The taxonomic positions and phylogenetic relationships of two new methylotrophic isolates from Lake Washington (USA) sediment, FAM5^Tand 500, and the previously described methylotrophic strain EHg5 isolated from contaminated soil in Estarreja (Portugal) were investigated. All three strains were facultative methylotrophs capable of growth on a variety of C₁and multicarbon compounds. Optimal growth occurred at pH 7.5–8 and 30–37 °C. The major fatty acids were C_16 : 1ω7cand C_16 : 0. The major quinone was ubiquinone Q8. Neither methanol dehydrogenase nor methanol oxidase activities were detectable in cells grown on methanol, suggesting an alternative, as-yet unknown, mechanism for methanol oxidation. The isolates assimilated C₁units at the level of formaldehyde, via the serine cycle. The DNA G+C content of the strains ranged between 64 and 65 mol%. 16S rRNA gene sequence similarity between the three new isolates was 99.85–100 %, but was below 94 % with other members of theBetaproteobacteria, indicating that the isolates represent a novel taxon. Based on physiological, phenotypic and genomic characteristics of the three isolates, a new genus,Methyloversatilisgen. nov., is proposed within the familyRhodocyclaceae. The type strain ofMethyloversatilis universalisgen. nov., sp. nov. is FAM5^T(=CCUG 52030^T=JCM 13912^T).

Three strains of methylotrophic Rhodocyclaceae (FAM1^T, RZ18-153 and RZ94) isolated from Lake Washington sediment samples were characterized. Based on phylogenetic analysis of 16S rRNA gene sequences the strains should be assigned to the genus Methyloversatilis. Similarly to other members of the family, the strains show broad metabolic capabilities and are able to utilize a number of organic acids, alcohols and aromatic compounds in addition to methanol and methylamine. The main fatty acids were 16:1ω7c (49–59 %) and 16:0 (32–29 %). Genomes of all isolates were sequenced, assembled and annotated in collaboration with the DOE Joint Genome Institute (JGI). Genome comparison revealed that the strains FAM1^T, RZ18-153 and RZ94 are closely related to each other and almost equally distant from two previously described species of the genus Methyloversatilis, Methyloversatilis universalis and Methyloversatilis thermotolerans. Like other methylotrophic species of the genus Methyloversatilis, all three strains possess one-subunit PQQ-dependent ethanol/methanol dehydrogenase (Mdh-2), the N-methylglutamate pathway and the serine cycle (isocitrate lyase/malate synthase, Icl/ms⁺ variant). Like M. universalis, strains FAM1^T, RZ18-153 and RZ94 have a quinohemoprotein amine dehydrogenase, a tungsten-containing formaldehyde ferredoxin oxidoreductase, phenol hydroxylase, and the complete Calvin cycle. Similarly to M. thermotolerans, the three strains possess two-subunit methanol dehydrogenase (MxaFI), monoamine oxidase (MAO) and nitrogenase. Based on the phenotypic and genomic data, the strains FAM1^T, RZ18-153 and RZ94 represent a novel species of the genus Methyloversatilis, for which the name Methyloversatilis discipulorum sp. nov. is proposed. The type strain is FAM1^T ( = JCM 30542^T = VKM = B-2888^T).

Thirty one novel methylotrophic bacterial strains were isolated from a range of soil and sediment sources (both pristine and polluted) under differen