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Biological nitrogen fixation is an essential function of acid mine drainage (AMD) microbial communities. However, most acidophiles in AMD environments are uncultured microorganisms and little is known about the diversity of nitrogen-fixing genes and structure of  nif  gene cluster in AMD microbial communities. In this study, we used metagenomic sequencing to isolate  nif  genes in the AMD microbial community from Dexing Copper Mine, China. Meanwhile, a metagenome microarray containing 7,776 large-insertion fosmids was constructed to screen novel  nif  gene clusters. Metagenomic analyses revealed that 742 sequences were identified as  nif  genes including structural subunit genes  nifH ,  nifD ,  nifK  and various additional genes. The AMD community is massively dominated by the genus  Acidithiobacillus . However, the phylogenetic diversity of nitrogen-fixing microorganisms is much higher than previously thought in the AMD community. Furthermore, a 32.5-kb genomic sequence harboring  nif ,  fix  and associated genes was screened by metagenome microarray. Comparative genome analysis indicated that most  nif  genes in this cluster are most similar to those of  Herbaspirillum seropedicae , but the organization of the  nif  gene cluster had significant differences from  H. seropedicae . Sequence analysis and reverse transcription PCR also suggested that distinct transcription units of  nif  genes exist in this gene cluster.  nifQ  gene falls into the same transcription unit with  fixABCX  genes, which have not been reported in other diazotrophs before. All of these results indicated that more novel diazotrophs survive in the AMD community.

Identification of Nitrogen-Fixing Genes and Gene Clusters from Metagenomic Library of Acid Mine Drainage