Murein hydrolase activity in the surface layer of Lactobacillus acidophilus ATCC 4356

Abstract

We describe a new enzymatic functionality for the surface layer (S-layer) of Lactobacillus acidophilus ATCC 4356, namely, an endopeptidase activity against the cell wall of Salmonella enterica serovar Newport, assayed via zymograms and identified by Western blotting. Based on amino acid sequence comparisons, the hydrolase activity was predicted to be located at the C terminus. Subsequent cloning and expression of the C-terminal domain in Bacillus subtilis resulted in the functional verification of the enzymatic activity.

FIG. 1.

SDS, Western blot, and zymography profiles of the S-layer obtained from L. acidophilus. (A) SDS-polyacrylamide gel electrophoresis after isolation with LiCl and staining with Coomassie blue; (B) Western blot detection with the anti-S-layer antibody; (C) zymogram of the S-layer showing lytic activity over the cell wall from Salmonella serovar Newport. Mk, molecular mass standard.

FIG. 2.

Hydrolase activities against isolated cell walls and viable cells of Salmonella serovar Newport. Mixtures of cell walls (0.5 mg/ml) (A) or whole cells (OD₆₆₀, 1) (B) and the S-layer at the indicated mass were incubated, and the OD₆₀₀ was measured.

FIG. 3.

Correlation between hydrolysis and increased levels of DNFB-treated free amino groups. (A) Hydrolysis with 0.1 mg of S-layer protein was followed by determination of the OD₆₀₀ (•) and the amount of DNF-Ala (▴) by TLC and densitometry. (B) TLC analysis of hydrolysis with two S-layer concentrations and different times (in hours) as indicated. DNF-amino acids (100 nmol) are Glu (E), Lys (K), and Ala (A). The arrow indicates the position of DNF-Ala.

FIG. 4.

Identities of the primary sequence of the S_A protein to those of other lytic enzymes analyzed by ClustalW. The cloned C-terminal portion of the S_A protein is underlined. Asterisks indicate residues identical in all sequences in the alignment; colons, conserved substitutions; periods, semiconserved substitutions. CAA61560, S_A protein (Lactobacillus acidophilus ATCC 4356); YP_618401, putative amidase (Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842); YP_812313, N-acetylmuramoyl-l-alanine amidase (L. delbrueckii subsp. bulgaricus ATCC BAA-365).

FIG. 5.

Heterologous expression. (A) Zymogram of the C terminus showing lytic activity over the Salmonella serovar Newport cell wall. (B) Western blot detection with anti-S-layer. Times (measured in hours) after the addition of 0.5 mM IPTG are shown.