Open AccessDevelopment of a specific fluorescent phage endolysin for in situ detection of Clostridium species associated with cheese spoilage
Author(s) -
GómezTorres Natalia,
Dunne Matthew,
Garde Sonia,
Meijers Rob,
Narbad Arjan,
Ávila Marta,
Mayer Melinda J.
Publication year - 2018
Publication title -
microbial biotechnology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.287
H-Index - 74
ISSN - 1751-7915
DOI - 10.1111/1751-7915.12883
Subject(s) - green fluorescent protein , clostridium , bacteriophage , food spoilage , microbiology and biotechnology , biochemistry , biology , clostridiaceae , chemistry , bacteria , gene , escherichia coli , enzyme , genetics
Summary Late blowing defect ( LBD ) is a major cause of spoilage in cheeses, caused by the growth of Clostridium spp. in the cheese matrix. We investigated the application of CTP 1L, a bacteriophage endolysin active against Clostridium tyrobutyricum , and its enzymatically active and cell wall‐binding domains ( EAD and CBD ) attached to green fluorescent protein ( GFP ) to detect dairy‐related Clostridium species by fluorescence microscopy. GFP ‐ CTP 1L and GFP ‐ CBD demonstrated specificity for Clostridium spp. by labelling 15 and 17 of 20 Clostridium strains, respectively, but neither bound to other members of the cheese microbiota. However, GFP ‐ EAD did not label any Clostridium strain tested. Unexpectedly, GFP ‐ CTP 1L and GFP ‐ CBD were also able to bind to clostridial spores. In addition, GFP ‐ CBD allowed us to visualize the vegetative cells of C. tyrobutyricum directly in the matrix of a LBD cheese. Site‐directed mutants of GFP ‐ CTP 1L and GFP ‐ CBD were made to examine the amino acids involved in binding and oligomer formation. Oligomerization was not essential for binding, but specific mutations in the CBD which affected oligomer formation also affected binding and lytic activity. We conclude that GFP ‐ CTP 1L and GFP ‐ CBD could be good biomarkers for rapid detection of Clostridium spores in milk, so measures can be taken for the prevention of LBD in cheese, and also provide effective tools to study the development of Clostridium populations during cheese ripening.