In the past few years, the field of methylotrophy has undergone a significant transformation in terms of discovery of novel types of methylotrophs, novel modes of methylotrophy, and novel metabolic pathways. This time has also been marked by the resolution of long-standing questions regarding methylotrophy and the challenge of long-standing dogmas. This chapter is not intended to provide a comprehensive review of metabolism of methylotrophic bacteria. Instead we focus on significant recent discoveries that are both refining and transforming the current understanding of methylotrophy as a metabolic phenomenon. We also review new directions in methylotroph ecology that improve our understanding of the role of methylotrophy in global biogeochemical processes, along with an outlook for the future challenges in the field.

Methanogenic and sulfate-reducing Archaea are considered to have an energy metabolism involving C ₁ transfer coenzymes and enzymes unique for this group of strictly anaerobic microorganisms. An aerobic methylotrophic bacterium, Methylobacterium extorquens AM1, was found to contain a cluster of genes that are predicted to encode some of these enzymes and was shown to contain two of the enzyme activities and one of the methanogenic coenzymes. Insertion mutants were all unable to grow on C ₁ compounds, suggesting that the archaeal enzymes function in aerobic C ₁ metabolism. Thus, methylotrophy and methanogenesis involve common genes that cross the bacterial/archaeal boundaries.

Active members of the bacterial community in the sediment of Lake Washington, with special emphasis on C ₁ utilizers, were identified by employing two complementary culture-independent approaches: reverse transcription of environmental mRNA and 16S rRNA combined with PCR (RT-PCR) and stable-isotope probing (SIP) of DNA with the ¹³ C-labeled C ₁ substrates methanol, methylamine, formaldehyde, and formate. Analysis of RT-PCR-amplified fragments of 16S rRNA-encoding genes revealed that gammaproteobacterial methanotrophs belonging to Methylobacter and Methylomonas dominate the active methylotroph population, while only one other known methylotrophic lineage, Methylophilaceae , was detected via this approach. Analysis of RT-PCR-amplified functional genes, pmoA and fae , allowed detection of alphaproteobacterial ( Methylosinus ) and gammaproteobacterial ( Methylobacter , Methylomonas , and Methylomicrobium ) methanotrophs, methylotrophs of the genus Methylobacterium , and yet-unidentified proteobacteria. SIP experiments allowed detection of a broad variety of groups actively metabolizing C ₁ compounds. Comparisons between 16S rRNA gene pools amplified from [ ¹³ C]DNA and from [ ¹² C]DNA revealed that the proportion of Methylophilus -related sequences increased in the presence of [ ¹³ C]methanol, [ ¹³ C]methylamine, and [ ¹³ C]formaldehyde; Novosphingobium -related sequences were enriched in the presence of [ ¹³ C]methanol; Gemmatimonadaceae -related sequences were enriched in the presence of [ ¹³ C]formaldehyde and [ ¹³ C]formate; and Xanthomonadaceae -related sequences were enriched in the presence of [ ¹³ C]formate. Analysis of fae genes amplified from [ ¹³ C]DNAs isolated from different microcosms revealed specific shifts in populations in response to a specific C ₁ compound: Methylosinus sequences dominated the [ ¹³ C]methanol microcosm pool, and beta- and gammaproteobacterial sequences dominated the [ ¹³ C]methylamine microcosm pool. The [ ¹³ C]formaldehyde microcosm was dominated by betaproteobacterial sequences and by sequences of a nonaffiliated group, while the [ ¹³ C]formate microcosm was dominated by alpha- and betaproteobacterial sequences. Overall, these data point toward the presence of a diverse population of active methylotrophs in Lake Washington sediments and toward the existence of yet-uncultivated organisms.