The cell lysis activity of the Streptococcus agalactiae bacteriophage B30 endolysin relies on the cysteine, histidine-dependent amidohydrolase/peptidase domain

Abstract

The Streptococcus agalactiae bacteriophage B30 endolysin contains three domains: cysteine, histidine-dependent amidohydrolase/peptidase (CHAP), Acm glycosidase, and the SH3b cell wall binding domain. Truncations and point mutations indicated that the Acm domain requires the SH3b domain for activity, while the CHAP domain is responsible for nearly all the cell lysis activity.

FIG. 1.

Phage B30 endolysin (443-amino-acid) conserved domains and deletion constructs. The schematic diagram at the top indicates the locations of the NH₂-terminal CHAP endopeptidase, Acm glycosidase, C-terminal SH3b cell wall binding domain, and the six-His tag (solid box) in the pET21a-derived construct. The numbers indicate the initial and final amino acids in the deletion constructs and the C-terminal six-His tag.

FIG. 2.

Plate lysis assay of the B30 endolysin and selected truncations. Ten-microliter portions of E. coli extracts harboring B30-derived proteins were spotted onto tryptic soy agar plates containing mid-log-phase cultures of the pathogens S. agalactiae (S. agal.), S. dysgalactiae (S. dysgal.), and S. uberis.

FIG. 3.

Protein purification analysis and turbidity assay with selected B30 endolysin truncated proteins. (A) Representative SDS-PAGE gel containing B30 endolysin and derived proteins purified with nickel affinity columns. Purified proteins were analyzed by standard 15% SDS-PAGE performed with Tris-glycine buffer at 131 V for 1.5 h in a Bio-Rad Mini-PROTEAN 3 gel apparatus according to the manufacturer's instructions. Gels were stained with BioSafe Coomassie blue stain (Bio-Rad) for 1 h and then rinsed in distilled water overnight. Lane M, molecular mass protein standards (Kaleidoscope protein standards; Bio-Rad); lane 1, 1-156; lane 2, 1-182; lane 3, 1-356; lane 4, full-length 1-443 protein. (B) Turbidity assay results with three bacterial strains obtained by using 100 μg of each B30 endolysin-derived protein. Black bars, 1-156; white bars, 1-182; plaid bars, 1-356; striped bars, full-length 1-443. The turbidity data are the results of three independent experiments performed with three unique preparations of purified protein. Specific activity is expressed in OD₆₀₀/hour/milligram. S. agal., S. agalactiae.

FIG. 4.

Protein purification analysis and turbidity assay with B30 endolysin site-directed mutants. (A) SDS-PAGE of the B30 endolysin and selected nickel column-purified mutant proteins. Lane M, protein standards; lane 1, C26S; lane 2, H91A; lane 3, D158A; lane 4, full-length 1-443 protein. (B) Turbidity assay results with S. agalactiae obtained by using 100 μg of each B30 endolysin and selected mutated proteins. The turbidity data are the results of two independent experiments. Specific activity is expressed in OD₆₀₀/hour/milligram.