A novel methane-oxidizing bacterium, strain Fw12E-Y T , was isolated from floodwater of a rice paddy field in Japan. Cells of strain Fw12E-Y T were Gram-negative, motile rods with a single polar flagellum and type I intracytoplasmic membrane arrangement. The strain grew only on methane or methanol as sole carbon and energy source. It was able to grow at 10-40 6C (optimum 30 6C), at pH 5.5-7.0 (optimum 6.5) and with 0-0.1 % (w/w) NaCl (no growth above 0.5 % NaCl). 16S rRNA gene sequence analysis showed that strain Fw12E-Y T is related most closely to members of the genus Methylomonas, but at low levels of similarity (95.0-95.4 %). Phylogenetic analysis of pmoA and mxaF genes indicated that the strain belongs to the genus Methylomonas (97 and 92 % deduced amino acid sequence identities to Methylomonas methanica S1

The diversity of cultivable methane-oxidizing bacteria (MOB) in the rice paddy field ecosystem was investigated by combined culture-dependent and fluorescence in situ hybridization (FISH) techniques. Seven microsites of a Japanese rice paddy field were the focus of the study: floodwater, surface soil, bulk soil, rhizosphere soil, root, basal stem of rice plant, and rice stumps of previous harvest. Based on pmoA gene analysis and transmission electron microscopy (TEM), four type I, and nine type II MOB isolates were obtained from the highest dilution series of enrichment cultures. The type I MOB isolates included a novel species in the genus Methylomonas from floodwater and this is the first type I MOB strain isolated from floodwater of a rice paddy field. In the type I MOB, two isolates from stumps were closely related to Methylomonas spp.; one isolate obtained from rhizosphere soil was most related to Methyloccocus-Methylocaldum-Methylogaea clade. Almost all the type II MOB isolates were related to Methylocystis methanotrophs. FISH confirmed the presence of both types I and II MOB in all the microsites and in the related enrichment cultures. The study reported, for the first time, the diversity of cultivable methanotrophs including a novel species of type I MOB in rice paddy field compartments. Refining growth media and culture conditions, in combination with molecular approaches, will allow us to broaden our knowledge on the MOB community in the rice paddy field ecosystem and consequently to implement strategies for mitigating CH 4 emission from this ecosystem.

Methylomagnum ishizawai gen. nov., sp. nov., a mesophilic type I methanotroph isolated from rice rhizosphere An aerobic, methane-oxidizing bacterium (strain RS11D-Pr T ) was isolated from rice rhizosphere. Cells of strain RS11D-Pr T were Gram-stain-negative, motile rods with a single polar flagellum and contained an intracytoplasmic membrane system typical of type I methanotrophs. The strain utilized methane and methanol as sole carbon and energy sources. It could grow at 20-37 8C (optimum 31-33 8C), at pH 6.8-7.4 (range 5.5-9.0) and with 0-0.2 % (w/v) NaCl (there was no growth at above 0.5 % NaCl). pmoA and mmoX genes were present. The ribulose monophosphate and/or ribulose bisphosphate pathways were used for carbon assimilation. Results of sequence analysis of 16S rRNA genes showed that strain RS11D-Pr T is related closely to the genera Methylococcus, Methylocaldum, Methyloparacoccus and Methylogaea in the family Methylococcaceae. The similarity was low (94.6 %) between strain RS11D-Pr T and the most closely related type strain (Methyloparacoccus murrellii R-49797 T ). The DNA G+C content was 64.1 mol%. Results of phylogenetic analysis of the pmoA gene and chemotaxonomic data regarding the major cellular fatty acids (C 16 : 1 v7c, C 16 : 0 and C 14 : 0 ) and the major respiratory quinone (MQ-8) also indicated the affiliation of strain RS11D-Pr T to the Methylococcus-Methylocaldum-MethyloparacoccusMethylogaea clade. On the basis of phenotypic, genotypic and phylogenetic characteristics, strain RS11D-Pr T is considered to represent a novel genus and species within the family Methylococcaceae, for which the name Methylomagnum ishizawai gen. nov., sp. nov. is proposed. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA, pmoA and mmoX gene sequences of strain RS11D-Pr T are AB669155, AB669168 and AB983338, respectively.A supplementary figure is available with the online Supplementary Material.