A novel high-throughput cell-based assay aimed at identifying inhibitors of DNA metabolism in bacteria
Bacterial biosensor strains can be useful tools for the discovery and characterization of antibacterial compounds. A plasmid-based reporter vector containing a transcriptional fusion between the recA promoter and green fluorescence protein gene was introduced into an Escherichia coli ΔtolC strain to...
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| Veröffentlicht in: | Antimicrobial agents and chemotherapy 2014-12, Vol.58 (12), p.7264-7272 |
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| container_title | Antimicrobial agents and chemotherapy |
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| creator | Fan, Jun de Jonge, Boudewijn L M MacCormack, Kathy Sriram, Shubha McLaughlin, Robert E Plant, Helen Preston, Marian Fleming, Paul R Albert, Robert Foulk, Melinda Mills, Scott D |
| description | Bacterial biosensor strains can be useful tools for the discovery and characterization of antibacterial compounds. A plasmid-based reporter vector containing a transcriptional fusion between the recA promoter and green fluorescence protein gene was introduced into an Escherichia coli ΔtolC strain to create a biosensor strain that selectively senses inhibitors of DNA metabolism via the SOS response. The strain was used to develop a high-throughput assay to identify new inhibitors of DNA metabolism. Screening of the AstraZeneca compound library with this strain identified known inhibitors of DNA metabolism, as well as novel chemotypes. The cellular target of one novel series was elucidated as DNA gyrase through genetic characterization of laboratory-generated resistant mutants followed by 50% inhibitory concentration measurements in a DNA gyrase activity assay. These studies validated the use of this antibiotic biosensor strain to identify novel selective inhibitors of DNA metabolism by high-throughput screening. |
| doi_str_mv | 10.1128/AAC.03475-14 |
| format | Article |
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A plasmid-based reporter vector containing a transcriptional fusion between the recA promoter and green fluorescence protein gene was introduced into an Escherichia coli ΔtolC strain to create a biosensor strain that selectively senses inhibitors of DNA metabolism via the SOS response. The strain was used to develop a high-throughput assay to identify new inhibitors of DNA metabolism. Screening of the AstraZeneca compound library with this strain identified known inhibitors of DNA metabolism, as well as novel chemotypes. The cellular target of one novel series was elucidated as DNA gyrase through genetic characterization of laboratory-generated resistant mutants followed by 50% inhibitory concentration measurements in a DNA gyrase activity assay. These studies validated the use of this antibiotic biosensor strain to identify novel selective inhibitors of DNA metabolism by high-throughput screening.</description><identifier>ISSN: 0066-4804</identifier><identifier>EISSN: 1098-6596</identifier><identifier>DOI: 10.1128/AAC.03475-14</identifier><identifier>PMID: 25246396</identifier><language>eng</language><publisher>United States: American Society for Microbiology</publisher><subject>Bacterial Outer Membrane Proteins - genetics ; Biosensing Techniques ; DNA Gyrase - genetics ; DNA Gyrase - metabolism ; DNA, Bacterial ; DNA, Bacterial - antagonists & inhibitors ; DNA, Bacterial - genetics ; DNA, Bacterial - metabolism ; Escherichia coli ; Escherichia coli - drug effects ; Escherichia coli - genetics ; Escherichia coli - metabolism ; Escherichia coli Proteins - genetics ; Gene Expression ; Genes, Reporter ; Green Fluorescent Proteins - genetics ; Green Fluorescent Proteins - metabolism ; High-Throughput Screening Assays ; Inhibitory Concentration 50 ; Mechanisms of Action: Physiological Effects ; Membrane Transport Proteins - deficiency ; Membrane Transport Proteins - genetics ; Nucleic Acid Synthesis Inhibitors ; Nucleic Acid Synthesis Inhibitors - chemistry ; Nucleic Acid Synthesis Inhibitors - pharmacology ; Plasmids - chemistry ; Plasmids - metabolism ; Promoter Regions, Genetic ; Rec A Recombinases - genetics ; Rec A Recombinases - metabolism ; Recombinant Fusion Proteins - genetics ; Recombinant Fusion Proteins - metabolism ; Small Molecule Libraries ; Small Molecule Libraries - chemistry ; Small Molecule Libraries - pharmacology ; SOS Response (Genetics) - drug effects</subject><ispartof>Antimicrobial agents and chemotherapy, 2014-12, Vol.58 (12), p.7264-7272</ispartof><rights>Copyright © 2014, American Society for Microbiology. All Rights Reserved.</rights><rights>Copyright © 2014, American Society for Microbiology. All Rights Reserved. 2014 American Society for Microbiology</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a517t-7054e42b757518ac60eeee5b5c51a275f1abb598b0426fa0d5d28526c5ad7f4b3</citedby><cites>FETCH-LOGICAL-a517t-7054e42b757518ac60eeee5b5c51a275f1abb598b0426fa0d5d28526c5ad7f4b3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4249574/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4249574/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,727,780,784,885,27923,27924,53790,53792</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/25246396$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Fan, Jun</creatorcontrib><creatorcontrib>de Jonge, Boudewijn L M</creatorcontrib><creatorcontrib>MacCormack, Kathy</creatorcontrib><creatorcontrib>Sriram, Shubha</creatorcontrib><creatorcontrib>McLaughlin, Robert E</creatorcontrib><creatorcontrib>Plant, Helen</creatorcontrib><creatorcontrib>Preston, Marian</creatorcontrib><creatorcontrib>Fleming, Paul R</creatorcontrib><creatorcontrib>Albert, Robert</creatorcontrib><creatorcontrib>Foulk, Melinda</creatorcontrib><creatorcontrib>Mills, Scott D</creatorcontrib><title>A novel high-throughput cell-based assay aimed at identifying inhibitors of DNA metabolism in bacteria</title><title>Antimicrobial agents and chemotherapy</title><addtitle>Antimicrob Agents Chemother</addtitle><addtitle>Antimicrob Agents Chemother</addtitle><description>Bacterial biosensor strains can be useful tools for the discovery and characterization of antibacterial compounds. A plasmid-based reporter vector containing a transcriptional fusion between the recA promoter and green fluorescence protein gene was introduced into an Escherichia coli ΔtolC strain to create a biosensor strain that selectively senses inhibitors of DNA metabolism via the SOS response. The strain was used to develop a high-throughput assay to identify new inhibitors of DNA metabolism. Screening of the AstraZeneca compound library with this strain identified known inhibitors of DNA metabolism, as well as novel chemotypes. The cellular target of one novel series was elucidated as DNA gyrase through genetic characterization of laboratory-generated resistant mutants followed by 50% inhibitory concentration measurements in a DNA gyrase activity assay. These studies validated the use of this antibiotic biosensor strain to identify novel selective inhibitors of DNA metabolism by high-throughput screening.</description><subject>Bacterial Outer Membrane Proteins - genetics</subject><subject>Biosensing Techniques</subject><subject>DNA Gyrase - genetics</subject><subject>DNA Gyrase - metabolism</subject><subject>DNA, Bacterial</subject><subject>DNA, Bacterial - antagonists & inhibitors</subject><subject>DNA, Bacterial - genetics</subject><subject>DNA, Bacterial - metabolism</subject><subject>Escherichia coli</subject><subject>Escherichia coli - drug effects</subject><subject>Escherichia coli - genetics</subject><subject>Escherichia coli - metabolism</subject><subject>Escherichia coli Proteins - genetics</subject><subject>Gene Expression</subject><subject>Genes, Reporter</subject><subject>Green Fluorescent Proteins - genetics</subject><subject>Green Fluorescent Proteins - metabolism</subject><subject>High-Throughput Screening Assays</subject><subject>Inhibitory Concentration 50</subject><subject>Mechanisms of Action: Physiological Effects</subject><subject>Membrane Transport Proteins - deficiency</subject><subject>Membrane Transport Proteins - genetics</subject><subject>Nucleic Acid Synthesis Inhibitors</subject><subject>Nucleic Acid Synthesis Inhibitors - chemistry</subject><subject>Nucleic Acid Synthesis Inhibitors - pharmacology</subject><subject>Plasmids - chemistry</subject><subject>Plasmids - metabolism</subject><subject>Promoter Regions, Genetic</subject><subject>Rec A Recombinases - genetics</subject><subject>Rec A Recombinases - metabolism</subject><subject>Recombinant Fusion Proteins - genetics</subject><subject>Recombinant Fusion Proteins - metabolism</subject><subject>Small Molecule Libraries</subject><subject>Small Molecule Libraries - chemistry</subject><subject>Small Molecule Libraries - pharmacology</subject><subject>SOS Response (Genetics) - drug effects</subject><issn>0066-4804</issn><issn>1098-6596</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2014</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp1kU1v1DAQhi0Eokvhxhn5SCVSbK_HTi5I0fIpVXCBszVOnI2rJF5sp9L-e7xsqeDAXDyjefXMeF5CXnJ2zbmo37bt7pptpYaKy0dkw1lTVwoa9ZhsGFOqkjWTF-RZSres1NCwp-RCgJBq26gNGVq6hDs30dHvxyqPMaz78bBm2rlpqiwm11NMCY8U_XzKM_W9W7Ifjn7ZU7-M3vocYqJhoO-_tnR2GW2YfJpLk1rssosen5MnA07Jvbh_L8mPjx--7z5XN98-fdm1NxUC17nSDKSTwmrQwGvsFHMlwEIHHIWGgaO10NSWSaEGZD30ogahOsBeD9JuL8m7M_ew2rJuVzaNOJlD9DPGownozb-dxY9mH-6MFLIBLQvg9T0ghp-rS9nMPp1ugYsLazJclclccy2K9M1Z2sWQUnTDwxjOzMkaU6wxv60x_ES-OssxzcLchjUu5RL_0776-xsP4D--bX8BfdKXng</recordid><startdate>20141201</startdate><enddate>20141201</enddate><creator>Fan, Jun</creator><creator>de Jonge, Boudewijn L M</creator><creator>MacCormack, Kathy</creator><creator>Sriram, Shubha</creator><creator>McLaughlin, Robert E</creator><creator>Plant, Helen</creator><creator>Preston, Marian</creator><creator>Fleming, Paul R</creator><creator>Albert, Robert</creator><creator>Foulk, Melinda</creator><creator>Mills, Scott D</creator><general>American Society for Microbiology</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>7QL</scope><scope>7T7</scope><scope>7TM</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>P64</scope><scope>5PM</scope></search><sort><creationdate>20141201</creationdate><title>A novel high-throughput cell-based assay aimed at identifying inhibitors of DNA metabolism in bacteria</title><author>Fan, Jun ; de Jonge, Boudewijn L M ; MacCormack, Kathy ; Sriram, Shubha ; McLaughlin, Robert E ; Plant, Helen ; Preston, Marian ; Fleming, Paul R ; Albert, Robert ; Foulk, Melinda ; Mills, Scott D</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a517t-7054e42b757518ac60eeee5b5c51a275f1abb598b0426fa0d5d28526c5ad7f4b3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2014</creationdate><topic>Bacterial Outer Membrane Proteins - genetics</topic><topic>Biosensing Techniques</topic><topic>DNA Gyrase - genetics</topic><topic>DNA Gyrase - metabolism</topic><topic>DNA, Bacterial</topic><topic>DNA, Bacterial - antagonists & inhibitors</topic><topic>DNA, Bacterial - genetics</topic><topic>DNA, Bacterial - metabolism</topic><topic>Escherichia coli</topic><topic>Escherichia coli - drug effects</topic><topic>Escherichia coli - genetics</topic><topic>Escherichia coli - metabolism</topic><topic>Escherichia coli Proteins - genetics</topic><topic>Gene Expression</topic><topic>Genes, Reporter</topic><topic>Green Fluorescent Proteins - genetics</topic><topic>Green Fluorescent Proteins - metabolism</topic><topic>High-Throughput Screening Assays</topic><topic>Inhibitory Concentration 50</topic><topic>Mechanisms of Action: Physiological Effects</topic><topic>Membrane Transport Proteins - deficiency</topic><topic>Membrane Transport Proteins - genetics</topic><topic>Nucleic Acid Synthesis Inhibitors</topic><topic>Nucleic Acid Synthesis Inhibitors - chemistry</topic><topic>Nucleic Acid Synthesis Inhibitors - pharmacology</topic><topic>Plasmids - chemistry</topic><topic>Plasmids - metabolism</topic><topic>Promoter Regions, Genetic</topic><topic>Rec A Recombinases - genetics</topic><topic>Rec A Recombinases - metabolism</topic><topic>Recombinant Fusion Proteins - genetics</topic><topic>Recombinant Fusion Proteins - metabolism</topic><topic>Small Molecule Libraries</topic><topic>Small Molecule Libraries - chemistry</topic><topic>Small Molecule Libraries - pharmacology</topic><topic>SOS Response (Genetics) - drug effects</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Fan, Jun</creatorcontrib><creatorcontrib>de Jonge, Boudewijn L M</creatorcontrib><creatorcontrib>MacCormack, Kathy</creatorcontrib><creatorcontrib>Sriram, Shubha</creatorcontrib><creatorcontrib>McLaughlin, Robert E</creatorcontrib><creatorcontrib>Plant, Helen</creatorcontrib><creatorcontrib>Preston, Marian</creatorcontrib><creatorcontrib>Fleming, Paul R</creatorcontrib><creatorcontrib>Albert, Robert</creatorcontrib><creatorcontrib>Foulk, Melinda</creatorcontrib><creatorcontrib>Mills, Scott D</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Nucleic Acids Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Antimicrobial agents and chemotherapy</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Fan, Jun</au><au>de Jonge, Boudewijn L M</au><au>MacCormack, Kathy</au><au>Sriram, Shubha</au><au>McLaughlin, Robert E</au><au>Plant, Helen</au><au>Preston, Marian</au><au>Fleming, Paul R</au><au>Albert, Robert</au><au>Foulk, Melinda</au><au>Mills, Scott D</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A novel high-throughput cell-based assay aimed at identifying inhibitors of DNA metabolism in bacteria</atitle><jtitle>Antimicrobial agents and chemotherapy</jtitle><stitle>Antimicrob Agents Chemother</stitle><addtitle>Antimicrob Agents Chemother</addtitle><date>2014-12-01</date><risdate>2014</risdate><volume>58</volume><issue>12</issue><spage>7264</spage><epage>7272</epage><pages>7264-7272</pages><issn>0066-4804</issn><eissn>1098-6596</eissn><abstract>Bacterial biosensor strains can be useful tools for the discovery and characterization of antibacterial compounds. A plasmid-based reporter vector containing a transcriptional fusion between the recA promoter and green fluorescence protein gene was introduced into an Escherichia coli ΔtolC strain to create a biosensor strain that selectively senses inhibitors of DNA metabolism via the SOS response. The strain was used to develop a high-throughput assay to identify new inhibitors of DNA metabolism. Screening of the AstraZeneca compound library with this strain identified known inhibitors of DNA metabolism, as well as novel chemotypes. The cellular target of one novel series was elucidated as DNA gyrase through genetic characterization of laboratory-generated resistant mutants followed by 50% inhibitory concentration measurements in a DNA gyrase activity assay. These studies validated the use of this antibiotic biosensor strain to identify novel selective inhibitors of DNA metabolism by high-throughput screening.</abstract><cop>United States</cop><pub>American Society for Microbiology</pub><pmid>25246396</pmid><doi>10.1128/AAC.03475-14</doi><tpages>9</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | Antimicrobial agents and chemotherapy, 2014-12, Vol.58 (12), p.7264-7272 |
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| source | MEDLINE; EZB-FREE-00999 freely available EZB journals; PubMed Central |
| subjects | Bacterial Outer Membrane Proteins - genetics Biosensing Techniques DNA Gyrase - genetics DNA Gyrase - metabolism DNA, Bacterial DNA, Bacterial - antagonists & inhibitors DNA, Bacterial - genetics DNA, Bacterial - metabolism Escherichia coli Escherichia coli - drug effects Escherichia coli - genetics Escherichia coli - metabolism Escherichia coli Proteins - genetics Gene Expression Genes, Reporter Green Fluorescent Proteins - genetics Green Fluorescent Proteins - metabolism High-Throughput Screening Assays Inhibitory Concentration 50 Mechanisms of Action: Physiological Effects Membrane Transport Proteins - deficiency Membrane Transport Proteins - genetics Nucleic Acid Synthesis Inhibitors Nucleic Acid Synthesis Inhibitors - chemistry Nucleic Acid Synthesis Inhibitors - pharmacology Plasmids - chemistry Plasmids - metabolism Promoter Regions, Genetic Rec A Recombinases - genetics Rec A Recombinases - metabolism Recombinant Fusion Proteins - genetics Recombinant Fusion Proteins - metabolism Small Molecule Libraries Small Molecule Libraries - chemistry Small Molecule Libraries - pharmacology SOS Response (Genetics) - drug effects |
| title | A novel high-throughput cell-based assay aimed at identifying inhibitors of DNA metabolism in bacteria |
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