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 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.

Adaptation of the symbiotic Mesorhizobium –chickpea relationship to phosphate deficiency relies on reprogramming of whole-plant metabolism

“…Previous reports have suggested that expression levels of nifD and nifK genes could be used as indicators of SNF capacity (9,(16)(17)(18). The higher levels of nifD and nifK expression under Pisufficient conditions observed in MmSWRI9-induced nodules relative to McCP-31-induced nodules suggest that the MmSWRI9-inoculated association may have higher SNF capacity than the McCP-31-inoculated association under Pi-sufficient conditions (Fig.…”

“…SNF capability of leguminous plants is controlled by the rhizobial nitrogenase complex, which catalyzes the reduction of N 2 into ammonia (15). The nifDK genes are the structural genes encoding the MoFe protein subunits of the nitrogenase complex, and assessing their expression levels has been shown to be a reliable method for evaluating the contribution of the N 2 fixing bacteria to plant N nutrition (9,(16)(17)(18). Thus, analysis of the expression levels of the nifDK genes will enable us to characterize the SNF capacity of the nodules induced by the tested strains under both normal and low Pi conditions (Fig.…”

Reconstruction and minimal gene requirements for the alternative iron-only nitrogenase in Escherichia coli

“…Recently, it was shown that an operon comprising nine genes derived from Paenibacillus sp. WLY78 is sufficient to express active molybdenum nitrogenase in E. coli (20). However, the minimal FeFe nitrogenase system described here has certain advantages, notably the simpler mode of FeFe-co biosynthesis and, in particular, the absence of a requirement for Mo, because this element is most probably limiting in plant organelles that lack molybdoenzymes.…”

“…We and others have established that Escherichia coli can provide a useful host for engineering diazotrophy (17)(18)(19), determining the minimum requirements for nitrogen fixation in evolutionary distant diazotrophs (20), and redesigning nif gene clusters for synthetic biology (21,22). Recently, we developed a modular operon-based system for functional analysis of the Klebsiella oxytoca nif gene cluster in E. coli (21).…”

Beneficial Bacillus Strains Improve Plant Resistance to Phytopathogens: A Review

“…are mentioned to be involved in nitrogen fixation. These will be an important step, of long-standing interest, for developing biofertilizers that can help non-legume crops to "fix" nitrogen at the same time with maintaining growth yields [14]. Phosphate solubilization is another highly important tool in plant growth promotion.…”