Analysis of the catalytic domain of the lysin of the lactococcal bacteriophage Tuc2009 by chimeric gene assembling

Abstract

An active chimeric cell wall lytic enzyme (Tsl) has been constructed by fusing the region coding for the N-terminal half of the lactococcal phage Tuc2009 lysin and the region coding for the C-terminal domain of the major pneumococcal autolysin. The chimeric enzyme exhibited a glycosidase activity capable of hydrolysing choline-containing pneumococcal cell walls. This experimental approach demonstrated that the Tuc2009 lysin possesses a modular structure and further supports the hypothesis that many cell wall lytic enzymes have evolved by the fusion of preexisting catalytic and peptidoglycan-binding domains.

Chimeric Ply187 endolysin killsStaphylococcus aureusmore effectively than the parental enzyme

“…Low et al, (2005) (Baba & Schneewind, 1996;Grundling & Schneewind, 2006;Lu et al, 2006). Despite the lack of a consensus binding site, SH3b domains and other CBDs have been used for decades to develop novel antimicrobials (Diaz et al, 1990(Diaz et al, , 1991Croux et al, 1993;Sheehan et al, 1996;Lopez et al, 1997). Recently, the Fischetti group used a non-SH3b CBD to generate a chimeric staphylolytic lysin using the Twort phage endolysin CHAP domain (Daniel et al, 2010).…”

Muralytic activity and modular structure of the endolysins of Pseudomonas aeruginosa bacteriophages φKZ and EL

“…Modular recombination of proteins is one of the driving forces in evolution that permits a rapid adaptation to new environmental conditions. This phenomenon has been observed for many enzymes degrading polymeric, insoluble substrates (Wren, 1991; Sheehan et al ., 1997) and is further confirmed by the in vitro construction of intergeneric functional chimeric endolysins (Croux et al ., 1993b; Sheehan et al ., 1996). Similarity searches of the peptidoglycan‐binding domains of KZ144 and EL188 reveal relatedness to many substrate‐binding modules of lytic enzymes encoded by Bacillus or Clostridium sp.…”

Bacteriophage Endolysins as Novel Antimicrobials

“…In a reverse approach, fusing the Lyc CBD to the EAD of LytA increased activity against clostridial cell walls 250-fold [137]. Similar results with inter-generic fusions were reported by Sheehan et al ., who combined an EAD from the lactococcal phage Tuc2009 with the ChBD of LytA [138], and more recently by Donovan et al ., who fused full-length and truncated versions of the Streptococcus agalactiae phage B30 endolysin to mature lysostaphin, yielding enzymes that were active against both streptococcal and staphylococcal cells [102]. The same laboratory created the aforementioned fusions (λSA2-E-LysK-SH3b and λSA2-E-Lyso-SH3b) of the endopeptidase of streptococcal phage λSA2 lysin with the Staphylococcus -specific SH3b CBDs of either the endolysin LysK or lysostaphin, in which the exchange of CBDs bestowed activity against staphylococci on the streptococcal enzyme, while still maintaining considerable streptolytic activity [42].…”