Development of a genetic system for a model manganese-oxidizing proteobacterium, Leptothrix discophora SS1
Understanding the molecular underpinnings of manganese oxidation in Leptothrix discophora SS1 has been hampered by the lack of a genetic system. In this report, we describe the development of a genetic system for L. discophora SS1. The antibiotic sensitivity was characterized, and a procedure for tr...
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| Veröffentlicht in: | Microbiology (Society for General Microbiology) 2014-11, Vol.160 (Pt 11), p.2396-2405 |
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| container_issue | Pt 11 |
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| container_title | Microbiology (Society for General Microbiology) |
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| creator | Bocioaga, Daniela El Gheriany, Iman A Lion, Leonard W Ghiorse, William C Shuler, Michael L Hay, Anthony G |
| description | Understanding the molecular underpinnings of manganese oxidation in Leptothrix discophora SS1 has been hampered by the lack of a genetic system. In this report, we describe the development of a genetic system for L. discophora SS1. The antibiotic sensitivity was characterized, and a procedure for transformation with exogenous DNA via conjugation was developed and optimized, resulting in a maximum transfer frequency of 5.2×10(-1) and a typical transfer frequency of the order of 1×10(-3) transconjugants per donor. Genetic manipulation of L. discophora SS1 was demonstrated by disrupting pyrF via chromosomal integration with a plasmid containing a R6Kγ origin of replication through homologous recombination. This resulted in resistance to 5-fluoroorotidine, which was abolished by complementation with an ectopically expressed copy of pyrF cloned into pBBR1MCS. This system is expected to be amenable to a systematic genetic analysis of L. discophora SS1, including those genes responsible for manganese oxidation. |
| doi_str_mv | 10.1099/mic.0.079459-0 |
| format | Article |
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In this report, we describe the development of a genetic system for L. discophora SS1. The antibiotic sensitivity was characterized, and a procedure for transformation with exogenous DNA via conjugation was developed and optimized, resulting in a maximum transfer frequency of 5.2×10(-1) and a typical transfer frequency of the order of 1×10(-3) transconjugants per donor. Genetic manipulation of L. discophora SS1 was demonstrated by disrupting pyrF via chromosomal integration with a plasmid containing a R6Kγ origin of replication through homologous recombination. This resulted in resistance to 5-fluoroorotidine, which was abolished by complementation with an ectopically expressed copy of pyrF cloned into pBBR1MCS. 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In this report, we describe the development of a genetic system for L. discophora SS1. The antibiotic sensitivity was characterized, and a procedure for transformation with exogenous DNA via conjugation was developed and optimized, resulting in a maximum transfer frequency of 5.2×10(-1) and a typical transfer frequency of the order of 1×10(-3) transconjugants per donor. Genetic manipulation of L. discophora SS1 was demonstrated by disrupting pyrF via chromosomal integration with a plasmid containing a R6Kγ origin of replication through homologous recombination. This resulted in resistance to 5-fluoroorotidine, which was abolished by complementation with an ectopically expressed copy of pyrF cloned into pBBR1MCS. This system is expected to be amenable to a systematic genetic analysis of L. discophora SS1, including those genes responsible for manganese oxidation.</description><subject>Anti-Bacterial Agents - pharmacology</subject><subject>Bacterial Proteins - genetics</subject><subject>Bacterial Proteins - metabolism</subject><subject>Conjugation, Genetic</subject><subject>Genetic Techniques</subject><subject>Leptothrix - drug effects</subject><subject>Leptothrix - genetics</subject><subject>Leptothrix - metabolism</subject><subject>Manganese - metabolism</subject><subject>Oxidation-Reduction</subject><subject>Recombination, Genetic</subject><subject>Replication Origin</subject><issn>1350-0872</issn><issn>1465-2080</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2014</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNo9kDtPxDAQhC0E4t1SIpcU5Fjbedgl4i2dRAHUUc5ZH0ZxHGwfOvj1GB1Q7Wp3ZvRpCDlhMGOg1IWzegYzaFRZqQK2yD4r66rgIGE776KCAmTD98hBjG8A-Qlsl-zxipWKyWafvF3jBw5-cjgm6g3t6BJHTFbT-BkTOmp8yEfnexyo68ZlN2LEwq9tb7_suKRT8An9otMJg125czrHKfn0Guya9jZqP7360NGnJ3ZEdkw3RDz-nYfk5fbm-eq-mD_ePVxdzgtdNiwVlRIoOBdQikagkVWNsgTFuWlAVT1o4JwZ3te6WihuuOwRuFwgGiVLxZU4JGeb3Iz2vsKYWpc5cBgyul_FltWccSZkLbJ0tpHq4GMMaNopWNeFz5ZB-9NvtuoW2k2_LWTD6W_2auGw_5f_FSq-AeBVdrk</recordid><startdate>201411</startdate><enddate>201411</enddate><creator>Bocioaga, Daniela</creator><creator>El Gheriany, Iman A</creator><creator>Lion, Leonard W</creator><creator>Ghiorse, William C</creator><creator>Shuler, Michael L</creator><creator>Hay, Anthony G</creator><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></search><sort><creationdate>201411</creationdate><title>Development of a genetic system for a model manganese-oxidizing proteobacterium, Leptothrix discophora SS1</title><author>Bocioaga, Daniela ; El Gheriany, Iman A ; Lion, Leonard W ; Ghiorse, William C ; Shuler, Michael L ; Hay, Anthony G</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c471t-593e322304373ef856e840922f7095d0c0221f2d6c5b92f28de028beef9849293</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2014</creationdate><topic>Anti-Bacterial Agents - pharmacology</topic><topic>Bacterial Proteins - genetics</topic><topic>Bacterial Proteins - metabolism</topic><topic>Conjugation, Genetic</topic><topic>Genetic Techniques</topic><topic>Leptothrix - drug effects</topic><topic>Leptothrix - genetics</topic><topic>Leptothrix - metabolism</topic><topic>Manganese - metabolism</topic><topic>Oxidation-Reduction</topic><topic>Recombination, Genetic</topic><topic>Replication Origin</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Bocioaga, Daniela</creatorcontrib><creatorcontrib>El Gheriany, Iman A</creatorcontrib><creatorcontrib>Lion, Leonard W</creatorcontrib><creatorcontrib>Ghiorse, William C</creatorcontrib><creatorcontrib>Shuler, Michael L</creatorcontrib><creatorcontrib>Hay, Anthony G</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>Microbiology (Society for General Microbiology)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Bocioaga, Daniela</au><au>El Gheriany, Iman A</au><au>Lion, Leonard W</au><au>Ghiorse, William C</au><au>Shuler, Michael L</au><au>Hay, Anthony G</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Development of a genetic system for a model manganese-oxidizing proteobacterium, Leptothrix discophora SS1</atitle><jtitle>Microbiology (Society for General Microbiology)</jtitle><addtitle>Microbiology</addtitle><date>2014-11</date><risdate>2014</risdate><volume>160</volume><issue>Pt 11</issue><spage>2396</spage><epage>2405</epage><pages>2396-2405</pages><issn>1350-0872</issn><eissn>1465-2080</eissn><abstract>Understanding the molecular underpinnings of manganese oxidation in Leptothrix discophora SS1 has been hampered by the lack of a genetic system. In this report, we describe the development of a genetic system for L. discophora SS1. The antibiotic sensitivity was characterized, and a procedure for transformation with exogenous DNA via conjugation was developed and optimized, resulting in a maximum transfer frequency of 5.2×10(-1) and a typical transfer frequency of the order of 1×10(-3) transconjugants per donor. Genetic manipulation of L. discophora SS1 was demonstrated by disrupting pyrF via chromosomal integration with a plasmid containing a R6Kγ origin of replication through homologous recombination. This resulted in resistance to 5-fluoroorotidine, which was abolished by complementation with an ectopically expressed copy of pyrF cloned into pBBR1MCS. This system is expected to be amenable to a systematic genetic analysis of L. discophora SS1, including those genes responsible for manganese oxidation.</abstract><cop>England</cop><pmid>25149187</pmid><doi>10.1099/mic.0.079459-0</doi><tpages>10</tpages><oa>free_for_read</oa></addata></record> |
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| source | MEDLINE; PubMed Central |
| subjects | Anti-Bacterial Agents - pharmacology Bacterial Proteins - genetics Bacterial Proteins - metabolism Conjugation, Genetic Genetic Techniques Leptothrix - drug effects Leptothrix - genetics Leptothrix - metabolism Manganese - metabolism Oxidation-Reduction Recombination, Genetic Replication Origin |
| title | Development of a genetic system for a model manganese-oxidizing proteobacterium, Leptothrix discophora SS1 |
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