Comparative Genomic Analysis of N ₂-Fixing and Non-N ₂-Fixing Paenibacillus spp.: Organization, Evolution and Expression of the Nitrogen Fixation Genes

摘要

We provide here a comparative genome analysis of 31 strains within the genus Paenibacillus including 11 new genomic sequences of N_(2)-fixing strains. The heterogeneity of the 31 genomes (15 N_(2)-fixing and 16 non-N_(2)-fixing Paenibacillus strains) was reflected in the large size of the shell genome, which makes up approximately 65.2% of the genes in pan genome. Large numbers of transposable elements might be related to the heterogeneity. We discovered that a minimal and compact nif cluster comprising nine genes nifB , nifH , nifD , nifK , nifE , nifN , nifX , hesA and nifV encoding Mo-nitrogenase is conserved in the 15 N_(2)-fixing strains. The nif cluster is under control of a σ~(70)-depedent promoter and possesses a GlnR/TnrA-binding site in the promoter. Suf system encoding [Fe–S] cluster is highly conserved in N_(2)-fixing and non-N_(2)-fixing strains. Furthermore, we demonstrate that the nif cluster enabled Escherichia coli JM109 to fix nitrogen. Phylogeny of the concatenated NifHDK sequences indicates that Paenibacillus and Frankia are sister groups. Phylogeny of the concatenated 275 single-copy core genes suggests that the ancestral Paenibacillus did not fix nitrogen. The N_(2)-fixing Paenibacillus strains were generated by acquiring the nif cluster via horizontal gene transfer (HGT) from a source related to Frankia . During the history of evolution, the nif cluster was lost, producing some non-N_(2)-fixing strains, and vnf encoding V-nitrogenase or anf encoding Fe-nitrogenase was acquired, causing further diversification of some strains. In addition, some N_(2)-fixing strains have additional nif and nif -like genes which may result from gene duplications. The evolution of nitrogen fixation in Paenibacillus involves a mix of gain, loss, HGT and duplication of nif/anf/vnf genes. This study not only reveals the organization and distribution of nitrogen fixation genes in Paenibacillus , but also provides insight into the complex evolutionary history of nitrogen fixation. Author Summary We sequenced the genomes of 11 N_(2)-fixing Paenibacillus strains and demonstrated the genomic diversity of the genus Paenibacillus by comparing these strains to each other and to 20 other strains (4 N_(2)-fixing and 16 non-N_(2)-fixing strains) that were sequenced previously. Phylogenetic analysis of the concatenated 275 single-copy core genes suggests that ancestral Paenibacillus did not fix nitrogen and the N_(2)-fixing strains fall into two sub-groups, which were likely originated from a N_(2)-fixing common ancestor. A minimal and compact nif cluster comprising nine nif genes encoding Mo-nitrogenase is highly conserved in the 15 N_(2)-fixing strains. Variations in the nif cluster and in the chromosomal regions surrounding the nif cluster between two sub-groups imply at least two independent acquisitions with insertion of distinct nif cluster variants in different genomic sites of Paenibacillus in early evolutionary history. Phylogeny of the concatenated NifHDK sequences suggests that Paenibacillus and Frankia are sister groups. The nif cluster, a functional unit for nitrogen fixation, was lost, producing some non-N_(2)-fixing strains. There were recent events of acquisition of vnf and anf genes, causing further diversification of some strains. The evolution of nitrogen fixation in Paenibacillus involves a mix of gain, loss, HGT and duplication of nif/anf/vnf genes.