Genomic Sequence of Bacteriophage ATCC 8074-B1 and Activity of Its Endolysin and Engineered Variants against Clostridium sporogenes

Abstract

ABSTRACTLytic bacteriophage ATCC 8074-B1 produces large plaques on its hostClostridium sporogenes. Sequencing of the 47,595-bp genome allowed the identification of 82 putative open reading frames, including those encoding proteins for head and tail morphogenesis and lysis. However, sequences commonly associated with lysogeny were absent. ORF 22 encodes an endolysin, CS74L, that shows homology toN-acetylmuramoyl-l-alanine amidases, and when expressed inEscherichia coli, the protein causes effective lysis ofC. sporogenescells when added externally. CS74L was also active onClostridium tyrobutyricumandClostridium acetobutylicum. The catalytic domain expressed alone (CS74L1–177) exhibited a similar activity and the same host range as the full-length endolysin. A chimeric endolysin consisting of the CS74L catalytic domain fused to the C-terminal domain of endolysin CD27L, derived fromClostridium difficilebacteriophage ΦCD27, was produced. This chimera (CSCD) lysedC. sporogenescells with an activity equivalent to that of the catalytic domain alone. In contrast, the CD27L C-terminal domain reduced the efficacy of the CS74L catalytic domain when tested againstC. tyrobutyricum. The addition of the CD27L C-terminal domain did not enable the lysin to targetC. difficileor other CD27L-sensitive bacteria.