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

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 ⁷⁰ (σ ^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.

Author Summary

Biological nitrogen fixation plays an essential role in the nitrogen cycle, sustaining agricultural productivity by providing a source of fixed nitrogen for plants and ultimately animals. The enzyme nitrogenase that catalyses the reduction of atmospheric dinitrogen to ammonia contains one of the most complex heterometal cofactors found in biology. Biosynthesis of nitrogenase and provision of support for its activity requires a large number of nitrogen fixation ( nif) genes, which vary according to the physiological lifestyle of the host organism. In this study, we identified a nif cluster with reduced genetic complexity, consisting of nine genes organized as a single operon in the genome of Paenibacillus sp. WLY78. When transferred to Escherichia coli, the Paenibacllus nif cluster enables synthesis of catalytically active nitrogenase, which is competent to reduce both acetylene and dinitrogen as substrates of the enzyme. Environmental regulation of nif gene expression in Paenibacillus, in response to either oxygen or fixed nitrogen, is circumvented when the nif operon is expressed from its native promoter in E. coli, suggesting that nif transcription in Paenibacillus is negatively regulated in response to these effectors.