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Phylogenetic positions, and genotypic and phenotypic characteristics of three novel methylotrophic isolates, strains 301T, 30S and SIP3-4, from sediment of Lake Washington, Seattle, USA, are described. The strains were restricted facultative methylotrophs capable of growth on single carbon compounds (methylamine and methanol) in addition to a limited range of multicarbon compounds. All strains used the N-methylglutamate pathway for methylamine oxidation. Strain SIP3-4 possessed the canonical (MxaFI) methanol dehydrogenase, but strains 301T and 30S did not. All three strains used the ribulose monophosphate pathway for C1 assimilation. The major fatty acids in the three strains were C16 : 0 and C16 : 1ω7c. The DNA G+C contents of strains 301T and SIP3-4 were 42.6 and 54.6 mol%, respectively. Based on 16S rRNA gene sequence phylogeny and the relevant phenotypic characteristics, strain SIP3-4 was assigned to the previously defined species Methylovorus glucosotrophus. Strains 301T and 30S were closely related to each other (100 % 16S rRNA gene sequence similarity) and shared 96.6 % 16S rRNA gene sequence similarity with a previously described isolate, Methylotenera mobilis JLW8T. Based on significant genomic and phenotypic divergence with the latter, strains 301T and 30S represent a novel species within the genus Methylotenera, for which the name Methylotenera versatilis sp. nov. is proposed; the type strain is 301T ( = VKM B-2679T = JCM 17579T). An emended description of the genus Methylotenera is provided.