Identification and genetic characterization of a novel proteinase, PrtR, from the human isolate Lactobacillus rhamnosus BGT10

Abstract

A novel proteinase, PrtR, produced by the human vaginal isolate Lactobacillus rhamnosus strain BGT10 was identified and genetically characterized. The prtR gene and flanking regions were cloned and sequenced. The deduced amino acid sequence of PrtR shares characteristics that are common for other cell envelope proteinases (CEPs) characterized to date, but in contrast to the other cell surface subtilisin-like serine proteinases, it has a smaller and somewhat different B domain and lacks the helix domain, and the anchor domain has a rare sorting signal sequence. Furthermore, PrtR lacks the insert domain, which otherwise is situated inside the catalytic serine protease domain of all CEPs, and has a different cell wall spacer (W) domain similar to that of the cell surface antigen I and II polypeptides expressed by oral and vaginal streptococci. Moreover, the PrtR W domain exhibits significant sequence homology to the consensus sequence that has been shown to be the hallmark of human intestinal mucin protein. According to its alpha(S1)- and beta-casein cleavage efficacy, PrtR is an efficient proteinase at pH 6.5 and is distributed throughout all L. rhamnosus strains tested. Proteinase extracts of the BGT10 strain obtained with Ca(2+)-free buffer at pH 6.5 were proteolytically active. The prtR promoter-like sequence was determined, and the minimal promoter region was defined by use of prtR-gusA operon fusions. The prtR expression is Casitone dependent, emphasizing that nitrogen depletion elevates its transcription. This is in correlation with the catalytic activity of the PrtR proteinase.

FIG. 1.

Proteolytic activity of L. rhamnosus BGT10. (A) Caseins hydrolyzed by whole cells collected from MCA plates. Lanes: α, α_S1-casein substrate; 1, hydrolyzed α_S1-casein; β, β-casein substrate; 2, hydrolyzed β-casein. (B) Nutrient-dependent proteolytic activity of L. rhamnosus BGT10. Lanes: β, β-casein substrate; 1 to 4, β-casein hydrolyzed by whole cells collected from CDM plates containing 0.1, 0.5, 1, and 2% Casitone, respectively; 5, whole cells collected from MRS plates; 6, whole cells collected from MCA plates. (C) Hydrolysis of β-casein by the proteinase extracts. Lanes: β, β-casein substrate; 1, β-casein hydrolyzed by extract obtained from cells grown on CDM plates containing 0.1% Casitone; 2, β-casein hydrolyzed by extract obtained from the cells grown on MCA plates. Arrows indicate α- or β-casein.

FIG. 2.

Schematic representation of PrtP as a prototype for the subtilases and the PrtR of L. rhamnosus strain BGT10, showing different domains according to the model of Siezen (50). PP, prepro domain; PR, protease domain; I, insert domain; A, A domain; B, B domain; H, helix domain; W, cell wall spacer domain; AN, anchor domain; black dot, sorting signal.

FIG. 3.

The A, B, and W domains of PrtR. (A) Alignment of the A-domain segments of L. rhamnosus PrtR with conserved residues from PrtP (L. lactis), PrtH (L. helveticus), PrtB (L. delbrueckii subsp. bulgaricus), Csp (S. agalactiae), and ScpA (S. pyogenes). Consensus residues were derived from an alignment of known CEPs; uppercase indicates identical residues, and lowercase indicates highly conserved residues (50). (B) Comparison of B-domain segments of PrtR with conserved sequences residues of PrtP and consensus residues derived from known CEPs. Consensus residues (Con.) are as described for panel A. Identical amino acids are shaded in black; homologous residues are shaded in grey. (C) Alignment of the W domain of PrtR from L. rhamnosus strain BGT10 and the C-terminal part of SspB from S. gordonii (8). Identical amino acids are shaded in black; homologous residues are shaded in grey.(D) Alignment of the W domain of PrtR from L. rhamnosus strain BGT10 and a fragment of the human intestinal mucin (IMuc) (18). Identical amino acids are shaded in black; homologous residues are shaded in grey.

FIG. 4.

Determination of the prtR transcription start point. (A) Primer extension analysis of the prtR promoter was performed with RNA from L. casei ATCC 393^T cells harboring pEB471. (B) L. casei ATCC 393^T cells harboring pEB640 (lane 1) and L. rhamnosus BGT10 (lane 2). The sequencing reactions (A, C, G, and T) were performed with the same primers as the corresponding primer extension reactions. The primer extension products are indicated with an arrow. (C) Interpretation of the primer extension results. The putative −35 and −10 prtR promoter haxamers (underlined), the transcription start point (+1, underlined), and the putative ribosome binding site (RBS) (boldface) are shown. The prtR coding sequence is in italic.

FIG. 5.

Schematic representation of different prtR-gusA operon fusions. The putative prtR promoter position (−35 and −10 hexanucleotides) is marked. Thick lines represent different fragments of the prtR regulatory region, and dashed lines represent deleted regions.