Evolution and Functional Analysis of orf1 Within nif Gene Cluster from Paenibacillus graminis RSA19.

Qin Li,Sanfeng Chen,Xiaomeng Liu,Haowei Zhang

doi:10.3390/ijms20051145

Qin Li, Sanfeng Chen + Show 2 more

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https://doi.org/10.3390/ijms20051145

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Abstract

Paenibacillus is a genus of Gram-positive, facultative anaerobic and endospore-forming bacteria. Genomic sequence analysis has revealed that a compact nif (nitrogen fixation) gene cluster comprising 9–10 genes nifBHDKENX(orf1)hesAnifV is conserved in diazotrophic Paenibacillus species. The evolution and function of the orf1 gene within the nif gene cluster of Paenibacillus species is unknown. In this study, a careful comparison analysis of the compositions of the nif gene clusters from various diazotrophs revealed that orf1 located downstream of nifENX was identified in anaerobic Clostridium ultunense, the facultative anaerobic Paenibacillus species and aerobic diazotrophs (e.g., Azotobacter vinelandii and Azospirillum brasilense). The predicted amino acid sequences encoded by the orf1 gene, part of the nif gene cluster nifBHDKENXorf1hesAnifV in Paenibacillus graminis RSA19, showed 60–90% identity with those of the orf1 genes located downstream of nifENX from different diazotrophic Paenibacillus species, but shared no significant identity with those of the orf1 genes from different taxa of diazotrophic organisms. Transcriptional analysis showed that the orf1 gene was expressed under nitrogen fixation conditions from the promoter located upstream from nifB. Mutational analysis suggested that the orf1 gene functions in nitrogen fixation in the presence of a high concentration of O2.

Highlights

Biological nitrogen fixation, the conversion of atmospheric N2 to NH3, plays an important role in the global nitrogen cycle and in world agriculture
The orf1 gene is a part of the nif gene cluster in P. graminis RSA19 and some other Paenibacillus species or strains
To investigate the evolution of orf1, we here performed a comparison of the nif gene clusters from the representatives of diazotrophic methanogenic archae and bacteria in six taxonomic phyla: Actinobacteria, Chlorobi, Chloroflexi, Cyanobacteria, Firmicutes and Proteobacteria

Summary

Introduction

Biological nitrogen fixation, the conversion of atmospheric N2 to NH3, plays an important role in the global nitrogen cycle and in world agriculture. The MoFe protein component is an α2β2 heterotetramer (encoded by nifD and nifK) that contains two metalloclusters: FeMo-co, a [Mo-7Fe-9S-C-homocitrate] cluster, which serves as the active site of substrate binding and reduction and the P-cluster, a [8Fe-7S] cluster which shuttles electrons to FeMo-co. The transition of nitrogenase from anaerobic to facultative anaerobic and aerobic organisms was accompanied by a substantial increase in the number of nif genes from a minimum of 7 to a maximum of 20 genes [4]. Genetic and biochemical studies on the two model diazotrophs K. oxytoca and A. vinelandii revealed that nifH, nifD and nifK genes encode the structural subunits, the nifE, nifN, nifX, nifB, nifQ, nifV, nifY, nifS, nifU and nifH contribute to the synthesis and insertion of FeMo-co into nitrogenase, and nifL and nifA are involved in the regulation of other nif gene transcription [3]. In archaea and some Gram-positive anaerobes, NifI1 and NifI2, the GlnB homologues, regulate nitrogenase activities at a post-transcriptional level by interacting with nifHDK in response to N availability [6]

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