No lysis of other B. flavum ATCC strains, B. lactofermentum BLOB or C. glutamicum RM3 was observed. Consequently, the same strains were used for lysis studies of BFK20 endolysin along with two controls –B. subtilis wt PY79 (Gram-positive control) and E. coli XL1 Blue (Gram-negative control). The corynebacteria and bacilli cells were prepared as described (Materials and methods). The purified BFK20 endolysin gp24′T (without His6Tag) and its catalytic domain gp24CD were used in the turbidity reduction assay. The lytic activity of BFK20 endolysin towards the host cells of B. flavum CCM 251 was not as strong (Fig. 4a) as might have been expected

based on the lytic activity of previously characterized endolysins (Low et al., 2005; Briers et al., 2007). Moreover, BFK20 endolysin selleck kinase inhibitor lysed the other six corynebacterial strains tested with higher efficiency than the original

host cells. Brevibacterium lactofermentum BLOB cells were lysed 20 times faster by the entire endolysin compared with lysis of B. flavum CCM 251 cells (Fig. 4c). Corynebacterium glutamicum RM3 and B. flavum ATCC strains 21474, 21128, 21127 and 21129 were also lysed more efficiently Selleck INCB024360 (Fig. 4a and c). Interestingly, the lytic activity of the catalytic domain alone (gp24CD) was 13 times higher on the host cell substrate than that of the entire endolysin and about eight times higher for the other corynebacterial strains (Fig. 4b and c). Possibly the antibacterial activity of BFK20 endolysin was increased by removing

the cell wall binding domain. Similarly, other lysins have been reported to exhibit higher antibacterial activity after removal of their C-terminal domains (Borysowski et al., 2006). The C-terminal domain is responsible for binding to the bacterial cell wall but it could also interact with the catalytic domain before the interaction with the cell wall substrate (Fischetti, Carnitine palmitoyltransferase II 2010). A high-affinity binding to the catalytic domain may aid in controlling diffusion of the endolysin molecules after the phage progeny is released from the host cell. Neighboring host cells that are not yet infected by phages may be killed by the released endolysin molecules and this might either prevent or reduce new infections. It is possible that a similar inhibition of the catalytic domain activity by an interaction with the cell wall binding domain occurs in the BFK20 endolysin. We confirmed that BFK20 endolysin and its catalytic domain possess antibacterial activity against Gram-positive bacteria (corynebacteria, B. subtilis) (Fig. 5a and b). No degradation of Gram-negative E. coli was observed (Fig. 5a and b). Amidases generally have been suggested to display a broader spectrum of antibacterial activity than the other classes of endolysins because of the very frequent occurrence of the amide bond between N-acetylmuramic acid and l-alanine in peptidoglycan.