Genomic Analysis Of Broad-host-range Salmonella Bacteriophage And Lytic Activity Of The Recombinant Phage-encoded Endolysin

Salmonella is Gram-negative, rod shaped bacterium, which are the importantzoonotic pathogens with an enormous social and economic impact, one of the leadingcauses of foodborne illness worldwide. In recent years, the emergence of extensivelyantibiotic-resistant Salmonella has become serious worldwide concern. As thetreatment options become increasing limited, the development and evaluation of newstrategies for the control of Salmonella is urgently needed. Bacteriophages arebacterial viruses that only infect and multiply within their specific hosts. They areubiquitously distributed in nature, representing the most abundant replicating unit onearth. Many studies illustrated that the broad-host range phages and their endolysinshave potential as effective antimicrobial agents.A novel lytic Salmonella phage STP4-a was isolated, and its major biological andgenomic characteristics were determined. STP4-a has the broad-host-range andstability lysis activity and no toxin genes, which shows the potential application asantibacterial agents. The full genome sequence of bacteriophage contains genes thatencode holin, anti-holin, endolysin and Rz/Rz1-like accessory proteins, which are5phage lysis proteins. The lysis mechanism of STP4-a was analyzed and the criticalenzyme of endolysin was expressed with the lysis activity. This work facilitates theabundance of the genome and lysis mechanism of Salmonella phage, and alsoprovides theoretical basis for the application of endolysin. The main results weresummarized as follows.Bacteriophage STP4-a was isolated from a sewage sample as a clearing zoneformer against strain S. Typhimurium ATCC14028. Electron microscopy of STP4-ashows that the phage with contractile tail structures belongs to the order Caudovirales and family Myoviridae. STP4-a has relatively broad host range, which can lysis fivedifferent Salmonella serotypes. The antimicrobial activity of STP4-a was stable overbroad pH and temperature ranges and was active from pH4.0to12and from40°C to50°C. After12months storage under-20°C, the acivity of phage also kept90%.The complete STP4-a genome was sequenced using pyrosequencing (Roche454GS FLX+Titanium sequencer) and assembled using the GS de novo assemblersoftware. Functional genomics analysis of STP4-a were carried out by DNAstar,Glimmer3.0, ORF Finder of NCBI, BLAST and ClustalX programs. The genome ofSTP4-a contains159,914bp of circular double-stranded DNA with an average GCcontent of36.86%. The bioinformatics analysis revealed a total of257ORFs and4tRNA gene and the average size of the ORFs is577bp, taking up to92.79%of thetotal coding capacity of the genome. Using the BLAST, the251ORFS was found theHomologous sequences,128of these were identified the function. The annotationresults show that STP4-a have no toxin factor, which show this novelbroad-host-range phage is safety in gene and has potential to use as therapeutic agent.After multiple sequence blast, it is certified that STP4-a is T4like virus and the firstlyPseudo T-evens Salmonella phage. The annotated genomic sequence of phage STP4-ais able from the NCBI under the accession number KJ000058.The full genome sequence of bacteriophage contains genes that encode holin,anti-holin, endolysin and Rz/Rz1-like accessory proteins, which are5phage lysisproteins. Across the similar sequence and motif blast and the feature of sequenceidentify, the lysis mechanism of STP4-a was induced by endolysin and regulated bythe other four protein. LysSTP4, like lysozyme, cleaves the glycosides bond ofpeptidoglycan, which shows the potential as antibacterial agent.To illustrate the potential application of endolysin, it was cloned into pET30avector and expressed in E.coli. The enzyme was purified by Ni-nitrilotracetic acidagarose and dialyzed against storage buffer using centrifugal filter tube. The finalconcentration of purified endolysin was400μg/mL. According to the turbidity andplate diffusion methods, the expression of endolysin was sufficient to cause lysis, which is the firstly report about endolysin encoded by T4like Salmonella phage.The host range test of endolysin revealed that it has a more broad inhibition rangeagainst member of the Salmonella and E.coli than STP4-a.The antimicrobial activityof endolysin was stable over broad pH and temperature ranges and was active frompH5.0to10and from30°C to50°C. After6months storage under-80°C, theacivity of endolysin also kept85%. Ethylenediaminetetraacetic acid (EDTA) was usedto increase outer membrane permeability, and it greatly enhanced the lytic activity ofLysSTP4endolysin. These results suggested that LysSTP4has potential as atherapeutic agent against Gram-negative bacteria.