PMID:21097580

Citation	Uchiyama, J, Takemura, I, Hayashi, I, Matsuzaki, S, Satoh, M, Ujihara, T, Murakami, M, Imajoh, M, Sugai, M and Daibata, M (2011) Characterization of lytic enzyme open reading frame 9 (ORF9) derived from Enterococcus faecalis bacteriophage phiEF24C. Appl. Environ. Microbiol. 77:580-5
Abstract	In bacteriophage (phage) therapy against Gram-positive bacteria, such as Staphylococcus aureus, Listeria monocytogenes, and Enterococcus faecalis, members of a genus of SPO1-like viruses are typically employed because of their extreme virulence and broad host spectrum. Phage φEF24C, which is a SPO1-like virus infecting E. faecalis, has previously been characterized as a therapeutic phage candidate. In addition to the phage itself, phage endolysin is also recognized as an effective antimicrobial agent. In this study, a putative endolysin gene (orf9) of E. faecalis phage φEF24C was analyzed in silico, and its activity was characterized using the recombinant form. First, bioinformatics analysis predicted that the open reading frame 9 (ORF9) protein is N-acetylmuramoyl-l-alanine amidase. Second, bacteriolytic and bactericidal activities of ORF9 against E. faecalis were confirmed by zymography, decrease of peptidoglycan turbidity, decrease of the viable count, and morphological analysis of ORF9-treated cells. Third, ORF9 did not appear to require Zn(2+) ions for its activity, contrary to the bioinformatics prediction of a Zn(2+) ion requirement. Fourth, the lytic spectrum was from 97.1% (34 out of 35 strains, including vancomycin-resistant strains) of E. faecalis strains to 60% (6 out of 10 strains) of Enterococcus faecium strains. Fifth, N-acetylmuramoyl-l-alanine amidase activity of ORF9 was confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and the subsequent MALDI-postsource decay (PSD) analyses. Finally, functional analysis using N- or C-terminally deleted ORF9 mutants suggested that a complete ORF9 molecule is essential for its activity. These results suggested that ORF9 is an endolysin of phage φEF24C and can be a therapeutic alternative to antibiotics.
Links	PubMed PMC3020548 Online version:10.1128/AEM.01540-10
Keywords	Amino Acid Sequence; Bacteriolysis; Bacteriophages/enzymology; Bacteriophages/genetics; Coenzymes/metabolism; Colony Count, Microbial; Computational Biology; Endopeptidases/genetics; Endopeptidases/metabolism; Enterococcus faecalis/virology; Microbial Viability/drug effects; Molecular Sequence Data; N-Acetylmuramoyl-L-alanine Amidase/genetics; N-Acetylmuramoyl-L-alanine Amidase/metabolism; Nephelometry and Turbidimetry; Open Reading Frames; Zinc/metabolism
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
BPPHE:A8E261		GO:0008745: N-acetylmuramoyl-L-alanine amidase activity	ECO:0000315:		F		Figure 5: It was previously proven, in the same paper, that orf9 is a N-acetylmuramoyl-L-alanine amidase, but it wasn't known what regions of orf9 was imporant for the lytic activity. To determine the regions of orf9 that are functionally important the researchers constructed eight mutants of the orf9 (of E. faecalis phage φEF24C) by deleting the N-terminal and C-terminal domains (Figure 1A). Only three of the C-terminal deleted mutants were viable (they were soluble), but none of the N-terminal regions were soluble which would indicate that the N-terminal region is required for stability. The C-terminal mutants had their lytic activities were tested by measuring the turbidity, but only the full length orf9, with a Histidine tag, displayed lytic activity. This demonstrated that the full length, N-terminal and C-terminal, orf9 is required for solubilty and lytic activity.	complete CACAO 12883

References

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