Nadia C. S. Mykytczuk

Bacterial growth at −15 °C; molecular insights from the permafrost bacterium Planococcus halocryophilus Or1

Planococcus halocryophilus strain Or1, isolated from high Arctic permafrost, grows and divides at −15 °C, the lowest temperature demonstrated to date, and is metabolically active at −25 °C in frozen permafrost microcosms. To understand how P. halocryophilus Or1 remains active under the subzero and osmotically dynamic conditions that characterize its native permafrost habitat, we investigated the genome, cell physiology and transcriptomes of growth at −15 °C and 18% NaCl compared with optimal (25 °C) temperatures. Subzero growth coincides with unusual cell envelope features of encrustations surrounding cells, while the cytoplasmic membrane is significantly remodeled favouring a higher ratio of saturated to branched fatty acids. Analyses of the 3.4 Mbp genome revealed that a suite of cold and osmotic-specific adaptive mechanisms are present as well as an amino acid distribution favouring increased flexibility of proteins. Genomic redundancy within 17% of the genome could enable P. halocryophilus Or1 to exploit isozyme exchange to maintain growth under stress, including multiple copies of osmolyte uptake genes (Opu and Pro genes). Isozyme exchange was observed between the transcriptome data sets, with selective upregulation of multi-copy genes involved in cell division, fatty acid synthesis, solute binding, oxidative stress response and transcriptional regulation. The combination of protein flexibility, resource efficiency, genomic plasticity and synergistic adaptation likely compensate against osmotic and cold stresses. These results suggest that non-spore forming P. halocryophilus Or1 is specifically suited for active growth in its Arctic permafrost habitat (ambient temp. ∼−16 °C), indicating that such cryoenvironments harbor a more active microbial ecosystem than previously thought.

Life at the Wedge: the Activity and Diversity of Arctic Ice Wedge Microbial Communities

The discovery of polygonal terrain on Mars underlain by ice heightens interest in the possibility that this water-bearing habitat may be, or may have been, a suitable habitat for extant life. The possibility is supported by the recurring detection of terrestrial microorganisms in subsurface ice environments, such as ice wedges found beneath tundra polygon features. A characterization of the microbial community of ice wedges from the high Arctic was performed to determine whether this ice environment can sustain actively respiring microorganisms and to assess the ecology of this extreme niche. We found that ice wedge samples contained a relatively abundant number of culturable cells compared to other ice habitats (∼10(5) CFU·mL(-1)). Respiration assays in which radio-labeled acetate and in situ measurement of CO(2) flux were used suggested low levels of microbial activity, though more sensitive techniques are required to confirm these findings. Based on 16S rRNA gene pyrosequencing, bacterial and archaeal ice wedge communities appeared to reflect surrounding soil communities. Two Pseudomonas sp. were the most abundant taxa in the ice wedge bacterial library (∼50%), while taxa related to ammonia-oxidizing Thaumarchaeota occupied 90% of the archaeal library. The tolerance of a variety of isolates to salinity and temperature revealed characteristics of a psychrotolerant, halotolerant community. Our findings support the hypothesis that ice wedges are capable of sustaining a diverse, plausibly active microbial community. As such, ice wedges, compared to other forms of less habitable ground ice, could serve as a reservoir for life on permanently cold, water-scarce, ice-rich extraterrestrial bodies and are therefore of interest to astrobiologists and ecologists alike. .

Characterization of the microbial acid mine drainage microbial community using culturing and direct sequencing techniques

We characterized the bacterial community from an AMD tailings pond using both classical culturing and modern direct sequencing techniques and compared the two methods. Acid mine drainage (AMD) is produced by the environmental and microbial oxidation of minerals dissolved from mining waste. Surprisingly, we know little about the microbial communities associated with AMD, despite the fundamental ecological roles of these organisms and large-scale economic impact of these waste sites. AMD microbial communities have classically been characterized by laboratory culturing-based techniques and more recently by direct sequencing of marker gene sequences, primarily the 16S rRNA gene. In our comparison of the techniques, we find that their results are complementary, overall indicating very similar community structure with similar dominant species, but with each method identifying some species that were missed by the other. We were able to culture the majority of species that our direct sequencing results indicated were present, primarily species within the Acidithiobacillus and Acidiphilium genera, although estimates of relative species abundance were only obtained from direct sequencing. Interestingly, our culture-based methods recovered four species that had been overlooked from our sequencing results because of the rarity of the marker gene sequences, likely members of the rare biosphere. Further, direct sequencing indicated that a single genus, completely missed in our culture-based study, Legionella, was a dominant member of the microbial community. Our results suggest that while either method does a reasonable job of identifying the dominant members of the AMD microbial community, together the methods combine to give a more complete picture of the true diversity of this environment.

Planococcus halocryophilus sp. nov., an extreme sub-zero species from high Arctic permafrost

A novel aerobic, Gram-positive, motile, coccoid bacterial strain, designated Or1(T), was isolated from permafrost active-layer soil collected from the Canadian high Arctic. Strain Or1(T) was capable of growth over a broad temperature range, including sub-zero growth (below -10 to 37 °C), and at high salinity (0-19% NaCl), growing optimally at 25 °C, at pH 7.0-8.0 and in the presence of 2% NaCl. Its taxonomic and phylogenetic position was determined by using a polyphasic approach, which indicated that strain Or1(T) was a member of the genus Planococcus. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain Or1(T) belonged to the genus Planococcus, differing by 0.4-3.6% from the type strains of all recognized Planococcus species, and was related most closely to Planococcus antarcticus CMS 26or(T) (98.8% similarity) and Planococcus donghaensis JH1(T) (99.6%). However, DNA-DNA hybridization experiments showed that strain Or1(T) had low genomic relatedness to Planococcus antarcticus CMS 26or(T) (18%) and Planococcus donghaensis JH1(T) (46%). The major menaquinones of strain Or1(T) were MK-7 (55%), MK-8 (36%) and MK-6 (9%) and the major fatty acids were anteiso-C(15:0), C(16:1)ω7c alcohol and anteiso-C(17:0). The DNA G+C content of strain Or1(T) was 40.5 mol%. Differential phenotypic, phylogenetic and genomic data suggest that strain Or1(T) represents a novel species of the genus Planococcus, for which the name Planococcus halocryophilus sp. nov. is proposed. The type strain is Or1(T) ( = DSM 24743(T) = JCM 17719(T)).

An active atmospheric methane sink in high Arctic mineral cryosols

Methane (CH4) emission by carbon-rich cryosols at the high latitudes in Northern Hemisphere has been studied extensively. In contrast, data on the CH4 emission potential of carbon-poor cryosols is limited, despite their spatial predominance. This work employs CH4 flux measurements in the field and under laboratory conditions to show that the mineral cryosols at Axel Heiberg Island in the Canadian high Arctic consistently consume atmospheric CH4. Omics analyses present the first molecular evidence of active atmospheric CH4-oxidizing bacteria (atmMOB) in permafrost-affected cryosols, with the prevalent atmMOB genotype in our acidic mineral cryosols being closely related to Upland Soil Cluster α. The atmospheric (atm) CH4 uptake at the study site increases with ground temperature between 0 °C and 18 °C. Consequently, the atm CH4 sink strength is predicted to increase by a factor of 5–30 as the Arctic warms by 5–15 °C over a century. We demonstrate that acidic mineral cryosols are a previously unrecognized potential of CH4 sink that requires further investigation to determine its potential impact on larger scales. This study also calls attention to the poleward distribution of atmMOB, as well as to the potential influence of microbial atm CH4 oxidation, in the context of regional CH4 flux models and global warming.

Proteomic insights into cold adaptation of psychrotrophic and mesophilic Acidithiobacillus ferrooxidans strains

Cold tolerant strains of Acidithiobacillus ferrooxidans play a role in metal leaching and acid mine drainage (AMD) production in northern latitude/boreal mining environments. In this study we used a proteomics and bioinformatics approach to decipher the proteome changes related to sustained growth at low temperatures to increase our understanding of cold adaptation mechanisms in A. ferrooxidans strains. Changes in protein abundance in response to low temperatures (5 and 15°C) were monitored and protein analyses of a psychrotrophic strain (D6) versus a mesophilic strain (F1) showed that both strains increased levels of 11 stress-related and metabolic proteins including survival protein SurA, trigger factor Tig, and AhpC-Tsa antioxidant proteins. However, a unique set of changes in the proteome of psychrotrophic strain D6 were observed. In particular, the importance of protein fate, membrane transport and structure for psychrotrophic growth were evident with increases in numerous chaperone and transport proteins including GroEL, SecB, ABC transporters and a capsule polysaccharide export protein. We also observed that low temperature iron oxidation coincides with a relative increase in the key iron metabolism protein rusticyanin, which was more highly expressed in strain D6 than in strain F1 at colder growth temperatures. We demonstrate that the psychrotrophic strain uses a global stress response and cold-active metabolism which permit growth of A. ferrooxidans in the extreme AMD environment in colder climates.

Stress and the microbiome: linking glucocorticoids to bacterial community dynamics in wild red squirrels

Bacterial diversity within animals is emerging as an essential component of health, but it is unknown how stress may influence the microbiome. We quantify a proximate link between the oral microbiome and hypothalamic–pituitary–adrenal (HPA) axis activity using faecal glucocorticoid metabolites (FGM) in wild red squirrels (Tamiasciurus hudsonicus). Not only was bacterial diversity lower at higher levels of FGM, but also between capture periods a change in bacterial relative abundance was related to an increase in FGM. These linkages between the HPA axis and microbiome communities represent a powerful capacity for stress to have multi-dimensional effects on health.

Cytoplasmic membrane fluidity and fatty acid composition of Acidithiobacillus ferrooxidans in response to pH stress

Strain variation in the acidophile Acidithiobacillus ferrooxidans was examined as a product of membrane adaptation in response to pH stress. We tested the effects of sub and supra-optimal pH in two type strains and four strains isolated from acid mine drainage water around Sudbury, Ontario, Canada. Growth rate, membrane fluidity and phase, determined from the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene, and fatty acid profiles were compared. The effect of pH 1.5 was the most pronounced compared to the other pH values of 1.8, 3.1, and 3.5. Three different types of response to lower pH were observed, the first of which appeared to maintain cellular homeostasis more effectively. This adaptive mode included a decrease in membrane fluidity and concomitant depression of the phase transition in two distinct membrane lipid components. This was explained through the increase in saturated fatty acids (predominantly 16:0 and cyclopropane 19:0 w8c) with a concomitant decrease in 18:1 w7c fatty acid. The other strains also showed common adaptive mechanisms of specific fatty acid remodeling increasing the abundance of short-chain fatty acids. However, we suspect membrane permeability was compromised due to potential phase separation, which may interfere with energy transduction and viability at pH 1.5. We demonstrate that membrane physiology permits differentiating pH tolerance in strains of this extreme acidophile.

Defining the Functional Potential and Active Community Members of a Sediment Microbial Community in a High-Arctic Hypersaline Subzero Spring

ABSTRACT The Lost Hammer (LH) Spring is the coldest and saltiest terrestrial spring discovered to date and is characterized by perennial discharges at subzero temperatures (−5�C), hypersalinity (salinity, 24%), and reducing (≈−165 mV), microoxic, and oligotrophic conditions. It is rich in sulfates (10.0%, wt/wt), dissolved H2S/sulfides (up to 25 ppm), ammonia (≈381 μM), and methane (11.1 g day−1). To determine its total functional and genetic potential and to identify its active microbial components, we performed metagenomic analyses of the LH Spring outlet microbial community and pyrosequencing analyses of the cDNA of its 16S rRNA genes. Reads related to Cyanobacteria (19.7%), Bacteroidetes (13.3%), and Proteobacteria (6.6%) represented the dominant phyla identified among the classified sequences. Reconstruction of the enzyme pathways responsible for bacterial nitrification/denitrification/ammonification and sulfate reduction appeared nearly complete in the metagenomic data set. In the cDNA profile of the LH Spring active community, ammonia oxidizers (Thaumarchaeota), denitrifiers (Pseudomonas spp.), sulfate reducers (Desulfobulbus spp.), and other sulfur oxidizers (Thermoprotei) were present, highlighting their involvement in nitrogen and sulfur cycling. Stress response genes for adapting to cold, osmotic stress, and oxidative stress were also abundant in the metagenome. Comparison of the composition of the functional community of the LH Spring to metagenomes from other saline/subzero environments revealed a close association between the LH Spring and another Canadian high-Arctic permafrost environment, particularly in genes related to sulfur metabolism and dormancy. Overall, this study provides insights into the metabolic potential and the active microbial populations that exist in this hypersaline cryoenvironment and contributes to our understanding of microbial ecology in extreme environments.

Methanogen community composition and rates of methane consumption in Canadian High Arctic permafrost soils

Increasing permafrost thaw, driven by climate change, has the potential to result in organic carbon stores being mineralized into carbon dioxide (CO2) and methane (CH4) through microbial activity. This study examines the effect of increasing temperature on community structure and metabolic activity of methanogens from the Canadian High Arctic, in an attempt to predict how warming will affect microbially controlled CH4 soil flux. In situ CO2 and CH4 flux, measured in 2010 and 2011 from ice-wedge polygons, indicate that these soil formations are a net source of CO2 emissions, but a CH4 sink. Permafrost and active layer soil samples were collected at the same sites and incubated under anaerobic conditions at warmer temperatures, with and without substrate amendment. Gas flux was measured regularly and indicated an increase in CH4 flux after extended incubation. Pyrosequencing was used to examine the effects of an extended thaw cycle on methanogen diversity and the results indicate that in situ methanogen diversity, based on the relative abundance of the 16S ribosomal ribonucleic acid (rRNA) gene associated with known methanogens, is higher in the permafrost than in the active layer. Methanogen diversity was also shown to increase in both the active layer and permafrost soil after an extended thaw. This study provides evidence that although High Arctic ice-wedge polygons are currently a sink for CH4, higher arctic temperatures and anaerobic conditions, a possible result of climate change, could result in this soil becoming a source for CH4 gas flux.