Streptococcus thermophilus cell wall-anchored proteinase: release, purification, and biochemical and genetic characterization

Abstract

Streptococcus thermophilus CNRZ 385 expresses a cell envelope proteinase (PrtS), which is characterized in the present work, both at the biochemical and genetic levels. Since PrtS is resistant to most classical methods of extraction from the cell envelopes, we developed a three-step process based on loosening of the cell wall by cultivation of the cells in the presence of glycine (20 mM), mechanical disruption (with alumina powder), and enzymatic treatment (lysozyme). The pure enzyme is a serine proteinase highly activated by Ca(2+) ions. Its activity was optimal at 37 degrees C and pH 7.5 with acetyl-Ala-Ala-Pro-Phe-paranitroanilide as substrate. The study of the hydrolysis of the chromogenic and casein substrates indicated that PrtS presented an intermediate specificity between the most divergent types of cell envelope proteinases from lactococci, known as the PI and PIII types. This result was confirmed by the sequence determination of the regions involved in substrate specificity, which were a mix between those of PI and PIII types, and also had unique residues. Sequence analysis of the PrtS encoding gene revealed that PrtS is a member of the subtilase family. It is a multidomain protein which is maturated and tightly anchored to the cell wall via a mechanism involving an LPXTG motif. PrtS bears similarities to cell envelope proteinases from pyogenic streptococci (C5a peptidase and cell surface proteinase) and lactic acid bacteria (PrtP, PrtH, and PrtB). The highest homologies were found with streptococcal proteinases which lack, as PrtS, one domain (the B domain) present in cell envelope proteinases from all other lactic acid bacteria.

FIG. 1

Specificity of PrtS from S. thermophilus CNRZ 385 and lactococcal PrtPs toward α_s1-casein-(1-23) fragment. The cleavage sites are indicated by arrows. The sizes of the arrows are related to relative cleavage rates. Abbreviations: PrtS, proteinase from S. thermophilus CNRZ 385; PrtPI and PrtPIII, proteinases from L. lactis, HP and AM1, respectively (20, 21).

FIG. 2

Genetic organization of prtS from S. thermophilus CNRZ 385 and its flanking regions and homologies with other CEPs from LAB and pyogenic streptococci. The oval indicates the Potential terminator of transcription. Abbreviations: SS, signal sequence; PP, propeptide; PR, catalytic domain; A, globular domain; B, PR stabilizing domain; H, helical domain; W, cell wall domain; AN, cell wall anchor; n.s., not significant.

FIG. 3

Multiple sequence alignment of substrate binding regions of PrtS from S. thermophilus CNRZ 385 and other CEPs from LAB and pyogenic streptococci. Boxed letters are amino acids of the substrate binding areas which are different in PrtP from L. lactis SK11 (PIII type) and Wg2 (PI type). Shaded letters are amino acids conserved in the majority of the sequences. Asterisks show amino acids involved in L. lactis PrtP substrate specificity as demonstrated by punctual mutagenesis (61). Residue numbering corresponds to that of mature L. lactis PrtP. Abbreviations: PrtS, proteinase from S. thermophilus CNRZ 385 (this work); PrtPI, proteinase from L. lactis Wg2 (36); PrtPIII, proteinase from L. lactis SK11 (69); PrtP Lb. casei, proteinase from L. casei NCDO 151 (33); PrtB, proteinase from L. delbrueckii subsp. bulgaricus NCDO 1489 (30); PrtH, proteinase from L. helveticus CNRZ 32 (48).