Identification of a Broad-Spectrum Peptidoglycan Hydrolase Associated with the Particle of <b><i>Xanthomonas oryzae</i></b> Phage Xop411

Virion-associated peptidoglycan hydrolases (VAPGH) in bacteriophages are potential antimicrobials. Xop411 is a syphophage infecting the Gram-negative <i>Xanthomonas oryzae</i> pv. oryzae that causes bacterial leaf blight in rice plants. The Xop411 gp21 protein was identified here as a peptidoglycan glycohydrolase by Western blotting and zymogram assay, and localized to the phage tail by immunogold-labelling electron microscopy. This protein showed an apparent molecular mass of 17 kDa in SDS-polyacrylamide gels, larger than that calculated from the amino acid sequence, 15 kDa with 130 residues. The recombinant gp21 expressed in <i>Escherichia coli</i> formed inclusion bodies, which gained enzyme activity after in-gel renaturation. In contrast, the secreted recombinant protein (s-gp21His) expressed in <i>Pichia pastoris</i> was soluble and enzymatically active. Plate assays showed that s-gp21His was capable of killing 3 species of <i>Xanthomonas</i>, a genus containing 27 closely related plant pathogenic species, as well as the opportunistic <i>Pseudomonas aeruginosa</i> and <i>Stenotrophomonas maltophilia</i> causing nosocomial infections. These results indicate that the Xop411 gp21 has possible wide applications as an antimicrobial against xanthomonads and at least 2 opportunistic bacteria. Several other VAPGH from <i>Xanthomonas</i> phages were also identified by bioinformatic analysis, with 1 being confirmed by Western blotting.