Cloning and characterization of a prolinase gene (pepR) from Lactobacillus rhamnosus

Abstract

A peptidase gene expressing L-proline-beta-naphthylamide-hydrolyzing activity was cloned from a gene library of Lactobacillus rhamnosus 1/6 isolated from cheese. Peptidase-expressing activity was localized in a 1.5-kb SacI fragment. A sequence analysis of the SacI fragment revealed the presence of one complete open reading frame (ORF1) that was 903 nucleotides long. The ORF1-encoded 34.2-kDa protein exhibited 68% identity with the PepR protein from Lactobacillus helveticus. Additional sequencing revealed the presence of another open reading frame (ORF2) following pepR; this open reading frame was 459 bp long. Northern (RNA) and primer extension analyses indicated that pepR is expressed both as a monocistronic transcriptional unit and as a dicistronic transcriptional unit with ORF2. Gene replacement was used to construct a PepR-negative strain of L. rhamnosus. PepR was shown to be the primary enzyme capable of hydrolyzing Pro-Leu in L. rhamnosus. However, the PepR-negative mutant did not differ from the wild type in its ability to grow and produce acid in milk. The cloned pepR expressed activity against dipeptides with N-terminal proline residues. Also, Met-Ala, Leu-Leu, and Leu-Gly-Gly and the chromogenic substrates L-leucine-beta-naphthylamide and L-phenylalanine-beta-naphthylamide were hydrolyzed by the PepR of L. rhamnosus.

FIG. 1

Nucleotide and deduced amino acid sequences of L. rhamnosus pepR. The predicted −35 and −10 hexanucleotides are underlined. The 5′ end of the pepR transcript, identified by primer extension, is indicated by a vertical arrow. RBS is the predicted ribosome binding site. The translation stop codon is indicated by boldface type, and the putative transcription terminator is indicated by dashed arrows. The conserved residues of the active site region of prolyl oligopeptidases are indicated by boldface type and asterisks.

FIG. 2

Partial restriction map of the L. rhamnosus 1/6 pepR region. The positions and orientations of pepR and ORF2 are indicated by arrows. The 3.5- and 1.5-kb inserts of plasmids pVS98 and pVS99, respectively, are shown below the map.

FIG. 3

Expression of the pepR gene and ORF2. (A) L. rhamnosus 1/6 grown in MRS at 37°C. The cell density is shown as a function of growth. OD₆₀₀, optical density at 600 nm. (B) Northern blot analysis performed with the 0.7-kb pepR-specific hybridization probe. (C) Northern blot analysis performed with the 0.4-kb ORF2-specific hybridization probe. Samples for total RNA isolation were taken 4, 6, and 8 h after cell inoculation. The numbers on the left indicate the sizes of RNA molecular mass markers (Gibco BRL).

FIG. 4

Analysis of L. rhamnosus 1/6 with a 227-bp chromosomal deletion in the pepR gene by Southern hybridization with a pepR-specific probe. Lanes 1, L. rhamnosus 1/6ΔpepR chromosomal DNA digested with SacI; 2, L. rhamnosus 1/6::pVS101 chromosomal DNA digested with SacI; 3, L. rhamnosus 1/6 chromosomal DNA digested with SacI.