Assessment of fungal spores and spore-like diversity in environmental samples by targeted lysis.

At particular stages during their life cycles, fungi use multiple strategies to form specialized structures to survive unfavorable environmental conditions. These strategies encompass sporulation, as well as cell-wall melanization, multicellular tissue formation or even dimorphism. The resulting structures are not only used to disperse to other environments, but also to survive long periods of time awaiting favorable growth conditions. As a result, these specialized fungal structures are part of the microbial seed bank, which is known to influence the microbial community composition and contribute to the maintenance of diversity. Despite the importance of the microbial seed bank in the environment, methods to study the diversity of fungal structures with improved resistance only target spores dispersing in the air, omitting the high diversity of these structures in terms of morphology and environmental distribution. In this study, we applied a separation method based on cell lysis to enrich lysis-resistant fungal structures (for instance, spores, sclerotia, melanized yeast) to obtain a proxy of the composition of the fungal seed bank. This approach was first evaluated in-vitro in selected species. The results obtained showed that DNA from fungal spores and from yeast was only obtained after the application of the enrichment method, while mycelium was always lysed. After validation, we compared the diversity of the total and lysis-resistant fractions in the polyextreme environment of the Salar de Huasco, a high-altitude athalassohaline wetland in the Chilean Altiplano. Environmental samples were collected from the salt flat and from microbial mats in small surrounding ponds. Both the lake sediments and microbial mats were dominated by Ascomycota and Basidiomycota, however, the diversity and composition of each environment differed at lower taxonomic ranks. Members of the phylum Chytridiomycota were enriched in the lysis-resistant fraction, while members of the phylum Rozellomycota were never detected in this fraction. Moreover, we show that the community composition of the lysis-resistant fraction reflects the diversity of life cycles and survival strategies developed by fungi in the environment. To the best of our knowledge this is the first time that the fungal diversity is explored in the Salar de Huasco. In addition, the method presented here provides a simple and culture independent approach to assess the diversity of fungal lysis-resistant cells in the environment.

Overview of strategies for developing high thermostability industrial enzymes: Discovery, mechanism, modification and challenges.

Biocatalysts such as enzymes are environmentally friendly and have substrate specificity, which are preferred in the production of various industrial products. However, the strict reaction conditions in industry including high temperature, organic solvents, strong acids and bases and other harsh environments often destabilize enzymes, and thus substantially compromise their catalytic functions, and greatly restrict their applications in food, pharmaceutical, textile, bio-refining and feed industries. Therefore, developing industrial enzymes with high thermostability becomes very important in industry as thermozymes have more advantages under high temperature. Discovering new thermostable enzymes using genome sequencing, metagenomics and sample isolation from extreme environments, or performing molecular modification of the existing enzymes with poor thermostability using emerging protein engineering technology have become an effective means of obtaining thermozymes. Based on the thermozymes as biocatalytic chips in industry, this review systematically analyzes the ways to discover thermostable enzymes from extreme environment, clarifies various interaction forces that will affect thermal stability of enzymes, and proposes different strategies to improve enzymes' thermostability. Furthermore, latest development in the thermal stability modification of industrial enzymes through rational design strategies is comprehensively introduced from structure-activity relationship point of view. Challenges and future research perspectives are put forward as well.

Seasonal Variation of Hypolithic Microbiomes in the Gobi Desert : Seasonal Variation of Hypolithic Microbiomes in the Gobi Desert.

Understanding how microbial communities adapt to environmental stresses is critical for interpreting ecological patterns and microbial diversity. In the case of the Gobi Desert, little is known on the environmental factors that explain hypolithic colonization under quartz stones. By analyzing nine hypolithic communities across an arid gradient and the effects of the season of the year in the Hexi Corridor of this desert, we found a significant decrease in hypolithic colonization rates (from 47.24 to 15.73%) with the increasing drought gradient and found two distinct communities in Hot and Cold samples, which survived or proliferated after a hot or a cold period. While Cold communities showed a greater species diversity and a predominance of Cyanobacteria, Hot communities showed a predominance of members of the Proteobacteria and the Firmicutes. In comparison, Cold communities also possessed stronger functions in the photosynthesis and carbon metabolism. Based on the findings of this study, we proposed that the hypolithic communities of the Hexi Corridor of the Gobi Desert might follow a seasonal developmental cycle in which temperature play an important role. Thus after a critical thermal threshold is crossed, heterotrophic microorganisms predominate in the hot period, while Cyanobacteria predominate in the cold period.

Metagenomics revealing molecular profiles of microbial community structure and metabolic capacity in Bamucuo lake, Tibet.

Microorganisms play critical ecological roles in the global biogeochemical cycles. However, extensive information on the microbial communities in Qinghai-Tibet Plateau (QTP), which is the highest plateau in the world, is still lacking, particularly in high elevation locations above 4500 m. Here, we performed a survey of th e soil and water microbial communities in Bamucuo Lake, Tibet, by using shotgun metagenomic methods. In the soil and water samples, we reconstructed 75 almost complete metagenomic assembly genomes, and 74 of the metagenomic assembly genomes from the water sample represented novel species. Proteobacteria and Actinobacteria were found to be the dominant bacterial phyla, while Euryarchaeota was the dominant archaeal phylum. The largest virus, Pandoravirus salinus, was found in the soil microbial community. We concluded that the microorganisms in Bamucuo Lake are most likely to fix carbon mainly through the 3-hydroxypropionic bi-cycle pathway. This study, for the first time, characterized the microbial community composition and metabolic capacity in QTP high-elevation locations with 4555 m, confirming that QTP is a vast and valuable resource pool, in which many microorganisms can be used to develop new bioactive substances and new antibiotics to which pathogenic microorganisms have not yet developed resistance.

Development and Regulation of the Extreme Biofilm Formation of Deinococcus radiodurans R1 under Extreme Environmental Conditions.

To grow in various harsh environments, extremophiles have developed extraordinary strategies such as biofilm formation, which is an extremely complex and progressive process. However, the genetic elements and exact mechanisms underlying extreme biofilm formation remain enigmatic. Here, we characterized the biofilm-forming ability of Deinococcus radiodurans in vitro under extreme environmental conditions and found that extremely high concentrations of NaCl or sorbitol could induce biofilm formation. Meantime, the survival ability of biofilm cells was superior to that of planktonic cells in different extreme conditions, such as hydrogen peroxide stress, sorbitol stress, and high UV radiation. Transcriptome profiles of D. radiodurans in four different biofilm development stages further revealed that only 13 matched genes, which are involved in environmental information processing, carbohydrate metabolism, or stress responses, share sequence homology with genes related to the biofilm formation of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. Overall, 64% of the differentially expressed genes are functionally unknown, indicating the specificity of the regulatory network of D. radiodurans. The mutation of the drRRA gene encoding a response regulator strongly impaired biofilm formation ability, implying that DrRRA is an essential component of the biofilm formation of D. radiodurans. Furthermore, transcripts from both the wild type and the drRRA mutant were compared, showing that the expression of drBON1 (Deinococcus radioduransBON domain-containing protein 1) significantly decreased in the drRRA mutant during biofilm development. Further analysis revealed that the drBON1 mutant lacked the ability to form biofilm and DrRRA, and as a facilitator of biofilm formation, could directly stimulate the transcription of the biofilm-related gene drBON1. Overall, our work highlights a molecular mechanism mediated by the response regulator DrRRA for controlling extreme biofilm formation and thus provides guidance for future studies to investigate novel mechanisms that are used by D. radiodurans to adapt to extreme environments.

A Parachlorella kessleri (Trebouxiophyceae, Chlorophyta) strain tolerant to high concentration of calcium chloride.

Members of coccoid green algae have been documented in various extreme environments. In this article, a unicellular green alga was found to slowly grow in high concentration (3.6 g/L) and pure calcium chloride solution in the laboratory. It was successfully cultured and a taxonomic study combined approaches of morphological and molecular methods was conducted to determine its classification attribution, which was followed by a preliminary physiology research to explore its unique tolerance characteristics against calcium chloride stress. The strain was identified as Parachlorella kessleri by very similar morphology and the same phylogenetic position. The morphological differences among the three species in genus Parachlorella were then discussed and the characteristic traits of absent or thin mucilaginous envelop and mantel-shaped chloroplast for P. kessleri were supported. In addition, the almost strictly spherical shape of adult cells could further distinguish the P. kessleri from the other two species. The tolerant characteristics to CaCl2  stress for this strain were confirmed and the limit concentration was revealed as between 2000 and 4000 times than the standard BG11 culture concentration. Therefore, this P. kessleri strain is expected to be a good material to explore the mechanism of resistance to calcium ions stress for eukaryotic microbiology.

Selection of ACC deaminase positive, thermohalotolerant and drought tolerance enhancing plant growth-promoting bacteria from rhizospheres of Cyamopsis tetragonoloba grown in arid regions.

Plant growth-promoting bacteria (PGPB) expressing 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity are widely acknowledged to have a role in mitigation of abiotic stress caused by extreme environmental conditions. Consequently, several studies have focused on the isolation of ACC deaminase positive PGPBs. However, the application of such strains in drought-prone arid regions has remained grossly under-exploited. In order to be used in arid agroecosystems, PGPBs need to have the dual capability: to express ACC deaminase and to have the ability to tolerate increased temperature and salt concentration. Conspicuously, to date, very few studies have reported about isolation and characterization of PGPBs with this kind of dual capability. Here we report the isolation of bacterial strains from rhizosphere(s) of Cyamopsis tetragonoloba, a commercial crop from arid regions of Rajasthan, India, and their characterization for ACC deaminase activity and thermohalotolerance. Isolates found positive for desired traits were subsequently assessed for plant growth promotion under simulated drought conditions. Our finding showed that although the bacterial diversity within the rhizosphere of C. tetragonoloba grown in the arid region is quite poor, multiple isolates are ACC deaminase positive. Four isolates were found to be ACC deaminase positive, thermohalotolerant, and successfully enhanced drought tolerance. These isolates were identified as strains belonging to genera Pseudomonas, Enterobacter, and Stenotrophomonas based on 16S rRNA sequence homology.

Genome of Crucihimalaya himalaica, a close relative of Arabidopsis, shows ecological adaptation to high altitude.

Crucihimalaya himalaica, a close relative of Arabidopsis and Capsella, grows on the Qinghai-Tibet Plateau (QTP) about 4,000 m above sea level and represents an attractive model system for studying speciation and ecological adaptation in extreme environments. We assembled a draft genome sequence of 234.72 Mb encoding 27,019 genes and investigated its origin and adaptive evolutionary mechanisms. Phylogenomic analyses based on 4,586 single-copy genes revealed that C. himalaica is most closely related to Capsella (estimated divergence 8.8 to 12.2 Mya), whereas both species form a sister clade to Arabidopsis thaliana and Arabidopsis lyrata, from which they diverged between 12.7 and 17.2 Mya. LTR retrotransposons in C. himalaica proliferated shortly after the dramatic uplift and climatic change of the Himalayas from the Late Pliocene to Pleistocene. Compared with closely related species, C. himalaica showed significant contraction and pseudogenization in gene families associated with disease resistance and also significant expansion in gene families associated with ubiquitin-mediated proteolysis and DNA repair. We identified hundreds of genes involved in DNA repair, ubiquitin-mediated proteolysis, and reproductive processes with signs of positive selection. Gene families showing dramatic changes in size and genes showing signs of positive selection are likely candidates for C. himalaica's adaptation to intense radiation, low temperature, and pathogen-depauperate environments in the QTP. Loss of function at the S-locus, the reason for the transition to self-fertilization of C. himalaica, might have enabled its QTP occupation. Overall, the genome sequence of C. himalaica provides insights into the mechanisms of plant adaptation to extreme environments.

Species and geographic specificity between endophytic fungi and host supported by parasitic Cynomorium songaricum and its host Nitraria tangutorum distributed in desert.

This study was aimed to investigate whether host plant species and lifestyles, and environmental conditions in the desert affect endophytic fungi composition. Endophytic fungal communities from parasitic plant Cynomorium songaricum and its host Nitraria tangutorum were investigated from three sites including Tonggu Naoer, Xilin Gaole, and Guazhou in Tengger and Badain Jaran Deserts in China using the next-generation sequencing of a ribosomal RNA gene region. Similarity and difference in endophytic fungal composition from different geographic locations were evaluated through multivariate statistical analysis. It showed that plant genetics was a deciding factor affecting endophytic fungal composition even when C. songaricum and N. tangutorum grow together tightly. Not only that, the fungal composition was also greatly affected by the local environment and rainfall. However, the distribution and richness of fungal species indicated that the geographical distance exerted little influence on characterizing the fungal composition. Overall, the findings suggested that plant species, parasitic or non-parasitic lifestyles of the plant, and local environment strongly affected the number and diversity of the endophytic fungal species, which may provide valuable insights into the microbe ecology, symbiosis specificity, and the tripartite relationship among parasitic plant, host, and endophytic fungi, especially under desert environment.

Phylogenetic Relationships and Adaptation in Deep-Sea Mussels: Insights from Mitochondrial Genomes.

Mitochondrial genomes (mitogenomes) are an excellent source of information for phylogenetic and evolutionary studies, but their application in marine invertebrates is limited. In the present study, we utilized mitogenomes to elucidate the phylogeny and environmental adaptation in deep-sea mussels (Mytilidae: Bathymodiolinae). We sequenced and assembled seven bathymodioline mitogenomes. A phylogenetic analysis integrating the seven newly assembled and six previously reported bathymodioline mitogenomes revealed that these bathymodiolines are divided into three well-supported clades represented by five Gigantidas species, six Bathymodiolus species, and two "Bathymodiolus" species, respectively. A Common interval Rearrangement Explorer (CREx) analysis revealed a gene order rearrangement in bathymodiolines that is distinct from that in other shallow-water mytilids. The CREx analysis also suggested that reversal, transposition, and tandem duplications with subsequent random gene loss (TDRL) may have been responsible for the evolution of mitochondrial gene orders in bathymodiolines. Moreover, a comparison of the mitogenomes of shallow-water and deep-sea mussels revealed that the latter lineage has experienced relaxed purifying selection, but 16 residues of the atp6, nad4, nad2, cob, nad5, and cox2 genes have underwent positive selection. Overall, this study provides new insights into the phylogenetic relationships and mitogenomic adaptations of deep-sea mussels.

Genome Study of a Novel Virulent Phage vB_SspS_KASIA and Mu-like Prophages of Shewanella sp. M16 Provides Insights into the Genetic Diversity of the Shewanella Virome.

Shewanella is a ubiquitous bacterial genus of aquatic ecosystems, and its bacteriophages are also isolated from aquatic environments (oceans, lakes, ice, and wastewater). In this study, the isolation and characterization of a novel virulent Shewanella phage vB_SspS_KASIA and the identification of three prophages of its host, Shewanella sp. M16, including a mitomycin-inducible Mu-like siphovirus, vB_SspS_MuM16-1, became the starting point for comparative analyses of phages infecting Shewanella spp. and the determination of their position among the known bacterial viruses. A similarity networking analysis revealed the high diversity of Shewanella phages in general, with vB_SspS_KASIA clustering exclusively with Colwellia phage 9A, with which it forms a single viral cluster composed of two separate viral subclusters. Furthermore, vB_SspS_MuM16-1 presented itself as being significantly different from the phages deposited in public databases, expanding the diversity of the known Mu-like phages and giving potential molecular markers for the identification of Mu-like prophages in bacterial genomes. Moreover, the functional analysis performed for vB_SspS_KASIA suggested that, despite the KASIA host, the M16 strain grows better in a rich medium and at 30 °C the phage replication cycle seems to be optimal in restrictive culture conditions mimicking their natural environment, the Zloty Stok gold and arsenic mine.

High-Throughput Griess Assay of Nitrite and Nitrate in Plasma and Red Blood Cells for Human Physiology Studies under Extreme Conditions.

The metabolism of nitric oxide plays an increasingly interesting role in the physiological response of the human body to extreme environmental conditions, such as underwater, in an extremely cold climate, and at low oxygen concentrations. Field studies need the development of analytical methods to measure nitrite and nitrate in plasma and red blood cells with high requirements of accuracy, precision, and sensitivity. An optimized spectrophotometric Griess method for nitrite-nitrate affords sensitivity in the low millimolar range and precision within ±2 µM for both nitrite and nitrate, requiring 100 µL of scarcely available plasma sample or less than 50 µL of red blood cells. A scheduled time-efficient procedure affords measurement of as many as 80 blood samples, with combined nitrite and nitrate measurement in plasma and red blood cells. Performance and usefulness were tested in pilot studies that use blood fractions deriving from subjects who dwelt in an Antarctica scientific station and on breath-holding and scuba divers who performed training at sea and in a land-based deep pool facility. The method demonstrated adequate to measure low basal concentrations of nitrite and high production of nitrate as a consequence of water column pressure-triggered vasodilatation in deep-water divers.

Submarine medicine: An overview of the unique challenges, medical concerns, and gaps.

Submariners face many challenges. For example, they "live where they work" and can be called to duty anytime. They have limited access to open space, natural settings, fresh air, fresh food, sunlight, privacy, exercise, and outside communication. They support a wider range of missions than occur aboard most other Navy vessels. At sea or on shore, submariners work long hours under conditions with little margin for error. They may traverse remote or disputed areas of the ocean far from rescue assets, and must remain vigilant for potential encounters with hostile forces, onboard fires, anomalies in the breathing atmosphere, leaks, undersea collisions, or radiation exposures. If any of these factors cause casualties, the Independent Duty Corpsman (with intermittent advice from shore-based medical personnel), must be ready to provide aid as long as necessary. The challenges of submarine service led to the growth of the unique field of submarine medicine, which has maintained an excellent record of health and safety. This review introduces the field of submarine medicine as practiced in the U.S. Navy, describing its major concerns, giving an overview of the operation of a submarine medical department, and identifying several medical gaps that researchers are working to fill. Submarine medicine already has a stellar record in terms of radiation and atmospheric safety and has made strides in fatigue management. Ongoing work will deliver improved psychological screening and support tools. This report summarizes developments in these and other areas of submarine medicine.

A chromosome-scale assembly of the smallest Dothideomycete genome reveals a unique genome compaction mechanism in filamentous fungi.

BACKGROUND: The wide variation in the size of fungal genomes is well known, but the reasons for this size variation are less certain. Here, we present a chromosome-scale assembly of ectophytic Peltaster fructicola, a surface-dwelling extremophile, based on long-read DNA sequencing technology, to assess possible mechanisms associated with genome compaction. RESULTS: At 18.99 million bases (Mb), P. fructicola possesses one of the smallest known genomes sequence among filamentous fungi. The genome is highly compact relative to other fungi, with substantial reductions in repeat content, ribosomal DNA copies, tRNA gene quantity, and intron sizes, as well as intergenic lengths and the size of gene families. Transposons take up just 0.05% of the entire genome, and no full-length transposon was found. We concluded that reduced genome sizes in filamentous fungi such as P. fructicola, Taphrina deformans and Pneumocystis jirovecii occurred through reduction in ribosomal DNA copy number and reduced intron sizes. These dual mechanisms contrast with genome reduction in the yeast fungus Saccharomyces cerevisiae, whose small and compact genome is associated solely with intron loss. CONCLUSIONS: Our results reveal a unique genomic compaction architecture of filamentous fungi inhabiting plant surfaces, and broaden the understanding of the mechanisms associated with compaction of fungal genomes.

Extremophile Microalgae: the potential for biotechnological application.

Microalgae are photosynthetic microorganisms that use sunlight as an energy source, and convert water, carbon dioxide, and inorganic salts into algal biomass. The isolation and selection of microalgae, which allow one to obtain large amounts of biomass and valuable compounds, is a prerequisite for their successful industrial production. This work provides an overview of extremophile algae, where their ability to grow under harsh conditions and the corresponding accumulation of metabolites are addressed. Emphasis is placed on the high-value products of some prominent algae. Moreover, the most recent applications of these microorganisms and their potential exploitation in the context of astrobiology are taken into account.

Design of a Large-Scale Piezoelectric Transducer Network Layer and Its Reliability Verification for Space Structures.

As an effective structural health monitoring (SHM) technology, the piezoelectric transducer (PZT) and guided wave-based monitoring methods have attracted growing interest in the space field. When facing the large-scale monitoring requirements of space structures, a lot of PZTs are needed and may cause problems regarding to additional weight of connection cables, placement efficiency and performance consistency. The PZT layer is a promising solution against these problems. However, the current PZT layers still face challenges from large-scale lightweight monitoring and the lack of reliability assessment under extreme space service conditions. In this paper, a large-scale PZT network layer (LPNL) design method is proposed to overcome these challenges, by adopting a large-scale lightweight PZT network design method and network splitting-recombination based integration strategy. The developed LPNL offers the advantages of being large size, lightweight, ultra-thin, flexible, customized in shape and highly reliable. A series of extreme environmental tests are performed to verify the reliability of the developed LPNL under space service environment, including extreme temperature conditions, vibration at different flying phases, landing impact, and flying overload. Results show that the developed LPNL can withstand these harsh environmental conditions and presents high reliability and functionality.

Ecophysiology of a lacertid community in the high Moroccan mountains suggests conservation guidelines.

Lizard species may differ in their ecophysiology due to adaptation, plasticity and/or phylogeny. In restrictive environments, ecophysiological differences of species living in sympatry are expected to reveal long-term evolutionary responses to the abiotic environment while competitive interactions should be limited. These influences can be disentangled by combining field monitoring with experimental tests. Here, three lacertid lizard species, Atlantolacerta andreanskyi, Scelarcis perspicillata and Podarcis vaucheri sharing high mountain habitats in Oukaimeden (High Atlas, Morocco), were studied. In the field, spatiotemporal variation of the thermal and hydric environment used by the lizards was monitored using data-loggers. In the lab, thermal and hydric ecophysiology was estimated through assessments of preferred temperatures (Tp) and water loss (WL) rates. Species differed in microhabitat use and, hence, in their exposure to variations in temperature and humidity. However, they only differed in their WL (A. andreanskyi > S. perspicillata > P. vaucheri) while their Tp were similar. Such partial differences of species in in the fundamental niche, likely derived from their long-term independent phylogenetic trajectories, can be used to predict their responses to climate and habitat shifts in this and other parts of their respective ranges. Results also confirm previous suggestions that, together with thermal physiology, hydric physiology plays a prominent role in the organisation of lizard communities in the temperate region.

Medical Ethics in Extreme and Austere Environments.

American society has a history of turning to physicians during times of extreme need, from plagues in the past to recent outbreaks of communicable diseases. This public instinct comes from a deep seated trust in physician duty that has been earned over the centuries through dedicated and selfless care, often in the face of personal risks. As dangers facing our communities include terroristic events physicians must be adequately prepared to respond, both medically and ethically. While the ethical principles that govern physician behavior-beneficence, nonmaleficence, autonomy, and social justice-are unchanging, fundamental doctrines must change with the new risks inherent to terroristic events. Responding to mass casualty disasters caused by terrorists, natural calamities, and combat continue to be challenging frontiers in medicine. Preparing physicians to deal with the consequences of a terroristic disease must include understanding the ethical challenges that can occur.

The bacterial diversity on steam vents from Paricutín and Sapichu volcanoes.

Vapor steam vents are prevailing structures on geothermal sites in which local geochemical conditions allow the development of extremophilic microorganisms. We describe the structure of the prokaryotic community able to grow on the walls and rocks of such microecosystems in two terrestrial Mexican volcanoes: Paricutín (PI and PII samples) and its satellite Sapichu (S sample). The investigated samples showed similar diversity indices, with few dominant OTUs (abundance > 1%): 21, 16 and 23, respectively for PI, PII and S. However, each steam vent showed a particular community profile: PI was dominated by photosynthetic bacteria (Cyanobacteria and Chloroflexia class), PII by Actinobacteria and Proteobacteria, and S by Ktedonobacteria class, Acidobacteria and Cyanobacteria phyla. Concerning the predicted metabolic potential, we found a dominance of cellular pathways, especially the ones for energy generation with metabolisms for sulfur respiration, nitrogen fixation, methanogenesis, carbon fixation, photosynthesis, and metals, among others. We suggest a different maturity stage for the three studied fumaroles, from the youngest (PI) to the oldest (S and PII), also influenced by the temperature and other geochemical parameters. Furthermore, four anaerobic strains were isolated, belonging to Clostridia class (Clostridium sphenoides, C. swellfunanium and Anaerocolumna cellulosilytica) and to Bacilli class (Paenibacillus azoreducens).