RESUMO
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.
Assuntos
Methylococcaceae/metabolismo , Fixação de Nitrogênio , Oxirredutases/genética , Rhizobiaceae/metabolismo , Sequência de Aminoácidos , Metano/metabolismo , Methylococcaceae/genética , Dados de Sequência Molecular , Oxirredutases/química , Oxirredutases/metabolismo , Filogenia , Reação em Cadeia da Polimerase , Rhizobiaceae/genética , Análise de Sequência de DNARESUMO
Profiles of dissolved O(2) and methane with increasing depth were generated for Lake Washington sediment, which suggested the zone of methane oxidation is limited to the top 0.8 cm of the sediment. Methane oxidation potentials were measured for 0.5-cm layers down to 1.5 cm and found to be relatively constant at 270 to 350 micromol/liter of sediment/h. Approximately 65% of the methane was oxidized to cell material or metabolites, a signature suggestive of type I methanotrophs. Eleven methanotroph strains were isolated from the lake sediment and analyzed. Five of these strains classed as type I, while six were classed as type II strains by 16S rRNA gene sequence analysis. Southern hybridization analysis with oligonucleotide probes detected, on average, one to two copies of pmoA and one to three copies of 16S rRNA genes. Only one restriction length polymorphism pattern was shown for pmoA genes in each isolate, and in cases where, sequencing was done, the pmoA copies were found to be almost identical. PCR primers were developed for mmoX which amplified 1.2-kb regions from all six strains that tested positive for cytoplasmic soluble methane mono-oxygenase (sMMO) activity. Phylogenetic analysis of the translated PCR products with published mmoX sequences showed that MmoX falls into two distinct clusters, one containing the orthologs from type I strains and another containing the orthologs from type II strains. The presence of sMMO-containing Methylomonas strains in a pristine freshwater lake environment suggests that these methanotrophs are more widespread than has been previously thought.
Assuntos
Água Doce/microbiologia , Sedimentos Geológicos/microbiologia , Metano/metabolismo , Proteobactérias/isolamento & purificação , Proteobactérias/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA/genética , DNA Bacteriano/genética , Cinética , Methylococcaceae/genética , Methylococcaceae/isolamento & purificação , Methylococcaceae/metabolismo , Methylomonas/genética , Methylomonas/isolamento & purificação , Methylomonas/metabolismo , Methylosinus/genética , Methylosinus/isolamento & purificação , Methylosinus/metabolismo , Dados de Sequência Molecular , Oxigênio/metabolismo , Oxigenases/genética , Oxigenases/metabolismo , Proteobactérias/genética , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Rhizobiaceae/genética , Rhizobiaceae/isolamento & purificação , Rhizobiaceae/metabolismo , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , WashingtonRESUMO
In the facultative serine cycle methylotroph Methylobacterium extorquens AM1, mxaB is required for regulation of methanol oxidation and is located at the end of a large cluster of methylotrophy genes that begins with mxaF. The sequence of mxaB has been obtained and indicates that the gene product is a member of the response regulator family. None of the open reading frames near mxaB showed sequence identity to sensor kinases. Complementation studies suggest a promoter may be located adjacent to mxaB. Another gene (mxaW) is present immediately upstream of mxaF, divergently transcribed from a methanol-inducible promoter. The sequence in the region of mxaW was also obtained. MxaW showed no identity to known proteins. Mutations in mxaW and in an adjacent open reading frame, OrfR, had no effect on growth of M. extorquens AM1 on methanol or other substrates. The MxaW mutant had normal methanol dehydrogenase activity and normal transcription of the mxaF promoter. Therefore, the function of mxaW is unknown.
