Antarctic lake phytoplankton and bacteria from near-surface waters exhibit high sensitivity to climate-driven disturbance.

The McMurdo Dry Valleys (MDVs), Antarctica, represent a cold, desert ecosystem poised on the threshold of melting and freezing water. The MDVs have experienced dramatic signs of climatic change, most notably a warm austral summer in 2001-2002 that caused widespread flooding, partial ice cover loss and lake level rise. To understand the impact of these climatic disturbances on lake microbial communities, we simulated lake level rise and ice-cover loss by transplanting dialysis-bagged communities from selected depths to other locations in the water column or to an open water perimeter moat. Bacteria and eukaryote communities residing in the surface waters (5 m) exhibited shifts in community composition when exposed to either disturbance, while microbial communities from below the surface were largely unaffected by the transplant. We also observed an accumulation of labile dissolved organic carbon in the transplanted surface communities. In addition, there were taxa-specific sensitivities: cryptophytes and Actinobacteria were highly sensitive particularly to the moat transplant, while chlorophytes and several bacterial taxa increased in relative abundance or were unaffected. Our results reveal that future climate-driven disturbances will likely undermine the stability and productivity of MDV lake phytoplankton and bacterial communities in the surface waters of this extreme environment.

Cyclic electron flow (CEF) and ascorbate pathway activity provide constitutive photoprotection for the photopsychrophile, Chlamydomonas sp. UWO 241 (renamed Chlamydomonas priscuii).

Under environmental stress, plants and algae employ a variety of strategies to protect the photosynthetic apparatus and maintain photostasis. To date, most studies on stress acclimation have focused on model organisms which possess limited to no tolerance to stressful extremes. We studied the ability of the Antarctic alga Chlamydomonas sp. UWO 241 (UWO 241) to acclimate to low temperature, high salinity or high light. UWO 241 maintained robust growth and photosynthetic activity at levels of temperature (2 °C) and salinity (700 mM NaCl) which were nonpermissive for a mesophilic sister species, Chlamydomonas raudensis SAG 49.72 (SAG 49.72). Acclimation in the mesophile involved classic mechanisms, including downregulation of light harvesting and shifts in excitation energy between photosystem I and II. In contrast, UWO 241 exhibited high rates of PSI-driven cyclic electron flow (CEF) and a larger capacity for nonphotochemical quenching (NPQ). Furthermore, UWO 241 exhibited constitutively high activity of two key ascorbate cycle enzymes, ascorbate peroxidase and glutathione reductase and maintained a large ascorbate pool. These results matched the ability of the psychrophile to maintain low ROS under short-term photoinhibition conditions. We conclude that tight control over photostasis and ROS levels are essential for photosynthetic life to flourish in a native habitat of permanent photooxidative stress. We propose to rename this organism Chlamydomonas priscuii.

Glycogen Metabolism Supports Photosynthesis Start through the Oxidative Pentose Phosphate Pathway in Cyanobacteria.

Cyanobacteria experience drastic changes in their carbon metabolism under daily light/dark cycles. During the day, the Calvin-Benson cycle fixes CO2 and diverts excess carbon into glycogen storage. At night, glycogen is degraded to support cellular respiration. The dark/light transition represents a universal environmental stress for cyanobacteria and other photosynthetic lifeforms. Recent studies revealed the essential genetic background necessary for the fitness of cyanobacteria during diurnal growth. However, the metabolic processes underlying the dark/light transition are not well understood. In this study, we observed that glycogen metabolism supports photosynthesis in the cyanobacterium Synechococcus elongatus PCC 7942 when photosynthesis reactions start upon light exposure. Compared with the wild type, the glycogen mutant ∆glgC showed a reduced photosynthetic efficiency and a slower P700+ rereduction rate when photosynthesis starts. Proteomic analyses indicated that glycogen is degraded through the oxidative pentose phosphate (OPP) pathway during the dark/light transition. We confirmed that the OPP pathway is essential for the initiation of photosynthesis and further showed that glycogen degradation through the OPP pathway contributes to the activation of key Calvin-Benson cycle enzymes by modulating NADPH levels. This strategy stimulates photosynthesis in cyanobacteria following dark respiration and stabilizes the Calvin-Benson cycle under fluctuating environmental conditions, thereby offering evolutionary advantages for photosynthetic organisms using the Calvin-Benson cycle for carbon fixation.

Ultrastructural and Single-Cell-Level Characterization Reveals Metabolic Versatility in a Microbial Eukaryote Community from an Ice-Covered Antarctic Lake.

UNLABELLED: The McMurdo Dry Valleys (MCM) of southern Victoria Land, Antarctica, harbor numerous ice-covered bodies of water that provide year-round liquid water oases for isolated food webs dominated by the microbial loop. Single-cell microbial eukaryotes (protists) occupy major trophic positions within this truncated food web, ranging from primary producers (e.g., chlorophytes, haptophytes, and cryptophytes) to tertiary predators (e.g., ciliates, dinoflagellates, and choanoflagellates). To advance the understanding of MCM protist ecology and the roles of MCM protists in nutrient and energy cycling, we investigated potential metabolic strategies and microbial interactions of key MCM protists isolated from a well-described lake (Lake Bonney). Fluorescence-activated cell sorting (FACS) of enrichment cultures, combined with single amplified genome/amplicon sequencing and fluorescence microscopy, revealed that MCM protists possess diverse potential metabolic capabilities and interactions. Two metabolically distinct bacterial clades (Flavobacteria and Methylobacteriaceae) were independently associated with two key MCM lake microalgae (Isochrysis and Chlamydomonas, respectively). We also report on the discovery of two heterotrophic nanoflagellates belonging to the Stramenopila supergroup, one of which lives as a parasite of Chlamydomonas, a dominate primary producer in the shallow, nutrient-poor layers of the lake. IMPORTANCE: Single-cell eukaryotes called protists play critical roles in the cycling of organic matter in aquatic environments. In the ice-covered lakes of Antarctica, protists play key roles in the aquatic food web, providing the majority of organic carbon to the rest of the food web (photosynthetic protists) and acting as the major consumers at the top of the food web (predatory protists). In this study, we utilized a combination of techniques (microscopy, cell sorting, and genomic analysis) to describe the trophic abilities of Antarctic lake protists and their potential interactions with other microbes. Our work reveals that Antarctic lake protists rely on metabolic versatility for their energy and nutrient requirements in this unique and isolated environment.

The Antarctic Chlamydomonas raudensis: an emerging model for cold adaptation of photosynthesis.

Permanently cold habitats dominate our planet and psychrophilic microorganisms thrive in cold environments. Environmental adaptations unique to psychrophilic microorganisms have been thoroughly described; however, the vast majority of studies to date have focused on cold-adapted bacteria. The combination of low temperatures in the presence of light is one of the most damaging environmental stresses for a photosynthetic organism: in order to survive, photopsychrophiles (i.e. photosynthetic organisms adapted to low temperatures) balance temperature-independent reactions of light energy capture/transduction with downstream temperature-dependent metabolic processes such as carbon fixation. Here, we review research on photopsychrophiles with a focus on an emerging model organism, Chlamydomonas raudensis UWO241 (UWO241). UWO241 is a psychrophilic green algal species and is a member of the photosynthetic microbial eukaryote community that provides the majority of fixed carbon for ice-covered lake ecosystems located in the McMurdo Dry Valleys, Antarctica. The water column exerts a range of environmental stressors on the phytoplankton community that inhabits this aquatic ecosystem, including low temperatures, extreme shade of an unusual spectral range (blue-green), high salinity, nutrient deprivation and extremes in seasonal photoperiod. More than two decades of work on UWO241 have produced one of our most comprehensive views of environmental adaptation in a cold-adapted, photosynthetic microbial eukaryote.

Aberrant light sensing and motility in the green alga Chlamydomonas priscuii from the ice-covered Antarctic Lake Bonney.

The Antarctic green alga Chlamydomonas priscuii is an obligate psychrophile and an emerging model for photosynthetic adaptation to extreme conditions. Endemic to the ice-covered Lake Bonney, this alga thrives at highly unusual light conditions characterized by very low light irradiance (<15 µmol m-2 s-1), a narrow wavelength spectrum enriched in blue light, and an extreme photoperiod. Genome sequencing of C. priscuii exposed an unusually large genome, with hundreds of highly similar gene duplicates and expanded gene families, some of which could be aiding its survival in extreme conditions. In contrast to the described expansion in the genetic repertoire in C. priscuii, here we suggest that the gene family encoding for photoreceptors is reduced when compared to related green algae. This alga also possesses a very small eyespot and exhibits an aberrant phototactic response, compared to the model Chlamydomonas reinhardtii. We also investigated the genome and behavior of the closely related psychrophilic alga Chlamydomonas sp. ICE-MDV, that is found throughout the photic zone of Lake Bonney and is naturally exposed to higher light levels. Our analyses revealed a photoreceptor gene family and a robust phototactic response similar to those in the model Chlamydomonas reinhardtii. These results suggest that the aberrant phototactic response in C. priscuii is a result of life under extreme shading rather than a common feature of all psychrophilic algae. We discuss the implications of these results on the evolution and survival of shade adapted polar algae.

Diversity and expression of RubisCO genes in a perennially ice-covered Antarctic lake during the polar night transition.

The autotrophic communities in the lakes of the McMurdo Dry Valleys, Antarctica, have generated interest since the early 1960s owing to low light transmission through the permanent ice covers, a strongly bimodal seasonal light cycle, constant cold water temperatures, and geographical isolation. Previous work has shown that autotrophic carbon fixation in these lakes provides an important source of organic matter to this polar desert. Lake Bonney has two lobes separated by a shallow sill and is one of several chemically stratified lakes in the dry valleys that support year-round biological activity. As part of an International Polar Year initiative, we monitored the diversity and abundance of major isoforms of RubisCO in Lake Bonney by using a combined sequencing and quantitative PCR approach during the transition from summer to polar winter. Form ID RubisCO genes related to a stramenopile, a haptophyte, and a cryptophyte were identified, while primers specific for form IA/B RubisCO detected a diverse autotrophic community of chlorophytes, cyanobacteria, and chemoautotrophic proteobacteria. Form ID RubisCO dominated phytoplankton communities in both lobes of the lake and closely matched depth profiles for photosynthesis and chlorophyll. Our results indicate a coupling between light availability, photosynthesis, and rbcL mRNA levels in deep phytoplankton populations. Regulatory control of rbcL in phytoplankton living in nutrient-deprived shallow depths does not appear to be solely light dependent. The distinct water chemistries of the east and west lobes have resulted in depth- and lobe-dependent variability in RubisCO diversity, which plays a role in transcriptional activity of the key gene responsible for carbon fixation.

Adaptation and acclimation of photosynthetic microorganisms to permanently cold environments.

Persistently cold environments constitute one of our world's largest ecosystems, and microorganisms dominate the biomass and metabolic activity in these extreme environments. The stress of low temperatures on life is exacerbated in organisms that rely on photoautrophic production of organic carbon and energy sources. Phototrophic organisms must coordinate temperature-independent reactions of light absorption and photochemistry with temperature-dependent processes of electron transport and utilization of energy sources through growth and metabolism. Despite this conundrum, phototrophic microorganisms thrive in all cold ecosystems described and (together with chemoautrophs) provide the base of autotrophic production in low-temperature food webs. Psychrophilic (organisms with a requirement for low growth temperatures) and psychrotolerant (organisms tolerant of low growth temperatures) photoautotrophs rely on low-temperature acclimative and adaptive strategies that have been described for other low-temperature-adapted heterotrophic organisms, such as cold-active proteins and maintenance of membrane fluidity. In addition, photoautrophic organisms possess other strategies to balance the absorption of light and the transduction of light energy to stored chemical energy products (NADPH and ATP) with downstream consumption of photosynthetically derived energy products at low temperatures. Lastly, differential adaptive and acclimative mechanisms exist in phototrophic microorganisms residing in low-temperature environments that are exposed to constant low-light environments versus high-light- and high-UV-exposed phototrophic assemblages.

Expression of Vibrio harveyi acyl-ACP synthetase allows efficient entry of exogenous fatty acids into the Escherichia coli fatty acid and lipid A synthetic pathways.

Although the Escherichia coli fatty acid synthesis (FAS) pathway is the best studied type II fatty acid synthesis system, a major experimental limitation has been the inability to feed intermediates into the pathway in vivo because exogenously supplied free fatty acids are not efficiently converted to the acyl-acyl carrier protein (ACP) thioesters required by the pathway. We report that expression of Vibrio harveyi acyl-ACP synthetase (AasS), a soluble cytosolic enzyme that ligates free fatty acids to ACP to form acyl-ACPs, allows exogenous fatty acids to enter the E. coli fatty acid synthesis pathway. The free fatty acids are incorporated intact and can be elongated or directly incorporated into complex lipids by acyltransferases specific for acyl-ACPs. Moreover, expression of AasS strains and supplementation with the appropriate fatty acid restored growth to E. coli mutant strains that lack essential fatty acid synthesis enzymes. Thus, this strategy provides a new tool for circumventing the loss of enzymes essential for FAS function.

Beyond the genome: functional studies of phototrophic sulfur oxidation.

The increasing availability of complete genomic sequences for cultured phototrophic bacteria and assembled metagenomes from environments dominated by phototrophs has reinforced the need for a "post-genomic" analytical effort to test models of cellular structure and function proposed from genomic data. Comparative genomics has produced a testable model for pathways of sulfur compound oxidation in the phototrophic bacteria. In the case of sulfide, two enzymes are predicted to oxidize sulfide: sulfide:quinone oxidoreductase and flavocytochrome c sulfide dehydrogenase. However, these models do not predict which enzyme is important under what conditions. In Chlorobaculum tepidum, a model green sulfur bacterium, a combination of genetics and physiological analysis of mutant strains has led to the realization that this organism contains at least two active sulfide:quinone oxidoreductases and that there is significant interaction between sulfide oxidation and light harvesting. In the case of elemental sulfur, an organothiol intermediate of unknown structure has been proposed to activate elemental sulfur for transport into the cytoplasm where it can be oxidized or assimilated, and recent approaches using classical metabolite analysis have begun to shed light on this issue both in C. tepidum and the purple sulfur bacterium Allochromatium vinosum.

Functional analysis of three sulfide:quinone oxidoreductase homologs in Chlorobaculum tepidum.

Sulfide:quinone oxidoreductase (SQR) catalyzes sulfide oxidation during sulfide-dependent chemo- and phototrophic growth in bacteria. The green sulfur bacterium Chlorobaculum tepidum (formerly Chlorobium tepidum) can grow on sulfide as the sole electron donor and sulfur source. C. tepidum contains genes encoding three SQR homologs: CT0117, CT0876, and CT1087. This study examined which, if any, of the SQR homologs possess sulfide-dependent ubiquinone reduction activity and are required for growth on sulfide. In contrast to CT0117 and CT0876, transcripts of CT1087 were detected only when cells actively oxidized sulfide. Mutation of CT0117 or CT1087 in C. tepidum decreased SQR activity in membrane fractions, and the CT1087 mutant could not grow with >or=6 mM sulfide. Mutation of both CT0117 and CT1087 in C. tepidum completely abolished SQR activity, and the double mutant failed to grow with >or=4 mM sulfide. A C-terminal His(6)-tagged CT1087 protein was membrane localized, as was SQR activity. Epitope-tagged CT1087 was detected only when sulfide was actively consumed by cells. Recombinantly produced CT1087 and CT0117 proteins had SQR activity, while CT0876 did not. In summary, we conclude that, under the conditions tested, both CT0117 and CT1087 function as SQR proteins in C. tepidum. CT0876 may support the growth of C. tepidum at low sulfide concentrations, but no evidence was found for SQR activity associated with this protein.

Influence of Environmental Drivers and Potential Interactions on the Distribution of Microbial Communities From Three Permanently Stratified Antarctic Lakes.

The McMurdo Dry Valley (MDV) lakes represent unique habitats in the microbial world. Perennial ice covers protect liquid water columns from either significant allochthonous inputs or seasonal mixing, resulting in centuries of stable biogeochemistry. Extreme environmental conditions including low seasonal photosynthetically active radiation (PAR), near freezing temperatures, and oligotrophy have precluded higher trophic levels from the food webs. Despite these limitations, diverse microbial life flourishes in the stratified water columns, including Archaea, bacteria, fungi, protists, and viruses. While a few recent studies have applied next generation sequencing, a thorough understanding of the MDV lake microbial diversity and community structure is currently lacking. Here we used Illumina MiSeq sequencing of the 16S and 18S rRNA genes combined with a microscopic survey of key eukaryotes to compare the community structure and potential interactions among the bacterial and eukaryal communities within the water columns of Lakes Bonney (east and west lobes, ELB, and WLB, respectively) and Fryxell (FRX). Communities were distinct between the upper, oxic layers and the dark, anoxic waters, particularly among the bacterial communities residing in WLB and FRX. Both eukaryal and bacterial community structure was influenced by different biogeochemical parameters in the oxic and anoxic zones. Bacteria formed complex interaction networks which were lake-specific. Several eukaryotes exhibit potential interactions with bacteria in ELB and WLB, while interactions between these groups in the more productive FRX were relatively rare.

Community response of microbial primary producers to salinity is primarily driven by nutrients in lakes.

Higher microbial diversity was frequently observed in saline than fresh waters, but the underlying mechanisms remains unknown, particularly in microbial primary producers (MPP). MPP abundance and activity are notably constrained by high salinity, but facilitated by high nutrients. It remains to be ascertained whether and how nutrients regulate the salinity constraints on MPP abundance and community structure. Here we investigated the impact of nutrients on salinity constraints on MPP abundance and diversity in undisturbed lakes with a wide salinity range on the Tibetan Plateau. MPP community was explored using quantitative PCR, terminal restriction fragment length polymorphism and sequencing of cloning libraries targeting form IC cbbL gene. The MPP community structure was sorted by salinity into freshwater (salinity<1‰), saline (1‰â€¯< salinity<29‰) and hypersaline (salinity>29‰) lakes. Furthermore, while MPP abundance, diversity and richness were significantly constrained with increasing salinity, these constraints were mitigated by enhancing total organic carbon (TOC) and total nitrogen (TN) contents in freshwater and saline lakes. In contrast, the MPP diversity increased significantly with the salinity in hypersaline lakes, due to the mitigation of enhancing TOC and TN contents and salt-tolerant MPP taxa. The mitigating effect of nutrients was more pronounced in saline than in freshwater and hypersaline lakes. The MPP compositions varied along salinity, with Betaproteobacteria dominating both the freshwater and saline lakes and Gammaproteobacteria dominating the hypersaline lakes. We concluded that high nutrients could mitigate the salinity constraining effects on MPP abundance, community richness and diversity. Our findings offer a novel insight into the salinity effects on primary producers and highlight the interactive effects of salinity and nutrients on MPP in lakes. These findings can be used as a baseline to illuminate the effects of increased anthropogenic activities altering nutrient dynamics on the global hydrological cycle and the subsequent responses thereof by MPP communities.

Autotrophic microbial community succession from glacier terminus to downstream waters on the Tibetan Plateau.

Glaciers harbour diverse microbes and autotrophic microbes play a key role in sustaining the glacial ecosystems by providing organic carbon. The succession of glacier-originated autotrophic microbes and their effects on downstream aquatic ecosystems remain unknown. We herein investigated the shift of autotrophic microbial communities in waters (not biofilms) along a glacier meltwater transect consisting of a glacier terminus outflow (subglacial), a glacial stream, two glacier-fed lakes (upper and lower) and their outflow on the Tibetan Plateau. The autotrophic community was characterized by cbbL gene using qPCR, T-RFLP and clone library/sequencing methods. The results demonstrated that form IC and ID autotrophic microbes exhibited a much higher abundance than form IAB in all waters along the transect. Form IAB autotrophic abundance in waters gradually decreased, while the form IC exhibited a substantial increase in the upper lake waters, and ID exhibited a substantial increase in the lower lake waters. The water form IC autotrophic community structure exhibited a distinguished shift from the glacier terminus outflow to the stream, while the form ID showed a dramatic shift from the stream to the lower lake. Our results revealed the succession patterns of glacier-originated autotrophic microbial communities and possible effects on downstream aquatic ecosystems.

The Lactococcus lactis FabF fatty acid synthetic enzyme can functionally replace both the FabB and FabF proteins of Escherichia coli and the FabH protein of Lactococcus lactis.

The genome of Lactococcus lactis encodes a single long chain 3-ketoacyl-acyl carrier protein synthase. This is in contrast to its close relative, Enterococcus faecalis, and to Escherichia coli, both of which have two such enzymes. In E. faecalis and E. coli, one of the two long chain synthases (FabO and FabB, respectively) has a role in unsaturated fatty acid synthesis that cannot be satisfied by FabF, the other long chain synthase. Since L. lactis has only a single long chain 3-ketoacyl-acyl carrier protein synthase (annotated as FabF), it seemed likely that this enzyme must function both in unsaturated fatty acid synthesis and in elongation of short chain acyl carrier protein substrates to the C18 fatty acids found in the cellular phospholipids. We report that this is the case. Expression of L. lactis FabF can functionally replace both FabB and FabF in E. coli, although it does not restore thermal regulation of phospholipid fatty acid composition to E. coli fabF mutant strains. The lack of thermal regulation was predictable because wild-type L. lactis was found not to show any significant change in fatty acid composition with growth temperature. We also report that overproduction of L. lactis FabF allows growth of an L. lactis mutant strain that lacks the FabH short chain 3-ketoacyl-acyl carrier protein synthase. The strain tested was a derivative (called the fabH bypass strain) of the original fabH deletion strain that had acquired the ability to grow when supplemented with octanoate. Upon introduction of a FabF overexpression plasmid into this strain, growth proceeded normally in the absence of fatty acid supplementation. Moreover, this strain had a normal rate of fatty acid synthesis and a normal fatty acid composition. Both the fabH bypass strain that overproduced FabF and the wild type strain incorporated much less exogenous octanoate into long chain phospholipid fatty acids than did the fabH bypass strain. Incorporation of octanoate and decanoate labeled with deuterium showed that these acids were incorporated intact as the distal methyl and methylene groups of the long chain fatty acids.

Diversity and succession of autotrophic microbial community in high-elevation soils along deglaciation chronosequence.

Global warming has resulted in substantial glacier retreats in high-elevation areas, exposing deglaciated soils to harsh environmental conditions. Autotrophic microbes are pioneering colonizers in the deglaciated soils and provide nutrients to the extreme ecosystem devoid of vegetation. However, autotrophic communities remain less studied in deglaciated soils. We explored the diversity and succession of the cbbL gene encoding the large subunit of form I RubisCO, a key CO2-fixing enzyme, using molecular methods in deglaciated soils along a 10-year deglaciation chronosequence on the Tibetan Plateau. Our results demonstrated that the abundance of all types of form I cbbL (IA/B, IC and ID) rapidly increased in young soils (0-2.5 years old) and kept stable in old soils. Soil total organic carbon (TOC) and total nitrogen (TN) gradually increased along the chronosequence and both demonstrated positive correlations with the abundance of bacteria and autotrophs, indicating that soil TOC and TN originated from autotrophs. Form IA/B autotrophs, affiliated with cyanobacteria, exhibited a substantially higher abundance than IC and ID. Cyanobacterial diversity and evenness increased in young soils (<6 years old) and then remained stable. Our findings suggest that cyabobacteria play an important role in accumulating TOC and TN in the deglaciated soils.

An integrated study of photochemical function and expression of a key photochemical gene (psbA) in photosynthetic communities of Lake Bonney (McMurdo Dry Valleys, Antarctica).

Lake Bonney is one of several permanently ice-covered lakes in the McMurdo Dry Valleys, Antarctica, which maintain the only year-round biological activity on the Antarctic continent. Vertically stratified populations of autotrophic microorganisms occupying the water columns are adapted to numerous extreme conditions, including very low light, hypersalinity, ultra-oligotrophy and low temperatures. In this study, we integrated molecular biology, microscopy, flow cytometry, and functional photochemical analyses of the photosynthetic communities residing in the east and west basins of dry valley Lake Bonney. Diversity and abundance of the psbA gene encoding a major protein of the photosystem II reaction center were monitored during the seasonal transition between Antarctic summer (24-h daylight) to winter (24-h darkness). Vertical trends through the photic zone in psbA abundance (DNA and mRNA) closely matched that of primary production in both lobes. Seasonal trends in psbA transcripts differed between the two lobes, with psbA expression in the west basin exhibiting a transient rise in early Fall. Last, using spectroscopic and flow cytometric analyses, we provide the first evidence that the Lake Bonney photosynthetic community is dominated by picophytoplankton that possess photosynthetic apparatus adapted to extreme shade.

Establishment of microbial eukaryotic enrichment cultures from a chemically stratified antarctic lake and assessment of carbon fixation potential.

Lake Bonney is one of numerous permanently ice-covered lakes located in the McMurdo Dry Valleys, Antarctica. The perennial ice cover maintains a chemically stratified water column and unlike other inland bodies of water, largely prevents external input of carbon and nutrients from streams. Biota are exposed to numerous environmental stresses, including year-round severe nutrient deficiency, low temperatures, extreme shade, hypersalinity, and 24-hour darkness during the winter (1). These extreme environmental conditions limit the biota in Lake Bonney almost exclusively to microorganisms (2). Single-celled microbial eukaryotes (called "protists") are important players in global biogeochemical cycling (3) and play important ecological roles in the cycling of carbon in the dry valley lakes, occupying both primary and tertiary roles in the aquatic food web. In the dry valley aquatic food web, protists that fix inorganic carbon (autotrophy) are the major producers of organic carbon for organotrophic organisms (4, 2). Phagotrophic or heterotrophic protists capable of ingesting bacteria and smaller protists act as the top predators in the food web (5). Last, an unknown proportion of the protist population is capable of combined mixotrophic metabolism (6, 7). Mixotrophy in protists involves the ability to combine photosynthetic capability with phagotrophic ingestion of prey microorganisms. This form of mixotrophy differs from mixotrophic metabolism in bacterial species, which generally involves uptake dissolved carbon molecules. There are currently very few protist isolates from permanently ice-capped polar lakes, and studies of protist diversity and ecology in this extreme environment have been limited (8, 4, 9, 10, 5). A better understanding of protist metabolic versatility in the simple dry valley lake food web will aid in the development of models for the role of protists in the global carbon cycle. We employed an enrichment culture approach to isolate potentially phototrophic and mixotrophic protists from Lake Bonney. Sampling depths in the water column were chosen based on the location of primary production maxima and protist phylogenetic diversity (4, 11), as well as variability in major abiotic factors affecting protist trophic modes: shallow sampling depths are limited for major nutrients, while deeper sampling depths are limited by light availability. In addition, lake water samples were supplemented with multiple types of growth media to promote the growth of a variety of phototrophic organisms. RubisCO catalyzes the rate limiting step in the Calvin Benson Bassham (CBB) cycle, the major pathway by which autotrophic organisms fix inorganic carbon and provide organic carbon for higher trophic levels in aquatic and terrestrial food webs (12). In this study, we applied a radioisotope assay modified for filtered samples (13) to monitor maximum carboxylase activity as a proxy for carbon fixation potential and metabolic versatility in the Lake Bonney enrichment cultures.

Evidence of form II RubisCO (cbbM) in a perennially ice-covered Antarctic lake.

The permanently ice-covered lakes of the McMurdo Dry Valleys, Antarctica, harbor microbially dominated food webs. These organisms are adapted to a variety of unusual environmental extremes, including low temperature, low light, and permanently stratified water columns with strong chemo- and oxy-clines. Owing to the low light levels during summer caused by thick ice cover as well as 6 months of darkness during the polar winter, chemolithoautotrophic microorganisms could play a key role in the production of new carbon for the lake ecosystems. We used clone library sequencing and real-time quantitative PCR of the gene encoding form II Ribulose 1, 5-bisphosphate carboxylase/oxygenase to determine spatial and seasonal changes in the chemolithoautotrophic community in Lake Bonney, a 40-m-deep lake covered by c. 4 m of permanent ice. Our results revealed that chemolithoautotrophs harboring the cbbM gene are restricted to layers just above the chemo- and oxi-cline (≤ 15 m) in the west lobe of Lake Bonney (WLB). Our data reveal that the WLB is inhabited by a unique chemolithoautotrophic community that resides in the suboxic layers of the lake where there are ample sources of alternative electron sources such as ammonium, reduced iron and reduced biogenic sulfur species.

Protist diversity in a permanently ice-covered Antarctic lake during the polar night transition.

The McMurdo Dry Valleys of Antarctica harbor numerous permanently ice-covered lakes, which provide a year-round oasis for microbial life. Microbial eukaryotes in these lakes occupy a variety of trophic levels within the simple aquatic food web ranging from primary producers to tertiary predators. Here, we report the first molecular study to describe the vertical distribution of the eukaryotic community residing in the photic zone of the east lobe (ELB) and west lobe (WLB) of the chemically stratified Lake Bonney. The 18S ribosomal RNA (rRNA) libraries revealed vertically stratified populations dominated by photosynthetic protists, with a cryptophyte dominating shallow populations (ELB-6 m; WLB-10 m), a haptophyte occupying mid-depths (both lobes 13 m) and chlorophytes residing in the deepest layers (ELB-18 and 20 m; WLB-15 and 20 m) of the photic zone. A previously undetected stramenopile occurred throughout the water column of both lobes. Temporal variation in the eukaryotic populations was examined during the transition from Antarctic summer (24-h sunlight) to polar night (complete dark). Protist diversity was similar between the two lobes of Lake Bonney due to exchange between the photic zones of the two basins via a narrow bedrock sill. However, vertical and temporal variation in protist distribution occurred, indicating the influence of the unique water chemistry on the biology of the two dry valley watersheds.