A Minimal Nitrogen Fixation Gene Cluster from Paenibacillus sp. WLY78 Enables Expression of Active Nitrogenase in Escherichia coli
Name
Wang-2013-A Minimal Nitrogen F.pdf
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Author(s)
Wang, Liying
Zhang, Lihong
Liu, Zhangzhi
Zhao, Dehua
Liu, Xiaomeng
Zhang, Bo
Xie, Jianbo
Hong, Yuanyuan
Li, Pengfei
Chen, Sanfeng
Date Issued
October 2013
Journal
PLoS Genetics
Publisher
Public Library of Science
Citation
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.
Version
Final published version
Abstract
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.
MIT Department
Massachusetts Institute of Technology. Department of Biological Engineering
Massachusetts Institute of Technology. Synthetic Biology Center
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DOI of Published Version
https://doi.org/10.1371/journal.pgen.1003865
