Genomic sequence and characterization of the virulent bacteriophage phiCTP1 from Clostridium tyrobutyricum and heterologous expression of its endolysin.
The growth of Clostridium tyrobutyricum in developing cheese leads to spoilage and cheese blowing. Bacteriophages or their specific lytic enzymes may provide a biological control method for eliminating such undesirable organisms without affecting other microflora. We isolated the virulent bacteriophage phiCTP1 belonging to the Siphoviridae and have shown that it is effective in causing lysis of sensitive strains. The double-stranded DNA genome of phiCTP1 is 59,199 bp, and sequence analysis indicated that it has 86 open reading frames. orf29 was identified as the gene coding for the phage endolysin responsible for cell wall degradation prior to virion release. We cloned and expressed the ctp1l gene in E. coli and demonstrated that the partially purified protein induced lysis of C. tyrobutyricum cells and reduced viable counts both in buffer and in milk. The endolysin was inactive against a range of clostridial species but did show lysis of Clostridium sporogenes, another potential spoilage organism. Removal of the C-terminal portion of the endolysin completely abolished lytic activity.
Mayer
Melinda J
MJ
Institute of Food Research, Norwich Research Park, Colney, Norwich, United Kingdom. Melinda.Mayer@BBSRC.ac.uk
Payne
John
J
Gasson
Michael J
MJ
Narbad
Arjan
A
D000595:Amino Acid Sequence; D001433:Bacteriolysis; D001435:Bacteriophages / Q000235:genetics* / Q000302:isolation & purification / Q000648:ultrastructure*; D003001:Cloning, Molecular; D048014:Clostridium tyrobutyricum / Q000821:virology*; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D010450:Endopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D023061:Gene Order; D005814:Genes, Viral; D016679:Genome, Viral; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D016366:Open Reading Frames; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D017906:Siphoviridae / Q000235:genetics / Q000302:isolation & purification / Q000648:ultrastructure; D014771:Virion / Q000648:ultrastructure
D004279:DNA, Viral; D050505:Mutant Proteins; D011994:Recombinant Proteins; D010450:Endopeptidases; C001143:endolysin