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

The perennial springs at Gypsum Hill (GH) and Colour Peak (CP), situated at nearly 80°N 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°C to 6.9°C, ∼7.5% NaCl, 0–20 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, phototrophic-independent metabolism in a high Arctic winter environment characterized by air temperatures commonly below −40°C and with an annual average air temperature of −15°C. 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.     

Stable Isotope Probing Analysis of the Diversity and Activity of Methanotrophic Bacteria in Soils from the Canadian High Arctic

The melting of permafrost and its potential impact on CH₄ emissions are major concerns in the context of global warming. Methanotrophic bacteria have the capacity to mitigate CH₄ emissions from melting permafrost. Here, we used quantitative PCR (qPCR), stable isotope probing (SIP) of DNA, denaturing gradient gel electrophoresis (DGGE) fingerprinting, and sequencing of the 16S rRNA and pmoA genes to study the activity and diversity of methanotrophic bacteria in active-layer soils from Ellesmere Island in the Canadian high Arctic. Results showed that most of the soils had the capacity to oxidize CH₄ at 4°C and at room temperature (RT), but the oxidation rates were greater at RT than at 4°C and were significantly enhanced by nutrient amendment. The DGGE banding patterns associated with active methanotrophic bacterial populations were also different depending on the temperature of incubation and the addition of nutrients. Sequencing of the 16S rRNA and pmoA genes indicated a low diversity of the active methanotrophic bacteria, with all methanotroph 16S rRNA and pmoA gene sequences being related to type I methanotrophs from Methylobacter and Methylosarcina. The dominance of type I methanotrophs over type II methanotrophs in the native soil samples was confirmed by qPCR of the 16S rRNA gene with primers specific for these two groups of bacteria. The 16S rRNA and pmoA gene sequences related to those of Methylobacter tundripaludum were found in all soils, regardless of the incubation conditions, and they might therefore play a role in CH₄ degradation in situ. This work is providing new information supporting the potential importance of Methylobacter spp. in Arctic soils found in previous studies and contributes to the limited body of knowledge on methanotrophic activity and diversity in this extreme environment.Permafrost regions occupy approximately 22% of the exposed land area of the Northern Hemisphere (63). In the past 100 years, the average temperatures in the arctic regions have increased at almost twice the average global rate (25). The melting of permafrost is one of the most important impacts of global warming on these high-latitude environments, and theoretical modeling suggests that as much as 90% of the permafrost could thaw by the end of the 21st century (29). While it has been generally reported that 15% of the total soil organic carbon is stocked in permafrost (42), a recent estimate indicates that it contains as much as 50% of the global belowground organic carbon pool (53). Carbon stocked in permafrost is now regarded as one of the most important carbon-climate feedbacks because of the size of the carbon pool and the intensity of climate change at high latitudes (46, 47). The presence of these large amounts of carbon in permafrost is raising serious concerns whether melting permafrost, and the resulting increase in microbial activity, might be a source of extensive emissions of the greenhouse gases carbon dioxide and methane (CH₄) to the atmosphere. The actual emissions of CH₄ from soils of high latitudes have been estimated to represent about 25% of the emissions from natural sources (19). Methane, which is 25 times more potent than carbon dioxide as a greenhouse gas (25), is produced by methanogenic archaea under anaerobic conditions. These microorganisms are known to inhabit permafrost environments (44, 49), and their capacity to produce methane at cold temperatures has been reported (20, 35, 44, 56). Their methanogenic activity is expected to increase as permafrost soil temperature increases (20). Moreover, large amounts of methane are stocked as methane hydrates in permafrost at an average depth of several hundred meters (33). Methane is also found in permafrost layers near the surface and could potentially be liberated to the atmosphere as permafrost melts (44).Methane can be oxidized in aerobic zones by aerobic methanotrophic bacteria or in anaerobic zones by anaerobic methanotrophic archaea (for a recent review, see reference 27). Anaerobic methane oxidizers were not covered in the context of this study, which focused exclusively on aerobic methanotrophs. These bacteria utilize methane as the sole carbon and energy source through the activity of the enzyme methane monooxygenase (MMO). Most known aerobic methanotrophs are divided into two major groups (type I and type II) based on phylogeny and carbon assimilation pathways (5). Type I methanotrophs, also known as Gammaproteobacteria methanotrophs (6) belong to the family Methylococcaceae within the Gammaproteobacteria subdivision, while type II methanotrophs (Alphaproteobacteria methanotrophs) belong to the family Methylocystaceae in the Alphaproteobacteria subdivision (5). Because of their capacity to oxidize methane, aerobic methanotrophs can significantly reduce methane emissions to the atmosphere and play an important role in the global methane cycle (12, 22). Methanotrophic activity has been observed in cold environments, and methanotrophs might contribute to the reduction of methane emissions from melting permafrost. Aerobic methanotrophic bacteria from cold environments have been reviewed in detail elsewhere (54).Most studies addressing methanotrophs from cold environments were conducted on soils from very few sites located in Northern Europe and Siberia (14, 30, 31, 40, 56-58), while methanotrophic bacterial populations in soils from the Canadian high Arctic remain mostly unexplored (41). In addition, most of these studies were conducted at low latitudes, and the pool of knowledge concerning the activity and diversity of methanotrophic bacterial populations in high Arctic soils is limited. The question being addressed in this study is whether there are active methanotrophs in the active-layer soil in the high Arctic. Therefore, the present work had two objectives: (i) to evaluate the methane oxidation capacity of three active-layer soils from the Canadian high Arctic under various incubation conditions and (ii) to identify and characterize the diversity of the active methanotrophs in these soils using stable isotope probing (SIP) of DNA (DNA-SIP) and sequencing of the 16S rRNA and pmoA genes. With this work, we identify for the first time active methanotrophs in high Arctic soils through the use of DNA-SIP.     

Cloning and expression of single chain antibody fragments in Escherichia coli and Pichia pastoris

The potential of recombinant antibody fragments is likely to be fulfilled only if they can be produced routinely at high concentrations. We have compared the ability of Escherichia coli and Pichia pastoris to produce functional recombinant single chain antibody (scAb) fragments. Two scAb fragments were expressed, an antihuman type V acid phosphatase (TRAP) and an anti-Pseudomonas aeruginosa lipoprotein I. We report here that, while expression from P. pastoris resulted in a significantly increased level of expression of the anti-TRAP scAb compared to E. coli, neither fragment was able to bind its target antigen as well as the bacterial product.     

Effects of ovariectomy on aggregation,secretion, and metalloproteinases in porcine platelets

Differences in the aggregation and release of growth factors including matrix metalloproteinases (MMPs) after loss of ovarian hormones could contribute to an exaggerated response to injury in arteries of ovariectomized animals. Therefore, experiments were designed to compare aggregation, dense granular ATP release, expression of MMPs (MMP-2, MMP-9, and MMP-14) and tissue inhibitors of metalloproteinase (TIMP-1 and TIMP-2) in circulating platelets from sexually mature (7 mo old) gonadally intact and ovariectomized (4 wk) female pigs. Numbers of circulating platelets did not change after ovariectomy, but the percentage of reticulated platelets increased significantly. Platelet aggregation and dense granular ATP secretion also increased significantly with ovariectomy. In platelet lysates, active MMP-2 increased, whereas MMP-14 significantly decreased, after ovariectomy; the expression of TIMP-1, TIMP-2, and P-selectin did not change. These results suggest that platelet turnover, aggregation, and ATP secretion increase with ovariectomy. Also, ovarian hormones selectively regulate the expression and activity of MMPs in porcine platelets. Increased platelet aggregation and activity of MMP-2 would alter platelet-platelet and platelet-vessel wall interactions, contributing to an exaggerated response to injury with loss of ovarian hormones.     

The ARF-like GTPases Arl1p and Arl3p act in a pathway that interacts with vesicle-tethering factors at the Golgi apparatus

The ARLs are a diverse family of GTPases that are related to ADP-ribosylation factors (ARFs), but whose function is poorly understood. There are at least ten ARLs in humans, two of which have homologs in the yeast Saccharomyces cerevisiae (ARL1/Arl1p and ARFRP1/Arl3p). The function of ARFRP1 is unknown, but mammalian ARL1 has recently been found to interact with a number of effectors including the GRIP domain that is present in a family of Golgi-localized long coiled-coil proteins. We find that in yeast, the intracellular targeting of Imh1p, the only yeast GRIP domain protein, is dependent on both Arl1p and Arl3p, but not on the ARF proteins. A recombinant form of the Imh1p GRIP domain binds to Arl1p in a GTP-dependent manner, but not to Arl3p. Yeast also contain a relative of SCOCO, a protein proposed to bind human ARL1, but this yeast protein, Slo1p, appears to bind Arl3p rather than Arl1p in vitro. However, Imh1p is not the sole effector of Arl1p since affinity chromatography of cytosol with immobilized Arl1p:GTP revealed an interaction with the GARP/VFT complex that is thought to act in the tethering of vesicles to the Golgi apparatus. Finally, we find that Arl3p is required in vivo for the targeting of Arl1p, explaining its requirement for the normal distribution of Imh1p.     

Indigenous Sediment Microbial Activity in Response to Nutrient Enrichment and Plant Growth Following a Controlled Oil Spill on a Freshwater Wetland

The population density and activity of a microbial community associated with the sediment and rhizosphere of an intertidal freshwater wetland dominated by Scirpus pungens was monitored before and following the application of weathered Mesa light crude oil and fertilizers. The influence of nutrient enrichment (fertilizers) and plant growth on oil degradation rates was determined from the resulting data. The study plots (four blocks of replicates) were subjected to five treatments: oil only (natural attenuation); oil plus ammonium nitrate and phosphate, with regular cropping of the plants; oil plus ammonium nitrate and phosphate; oil plus sodium nitrate and phosphate; no oil, ammonium nitrate and phosphate. The plots were regularly monitored in the field for gas production (carbon dioxide and nitrous oxide), and samples were collected for laboratory analysis of denitrification activity, aliphatic and aromatic hydrocarbon degradation activity, and total heteroptrophic bacteria. The viable bacterial population density increased during the first 4 weeks in oiled and unoiled experimental plots that were fertilized. In contrast, population densities in untreated areas remained relatively unchanged throughout the monitoring period. The microbial population demonstrated a rapid and sustained increase in naphthalene mineralization activity in plots that were both fertilized and oiled. Hexadecane mineralization activity increased in response to fertilizer application, with ammonium nitrate causing a larger increase than sodium nitrate. A very significant difference observed in the mineralization of hexadecane was that the surface sediments were much more active than the subsurface sediments. This difference became even more pronounced in the second year of monitoring, even though the treatment regime had been discontinued. This compartmentalization of mineralization activity was not observed for naphthalene. Following fertilizer application, field and laboratory evaluation of nitrogen metabolism in the sediments indicated significant denitrification activity that was not adversely affected by oiling. The results demonstrated that the application of fertilizers stimulated the activities of indigenous hydrocarbon-degrading and denitrifying bacteria, and the presence of oil either enhanced or had no detrimental effect on these activities. As a remediation strategy, the application of fertilizers to a wetland shoreline following an oil spill would promote the growth of indigenous plants and their associated microbial flora, resulting in increased metabolic activity and the potential for increased oil degradation activity.