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

Abstract

Some methane-oxidizing bacteria (methanotrophs) are known to be capable of expressing nitrogenase and utilizing N2 as a nitrogen source. However, no sequences are available for nif genes in these strains, and the known nitrogen-fixing methanotrophs are confined mainly to a few genera. The purpose of this work was to assess the nitrogen-fixing capabilities of a variety of methanotroph strains. nifH gene fragments from four type I methanotrophs and 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. Sequence analysis shows that the nifH sequences from the type I and type II strains cluster with nifH sequences from other gamma proteobacteria and alpha proteobacteria, respectively. The translated nifH sequences from three Methylomonas strains show high identity (95 to 99%) to several published translated environmental nifH sequences PCR amplified from rice roots and a freshwater 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 fixation in a broad range of methanotrophs and suggest that nitrogen-fixing methanotrophs may be widespread and important in the nitrogen cycling of many environments.

FIG. 1

Alignment of deduced amino acid sequences of the approximately 360-bp partial nifH genes from Methylobacter marinus A45, Methylomonas sp. strain LW13, Methylomonas sp. strain LW15, M. methanica S1, A. chroococcum (accession no. M73020), A. vinelandii (M11579), K. pneumoniae (J01740), P. stutzeri (AJ297529), V. diazotrophicus (AF111110), A. faecalis (X96609), H. seropedicae (Z54207), Methylocystis sp. strain LW2, Methylocystis sp. strain LW5, Methylosinus sp. strain LW3, Methylosinus sp. strain LW4, Methylosinus sp. strain LW8, Methylosinus sp. strain PW1, M. trichosporium OB3b, A. brasilense (M64344), B. japonicum (E00713), B. japonicum USDA 110 (K01620), R. meliloti (V01215), R. phaseoli (M10587), Rhizoboium sp. strain ORS571 (M16710), P. rhizobium (K00487), R. capsulatus (M15270), and R. rubrum (M33774). Identical residues are in black boxes, and similar residues are in gray boxes. Methanotroph strain names are in bold face. Solid triangles indicate signature amino acids for Methylomonas strains, while open triangles indicate signature amino acids for type II strains.

FIG. 2

Phylogenetic analysis of the derived amino acid sequences of nifH genes. Bootstrap values of >50% are shown near the clades. The bar represents 10% sequence divergence, as determined by the lengths of the horizontal lines connecting any two species. The tree includes sequences shown in Fig. 1 as well as F. alni L41344, Synechococcus sp. strain U22146, A. variabilis U89346, and P. boryanum D00666.