A Minimal Nitrogen Fixation Gene Cluster from Paenibacillus sp. WLY78 Enables Expression of Active Nitrogenase in Escherichia coli

Liying Wang,Xiaomeng Liu,Ray Dixon,Jilun Li,Lihong Zhang,Sanfeng Chen,Pengfei Li,Yuanyuan Hong,Zhangzhi Liu,Jianbo Xie,Bo Zhang,Dehua Zhao,Josep Casadesús

doi:10.1371/journal.pgen.1003865

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 σ70 (σA)-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.

Highlights

Fixed nitrogen plays a critical role in the global food supply, overuse of chemical nitrogen fertilizers has led to increased costs for farmers and harmful consequences for the environment and human health
The gene designated as hesA, which is located between nifX and nifV is found in other nif clusters (Figure 1) and the predicted product shares,45% identity with the putative molybdenum cofactor biosynthesis protein HesA of Frankia alni ACN14a [16] and Cyanothece sp
The conserved nature of the structural genes and the assembly pathway for FeMoco biosynthesis dictates the presence of a common core of nif genes, other functions may be provided by protein counterparts encoded elsewhere in the genome

Summary

Introduction

Fixed nitrogen plays a critical role in the global food supply, overuse of chemical nitrogen fertilizers has led to increased costs for farmers and harmful consequences for the environment and human health. Apart from the structural subunits encoded by nifH, nifD and nifK, several genes are required for the biosynthesis of the metalloclusters, in addition to other gene products necessary to produce a fully functional enzyme. It is well established from genetic and biochemical analysis that nifE nifN, nifX nifB, nifQ, nifV, nifY and nifH contribute to the synthesis and insertion of FeMo-co into nitrogenase, that nifU nifS and nifZ play an important role in synthesis of metalloclusters and that nifM is required for proper folder of nitrogenase Fe protein [4]–[7]

Methods

Results

Conclusion