Along with methane, methanol and methylated amines represent important biogenic atmospheric constituents; thus, not only methanotrophs but also nonmethanotrophic methylotrophs play a significant role in global carbon cycling. The complete genome of a model obligate methanol and methylamine utilizer, Methylobacillus flagellatus (strain KT) was sequenced. The genome is represented by a single circular chromosome of approximately 3 Mbp, potentially encoding a total of 2,766 proteins. Based on genome analysis as well as the results from previous genetic and mutational analyses, methylotrophy is enabled by methanol and methylamine dehydrogenases and their specific electron transport chain components, the tetrahydromethanopterin-linked formaldehyde oxidation pathway and the assimilatory and dissimilatory ribulose monophosphate cycles, and by a formate dehydrogenase. Some of the methylotrophy genes are present in more than one (identical or nonidentical) copy. The obligate dependence on single-carbon compounds appears to be due to the incomplete tricarboxylic acid cycle, as no genes potentially encoding alpha-ketoglutarate, malate, or succinate dehydrogenases are identifiable. The genome of M. flagellatus was compared in terms of methylotrophy functions to the previously sequenced genomes of three methylotrophs, Methylobacterium extorquens (an alphaproteobacterium, 7 Mbp), Methylibium petroleiphilum (a betaproteobacterium, 4 Mbp), and Methylococcus capsulatus (a gammaproteobacterium, 3.3 Mbp). Strikingly, metabolically and/or phylogenetically, the methylotrophy functions in M. flagellatus were more similar to those in M. capsulatus and M. extorquens than to the ones in the more closely related M. petroleiphilum species, providing the first genomic evidence for the polyphyletic origin of methylotrophy in Betaproteobacteria.Methylotrophy is the metabolic capacity to grow on reduced carbon compounds containing no COC bonds, such as methane, methanol, methylated amines, etc. (1, 39). While the role of methanotrophs in the reduction of global emissions of methane has been well recognized (27), less attention has been paid to nonmethanotrophic methylotrophs as participants in the global carbon cycle. However, recent models estimate methanol emissions into the atmosphere at 82 to 273 teragrams (Tg) year Ϫ1 (with living plants as the major source [19,25]), putting methanol emission on a scale similar to that of methane emissions (approximately 600 Tg year Ϫ1 [36]) and pointing toward the global role of nonmethanotrophic methanol utilizers. While no global modeling has been attempted to characterize the production of methylated amines, they are known to be abundant in marine and freshwater environments and represent dynamic constituents of not only carbon but also nitrogen global cycles (44). So far, only nonmethanotrophic methylotrophs have been implicated in utilizing methylated amines (1, 39).Methylobacillus flagellatus strain KT utilizes methanol and methylated amines as the sole sources of carbon and energy and is classifi...