Irina Alekhina

DNA signature of thermophilic bacteria from the aged accretion ice of Lake Vostok, Antarctica: implications for searching for life in extreme icy environments

We have used 16S ribosomal genes to estimate the bacterial contents of Lake Vostok accretion ice samples at 3551 m and 3607 m, both containing sediment inclusions and formed 20000–15000 yr ago. Decontamination proved to be a critical issue, and we used stringent ice chemistry-based procedures and comprehensive biological controls in order to restrain contamination. As a result, up to now we have only recognized one 16S rDNA bacterial phylotype with confident relevance to the lake environment. It was found in one sample at 3607 m depth and represents the extant thermophilic facultative chemolithoautotroph Hydrogenophilus thermoluteolus of beta- Proteobacteria , and until now had only been found in hot springs. No confident findings were detected in the sample at 3551 m, and all other phylotypes revealed (a total of 16 phylotypes, 336 clones including controls) are presumed to be contaminants. It seems that the Lake Vostok accretion ice is actually microbe-free, indicating that the water body should also be hosting a highly sparse life. The message of thermophilic bacteria suggests that a geothermal system exists beneath the cold water body of Lake Vostok, what is supported by the geological setting, the long-term seismotectonic evidence from 4 He degassing and the ‘ 18 O shift’ of the Vostok accretion ice. The seismotectonic activity that seems to operate in deep faults beneath the lake could sustain thermophilic chemolithoautotrophic microbial communities. Such a primary production scenario for Lake Vostok may have relevance for icy planets and the approaches used for estimating microbial contents in accretion ice are clearly relevant for searching for extraterrestrial life.

Identification of a Universally Primed-PCR-Derived Sequence-Characterized Amplified Region Marker for an Antagonistic Strain of Clonostachys rosea and Development of a Strain-Specific PCR Detection Assay

ABSTRACT We developed a PCR detection method that selectively recognizes a single biological control agent and demonstrated that universally primed PCR (UP-PCR) can identify strain-specific markers. Antagonistic strains of Clonostachys rosea (syn. Gliocladium roseum) were screened by UP-PCR, and a strain-specific marker was identified for strain GR5. No significant sequence homology was found between this marker and any other sequences in the databases. Southern blot analysis of the PCR product revealed that the marker represented a single-copy sequence specific for strain GR5. The marker was converted into a sequence-characterized amplified region (SCAR), and a specific PCR primer pair was designed. Eighty-two strains, isolated primarily from Danish soils, and 31 soil samples, originating from different localities, were tested, and this specificity was confirmed. Two strains responded to the SCAR primers under suboptimal PCR conditions, and the amplified sequences from these strains were similar, but not identical, to the GR5 marker. Soil assays in which total DNA was extracted from GR5-infested and noninoculated field soils showed that the SCAR primers could detect GR5 in a pool of mixed DNA and that no other soil microorganisms present contained sequences amplified by the primers. The assay developed will be useful for monitoring biological control agents released into natural field soil.

Molecular, morphological and phylogenetic analysis of the Fusarium avenaceum/F. arthrosporioides/F. tricinctum species complex – a polyphasic approach

Differences in morphology, ITS, IGS, mtSSU and β-tubulin sequences and UP-PCR hybridization were compared between morphologically identified F. avenaceum, F. arthrosporioides, F. anguioides, F. tricinctum, F. graminum and F. acuminatum strains. According to the combined β-tubulin, IGS and ITS tree, the strains of the Fusarium avenaceum/F. arthrosporioides/F. tricinctum species complex species can be divided into seven clusters supported by bootstrap values higher than 50%. The two main groups of European F. avenaceum , which cannot be distinguished by morphology, were separated in the tree based on β-tubulin sequences and less clearly in trees based on IGS and ITS sequences. MtSSU sequences were identical in all F. avenaceum and F. tricinctum strains studied. The European F. avenaceum strains of main group II had identical β-tubulin sequences with one American F. avenaceum strain and four European F. arthrosporioides strains, while F. avenaceum strains of main group I were closely related to two European F. arthrosporioides strains and to one Japanese F. anguioides strain. According to the combined β-tubulin/IGS/ITS sequence tree, European F. arthrosporioides strains were divided into four groups; F. tricinctum strains formed a wellsupported cluster, in which two European clusters were separated from one African isolate. In the IGS sequence tree two European F. acuminatum strains together with one American F. acuminatum strain formed a cluster, which was separate from another American F. acuminatum strain. The F. acuminatum cluster was nested within the large F. tricinctum cluster together with one F. reticulatum strain in the combined IGS/β-tubulin tree. Several strains may be intermediates between the F. avenaceum/F. arthrosporioides/F. anguioides and F. tricinctum clusters and represent their own species. These results are partially supported by the results of UP-PCR hybridization analysis. Thus the molecular results may be helpful in future revision in the taxonomy of these species.

Delineation of Trichoderma harzianum into two different genotypic groups by a highly robust fingerprinting method, UP-PCR, and UP-PCR product cross-hybridization

Strains of Trichoderma harzianum possess biocontrol capabilities. As background for identification of strain-specific markers for monitoring strains of interest, the relationship of strains designated as T. harzianum , including representatives of three biological forms Th1, Th2 and Th3, were analysed by Universally Primed PCR, UP-PCR, using UP and random primers. Cross dot blot hybridization of UP-PCR products generated with either of two different UP primers or a random primer showed unequivocal differences among strains. Using this approach, T. harzianum strains were distributed into two different genotypic groups. One of the T. harzianum groups included forms Th1 and Th2 while the other group accounted for the Th3 form. The relatedness of strains of each group was estimated by UPGMA analysis based on markers revealed with three primers. It was found that both genotypic groups are heterogeneous, and Th2 form strains definitely cluster together with those of Th1. Division of the three biological forms of T. harzianum into two groups was also supported by rDNA-ITS1 analysis, where Sau 3A digestion of the amplified ITS1 region gave a restriction fragment profile specific for each genotypic group. Two strains with known biocontrol capabilities were found to relate to the genotypic group containing Th1 and Th2 forms and, based on variation within this group, to belong to a homogeneous group of form Th1 strains. The robustness and reliability of UP-PCR fingerprinting were demonstrated by obtaining identical banding profiles using different conditions for PCR in different laboratories.

Quantification of Dissolved Organic Carbon at Very Low Levels in Natural Ice Samples by a UV-Induced Oxidation Method

The study of chemical impurities trapped in solid precipitation and accumulated in polar ice sheets and high-elevation, midlatitude cold glaciers over the last several hundreds of years provides a unique way to reconstruct our changing atmosphere from the preindustrial era to the present day. Numerous ice core studies of inorganic species have already evaluated the effects of growing anthropogenic emissions of SO(2) or NO(x) on the chemical composition of the atmosphere in various regions of the world. While it was recently shown that organic species dominate the atmospheric aerosol mass, the contribution of anthropogenic emissions to their budget remains poorly understood. The study of organics in ice is at the infancy stage, and it still is difficult to draw a consistent picture of the organic content of polar ice from sparse available data. A UV oxidation method and IR quantification of CO(2) was optimized to obtain measurements of dissolved organic carbon content as low as a few ppbC. Stringent working conditions were defined to prevent contamination during the cleaning of ice. Measurements in various ice cores corresponding to preindustrial times revealed dissolved organic carbon content of less than 10 ppbC in Antarctica and up to 75 ppbC in alpine ice.

Aerobiology over Antarctica – a new initiative for atmospheric ecology

The role of aerial dispersal in shaping patterns of biodiversity remains poorly understood, mainly due to a lack of coordinated efforts in gathering data at appropriate temporal and spatial scales. It has been long known that the rate of dispersal to an ecosystem can significantly influence ecosystem dynamics, and that aerial transport has been identified as an important source of biological input to remote locations. With the considerable effort devoted in recent decades to understanding atmospheric circulation in the south-polar region, a unique opportunity has emerged to investigate the atmospheric ecology of Antarctica, from regional to continental scales. This concept note identifies key questions in Antarctic microbial biogeography and the need for standardized sampling and analysis protocols to address such questions. A consortium of polar aerobiologists is established to bring together researchers with a common interest in the airborne dispersion of microbes and other propagules in the Antarctic, with opportunities for comparative studies in the Arctic.

Three events of Saharan dust deposition on the Mont Blanc glacier associated with different snow-colonizing bacterial phylotypes

A preliminary study has demonstrated that the structure and species composition of microbial communities associated with events of dust deposition from the Sahara Desert to the Mont Blanc glacier varied considerably between samples originating from different time periods. Even for depositions within a single month, the dominant microbial phylotypes and candidates to colonize the snow pack were different. It is therefore highly probable that the structure and species composition of microbial communities will be different between any events of the kind. Apparently, the phenomenon does not correlate with the time the dust stays in the snow cover and consequently with the probable development of microorganisms in situ (three months, one month, and one week). The reasons for the variation may be the differences in conditions in the epicenter of a specific North African dust storm, as well as the history of the dust transport in the atmosphere. The candidates for joining the snow biome of Mont Blanc turned out to be different for three dust events (DEs) and belong to different, mostly minor, phylotypes related to Crossiella cryophilus (Actinobacteria), Devosia limi (α-Proteobacteria), Deinococcus claudionis Deinococcus-Thermus), Anabaena sp. (Cyanobacteria), and Hymenobacter soli (Bacteroidetes). Since all these phylotypes have been previously isolated from soil samples of the Antarctic and Arctic, Arctic snow and ice, and the Alpine belt soils and sedimentary rocks of the glacier bed, they were tentatively ascribed to the group of snow pack colonizers.

Microbial sulfur transformations in sediments from Subglacial Lake Whillans

Diverse microbial assemblages inhabit subglacial aquatic environments. While few of these environments have been sampled, data reveal that subglacial organisms gain energy for growth from reduced minerals containing nitrogen, iron, and sulfur. Here we investigate the role of microbially mediated sulfur transformations in sediments from Subglacial Lake Whillans (SLW), Antarctica, by examining key genes involved in dissimilatory sulfur oxidation and reduction. The presence of sulfur transformation genes throughout the top 34 cm of SLW sediments changes with depth. SLW surficial sediments were dominated by genes related to known sulfur-oxidizing chemoautotrophs. Sequences encoding the adenosine-5′-phosphosulfate (APS) reductase gene, involved in both dissimilatory sulfate reduction and sulfur oxidation, were present in all samples and clustered into 16 distinct operational taxonomic units. The majority of APS reductase sequences (74%) clustered with known sulfur oxidizers including those within the “Sideroxydans” and Thiobacillus genera. Reverse-acting dissimilatory sulfite reductase (rDSR) and 16S rRNA gene sequences further support dominance of “Sideroxydans” and Thiobacillus phylotypes in the top 2 cm of SLW sediments. The SLW microbial community has the genetic potential for sulfate reduction which is supported by experimentally measured low rates (1.4 pmol cm-3d-1) of biologically mediated sulfate reduction and the presence of APS reductase and DSR gene sequences related to Desulfobacteraceae and Desulfotomaculum. Our results also infer the presence of sulfur oxidation, which can be a significant energetic pathway for chemosynthetic biosynthesis in SLW sediments. The water in SLW ultimately flows into the Ross Sea where intermediates from subglacial sulfur transformations can influence the flux of solutes to the Southern Ocean.

Antarctic subglacial lake exploration: first results and future plans.

After more than a decade of planning, three attempts were made in 2012–2013 to access, measure in situ properties and directly sample subglacial Antarctic lake environments. First, Russian scientists drilled into the top of Lake Vostok, allowing lake water to infiltrate, and freeze within, the lower part of the ice-core borehole, from which further coring would recover a frozen sample of surface lake water. Second, UK engineers tried unsuccessfully to deploy a clean-access hot-water drill, to sample the water column and sediments of subglacial Lake Ellsworth. Third, a US mission successfully drilled cleanly into subglacial Lake Whillans, a shallow hydraulically active lake at the coastal margin of West Antarctica, obtaining samples that would later be used to prove the existence of microbial life and active biogeochemical cycling beneath the ice sheet. This article summarizes the results of these programmes in terms of the scientific results obtained, the operational knowledge gained and the engineering challenges revealed, to collate what is known about Antarctic subglacial environments and how to explore them in future. While results from Lake Whillans testify to subglacial lakes as being viable biological habitats, the engineering challenges to explore deeper more isolated lakes where unique microorganisms and climate records may be found, as exemplified in the Lake Ellsworth and Vostok missions, are considerable. Through international cooperation, and by using equipment and knowledge of the existing subglacial lake exploration programmes, it is possible that such environments could be explored thoroughly, and at numerous sites, in the near future.

[Microbial communities of water column of Lake Radok, East Antarctica, dominated by abundant actinobacterium “Candidatus Planktophila limnetica”].

The icefree regions of the Antarctic continent(Antarctic oases [1]), have attracted the special attention of researchers from the very first studies, asregions more likely to harbor life. A prominent featureof their landscape is the presence of numerous different lakes. One such Lake is Lake Radok, located in theAmery Oasis (a socalled “mountain” oasis [2]) inEast Antarctica.Antarctic lakes are unique ecosystems which arecharacterized by lowtemperature and, mainly, oligotrophic conditions supporting rather simple truncated food webs [3]. Studying the biodiversity andcommunity structure of such lakes affords a greatopportunity to understand the mechanisms of adaptation and evolution of microorganisms, as well as in thesearch for new species [4] which may have biotechnological application.Lake Radok is covered with ice, up to 3 m in thickness through the year. However, during the summerseason, the lake surface is partially open, forming bothpolynyas and shore ice. The water catchment areaaverages 20.1 km