Multiple-site mutations of phage Bp7 endolysin improves its activities against target bacteria.
The widespread use of antibiotics has caused serious drug resistance. Bacteria that were once easily treatable are now extremely difficult to treat. Endolysin can be used as an alternative to antibiotics for the treatment of drug-resistant bacteria. To analyze the antibacterial activity of the endolysin of phage Bp7 (Bp7e), a 489-bp DNA fragment of endolysin Bp7e was PCR-amplified from a phage Bp7 genome and cloned, and then a pET28a-Bp7e prokaryotic expression vector was constructed. Two amino acids were mutated (L99A, M102E) to construct pET28a-Bp7Δe, with pET28a-Bp7e as a template. Phylogenetic analysis suggested that BP7e belongs to a T4-like phage endolysin group. Bp7e and its mutant Bp7Δe were expressed in Escherichia coli BL21(DE3) as soluble proteins. They were purified by affinity chromatography, and then their antibacterial activities were analyzed. The results demonstrated that the recombinant proteins Bp7e and Bp7Δe showed obvious antibacterial activity against Micrococcus lysodeikticus but no activity against Staphylococcus aureus. In the presence of malic acid, Bp7e and Bp7Δe exhibited an effect on most E. coli strains which could be lysed by phage Bp7, but no effect on Salmonella paratyphi or Pseudomonas aeruginosa. Moreover, Bp7Δe with double-site mutations showed stronger antibacterial activity and a broader lysis range than Bp7e.
College of Animal Husbandry and Veterinary Medicine, Qingdao Agricultural University, Qingdao, 266109, China.
Can
C
Zhang
College of Animal Husbandry and Veterinary Medicine, Qingdao Agricultural University, Qingdao, 266109, China.
Yuanchao
Y
Wang
College of Animal Husbandry and Veterinary Medicine, Qingdao Agricultural University, Qingdao, 266109, China.
Huzhi
H
Sun
College of Animal Husbandry and Veterinary Medicine, Qingdao Agricultural University, Qingdao, 266109, China. renren0228@sina.com.
Huiying
H
Ren
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001435:Bacteriophages / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D024881:Drug Resistance, Bacterial; D010450:Endopeptidases / Q000235:genetics* / Q000494:pharmacology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism / Q000821:virology; D016679:Genome, Viral; D008826:Microbial Sensitivity Tests; D008837:Micrococcus / Q000187:drug effects; D009154:Mutation; D010802:Phylogeny; D011550:Pseudomonas aeruginosa / Q000187:drug effects; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000494:pharmacology; D012482:Salmonella paratyphi A / Q000187:drug effects; D013211:Staphylococcus aureus / Q000187:drug effects; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D000900:Anti-Bacterial Agents; D011994:Recombinant Proteins; D014764:Viral Proteins; D010450:Endopeptidases; C001143:endolysin
Bp7 endolysin (Bp7e); Bp7 endolysin mutant (Bp7Δe); antibacterial activity; bacteriophage; prokaryotic expression