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Required Gene Set for Autotrophic Growth of Clostridium autoethanogenum
The majority of the genes present in bacterial genomes remain poorly characterized, with up to one-third of those that are protein encoding having no definitive function. Transposon insertion sequencing represents a high-throughput technique that can help rectify this deficiency. The technology, how...
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| Published in: | Applied and environmental microbiology 2022-04, Vol.88 (7), p.e0247921-e0247921 |
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| container_title | Applied and environmental microbiology |
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| creator | Woods, Craig Humphreys, Christopher M Tomi-Andrino, Claudio Henstra, Anne M Köpke, Michael Simpson, Sean D Winzer, Klaus Minton, Nigel P |
| description | The majority of the genes present in bacterial genomes remain poorly characterized, with up to one-third of those that are protein encoding having no definitive function. Transposon insertion sequencing represents a high-throughput technique that can help rectify this deficiency. The technology, however, can only be realistically applied to those species in which high rates of DNA transfer can be achieved. Here, we have developed a number of approaches that overcome this barrier in the autotrophic species Clostridium autoethanogenum by using a
-based transposon system. The inherent instability of such systems in the Escherichia coli conjugation donor due to transposition events was counteracted through the incorporation of a conditionally lethal
marker on the plasmid backbone. Relatively low frequencies of transformation of the plasmid into
were circumvented through the use of a plasmid that is conditional for replication coupled with the routine implementation of an Illumina library preparation protocol that eliminates plasmid-based reads. A transposon library was then used to determine the essential genes needed for growth using carbon monoxide as the sole carbon and energy source.
Although microbial genome sequences are relatively easily determined, assigning gene function remains a bottleneck. Consequently, relatively few genes are well characterized, leaving the function of many as either hypothetical or entirely unknown. High-throughput transposon sequencing can help remedy this deficiency, but is generally only applicable to microbes with efficient DNA transfer procedures. These exclude many microorganisms of importance to humankind either as agents of disease or as industrial process organisms. Here, we developed approaches to facilitate transposon insertion sequencing in the acetogen Clostridium autoethanogenum, a chassis being exploited to convert single-carbon waste gases CO and CO
into chemicals and fuels at an industrial scale. This allowed the determination of gene essentiality under heterotrophic and autotrophic growth, providing insights into the utilization of CO as a sole carbon and energy source. The strategies implemented are translatable and will allow others to apply transposon insertion sequencing to other microbes where DNA transfer has until now represented a barrier to progress. |
| doi_str_mv | 10.1128/aem.02479-21 |
| format | article |
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-based transposon system. The inherent instability of such systems in the Escherichia coli conjugation donor due to transposition events was counteracted through the incorporation of a conditionally lethal
marker on the plasmid backbone. Relatively low frequencies of transformation of the plasmid into
were circumvented through the use of a plasmid that is conditional for replication coupled with the routine implementation of an Illumina library preparation protocol that eliminates plasmid-based reads. A transposon library was then used to determine the essential genes needed for growth using carbon monoxide as the sole carbon and energy source.
Although microbial genome sequences are relatively easily determined, assigning gene function remains a bottleneck. Consequently, relatively few genes are well characterized, leaving the function of many as either hypothetical or entirely unknown. High-throughput transposon sequencing can help remedy this deficiency, but is generally only applicable to microbes with efficient DNA transfer procedures. These exclude many microorganisms of importance to humankind either as agents of disease or as industrial process organisms. Here, we developed approaches to facilitate transposon insertion sequencing in the acetogen Clostridium autoethanogenum, a chassis being exploited to convert single-carbon waste gases CO and CO
into chemicals and fuels at an industrial scale. This allowed the determination of gene essentiality under heterotrophic and autotrophic growth, providing insights into the utilization of CO as a sole carbon and energy source. The strategies implemented are translatable and will allow others to apply transposon insertion sequencing to other microbes where DNA transfer has until now represented a barrier to progress.</description><identifier>ISSN: 0099-2240</identifier><identifier>EISSN: 1098-5336</identifier><identifier>DOI: 10.1128/aem.02479-21</identifier><identifier>PMID: 35285680</identifier><language>eng</language><publisher>United States: American Society for Microbiology</publisher><subject>Applied and Industrial Microbiology ; Autotrophic Processes ; Bacteria ; Carbon monoxide ; Carbon Monoxide - metabolism ; Clostridium ; Clostridium - metabolism ; Conjugation ; Deoxyribonucleic acid ; DNA ; DNA Transposable Elements ; E coli ; Energy sources ; Genes ; Genetics and Molecular Biology ; Genome, Bacterial ; Genomes ; Libraries ; Mutagenesis, Insertional ; Sequences ; Transposition ; Transposons</subject><ispartof>Applied and environmental microbiology, 2022-04, Vol.88 (7), p.e0247921-e0247921</ispartof><rights>Copyright © 2022 Woods et al.</rights><rights>Copyright American Society for Microbiology Apr 2022</rights><rights>Copyright © 2022 Woods et al. 2022 Woods et al.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a446t-8eda71dffcc4ac38149ac9bcf4135b5edc8138c607c7818a9a9da3cbcd9f6de73</citedby><cites>FETCH-LOGICAL-a446t-8eda71dffcc4ac38149ac9bcf4135b5edc8138c607c7818a9a9da3cbcd9f6de73</cites><orcidid>0000-0002-9277-1261 ; 0000-0002-3956-8096</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://journals.asm.org/doi/pdf/10.1128/aem.02479-21$$EPDF$$P50$$Gasm2$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://journals.asm.org/doi/full/10.1128/aem.02479-21$$EHTML$$P50$$Gasm2$$Hfree_for_read</linktohtml><link.rule.ids>230,315,733,786,790,891,3207,27957,27958,52786,52787,52788,53827,53829</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/35285680$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Nojiri, Hideaki</contributor><creatorcontrib>Woods, Craig</creatorcontrib><creatorcontrib>Humphreys, Christopher M</creatorcontrib><creatorcontrib>Tomi-Andrino, Claudio</creatorcontrib><creatorcontrib>Henstra, Anne M</creatorcontrib><creatorcontrib>Köpke, Michael</creatorcontrib><creatorcontrib>Simpson, Sean D</creatorcontrib><creatorcontrib>Winzer, Klaus</creatorcontrib><creatorcontrib>Minton, Nigel P</creatorcontrib><title>Required Gene Set for Autotrophic Growth of Clostridium autoethanogenum</title><title>Applied and environmental microbiology</title><addtitle>Appl Environ Microbiol</addtitle><addtitle>Appl Environ Microbiol</addtitle><description>The majority of the genes present in bacterial genomes remain poorly characterized, with up to one-third of those that are protein encoding having no definitive function. Transposon insertion sequencing represents a high-throughput technique that can help rectify this deficiency. The technology, however, can only be realistically applied to those species in which high rates of DNA transfer can be achieved. Here, we have developed a number of approaches that overcome this barrier in the autotrophic species Clostridium autoethanogenum by using a
-based transposon system. The inherent instability of such systems in the Escherichia coli conjugation donor due to transposition events was counteracted through the incorporation of a conditionally lethal
marker on the plasmid backbone. Relatively low frequencies of transformation of the plasmid into
were circumvented through the use of a plasmid that is conditional for replication coupled with the routine implementation of an Illumina library preparation protocol that eliminates plasmid-based reads. A transposon library was then used to determine the essential genes needed for growth using carbon monoxide as the sole carbon and energy source.
Although microbial genome sequences are relatively easily determined, assigning gene function remains a bottleneck. Consequently, relatively few genes are well characterized, leaving the function of many as either hypothetical or entirely unknown. High-throughput transposon sequencing can help remedy this deficiency, but is generally only applicable to microbes with efficient DNA transfer procedures. These exclude many microorganisms of importance to humankind either as agents of disease or as industrial process organisms. Here, we developed approaches to facilitate transposon insertion sequencing in the acetogen Clostridium autoethanogenum, a chassis being exploited to convert single-carbon waste gases CO and CO
into chemicals and fuels at an industrial scale. This allowed the determination of gene essentiality under heterotrophic and autotrophic growth, providing insights into the utilization of CO as a sole carbon and energy source. The strategies implemented are translatable and will allow others to apply transposon insertion sequencing to other microbes where DNA transfer has until now represented a barrier to progress.</description><subject>Applied and Industrial Microbiology</subject><subject>Autotrophic Processes</subject><subject>Bacteria</subject><subject>Carbon monoxide</subject><subject>Carbon Monoxide - metabolism</subject><subject>Clostridium</subject><subject>Clostridium - metabolism</subject><subject>Conjugation</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>DNA Transposable Elements</subject><subject>E coli</subject><subject>Energy sources</subject><subject>Genes</subject><subject>Genetics and Molecular Biology</subject><subject>Genome, Bacterial</subject><subject>Genomes</subject><subject>Libraries</subject><subject>Mutagenesis, Insertional</subject><subject>Sequences</subject><subject>Transposition</subject><subject>Transposons</subject><issn>0099-2240</issn><issn>1098-5336</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2022</creationdate><recordtype>article</recordtype><recordid>eNp1kUtLxDAURoMoOj52rqXgRsFqHk2abAQZdBQEwcc6ZNJbp9I2Y5Iq_nuj4xtcXbj3cLgfH0LbBB8SQuWRge4Q06JUOSVLaESwkjlnTCyjEcYqbWmB19B6CA8Y4wILuYrWGKeSC4lHaHINj0Pjocom0EN2AzGrnc9Ohuiid_NZY7OJd89xlrk6G7cuRN9UzdBlJhEQZ6Z399AP3SZaqU0bYOtjbqC7s9Pb8Xl-eTW5GJ9c5qYoRMwlVKYkVV1bWxjLJCmUsWpq64IwPuVQWUmYtAKXtpREGmVUZZid2krVooKSbaDjhXc-TLuEQx-9afXcN53xL9qZRv--9M1M37snrVJ4xkUS7H0IvHscIETdNcFC25oe3BA0FUwqLqWgCd39gz64wfcpXqI4F0Txd-HBgrLeheCh_nqGYP3WkE4N6feGNCUJ31_gJnT0W_gPu_Mz7Jf4sz72Cq6lmz0</recordid><startdate>20220412</startdate><enddate>20220412</enddate><creator>Woods, Craig</creator><creator>Humphreys, Christopher M</creator><creator>Tomi-Andrino, Claudio</creator><creator>Henstra, Anne M</creator><creator>Köpke, Michael</creator><creator>Simpson, Sean D</creator><creator>Winzer, Klaus</creator><creator>Minton, Nigel P</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>7QO</scope><scope>7SN</scope><scope>7SS</scope><scope>7ST</scope><scope>7T7</scope><scope>7TM</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope><scope>SOI</scope><scope>7X8</scope><scope>5PM</scope><orcidid>https://orcid.org/0000-0002-9277-1261</orcidid><orcidid>https://orcid.org/0000-0002-3956-8096</orcidid></search><sort><creationdate>20220412</creationdate><title>Required Gene Set for Autotrophic Growth of Clostridium autoethanogenum</title><author>Woods, Craig ; 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Transposon insertion sequencing represents a high-throughput technique that can help rectify this deficiency. The technology, however, can only be realistically applied to those species in which high rates of DNA transfer can be achieved. Here, we have developed a number of approaches that overcome this barrier in the autotrophic species Clostridium autoethanogenum by using a
-based transposon system. The inherent instability of such systems in the Escherichia coli conjugation donor due to transposition events was counteracted through the incorporation of a conditionally lethal
marker on the plasmid backbone. Relatively low frequencies of transformation of the plasmid into
were circumvented through the use of a plasmid that is conditional for replication coupled with the routine implementation of an Illumina library preparation protocol that eliminates plasmid-based reads. A transposon library was then used to determine the essential genes needed for growth using carbon monoxide as the sole carbon and energy source.
Although microbial genome sequences are relatively easily determined, assigning gene function remains a bottleneck. Consequently, relatively few genes are well characterized, leaving the function of many as either hypothetical or entirely unknown. High-throughput transposon sequencing can help remedy this deficiency, but is generally only applicable to microbes with efficient DNA transfer procedures. These exclude many microorganisms of importance to humankind either as agents of disease or as industrial process organisms. Here, we developed approaches to facilitate transposon insertion sequencing in the acetogen Clostridium autoethanogenum, a chassis being exploited to convert single-carbon waste gases CO and CO
into chemicals and fuels at an industrial scale. This allowed the determination of gene essentiality under heterotrophic and autotrophic growth, providing insights into the utilization of CO as a sole carbon and energy source. The strategies implemented are translatable and will allow others to apply transposon insertion sequencing to other microbes where DNA transfer has until now represented a barrier to progress.</abstract><cop>United States</cop><pub>American Society for Microbiology</pub><pmid>35285680</pmid><doi>10.1128/aem.02479-21</doi><tpages>14</tpages><orcidid>https://orcid.org/0000-0002-9277-1261</orcidid><orcidid>https://orcid.org/0000-0002-3956-8096</orcidid><oa>free_for_read</oa></addata></record> |
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| subjects | Applied and Industrial Microbiology Autotrophic Processes Bacteria Carbon monoxide Carbon Monoxide - metabolism Clostridium Clostridium - metabolism Conjugation Deoxyribonucleic acid DNA DNA Transposable Elements E coli Energy sources Genes Genetics and Molecular Biology Genome, Bacterial Genomes Libraries Mutagenesis, Insertional Sequences Transposition Transposons |
| title | Required Gene Set for Autotrophic Growth of Clostridium autoethanogenum |
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