Functional Characterization of Bacteria Isolated from Ancient Arctic Soil Exposes Diverse Resistance Mechanisms to Modern Antibiotics

Using functional metagenomics to study the resistomes of bacterial communities isolated from different layers of the Canadian high Arctic permafrost, we show that microbial communities harbored diverse resistance mechanisms at least 5,000 years ago. Among bacteria sampled from the ancient layers of a permafrost core, we isolated eight genes conferring clinical levels of resistance against aminoglycoside, $\beta$-lactam and tetracycline antibiotics that are naturally produced by microorganisms. Among these resistance genes, four also conferred resistance against amikacin, a modern semi-synthetic antibiotic that does not naturally occur in microorganisms. In bacteria sampled from the overlaying active layer, we isolated ten different genes conferring resistance to all six antibiotics tested in this study, including aminoglycoside, $\beta$-lactam and tetracycline variants that are naturally produced by microorganisms as well as semi-synthetic variants produced in the laboratory. On average, we found that resistance genes found in permafrost bacteria conferred lower levels of resistance against clinically relevant antibiotics than resistance genes sampled from the active layer. Our results demonstrate that antibiotic resistance genes were functionally diverse prior to the anthropogenic use of antibiotics, contributing to the evolution of natural reservoirs of resistance genes.Organismic and Evolutionary Biolog

The functional potential of high Arctic permafrost revealed by metagenomic sequencing, qPCR and microarray analyses

The fate of the carbon stocked in permafrost following global warming and permafrost thaw is of major concern in view of the potential for increased CH4 and CO2 emissions from these soils. Complex carbon compound degradation and greenhouse gas emissions are due to soil microbial communities, but no comprehensive study has yet addressed their composition and functional potential in permafrost. Here, a 2-m deep permafrost sample and its overlying active layer soil were subjected to metagenomic sequencing, quantitative PCR (qPCR) and microarray analyses

Comparative Transcriptomics of Cold Growth and Adaptive Features of a Eury- and Steno-Psychrophile

Permafrost subzero environments harbor diverse, active communities of microorganisms. However, our understanding of the subzero growth, metabolisms, and adaptive properties of these microbes remains very limited. We performed transcriptomic analyses on two subzero-growing permafrost isolates with different growth profiles in order to characterize and compare their cold temperature growth and cold-adaptive strategies. The two organisms, Rhodococcus sp. JG3 (-5 to 30°C) and Polaromonas sp. Eur3 1.2.1 (-5 to 22°C), shared several common responses during low temperature growth, including induction of translation and ribosomal processes, upregulation of nutrient transport, increased oxidative and osmotic stress responses, and stimulation of polysaccharide capsule synthesis. Recombination appeared to be an important adaptive strategy for both isolates at low temperatures, likely as a mechanism to increase genetic diversity and the potential for survival in cold systems. While Rhodococcus sp. JG3 favored upregulating iron and amino acid transport, sustaining redox potential, and modulating fatty acid synthesis and composition during growth at -5°C compared to 25°C, Polaromonas sp. Eur3 1.2.1 increased the relative abundance of transcripts involved in primary energy metabolism and the electron transport chain, in addition to signal transduction and peptidoglycan synthesis at 0°C compared to 20°C. The increase in energy metabolism may explain why Polaromonas sp. Eur3 1.2.1 is able to sustain growth rates at 0°C comparable to those at higher temperatures. For Rhodococcus sp. JG3, flexibility in use of carbon sources, iron acquisition, control of membrane fatty acid composition, and modulating redox and co-factor potential may be ways in which this organism is able to sustain growth over a wider range of temperatures. Increasing our understanding of the microbes in these habitats helps us better understand active pathways and metabolisms in extreme environments. Identifying novel, thermolabile, and cold-active enzymes from studies such as this is also of great interest to the biotechnology and food industries

Distinct gut microbiomes in two polar bear subpopulations inhabiting different sea ice ecoregions

Gut microbiomes were analyzed by 16S rRNA gene metabarcoding for polar bears (Ursus maritimus) from the southern Beaufort Sea (SB), where sea ice loss has led to increased use of land-based food resources by bears, and from East Greenland (EG), where persistent sea ice has allowed hunting of ice-associated prey nearly year-round. SB polar bears showed a higher number of total (940 vs. 742) and unique (387 vs. 189) amplicon sequence variants and higher inter-individual variation compared to EG polar bears. Gut microbiome composition differed significantly between the two subpopulations and among sex/age classes, likely driven by diet variation and ontogenetic shifts in the gut microbiome. Dietary tracer analysis using fatty acid signatures for SB polar bears showed that diet explained more intrapopulation variation in gut microbiome composition and diversity than other tested variables, i.e., sex/age class, body condition, and capture year. Substantial differences in the SB gut microbiome relative to EG polar bears, and associations between SB gut microbiome and diet, suggest that the shifting foraging habits of SB polar bears tied to sea ice loss may be altering their gut microbiome, with potential consequences for nutrition and physiology

Arctic microbial ecosystems and impacts of extreme warming during the International Polar Year

As a contribution to the International Polar Year program MERGE (Microbiological and Ecological Responses to Global Environmental change in polar regions), studies were conducted on the terrestrial and aquatic microbial ecosystems of northern Canada (details at: http://www.cen.ulaval.ca/merge/). The habitats included permafrost soils, saline coldwater springs, supraglacial lakes on ice shelves, epishelf lakes in fjords, deep meromictic lakes, and shallow lakes, ponds and streams. Microbiological samples from each habitat were analysed by HPLC pigment assays, light and fluorescence microscopy, and DNA sequencing. The results show a remarkably diverse microflora of viruses, Archaea (including ammonium oxidisers and methanotrophs), Bacteria (including filamentous sulfur-oxidisers in a saline spring and benthic mats of Cyanobacteria in many waterbodies), and protists (including microbial eukaryotes in snowbanks and ciliates in ice-dammed lakes). In summer 2008, we recorded extreme warming at Ward Hunt Island and vicinity, the northern limit of the Canadian high Arctic, with air temperatures up to 20.5 \ub0C. This was accompanied by pronounced changes in microbial habitats: deepening of the permafrost active layer; loss of perennial lake ice and sea ice; loss of ice-dammed freshwater lakes; and 23% loss of total ice shelf area, including complete break-up and loss of the Markham Ice Shelf cryo-ecosystem. These observations underscore the vulnerability of Arctic microbial ecosystems to ongoing climate change.Peer reviewed: YesNRC publication: Ye

Arctic microbial ecosystems and impacts of extreme warming during the International Polar Year

As a contribution to the International Polar Year program MERGE (Microbiological and Ecological Responses to Global Environmental change in polar regions), studies were conducted on the terrestrial and aquatic microbial ecosystems of northern Canada (details at: http://www.cen.ulaval.ca/merge/). The habitats included permafrost soils, saline coldwater springs, supraglacial lakes on ice shelves, epishelf lakes in fjords, deep meromictic lakes, and shallow lakes, ponds and streams. Microbiological samples from each habitat were analysed by HPLC pigment assays, light and fluorescence microscopy, and DNA sequencing. The results show a remarkably diverse microflora of viruses, Archaea (including ammonium oxidisers and methanotrophs), Bacteria (including filamentous sulfur-oxidisers in a saline spring and benthic mats of Cyanobacteria in many waterbodies), and protists (including microbial eukaryotes in snowbanks and ciliates in ice-dammed lakes). In summer 2008, we recorded extreme warming at Ward Hunt Island and vicinity, the northern limit of the Canadian high Arctic, with air temperatures up to 20.5 \ub0C. This was accompanied by pronounced changes in microbial habitats: deepening of the permafrost active layer; loss of perennial lake ice and sea ice; loss of ice-dammed freshwater lakes; and 23% loss of total ice shelf area, including complete break-up and loss of the Markham Ice Shelf cryo-ecosystem. These observations underscore the vulnerability of Arctic microbial ecosystems to ongoing climate change.Peer reviewed: YesNRC publication: Ye

Geomicrobiological heterogeneity of lithic habitats in the extreme environment of Antarctic nunataks: a potential early Mars analog

Nunataks are permanent ice-free rocky peaks that project above ice caps in polar regions, thus being exposed to extreme climatic conditions throughout the year. They undergo extremely low temperatures and scarcity of liquid water in winter, while receiving high incident and reflected (albedo) UVA-B radiation in summer. Here, we investigate the geomicrobiology of the permanently exposed lithic substrates of nunataks from Livingston Island (South Shetlands, Antarctic Peninsula), with focus on prokaryotic community structure and their main metabolic traits. Contrarily to first hypothesis, an extensive sampling based on different gradients and multianalytical approaches demonstrated significant differences for most geomicrobiological parameters between the bedrock, soil, and loose rock substrates, which overlapped any other regional variation. Brevibacillus genus dominated on bedrock and soil substrates, while loose rocks contained a diverse microbial community, including Actinobacteria, Alphaproteobacteria and abundant Cyanobacteria inhabiting the milder and diverse microhabitats within. Archaea, a domain never described before in similar Antarctic environments, were also consistently found in the three substrates, but being more abundant and potentially more active in soils. Stable isotopic ratios of total carbon (δ 13C) and nitrogen (δ 15N), soluble anions concentrations, and the detection of proteins involved in key metabolisms via the Life Detector Chip (LDChip), suggest that microbial primary production has a pivotal role in nutrient cycling at these exposed areas with limited deposition of nutrients. Detection of stress-resistance proteins, such as molecular chaperons, suggests microbial molecular adaptation mechanisms to cope with these harsh conditions. Since early Mars may have encompassed analogous environmental conditions as the ones found in these Antarctic nunataks, our study also contributes to the understanding of the metabolic features and biomarker profiles of a potential Martian microbiota, as well as the use of LDChip in future life detection missions.This project has been funded by the Spanish Ministry of Science and Innovation (MICINN)/European Regional Development Fund (FEDER) project no. RTI2018-094368-B-I00; the European Research Council Consolidator grant no. 818602; and the Spanish State Research Agency (AEI) project no. MDM-2017-0737, Unidad de Excelencia “María de Maeztu” to Centro de Astrobiología

Biosignature detection by Mars rover equivalent instruments in samples from the CanMars Mars Sample Return Analogue Deployment

The University of Winnipeg's HOSERLab was established with funding from the Canada Foundation for Innovation, the Manitoba Research Innovations Fund and the Canadian Space Agency, whose support is gratefully acknowledged. This study was supported with grants from the Canadian Space Agency through their FAST program, NSERC, and UWinnipeg.This work details the laboratory analysis of a suite of 10 samples collected from an inverted fluvial channel near Hanksville, Utah, USA as a part of the CanMars Mars Sample Return Analogue Deployment (MSRAD). The samples were acquired along the rover traverse for detailed off-site analysis to evaluate the TOC and astrobiological significance of the samples selected based on site observations, and to address one of the science goals of the CanMars mission: to evaluate the ability of different analytical techniques being employed by the Mars2020 mission to detect and characterize any present biosignatures. Analytical techniques analogous to those on the ExoMars, MSL and the MER rovers were also applied to the samples. The total organic carbon content of the samples was <0.02% for all but 4 samples, and organic biosignatures were detected in multiple samples by UV–Vis–NIR reflectance spectroscopy and Raman spectroscopy (532 nm, time-resolved, and UV), which was the most effective of the techniques. The total carbon content of the samples is < 0.3 wt% for all but one calcite rich sample, and organic C was not detectable by FTIR. Carotene and chlorophyll were detected in two samples which also contained gypsum and mineral phases of astrobiological importance for paleoenvironment/habitability and biomarker preservation (clays, gypsum, calcite) were detected and characterized by multiple techniques, of which passive reflectance was most effective. The sample selected in the field (S2) as having the highest potential for TOC did not have the highest TOC values, however, when considering the sample mineralogy in conjunction with the detection of organic carbon, it is the most astrobiologically relevant. These results highlight importance of applying multiple techniques for sample characterization and provide insights into their strengths and limitations.PostprintPeer reviewe

Novel sulfur-oxidizing streamers thriving in a perennial cold saline springs of the Canadian high Arctic

The perennial springs at Gypsum Hill (GH) and Colour Peak (CP), situated at nearly 80\ub0N on Axel Heiberg Island in the Canadian high Arctic, are one of the few known examples of cold springs in thick permafrost on Earth. The springs emanate from deep saline aquifers and discharge cold anoxic brines rich in both sulfide and sulfate. Grey-coloured microbial streamers form during the winter months in snow-covered regions of the GH spring run-off channels (-1.3\ub0C to 6.9\ub0C, ~7.5% NaCl, 0\u201320 p.p.m. dissolved sulfide, 1 p.p.m. dissolved oxygen) but disappear during the Arctic summer. Culture- and molecular-based analyses of the 16S rRNA gene (FISH, DGGE and clone libraries) indicated that the streamers were uniquely dominated by chemolithoautotrophic sulfur-oxidizing Thiomicrospira species. The streamers oxidized both sulfide and thiosulfate and fixed CO2 under in situ conditions and a Thiomicrospira strain isolated from the streamers also actively oxidized sulfide and thiosulfate and fixed CO2 under cold, saline conditions. Overall, the snow-covered spring channels appear to represent a unique polar saline microhabitat that protects and allows Thiomicrospira streamers to form and flourish via chemolithoautrophic, phototrophicindependent metabolism in a high Arctic winter environment characterized by air temperatures commonly below -40\ub0C and with an annual average air temperature of -15\ub0C. These results broaden our knowledge of the physical and chemical boundaries that define life on Earth and have astrobiological implications for the possibility of life existing under similar Martian conditions.NRC publication: Ye