The Novel Shewanella Putrefaciens-infecting Bacteriophage Spp001: Genome Sequence And Lytic Activity

Shewanella putrefaciens has been identified as a specific spoilage organism(SSO) commonly found in chilled fresh fish, which contributes to the spoilage of fishproducts. Limiting S. putrefaciens growth can extend the shelf-life of chilled fish.Endolysins, which efficiently lyse the bacterial host cell wall in the late stages of thephage’s lytic replication cycle to release the viral progeny to the extracellularenvironment, have been considered an alternative to control bacterial growth, andhave been useful in various applications, including food preservation. The approachof using SSO-targeting biopreservative which do not impact the organolepticproperties of the food was not studied for food preservation. Thus SSO-targetingendolysins instead of chemical preservative will apply to prevent bacterial infectionand control bacterial growth.Phage Spp001could extent the shelf life of chilled flounder fillet withoutaffecting the aesthetic quality. To further examine the lytic activity of Spp001, wehave analyzed the complete genome sequence of phage Spp001, extracted the lyticenzyme-containing fraction from phages released at the end of the phage lytic cycle inS putrefaciens, established detection of lytic activity assays, and confirmed thecharacter of partially purified lytic enzymes.We report here, for the first time, the complete genome sequence of a novelphage Spp001, which lyses S. putrefaciens Sp225. The Spp001genome comprises a54,789-bp DNA molecule with67open reading frames (ORF) and an average totalG+C content of49.42%. In silico analysis revealed that the Spp001ORF encodevarious putative functional proteins, including an endolysin (ORF62); however, nosequence for genes encoding the holin polypeptides, which work in concert withendolysins, was identified.The lytic enzyme-containing fraction from phages released at the end of the phage lytic cycle in S putrefaciens was performed through diffusion and turbidimetricassays. The results show that the partially purified extract have high hydrolyticactivity due to endolysin, and the OD590of bacterial substrate decrease by0.160in15min. Compared with phage and lysozyme, partially purified lytic enzymes of Spp001had better lytic activity, which could efficiently lyse the bacterial peptidoglycan, diedcells and living cells within short time (5min). Partially purified lytic enzymes ofSpp001could destroy the cell wall even cell membrane, which was observed bytransmission electron microscope and alkaline phosphatase (AKP) content.The effects of the environment parameters on the crude lytic enzymes of Spp001were as follows, it has the highest lytic activity at pH6-8(optimum pH7.0). Theenzymes were active to lyse the bacteria between20to30°C (optimum30°C). Theenzyme had good salt resistance. Metal ion did not take effect on activity. At optimumconditions, the lytic activity increased from160U/mL to380U/mL. The enzyme isthermally stability, and showing activity even after incubation at50°C for24h. Whenpermeabilization agent (0.1%Triton-X-100) was added to the enzymes, its enzymaticactivity could increase to404U/mL. Crude lytic enzymes would loss activity at4°Cand lost73.7%at-80°C for9days.Spp001was a novel Shewanella putrefaciens-infecting bacteriophage, whichhave high encoded endolysin with strong lytic activity. The results will allow furtherthe investigation of the purification of natural Spp001endolysin, the extension ofSpp001host range, and the applications of the phage-encoded enzymes.