Table 1.
detection of nif genes in metagenomic sequencing of acid mine drainage.
Table 2.
List of ORFs from fosmid DX-1A-14, gene length, and similar genes in GenBank.
Table 3.
Gene products in the nif-fix cluster of fosmid DX-1A-14.
Figure 1.
Classification of total nif genes obtained from metagenomic sequencing reads.
742 nif sequences were classified into different phyla based on the similarities to the known sequences. The community is massively dominated by γ-proteobacteria, followed in smaller amounts by α-, β-proteobacteria and Verrucomicrobia.
Figure 2.
Phylogenetic tree of nifK genes.
The phylogenetic tree was constructed by the neighbor-joining method using MEGA, version 4.0 with 1000 bootstrap repetitions. The sequences obtained from metagenomic sequencing of acid mine drainage are designated DX_SY_, followed by their number in library. These sequences are shown in bold. Only some representatives of 93 nifK sequences are shown here. The scale represents the number of amino acid substitutions per site.
Figure 3.
Comparison of the physical organization of nif, fix, and associated genes from fosmid DX-1A-14 with those from three closest organisms.
Organisms: Azoarcus sp., Herbaspirillum seropedicae, Acetobacter diazotrophicus. The nif genes are highlighted in bold. nrf, nif associated genes; orf, hypothetical protein. The structure of nif gene cluster differs greatly from those of unknown microorganisms. The nif gene cluster does not contain modABC genes occurring in H. seropedicae, and RubisCO gene cluster can not be identified in the nif gene cluster region of H. seropedicae. nifQ gene is clustered together with nifENX and fdxB genes in A. diazotrophicus, while nifV, nifW, fixABCX, and nifQ genes constitute a single operon in fosmid DX-1A-14.
Figure 4.
Reverse transcription PCR of fixX and nifQ gene fragment.
Lane M, 100; lane 1, PCR products with the template of total RNA extraction; lane 2, PCR products with the template of total RNA extracting digested by RNase.