nfxB as a novel target for analysis of mutation spectra in Pseudomonas aeruginosa
nfxB encodes a negative regulator of the mexCD-oprJ genes for drug efflux in the opportunistic pathogen Pseudomonas aeruginosa. Inactivating mutations in this transcriptional regulator constitute one of the main mechanisms of resistance to ciprofloxacin (Cip(r)). In this work, we evaluated the use o...
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| description | nfxB encodes a negative regulator of the mexCD-oprJ genes for drug efflux in the opportunistic pathogen Pseudomonas aeruginosa. Inactivating mutations in this transcriptional regulator constitute one of the main mechanisms of resistance to ciprofloxacin (Cip(r)). In this work, we evaluated the use of nfxB/Cip(r) as a new test system to study mutation spectra in P. aeruginosa. The analysis of 240 mutations in nfxB occurring spontaneously in the wild-type and mutator backgrounds or induced by mutagens showed that nfxB/Cip(r) offers several advantages compared with other mutation detection systems. Identification of nfxB mutations was easy since the entire open reading frame and its promoter region were sequenced from the chromosome using a single primer. Mutations detected in nfxB included all transitions and transversions, 1-bp deletions and insertions, >1-bp deletions and duplications. The broad mutation spectrum observed in nfxB relies on the selection of loss-of-function changes, as we confirmed by generating a structural model of the NfxB repressor and evaluating the significance of each detected mutation. The mutation spectra characterized in the mutS, mutT, mutY and mutM mutator backgrounds or induced by the mutagenic agents 2-aminopurine, cisplatin and hydrogen peroxide were in agreement with their predicted mutational specificities. Additionally, this system allowed the analysis of sequence context effects since point mutations occurred at 85 different sites distributed over the entire nfxB. Significant hotspots and preferred sequence contexts were observed for spontaneous and mutagen-induced mutation spectra. Finally, we demonstrated the utility of a luminescence-based reporter for identification of nfxB mutants previous to sequencing analysis. Thus, the nfxB/Cip(r) system in combination with the luminescent reporter may be a valuable tool for studying mutational processes in Pseudomonas spp. wherein the genes encoding the NfxB repressor and the associated efflux pump are conserved. |
| doi_str_mv | 10.1371/journal.pone.0066236 |
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Inactivating mutations in this transcriptional regulator constitute one of the main mechanisms of resistance to ciprofloxacin (Cip(r)). In this work, we evaluated the use of nfxB/Cip(r) as a new test system to study mutation spectra in P. aeruginosa. The analysis of 240 mutations in nfxB occurring spontaneously in the wild-type and mutator backgrounds or induced by mutagens showed that nfxB/Cip(r) offers several advantages compared with other mutation detection systems. Identification of nfxB mutations was easy since the entire open reading frame and its promoter region were sequenced from the chromosome using a single primer. Mutations detected in nfxB included all transitions and transversions, 1-bp deletions and insertions, >1-bp deletions and duplications. The broad mutation spectrum observed in nfxB relies on the selection of loss-of-function changes, as we confirmed by generating a structural model of the NfxB repressor and evaluating the significance of each detected mutation. The mutation spectra characterized in the mutS, mutT, mutY and mutM mutator backgrounds or induced by the mutagenic agents 2-aminopurine, cisplatin and hydrogen peroxide were in agreement with their predicted mutational specificities. Additionally, this system allowed the analysis of sequence context effects since point mutations occurred at 85 different sites distributed over the entire nfxB. Significant hotspots and preferred sequence contexts were observed for spontaneous and mutagen-induced mutation spectra. Finally, we demonstrated the utility of a luminescence-based reporter for identification of nfxB mutants previous to sequencing analysis. Thus, the nfxB/Cip(r) system in combination with the luminescent reporter may be a valuable tool for studying mutational processes in Pseudomonas spp. wherein the genes encoding the NfxB repressor and the associated efflux pump are conserved.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0066236</identifier><identifier>PMID: 23762483</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>2-Aminopurine ; Amino Acid Sequence ; Amino Acid Substitution - genetics ; Amino acids ; Bacteria ; Bacterial Proteins - chemistry ; Bacterial Proteins - genetics ; Biology ; Ciprofloxacin ; Ciprofloxacin - pharmacology ; Cisplatin ; Comparative analysis ; Cystic fibrosis ; Deoxyribonucleic acid ; Detection equipment ; DNA ; DNA-Binding Proteins - chemistry ; DNA-Binding Proteins - genetics ; Drug Resistance, Bacterial - drug effects ; E coli ; Efflux ; Escherichia coli ; Gene mutation ; Genes ; Genes, Reporter ; Genetic aspects ; Genetic research ; Hydrogen ; Hydrogen peroxide ; Laboratories ; Luminescence ; Metabolism ; Models, Molecular ; Molecular Sequence Data ; Mutagenesis ; Mutagens ; Mutagens - toxicity ; Mutants ; Mutation ; Mutation - genetics ; Mutation Rate ; Opportunist infection ; Phenols ; Plasmids ; Proteins ; Pseudomonas ; Pseudomonas aeruginosa ; Pseudomonas aeruginosa - drug effects ; Pseudomonas aeruginosa - genetics ; RNA polymerase ; Sequence Analysis, DNA ; Sequences ; Spectra ; Transcription ; Transcription (Genetics) ; Transcription Factors - chemistry ; Transcription Factors - genetics</subject><ispartof>PloS one, 2013-06, Vol.8 (6), p.e66236</ispartof><rights>COPYRIGHT 2013 Public Library of Science</rights><rights>2013 Monti et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License: https://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2013 Monti et al 2013 Monti et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c692t-1524ae33894a9e97027e9bec6d6897a7a4f275043425e16644cbebde3b062e4e3</citedby><cites>FETCH-LOGICAL-c692t-1524ae33894a9e97027e9bec6d6897a7a4f275043425e16644cbebde3b062e4e3</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/PMC3676378/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3676378/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,864,885,2100,2919,23857,27915,27916,53782,53784,79361,79362</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/23762483$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Freitag, Nancy E.</contributor><creatorcontrib>Monti, Mariela R</creatorcontrib><creatorcontrib>Morero, Natalia R</creatorcontrib><creatorcontrib>Miguel, Virginia</creatorcontrib><creatorcontrib>Argaraña, Carlos E</creatorcontrib><title>nfxB as a novel target for analysis of mutation spectra in Pseudomonas aeruginosa</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>nfxB encodes a negative regulator of the mexCD-oprJ genes for drug efflux in the opportunistic pathogen Pseudomonas aeruginosa. Inactivating mutations in this transcriptional regulator constitute one of the main mechanisms of resistance to ciprofloxacin (Cip(r)). In this work, we evaluated the use of nfxB/Cip(r) as a new test system to study mutation spectra in P. aeruginosa. The analysis of 240 mutations in nfxB occurring spontaneously in the wild-type and mutator backgrounds or induced by mutagens showed that nfxB/Cip(r) offers several advantages compared with other mutation detection systems. Identification of nfxB mutations was easy since the entire open reading frame and its promoter region were sequenced from the chromosome using a single primer. Mutations detected in nfxB included all transitions and transversions, 1-bp deletions and insertions, >1-bp deletions and duplications. The broad mutation spectrum observed in nfxB relies on the selection of loss-of-function changes, as we confirmed by generating a structural model of the NfxB repressor and evaluating the significance of each detected mutation. The mutation spectra characterized in the mutS, mutT, mutY and mutM mutator backgrounds or induced by the mutagenic agents 2-aminopurine, cisplatin and hydrogen peroxide were in agreement with their predicted mutational specificities. Additionally, this system allowed the analysis of sequence context effects since point mutations occurred at 85 different sites distributed over the entire nfxB. Significant hotspots and preferred sequence contexts were observed for spontaneous and mutagen-induced mutation spectra. Finally, we demonstrated the utility of a luminescence-based reporter for identification of nfxB mutants previous to sequencing analysis. 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toxicity</subject><subject>Mutants</subject><subject>Mutation</subject><subject>Mutation - genetics</subject><subject>Mutation Rate</subject><subject>Opportunist infection</subject><subject>Phenols</subject><subject>Plasmids</subject><subject>Proteins</subject><subject>Pseudomonas</subject><subject>Pseudomonas aeruginosa</subject><subject>Pseudomonas aeruginosa - drug effects</subject><subject>Pseudomonas aeruginosa - genetics</subject><subject>RNA polymerase</subject><subject>Sequence Analysis, DNA</subject><subject>Sequences</subject><subject>Spectra</subject><subject>Transcription</subject><subject>Transcription (Genetics)</subject><subject>Transcription Factors - chemistry</subject><subject>Transcription Factors - genetics</subject><issn>1932-6203</issn><issn>1932-6203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2013</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><sourceid>DOA</sourceid><recordid>eNqNkl2L3CAYhUNp6X60_6C0QmGhFzM1ajTeFLZLPwYWtt-3YpLXjEMSp2qW3X9fZye7TKCF4oXy-pyjHE6WvcjxMqcif7txox90t9y6AZYYc04of5Qd55KSBSeYPj44H2UnIWwwLmjJ-dPsiFDBCSvpcfZ1MDfvkQ5Io8FdQ4ei9i1EZJxHOtnfBhuQM6gfo47WDShsoY5eIzugLwHGxvVu2MnBj60dXNDPsidGdwGeT_tp9vPjhx8XnxeXV59WF-eXi5pLEhd5QZgGSkvJtAQpMBEgK6h5w0sptNDMEFFgRhkpIOecsbqCqgFaYU6AAT3NXu19t50LakojqJzyosRCSJKI1Z5onN6orbe99rfKaavuBs63Svto6w5UVWDMtClBsopRwyojG6ElpiUAMaJMXu-m18aqh6aGIYXQzUznN4Ndq9ZdK8oFp3cGrycD736PEOI_vjxRrU6_soNxyazubajVORMlwzmmNFHLv1BpNdDbOvXB2DSfCd7MBImJcBNbPYagVt-__T979WvOnh2wa9BdXAfXjbumhDnI9mDtXQgezENyOVa7Ot-noXZ1VlOdk-zlYeoPovv-0j9Sy-9s</recordid><startdate>20130607</startdate><enddate>20130607</enddate><creator>Monti, Mariela R</creator><creator>Morero, Natalia R</creator><creator>Miguel, Virginia</creator><creator>Argaraña, Carlos E</creator><general>Public Library of Science</general><general>Public Library of Science (PLoS)</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>IOV</scope><scope>ISR</scope><scope>3V.</scope><scope>7QG</scope><scope>7QL</scope><scope>7QO</scope><scope>7RV</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TG</scope><scope>7TM</scope><scope>7U9</scope><scope>7X2</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>ARAPS</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>D1I</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB.</scope><scope>KB0</scope><scope>KL.</scope><scope>L6V</scope><scope>LK8</scope><scope>M0K</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>M7S</scope><scope>NAPCQ</scope><scope>P5Z</scope><scope>P62</scope><scope>P64</scope><scope>PATMY</scope><scope>PDBOC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>PTHSS</scope><scope>PYCSY</scope><scope>RC3</scope><scope>5PM</scope><scope>DOA</scope></search><sort><creationdate>20130607</creationdate><title>nfxB as a novel target for analysis of mutation spectra in Pseudomonas aeruginosa</title><author>Monti, Mariela R ; Morero, Natalia R ; Miguel, Virginia ; Argaraña, Carlos E</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c692t-1524ae33894a9e97027e9bec6d6897a7a4f275043425e16644cbebde3b062e4e3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2013</creationdate><topic>2-Aminopurine</topic><topic>Amino Acid Sequence</topic><topic>Amino Acid Substitution - genetics</topic><topic>Amino acids</topic><topic>Bacteria</topic><topic>Bacterial Proteins - chemistry</topic><topic>Bacterial Proteins - genetics</topic><topic>Biology</topic><topic>Ciprofloxacin</topic><topic>Ciprofloxacin - pharmacology</topic><topic>Cisplatin</topic><topic>Comparative analysis</topic><topic>Cystic fibrosis</topic><topic>Deoxyribonucleic acid</topic><topic>Detection equipment</topic><topic>DNA</topic><topic>DNA-Binding Proteins - chemistry</topic><topic>DNA-Binding Proteins - genetics</topic><topic>Drug Resistance, Bacterial - drug effects</topic><topic>E coli</topic><topic>Efflux</topic><topic>Escherichia coli</topic><topic>Gene mutation</topic><topic>Genes</topic><topic>Genes, Reporter</topic><topic>Genetic aspects</topic><topic>Genetic research</topic><topic>Hydrogen</topic><topic>Hydrogen peroxide</topic><topic>Laboratories</topic><topic>Luminescence</topic><topic>Metabolism</topic><topic>Models, Molecular</topic><topic>Molecular Sequence Data</topic><topic>Mutagenesis</topic><topic>Mutagens</topic><topic>Mutagens - toxicity</topic><topic>Mutants</topic><topic>Mutation</topic><topic>Mutation - genetics</topic><topic>Mutation Rate</topic><topic>Opportunist infection</topic><topic>Phenols</topic><topic>Plasmids</topic><topic>Proteins</topic><topic>Pseudomonas</topic><topic>Pseudomonas aeruginosa</topic><topic>Pseudomonas aeruginosa - drug effects</topic><topic>Pseudomonas aeruginosa - genetics</topic><topic>RNA polymerase</topic><topic>Sequence Analysis, DNA</topic><topic>Sequences</topic><topic>Spectra</topic><topic>Transcription</topic><topic>Transcription (Genetics)</topic><topic>Transcription Factors - chemistry</topic><topic>Transcription Factors - genetics</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Monti, Mariela R</creatorcontrib><creatorcontrib>Morero, Natalia R</creatorcontrib><creatorcontrib>Miguel, Virginia</creatorcontrib><creatorcontrib>Argaraña, Carlos E</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Gale In Context: Opposing Viewpoints</collection><collection>Gale In Context: Science</collection><collection>ProQuest Central (Corporate)</collection><collection>Animal Behavior Abstracts</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Biotechnology Research Abstracts</collection><collection>Nursing & Allied Health Database</collection><collection>Ecology Abstracts</collection><collection>Entomology Abstracts (Full archive)</collection><collection>Immunology Abstracts</collection><collection>Meteorological & Geoastrophysical Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Agricultural Science Collection</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Public Health Database</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Technology Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>Materials Science & Engineering Collection</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest One Sustainability</collection><collection>ProQuest Central UK/Ireland</collection><collection>Advanced Technologies & Aerospace Collection</collection><collection>Agricultural & Environmental Science Collection</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Technology Collection</collection><collection>Natural Science Collection</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Materials Science Collection</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Materials Science Database</collection><collection>Nursing & Allied Health Database (Alumni Edition)</collection><collection>Meteorological & Geoastrophysical Abstracts - 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Inactivating mutations in this transcriptional regulator constitute one of the main mechanisms of resistance to ciprofloxacin (Cip(r)). In this work, we evaluated the use of nfxB/Cip(r) as a new test system to study mutation spectra in P. aeruginosa. The analysis of 240 mutations in nfxB occurring spontaneously in the wild-type and mutator backgrounds or induced by mutagens showed that nfxB/Cip(r) offers several advantages compared with other mutation detection systems. Identification of nfxB mutations was easy since the entire open reading frame and its promoter region were sequenced from the chromosome using a single primer. Mutations detected in nfxB included all transitions and transversions, 1-bp deletions and insertions, >1-bp deletions and duplications. The broad mutation spectrum observed in nfxB relies on the selection of loss-of-function changes, as we confirmed by generating a structural model of the NfxB repressor and evaluating the significance of each detected mutation. The mutation spectra characterized in the mutS, mutT, mutY and mutM mutator backgrounds or induced by the mutagenic agents 2-aminopurine, cisplatin and hydrogen peroxide were in agreement with their predicted mutational specificities. Additionally, this system allowed the analysis of sequence context effects since point mutations occurred at 85 different sites distributed over the entire nfxB. Significant hotspots and preferred sequence contexts were observed for spontaneous and mutagen-induced mutation spectra. Finally, we demonstrated the utility of a luminescence-based reporter for identification of nfxB mutants previous to sequencing analysis. Thus, the nfxB/Cip(r) system in combination with the luminescent reporter may be a valuable tool for studying mutational processes in Pseudomonas spp. wherein the genes encoding the NfxB repressor and the associated efflux pump are conserved.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>23762483</pmid><doi>10.1371/journal.pone.0066236</doi><tpages>e66236</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | 2-Aminopurine Amino Acid Sequence Amino Acid Substitution - genetics Amino acids Bacteria Bacterial Proteins - chemistry Bacterial Proteins - genetics Biology Ciprofloxacin Ciprofloxacin - pharmacology Cisplatin Comparative analysis Cystic fibrosis Deoxyribonucleic acid Detection equipment DNA DNA-Binding Proteins - chemistry DNA-Binding Proteins - genetics Drug Resistance, Bacterial - drug effects E coli Efflux Escherichia coli Gene mutation Genes Genes, Reporter Genetic aspects Genetic research Hydrogen Hydrogen peroxide Laboratories Luminescence Metabolism Models, Molecular Molecular Sequence Data Mutagenesis Mutagens Mutagens - toxicity Mutants Mutation Mutation - genetics Mutation Rate Opportunist infection Phenols Plasmids Proteins Pseudomonas Pseudomonas aeruginosa Pseudomonas aeruginosa - drug effects Pseudomonas aeruginosa - genetics RNA polymerase Sequence Analysis, DNA Sequences Spectra Transcription Transcription (Genetics) Transcription Factors - chemistry Transcription Factors - genetics |
| title | nfxB as a novel target for analysis of mutation spectra in Pseudomonas aeruginosa |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-15T04%3A07%3A12IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=nfxB%20as%20a%20novel%20target%20for%20analysis%20of%20mutation%20spectra%20in%20Pseudomonas%20aeruginosa&rft.jtitle=PloS%20one&rft.au=Monti,%20Mariela%20R&rft.date=2013-06-07&rft.volume=8&rft.issue=6&rft.spage=e66236&rft.pages=e66236-&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0066236&rft_dat=%3Cgale_plos_%3EA478401033%3C/gale_plos_%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=1365807792&rft_id=info:pmid/23762483&rft_galeid=A478401033&rft_doaj_id=oai_doaj_org_article_b5004af8e94b43f4bf9d7a9038ee2f78&rfr_iscdi=true |