Here we report the complete genome sequence of Teredinibacter turnerae T7901. T. turnerae is a marine gamma proteobacterium that occurs as an intracellular endosymbiont in the gills of wood-boring marine bivalves of the family Teredinidae (shipworms). This species is the sole cultivated member of an endosymbiotic consortium thought to provide the host with enzymes, including cellulases and nitrogenase, critical for digestion of wood and supplementation of the host's nitrogen-deficient diet. T. turnerae is closely related to the free-living marine polysaccharide degrading bacterium Saccharophagus degradans str. 2–40 and to as yet uncultivated endosymbionts with which it coexists in shipworm cells. Like S. degradans, the T. turnerae genome encodes a large number of enzymes predicted to be involved in complex polysaccharide degradation (>100). However, unlike S. degradans, which degrades a broad spectrum (>10 classes) of complex plant, fungal and algal polysaccharides, T. turnerae primarily encodes enzymes associated with deconstruction of terrestrial woody plant material. Also unlike S. degradans and many other eubacteria, T. turnerae dedicates a large proportion of its genome to genes predicted to function in secondary metabolism. Despite its intracellular niche, the T. turnerae genome lacks many features associated with obligate intracellular existence (e.g. reduced genome size, reduced %G+C, loss of genes of core metabolism) and displays evidence of adaptations common to free-living bacteria (e.g. defense against bacteriophage infection). These results suggest that T. turnerae is likely a facultative intracellular ensosymbiont whose niche presently includes, or recently included, free-living existence. As such, the T. turnerae genome provides insights into the range of genomic adaptations associated with intracellular endosymbiosis as well as enzymatic mechanisms relevant to the recycling of plant materials in marine environments and the production of cellulose-derived biofuels.

RimO, encoded by the yliG gene in Escherichia coli, has been recently identified in vivo as the enzyme responsible for the attachment of a methylthio group on the β-carbon of Asp88 of the small ribosomal protein S12 [Anton, B. P., Saleh, L., Benner, J. S., Raleigh, E. A., Kasif, S., and Roberts, R. J. (2008) Proc. Natl. Acad. Sci. USA, 105, 1826USA, 105, -1831. To date, it is the only enzyme known to catalyze methylthiolation of a protein substrate; the four other naturally occurring methylthio modifications have been observed on tRNA. All members of the methylthiotransferase (MTTase) family, to which RimO belongs, have been shown to contain the canonical CxxxCxxC motif in their primary structures that is typical of the radical S-adenosylmethionine (SAM) family of proteins. MiaB, the only characterized MTTase, and the enzyme experimentally shown to be responsible for methylthiolation of N 6 -isopentenyladenosine of tRNA in E. coli and Thermotoga maritima, has been demonstrated to harbor two distinct clusters. Herein, we report in vitro biochemical, and spectroscopic characterization of RimO. We show by analytical and spectroscopic methods that RimO, heterologously overproduced in E. coli in the presence of iron-sulfur cluster biosynthesis proteins from Azotobacter vinelandii, contains one [4Fe-4S] 2+ cluster. Reconstitution of this form of RimO (RimO rcn ) with 57 Fe and sodium sulfide results in a protein that contains two [4Fe-4S] 2+ clusters, similar to MiaB. We also show by mass spectrometry that RimO rcn catalyzes the attachment of a methylthio group to a peptide substrate analog that mimics the loop structure bearing aspartyl 88 of the S12 ribosomal protein from E. coli. Kinetic analysis of this reaction shows that the activity of RimO rcn in the presence of the substrate analog does not support a complete turnover. We discuss the possible requirement for an assembled ribosome for fully active RimO in vitro. Our findings are † This work was supported by NIH Grant GM-63847 and NSF Grant MCB-0133826 (S.J.B.), the Dreyfus Foundation (Teacher Scholar Award to C.K.), the Beckman Foundation (Young Investigator Award to C.K.), and New England Biolabs. * To whom correspondence should be addressed. Squire J. Booker, 104 Chemistry Building, The Pennsylvania State University, University Park, PA 16802. Phone: 814-865-8793. Fax: 814-865-2927. Squire@psu.edu. Carsten Krebs, 104 Chemistry Building, The Pennsylvania State University, University Park, PA 16802. Phone: 814-865-6089. Fax: 814-865-2927. ckrebs@psu.edu. Lana Saleh, 240 County Road, Ipswich, MA 01908. Phone: 978-380-7446. Fax: 978-921-1350. Saleh@neb.com. Δ These authors contributed equally to this work 3 The EPR and Mössbauer spectroscopic features of RimO ai are consistent with the presence of a small amount of the SAM-bound form, because the spectral features of the SAM-bound and SAM-free forms overlap heavily. Supporting Information AvailableFigures S1, S2, S3, S4, S5, and S6. This material is available free of charge via the Internet at http...

tRNA His guanylyltransferase (Thg1) post-transcriptionally adds a G (position ؊1) to the 5 -terminus of tRNA His . The Methanosarcina acetivorans Thg1 (MaThg1) gene contains an in-frame TAG (amber) codon. Although a UAG codon typically directs translation termination, its presence in Methanosarcina mRNA may lead to pyrrolysine (Pyl) incorporation achieved by Pyl-tRNA Pyl , the product of pyrrolysyl-tRNA synthetase. Sequencing of the MaThg1 gene and transcript confirmed the amber codon. Translation of MaThg1 mRNA led to a full-length, Pyl-containing, active enzyme as determined by immunoblotting, mass spectrometry, and biochemical analysis. The nature of the inserted amino acid at the position specified by UAG is not critical, as Pyl or Trp insertion yields active MaThg1 variants in M. acetivorans and equal amounts of fulllength protein. These data suggest that Pyl insertion is akin to natural suppression and unlike the active stop codon reassignment that is required for selenocysteine insertion. Only three Pyl-containing proteins have been characterized previously, a set of methylamine methyltransferases in which Pyl is assumed to have specifically evolved to be a key active-site constituent. In contrast, Pyl in MaThg1 is a dispensable residue that appears to confer no selective advantage. Phylogenetic analysis suggests that Thg1 is becoming dispensable in the archaea, and furthermore supports the hypothesis that Pyl appeared in MaThg1 as the result of neutral evolution. This indicates that even the most unusual amino acid can play an ordinary role in proteins.amber codon ͉ Methanosarcina acetivorans ͉ natural supression

Galactofuranose (Gal(f)), the furanoic form of d-galactose produced by UDP-galactopyranose mutases (UGMs), is present in surface glycans of some prokaryotes and lower eukaryotes. Absence of the Gal(f) biosynthetic pathway in vertebrates and its importance in several pathogens make UGMs attractive drug targets. Since the existence of Gal(f) in nematodes has not been established, we investigated the role of the Caenorhabditis elegans UGM homolog glf-1 in worm development. glf-1 mutants display significant late embryonic and larval lethality, and other phenotypes indicative of defective surface coat synthesis, the glycan-rich outermost layer of the nematode cuticle. The glf homolog from the protozoan Leishmania major partially complements C. elegans glf-1. glf-1 mutants rescued by L. major glf, which behave as glf-1 hypomorphs, display resistance to infection by Microbacterium nematophilum, a pathogen of rhabditid nematodes thought to bind to surface coat glycans. To confirm the presence of Gal(f) in C. elegans, we analyzed C. elegans nucleotide sugar pools using online electrospray ionization-mass spectrometry (ESI-MS). UDP-Gal(f) was detected in wild-type animals while absent in glf-1 deletion mutants. Our data indicate that Gal(f) likely has a pivotal role in maintenance of surface integrity in nematodes, supporting investigation of UGM as a drug target in parasitic species.

A proteomic analysis was performed on spent fermentation medium following bioreactor propagation of a wild-type industrial strain to identify proteins naturally secreted by Kluyveromyces lactis cells. Here, we report changes detected in the K. lactis secretome as a result of growth in three different carbon sources: glucose, galactose and glycerol. A total of 151 secreted proteins were detected by multi-dimensional separations and reversed-phase online nanoESI-MS/MS analysis. From these, we were able to identify 63 proteins (termed the "base secretome") that were common to all three fermentation conditions. The majority of base secretome proteins, 79%, possessed general secretory pathway (GSP) sequences and were involved with cell wall structure, glycosylation, carbohydrate metabolism and proteolysis. There was little variation in the functional groupings of base secretome GSP proteins and GSP proteins that were not part of the base secretome. In contrast, the majority of non-GSP proteins detected were not part of the base secretome and the functions of these proteins varied significantly. Finally, through further identification of non-GSP proteins in carbon sources not originally tested, we have gained further evidence of a protein export mechanism separate from the GSP in K. lactis.