Structure of the O-specific polysaccharide from the lipopolysaccharide of Psychrobacter cryohalolentis K5(T) containing a 2,3,4-triacetamido-2,3,4-trideoxy-L-arabinose moiety.

A novel constituent of bacterial polysaccharides, 2,3,4-triacetamido-2,3,4-trideoxy-L-arabinose, was found in the O-specific polysaccharide from the lipopolysaccharide of Psychrobacter cryohalolentis K5(T) and identified by 1D and 2D (1)H and (13)C NMR studies of the polysaccharide and a disaccharide obtained by solvolysis of the polysaccharide with triflic acid. The following structure of the branched polysaccharide was established by sugar analysis, triflic acid solvolysis, Smith degradation, and 2D NMR spectroscopy.

Crosslinks rather than strand breaks determine access to ancient DNA sequences from frozen sediments.

Diagenesis was studied in DNA obtained from Siberian permafrost (permanently frozen soil) ranging from 10,000 to 400,000 years in age. Despite optimal preservation conditions, we found the sedimentary DNA to be severely modified by interstrand crosslinks; single- and double-stranded breaks; and freely exposed sugar, phosphate, and hydroxyl groups. Intriguingly, interstrand crosslinks were found to accumulate approximately 100 times faster than single-stranded breaks, suggesting that crosslinking rather than depurination is the primary limiting factor for ancient DNA amplification under frozen conditions. The results question the reliability of the commonly used models relying on depurination kinetics for predicting the long-term survival of DNA under permafrost conditions and suggest that new strategies for repair of ancient DNA must be considered if the yield of amplifiable DNA from permafrost sediments is to be significantly increased. Using the obtained rate constant for interstrand crosslinks the maximal survival time of amplifiable 120-bp fragments of bacterial 16S ribosomal DNA was estimated to be approximately 400,000 years. Additionally, a clear relationship was found between DNA damage and sample age, contradicting previously raised concerns about the possible leaching of free DNA molecules between permafrost layers.

Bacterial community in ancient Siberian permafrost as characterized by culture and culture-independent methods.

The microbial composition of ancient permafrost sediments from the Kolyma lowland of Northeast Eurasia was examined through culture and culture-independent approaches. These sediments have been continuously frozen for 5,000 to 2-3 million years. A total of 265 Bacteria 16S rRNA gene sequences were amplified from the permafrost total-community genomic DNA and screened by amplified ribosomal 16S rRNA restriction analysis. Members of three major lineages were found: gamma-Proteobacteria (mostly Xanthomonadaceae), Actinobacteria, and Firmicutes. We also determined partial 16S rRNA gene sequences of 49 isolates from a collection of 462 aerobes isolated from these sediments. The bacteria included Actinomycetales (Arthrobacter and Microbacteriaceae); followed by the Firmicutes (Exiguobacterium and Planomicrobium); the Bacteroidetes (Flavobacterium); the gamma-Proteobacteria (Psychrobacter); and the alpha-Proteobacteria (Sphingomonas). Both culture and culture-independent approaches showed the presence of high and low G+C Gram-positive bacteria and gamma-Proteobacteria. Some of the 16S rRNA gene sequences of environmental clones matched those of Arthrobacter isolates. Two-thirds of the isolates grew at -2.5 degrees C, indicating that they are psychroactive, and all are closely related to phylogenetic groups with strains from other cold environments, mostly commonly from Antarctica. The culturable and non-culturable microorganisms found in the terrestrial permafrost provide a prototype for possible life on the cryogenic planets of the Solar System.

Cryptic diversity within the choanoflagellate morphospecies complex Codosiga botrytis - phylogeny and morphology of ancient and modern isolates.

Choanoflagellates are closely related to metazoans and fungi according to recent phylogenetic studies; therefore the systematics of these organisms is of particular interest. The choanoflagellate morphospecies Codosiga botrytis is the first described choanoflagellate, and is one of the most frequently reported choanoflagellate species. In this study we present phylogenetic and morphological data on eight different strains of Codosiga botrytis. Among these there are five ancient strains; these cultures have been established from up to 43,000 years old cysts from Siberian permafrost. We found that based on the variable V4 region of the small subunit (SSU) of the rDNA, all the investigated freshwater isolates of Codosiga botrytis, together with Sphaeroeca volvox, form a cluster at the base of all other choanoflagellate species. Moreover, the morphospecies described classically as Codosiga botrytis contains at least four different genotypes separated by considerably high genetic distance. All these 'cryptic species' have identical general morphology and cell structure. Strains have a similar life cycle with several different life forms and large morphological plasticity. For the first time we were able to establish cultures from cryo-conserved cysts of choanoflagellates. The ancient strains did not differ significantly in partial SSU rDNA from the modern ones. Besides, no biogeographically pattern could be established. This fact and the low genetic distances of some strains from remote locations support the distribution of dormant stages via air.

Structure of the O-polysaccharide chain of the lipopolysaccharide of Psychrobacter muricolla 2pS(T) isolated from overcooled water brines within permafrost.

Psychrotrophic bacteria of the genus Psychrobacter have not been studied in respect to lipopolysaccharide structure. In this work, we determined the structure of the O-specific polysaccharide of the lipopolysaccharide of Psychrobacter muricolla 2pS(T) isolated from overcooled (-9°C) water brines within permafrost. The polysaccharide was found to be acidic due to the presence of an amide of 2-acetamido-2-deoxy-l-guluronic acid with glycine (l-GulNAcA6Gly), which has not been hitherto found in nature. The following structure of the disaccharide repeating unit of the polysaccharide was established using composition analysis along with 1D and 2D (1)H and (13)C NMR spectroscopy: →4)-α-l-GulpNAcA6Gly-(1→3)-ß-d-GlcpNAc-(1→

Structure of an acidic polysaccharide isolated from Psychrobacter maritimus 3pS containing a bacillosamine derivative.

An acidic polysaccharide was obtained from Psychrobacter maritimus 3pS isolated from a Siberian cryopeg sample (Kolyma lowland). The following structure of the tetrasaccharide repeating unit of the polysaccharide was established by sugar analysis along with (1)H and (13)C NMR spectroscopy: →2)-α-L-Rhap-(1→4)-α-D-GalpNAcA-(1→3)-α-D-QuipNAc4NHb-(1→3)-ß-D-QuipNAc4NHb-(1→ where D-GalNAcA indicates 2-acetamido-2-deoxy-D-galacturonic acid and d-QuiNAc4NHb indicates 2-acetamido-2,4,6-trideoxy-4-[(S)-3-hydroxybutanoyl]amino-D-glucose.

Characterization of a bacterial community from a Northeast Siberian seacoast permafrost sample.

A combination of culture-dependent and -independent techniques was used to characterize a bacterial community, examine cold adaptation of isocitrate lyase (icl) genes, and detect genes with important ecological functions in a permafrost sample from the Bykovsky Peninsula on the Laptev Sea coast of northeast Siberia. According to the 16S rRNA gene sequence, 47 of the cultured isolates were members of the phyla Firmicutes, Proteobacteria, and Actinobacteria, with 85% of the isolates belonging to the genera Arthrobacter and Planococcus. The 16S rRNA gene clone library derived from DNA from the same permafrost sample contained sequences from the same phyla plus a few from Acidobacteria, but favored the Firmicutes at the cost of the Actinobacteria. A partial sequence of the icl gene, a proposed marker for cold adaptation, was determined for 25 isolates that grew at 0 °C. Two Psychrobacter isolates contained two of the four residues shown to be important for low-temperature activity in Colwellia maris or Colwellia psychrerythreaea. The presence in the permafrost DNA of genes with ecosystem functions was determined using geochip 2.0. The highest number of genes identified was from the categories of aromatic and natural polymer degradation genes, perhaps reflecting selection for the use of tundra vegetation-produced carbon.

Clostridium tagluense sp. nov., a psychrotolerant, anaerobic, spore-forming bacterium from permafrost.

A strictly anaerobic, Gram-positive, psychrotolerant, endospore-forming bacterium (strain A121(T)) was isolated from a permafrost sample collected in the Canadian High Arctic. Phylogenetic analysis of the 16S rRNA gene sequence of strain A121(T) showed its affiliation with the group of psychrophilic and psychrotolerant members of cluster I of the genus Clostridium, Clostridium bowmanii DSM 14206(T) being the closest relative (sequence similarity 98.5 %). Levels of DNA-DNA relatedness between strain A121(T) and the type strains of phylogenetically related species ranged from 33 to 52 %. Strain A121(T) grew in PY broth at temperatures between 4 and 28 degrees C (optimum 15-20 degrees C), at pH 6.0-8.0 (optimum pH 6.5-7.2) and in NaCl concentrations of 0-10.0 % (optimum 0-2.0 %). The strain utilized a narrow range of carbohydrates as sources of carbon and energy, including glucose, fructose, trehalose, maltose and starch; it also hydrolysed gelatin. Predominant fatty acids were C(16 : 1) cis9, C(16 : 1) cis9 DMA, C(16 : 0) and C(14 : 0). The DNA G+C content was 31.5 mol%. On the basis of its overall genotypic and phenotypic characteristics, strain A121(T) is classified within a novel species of the genus Clostridium, Clostridium tagluense sp. nov. The type strain is A121(T) (=VKM B-2369(T) =DSM 17763(T)).

Beringian paleoecology inferred from permafrost-preserved fungal DNA.

The diversity of fungi in permanently frozen soil from northeastern Siberia was studied by culture-independent PCR amplification of diverse environmental 18S rRNA genes. Elaborate protocols to avoid contamination during drilling, sampling, and amplification were used. A broad diversity of eukaryotic DNA sequences that were 510 bp long, including sequences of various fungi, plants, and invertebrates, could be obtained reproducibly from samples that were up to 300,000 to 400,000 years old. The sequences revealed that ancient fungal communities included a diversity of cold-adapted yeasts, dark-pigmented fungi, plant-parasitic fungi, and lichen mycobionts. DNA traces of tree-associated macrofungi in a modern tundra sample indicated that there was a shift in fungal diversity following the last ice age and supported recent results showing that there was a severe change in the plant composition in northeastern Siberia during this period. Interestingly, DNA sequences with high homology to sequences of coprophilic and keratinophilic fungi indicated that feces, hair, skin, and nails could have been sources of ancient megafauna DNA recently reported to be present in small amounts of Siberian permafrost sediments.

Reproduction and metabolism at - 10 degrees C of bacteria isolated from Siberian permafrost.

We report the isolation and properties of several species of bacteria from Siberian permafrost. Half of the isolates were spore-forming bacteria unable to grow or metabolize at subzero temperatures. Other Gram-positive isolates metabolized, but never exhibited any growth at - 10 degrees C. One Gram-negative isolate metabolized and grew at - 10 degrees C, with a measured doubling time of 39 days. Metabolic studies of several isolates suggested that as temperature decreased below + 4 degrees C, the partitioning of energy changes with much more energy being used for cell maintenance as the temperature decreases. In addition, cells grown at - 10 degrees C exhibited major morphological changes at the ultrastructural level.

Diverse plant and animal genetic records from Holocene and Pleistocene sediments.

Genetic analyses of permafrost and temperate sediments reveal that plant and animal DNA may be preserved for long periods, even in the absence of obvious macrofossils. In Siberia, five permafrost cores ranging from 400,000 to 10,000 years old contained at least 19 different plant taxa, including the oldest authenticated ancient DNA sequences known, and megafaunal sequences including mammoth, bison, and horse. The genetic data record a number of dramatic changes in the taxonomic diversity and composition of Beringian vegetation and fauna. Temperate cave sediments in New Zealand also yielded DNA sequences of extinct biota, including two species of ratite moa, and 29 plant taxa characteristic of the prehuman environment. Therefore, many sedimentary deposits may contain unique, and widespread, genetic records of paleoenvironments.