The Chloroflexi CL500-11 clade contributes a large proportion of the bacterial biomass in the oxygenated hypolimnia of deep lakes worldwide, including the world's largest freshwater system, the Laurentian Great Lakes. Traits that allow CL500-11 to thrive and its biogeochemical role in these environments are currently unknown. Here, we found that a CL500-11 population was present mostly in offshore waters along a transect in ultraoligotrophic Lake Michigan (a Laurentian Great Lake). It occurred throughout the water column in spring and only in the hypolimnion during summer stratification, contributing up to 18.1% of all cells. Genome reconstruction from metagenomic data suggested an aerobic, motile, heterotrophic lifestyle, with additional energy being gained through carboxidovory and methylovory. Comparisons to other available streamlined freshwater genomes revealed that the CL500-11 genome contained a disproportionate number of cell wall/capsule biosynthesis genes and the most diverse spectrum of genes involved in the uptake of dissolved organic matter (DOM) substrates, particularly peptides. In situ expression patterns indicated the importance of DOM uptake and protein/peptide turnover, as well as type I and type II carbon monoxide dehydrogenase and flagellar motility. Its location in the water column influenced its gene expression patterns the most. We observed increased bacteriorhodopsin gene expression and a response to oxidative stress in surface waters compared to its response in deep waters. While CL500-11 carries multiple adaptations to an oligotrophic lifestyle, its investment in motility, its large cell size, and its distribution in both oligotrophic and mesotrophic lakes indicate its ability to thrive under conditions where resources are more plentiful. Our data indicate that CL500-11 plays an important role in nitrogen-rich DOM mineralization in the extensive deep-lake hypolimnion habitat. Freshwater lakes are disproportionally active sites of carbon cycling relative to the surface area that they cover due to strong linkages to the surrounding land, from which they receive inorganic nutrients as well as organic carbon (1, 2). Of the estimated 1.9 Pg of terrestrial organic carbon that freshwater systems process per year, nearly half is respired by bacteria (3-5). When soil dissolved organic carbon outgassing is included, net freshwater carbon emissions are of the same order of magnitude as net oceanic uptake (2). While photochemical mineralization of organic carbon can predominate in lake habitats with high levels of photosynthetically active radiation (6), bacterial contributions to dissolved organic matter (DOM) mineralization are important as well (7).Nevertheless, linkages between the metabolism of organic carbon and specific populations remain limited, particularly in the less-studied hypolimnia of lakes, even for ubiquitous and highly abundant taxa, due to challenges with the isolation of representatives of these taxa (8). In recent years, the use of culture-independent methods has provided ins...

Bias is a pervasive problem when characterizing microbial communities. An important source is the difference in lysis efficiencies of different populations, which vary depending on the extraction protocol used. To avoid such biases impacting comparisons between gene and transcript abundances in the environment, the use of one protocol that simultaneously extracts both types of nucleic acids from microbial community samples has gained popularity. However, knowledge regarding tradeoffs to combined nucleic acid extraction protocols is limited, particularly regarding yield and biases in the observed community composition. Here, we evaluated a commercially available protocol for simultaneous extraction of DNA and RNA, which we adapted for freshwater microbial community samples that were collected on filters. DNA and RNA yields were comparable to other commonly used, but independent DNA and RNA extraction protocols. RNA protection agents benefited RNA quality, but decreased DNA yields significantly. Choice of extraction protocol influenced the perceived bacterial community composition, with strong method-dependent biases observed for specific phyla such as the Verrucomicrobia. The combined DNA/RNA extraction protocol detected significantly higher levels of Verrucomicrobia than the other protocols, and those higher numbers were confirmed by microscopic analysis. Use of RNA protection agents as well as independent sequencing runs caused a significant shift in community composition as well, albeit smaller than the shift caused by using different extraction protocols. Despite methodological biases, sample origin was the strongest determinant of community composition. However, when the abundance of specific phylogenetic groups is of interest, researchers need to be aware of the biases their methods introduce. This is particularly relevant if different methods are used for DNA and RNA extraction, in addition to using RNA protection agents only for RNA samples.

Hibernation is a mammalian strategy that uses metabolic plasticity to reduce energy demands and enable long-term fasting. Fasting mitigates winter food scarcity but eliminates dietary nitrogen, jeopardizing body protein balance. Here, we reveal gut microbiome–mediated urea nitrogen recycling in hibernating thirteen-lined ground squirrels ( Ictidomys tridecemlineatus ). Ureolytic gut microbes incorporate urea nitrogen into metabolites that are absorbed by the host, with the nitrogen reincorporated into the squirrel’s protein pool. Urea nitrogen recycling is greatest after prolonged fasting in late winter, when urea transporter abundance in gut tissue and urease gene abundance in the microbiome are highest. These results reveal a functional role for the gut microbiome during hibernation and suggest mechanisms by which urea nitrogen recycling may contribute to protein balance in other monogastric animals.

The bacterial phylum Verrucomicrobia was formally described two decades ago and originally believed to be a minor member of many ecosystems; however, it is now recognized as ubiquitous and abundant in both soil and aquatic systems. Nevertheless, knowledge of the drivers of its relative abundance and within-phylum habitat preferences remains sparse, especially in lake systems. Here, we documented the distribution of Verrucomicrobia in 12 inland lakes in Southeastern Michigan, a Laurentian Great Lake (Lake Michigan), and a freshwater estuary, which span a gradient in lake sizes, depths, residence times, and trophic states. A wide range of physical and geochemical parameters was covered by sampling seasonally from the surface and bottom of each lake, and by separating samples into particle-associated and free-living fractions. On average, Verrucomicrobia was the 4th most abundant phylum (range 1.7–41.7%). Fraction, season, station, and depth explained up to 70% of the variance in Verrucomicrobia community composition and preference for these habitats was phylogenetically conserved at the class-level. When relative abundance was linearly modeled against environmental data, Verrucomicrobia and non-Verrucomicrobia bacterial community composition correlated to similar quantitative environmental parameters, although there were lake system-dependent differences and > 55% of the variance remained unexplained. A majority of the phylum exhibited preference for the particle-associated fraction and two classes (Opitutae and Verrucomicrobiae) were identified to be more abundant during the spring season. This study highlights the high relative abundance of Verrucomicrobia in north temperate lake systems and expands insights into drivers of within-phylum habitat preferences of the Verrucomicrobia.

Artificial sweeteners have been shown to induce glucose intolerance by altering the gut microbiota; however, little is known about the effect of stevia. Here, we investigate whether stevia supplementation induces glucose intolerance by altering the gut microbiota in mice, hypothesizing that stevia would correct high fat diet-induced glucose intolerance and alter the gut microbiota. Mice were split into four treatment groups: low fat, high fat, high fat + saccharin and high fat + stevia. After 10 weeks of treatment, mice consuming a high fat diet (60% kcal from fat) developed glucose intolerance and gained more weight than mice consuming a low fat diet. Stevia supplementation did not impact body weight or glucose intolerance. Differences in species richness and relative abundances of several phyla were observed in low fat groups compared to high fat, stevia and saccharin. We identified two operational taxonomic groups that contributed to differences in beta-diversity between the stevia and saccharin groups: Lactococcus and Akkermansia in females and Lactococcus in males. Our results demonstrate that stevia does not rescue high fat diet-induced changes in glucose tolerance or the microbiota, and that stevia results in similar alterations to the gut microbiota as saccharin when administered in concordance with a high fat diet.