Assessment of the plasmidome of an extremophilic microbial community from the Diamante Lake, Argentina.

Diamante Lake located at 4589 m.a.s.l. in the Andean Puna constitutes an extreme environment. It is exposed to multiple extreme conditions such as an unusually high concentration of arsenic (over 300 mg L-1) and low oxygen pressure. Microorganisms thriving in the lake display specific genotypes that facilitate survival, which include at least a multitude of plasmid-encoded resistance traits. Hence, the genetic information provided by the plasmids essentially contributes to understand adaptation to different stressors. Though plasmids from cultivable organisms have already been analyzed to the sequence level, the impact of the entire plasmid-borne genetic information on such microbial ecosystem is not known. This study aims at assessing the plasmidome from Diamante Lake, which facilitates the identification of potential hosts and prediction of gene functions as well as the ecological impact of mobile genetic elements. The deep-sequencing analysis revealed a large fraction of previously unknown DNA sequences of which the majority encoded putative proteins of unknown function. Remarkably, functions related to the oxidative stress response, DNA repair, as well as arsenic- and antibiotic resistances were annotated. Additionally, all necessary capacities related to plasmid replication, mobilization and maintenance were detected. Sequences characteristic for megaplasmids and other already known plasmid-associated genes were identified as well. The study highlights the potential of the deep-sequencing approach specifically targeting plasmid populations as it allows to evaluate the ecological impact of plasmids from (cultivable and non-cultivable) microorganisms, thereby contributing to the understanding of the distribution of resistance factors within an extremophilic microbial community.

A review of extracellular polysaccharides from extreme niches: An emerging natural source for the biotechnology. From the adverse to diverse!

Every year, new organisms that survive and colonize adverse environments are discovered and isolated. Those organisms, called extremophiles, are distributed throughout the world, both in aquatic and terrestrial environments, such as sulfurous marsh waters, hydrothermal springs, deep waters, volcanos, terrestrial hot springs, marine saltern, salt lakes, among others. According to the ecosystem inhabiting, extremophiles are categorized as thermophiles, psychrophiles, halophiles, acidophiles, alkalophilic, piezophiles, saccharophiles, metallophiles and polyextremophiles. They have developed chemical adaptation strategies that allow them to maintain their cellular integrity, altering physiology or improving repair capabilities; one of them is the biosynthesis of extracellular polysaccharides (EPS), which constitute a slime and hydrated matrix that keep the cells embedded, protecting from environmental stress (desiccation, salinity, temperature, radiation). EPS have gained interest; they are explored by their unique properties such as structural complexity, biodegradability, biological activities, and biocompatibility. Here, we present a review concerning the biosynthesis, characterization, and potential EPS applications produced by extremophile microorganisms, namely, thermophiles, halophiles, and psychrophiles. A bibliometric analysis was conducted, considering research articles published within the last two decades. Besides, an overview of the culture conditions used for extremophiles, the main properties and multiple potential applications of their EPS is also presented.

Molecular Characterization of a Date Palm Vascular Highway 1-Interacting Kinase (PdVIK) Under Abiotic Stresses.

The date palm (Khalas) is an extremophile plant that can adapt to various abiotic stresses including drought and salinity. Salinity tolerance is a complex trait controlled by numerous genes. Identification and functional characterization of salt-responsive genes from the date palm is fundamental to understand salinity tolerance at the molecular level in this plant species. In this study, a salt-inducible vascular highway 1-interacting kinase (PdVIK) that is a MAP kinase kinase kinase (MAPKKK) gene from the date palm, was functionally characterized using in vitro and in vivo strategies. PdVIK, one of the 597 kinases encoded by the date palm genome possesses an ankyrin repeat domain and a kinase domain. The recombinant PdVIK protein exhibited phosphotyrosine activity against myelin basic protein (MBP) substrate. Overexpression of PdVIK in yeast significantly improved its tolerance to salinity, LiCl, and oxidative stresses. Transgenic Arabidopsis seedlings overexpressing PdVIK displayed improved tolerance to salinity, osmotic, and oxidative stresses as assessed by root growth assay. The transgenic lines grown in the soil also displayed modulated salt response, compared to wild-type controls as evaluated by the overall plant growth and proline levels. Likewise, the transgenic lines exhibited drought tolerance by maintaining better relative water content (RWC) compared to non-transgenic control plants. Collectively, these results implicate the involvement of PdVIK in modulating the abiotic stress response of the date palm.

Halophilic microbial community compositional shift after a rare rainfall in the Atacama Desert.

Understanding the mechanisms underlying microbial resistance and resilience to perturbations is essential to predict the impact of climate change on Earth's ecosystems. However, the resilience and adaptation mechanisms of microbial communities to natural perturbations remain relatively unexplored, particularly in extreme environments. The response of an extremophile community inhabiting halite (salt rocks) in the Atacama Desert to a catastrophic rainfall provided the opportunity to characterize and de-convolute the temporal response of a highly specialized community to a major disturbance. With shotgun metagenomic sequencing, we investigated the halite microbiome taxonomic composition and functional potential over a 4-year longitudinal study, uncovering the dynamics of the initial response and of the recovery of the community after a rainfall event. The observed changes can be recapitulated by two general modes of community shifts-a rapid Type 1 shift and a more gradual Type 2 adjustment. In the initial response, the community entered an unstable intermediate state after stochastic niche re-colonization, resulting in broad predicted protein adaptations to increased water availability. In contrast, during recovery, the community returned to its former functional potential by a gradual shift in abundances of the newly acquired taxa. The general characterization and proposed quantitation of these two modes of community response could potentially be applied to other ecosystems, providing a theoretical framework for prediction of taxonomic and functional flux following environmental changes.

Diversity and distribution of hidden cultivable fungi associated with marine animals of Antarctica.

In the present study, we surveyed the distribution and diversity of fungal assemblages associated with 10 species of marine animals from Antarctica. The collections yielded 83 taxa from 27 distinct genera, which were identified using molecular biology methods. The most abundant taxa were Cladosporium sp. 1, Debaryomyces hansenii, Glaciozyma martinii, Metschnikowia australis, Pseudogymnoascus destructans, Thelebolus cf. globosus, Pseudogymnoascus pannorum, Tolypocladium tundrense, Metschnikowia australis, and different Penicillium species. The diversity, richness, and dominance of fungal assemblages ranged among the host; however, in general, the fungal community, which was composed of endemic and cold-adapted cosmopolitan taxa distributed across the different sites of Antarctic Peninsula, displayed high diversity, richness, and dominance indices. Our results contribute to knowledge about fungal diversity in the marine environment across the Antarctic Peninsula and their phylogenetic relationships with species that occur in other cold, temperate, and tropical regions of the World. Additionally, despite their extreme habitats, marine Antarctic animals shelter cryptic and complex fungal assemblages represented by endemic and cosmopolitan cold-adapted taxa, which may represent interesting models to study different symbiotic associations between fungi and their animal hosts in the extreme conditions of Antarctica.

Communication mechanisms in extremophiles: Exploring their existence and industrial applications.

Quorum sensing plays important roles in the regulation of physiological and virulence processes in most bacteria, but its role in extremophiles is largely unknown. Comparative genomic, phylogenic, structural and signaling pathways analyses and deletion mutant studies have suggested the presence of three major quorum sensing systems (AI-1, Peptide based and AI-2) in extremophiles. Autoinducer-1(AI-1) system was found to be most prevalent (except in thermophiles where it is autoinducer-2) while peptide based system was least prevalent in extremophiles. Some unknown mechanisms of quorum sensing have also been reported which need further exploration. Quorum sensing is utilized by extremophiles for processes like cold adaptation, lowering the freezing point, biofilm formation, oxidative stress resistance and persister cell formation. Explication of quorum sensing in extreme environments may provide discernment regarding the role and functional strategies for survival of extremophiles. Here the role of quorum sensing in different classes of extremophiles and also in their survival strategies has been reviewed. Further, the applications and problems caused by quorum sensing regulated factors in extreme environments are discussed.

A thermophilic microorganism from Deception Island, Antarctica with a thermostable glutamate dehydrogenase activity.

BACKGROUND: The Antarctic continent is a source of extreme microorganisms. Millions of years of isolation have produced unique biodiversity with adaptive responses to its extreme environment. Although the Antarctic climate is mainly cold, the presence of several geothermal sites, including thermal springs, fumaroles, hot soils and hydrothermal vents, provides ideal environments for the development of thermophilic and hyperthermophilic microorganisms. Their enzymes, called thermoenzymes, are the focus of interest in both academic and industrial research, mainly due to their high thermal activity and stability. Glutamate dehydrogenase, is an enzyme that plays a key role in the metabolism of carbon and nitrogen catalyzing reversibly the oxidative deamination of glutamate to alpha-ketoglutarate and ammonium. It belongs to the family of oxidoreductases, is widely distributed and it has been highly regarded for use as biosensors, particularly for their specificity and ability to operate in photochemical and electrochemical systems. However, the use of enzymes as biosensors is relatively problematic due to their instability to high temperatures, organic solvents and denaturing agents. The purpose of this study is to present the partial characterization of a thermophilic microorganism isolated from Deception Island, Antarctica, that displays glutamate dehydrogenase activity. RESULTS: In this work, we report the isolation of a thermophilic microorganism called PID15 from samples of Deception Island collected during the Antarctic Scientific Expedition ECA 46. This microorganism is a thermophile that grows optimally at 50 °C and pH 8.0. Scanning electron microscopy shows rod cells of 2.0 to 8.0 µm of length. Phylogenetic analysis of 16S rRNA gene revealed that this microorganism is closely related to Bacillus gelatini. This microorganism contains a thermostable glutamate dehydrogenase with optimal activity at pH 8.0 and temperatures for its activity from 37 to 50 °C, range of temperature of interest for biotechnological applications. This glutamate dehydrogenase is a highly thermostable enzyme. CONCLUSION: This is the first report of a microorganism from Antarctica containing a thermostable glutamate dehydrogenase that maintains its activity in a broad range of temperatures making it of potential interest for biotechnological applications.

Transcriptomic view of survival during early seedling growth of the extremophyte Haloxylon ammodendron.

Seedling establishment in an extreme environment requires an integrated genomic and physiological response to survive multiple abiotic stresses. The extremophyte, Haloxylon ammodendron is a pioneer species capable of colonizing temperate desert sand dunes. We investigated the induced and basal transcriptomes in H. ammodendron under water-deficit stress during early seedling establishment. We find that not only drought-responsive genes, but multiple genes in pathways associated with salt, osmotic, cold, UV, and high-light stresses were induced, suggesting an altered regulatory stress response system. Additionally, H. ammodendron exhibited enhanced biotic stress tolerance by down-regulation of genes that were generally up-regulated during pathogen entry in susceptible plants. By comparing the H. ammodendron basal transcriptome to six closely related transcriptomes in Amaranthaceae, we detected enriched basal level transcripts in H. ammodendron that shows preadaptation to abiotic stress and pathogens. We found transcripts that were generally maintained at low levels and some induced only under abiotic stress in the stress-sensitive model, Arabidopsis thaliana to be highly expressed under basal conditions in the Amaranthaceae transcriptomes including H. ammodendron. H. ammodendron shows coordinated expression of genes that regulate stress tolerance and seedling development resource allocation to support survival against multiple stresses in a sand dune dominated temperate desert environment.

Survival of Extremophilic Yeasts in the Stratospheric Environment during Balloon Flights and in Laboratory Simulations.

The high-altitude atmosphere is a harsh environment with extremely low temperatures, low pressure, and high UV irradiation. For this reason, it has been proposed as an analogue for Mars, presenting deleterious factors similar to those on the surface of that planet. We evaluated the survival of extremophilic UV-resistant yeasts isolated from a high-elevation area in the Atacama Desert under stratospheric conditions. As biological controls, intrinsically resistant Bacillus subtilis spores were used. Experiments were performed in two independent stratospheric balloon flights and with an environmental simulation chamber. The three following different conditions were evaluated: (i) desiccation, (ii) desiccation plus exposure to stratospheric low pressure and temperature, and (3) desiccation plus exposure to the full stratospheric environment (UV, low pressure, and temperature). Two strains, Naganishia (Cryptococcus) friedmannii 16LV2 and Exophiala sp. strain 15LV1, survived full exposures to the stratosphere in larger numbers than did B. subtilis spores. Holtermanniella watticus (also known as Holtermanniella wattica) 16LV1, however, suffered a substantial loss in viability upon desiccation and did not survive the stratospheric UV exposure. The remarkable resilience of N. friedmannii and Exophiala sp. 15LV1 under the extreme Mars-like conditions of the stratosphere confirms its potential as a eukaryotic model for astrobiology. Additionally, our results with N. friedmannii strengthen the recent hypothesis that yeasts belonging to the Naganishia genus are fit for aerial dispersion, which might account for the observed abundance of this species in high-elevation soils.IMPORTANCE Studies of eukaryotic microorganisms under conditions of astrobiological relevance, as well as the aerial dispersion potential of extremophilic yeasts, are still lacking in the literature compared to works with bacteria. Using stratospheric balloon flights and a simulation chamber, we demonstrate that yeasts isolated from an extreme environment are capable of surviving all stressors found in the stratosphere, including intense UV irradiation, scoring an even higher survival than B. subtilis spores. Notably, the yeast N. friedmannii, which displayed one of the highest tolerances to the stratospheric environment in the experiments, was recently proposed to be adapted to airborne transportation, although such a hypothesis had not yet been tested. Our results strengthen such an assumption and can help explain the observed distribution and ecology of this particular yeast species.

A thermophilic microorganism from Deception Island, Antarctica with a thermostable glutamate dehydrogenase activity

BACKGROUND: The Antarctic continent is a source of extreme microorganisms. Millions of years of isolation have produced unique biodiversity with adaptive responses to its extreme environment. Although the Antarctic climate is mainly cold, the presence of several geothermal sites, including thermal springs, fumaroles, hot soils and hydrothermal vents, provides ideal environments for the development of thermophilic and hyperthermophilic microorganisms. Their enzymes, called thermoenzymes, are the focus of interest in both academic and industrial research, mainly due to their high thermal activity and stability. Glutamate dehydrogenase, is an enzyme that plays a key role in the metabolism of carbon and nitrogen catalyzing reversibly the oxidative deamination of glutamate to alpha-ketoglutarate and ammonium. It belongs to the family of oxidoreductases, is widely distributed and it has been highly regarded for use as biosensors, particularly for their specificity and ability to operate in photochemical and electrochemical systems. However, the use of enzymes as biosensors is relatively problematic due to their instability to high temperatures, organic solvents and denaturing agents. The purpose of this study is to present the partial characterization of a thermophilic microorganism isolated from Deception Island, Antarctica, that displays glutamate dehydrogenase activity. RESULTS: In this work, we report the isolation of a thermophilic microorganism called PID15 from samples of Deception Island collected during the Antarctic Scientific Expedition ECA 46. This microorganism is a thermophile that grows optimally at 50 °C and pH 8.0. Scanning electron microscopy shows rod cells of 2.0 to 8.0 µm of length. Phylogenetic analysis of 16S rRNA gene revealed that this microorganism is closely related to Bacillus gelatini. This microorganism contains a thermostable glutamate dehydrogenase with optimal activity at pH 8.0 and temperatures for its activity from 37 to 50 °C, range of temperature of interest for biotechnological applications. This glutamate dehydrogenase is a highly thermostable enzyme. CONCLUSION: This is the first report of a microorganism from Antarctica containing a thermostable glutamate dehydrogenase that maintains its activity in a broad range of temperatures making it of potential interest for biotechnological applications.

Transcriptome Signatures of Selection, Drift, Introgression, and Gene Duplication in the Evolution of an Extremophile Endemic Plant.

Plants on serpentine soils provide extreme examples of adaptation to environment, and thus offer excellent models for the study of evolution at the molecular and genomic level. Serpentine outcrops are derived from ultramafic rock and have extremely low levels of essential plant nutrients (e.g., N, P, K, and Ca), as well as toxic levels of heavy metals (e.g., Ni, Cr, and Co) and low moisture availability. These outcrops provide habitat to a number of endemic plant species, including the annual mustard Caulanthus amplexicaulis var. barbarae (Cab) (Brassicaceae). Its sister taxon, C. amplexicaulis var. amplexicaulis (Caa), is intolerant to serpentine soils. Here, we assembled and annotated comprehensive reference transcriptomes of both Caa and Cab for use in protein coding sequence comparisons. A set of 29,443 reciprocal best Blast hit (RBH) orthologs between Caa and Cab was compared with identify coding sequence variants, revealing a high genome-wide dN/dS ratio between the two taxa (mean = 0.346). We show that elevated dN/dS likely results from the composite effects of genetic drift, positive selection, and the relaxation of negative selection. Further, analysis of paralogs within each taxon revealed the signature of a period of elevated gene duplication (∼10 Ma) that is shared with other species of the tribe Thelypodieae, and may have played a role in the striking morphological and ecological diversity of this tribe. In addition, distribution of the synonymous substitution rate, dS, is strongly bimodal, indicating a history of reticulate evolution that may have contributed to serpentine adaptation.

Comparative genomics of the tardigrades Hypsibius dujardini and Ramazzottius varieornatus.

Tardigrada, a phylum of meiofaunal organisms, have been at the center of discussions of the evolution of Metazoa, the biology of survival in extreme environments, and the role of horizontal gene transfer in animal evolution. Tardigrada are placed as sisters to Arthropoda and Onychophora (velvet worms) in the superphylum Panarthropoda by morphological analyses, but many molecular phylogenies fail to recover this relationship. This tension between molecular and morphological understanding may be very revealing of the mode and patterns of evolution of major groups. Limnoterrestrial tardigrades display extreme cryptobiotic abilities, including anhydrobiosis and cryobiosis, as do bdelloid rotifers, nematodes, and other animals of the water film. These extremophile behaviors challenge understanding of normal, aqueous physiology: how does a multicellular organism avoid lethal cellular collapse in the absence of liquid water? Meiofaunal species have been reported to have elevated levels of horizontal gene transfer (HGT) events, but how important this is in evolution, and particularly in the evolution of extremophile physiology, is unclear. To address these questions, we resequenced and reassembled the genome of H. dujardini, a limnoterrestrial tardigrade that can undergo anhydrobiosis only after extensive pre-exposure to drying conditions, and compared it to the genome of R. varieornatus, a related species with tolerance to rapid desiccation. The 2 species had contrasting gene expression responses to anhydrobiosis, with major transcriptional change in H. dujardini but limited regulation in R. varieornatus. We identified few horizontally transferred genes, but some of these were shown to be involved in entry into anhydrobiosis. Whole-genome molecular phylogenies supported a Tardigrada+Nematoda relationship over Tardigrada+Arthropoda, but rare genomic changes tended to support Tardigrada+Arthropoda.

Molecular Mechanisms Contributing to the Growth and Physiology of an Extremophile Cultured with Dielectric Heating.

The effect of microwave frequency electromagnetic fields on living microorganisms is an active and highly contested area of research. One of the major drawbacks to using mesophilic organisms to study microwave radiation effects is the unavoidable heating of the organism, which has limited the scale (<5 ml) and duration (<1 h) of experiments. However, the negative effects of heating a mesophile can be mitigated by employing thermophiles (organisms able to grow at temperatures of >60°C). This study identified changes in global gene expression profiles during the growth of Thermus scotoductus SA-01 at 65°C using dielectric (2.45 GHz, i.e., microwave) heating. RNA sequencing was performed on cultures at 8, 14, and 24 h after inoculation to determine the molecular mechanisms contributing to long-term cellular growth and survival under microwave heating conditions. Over the course of growth, genes associated with amino acid metabolism, carbohydrate metabolism, and defense mechanisms were upregulated; the number of repressed genes with unknown function increased; and at all time points, transposases were upregulated. Genes involved in cell wall biogenesis and elongation were also upregulated, consistent with the distinct elongated cell morphology observed after 24 h using microwave heating. Analysis of the global differential gene expression data enabled the identification of molecular processes specific to the response of T. scotoductus SA-01 to dielectric heating during growth. IMPORTANCE: The residual heating of living organisms in the microwave region of the electromagnetic spectrum has complicated the identification of radiation-only effects using microorganisms for 50 years. A majority of the previous experiments used either mature cells or short exposure times with low-energy high-frequency radiation. Using global differential gene expression data, we identified molecular processes unique to dielectric heating using Thermus scotoductus SA-01 cultured over 30 h in a commercial microwave digestor. Genes associated with amino acid metabolism, carbohydrate metabolism, and defense mechanisms were upregulated; the number of repressed genes with unknown function increased; and at all time points, transposases were upregulated. These findings serve as a platform for future studies with mesophiles in order to better understand the response of microorganisms to microwave radiation.