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

<div><p>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 <i>nif</i> genes that encode the structural subunits of the enzyme and components required to synthesize the metalloclusters. However, the complement of <i>nif</i> genes required to enable diazotrophic growth varies significantly amongst nitrogen fixing bacteria and archaea. In this study, we identified a minimal <i>nif</i> gene cluster consisting of nine <i>nif</i> genes in the genome of <i>Paenibacillus</i> sp. WLY78, a gram-positive, facultative anaerobe isolated from the rhizosphere of bamboo. We demonstrate that the <i>nif</i> genes in this organism are organized as an operon comprising <i>nifB</i>, <i>nifH</i>, <i>nifD</i>, <i>nifK</i>, <i>nifE</i>, <i>nifN</i>, <i>nifX</i>, <i>hesA</i> and <i>nifV</i> and that the <i>nif</i> cluster is under the control of a σ<sup>70</sup> (σ<sup>A</sup>)-dependent promoter located upstream of <i>nifB</i>. To investigate genetic requirements for diazotrophy, we transferred the <i>Paenibacillus nif</i> cluster to <i>Escherichia coli</i>. The minimal <i>nif</i> gene cluster enables synthesis of catalytically active nitrogenase in this host, when expressed either from the native <i>nifB</i> promoter or from the T7 promoter. Deletion analysis indicates that in addition to the core <i>nif</i> genes, <i>hesA</i> plays an important role in nitrogen fixation and is responsive to the availability of molybdenum. Whereas <i>nif</i> transcription in <i>Paenibacillus</i> is regulated in response to nitrogen availability and by the external oxygen concentration, transcription from the <i>nifB</i> promoter is constitutive in <i>E. coli</i>, indicating that negative regulation of <i>nif</i> 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 <i>nif</i> genes.</p></div>