Development of a specific fluorescent phage endolysin for in situ detection of Clostridium species associated with cheese spoilage

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 CTP1L, a bacteriophage endolysin active against Clostridium tyrobutyricum, and its enzymatically active and cell wall‐binding d...

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Bibliographic Details
Published in:Microbial biotechnology 2018-03, Vol.11 (2), p.332-345
Main Authors: Gómez‐Torres, Natalia, Dunne, Matthew, Garde, Sonia, Meijers, Rob, Narbad, Arjan, Ávila, Marta, Mayer, Melinda J.
Format: Article
Language:English
Subjects:
Acids
Amino acids
Bacteria
Binding
Biomarkers
Cell Wall - metabolism
Cell walls
Cheese
Cheese - microbiology
Clostridium
Clostridium tyrobutyricum - genetics
Clostridium tyrobutyricum - growth & development
Crystal structure
Dairy products
Domain walls
Domains
Endopeptidases - genetics
Endopeptidases - metabolism
Fluorescence
Fluorescence microscopy
Food safety
Green fluorescent protein
Labeling
Ligands
Microbiota
Microscopy
Microscopy, Fluorescence
Milk
Mutants
Mutation
Oligomerization
Phages
Protein Binding
Proteins
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - metabolism
Ripening
Spoilage
Spores
Staining and Labeling - methods
Vegetative cells
Viral Proteins - genetics
Viral Proteins - metabolism
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Summary: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 CTP1L, 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‐CTP1L 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‐CTP1L 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‐CTP1L 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‐CTP1L 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. A fluorescently labelled bacteriophage endolysin was shown to be specific for Clostridium species and was able to detect cheese spoilage strains in the cheese matrix, and to bind to spores. The lysin represents a tool for detection, for understanding clostridial development during cheesemaking and for investigating links between lysin structure and function.
ISSN:1751-7915
1751-7915
DOI:10.1111/1751-7915.12883