Pyrrolysine, the 22nd amino acid to be found in the natural genetic code1–4, is necessary for all known pathways of methane formation from methylamines5,6. The residue is comprised of a methylated pyrroline carboxylate in amide linkage to the ε-amino group of L-lysine2,7,8. The three different methyltransferases that initiate methanogenesis from different methylamines9–11 have genes with an in-frame amber codon12,13 translated as pyrrolysine2,7,8. E. coli transformed with pylTSBCD from methanogenic Archaea can incorporate endogenously biosynthesized pyrrolysine into protein14. The decoding of UAG as pyrrolysine requires pylT1,6 which produces tRNAPyl (also called tRNACUA), and pylS1 encoding a pyrrolysyl-tRNA synthetase4,15,16. The pylBCD genes1 are each required for tRNA-independent pyrrolysine synthesis14. Pyrrolysine has been the last remaining genetically encoded amino acid with an unknown biosynthetic pathway. Here, we provide genetic and mass spectroscopic evidence for a pylBCD-dependent pathway in which pyrrolysine arises from two lysines. We show that a new UAG encoded residue, desmethylpyrrolysine, is made from lysine and exogenous D-ornithine in a pylC, then a pylD, dependent process, but is not further converted to pyrrolysine. These results indicate that the radical S-adenosyl-methionine (SAM) protein PylB mediates a lysine mutase reaction producing 3-methylornithine, which is then ligated to a second molecule of lysine by PylC before oxidation by PylD results in pyrrolysine. The discovery of lysine as sole precursor to pyrrolysine will further inform discussions of the evolution the genetic code and amino acid biosynthetic pathways, while intermediates of the pathway may provide new avenues by which the pyl system may be exploited for production of recombinant proteins with useful modified residues.

Summary In Methanosarcina spp., amber codons in methylamine methyltransferase genes are translated as the 22nd amino acid, pyrrolysine. The responsible pyl genes plus amber-codon containing methyltransferase genes have been identified in four archaeal and five bacterial genera, including one human pathogen. In E. coli, the recombinant pylBCD gene products biosynthesize pyrrolysine from two lysine and the pylTS gene products direct pyrrolysine incorporation into protein. In the proposed biosynthetic pathway, PylB forms methylornithine from lysine, which is joined to another lysine by PylC, and oxidized to pyrrolysine by PylD. Structures of the catalytic domain of pyrrolysyl-tRNA synthetase (archaeal PylS or bacterial PylSc) revealed binding sites for tRNAPyl and pyrrolysine. PylS and tRNAPyl are now being exploited as an orthogonal pair in recombinant systems for introduction of useful modified amino acids into proteins.

Sialic acid reduced and stabilized gold nanoparticles (d = 20.1 ± 1.8 nm) were synthesized by a simple one-pot, green method without chemically modifying sialic acid for colorimetric detection of influenza virus. The gold nanoparticles showed target-specific aggregation with viral particles via hemagglutinin-sialic acid binding. A linear correlation was observed between the change in optical density and dilution of chemically inactivated influenza B/Victoria and influenza B/Yamagata. Virus dilution (hemagglutinination assay titer, 512) of 0.156 vol% was readily detected. The upper limit of the linearity can be extended with the use of more sialic acid-gold nanoparticles.

Shiga toxin (Stx), the main virulence factor of Shiga toxin producing Escherichia coli, is a major public health threat, causing hemorrhagic colitis and hemolytic uremic syndrome. Currently, there are no approved therapeutics for these infections; however manganese has been reported to provide protection from the Stx1 variant isolated from Shigella dysenteriae (Stx1-S) both in vitro and in vivo. We investigated the efficacy of manganese protection from Stx1-S and the more potent Stx2a isoform, using experimental systems well-established for studying Stx: in vitro responses of Vero monkey kidney cells, and in vivo toxicity to CD-1 outbred mice. Manganese treatment at the reported therapeutic concentration was toxic to Vero cells in culture and to CD-1 mice. At lower manganese concentrations that were better tolerated, we observed no protection from Stx1-S or Stx2a toxicity. The ability of manganese to prevent the effects of Stx may be particular to certain cell lines, mouse strains, or may only be manifested at high, potentially toxic manganese concentrations.

Biosensor for the detection of virus was developed by utilizing plasmonic peak shift phenomenon of the gold nanoparticles and viral infection mechanism of hemagglutinin on virus and sialic acid on animal cells. The plasmonic peak of the colloidal gold nanoparticles changes with the aggregation of the particles due to the plasmonic interaction between nearby particles and the color of the colloidal nanoparticle solution changes from wine red to purple. Sialic acid reduced and stabilized colloidal gold nanoparticle aggregation is induced by the addition of viral particles in the solution due to the hemagglutinin-sialic acid interaction. In this work, sialic acid reduced and stabilized gold nanoparticles (d = 20.1 ± 1.8 nm) were synthesized by a simple one-pot, green method without chemically modifying sialic acid. The gold nanoparticles showed target-specific aggregation with viral particles via hemagglutinin-sialic acid binding. A linear correlation was observed between the change in optical density and dilution of chemically inactivated influenza B virus species. The detection limit of the virus dilution (hemagglutinination assay titer, 512) was shown to be 0.156 vol% and the upper limit of the linearity can be extended with the use of more sialic acid-gold nanoparticles.