title: Characteristics of a broad lytic spectrum endolysin from phage BtCS33 of Bacillus thuringiensis.
abstract: BACKGROUND
  Endolysins produced by bacteriophages lyse bacteria, and are thus considered a novel type of antimicrobial agent. Several endolysins from Bacillus phages or prophages have previously been characterized and used to target Bacillus strains that cause disease in animals and humans. B. thuringiensis phage BtCS33 is a Siphoviridae family phage and its genome has been sequenced and analyzed. In the BtCS33 genome, orf18 was found to encode an endolysin protein (PlyBt33).
  
  
  RESULTS
  Bioinformatic analyses showed that endolysin PlyBt33 was composed of two functional domains, the N-terminal catalytic domain and the C-terminal cell wall binding domain. In this study, the entire endolysin PlyBt33, and both the N- and C-termini,were expressed in Escherichia coli and then purified. The lytic activities of PlyBt33 and its N-terminus were tested on bacteria. Both regions exhibited lytic activity, although PlyBt33 showed a higher lytic activity than the N-terminus. PlyBt33 exhibited activity against all Bacillus strains tested from five different species, but was not active against Gram-negative bacteria. Optimal conditions for PlyBt33 reactivity were pH 9.0 and 50 °C. PlyBt33 showed high thermostability, with 40% of initial activity remaining following 1 h of treatment at 60 °C. The C-terminus of PlyBt33 bound to B. thuringiensis strain HD-73 and Bacillus subtilis strain 168. This cell wall binding domain might be novel, as its amino acid sequence showed little similarity to previously reported endolysins.
  
  
  CONCLUSIONS
  PlyBt33 showed potential as a novel antimicrobial agent at a relatively high temperature and had a broad lytic spectrum within the Bacillus genus. The C-terminus of PlyBt33 might be a novel kind of cell wall binding domain.
authors.affiliation: Key Laboratory of Agricultural and Environmental Microbiology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, PR, China. mygao@wh.iov.cn
authors.forename: Yihui
authors.initials: Y
authors.lastname: Yuan
authors.forename: Qin
authors.initials: Q
authors.lastname: Peng
authors.forename: Meiying
authors.initials: M
authors.lastname: Gao
mesh_terms: D000595:Amino Acid Sequence; D017103:Bacillus Phages / Q000201:enzymology* / Q000302:isolation & purification / Q000502:physiology; D001412:Bacillus subtilis / Q000187:drug effects / Q000378:metabolism; D001413:Bacillus thuringiensis / Q000821:virology*; D001433:Bacteriolysis; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011485:Protein Binding; D055550:Protein Stability; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; D010450:Endopeptidases; C001143:endolysin