Isolation, Characterization And Functional Study Of Partial Genes Of Lysogenic Phage W001of Listeria Monocytogenes

Listeria monocytogenes is an important zoonotic pathogen. phage is avirus that can specifically infect bacteria, and may have a lytic cycle or alysogenic cycle. The results of PCR and PCR products sequences showedthat16of38Listeria monocytogenes isolates carry pro-phages at tRNAArgattachment sites (attBB’). Mitomycin C was used to induce lysogenicphages from the above16Listeria monocytogenes isolates, and a phageisolate with typical shape of Siphovirus was observed under transmissionelectronic microscope, and named W001. The phage nucleic acid wasconfirmed to be double-stranded DNA (dsDNA) after digested with DNaseI、 RNase A and Mung Bean nuclease， respectively. The sequence of theentire W001dsDNA genome was determined using a shot-gun approach,the COS site sequence5’-CGGTGTGGGG-3’ at genome ends wasobtained by run-off method, the complete genome has42,227bp and66open reading frames, the core sequence5’-AATCCCTCTCAGGACG-3’of phage attachment site (attPP’) is immediately downstream integrase.Endolysin of phage W001(PLYW001) with full length of281aminoacids is predicted to be composed of NH2terminal EAD（EnzymaticallyActive Domain） and COOH terminal CBD (Cell-wall Binding Domain),the PLYW001polypeptide was expressed by E. Coli, and purified with Nichromatography column, the concentration of the purified PLYW001wasdetermined by Bradford protein assay. The lytic activities and spectrum ofPLYW001was measured by turbidimetric determination of cell lysis, andthe results showed that PLYW001can lyse1/2a serotype Listeriamonocytogenes including phage W001host HA1073, EGDe and10403s. Amino acids substitutions proved that the7amino acids (45R,50Q,64S,66H,73D,115D and118H) of EAD were very important for lysis activity.DNA fragments coding for EGFP or phage lysin CBD were amplifiedby PCR, and inserted into pET28a, resulting vector pET28a-EGFP-CBD,the fused EGFP-CBD polypeptide was expressed by E. Coli BL21withIPTG induction, and purified by Ni chromatography. Binding ofEGFP-tagged CBD of PLYW001to bacterial cell surface was observedunder the confocal laser scanning microscope.After infecting the host cell, lysogenic phage DNA would integrateonto bacterial genome at attBB’ site via phage attPP’ site and integrase.The attPP’-integrase DNA fragment was amplified by PCR, and clonedonto pHAL1plasmid, resulting recombinant plasmid pHAL2, pHAL2wasthen electroporated into competent cell of LM1003with actA/plcB deleted.The sequences of PCR products of candidate strains showed that pHAL2specifically integrate onto LM1003genome DNA at attBB’ site, this site-specific integrated plasmid can be used for gene complement for genedeleted strain.