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

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.