A Minimal Nitrogen Fixation Gene Cluster from Paenibacillus sp. WLY78 Enables Expression of Active Nitrogenase in Escherichia coli
Most biological nitrogen fixation is catalyzed by molybdenum-dependent nitrogenase, an enzyme complex comprising two component proteins that contains three different metalloclusters. Diazotrophs contain a common core of nitrogen fixation nif genes that encode the structural subunits of the enzyme an...
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| Format: | Article |
| Language: | en_US |
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Public Library of Science
2013
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| Online Access: | http://hdl.handle.net/1721.1/83411 |
| _version_ | 1826211708400566272 |
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| author | Wang, Liying Zhang, Lihong Liu, Zhangzhi Zhao, Dehua Liu, Xiaomeng Zhang, Bo Xie, Jianbo Hong, Yuanyuan Li, Pengfei Chen, Sanfeng Dixon, Ray Li, Jilun |
| author2 | Massachusetts Institute of Technology. Department of Biological Engineering |
| author_facet | Massachusetts Institute of Technology. Department of Biological Engineering Wang, Liying Zhang, Lihong Liu, Zhangzhi Zhao, Dehua Liu, Xiaomeng Zhang, Bo Xie, Jianbo Hong, Yuanyuan Li, Pengfei Chen, Sanfeng Dixon, Ray Li, Jilun |
| author_sort | Wang, Liying |
| collection | MIT |
| description | Most biological nitrogen fixation is catalyzed by molybdenum-dependent nitrogenase, an enzyme complex comprising two component proteins that contains three different metalloclusters. Diazotrophs contain a common core of nitrogen fixation nif genes that encode the structural subunits of the enzyme and components required to synthesize the metalloclusters. However, the complement of nif genes required to enable diazotrophic growth varies significantly amongst nitrogen fixing bacteria and archaea. In this study, we identified a minimal nif gene cluster consisting of nine nif genes in the genome of Paenibacillus sp. WLY78, a gram-positive, facultative anaerobe isolated from the rhizosphere of bamboo. We demonstrate that the nif genes in this organism are organized as an operon comprising nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV and that the nif cluster is under the control of a σ[superscript 70] (σ[superscript A])-dependent promoter located upstream of nifB. To investigate genetic requirements for diazotrophy, we transferred the Paenibacillus nif cluster to Escherichia coli. The minimal nif gene cluster enables synthesis of catalytically active nitrogenase in this host, when expressed either from the native nifB promoter or from the T7 promoter. Deletion analysis indicates that in addition to the core nif genes, hesA plays an important role in nitrogen fixation and is responsive to the availability of molybdenum. Whereas nif transcription in Paenibacillus is regulated in response to nitrogen availability and by the external oxygen concentration, transcription from the nifB promoter is constitutive in E. coli, indicating that negative regulation of nif transcription is bypassed in the heterologous host. This study demonstrates the potential for engineering nitrogen fixation in a non-nitrogen fixing organism with a minimum set of nine nif genes. |
| first_indexed | 2024-09-23T15:10:16Z |
| format | Article |
| id | mit-1721.1/83411 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T15:10:16Z |
| publishDate | 2013 |
| publisher | Public Library of Science |
| record_format | dspace |
| spelling | mit-1721.1/834112022-09-29T13:09:26Z A Minimal Nitrogen Fixation Gene Cluster from Paenibacillus sp. WLY78 Enables Expression of Active Nitrogenase in Escherichia coli Wang, Liying Zhang, Lihong Liu, Zhangzhi Zhao, Dehua Liu, Xiaomeng Zhang, Bo Xie, Jianbo Hong, Yuanyuan Li, Pengfei Chen, Sanfeng Dixon, Ray Li, Jilun Massachusetts Institute of Technology. Department of Biological Engineering Massachusetts Institute of Technology. Synthetic Biology Center Zhao, Dehua Most biological nitrogen fixation is catalyzed by molybdenum-dependent nitrogenase, an enzyme complex comprising two component proteins that contains three different metalloclusters. Diazotrophs contain a common core of nitrogen fixation nif genes that encode the structural subunits of the enzyme and components required to synthesize the metalloclusters. However, the complement of nif genes required to enable diazotrophic growth varies significantly amongst nitrogen fixing bacteria and archaea. In this study, we identified a minimal nif gene cluster consisting of nine nif genes in the genome of Paenibacillus sp. WLY78, a gram-positive, facultative anaerobe isolated from the rhizosphere of bamboo. We demonstrate that the nif genes in this organism are organized as an operon comprising nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV and that the nif cluster is under the control of a σ[superscript 70] (σ[superscript A])-dependent promoter located upstream of nifB. To investigate genetic requirements for diazotrophy, we transferred the Paenibacillus nif cluster to Escherichia coli. The minimal nif gene cluster enables synthesis of catalytically active nitrogenase in this host, when expressed either from the native nifB promoter or from the T7 promoter. Deletion analysis indicates that in addition to the core nif genes, hesA plays an important role in nitrogen fixation and is responsive to the availability of molybdenum. Whereas nif transcription in Paenibacillus is regulated in response to nitrogen availability and by the external oxygen concentration, transcription from the nifB promoter is constitutive in E. coli, indicating that negative regulation of nif transcription is bypassed in the heterologous host. This study demonstrates the potential for engineering nitrogen fixation in a non-nitrogen fixing organism with a minimum set of nine nif genes. 2013-12-30T21:02:20Z 2013-12-30T21:02:20Z 2013-10 2013-05 Article http://purl.org/eprint/type/JournalArticle 1553-7404 1553-7390 http://hdl.handle.net/1721.1/83411 Wang, Liying, Lihong Zhang, Zhangzhi Liu, Dehua Zhao, Xiaomeng Liu, Bo Zhang, Jianbo Xie, et al. “A Minimal Nitrogen Fixation Gene Cluster from Paenibacillus sp. WLY78 Enables Expression of Active Nitrogenase in Escherichia coli.” Edited by Josep Casadesús. PLoS Genetics 9, no. 10 (October 17, 2013): e1003865. en_US http://dx.doi.org/10.1371/journal.pgen.1003865 PLoS Genetics http://creativecommons.org/licenses/by/2.5/ application/pdf Public Library of Science PLoS |
| spellingShingle | Wang, Liying Zhang, Lihong Liu, Zhangzhi Zhao, Dehua Liu, Xiaomeng Zhang, Bo Xie, Jianbo Hong, Yuanyuan Li, Pengfei Chen, Sanfeng Dixon, Ray Li, Jilun A Minimal Nitrogen Fixation Gene Cluster from Paenibacillus sp. WLY78 Enables Expression of Active Nitrogenase in Escherichia coli |
| title | A Minimal Nitrogen Fixation Gene Cluster from Paenibacillus sp. WLY78 Enables Expression of Active Nitrogenase in Escherichia coli |
| title_full | A Minimal Nitrogen Fixation Gene Cluster from Paenibacillus sp. WLY78 Enables Expression of Active Nitrogenase in Escherichia coli |
| title_fullStr | A Minimal Nitrogen Fixation Gene Cluster from Paenibacillus sp. WLY78 Enables Expression of Active Nitrogenase in Escherichia coli |
| title_full_unstemmed | A Minimal Nitrogen Fixation Gene Cluster from Paenibacillus sp. WLY78 Enables Expression of Active Nitrogenase in Escherichia coli |
| title_short | A Minimal Nitrogen Fixation Gene Cluster from Paenibacillus sp. WLY78 Enables Expression of Active Nitrogenase in Escherichia coli |
| title_sort | minimal nitrogen fixation gene cluster from paenibacillus sp wly78 enables expression of active nitrogenase in escherichia coli |
| url | http://hdl.handle.net/1721.1/83411 |
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