Browsing by Author "Li, Yanru"

Phage lysins are enzymes that degrade bacterial cell wall. A wild-type Lactococcus lactis strain LAC460 secretes three phage lysins, LysL, LysP, and LysT, encoded by three different prophages. Unlike common phage lysins, these enzymes do not break down the host's cell wall. Therefore, these lysins can attack other L. lactis strains and behave like bacteriocins, antimicrobial proteins. The binding of a phage lysin to bacterial cell wall requires a specific cell wall binding domain (CBD) in the lysin. However, nothing about the CBDs of LysL, LysP and LysT is known. This study aimed to determine the CBDs of these three lysins and the target specificity of the lysins. Putative CBD regions of the lysins were fused with green fluorescent protein (GFPuv). GFPuv-CBD-LysL and GFPuv-CBD-LysT were ligated into the pASG-IBA4 vector and cloned in Escherichia coli DH5α. After all, only the construction of the GFPuv-CBD- LysL was successful resulting in fluorescent transformants. To analyse the binding of GFPuv- CBD-LysL to cells of different L. lactis strains, the fusion proteins were mixed with the LysL sensitive L. lactis MG1614, LysL resistant L. lactis LM0230, and the LysL producing LAC460 cells. With fluorescence microscope it could be seen that the GFPuv-CBD-LysL decorated the cell surface of L. lactis MG1614 with green fluorescence, but LM0230 and LAC460 cells remained non-fluorescent. The fluorescence of the cells was also measured with a fluorometer, showing strong fluorescence from MG1614, but nothing from the other two strains. This showed that the fusion protein specifically bound to the MG1614 cell surface, but it did not bind to the LysL resistant strain LM0230 or the LysL producer LAC460 cell. In conclusion, the results demonstrate that the C-terminus of LysL contains a specific cell wall binding domain. In addition, the results provide an explanation for how LAC460 can secrete LysL without autolysis, as phage lysins not able to bind onto peptidoglycan are unable to lyse cells.