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The preservation of nucleic acids for high-throughput sequencing is an ongoing challenge for field scientists. In particular, samples that are low biomass, or that have to be collected and preserved in logistically challenging environments (such as remote sites or during long sampling campaigns) can pose exceptional difficulties. With this work, we compare and assess the effectiveness of three preservation methods for DNA and RNA extracted from microbial communities of glacial snow and ice samples. Snow and ice samples were melted and filtered upon collection in Iceland, and filters were preserved using: (i) liquid nitrogen flash freezing, (ii) storage in RNAlater, or (iii) storage in Zymo DNA/RNA Shield. Comparative statistics covering nucleic acid recovery, sequencing library preparation, genome assembly, and taxonomic diversity were used to determine best practices for the preservation of DNA and RNA samples from these environments. Our results reveal that microbial community composition based on DNA was comparable at the class level across preservation types. Based on extracted RNA, the taxonomic composition of the active community was primarily driven by the filtered sample volume (i.e., biomass content). In low biomass samples (where <200 ml of sample volume was filtered) the taxonomic and functional signatures trend toward the composition of the control samples, while in samples where a larger volume (more biomass) was filtered our data showed comparable results independent of preservation type. Based on all comparisons our data suggests that flash freezing of filters containing low biomass is the preferred method for preserving DNA and RNA (notwithstanding the difficulties of accessing liquid nitrogen in remote glacial field sites). Generally, RNAlater and Zymo DNA/RNA Shield solutions work comparably well, especially for DNA from high biomass samples, but Zymo DNA/RNA Shield is favored due to its higher yield of preserved RNA. Biomass quantity from snow and ice samples appears to be the most important factor in regards to the collection and preservation of samples from glacial environments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Trivedi, Keuschnig, Larose, Rissi, Mourot, Bradley, Winkel and Benning.)

Blooms of microalgae on glaciers and ice sheets are amplifying surface ice melting rates, which are already affected by climate change. Most studies on glacial microorganisms (including snow and glacier ice algae) have so far focused on the spring and summer melt season, leading to a temporal bias, and a knowledge gap in our understanding of the variations in microbial diversity, productivity, and physiology on glacier surfaces year-round. Here, we investigated the microbial communities from Icelandic glacier surface snow and bare ice habitats, with sampling spanning two consecutive years and carried out in both winter and two summer seasons. We evaluated the seasonal differences in microbial community composition using Illumina sequencing of the 16S rRNA, 18S rRNA, and ITS marker genes and correlating them with geochemical signals in the snow and ice. During summer, Chloromonas , Chlainomonas , Raphidonema , and Hydrurus dominated surface snow algal communities, while Ancylonema and Mesotaenium dominated the surface bare ice habitats. In winter, algae could not be detected, and the community composition was dominated by bacteria and fungi. The dominant bacterial taxa found in both winter and summer samples were Bacteriodetes , Actinobacteria , Alphaproteobacteria , and Gammaproteobacteria . The winter bacterial communities showed high similarities to airborne and fresh snow bacteria reported in other studies. This points toward the importance of dry and wet deposition as a wintertime source of microorganisms to the glacier surface. Winter samples were also richer in nutrients than summer samples, except for dissolved organic carbon-which was highest in summer snow and ice samples with blooming microalgae, suggesting that nutrients are accumulated during winter but primarily used by the microbial communities in the summer. Overall, our study shows that glacial snow and ice microbial communities are highly variable on a seasonal basis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Winkel, Trivedi, Mourot, Bradley, Vieth-Hillebrand and Benning.)

Large, colorless sulfur-oxidizing bacteria (LSB) of the family Beggiatoaceae form thick mats at sulfidic sediment surfaces, where they efficiently detoxify sulfide before it enters the water column. The genus Thiomargarita harbors the largest known free-living bacteria with cell sizes of up to 750 μm in diameter. In addition to their ability to oxidize reduced sulfur compounds, some Thiomargarita spp. are known to store large amounts of nitrate, phosphate and elemental sulfur internally. To date little is known about their energy yielding metabolic pathways, and how these pathways compare to other Beggiatoaceae. Here, we present a draft single-cell genome of a chain-forming "Candidatus Thiomargarita nelsonii Thio36", and conduct a comparative analysis to five draft and one full genome of other members of the Beggiatoaceae. "Ca. T. nelsonii Thio36" is able to respire nitrate to both ammonium and dinitrogen, which allows them to flexibly respond to environmental changes. Genes for sulfur oxidation and inorganic carbon fixation confirmed that "Ca. T. nelsonii Thio36" can function as a chemolithoautotroph. Carbon can be fixed via the Calvin-Benson-Bassham cycle, which is common among the Beggiatoaceae. In addition we found key genes of the reductive tricarboxylic acid cycle that point toward an alternative CO2 fixation pathway. Surprisingly, "Ca. T. nelsonii Thio36" also encodes key genes of the C2-cycle that convert 2-phosphoglycolate to 3-phosphoglycerate during photorespiration in higher plants and cyanobacteria. Moreover, we identified a novel trait of a flavin-based energy bifurcation pathway coupled to a Na(+)-translocating membrane complex (Rnf). The coupling of these pathways may be key to surviving long periods of anoxia. As other Beggiatoaceae "Ca. T. nelsonii Thio36" encodes many genes similar to those of (filamentous) cyanobacteria. In summary, the genome of "Ca. T. nelsonii Thio36" provides additional insight into the ecology of giant sulfur-oxidizing bacteria, and reveals unique genomic features for the Thiomargarita lineage within the Beggiatoaceae.

The genus Thiomargarita includes the world's largest bacteria. But as uncultured organisms, their physiology, metabolism, and basis for their gigantism are not well understood. Thus, a genomics approach, applied to a single Candidatus Thiomargarita nelsonii cell was employed to explore the genetic potential of one of these enigmatic giant bacteria. The Thiomargarita cell was obtained from an assemblage of budding Ca. T. nelsonii attached to a provannid gastropod shell from Hydrate Ridge, a methane seep offshore of Oregon, USA. Here we present a manually curated genome of Bud S10 resulting from a hybrid assembly of long Pacific Biosciences and short Illumina sequencing reads. With respect to inorganic carbon fixation and sulfur oxidation pathways, the Ca. T. nelsonii Hydrate Ridge Bud S10 genome was similar to marine sister taxa within the family Beggiatoaceae. However, the Bud S10 genome contains genes suggestive of the genetic potential for lithotrophic growth on arsenite and perhaps hydrogen. The genome also revealed that Bud S10 likely respires nitrate via two pathways: a complete denitrification pathway and a dissimilatory nitrate reduction to ammonia pathway. Both pathways have been predicted, but not previously fully elucidated, in the genomes of other large, vacuolated, sulfur-oxidizing bacteria. Surprisingly, the genome also had a high number of unusual features for a bacterium to include the largest number of metacaspases and introns ever reported in a bacterium. Also present, are a large number of other mobile genetic elements, such as insertion sequence (IS) transposable elements and miniature inverted-repeat transposable elements (MITEs). In some cases, mobile genetic elements disrupted key genes in metabolic pathways. For example, a MITE interrupts hupL, which encodes the large subunit of the hydrogenase in hydrogen oxidation. Moreover, we detected a group I intron in one of the most critical genes in the sulfur oxidation pathway, dsrA. The dsrA group I intron also carried a MITE sequence that, like the hupL MITE family, occurs broadly across the genome. The presence of a high degree of mobile elements in genes central to Thiomargarita's core metabolism has not been previously reported in free-living bacteria and suggests a highly mutable genome.