Our current knowledge of host–virus interactions in biofilms is limited to computational predictions based on laboratory experiments with a small number of cultured bacteria. However, natural biofilms are diverse and chiefly composed of uncultured bacteria and archaea with no viral infection patterns and lifestyle predictions described to date. Herein, we predict the first DNA sequence-based host–virus interactions in a natural biofilm. Using single-cell genomics and metagenomics applied to a hot spring mat of the Cone Pool in Mono County, California, we provide insights into virus–host range, lifestyle and distribution across different mat layers. Thirty-four out of 130 single cells contained at least one viral contig (26%), which, together with the metagenome-assembled genomes, resulted in detection of 59 viruses linked to 34 host species. Analysis of single-cell amplification kinetics revealed a lack of active viral replication on the single-cell level. These findings were further supported by mapping metagenomic reads from different mat layers to the obtained host–virus pairs, which indicated a low copy number of viral genomes compared to their hosts. Lastly, the metagenomic data revealed high layer specificity of viruses, suggesting limited diffusion to other mat layers. Taken together, these observations indicate that in low mobility environments with high microbial abundance, lysogeny is the predominant viral lifestyle, in line with the previously proposed “Piggyback-the-Winner” theory.

Large-scale deposits of elemental sulfur form annually on a glacier's surface at Borup Fiord Pass in the Canadian High Arctic. However, the mechanisms of mineralization and stabilization of elemental sulfur at this site are currently unknown. Here we show that x-ray diffraction (XRD) data for fresh sulfur precipitates collected from the surface of a melt pool over sulfide-rich ice reveal the presence of three sulfur allotropes, α-S 8 , β-S 8 , and γ-S 8 (the three solid forms of cyclooctasulfur (S 8 )). The detection of the β-S 8 allotrope of elemental sulfur is notable, since β-S 8 typically only forms in high temperature environments (>96°C). The γ-S 8 allotrope is also rare in natural settings and has previously been implicated as a signature of microbial sulfur cycling. Using combustion and infrared spectroscopy approaches, organic carbon is also detected within the sample bearing the three allotropes of elemental sulfur. Electron microscopy and scanning transmission x-ray microscopy (STXM) at the C K-edge show that the sulfur precipitates are intimately associated with the organic carbon at the submicron scale. The occurrence of β-S 8 and γ-S 8 in this low-temperature setting indicates that there are unknown pathways for the formation and stabilization of these rare allotropes of elemental sulfur. In particular, we infer that the occurrence of these allotropes is related to their association with organic carbon. The formation of carbon-associated sulfur globules may not be a direct by-product of microbial activity; however, a potential role of direct or indirect microbial mediation in the formation and stabilization of β-S 8 and γ-S 8 remains to be assessed. 48 of sulfide (e.g. Helz, 2014;Pjevac et al., 2014) and can also serve as a reactant for microbially 49 mediated sulfur reduction, oxidation, and disproportionation reactions (e.g. Zopfi et al., 2004; 50 Boyd and Druschel, 2013;Pjevac et al., 2014). Even though S 0 has a low solubility in water 51 (Kamyshny, 2009) and is far less reactive than sulfides, polysulfides, and other sulfur 52 intermediates, the range of environments in which S 0 is stable are limited (e.g. anoxic marine 53 settings, volcanic and fumarolic systems, and in caprock deposits overlying salt domes; Davis 54 and Kirkland, 1979;Zopfi et al., 2004;Sievert et al., 2007;and Mandeville, 2010). 55

Biological sulfur cycling in polar, low-temperature ecosystems is an understudied phenomenon in part due to difficulty of access and the dynamic nature of glacial environments. One such environment where sulfur cycling is known to play an important role in microbial metabolisms is located at Borup Fiord Pass (BFP) in the Canadian High Arctic. Here, transient springs emerge from ice near the terminus of a glacier, creating a large area of proglacial aufeis (spring-derived ice) that is often covered in bright yellow/white sulfur, sulfate, and carbonate mineral precipitates accompanied by a strong odor of hydrogen sulfide. Metagenomic sequencing of samples from multiple sites and of various sample types across the BFP glacial system produced 31 metagenome-assembled genomes (MAGs) that were queried for sulfur, nitrogen, and carbon cycling/metabolism genes. An abundance of sulfur cycling genes was widespread across the isolated MAGs and sample metagenomes taxonomically associated with the bacterial classes Alphaproteobacteria and Gammaproteobacteria and Campylobacteria (formerly the Epsilonproteobacteria). This corroborates previous research from BFP implicating Campylobacteria as the primary class responsible for sulfur oxidation; however, data reported here suggested putative sulfur oxidation by organisms in both the alphaproteobacterial and gammaproteobacterial classes that was not predicted by previous work. These findings indicate that in low-temperature, sulfur-based environments, functional redundancy may be a key mechanism that microorganisms use to enable coexistence whenever energy is limited and/or focused by redox chemistry. IMPORTANCE A unique environment at Borup Fiord Pass is characterized by a sulfur-enriched glacial ecosystem in the low-temperature Canadian High Arctic. BFP represents one of the best terrestrial analog sites for studying icy, sulfur-rich worlds outside our own, such as Europa and Mars. The site also allows investigation of sulfur-based microbial metabolisms in cold environments here on Earth. Here, we report whole-genome sequencing data that suggest that sulfur cycling metabolisms at BFP are more widely used across bacterial taxa than predicted. From our analyses, the metabolic capability of sulfur oxidation among multiple community members appears likely due to functional redundancy present in their genomes. Functional redundancy, with respect to sulfur-oxidation at the BFP sulfur-ice environment, may indicate that this dynamic ecosystem hosts microorganisms that are able to use multiple sulfur electron donors alongside other metabolic pathways, including those for carbon and nitrogen.

A sulfur-dominated supraglacial spring system found at Borup Fiord Pass (BFP), Ellesmere Island, Nunavut, Canada, is a unique sulfur-on-ice system expressed along the toe of a glacier. BFP has an intermittent flowing, subsurface-derived, glacial spring that creates a large white-yellow icing (aufeis) that extends down-valley. Over field campaigns in 2014, 2016, and 2017, numerous samples were collected and analyzed for both microbial community composition and aqueous geochemistry. Samples were collected from multiple site types: spring discharge fluid, aufeis (spring-derived ice), melt pools with sedimented cryoconite material, and mineral precipitate scrapings, to probe how microbial communities differed between site types in a dynamic freeze/thaw sulfur-rich system. Dissolved sulfate varied between 0.07 and 11.6 mM and was correlated with chloride concentrations, where the fluids were saltiest among spring fluids. The highest sulfate samples exhibited high dissolved sulfide values between 0.22 and 2.25 mM. 16S rRNA gene sequencing from melt pool and aufeis samples from the 2014 campaign were highly abundant in operational taxonomic units (OTUs) closely related to sulfur-oxidizing microorganisms (SOM; Sulfurimonas, Sulfurovum, and Sulfuricurvum). Subsequent sampling 2 weeks later had fewer SOMs and showed an increased abundance of the genus Flavobacterium. Desulfocapsa, an organism that specializes in the disproportionation of inorganic sulfur compounds was also found. Samples from 2016 and 2017 revealed that microorganisms present were highly similar in community composition to 2014 samples, primarily echoed by the continued presence of Flavobacterium sp. Results suggest that while there may be acute events where sulfur cycling organisms dominate, a basal community structure appears to dominate over time and site type. These results further enhance our knowledge of low-temperature sulfur systems on Earth, and help to guide the search for potential life on extraterrestrial worlds, such as Europa, where similar low-temperature sulfur-rich conditions may exist.

Ancient putative microbial structures that appear in the rock record commonly serve as evidence of early life on Earth, but the details of their formation remain unclear. The study of modern microbial mat structures can help inform the properties of their ancient counterparts, but modern mineralizing mat systems with morphological similarity to ancient structures are rare. Here, we characterize partially lithified microbial mats containing cm-scale dendrolitic coniform structures from a geothermal pool (“Cone Pool”) at Little Hot Creek, California, that if fully lithified, would resemble ancient dendrolitic structures known from the rock record. Light and electron microscopy revealed that the cm-scale ‘dendrolitic cones’ were comprised of intertwined microbial filaments and grains of calcium carbonate. The degree of mineralization (carbonate content) increased with depth in the dendrolitic cones. Sequencing of 16S rRNA gene libraries revealed that the dendrolitic cone tips were enriched in OTUs most closely related to the genera Phormidium, Leptolyngbya, and Leptospira, whereas mats at the base and adjacent to the dendrolitic cones were enriched in Synechococcus. We hypothesize that the consumption of nutrients during autotrophic and heterotrophic growth may promote movement of microbes along diffusive nutrient gradients, and thus microbialite growth. Hour-glass shaped filamentous structures present in the dendrolitic cones may have formed around photosynthetically-produced oxygen bubbles—suggesting that mineralization occurs rapidly and on timescales of the lifetime of a bubble. The dendrolitic-conical structures in Cone Pool constitute a modern analog of incipient microbialite formation by filamentous microbiota that are morphologically distinct from any structure described previously. Thus, we provide a new model system to address how microbial mats may be preserved over geological timescales.