nifH sequences and nitrogen fixation in type I and type II methanotrophs

Some methane-oxidizing bacteria (methanotrophs) are known to be capable of expressing nitrogenase and utilizing N-2 as a nitrogen source. However, no sequences are available for nif genes in these strains, and the known nitro gen-fixing methanotrophs are confined mainly to a few genera. The purpose o f this work was to assess the nitrogen-fixing capabilities of a variety of methanotroph strains. nifH gene fragments from four type I methanotrophs an d seven type II methanotrophs were PCR amplified and sequenced. Nitrogenase activity was confirmed in selected type I and type II strains by acetylene reduction. Activities ranged from 0.4 to 3.3 nmol/min/mg of protein. Seque nce analysis shows that the nifH sequences from the type I and type II stra ins cluster with nifH sequences from other gamma proteobacteria and alpha p roteobacteria, respectively. The translated nifH sequences from three Methy lomonas strains show high identity (95 to 99%) to several published transla ted environmental nifH sequences PCR amplified from rice roots and a freshw ater lake. The translated nifH sequences from the type II strains show high identity (94 to 99%) to published translated nifH sequences from a variety of environments, including rice roots, a freshwater lake, an oligotrophic ocean, and forest soil. These results provide evidence for nitrogen fixatio n in a broad range of methanotrophs and suggest that nitrogen-fixing methan otrophs may be widespread and important in the nitrogen cycling of many env ironments.