Use of prophage free host for achieving homogenous population of bacteriophages: New findings
► A new methodology of making pure bacteriophage preparation using prophage free hosts is proposed. ► The role of phage endolysin in dictating host range of a phage is demonstrated. ► Release of prophages during phage multiplication in a prophage containing host is highlighted. ► We report efficienc...
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| Veröffentlicht in: | Virus research 2012-10, Vol.169 (1), p.182-187 |
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| creator | Nirmal Kumar, G.P. Sundarrajan, Sudarson Paul, Vivek Daniel Nandini, S. Saravanan, R. Sanjeev Hariharan, Sukumar Sriram, Bharathi Padmanabhan, Sriram |
| description | ► A new methodology of making pure bacteriophage preparation using prophage free hosts is proposed. ► The role of phage endolysin in dictating host range of a phage is demonstrated. ► Release of prophages during phage multiplication in a prophage containing host is highlighted. ► We report efficiency of phage endolysin to play a role in effective phage propagation. ► Strength of endolysin for phage release in identification of a potent phage is speculated.
We demonstrate that the prophage status of bacteria plays a critical role in achieving homogenous population of a phage preparation. When a lytic Staphylococcus bacteriophage 44AHJD was propagated in a Staphylococcus clinical isolate, the enriched phage showed 44AHJD phage virions along with the released prophages from the baiting host. The released prophage was identified as a siphophage by transmission electron microscopy. To obtain a phage preparation free of prophages, when we carried out multiplication of the 44AHJD phage in a prophage free Staphyloccoccus aureus host namely RN4220, we were surprised not to see any phage plaques in spite of the phage exhibiting >99.9% adsorption to such cells. Since RN4220 host is devoid of restriction modification system and prophages, we hypothesized that in spite of successful infection and multiplication, the phage virions might have failed to show plaques due to its insignificant release from the cell possibly due to insufficient endolysin expressed from phage virions during phage development and assembly. Our hypothesis was confirmed when we observed plaques of 44AHJD phage in RN4220 cells where additional phage endolysin protein was supplemented via a plasmid. Endolysin protein from various types of Staphylococcus phages showed plaques of 44AHJD in RN4220 cells confirming our hypothesis. Also, we demonstrate for the first time that propagation of 44AHJD phage with endolysin supplementation in prophage free RN4220 host yields pure phage preparation. |
| doi_str_mv | 10.1016/j.virusres.2012.07.026 |
| format | Article |
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We demonstrate that the prophage status of bacteria plays a critical role in achieving homogenous population of a phage preparation. When a lytic Staphylococcus bacteriophage 44AHJD was propagated in a Staphylococcus clinical isolate, the enriched phage showed 44AHJD phage virions along with the released prophages from the baiting host. The released prophage was identified as a siphophage by transmission electron microscopy. To obtain a phage preparation free of prophages, when we carried out multiplication of the 44AHJD phage in a prophage free Staphyloccoccus aureus host namely RN4220, we were surprised not to see any phage plaques in spite of the phage exhibiting >99.9% adsorption to such cells. Since RN4220 host is devoid of restriction modification system and prophages, we hypothesized that in spite of successful infection and multiplication, the phage virions might have failed to show plaques due to its insignificant release from the cell possibly due to insufficient endolysin expressed from phage virions during phage development and assembly. Our hypothesis was confirmed when we observed plaques of 44AHJD phage in RN4220 cells where additional phage endolysin protein was supplemented via a plasmid. Endolysin protein from various types of Staphylococcus phages showed plaques of 44AHJD in RN4220 cells confirming our hypothesis. Also, we demonstrate for the first time that propagation of 44AHJD phage with endolysin supplementation in prophage free RN4220 host yields pure phage preparation.</description><identifier>ISSN: 0168-1702</identifier><identifier>EISSN: 1872-7492</identifier><identifier>DOI: 10.1016/j.virusres.2012.07.026</identifier><identifier>PMID: 22917718</identifier><language>eng</language><publisher>Netherlands: Elsevier B.V</publisher><subject>Adsorption ; Animals ; bacteria ; Bacteriolysis ; Bacteriophages ; Bacteriophages - genetics ; Bacteriophages - isolation & purification ; Baiting ; Clinical isolates ; Complementation ; Endolysin ; Endopeptidases - genetics ; Endopeptidases - metabolism ; Humans ; Infection ; Microscopy, Electron, Transmission ; Phages ; Plaques ; Plasmids ; Prophages ; Prophages - genetics ; Prophages - isolation & purification ; Prophages - ultrastructure ; restriction endonucleases ; RN4220 ; Staphylococcal Infections - microbiology ; Staphylococcus ; Staphylococcus aureus - genetics ; Staphylococcus aureus - isolation & purification ; Staphylococcus aureus - virology ; Superinfection immunity ; Supplementation ; Transmission electron microscopy ; VegII promoter ; virion ; Virion - ultrastructure ; Virions</subject><ispartof>Virus research, 2012-10, Vol.169 (1), p.182-187</ispartof><rights>2012 Elsevier B.V.</rights><rights>Copyright © 2012 Elsevier B.V. All rights reserved.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c425t-ab127b9627d1f358957b8ff646e4a54746f1e3c6c7788226aa6d2f8e475f8a633</citedby><cites>FETCH-LOGICAL-c425t-ab127b9627d1f358957b8ff646e4a54746f1e3c6c7788226aa6d2f8e475f8a633</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.virusres.2012.07.026$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>315,781,785,3551,27928,27929,45999</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/22917718$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Nirmal Kumar, G.P.</creatorcontrib><creatorcontrib>Sundarrajan, Sudarson</creatorcontrib><creatorcontrib>Paul, Vivek Daniel</creatorcontrib><creatorcontrib>Nandini, S.</creatorcontrib><creatorcontrib>Saravanan, R. Sanjeev</creatorcontrib><creatorcontrib>Hariharan, Sukumar</creatorcontrib><creatorcontrib>Sriram, Bharathi</creatorcontrib><creatorcontrib>Padmanabhan, Sriram</creatorcontrib><title>Use of prophage free host for achieving homogenous population of bacteriophages: New findings</title><title>Virus research</title><addtitle>Virus Res</addtitle><description>► A new methodology of making pure bacteriophage preparation using prophage free hosts is proposed. ► The role of phage endolysin in dictating host range of a phage is demonstrated. ► Release of prophages during phage multiplication in a prophage containing host is highlighted. ► We report efficiency of phage endolysin to play a role in effective phage propagation. ► Strength of endolysin for phage release in identification of a potent phage is speculated.
We demonstrate that the prophage status of bacteria plays a critical role in achieving homogenous population of a phage preparation. When a lytic Staphylococcus bacteriophage 44AHJD was propagated in a Staphylococcus clinical isolate, the enriched phage showed 44AHJD phage virions along with the released prophages from the baiting host. The released prophage was identified as a siphophage by transmission electron microscopy. To obtain a phage preparation free of prophages, when we carried out multiplication of the 44AHJD phage in a prophage free Staphyloccoccus aureus host namely RN4220, we were surprised not to see any phage plaques in spite of the phage exhibiting >99.9% adsorption to such cells. Since RN4220 host is devoid of restriction modification system and prophages, we hypothesized that in spite of successful infection and multiplication, the phage virions might have failed to show plaques due to its insignificant release from the cell possibly due to insufficient endolysin expressed from phage virions during phage development and assembly. Our hypothesis was confirmed when we observed plaques of 44AHJD phage in RN4220 cells where additional phage endolysin protein was supplemented via a plasmid. Endolysin protein from various types of Staphylococcus phages showed plaques of 44AHJD in RN4220 cells confirming our hypothesis. Also, we demonstrate for the first time that propagation of 44AHJD phage with endolysin supplementation in prophage free RN4220 host yields pure phage preparation.</description><subject>Adsorption</subject><subject>Animals</subject><subject>bacteria</subject><subject>Bacteriolysis</subject><subject>Bacteriophages</subject><subject>Bacteriophages - genetics</subject><subject>Bacteriophages - isolation & purification</subject><subject>Baiting</subject><subject>Clinical isolates</subject><subject>Complementation</subject><subject>Endolysin</subject><subject>Endopeptidases - genetics</subject><subject>Endopeptidases - metabolism</subject><subject>Humans</subject><subject>Infection</subject><subject>Microscopy, Electron, Transmission</subject><subject>Phages</subject><subject>Plaques</subject><subject>Plasmids</subject><subject>Prophages</subject><subject>Prophages - genetics</subject><subject>Prophages - isolation & purification</subject><subject>Prophages - ultrastructure</subject><subject>restriction endonucleases</subject><subject>RN4220</subject><subject>Staphylococcal Infections - microbiology</subject><subject>Staphylococcus</subject><subject>Staphylococcus aureus - genetics</subject><subject>Staphylococcus aureus - isolation & purification</subject><subject>Staphylococcus aureus - virology</subject><subject>Superinfection immunity</subject><subject>Supplementation</subject><subject>Transmission electron microscopy</subject><subject>VegII promoter</subject><subject>virion</subject><subject>Virion - ultrastructure</subject><subject>Virions</subject><issn>0168-1702</issn><issn>1872-7492</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2012</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkcFv2yAYxVG1qsm6_gsdx13sAbYB77QpWrtJUXvocqwQxh8JUWI8sDP1vy-W213DBQn93vs-3kPolpKcEsq_7vOTC2MMEHNGKMuJyAnjF2hJpWCZKGv2AS0TKDMqCFugjzHuCSG8EPwKLRirqRBULtHzJgL2FvfB9zu9BWwDAN75OGDrA9Zm5-Dkum16OvotdH6MuPf9eNCD892kbLQZILhZHr_hB_iHrevaJIqf0KXVhwg3b_c12tz9_LP6la0f73-vfqwzU7JqyHRDmWhqzkRLbVHJuhKNtJaXHEpdlaLklkJhuBFCSsa41rxlVkIpKis1L4pr9GX2Td_4O0Ic1NFFA4eD7iBtrCilBZtOeR4ldZUcZcUSymfUBB9T1Fb1wR11eEmQmlpQe_XegppaUESo1EIS3r7NGJsjtP9l77En4PMMWO2V3gYX1eYpOfBUUSLkNPv7TECK7eQgqGgcdAZaF8AMqvXu3BavndOlsQ</recordid><startdate>20121001</startdate><enddate>20121001</enddate><creator>Nirmal Kumar, G.P.</creator><creator>Sundarrajan, Sudarson</creator><creator>Paul, Vivek Daniel</creator><creator>Nandini, S.</creator><creator>Saravanan, R. 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Sanjeev ; Hariharan, Sukumar ; Sriram, Bharathi ; Padmanabhan, Sriram</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c425t-ab127b9627d1f358957b8ff646e4a54746f1e3c6c7788226aa6d2f8e475f8a633</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2012</creationdate><topic>Adsorption</topic><topic>Animals</topic><topic>bacteria</topic><topic>Bacteriolysis</topic><topic>Bacteriophages</topic><topic>Bacteriophages - genetics</topic><topic>Bacteriophages - isolation & purification</topic><topic>Baiting</topic><topic>Clinical isolates</topic><topic>Complementation</topic><topic>Endolysin</topic><topic>Endopeptidases - genetics</topic><topic>Endopeptidases - metabolism</topic><topic>Humans</topic><topic>Infection</topic><topic>Microscopy, Electron, Transmission</topic><topic>Phages</topic><topic>Plaques</topic><topic>Plasmids</topic><topic>Prophages</topic><topic>Prophages - genetics</topic><topic>Prophages - isolation & purification</topic><topic>Prophages - ultrastructure</topic><topic>restriction endonucleases</topic><topic>RN4220</topic><topic>Staphylococcal Infections - microbiology</topic><topic>Staphylococcus</topic><topic>Staphylococcus aureus - genetics</topic><topic>Staphylococcus aureus - isolation & purification</topic><topic>Staphylococcus aureus - virology</topic><topic>Superinfection immunity</topic><topic>Supplementation</topic><topic>Transmission electron microscopy</topic><topic>VegII promoter</topic><topic>virion</topic><topic>Virion - ultrastructure</topic><topic>Virions</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Nirmal Kumar, G.P.</creatorcontrib><creatorcontrib>Sundarrajan, Sudarson</creatorcontrib><creatorcontrib>Paul, Vivek Daniel</creatorcontrib><creatorcontrib>Nandini, S.</creatorcontrib><creatorcontrib>Saravanan, R. 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Sanjeev</au><au>Hariharan, Sukumar</au><au>Sriram, Bharathi</au><au>Padmanabhan, Sriram</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Use of prophage free host for achieving homogenous population of bacteriophages: New findings</atitle><jtitle>Virus research</jtitle><addtitle>Virus Res</addtitle><date>2012-10-01</date><risdate>2012</risdate><volume>169</volume><issue>1</issue><spage>182</spage><epage>187</epage><pages>182-187</pages><issn>0168-1702</issn><eissn>1872-7492</eissn><abstract>► A new methodology of making pure bacteriophage preparation using prophage free hosts is proposed. ► The role of phage endolysin in dictating host range of a phage is demonstrated. ► Release of prophages during phage multiplication in a prophage containing host is highlighted. ► We report efficiency of phage endolysin to play a role in effective phage propagation. ► Strength of endolysin for phage release in identification of a potent phage is speculated.
We demonstrate that the prophage status of bacteria plays a critical role in achieving homogenous population of a phage preparation. When a lytic Staphylococcus bacteriophage 44AHJD was propagated in a Staphylococcus clinical isolate, the enriched phage showed 44AHJD phage virions along with the released prophages from the baiting host. The released prophage was identified as a siphophage by transmission electron microscopy. To obtain a phage preparation free of prophages, when we carried out multiplication of the 44AHJD phage in a prophage free Staphyloccoccus aureus host namely RN4220, we were surprised not to see any phage plaques in spite of the phage exhibiting >99.9% adsorption to such cells. Since RN4220 host is devoid of restriction modification system and prophages, we hypothesized that in spite of successful infection and multiplication, the phage virions might have failed to show plaques due to its insignificant release from the cell possibly due to insufficient endolysin expressed from phage virions during phage development and assembly. Our hypothesis was confirmed when we observed plaques of 44AHJD phage in RN4220 cells where additional phage endolysin protein was supplemented via a plasmid. Endolysin protein from various types of Staphylococcus phages showed plaques of 44AHJD in RN4220 cells confirming our hypothesis. Also, we demonstrate for the first time that propagation of 44AHJD phage with endolysin supplementation in prophage free RN4220 host yields pure phage preparation.</abstract><cop>Netherlands</cop><pub>Elsevier B.V</pub><pmid>22917718</pmid><doi>10.1016/j.virusres.2012.07.026</doi><tpages>6</tpages></addata></record> |
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| subjects | Adsorption Animals bacteria Bacteriolysis Bacteriophages Bacteriophages - genetics Bacteriophages - isolation & purification Baiting Clinical isolates Complementation Endolysin Endopeptidases - genetics Endopeptidases - metabolism Humans Infection Microscopy, Electron, Transmission Phages Plaques Plasmids Prophages Prophages - genetics Prophages - isolation & purification Prophages - ultrastructure restriction endonucleases RN4220 Staphylococcal Infections - microbiology Staphylococcus Staphylococcus aureus - genetics Staphylococcus aureus - isolation & purification Staphylococcus aureus - virology Superinfection immunity Supplementation Transmission electron microscopy VegII promoter virion Virion - ultrastructure Virions |
| title | Use of prophage free host for achieving homogenous population of bacteriophages: New findings |
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