DNA extraction and phylogenetic analysis based on 16S rRNA and nifH genes

DNA was isolated from bacterial cells following a combined protocol of mechanical disruption (bead beating) and chloroform phenol extraction, as described by Noll et al. (30). The 16S rRNA genes and putative nifH genes of bacterial isolates were amplified using the 16S rRNA gene primers 27F2/1492R2 (20, 21) and nifH gene primers PolF/PolR (33) under the standard PCR conditions given in the respective references. PCR was performed using ExTaq polymerase (Takara, Kusatsu, Japan) as described previously (28, 29). Purified PCR products were prepared and sequenced using BigDye terminator kit v3.1 on an ABI 3130 Genetic Analyzer (Applied Biosystems, Foster city, CA, USA) according to the standard protocol. nifH gene sequences were translated into amino acid sequences using the standard code in MEGA7 (19). The deduced NifH sequences were confirmed to contain one required residue, Cys 97 (protein numbering for NifH in Azotobacter vinelandii; PCR products were of insufficient lengths to contain both Cys 97 and Cys 132), which is a 4Fe-4S iron sulfur cluster ligating cysteine, and were used to construct a phylogenetic tree as previously reported (9, 29). NifH and 16S rRNA gene sequences were aligned in ClustalW with default settings implemented in MEGA7 (19). Phylogenetic trees for the 16S rRNA gene and NifH sequences were reconstructed using the Maximum Likelihood method with the Tamura-Nei model in MEGA7 (19) and the WAG model in the ARB program package (23), respectively. The robustness of the tree topologies was tested with 500 (for 16S rRNA) or 100 (for NifH) bootstrap replicates.