Recombinant production of A1S_0222 from Acinetobacter baumannii ATCC 17978 and confirmation of its DNA-(adenine N6)-methyltransferase activity
Acinetobacter baumannii appears as an often multidrug-resistant nosocomial pathogen in hospitals worldwide. Its remarkable persistence in the hospital environment is probably due to intrinsic and acquired resistance to disinfectants and antibiotics, tolerance to desiccation stress, capability to for...
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| Veröffentlicht in: | Protein expression and purification 2018-11, Vol.151, p.78-85 |
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| creator | Blaschke, Ulrike Suwono, Beneditta Zafari, Sachli Ebersberger, Ingo Skiebe, Evelyn Jeffries, Cy M. Svergun, Dmitri I. Wilharm, Gottfried |
| description | Acinetobacter baumannii appears as an often multidrug-resistant nosocomial pathogen in hospitals worldwide. Its remarkable persistence in the hospital environment is probably due to intrinsic and acquired resistance to disinfectants and antibiotics, tolerance to desiccation stress, capability to form biofilms, and is possibly facilitated by surface-associated motility. Our attempts to elucidate surface-associated motility in A. baumannii revealed a mutant inactivated in a putative DNA-(adenine N6)-methyltransferase, designated A1S_0222 in strain ATCC 17978. We recombinantly produced A1S_0222 as a glutathione S-transferase (GST) fusion protein and purified it to near homogeneity through a combination of GST affinity chromatography, cation exchange chromatography and PD-10 desalting column. Furthermore we demonstrate A1S_0222-dependent adenine methylation at a GAATTC site. We propose the name AamA (Acinetobacteradenine methyltransferase A) in addition to the formal names M.AbaBGORF222P/M.Aba17978ORF8565P. Small angle X-ray scattering (SAXS) revealed that the protein is monomeric and has an extended and likely two-domain shape in solution.
•First purification of a m6A DNA methyltransferase from Acinetobacter baumannii (AamA).•Verification of methyltransferase activity of AamA in vitro.•Monomeric structure of AamA confirmed by SAXS.•AamA is ubiquitously present in Acinetobacter spp. |
| doi_str_mv | 10.1016/j.pep.2018.06.009 |
| format | Article |
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•First purification of a m6A DNA methyltransferase from Acinetobacter baumannii (AamA).•Verification of methyltransferase activity of AamA in vitro.•Monomeric structure of AamA confirmed by SAXS.•AamA is ubiquitously present in Acinetobacter spp.</description><identifier>ISSN: 1046-5928</identifier><identifier>EISSN: 1096-0279</identifier><identifier>DOI: 10.1016/j.pep.2018.06.009</identifier><identifier>PMID: 29908915</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>AamA ; Acinetobacter baumannii ; Acinetobacter baumannii - genetics ; Bacterial Proteins - biosynthesis ; Bacterial Proteins - genetics ; DNA Methylation ; DNA-adenine-methyltransferase ; E.coli ; Epigenetic ; Escherichia coli - genetics ; Escherichia coli - metabolism ; Glutathione Transferase - genetics ; Glutathione Transferase - metabolism ; M.AbaBGORF222P ; Methyltransferases - biosynthesis ; Methyltransferases - genetics ; Mutation ; Protein Binding ; Protein Conformation ; Recombinant ; Recombinant Fusion Proteins - biosynthesis ; Recombinant Fusion Proteins - genetics</subject><ispartof>Protein expression and purification, 2018-11, Vol.151, p.78-85</ispartof><rights>2018 Elsevier Inc.</rights><rights>Copyright © 2018 Elsevier Inc. All rights reserved.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c462t-d7d495bd50a77e0fc4385e58aef0a305e5cd41406bb6d125ae40b160bb3eaf283</citedby><cites>FETCH-LOGICAL-c462t-d7d495bd50a77e0fc4385e58aef0a305e5cd41406bb6d125ae40b160bb3eaf283</cites><orcidid>0000-0002-1771-6799 ; 0000-0001-8187-9253 ; 0000-0003-3054-1935</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.pep.2018.06.009$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3550,27924,27925,45995</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/29908915$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Blaschke, Ulrike</creatorcontrib><creatorcontrib>Suwono, Beneditta</creatorcontrib><creatorcontrib>Zafari, Sachli</creatorcontrib><creatorcontrib>Ebersberger, Ingo</creatorcontrib><creatorcontrib>Skiebe, Evelyn</creatorcontrib><creatorcontrib>Jeffries, Cy M.</creatorcontrib><creatorcontrib>Svergun, Dmitri I.</creatorcontrib><creatorcontrib>Wilharm, Gottfried</creatorcontrib><title>Recombinant production of A1S_0222 from Acinetobacter baumannii ATCC 17978 and confirmation of its DNA-(adenine N6)-methyltransferase activity</title><title>Protein expression and purification</title><addtitle>Protein Expr Purif</addtitle><description>Acinetobacter baumannii appears as an often multidrug-resistant nosocomial pathogen in hospitals worldwide. Its remarkable persistence in the hospital environment is probably due to intrinsic and acquired resistance to disinfectants and antibiotics, tolerance to desiccation stress, capability to form biofilms, and is possibly facilitated by surface-associated motility. Our attempts to elucidate surface-associated motility in A. baumannii revealed a mutant inactivated in a putative DNA-(adenine N6)-methyltransferase, designated A1S_0222 in strain ATCC 17978. We recombinantly produced A1S_0222 as a glutathione S-transferase (GST) fusion protein and purified it to near homogeneity through a combination of GST affinity chromatography, cation exchange chromatography and PD-10 desalting column. Furthermore we demonstrate A1S_0222-dependent adenine methylation at a GAATTC site. We propose the name AamA (Acinetobacteradenine methyltransferase A) in addition to the formal names M.AbaBGORF222P/M.Aba17978ORF8565P. Small angle X-ray scattering (SAXS) revealed that the protein is monomeric and has an extended and likely two-domain shape in solution.
•First purification of a m6A DNA methyltransferase from Acinetobacter baumannii (AamA).•Verification of methyltransferase activity of AamA in vitro.•Monomeric structure of AamA confirmed by SAXS.•AamA is ubiquitously present in Acinetobacter spp.</description><subject>AamA</subject><subject>Acinetobacter baumannii</subject><subject>Acinetobacter baumannii - genetics</subject><subject>Bacterial Proteins - biosynthesis</subject><subject>Bacterial Proteins - genetics</subject><subject>DNA Methylation</subject><subject>DNA-adenine-methyltransferase</subject><subject>E.coli</subject><subject>Epigenetic</subject><subject>Escherichia coli - genetics</subject><subject>Escherichia coli - metabolism</subject><subject>Glutathione Transferase - genetics</subject><subject>Glutathione Transferase - metabolism</subject><subject>M.AbaBGORF222P</subject><subject>Methyltransferases - biosynthesis</subject><subject>Methyltransferases - genetics</subject><subject>Mutation</subject><subject>Protein Binding</subject><subject>Protein Conformation</subject><subject>Recombinant</subject><subject>Recombinant Fusion Proteins - biosynthesis</subject><subject>Recombinant Fusion Proteins - genetics</subject><issn>1046-5928</issn><issn>1096-0279</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2018</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kc1u1DAUhSMEoqXwAGyQl2WRcO0kTiJW0fBXqWqlUtaWf66FRxN7sJ1K8xI8Mx5Ny7KrexbnfNK5p6reU2goUP5p2-xx3zCgYwO8AZheVOcUJl4DG6aXR93xup_YeFa9SWkLQCmH_nV1xqYJxon259XfO9RhUc5Ln8k-BrPq7IInwZKZ_hTAGCM2hoXM2nnMQUmdMRIl10V67xyZ7zcbQodpGIn0hujgrYuLfIK4nMiXm7m-lAZ9IZAb_rFeMP8-7HKUPlmMMiEpVPfg8uFt9crKXcJ3j_ei-vXt6_3mR319-_1qM1_XuuMs12Yw3dQr04McBgSru3bssR8lWpAtFKlNRzvgSnFDWS-xA1W6K9WitGxsL6rLE7dU_rNiymJxSeNuJz2GNQkGPR94SwdWrPRk1TGkFNGKfXSLjAdBQRxnEFtRZhDHGQRwUWYomQ-P-FUtaP4nnv5eDJ9PBiwlHxxGkbRDr9G4iDoLE9wz-H8sPphL</recordid><startdate>201811</startdate><enddate>201811</enddate><creator>Blaschke, Ulrike</creator><creator>Suwono, Beneditta</creator><creator>Zafari, Sachli</creator><creator>Ebersberger, Ingo</creator><creator>Skiebe, Evelyn</creator><creator>Jeffries, Cy M.</creator><creator>Svergun, Dmitri I.</creator><creator>Wilharm, Gottfried</creator><general>Elsevier Inc</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope><orcidid>https://orcid.org/0000-0002-1771-6799</orcidid><orcidid>https://orcid.org/0000-0001-8187-9253</orcidid><orcidid>https://orcid.org/0000-0003-3054-1935</orcidid></search><sort><creationdate>201811</creationdate><title>Recombinant production of A1S_0222 from Acinetobacter baumannii ATCC 17978 and confirmation of its DNA-(adenine N6)-methyltransferase activity</title><author>Blaschke, Ulrike ; Suwono, Beneditta ; Zafari, Sachli ; Ebersberger, Ingo ; Skiebe, Evelyn ; Jeffries, Cy M. ; Svergun, Dmitri I. ; Wilharm, Gottfried</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c462t-d7d495bd50a77e0fc4385e58aef0a305e5cd41406bb6d125ae40b160bb3eaf283</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2018</creationdate><topic>AamA</topic><topic>Acinetobacter baumannii</topic><topic>Acinetobacter baumannii - genetics</topic><topic>Bacterial Proteins - biosynthesis</topic><topic>Bacterial Proteins - genetics</topic><topic>DNA Methylation</topic><topic>DNA-adenine-methyltransferase</topic><topic>E.coli</topic><topic>Epigenetic</topic><topic>Escherichia coli - genetics</topic><topic>Escherichia coli - metabolism</topic><topic>Glutathione Transferase - genetics</topic><topic>Glutathione Transferase - metabolism</topic><topic>M.AbaBGORF222P</topic><topic>Methyltransferases - biosynthesis</topic><topic>Methyltransferases - genetics</topic><topic>Mutation</topic><topic>Protein Binding</topic><topic>Protein Conformation</topic><topic>Recombinant</topic><topic>Recombinant Fusion Proteins - biosynthesis</topic><topic>Recombinant Fusion Proteins - genetics</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Blaschke, Ulrike</creatorcontrib><creatorcontrib>Suwono, Beneditta</creatorcontrib><creatorcontrib>Zafari, Sachli</creatorcontrib><creatorcontrib>Ebersberger, Ingo</creatorcontrib><creatorcontrib>Skiebe, Evelyn</creatorcontrib><creatorcontrib>Jeffries, Cy M.</creatorcontrib><creatorcontrib>Svergun, Dmitri I.</creatorcontrib><creatorcontrib>Wilharm, Gottfried</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Protein expression and purification</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Blaschke, Ulrike</au><au>Suwono, Beneditta</au><au>Zafari, Sachli</au><au>Ebersberger, Ingo</au><au>Skiebe, Evelyn</au><au>Jeffries, Cy M.</au><au>Svergun, Dmitri I.</au><au>Wilharm, Gottfried</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Recombinant production of A1S_0222 from Acinetobacter baumannii ATCC 17978 and confirmation of its DNA-(adenine N6)-methyltransferase activity</atitle><jtitle>Protein expression and purification</jtitle><addtitle>Protein Expr Purif</addtitle><date>2018-11</date><risdate>2018</risdate><volume>151</volume><spage>78</spage><epage>85</epage><pages>78-85</pages><issn>1046-5928</issn><eissn>1096-0279</eissn><abstract>Acinetobacter baumannii appears as an often multidrug-resistant nosocomial pathogen in hospitals worldwide. Its remarkable persistence in the hospital environment is probably due to intrinsic and acquired resistance to disinfectants and antibiotics, tolerance to desiccation stress, capability to form biofilms, and is possibly facilitated by surface-associated motility. Our attempts to elucidate surface-associated motility in A. baumannii revealed a mutant inactivated in a putative DNA-(adenine N6)-methyltransferase, designated A1S_0222 in strain ATCC 17978. We recombinantly produced A1S_0222 as a glutathione S-transferase (GST) fusion protein and purified it to near homogeneity through a combination of GST affinity chromatography, cation exchange chromatography and PD-10 desalting column. Furthermore we demonstrate A1S_0222-dependent adenine methylation at a GAATTC site. We propose the name AamA (Acinetobacteradenine methyltransferase A) in addition to the formal names M.AbaBGORF222P/M.Aba17978ORF8565P. Small angle X-ray scattering (SAXS) revealed that the protein is monomeric and has an extended and likely two-domain shape in solution.
•First purification of a m6A DNA methyltransferase from Acinetobacter baumannii (AamA).•Verification of methyltransferase activity of AamA in vitro.•Monomeric structure of AamA confirmed by SAXS.•AamA is ubiquitously present in Acinetobacter spp.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>29908915</pmid><doi>10.1016/j.pep.2018.06.009</doi><tpages>8</tpages><orcidid>https://orcid.org/0000-0002-1771-6799</orcidid><orcidid>https://orcid.org/0000-0001-8187-9253</orcidid><orcidid>https://orcid.org/0000-0003-3054-1935</orcidid><oa>free_for_read</oa></addata></record> |
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| subjects | AamA Acinetobacter baumannii Acinetobacter baumannii - genetics Bacterial Proteins - biosynthesis Bacterial Proteins - genetics DNA Methylation DNA-adenine-methyltransferase E.coli Epigenetic Escherichia coli - genetics Escherichia coli - metabolism Glutathione Transferase - genetics Glutathione Transferase - metabolism M.AbaBGORF222P Methyltransferases - biosynthesis Methyltransferases - genetics Mutation Protein Binding Protein Conformation Recombinant Recombinant Fusion Proteins - biosynthesis Recombinant Fusion Proteins - genetics |
| title | Recombinant production of A1S_0222 from Acinetobacter baumannii ATCC 17978 and confirmation of its DNA-(adenine N6)-methyltransferase activity |
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