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TRP channel-associated factor 1/2 (TCAF1/TCAF2) proteins antagonistically regulate the cold-sensor protein TRPM8 in multiple human tissues. Understanding their significance has been complicated given the locus spans a gap-ridden region with complex segmental duplications in GRCh38. Using long-read sequencing, we sequence-resolve the locus, annotate full-length TCAF models in human and nonhuman primate genomes, and show substantial human-specific TCAF copy number variation. We identify two human super haplogroups, H4 and H5, and establish that TCAF duplications originated ~1.7 million years ago but diversified only in Homo sapiens by recurrent structural mutations that altered TCAF copy number and regulation. Conversely, in all archaic-hominin samples the fixation for a specific H4 haplotype without duplication is likely due to positive selection. The significant, positive effect of H4 on TCAF2 expression in modern-day humans with candidate associations for hypothyroidism, nerve compression, and diabetes suggests TCAF diversification among hominins potentially in response to cold or dietary adaptations.

The North American beaver (Castor canadensis) has long been considered an engineering marvel, transforming landscapes and shaping biological diversity through its dam building behavior. While the beaver possesses conspicuous morphological features uniquely adapted for the use of woody plants as construction materials and dietary staples, relatively little is known about the specialized microorganisms inhabiting the beaver gastrointestinal tract and their functional roles in determining host nutrition. Here we use a combination of shotgun metagenomics, functional screening and carbohydrate biochemistry to chart the community structure and metabolic power of the beaver fecal microbiome. We relate this information to the metabolic capacity of other wood feeding and hindgut fermenting organisms and profile the functional repertoire of glycoside hydrolase (GH) families distributed among and between population genome bins. Metagenomic screening revealed novel mechanisms of xylan oligomer degradation involving GH43 enzymes from uncharacterized subfamilies and divergent polysaccharide utilization loci, indicating the potential for synergistic biomass deconstruction. Together, these results open a functional metagenomic window on less conspicuous adaptations enabling the beaver microbiome to efficiently convert woody plants into host nutrition and point toward rational design of enhanced enzyme mixtures for biorefining process streams.

The Scientific Committee on Oceanographic Research (SCOR) Working Group 144 Microbial Community Responses to Ocean Deoxygenation workshop held in Vancouver, B.C on July 2014 had the primary objective of initiating a process to standardize operating procedures for compatible process rate and multi-omic (DNA, RNA, protein and metabolite) data collection in marine oxygen minimum zones (OMZs) and other oxygen depleted waters. Workshop attendees participated in practical sampling and experimental activities in Saanich Inlet, British Columbia, a seasonally anoxic fjord. Experiments were designed to compare and cross-calibrate in situ versus bottle sampling methods to determine effects on microbial community structure and potential activity when using different filter combinations, filtration methods and sample volumes. Resulting biomass was preserved for small subunit ribosomal RNA (SSU or 16S rRNA) and SSU rRNA gene (rDNA) amplicon sequencing followed by downstream statistical and visual analyses. Results from these analyses showed that significant community shifts occurred between in situ versus on ship processed samples. For example, Bacteroidetes, Alphaproteobacteria and Opisthokonta associated with on-ship filtration onto 0.4 µm filters increased 5-fold compared to on-ship in-line 0.22 µm filters or 0.4 µm filters processed and preserved in situ. In contrast, Planctomycetes associated with 0.4 µm in situ filters increased 5-fold compared to on-ship filtration onto 0.4 µm filters and on-ship in-line 0.22 µm filters. In addition, candidate divisions and Chloroflexi were primarily recovered when filtered onto 0.4 µm filters in situ. Results based on rRNA:rDNA ratios for microbial indicator groups revealed previously unrecognized roles of candidate divisions, Desulfarculales, and Desulfuromandales in sulfur cycling, carbon fixation and fermentation within anoxic basin waters. Taken together, filter size and in situ versus on-ship filtration had the largest impact on recovery of microbial groups with the potential to influence downstream metabolic reconstruction and process rate measurements. These observations highlight the need for establishing standardized and reproducible techniques that facilitate cross-scale comparisons and more accurately assess in situ activities of microbial communities.

Extensive and expanding oxygen minimum zones (OMZs) exist at variable depths in coastal and open ocean waters. As oxygen levels decline, nutrients and energy are increasingly diverted away from higher trophic levels into microbial community metabolism, resulting in fixed nitrogen loss and production of climate active trace gases including nitrous oxide and methane. While ocean deoxygenation has been reported on a global scale, our understanding of OMZ biology and geochemistry is limited by a lack of time-resolved data sets. Here, we present a historical dataset of oxygen concentrations spanning fifty years and nine years of monthly geochemical time series observations in Saanich Inlet, a seasonally anoxic fjord on the coast of Vancouver Island, British Columbia, Canada that undergoes recurring changes in water column oxygenation status. This compendium provides a unique geochemical framework for evaluating long-term trends in biogeochemical cycling in OMZ waters.

Extensive and expanding oxygen minimum zones (OMZs) exist at variable depths in coastal and open ocean waters. As oxygen levels decline, nutrients and energy are increasingly diverted away from higher trophic levels into microbial community metabolism, resulting in fixed nitrogen loss and production of climate active trace gases including nitrous oxide and methane. While ocean deoxygenation has been reported on a global scale, our understanding of OMZ biology and geochemistry is limited by a lack of time-resolved data sets. Here, we present a historical dataset of oxygen concentrations spanning fifty years and nine years of monthly geochemical time series observations in Saanich Inlet, a seasonally anoxic fjord on the coast of Vancouver Island, British Columbia, Canada that undergoes recurring changes in water column oxygenation status. This compendium provides a unique geochemical framework for evaluating long-term trends in biogeochemical cycling in OMZ waters. Machine-accessible metadata file describing the reported data (ISA-Tab format)

Directed evolution methods were developed for Cu-containing nitrite reductase (NiR) from Alcaligenes faecalis S-6. The PCR cloning strategy allows for the efficient production of libraries of 100 000 clones by a modification of a megaprimer-based whole-plasmid synthesis reaction. The high-throughput screen includes colony lift onto a nylon membrane and subsequent lysis of NiR-expressing colonies in the presence of Cu(2+) ions for copper incorporation into intracellularly expressed NiR. Addition of a chromogenic substrate, 3, 3'-diaminobenzidine (DAB), results in deposition of red, insoluble color at the site of oxidation by functional NiR. Twenty-thousand random variants of NiR were screened for improved function with DAB as a reductant, and five variants were identified. These variants were shuffled and screened, yielding two double variants. An analog of the DAB substrate, o-dianisidine, which is oxidized to a water-soluble product was used for functional characterization. The double variant M150L/F312C was most proficient at o-dianisidine oxidation with dioxygen as the electron acceptor (5.5X wt), and the M150L single variant was most proficient at o-dianisidine oxidation with nitrite as the electron acceptor (8.5X wt). The library generation and screening method can be employed for evolving new reductase functions in NiR and for screening of efficient folding of engineered NiRs.

Viruses modulate microbial communities and alter ecosystem functions. However, due to cultivation bottlenecks, specific virus–host interaction dynamics remain cryptic. In this study, we examined 127 single-cell amplified genomes (SAGs) from uncultivated SUP05 bacteria isolated from a model marine oxygen minimum zone (OMZ) to identify 69 viral contigs representing five new genera within dsDNA Caudovirales and ssDNA Microviridae. Infection frequencies suggest that ∼1/3 of SUP05 bacteria is viral-infected, with higher infection frequency where oxygen-deficiency was most severe. Observed Microviridae clonality suggests recovery of bloom-terminating viruses, while systematic co-infection between dsDNA and ssDNA viruses posits previously unrecognized cooperation modes. Analyses of 186 microbial and viral metagenomes revealed that SUP05 viruses persisted for years, but remained endemic to the OMZ. Finally, identification of virus-encoded dissimilatory sulfite reductase suggests SUP05 viruses reprogram their host's energy metabolism. Together, these results demonstrate closely coupled SUP05 virus–host co-evolutionary dynamics with the potential to modulate biogeochemical cycling in climate-critical and expanding OMZs.

Campylobacter jejuni, a major cause of acute bacterial diarrhea in humans, expresses numerous proteins to import diverse forms of essential iron. The expression of p19 and an adjacent iron transporter homologue (ftr1) is strongly induced upon iron limitation, suggesting a function in iron acquisition. Here, we show that the loss of P19 alone is detrimental to growth on iron-restricted media. Furthermore, metal binding analysis demonstrates that recombinant P19 has distinct copper and iron binding sites. Crystal structures of P19 have been solved to 1.41 A resolution, revealing an immunoglobulin-like fold. A P19 homodimer in which both monomers contribute ligands to two equivalent copper sites located adjacent to methionine-rich patches is observed. Copper coordination occurs via three histidine residues (His42, His95, and His132) and Met88. A solvent channel lined with conserved acidic residues leads to the copper site. Soaking crystals with a solution of manganese as iron analog reveals a second metal binding site in this solvent channel (metal-metal distance, 7.7 A). Glu44 lies between the metal sites and displays multiple conformations in the crystal structures, suggesting a role in regulating metal-metal interaction. Dimerization is shown to be metal dependent in vitro and is detected in vivo by cross-linking.