Pyrrolysine is a lysine derivative encoded by the UAG codon in methylamine methyltransferase genes of Methanosarcina barkeri. Near a methyltransferase gene cluster is the pylT gene, which encodes an unusual transfer RNA (tRNA) with a CUA anticodon. The adjacent pylS gene encodes a class II aminoacyl-tRNA synthetase that charges the pylT-derived tRNA with lysine but is not closely related to known lysyl-tRNA synthetases. Homologs of pylS and pylT are found in a Gram-positive bacterium. Charging a tRNA(CUA) with lysine is a likely first step in translating UAG amber codons as pyrrolysine in certain methanogens. Our results indicate that pyrrolysine is the 22nd genetically encoded natural amino acid.

Methanogenesis, the biological production of methane, plays a pivotal role in the global carbon cycle and contributes significantly to global warming. The majority of methane in nature is derived from acetate. Here we report the complete genome sequence of an acetate-utilizing methanogen, Methanosarcina acetivorans C2A. Methanosarcineae are the most metabolically diverse methanogens, thrive in a broad range of environments, and are unique among the Archaea in forming complex multicellular structures. This diversity is reflected in the genome of M. acetivorans.

Hydraulic fracturing is the industry standard for extracting hydrocarbons from shale formations. Attention has been paid to the economic benefits and environmental impacts of this process, yet the biogeochemical changes induced in the deep subsurface are poorly understood. Recent single-gene investigations revealed that halotolerant microbial communities were enriched after hydraulic fracturing. Here, the reconstruction of 31 unique genomes coupled to metabolite data from the Marcellus and Utica shales revealed that many of the persisting organisms play roles in methylamine cycling, ultimately supporting methanogenesis in the deep biosphere. Fermentation of injected chemical additives also sustains long-term microbial persistence, while thiosulfate reduction could produce sulfide, contributing to reservoir souring and infrastructure corrosion. Extensive links between viruses and microbial hosts demonstrate active viral predation, which may contribute to the release of labile cellular constituents into the extracellular environment. Our analyses show that hydraulic fracturing provides the organismal and chemical inputs for colonization and persistence in the deep terrestrial subsurface. S hale gas accounts for one-third of natural gas energy resources worldwide. It has been estimated that shale gas will provide half of the natural gas in the USA, annually, by 2040, with the Marcellus shale in the Appalachian Basin projected to produce three times more than any other formation 1 . Recovery of these hydrocarbons is dependent on hydraulic fracturing technologies, where the high-pressure injection of water and chemical additives generates extensive fractures in the shale matrix. Hydrocarbons trapped in tiny pore spaces are subsequently released and collected at the wellpad surface, together with a portion of the injected fluids that have reacted with the shale formation. The mixture of injected fluids and hydrocarbons collected is referred to as 'produced fluids'.Microbial metabolism and growth in hydrocarbon reservoirs has both positive and negative impacts on energy recovery. Whereas stimulation of methanogens in coal beds enhances energy recovery 2 , bacterial hydrogen sulfide production ('reservoir souring') decreases profits and contributes to corrosion and the risk of environmental contamination 3 . Additionally, biomass accumulation within newly generated fractures may reduce their permeability, decreasing natural gas recovery. Despite these potential microbial impacts, little is known about the function and activity of microorganisms in hydraulically fractured shale.Initial work by our group and others 4-9 used single marker gene analyses to identify microorganisms from several geographically distinct shale formations. These analyses showed similar halotolerant taxa in produced fluids several months after hydraulic fracturing. To assign functional roles to these organisms, we conducted metagenomic and metabolite analyses on input and produced fluids up to a year after hydraulic fracturing (HF) from two Appala...

Genes encoding methanogenic methylamine methyltransferases all contain an in-frame amber (UAG) codon that is read through during translation. We have identified the UAG-encoded residue in a 1.55 angstrom resolution structure of the Methanosarcina barkeri monomethylamine methyltransferase (MtmB). This structure reveals a homohexamer comprised of individual subunits with a TIM barrel fold. The electron density for the UAG-encoded residue is distinct from any of the 21 natural amino acids. Instead it appears consistent with a lysine in amide-linkage to (4R,5R)-4-substituted-pyrroline-5-carboxylate. We suggest that this amino acid be named l-pyrrolysine.

Pyrrolysine is the 22nd amino acid. An unresolved question has been how this atypical genetically encoded residue is inserted into proteins, because all previously described naturally occurring aminoacyl-tRNA synthetases are specific for one of the 20 universally distributed amino acids. Here we establish that synthetic L-pyrrolysine is attached as a free molecule to tRNA(CUA) by PylS, an archaeal class II aminoacyl-tRNA synthetase. PylS activates pyrrolysine with ATP and ligates pyrrolysine to tRNA(CUA) in vitro in reactions specific for pyrrolysine. The addition of pyrrolysine to Escherichia coli cells expressing pylT (encoding tRNA(CUA)) and pylS results in the translation of UAG in vivo as a sense codon. This is the first example from nature of direct aminoacylation of a tRNA with a non-canonical amino acid and shows that the genetic code of E. coli can be expanded to include UAG-directed pyrrolysine incorporation into proteins.