Methylothermus thermalis gen. nov., sp. nov., a novel moderately thermophilic obligate methanotroph from a hot spring in Japan

1 Energy and Technology Laboratories, Osaka Gas Co., Ltd, 6-19-9 Torishima Konohana-ku, Osaka 554-0051, Japan 2 G. K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia Correspondence Yuri A. Trotsenko trotsenko{at}ibpm.pushchino.ru A novel moderately thermophilic methanotroph, strain MYHT T , was isolated from a hot spring in Japan. The isolate grew on methane or methanol at 37–67 °C, and optimally at 57–59 °C. It was found to be a Gram-negative aerobe, with colourless colonies of non-motile coccoid cells, possessing type I intracytoplasmic membranes and regularly arranged surface layers of linear ( p 2) symmetry. Strain MYHT T expressed only the particulate methane monooxygenase and employed the ribulose monophosphate pathway for formaldehyde assimilation. It is a neutrophilic and halotolerant organism capable of growth at pH 6·5–7·5 (optimum pH 6·8) and in up to 3 % NaCl (optimum 0·5–1 % NaCl). Phylogenetic analysis based on 16S rRNA gene sequence analysis indicated that strain MYHT T is most closely related to the thermophilic undescribed methanotroph ‘ Methylothermus ’ HB (91 % identity) and the novel halophilic methanotroph Methylohalobius crimeensis 10Ki T (90 % identity). Comparative sequence analysis of particulate methane monooxygenase ( pmoA ) genes also confirmed the clustering of strain MYHT T with ‘ Methylothermus ’ HB and Methylohalobius crimeensis 10Ki T (98 and 92 % derived amino acid sequence identity, respectively). The DNA G+C content was 62·5 mol%. The major cellular fatty acids were C 16 : 0 (37·2 %) and C 18 : 1 9 c (35·2 %) and the major polar lipids were phosphatidylethanolamine and phosphatidylglycerol. The major ubiquinone was Q-8. On the basis of comparative phenotypic and genotypic characteristics, a new genus and species, Methylothermus thermalis gen. nov., sp. nov., is proposed, with MYHT T as the type strain (=VKM B-2345 T =IPOD FERM P-19714 T ). Abbreviations: ICM, intracytoplasmic membrane; PLFA, phospholipid fatty acid; pMMO and sMMO, particulate and soluble methane monooxygenase, respectively; RuMP, ribulose monophosphate The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and partial sequence of the pmoA gene of Methylothermus thermalis strain MYHT T are AY829009 and AY829010 , respectively. A figure showing the oxidation and assimilation of 14 C-methane by cells of strain MYHT T is available as supplementary materi