Isolation, Identification And Comparative Genomics Of Nitrogen-fixing Paenibacillus

Paenibacillus is a large genus of Gram-positive, facultative anaerobic, endospore-forming bacteria with high genetic diversity and phenotypic diversity. Among them, some have nitrogen-fixation ability. Though these N2-fixing strains have great potential for agricultural use, genomic information to date is limited and the genetic and evolution of nitrogen-fixation of these diazotrophs are unknow.To isolate and identify more nitrogen-fixing strains with high nitrogenase activities, different soil samples were collected from different plant rhizospheres covering vast areas, for example, Beijing, Heibei, Gansu, Shandong, Yunnan. After16S rRNA gene analysis, gs65showed97.5%,97.3%,97.0%,96.9%sequence similarity with Paenibacillus borealis DSM13188T, Paenibacillus odorifer ATCC BAA-93T, Paenibacillus durus DSM1735T and Paenibacillus sophorae DSM23020T, respectively, and below97%with other recognized members of the genus. The level of DNA-DNA relatedness between strain gs65and the type strains was below40%. On the basis of its phenotypic characteristics and the level of DNA-DNA hybridization, strain gs65is considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus taohuashanense sp. nov. is proposed. The type strain is gs65T (=CGMCC1.121751=DSM25809T).To discover the evolutionary history of nitrogen fixation, comparative genomic analysis of the31genomes (15N2-fixing and16non-N2-fixing Paenibacillus strains) was conducted. The shell genome, which makes up approximately65.2%of the genes in pan genome, illustrating a large degree of genomic diversity. Large number of prophage remnants and transposable elements might be related to the heterogeneity, indicating their adaptation to the complex environment. A minimal and compact nif cluster nifBHDKENXhesAni/V is conserved in the15N2-fixing strains, and the nif’cluster is under controlled of a σ70-dependent promoter and possesses a GlnR/TnrA-binding site in the promoter region. Furthermore, we demonstrate that the nif cluster enabled Escherichia coli JM109to fix nitrogen. Phylogeny of the concatenated NifHDK sequence indicates that the Paenibacillus and Frankia have a last common ancestor. High-resolution species tree was constructed using439single copy genes.Through the comparative genomics of31genomes, our results suggest that ancestral Paenibacillus did not fix nitrogen, the N2-fixing strains was generated from acquiring the inf cluster via horization gene transfer (HGT) from a source related to Frankia. During the history of evolution, the inf cluster was lost, producing some non-N2-fixing strains and V-nitrogenase or Fe nitrogenase was acquired, further diversifing some strains. Additionally, several other copies of nif or nif-like genes might be generated from gene duplication. The evolution of nitrogen fixation in Paenibacillus involves a mix of gain, loss, HGT and duplication of nif/anf/vnf genes. Our study not only supports the methanogen origin hypothesis, but also provides insight into the complex evolutionary history of nitrogen fixation in genus Paenibacillus for the first time.