Genomic and in-situ Transcriptomic Characterization of the Candidate Phylum NPL-UPL2 From Highly Alkaline Highly Reducing Serpentinized Groundwater

Serpentinization is a process whereby water interacts with reduced mantle rock called peridotite to produce a new suite of minerals (e.g., serpentine), a highly alkaline fluid, and hydrogen. In previous reports, we identified abundance of microbes of the candidate phylum NPL-UPA2 in a serpentinization site called The Cedars. Here, we report the first metagenome assembled genome (MAG) of the candidate phylum as well as the in-situ gene expression. The MAG of the phylum NPL-UPA2, named Unc8, is only about 1 Mbp and its biosynthetic properties suggest it should be capable of independent growth. In keeping with the highly reducing niche of Unc8, its genome encodes none of the known oxidative stress response genes including superoxide dismutases. With regard to energy metabolism, the MAG of Unc8 encodes all enzymes for Wood-Ljungdahl acetogenesis pathway, a ferredoxin:NAD+ oxidoreductase (Rnf) and electron carriers for flavin-based electron bifurcation (Etf, Hdr). Furthermore, the transcriptome of Unc8 in the waters of The Cedars showed enhanced levels of gene expression in the key enzymes of the Wood-Ljungdahl pathway [e.g., Carbon monoxide dehydrogenase /Acetyl-CoA synthase complex (CODH/ACS), Rnf, Acetyl-CoA synthetase (Acd)], which indicated that the Unc8 is an acetogen. However, the MAG of Unc8 encoded no well-known hydrogenase genes, suggesting that the energy metabolism of Unc8 might be focused on CO as the carbon and energy sources for the acetate formation. Given that CO could be supplied via abiotic reaction associated with deep subsurface serpentinization, while available CO2 would be at extremely low concentrations in this high pH environment, CO-associated metabolism could provide advantageous approach. The CODH/ACS in Unc8 is a Bacteria/Archaea hybrid type of six-subunit complex and the electron carriers, Etf and Hdr, showed the highest similarity to those in Archaea, suggesting that archaeal methanogenic energy metabolism was incorporated into the bacterial acetogenesis in NPL-UPA2. Given that serpentinization systems are viewed as potential habitats for early life, and that acetogenesis via the Wood-Ljungdahl pathway is proposed as an energy metabolism of Last Universal Common Ancestor, a phylogenetically distinct acetogen from an early earth analog site may provide important insights in primordial lithotrophs and their habitat

Cell Wall Trapping of Autocrine Peptides for Human G-Protein-Coupled Receptors on the Yeast Cell Surface

G-protein-coupled receptors (GPCRs) regulate a wide variety of physiological processes and are important pharmaceutical targets for drug discovery. Here, we describe a unique concept based on yeast cell-surface display technology to selectively track eligible peptides with agonistic activity for human GPCRs (Cell Wall Trapping of Autocrine Peptides (CWTrAP) strategy). In our strategy, individual recombinant yeast cells are able to report autocrine-positive activity for human GPCRs by expressing a candidate peptide fused to an anchoring motif. Following expression and activation, yeast cells trap autocrine peptides onto their cell walls. Because captured peptides are incapable of diffusion, they have no impact on surrounding yeast cells that express the target human GPCR and non-signaling peptides. Therefore, individual yeast cells can assemble the autonomous signaling complex and allow single-cell screening of a yeast population. Our strategy may be applied to identify eligible peptides with agonistic activity for target human GPCRs

Microbial Fuel Cells and Microbial Ecology: Applications in Ruminant Health and Production Research

Microbial fuel cell (MFC) systems employ the catalytic activity of microbes to produce electricity from the oxidation of organic, and in some cases inorganic, substrates. MFC systems have been primarily explored for their use in bioremediation and bioenergy applications; however, these systems also offer a unique strategy for the cultivation of synergistic microbial communities. It has been hypothesized that the mechanism(s) of microbial electron transfer that enable electricity production in MFCs may be a cooperative strategy within mixed microbial consortia that is associated with, or is an alternative to, interspecies hydrogen (H2) transfer. Microbial fermentation processes and methanogenesis in ruminant animals are highly dependent on the consumption and production of H2in the rumen. Given the crucial role that H2 plays in ruminant digestion, it is desirable to understand the microbial relationships that control H2 partial pressures within the rumen; MFCs may serve as unique tools for studying this complex ecological system. Further, MFC systems offer a novel approach to studying biofilms that form under different redox conditions and may be applied to achieve a greater understanding of how microbial biofilms impact animal health. Here, we present a brief summary of the efforts made towards understanding rumen microbial ecology, microbial biofilms related to animal health, and how MFCs may be further applied in ruminant research

Isolation and Polyphasic Characterization of Desulfuromonas versatilis sp. Nov., an Electrogenic Bacteria Capable of Versatile Metabolism Isolated from a Graphene Oxide-Reducing Enrichment Culture

In this study, a novel electrogenic bacterium denoted as strain NIT-T3 of the genus Desulfuromonas was isolated from a graphene-oxide-reducing enrichment culture that was originally obtained from a mixture of seawater and coastal sand. Strain NIT-T3 utilized hydrogen and various organic acids as electron donors and exhibited respiration using electrodes, ferric iron, nitrate, and elemental sulfur. The strain contained C16:1ω7c, C16:0, and C15:0 as major fatty acids and MK-8, 9, and 7 as the major respiratory quinones. Strain NIT-T3 contained four 16S rRNA genes and showed 95.7% similarity to Desulfuromonasmichiganensis BB1T, the closest relative. The genome was 4.7 Mbp in size and encoded 76 putative c-type cytochromes, which included 6 unique c-type cytochromes (<40% identity) compared to those in the database. Based on the physiological and genetic uniqueness, and wide metabolic capability, strain NIT-T3 is proposed as a type strain of ‘Desulfuromonas versatilis’ sp. nov

Viruses, Vesicles, and other Biological Nanoparticles: The Sub-cellular Biosphere of a Deeply Buried 2km-Deep, 20-Million-Year-Old Coalbed Community

Horizontal gene transfer is an important driver of adaptation and evolution in microorganisms. Transducing biological nanoparticles such as viral particles are believed to be key facilitators of horizontal gene transfer. In deep subseafloor sediments, energy can be highly limiting, supporting only extremely slow metabolisms. In such low-energy, isolated environments where communities may subsist for millions of years, the mechanisms of subsurface microbial adaptation and evolution remain a mystery. Virus particles have been found everywhere that life has been found, including deep subsurface environments. Although microorganisms are abundant and active in the Earth's subsurface, the role of viruses in shaping and influencing these slow-growing communities is only recently starting to be explored. Here, we analyzed the deeply buried microbial community from a lignite coalbed layer 2km below the seafloor offshore Shimokita, Japan (IODP Expedition 337) that had been buried for 20 million years. We harvested cells (>0.2µm) and biological nanoparticles (<0.2µm) from a bioreactor enrichment seeded by lignite core samples. We sequenced DNA from the cells and nanoparticles and subsequently analyzed the metagenomes. Within the nanoparticle metagenome, numerous complete novel virus genomes were reconstructed. Comparison of the virus genomes to the prokaryotic MAGs (metagenome assembled genomes) revealed that many of the virus genomes had been integrated prophage within bacterial genomes, suggesting the potential for virus-host interactions to occur in the deep subseafloor. Additionally, lysogeny may be an important survival mechanism for viruses in deeply buried, low-energy environments. Host genes were found to be packaged by viral particles, demonstrating the potential for specialized and general transduction by viruses. Not only viral particles, but there was also evidence that membrane vesicles and gene transfer agents may participate in transduction in this deep subsurface community. Horizontal gene transfer mediated by biological nanoparticles may be an important mechanism of adaptation for deep subsurface microbial communities and may provide insight into possible evolutionary processes shaping microbial communities in the deep subsurface. These results may also shed some light onto the nature of viral infection in the subsurface, potentially revealing insights about the long-term persistence of life under extreme energy limitation and how viruses may survive this over geological timescales

Evaluation of the CWTrAP system using α-factor peptide for yeast endogenous Ste2 receptor.

<p>(A) Pheromone signaling assays of α-factor-displaying yeast strains. Error bars represent the standard deviation of three independent experiments. (B) Immunofluorescence staining of α-factor displaying yeast strains. Anti-Flag antibody and Alexa Fluor 546-conjugated secondary antibody were used for detection of secreted α-factor or α-factor-anchor fusion proteins. IMG-4 was used as the host strain. The transformants used in these experiments are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037136#pone.0037136.s008" target="_blank">Table S3</a>. Sec: free, secreted form of α-factor. AG: C-terminal half of α-agglutinin anchor.</p

Confirmation of protein expression.

<p>Western blots of extracts from somatostatin displaying yeast strains. Lane 1: Mock/Mock, 2: SSTR5/Mock, 3: SSTR5/Flag–Flo42, 4: SSTR5/S-14–Flag–Flo42, 5: Mock/S-14–Flag–Flo42. Anti-β-actin antibody was used as loading control. Anti-HA antibody was used for detection of SSTR5 receptor. Anti-Flag antibody was used for detection of Flag–Flo42 anchor or S-14–Flag–Flo42 fusion proteins. IMFD-70 was used as the host strain. The transformants used in these experiments are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037136#pone.0037136.s008" target="_blank">Table S3</a>.</p

Evaluation of the CWTrAP system using somatostatin peptide for the human SSTR5 receptor.

<p>(A) SSTR5 signaling assays of the cyclic somatostatin peptide displaying yeast strain and control strains. (B) SSTR5 signaling assays of non-target peptide displaying yeast strains. IMFD-70 was used as the host strain. The transformants used in these experiments are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037136#pone.0037136.s008" target="_blank">Table S3</a>. S-14 indicates 14 aa of somatostatin cyclic peptide, α-factor indicates 13 aa of yeast pheromone peptide, AII indicates 8 aa of angiotensin II peptide, and ET1 indicates 21 aa of endothelin-1 peptide.</p

Schematic illustration of our concept using yeast cell-surface display technology to selectively track eligible agonistic peptides for human GPCRs by assembling the autonomous signaling complex within individual cells (cell wall trapping of autocrine peptides (CWTrAP) strategy).

<p>The candidate autocrine peptides fused with the anchoring proteins are processed via secretion pathways in engineered yeast cells. Their agonistic activities for heterologously-expressed human GPCRs are discerned on yeast cell membranes. Only when the peptide possesses objective agonistic activity, membrane-peripheral G-proteins promote intracellular signaling and induce transcription of the GFP reporter gene. Because the autocrine peptides are automatically trapped onto individual yeast cell walls, the captured peptides are unable to diffuse toward surrounding yeast cells that express the target human GPCR and any other peptides. T.F. indicates transcription factor.</p