sequenced and analyzed. Only one open reading frame (ORF) of 2532 base pairs with a coding capacity for a 95.9 kDa protein could be found. The deduced amino acid sequence of the 95.9 kDa protein showed homology to PepN proteins from other lactic acid bacteria and carried the conserved catalytic and zinc binding sites of the neutral zinc metallo-peptidase family confirming the identity of the pepN gene. A 2.75 kb transcript and two transcription start sites were identified with mRNA analyses. Expression of pepN in L. helveticus, studied as the function of growth, revealed a high level of pepN transcripts throughout the growth, in contrast to the steady state levels of other peptidase mRNAs from L. helveticus analyzed in our laboratory.