Prokaryotic community in Pleistocene ice wedges of Mammoth Mountain.

Ice wedges differ from other types of surface and underground glacial bodies and are widely spread in perennially frozen sub-Arctic regions, but the bacterial and archaeal diversity in these permafrost features remains poorly studied. Here, we compared the prokaryotic community composition in the active layer and ancient, 13-19 kyr BP and ~ 40 kyr BP, ice wedge horizons from the same exposure profile of the Mammoth Mountain, using pyrosequencing 16S rRNA gene. The most abundant OTUs in the active layer were affiliated with Acidobacteria (31.81%) followed by Actinobacteria (18.29%), Proteobacteria (18.14%), Gemmatimonadetes (7.3%), Parcubacteria (7.13%) and Bacteroidetes (6.49%). The prokaryotic community in 13-19 kyr BP ice wedge differed at the phylum level by the predominance of Actinobacteria (29.15%) over Acidobacteria (19.52%), Proteobacteria (18.45%), Verrumicrobia (5.88%), Firmicutes (2.98%) and Gemmatimonadetes (2.87%). In contrast, the oldest (~ 40 kyr BP) ice wedge prokaryotic community was rather poor, and only three phyla Firmicutes (54.48%), Proteobacteria (31.42%) and Bacteroidetes (7.92%) constituted the major fraction of reads. Archaeal sequences contributed with no more than 0.6% to total reads in all studied samples. Apparently, the Mammoth Mountain exposure profile harbors insular microbial communities with specific structure that reflects the stratigraphy, properties and age.

Ancient permafrost staphylococci carry antibiotic resistance genes.

Background: Permafrost preserves a variety of viable ancient microorganisms. Some of them can be cultivated after being kept at subzero temperatures for thousands or even millions of years. Objective: To cultivate bacterial strains from permafrost. Design: We isolated and cultivated two bacterial strains from permafrost that was obtained at Mammoth Mountain in Siberia and attributed to the Middle Miocene. Bacterial genomic DNA was sequenced with 40-60× coverage and high-quality contigs were assembled. The first strain was assigned to Staphylococcus warneri species (designated MMP1) and the second one to Staphylococcus hominis species (designated MMP2), based on the classification of 16S ribosomal RNA genes and genomic sequences. Results: Genomic sequence analysis revealed the close relation of the isolated ancient bacteria to the modern bacteria of this species. Moreover, several genes associated with resistance to different groups of antibiotics were found in the S. hominis MMP2 genome. Conclusions: These findings supports a hypothesis that antibiotic resistance has an ancient origin. The enrichment of cultivated bacterial communities with ancient permafrost strains is essential for the analysis of bacterial evolution and antibiotic resistance.

Glaciimonas frigoris sp. nov., a psychrophilic bacterium isolated from ancient Siberian permafrost sediment, and emended description of the genus Glaciimonas.

The bacterial strain N1-38T was isolated from ancient Siberian permafrost sediment. The strain was Gram-reaction-negative, motile by gliding, rod-shaped and psychrophilic, and showed good growth over a temperature range of - 5 to 25 °C. Phylogenetic analysis of 16S rRNA gene sequences revealed that strain N1-38T was most closely related to members of the genus Glaciimonas and shared the highest 16S rRNA gene sequence similarities with the type strains of Glaciimonas alpina (99.3 %), Glaciimonas immobilis (98.9 %) and Glaciimonas singularis (96.5 %). The predominant cellular fatty acids of strain N1-38T were summed feature 3 (C16 : 1ω7c and/or iso-C15 : 0 2-OH), C16 : 0 and C18 : 1ω7c. The major respiratory quinone was ubiquinone 8 and the major polar lipids were phosphatidylethanolamine and diphosphatidylglycerol. The genomic DNA G+C content was 53.0 mol%. Combined data of phenotypic, phylogenetic and DNA-DNA relatedness studies demonstrated that strain N1-38T represents a novel species of the genus Glaciimonas, for which the name Glaciimonas frigoris sp. nov. is proposed. The type strain is N1-38T ( = LMG 28868T = CCOS 838T). An emended description of the genus Glaciimonas is also provided.

Different Scenarios for the Origin and the Subsequent Succession of a Hypothetical Microbial Community in the Cloud Layer of Venus.

The possible existence of a microbial community in the venusian clouds is one of the most intriguing hypotheses in modern astrobiology. Such a community must be characterized by a high survivability potential under severe environmental conditions, the most extreme of which are very low pH levels and water activity. Considering different scenarios for the origin of life and geological history of our planet, a few of these scenarios are discussed in the context of the origin of hypothetical microbial life within the venusian cloud layer. The existence of liquid water on the surface of ancient Venus is one of the key outstanding questions influencing this possibility. We link the inherent attributes of microbial life as we know it that favor the persistence of life in such an environment and review the possible scenarios of life's origin and its evolution under a strong greenhouse effect and loss of water on Venus. We also propose a roadmap and describe a novel methodological approach for astrobiological research in the framework of future missions to Venus with the intent to reveal whether life exists today on the planet.

Tomitella biformata gen. nov., sp. nov., a new member of the suborder Corynebacterineae isolated from a permafrost ice wedge.

Gram-reaction-positive, aerobic, non-spore-forming, irregular rod-shaped bacteria, designated AHU1821(T) and AHU1820, were isolated from an ice wedge in the Fox permafrost tunnel, Alaska. The strains were psychrophilic, growing at -5 to 27°C. Phylogenetic analysis of the 16S rRNA and gyrB gene sequences indicated that the ice-wedge isolates formed a clade distinct from other mycolic-acid-containing bacteria within the suborder Corynebacterineae. The cell wall of strains AHU1821(T) and AHU1820 contained meso-diaminopimelic acid, arabinose and galactose, indicating chemotype IV. The muramic acids in the peptidoglycan were glycolated. The predominant menaquinone was MK-9(H(2)). The polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylinositol mannosides and an unidentified glycolipid. The major fatty acids were hexadecenoic acid (C(16 : 1)), hexadecanoic acid (C(16 : 0)), octadecenoic acid (C(18 : 1)) and tetradecanoic acid (C(14 : 0)). Tuberculostearic acid was present in relatively small amounts (1 %). Strains AHU1821(T) and AHU1820 contained mycolic acids with 42-52 carbons. The DNA G+C content of the two strains was 69.3-71.6 mol% (T(m)). 16S rRNA, rpoB and recA gene sequences were identical between strains AHU1821(T) and AHU1820 and those of the gyrB gene showed 99.9 % similarity. Based on phylogenetic and phenotypic evidence, strains AHU1821(T) and AHU1820 represent a single novel species of a novel genus, for which the name Tomitella biformata gen. nov., sp. nov. is proposed. The type strain of Tomitella biformata is AHU1821(T) (=DSM 45403(T) =NBRC 106253(T)).

Pristine and Antibiotic-Loaded Nanosheets/Nanoneedles-Based Boron Nitride Films as a Promising Platform to Suppress Bacterial and Fungal Infections.

In recent years, bacteria inactivation during their direct physical contact with surface nanotopography has become one of the promising strategies for fighting infection. Contact-killing ability has been reported for several nanostructured surfaces, e.g., black silicon, carbon nanotubes, zinc oxide nanorods, and copper oxide nanosheets. Herein, we demonstrate that Gram-negative antibiotic-resistant Escherichia coli (E. coli) bacteria are killed as a result of their physical destruction while contacting nanostructured h-BN surfaces. BN films, made of spherical nanoparticles formed by numerous nanosheets and nanoneedles with a thickness <15 nm, have been obtained through a reaction of ammonia with amorphous boron. The contact-killing bactericidal effect of BN nanostructures has been compared with a toxic effect of gentamicin released from them. For a wider protection against bacterial and fungal infection, the films have been saturated with a mixture of gentamicin and amphotericin B. Such BN films demonstrate a high antibiotic/antimycotic agent loading capacity and a fast initial and sustained release of therapeutic agents for 170-260 h depending on the loaded dose. The pristine BN films possess high antibacterial activity against E. coli K-261 strain at their initial concentration of 104 cells/mL, attaining >99% inactivation of colony forming units after 24 h, same as gentamicin-loaded (150 µg/cm2) BN sample. The BN films loaded with a mixture of gentamicin (150 and 300 µg/cm2) and amphotericin B (100 µg/cm2) effectively inhibit the growth of E. coli K-261 and Neurospora crassa strains. During immersion in the normal saline solution, the BN film generates reactive oxygen species (ROS), which can lead to accelerated oxidative stress at the site of physical cell damage. The obtained results are valuable for further development of nanostructured surfaces having contact killing, ROS, and biocide release abilities.

Oncolytic Bacteria and their potential role in bacterium-mediated tumour therapy: a conceptual analysis.

As the human microbiota has been confirmed to be of great significance in maintaining health, the dominant bacteria in them have been applied as probiotics to treat various diseases. After the detection of bacteria in tumours, which had previously been considered a sterile region, these bacteria have been isolated and genetically modified for use in tumour therapy. In this review, we sum up the main types of bacteria used in tumour therapy and reveal the mechanisms of both wild type and engineered bacteria in eliminating tumour cells, providing potential possibilities for newly detected, genetically modified, tumour-associated bacteria in anti-tumour therapy.

Bacterial community in ancient permafrost alluvium at the Mammoth Mountain (Eastern Siberia).

Permanently frozen (approx. 3.5Ma) alluvial Neogene sediments exposed in the Aldan river valley at the Mammoth Mountain (Eastern Siberia) are unique, ancient, and poorly studied permafrost environments. So far, the structure of the indigenous bacterial community has remained unknown. Use of 16S metagenomic analysis with total DNA isolation using DNA Spin Kit for Soil (MO-Bio) and QIAamp DNA Stool Mini Kit (Qiagen) has revealed the major and minor bacterial lineages in the permafrost alluvium sediments. In sum, 61 Operational Taxonomic Units (OTUs) with 31,239 reads (Qiagen kit) and 15,404 reads (Mo-Bio kit) could be assigned to the known taxa. Only three phyla, Bacteroidetes, Proteobacteria and Firmicutes, comprised >5% of the OTUs abundance and accounted for 99% of the total reads. OTUs pertaining to the top families (Chitinophagaceae, Caulobacteraceae, Sphingomonadaceae, Bradyrhizobiaceae, Halomonadaceae) held >90% of reads. The abundance of Actinobacteria was less (0.7%), whereas members of other phyla (Deinococcus-Thermus, Cyanobacteria/Chloroplast, Fusobacteria, and Acidobacteria) constituted a minor fraction of reads. The bacterial community in the studied ancient alluvium differs from other permafrost sediments, mainly by predominance of Bacteroidetes (>52%). The diversity of this preserved bacterial community has the potential to cause effects unknown if prompted to thaw and spread with changing climate. Therefore, this study elicits further reason to study how reintroduction of these ancient bacteria could affect the surrounding ecosystem, including current bacterial species.

Isolation and characterization of bacteria from ancient siberian permafrost sediment.

In this study, we isolated and characterized bacterial strains from ancient (Neogene) permafrost sediment that was permanently frozen for 3.5 million years. The sampling site was located at Mammoth Mountain in the Aldan river valley in Central Yakutia in Eastern Siberia. Analysis of phospolipid fatty acids (PLFA) demonstrated the dominance of bacteria over fungi; the analysis of fatty acids specific for Gram-positive and Gram-negative bacteria revealed an approximately twofold higher amount of Gram-negative bacteria compared to Gram-positive bacteria. Direct microbial counts after natural permafrost enrichment showed the presence of (4.7 ± 1.5) × 108 cells g-1 sediment dry mass. Viable heterotrophic bacteria were found at 0 °C, 10 °C and 25 °C, but not at 37 °C. Spore-forming bacteria were not detected. Numbers of viable fungi were low and were only detected at 0 °C and 10 °C. Selected culturable bacterial isolates were identified as representatives of Arthrobacter phenanthrenivorans, Subtercola frigoramans and Glaciimonas immobilis. Representatives of each of these species were characterized with regard to their growth temperature range, their ability to grow on different media, to produce enzymes, to grow in the presence of NaCl, antibiotics, and heavy metals, and to degrade hydrocarbons. All strains could grow at -5 °C; the upper temperature limit for growth in liquid culture was 25 °C or 30 °C. Sensitivity to rich media, antibiotics, heavy metals, and salt increased when temperature decreased (20 °C > 10 °C > 1 °C). In spite of the ligninolytic activity of some strains, no biodegradation activity was detected.

Draft Genome Sequence of Bacillus cereus Strain F, Isolated from Ancient Permafrost.

Bacillus cereus strain F was isolated and cultured from a sample of permafrost, aged presumably about 3 million years, on the Mammoth Mountain (62°56'N, 133°59'E). These genome data provide the basis to investigate Bacillus cereus F, identified as a long-term survivor of the extremely cold and close environment.

Glaciibacter superstes gen. nov., sp. nov., a novel member of the family Microbacteriaceae isolated from a permafrost ice wedge.

Gram-positive, aerobic, non-spore-forming, irregular rod-shaped bacteria, designated strains AHU1791(T) and AHU1810, were isolated from a permafrost ice wedge in Alaska. Cells were motile by means of a polar flagellum. The strains were psychrophilic, growing at -5 to 25 degrees C. Phylogenetic analysis of 16S rRNA gene sequences indicated that the ice-wedge isolates formed a clade distinct from other genera affiliated with the family Microbacteriaceae. The novel strains showed highest levels of 16S rRNA gene sequence similarity with members of the genera Agreia and Subtercola (95.6-95.9 %). The level of 16S rRNA gene sequence similarity between strains AHU1791(T) and AHU1810 was 99.8 %. The cell-wall peptidoglycan type of the two strains was B2gamma, containing 2,4-diaminobutyric acid as the diagnostic amino acid. The predominant menaquinones were MK-12 and MK-13 (strain AHU1791(T)) and MK-11 and MK-12 (strain AHU1810). The major fatty acids of the two strains were 12-methyl tetradecanoic acid (anteiso-C(15 : 0)), 14-methyl hexadecanoic acid (anteiso-C(17 : 0)), 14-methyl pentadecanoic acid (iso-C(16 : 0)) and 13-methyl tetradecanoic acid (iso-C(15 : 0)). The DNA G+C contents of strains AHU1791(T) and AHU1810 were approximately 65 mol%. These phenotypic characteristics differentiated the ice-wedge strains from their closest phylogenetic neighbours, namely Subtercola boreus and the two recognized species of the genus Agreia. The sequences of the housekeeping genes coding for DNA gyrase subunit B (gyrB), RNA polymerase subunit B (rpoB) and recombinase A (recA) were almost identical between strains AHU1791(T) and AHU1810. Although the predominant menaquinones found in strains AHU1791(T) and AHU1810 were different, no other distinct differences were found with regard to other phenotypic and genotypic characteristics, indicating that the two strains were members of the same species. Accordingly, strains AHU1791(T) and AHU1810 are considered to represent a single novel species of a new genus, for which the name Glaciibacter superstes gen. nov., sp. nov. is proposed. The type strain of Glaciibacter superstes is AHU1791(T) (=DSM 21135(T) =NBRC 104264(T)).

Phylogenetic analysis of bacteria preserved in a permafrost ice wedge for 25,000 years.

Phylogenetic analysis of bacteria preserved within an ice wedge from the Fox permafrost tunnel was undertaken by cultivation and molecular techniques. The radiocarbon age of the ice wedge was determined. Our results suggest that the bacteria in the ice wedge adapted to the frozen conditions have survived for 25,000 years.