Mutational and biochemical analyses of the endolysin LysgaY encoded by the Lactobacillus gasseri JCM 1131T phage φgaY

The LysgaY of Lactobacillus gasseri JCM 1131T phage φgaY endolysin was purified to homogeneity using the Escherichia coli/His·Tag system. Zymographic and spectrophotometric assays showed that LysgaY lysed over 20 heated Gram-positive bacterial species as the substrates, including lactobacilli, lactococci, enterococci, micrococci, and staphylococci. The enzymatic activity had the pH and temperature optima of about 6.5 and 37°C, respectively. Amino-acid substitution analysis revealed that 13 residues of LysgaY were involved in cell-lytic activity: in the β/αgaY domain, G10, D12, E33, D36, H60, Y61, D96, E98, V124, L132, and D198; in the SH3bgaY domain, Y272 and W284. In addition, deletion analysis demonstrated that the β/αgaY domain of N-terminal 216 residues is the core enzyme portion, although the cell-lytic ability is lower than that of LysgaY. These mutational experiments suggested that β/αgaY (in which two acidic residues of D12 and E98 likely act as catalytic residues) is responsible for cell-lytic activity, and SH3bgaY promotes β/αgaY possibly through cell-wall binding function. The purified His-tagged SH3bgaY domain containing 94 residues from S217 to K310 (i) bound to Gram-positive bacteria susceptible to LysgaY, (ii) induced aggregation of exponentially growing cells of L. gasseri JCM 1131T, L. casei IAM 1045, Lactococcus lactis C2, L. lactis MG 1363, and Enterococcus hirae IAM 1262 by forming thread-like chained cells, (iii) inhibited lytic activity of LysgaY, and (iv) impeded autolysis of L. gasseri JCM 1131T in buffer systems. A truncated protein HΔSH3bgaY lacking in N-terminal 21 residues (from S217 to E237) of SH3bgaY and an amino-acid substituted protein HSH3bgaYG (W284G) lost the activities of HSH3bgaY, showing that the N-terminal region and W284 probably play important roles in the SH3bgaY function(s).