We present an unconventional approach to antiviral drug discovery, which is used to identify potent small molecules against rabies virus. First, we conceptualized viral capsid assembly as occurring via a host-catalyzed biochemical pathway, in contrast to the classical view of capsid formation by self-assembly. This suggested opportunities for antiviral intervention by targeting previously unappreciated catalytic host proteins, which were pursued. Second, we hypothesized these host proteins to be components of heterogeneous, labile, and dynamic multi-subunit assembly machines, not easily isolated by specific target protein-focused methods. This suggested the need to identify active compounds before knowing the precise protein target. A cell-free translation-based small molecule screen was established to recreate the hypothesized interactions involving newly synthesized capsid proteins as host assembly machine substrates. Hits from the screen were validated by efficacy against infectious rabies virus in mammalian cell culture. Used as affinity ligands, advanced analogs were shown to bind a set of proteins that effectively reconstituted drug sensitivity in the cell-free screen and included a small but discrete subfraction of cellular ATP-binding cassette family E1 (ABCE1), a host protein previously found essential for HIV capsid formation. Taken together, these studies advance an alternate view of capsid formation (as a host-catalyzed biochemical pathway), a different paradigm for drug discovery (whole pathway screening without knowledge of the target), and suggest the existence of labile assembly machines that can be rendered accessible as next-generation drug targets by the means described.

Ooids are a common component of carbonate successions of all ages and present significant potential as paleoenvironmental proxies, if the mechanisms that control their formation and growth can be understood quantitatively. There are a number of hypotheses about the controls on ooid growth, each offering different ideas on where and how ooids accrete and what role, if any, sediment transport and abrasion might play. These hypotheses have not been well tested in the field, largely due to the inherent challenges of tracking individual grains over long timescales. This study presents a detailed field test of ooid-growth hypotheses on Little Ambergris Cay in the Turks and Caicos Islands, British Overseas Territories. This field site is characterized by westward net sediment transport from waves driven by persistent easterly trade winds. This configuration makes it possible to track changes in ooid properties along their transport path as a proxy for changes in time. Ooid size, shape, and radiocarbon age were compared along this path to determine in which environments ooids are growing or abrading. Ooid surface textures, petrographic fabrics, stable-isotope compositions (δ¹³C, δ¹⁸O, and δ³⁴S), lipid geochemistry, and genetic data were compared to characterize mechanisms of precipitation and degradation and to determine the relative contributions of abiotic (e.g., abiotic precipitation, physical abrasion) and biologically influenced processes (e.g., biologically mediated precipitation, fabric destruction through microbial microboring and micritization) to grain size and character. A convergence of evidence shows that active ooid growth occurs along the transport path in a high-energy shoal environment characterized by frequent suspended-load transport: median ooid size increases by more than 100 μm and bulk radiocarbon ages decrease by 360 yr westward along the ∼ 20 km length of the shoal crest. Lipid and 16S rRNA data highlight a spatial disconnect between the environments with the most extensive biofilm colonization and environments with active ooid growth. Stable-isotope compositions are indistinguishable among samples, and are consistent with abiotic precipitation of aragonite from seawater. Westward increases in ooid sphericity and the abundance of well-polished ooids illustrate that ooids experience subequal amounts of growth and abrasion—in favor of net growth—as they are transported along the shoal crest. Overall, these results demonstrate that, in the Ambergris system, the mechanism of ooid growth is dominantly abiotic and the loci of ooid growth is determined by both carbonate saturation and sediment transport mode. Microbes play a largely destructive, rather than constructive, role in ooid size and fabric.

Desert varnish is a dark rock coating that forms in arid environments worldwide. It is highly and selectively enriched in manganese, the mechanism for which has been a long-standing geological mystery. We collected varnish samples from diverse sites across the western United States, examined them in petrographic thin section using microscale chemical imaging techniques, and investigated the associated microbial communities using 16S amplicon and shotgun metagenomic DNA sequencing. Our analyses described a material governed by sunlight, water, and manganese redox cycling that hosts an unusually aerobic microbial ecosystem characterized by a remarkable abundance of photosynthetic Cyanobacteria in the genus Chroococcidiopsis as the major autotrophic constituent. We then showed that diverse Cyanobacteria, including the relevant Chroococcidiopsis taxon, accumulate extraordinary amounts of intracellular manganese—over two orders of magnitude higher manganese content than other cells. The speciation of this manganese determined by advanced paramagnetic resonance techniques suggested that the Cyanobacteria use it as a catalytic antioxidant—a valuable adaptation for coping with the substantial oxidative stress present in this environment. Taken together, these results indicated that the manganese enrichment in varnish is related to its specific uptake and use by likely founding members of varnish microbial communities. Significance Rock varnish is a prominent feature of desert landscapes and the canvas for many prehistoric petroglyphs. How it forms—and, in particular, the basis for its extremely high manganese content—has been an enduring mystery. The work presented here establishes a biological mechanism for this manganese enrichment, underpinned by an apparent antioxidant strategy that enables microbes to survive in the harsh environments where varnish forms. The understanding that varnish is the residue of life using manganese to thrive in the desert illustrates that, even in extremely stark environments, the imprint of life is omnipresent on the landscape.

Nitrous oxide is a potent greenhouse gas whose production is catalyzed by nitric oxide reductase (NOR) members of the heme-copper oxidoreductase (HCO) enzyme superfamily. We identified several previously uncharacterized HCO families, four of which (eNOR, sNOR, gNOR, and nNOR) appear to perform NO reduction. These families have novel active-site structures and several have conserved proton channels, suggesting that they might be able to couple NO reduction to energy conservation. We isolated and biochemically characterized a member of the eNOR family from the bacterium Rhodothermus marinus and found that it performs NO reduction. These recently identified NORs exhibited broad phylogenetic and environmental distributions, greatly expanding the diversity of microbes in nature capable of NO reduction. Phylogenetic analyses further demonstrated that NORs evolved multiple times independently from oxygen reductases, supporting the view that complete denitrification evolved after aerobic respiration.

Microbial mats are taxonomically and metabolically diverse microbial ecosystems, with a characteristic layering that reflects vertical gradients in light and oxygen availability. Silicified microbial mats in Proterozoic carbonate successions are generally interpreted in terms of the surficial, mat building community. However, information about biodiversity in the once-surface-layer can be lost through decay as the mats accrete. To better understand how information about surface microbial communities is impacted by processes of decay within the mat, we studied microbial mats from Little Ambergris Cay, Turks and Caicos Islands. We used molecular techniques, microscopy and geochemistry to investigate microbial mat taphonomy— how processes of degradation affect biological signatures in sedimentary rocks, including fossils, molecular fossils and isotopic records. The top <1 cm of these mats host cyanobacteria-rich communities overlying and admixed with diverse bacterial and eukaryotic taxa. Lower layers contain abundant, often empty, sheaths of large filamentous cyanobacteria, preserving their record as key mat-builders. Morphological remains and free lipid biomarkers of several bacterial groups, as well as diatoms, arthropods, and other eukaryotes also persist in lower mat layers, although at lower abundances than in surface layers. Carbon isotope signatures of organic matter were consistent with the majority of the biomass being sourced from CO2-limited cyanobacteria. Porewater sulfide sulfur isotope values were lower than seawater sulfate sulfur isotope values by ~ 45 to 50‰, consistent with microbial sulfate reduction under sulfate-replete conditions. Our findings provide insight into how processes of degradation and decay bias biosignatures in the geological record of microbial mats, especially mats that formed widely during the Proterozoic (2,500-541 million years ago) Eon. Cyanobacteria were the key mat-builders, their robust and cohesive fabric retained at depth. Additionally, eukaryotic remains and eukaryotic biosignatures were preserved at depth, which suggests that microbial mats are not inherently biased against eukaryote preservation, either today or in the past.