Psychrophiles: A journey of hope.

Psychrophiles are organisms living in extremely cold conditions within the temperature range of -20°C to +10°C. These organisms survive in harsh environment by modulating their genetic make-up to thrive in extremely cold conditions. These cold-adaptations are closely associated with changes in the life forms, gene expression, and proteins, enzymes, lipids, etc. This review gives a brief description of the life and genetic adaptations of psychrophiles for their survival in extreme conditions as well as the bioactive compounds that are potential antimicrobials.

Characterisation of a synthetic Archeal membrane reveals a possible new adaptation route to extreme conditions.

It has been proposed that adaptation to high temperature involved the synthesis of monolayer-forming ether phospholipids. Recently, a novel membrane architecture was proposed to explain the membrane stability in polyextremophiles unable to synthesize such lipids, in which apolar polyisoprenoids populate the bilayer midplane and modify its physico-chemistry, extending its stability domain. Here, we have studied the effect of the apolar polyisoprenoid squalane on a model membrane analogue using neutron diffraction, SAXS and fluorescence spectroscopy. We show that squalane resides inside the bilayer midplane, extends its stability domain, reduces its permeability to protons but increases that of water, and induces a negative curvature in the membrane, allowing the transition to novel non-lamellar phases. This membrane architecture can be transposed to early membranes and could help explain their emergence and temperature tolerance if life originated near hydrothermal vents. Transposed to the archaeal bilayer, this membrane architecture could explain the tolerance to high temperature in hyperthermophiles which grow at temperatures over 100 °C while having a membrane bilayer. The induction of a negative curvature to the membrane could also facilitate crucial cell functions that require high bending membranes.

Cryptic Biosynthesis of the Berkeleypenostatins from Coculture of Extremophilic Penicillium sp.

Coculture fermentation of Penicillium fuscum and P. camembertii/clavigerum yielded berkeleypenostatins A-G (1-7) as well as the previously reported berkeleylactones A-H, the known macrolide A26771B, citrinin, and patulin. As was true with the berkeleylactones, there was no evidence of the berkeleypenostatins in either axenic culture. The structures were deduced from analyses of spectral data, and the absolute configuration of berkeleypenostatin A (1) was determined by single-crystal X-ray crystallography. Berkeleypenostatins A (1) and E (5) inhibited migration of human pancreatic carcinoma cells (HPAF-II). Both compounds were tested by the NCI Developmental Therapeutics Program. In the NCI 60 cell five-dose screen, berkeleypenostatin E (5) was the more active of the two, with 1-10 µM total growth inhibition (TGI) of all leukemia cell lines, as well as the majority of colon, CNS, melanoma, ovarian, prostate, renal, and breast cancer cell lines.

Computational Analysis of Thermal Adaptation in Extremophilic Chitinases: The Achilles' Heel in Protein Structure and Industrial Utilization.

Understanding protein stability is critical for the application of enzymes in biotechnological processes. The structural basis for the stability of thermally adapted chitinases has not yet been examined. In this study, the amino acid sequences and X-ray structures of psychrophilic, mesophilic, and hyperthermophilic chitinases were analyzed using computational and molecular dynamics (MD) simulation methods. From the findings, the key features associated with higher stability in mesophilic and thermophilic chitinases were fewer and/or shorter loops, oligomerization, and less flexible surface regions. No consistent trends were observed between stability and amino acid composition, structural features, or electrostatic interactions. Instead, unique elements affecting stability were identified in different chitinases. Notably, hyperthermostable chitinase had a much shorter surface loop compared to psychrophilic and mesophilic homologs, implying that the extended floppy surface region in cold-adapted and mesophilic chitinases may have acted as a "weak link" from where unfolding was initiated. MD simulations confirmed that the prevalence and flexibility of the loops adjacent to the active site were greater in low-temperature-adapted chitinases and may have led to the occlusion of the active site at higher temperatures compared to their thermostable homologs. Following this, loop "hot spots" for stabilizing and destabilizing mutations were also identified. This information is not only useful for the elucidation of the structure-stability relationship, but will be crucial for designing and engineering chitinases to have enhanced thermoactivity and to withstand harsh industrial processing conditions.

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.

MALDI-TOF analysis of a novel extremophile peptide purified from Halarchaeum acidiphilum ASDL78 with antiarchaeal and antibacterial activities.

In hypersaline environments, halophilic archaea synthesize antimicrobial substances called halocins. There is a promise to make new drugs for antibiotic-resistant strains. Here, we report the antibacterial activity of a new haloarchaea selected from Lut Desert, Iran. A total of 38 isolated halophilic bacteria and archaea were screened for the antagonistic activity test of each strain against other bacterial and archaeal strains. Finally, a strain, recognized as Halarchaeum acidiphilum, with a fast grown strain and high antagonistic potential against different strains was identified by morphological, physiological, and molecular characteristics. The halocin was produced in a semisolid submerge medium and partially purified by heat treatments and molecular weight ultrafiltration cutoff (3, 50, and 10 kDa). It was a cell-free, heat-resistant (85°C for 2 h) protein with a molecular mass near to 20 kDa produced at the endpoint of logarithmic growth. The molecular weight of halocin was 17 kDa, and indicated no apparent homology with known halocins, suggesting that this might be a new halocin. Therefore, a new strain belonging to Halarchaeum genus was isolated and characterized here that produced an antimicrobial and anti-haloarchaea halocin.

Efficient One-Pot Synthesis of Cytidine 5'-Monophosphate Using an Extremophilic Enzyme Cascade System.

A rapid in vitro enzymatic biosynthesis system has been developed as a biological manufacturing platform with potential industrial uses. Cytidine 5'-monophosphate (5'-CMP) is a key intermediate in the preparation of several nucleotide derivatives and is widely used in food and pharmaceutical industries. In this study, a highly efficient biosynthesis system was constructed for manufacturing 5'-CMP in vitro. Cytidine kinase (CK) was used for the biotransformation of cytidine to 5'-CMP, while polyphosphate kinase (PPK) was coupled for adenosine triphosphate regeneration. Both CK and PPK were selected from extremophiles, possessing great potential for biocatalytic synthesis. The effects of temperature, substrate concentration, and enzyme ratios were investigated to enhance the titer and yield of 5'-CMP. After optimization, 96 mM 5'-CMP was produced within 6 h, and the yield reached nearly 100%. This work highlights the ease of 5'-CMP production by an in vitro biomanufacturing platform and provides a green and efficient approach for the industrial synthesis of 5'-CMP.

Exploring the potentials of halophilic prokaryotes from a solar saltern for synthesizing nanoparticles: The case of silver and selenium.

Halophiles are the organisms that thrive in extreme high salt environments. Despite the extensive studies on their biotechnological potentials, the ability of halophilic prokaryotes for the synthesis of nanoparticles has remained understudied. In this study, the archaeal and bacterial halophiles from a solar saltern were investigated for the intracellular/extracellular synthesis of silver and selenium nanoparticles. Silver nanoparticles were produced by the archaeal Haloferax sp. (AgNP-A, intracellular) and the bacterial Halomonas sp. (AgNP-B, extracellular), while the intracellular selenium nanoparticles were produced by the archaeal Halogeometricum sp. (SeNP-A) and the bacterial Bacillus sp. (SeNP-B). The nanoparticles were characterized by various techniques including UV-Vis spectroscopy, XRD, DLS, ICP-OES, Zeta potentials, FTIR, EDX, SEM, and TEM. The average particle size of AgNP-A and AgNP-B was 26.34 nm and 22 nm based on TEM analysis. Also, the characteristic Bragg peaks of face-centered cubic with crystallite domain sizes of 13.01 nm and 6.13 nm were observed in XRD analysis, respectively. Crystallographic characterization of SeNP-A and SeNP-B strains showed a hexagonal crystallite structure with domain sizes of 30.63 nm and 29.48 nm and average sizes of 111.6 nm and 141.6 nm according to TEM analysis, respectively. The polydispersity index of AgNP-A, AgNP-B, SeNP-A, and SeNP-B was determined as 0.26, 0.28, 0.27, and 0.36 and revealed high uniformity of the nanoparticles. All of the synthesized nanoparticles were stable and their zeta potentials were calculated as (mV): -33.12, -35.9, -31.2, and -29.34 for AgNP-A, AgNP-B, SeNP-A, and SeNP-B, respectively. The nanoparticles showed the antibacterial activity against various bacterial pathogens. The results of this study suggested that the (extremely) halophilic prokaryotes have great potentials for the green synthesis of nanoparticles.

Plastic Silica Conglomerate with an Extremophile Microbial Matrix in a Hot-Water Stream Paleoenvironment.

A new and unusual type of fossil, siliceous hot-spring deposit (sinter)-comprising monomictic, quartzose conglomerate encrusted with silicified microbial laminates-has been recognized in distal portions of Jurassic and Miocene paleo-geothermal fields of South and North America, respectively. The siliceous clasts are inferred to have originated as conduit-delivered hydrothermal silica gel, owing to their general plastic morphologies, which were then locally reworked and redistributed in geothermally influenced stream paleoenvironments. Today, hot-spring-fed streams and creeks, in places with silica-armored pavements, host microbial mats coating streambeds and/or growing over, and silicifying at, stream air-water interfaces, for example, in Yellowstone National Park (USA) and Waimangu Volcanic Valley (New Zealand). However, the modern deposits do not contain the plastically deformed silica cobbles evident in Mesozoic and Cenozoic examples described herein. Moreover, the fossil microbial laminates of this study are relatively dense and strongly coat the silica cobbles, suggesting the mats stabilized the clasts under fully submerged and hot, high-energy conditions. Thus, this new sinter facies, typically found a few kilometers from main spring-vent areas, is a perhaps unexpected extreme environment in which life took hold in hydrothermal-fluvial settings of the past, and may serve as an additional target in the search for fossil biosignatures of early Earth and possibly Mars.

Characterization and DNA methylation modulatory activity of gold nanoparticles synthesized by Pseudoalteromonas strain.

Marine extremophiles are shown to tolerate extreme environmental conditions and have high metal reducing properties. Here, we report intracellular synthesis of gold nanoparticles (AuNP) by marine extremophilic bacteria Pseudoalteromonas sp. Bac178 which was isolated from the OMZ of Arabian Sea. Preliminary observations suggest that these bacteria use different pathways which may involves the membrane as well as intracellular proteins for the gold salt reduction. Characterization of the biosynthesised nanoparticles by various techniques such as Scanning electron microscopy (SEM), Transmission electron microscopy (TEM), X-ray diffraction (XRD) and Energy-dispersive X-ray spectroscopy (EDS) confirmed the presence of crystalline gold. These biologically synthesized AuNP were investigated for cytotoxicity and oxidative stress generation in human normal fibroblast and melanoma cells (A375). As AuNP are envisaged to find many applications in the medical field, it was of interest to study the effect of AuNP at the epigenetic level. They were found to be non-cytotoxic, non-genotoxic and non-oxidative stress generating over a range of concentrations. Exposure to these AuNP is observed to cause alterations in global DNA methylation as well as in the expression of DNA methyltransferase (DNMT) genes. Since biosynthesized AuNP are being used in various applications and therapies, their epigenetic modulatory activity needs careful consideration.

Global effect of the lack of inorganic polyphosphate in the extremophilic archaeon Sulfolobus solfataricus: A proteomic approach.

Inorganic polyphosphates (polyP) are present in all living cells and several important functions have been described for them. They are involved in the response to stress conditions, such as nutrient depletion, oxidative stress and toxic metals amongst others. A recombinant strain of Sulfolobus solfataricus unable to accumulate polyP was designed by the overexpression of its endogenous ppx gene. The overall impact of the lack of polyP on this S. solfataricus polyP (-) strain was analyzed by using quantitative proteomics (isotope-coded protein label, ICPL). Stress-related proteins, such as peroxiredoxins and heat shock proteins, proteins involved in metabolism and several others were produced at higher levels in the ppx expression strain. The polyP deficient strain showed an increased copper sensitivity and an earlier transcriptional up-regulation of copA gene coding for the P-type copper-exporting ATPase. This implies a complementary function of both copper resistance systems. These results strongly suggests that the lack of polyP makes this hyperthermophilic archaeon more sensitive to toxic conditions, such as an exposure to metals or other harmful stimuli, emphasizing the importance of this inorganic phosphate polymers in the adaptations to live in the environmental conditions in which thermoacidophilic archaea thrive. SIGNIFICANCE: Inorganic polyphosphate (polyP) are ubiquitous molecules with many functions in living organisms. Few studies related to these polymers have been made in archaea. The construction of a polyP deficient recombinant strain of Sulfolobus solfataricus allowed the study of the global changes in the proteome of this thermoacidophilic archaeon in the absence of polyP compared with the wild type strain. The results obtained using quantitative proteomics suggest an important participation of polyP in the oxidative stress response of the cells and as having a possible metabolic role in the cell, as previously described in bacteria. The polyP deficient strain also showed an increased copper sensitivity and an earlier transcriptional up-regulation of copA, implying a complementary role of both copper resistance systems.

First report of the in vitro antileishmanial properties of extremophile plants from the Algarve Coast.

This work reports for the first time the in vitro anti Leishmania infantum activity of acetone and dichloromethane (DCM) extracts from 25 extremophile plants from Southern Portugal. DCM extracts from Inula chritmoides and Spergularia rubra were active against axenic promastigotes and intracellular amastigotes, had anti-inflammatory properties on lipopolysaccharide (LPS)-stimulated macrophages, inhibited acetylcholinesterase and had no haemolytic activity on human erythrocytes. Eleven phenolics were identified by high-performance liquid chromatography with diode-array detection (HPLC-DAD) in I. crithmoides and one phenolic in S. rubra. Isolation and identification of the active molecules is in progress.

The Deep-Sea Polyextremophile Halobacteroides lacunaris TB21 Rough-Type LPS: Structure and Inhibitory Activity towards Toxic LPS.

The structural characterization of the lipopolysaccharide (LPS) from extremophiles has important implications in several biomedical and therapeutic applications. The polyextremophile Gram-negative bacterium Halobacteroideslacunaris TB21, isolated from one of the most extreme habitats on our planet, the deep-sea hypersaline anoxic basin Thetis, represents a fascinating microorganism to investigate in terms of its LPS component. Here we report the elucidation of the full structure of the R-type LPS isolated from H. lacunaris TB21 that was attained through a multi-technique approach comprising chemical analyses, NMR spectroscopy, and Matrix-Assisted Laser Desorption Ionization (MALDI) mass spectrometry. Furthermore, cellular immunology studies were executed on the pure R-LPS revealing a very interesting effect on human innate immunity as an inhibitor of the toxic Escherichia coli LPS.

Derivative of Extremophilic 50S Ribosomal Protein L35Ae as an Alternative Protein Scaffold.

Small antibody mimetics, or alternative binding proteins (ABPs), extend and complement antibody functionality with numerous applications in research, diagnostics and therapeutics. Given the superiority of ABPs, the last two decades have witnessed development of dozens of alternative protein scaffolds (APSs) for the design of ABPs. Proteins from extremophiles with their high structural stability are especially favorable for APS design. Here, a 10X mutant of the 50S ribosomal protein L35Ae from hyperthermophilic archaea Pyrococcus horikoshii has been probed as an APS. A phage display library of L35Ae 10X was generated by randomization of its three CDR-like loop regions (repertoire size of 2×108). Two L35Ae 10X variants specific to a model target, the hen egg-white lysozyme (HEL), were isolated from the resulting library using phage display. The affinity of these variants (L4 and L7) to HEL ranges from 0.10 µM to 1.6 µM, according to surface plasmon resonance data. While L4 has 1-2 orders of magnitude lower affinity to HEL homologue, bovine α-lactalbumin (BLA), L7 is equally specific to HEL and BLA. The reference L35Ae 10X is non-specific to both HEL and BLA. L4 and L7 are more resistant to denaturation by guanidine hydrochloride compared to the reference L35Ae 10X (mid-transition concentration is higher by 0.1-0.5 M). Chemical crosslinking experiments reveal an increased propensity of L4 and L7 to multimerization. Overall, the CDR-like loop regions of L35Ae 10X represent a proper interface for generation of functional ABPs. Hence, L35Ae is shown to extend the growing family of protein scaffolds dedicated to the design of novel binding proteins.

Pnserpin: A Novel Serine Protease Inhibitor from Extremophile Pyrobaculum neutrophilum.

Serine protease inhibitors (serpins) are native inhibitors of serine proteases, constituting a large protein family with members spread over eukaryotes and prokaryotes. However, only very few prokaryotic serpins, especially from extremophiles, have been characterized to date. In this study, Pnserpin, a putative serine protease inhibitor from the thermophile Pyrobaculum neutrophilum, was overexpressed in Escherichia coli for purification and characterization. It irreversibly inhibits chymotrypsin-, trypsin-, elastase-, and subtilisin-like proteases in a temperature range from 20 to 100 °C in a concentration-dependent manner. The stoichiometry of inhibition (SI) of Pnserpin for proteases decreases as the temperature increases, indicating that the inhibitory activity of Pnserpin increases with the temperature. SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) showed that Pnserpin inhibits proteases by forming a SDS-resistant covalent complex. Homology modeling and molecular dynamic simulations predicted that Pnserpin can form a stable common serpin fold. Results of the present work will help in understanding the structural and functional characteristics of thermophilic serpin and will broaden the current knowledge about serpins from extremophiles.

SOD activity and extremophilicity: a screening of various plant species.

All aerobes are dependent on enzymatic and non-enzymatic antioxidants to withstand the presence of reactive oxygen species (ROS). Superoxide dismutase (SOD) is a part of the enzymatic antioxidant system. It is one of the most important antioxidant enzymes, enabling organisms to survive in an oxygen containing atmosphere. A disorder in the oxidative and antioxidative balance can be associated with the occurrence of diseases in human organisms. Little data exist on the relevance of SOD in plants. Moreover, it is not known whether there is any association between a plant's origin and its SOD activity. Our screening of 27 different plant species was intended to expose whether there is a connection. The highest SOD activities were found for extremophile plants. Especially the Crassulaceae Aeonium haworthii Salm-Dyck Ex Webb & Berthel. and Crassula multiflora Schönland & Baker F. were highly active. Nevertheless, we did not find unambiguous evidence for a correlation between extremophilicity and SOD activity.

Extremophiles as sources of inorganic bio-nanoparticles.

Industrial use of nanotechnology in daily life has produced an emphasis on the safe and efficient production of nanoparticles (NPs). Traditional chemical oxidation and reduction methods are seen as inefficient, environmentally unsound, and often dangerous to those exposed and involved in NP manufacturing. However, utilizing microorganisms for biosynthesis of NPs allows efficient green production of a range of inorganic NPs, while maintaining specific size, shape, stability, and dispersity. Microorganisms living under harsh environmental conditions, called "Extremophiles," are one group of microorganisms being utilized for this biosynthesis. Extremophiles' unique living conditions have endowed them with various processes that enable NP biosynthesis. This includes a range of extremophiles: thermophiles, acidophilus, halophiles, psychrophiles, anaerobes, and some others. Fungi, bacteria, yeasts, and archaea, i.e. Ureibacillus thermosphaericus, and Geobacillus stearothermophilus, among others, have been established for NP biosynthesis. This article highlights the extremophiles and methods found to be viable candidates for the production of varying types of NPs, as well as interpreting selective methods used by the organisms to synthesize NPs.

Trading off stability against activity in extremophilic aldolases.

Understanding enzyme stability and activity in extremophilic organisms is of great biotechnological interest, but many questions are still unsolved. Using 2-deoxy-D-ribose-5-phosphate aldolase (DERA) as model enzyme, we have evaluated structural and functional characteristics of different orthologs from psychrophilic, mesophilic and hyperthermophilic organisms. We present the first crystal structures of psychrophilic DERAs, revealing a dimeric organization resembling their mesophilic but not their thermophilic counterparts. Conversion into monomeric proteins showed that the native dimer interface contributes to stability only in the hyperthermophilic enzymes. Nevertheless, introduction of a disulfide bridge in the interface of a psychrophilic DERA did confer increased thermostability, suggesting a strategy for rational design of more durable enzyme variants. Constraint network analysis revealed particularly sparse interactions between the substrate pocket and its surrounding α-helices in psychrophilic DERAs, which indicates that a more flexible active center underlies their high turnover numbers.