Archivory in hypersaline aquatic environments: haloarchaea as a dietary source for the brine shrimp Artemia.

Archaea have been the most overlooked and enigmatic of the three domains of life for decades. Knowledge of key ecological interactions, such as trophic links between this domain and higher level organisms, remains extremely limited. The co-occurrence of halophilic Archaea (haloarchaea) and the non-selective filter feeder, brine shrimp Artemia under the unique ecological characteristics of hypersaline aquatic environments, constitutes an excellent opportunity to further unravel the ecological role of the Archaea domain as a source of food to zooplankton metazoans. In the present study, we combine the use of haloarchaea biomass assimilation experiments using 13C isotope as tracer, with gnotobiotic Artemia culture tests using haloarchaea mono-diets, to investigate potential trophic links between the organisms. Our results demonstrated the ability of Artemia to assimilate nutrients from mono-diets of haloarchaea biomass in order to survive and grow, providing clear indications that archivory may occur in hypersaline aquatic environments. Additionally, our study highlights the use of stable isotopes labelling as a potential tool to further disentangle the specific pathways by which archaeal cellular constituents are digested by consumers.

Halophiles and Their Biomolecules: Recent Advances and Future Applications in Biomedicine.

The organisms thriving under extreme conditions better than any other organism living on Earth, fascinate by their hostile growing parameters, physiological features, and their production of valuable bioactive metabolites. This is the case of microorganisms (bacteria, archaea, and fungi) that grow optimally at high salinities and are able to produce biomolecules of pharmaceutical interest for therapeutic applications. As along as the microbiota is being approached by massive sequencing, novel insights are revealing the environmental conditions on which the compounds are produced in the microbial community without more stress than sharing the same substratum with their peers, the salt. In this review are reported the molecules described and produced by halophilic microorganisms with a spectrum of action in vitro: antimicrobial and anticancer. The action mechanisms of these molecules, the urgent need to introduce alternative lead compounds and the current aspects on the exploitation and its limitations are discussed.

Ecology and physiology of halophilic microorganisms - Thematic issue based on papers presented at Halophiles 2019 - 12th International Conference on Halophilic Microorganisms, Cluj-Napoca, Romania, 24-28 June, 2019.

In June 2019, the 12th International Conference on Halophilic Microorganisms - Halophiles 2019, was held in Cluj-Napoca, Romania. This thematic issue of FEMS Microbiology Letters contains papers based on lectures and posters presented at the conference. We here provide a short overview of past research on hypersaline environments in Romania and the microorganisms inhabiting them, and briefly present the papers published in this thematic issue.

Biochemical and thermodynamic analyses of energy conversion in extremophiles.

A variety of extreme environments, characterized by extreme values of various physicochemical parameters (temperature, pressure, salinity, pH, and so on), are found on Earth. Organisms that favorably live in such extreme environments are called extremophiles. All living organisms, including extremophiles, must acquire energy to maintain cellular homeostasis, including extremophiles. For energy conversion in harsh environments, thermodynamically useful reactions and stable biomolecules are essential. In this review, I briefly summarize recent studies of extreme environments and extremophiles living in these environments and describe energy conversion processes in various extremophiles based on my previous research. Furthermore, I discuss the correlation between the biological system of electrotrophy, a third biological energy acquisition system, and the mechanism underlying microbiologically influenced corrosion. These insights into energy conversion in extremophiles may improve our understanding of the "limits of life". Abbreviations: PPi: pyrophosphate; PPase: pyrophosphatase; ITC: isothermal titration microcalorimetry; SVNTase: Shewanella violacea 5'-nucleotidase; SANTase: Shewanella amazonensis 5'-nucleotidase.

Fructans of the saline world.

Saline and hypersaline environments make up the largest ecosystem on earth and the organisms living in such water-restricted environments have developed unique ways to cope with high salinity. As such these organisms not only carry significant industrial potential in a world where freshwater supplies are rapidly diminishing, but they also shed light upon the origins and extremes of life. One largely overlooked and potentially important feature of many salt-loving organisms is their ability to produce fructans, fructose polymers widely found in various mesophilic Eubacteria and plants, with potential functions as storage carbohydrates, aiding stress tolerance, and acting as virulence factors or signaling molecules. Intriguingly, within the whole archaeal domain of life, Archaea possessing putative fructan biosynthetic enzymes were found to belong to the extremely halophilic class of Halobacteria only, indicating a strong, yet unexplored link between the fructan syndrome and salinity. In fact, this link may indeed lead to novel strategies in fighting the global salinization problem. Hence this review explores the unknown world of fructanogenic salt-loving organisms, where water scarcity is the main stress factor for life. Within this scope, prokaryotes and plants of the saline world are discussed in detail, with special emphasis on their salt adaptation mechanisms, the potential roles of fructans and fructosyltransferase enzymes in adaptation and survival as well as future aspects for all fructanogenic salt-loving domains of life.

Halophilic Protein Adaptation Results from Synergistic Residue-Ion Interactions in the Folded and Unfolded States.

Halophilic organisms thrive in environments with extreme salt concentrations and have adapted by allowing molar quantities of cosolutes, mainly KCl, to accumulate in their cytoplasm. To cope with this high intracellular salinity, halophilic organisms modified the chemical composition of their proteins to enrich their surface with acidic and short polar side chains, while lysines and bulky hydrophobic residues got depleted. We have emulated the evolutionary process of haloadaptation with natural and designed halophilic polypeptides and applied novel nuclear magnetic resonance (NMR) methodology to study the different mechanisms contributing to protein haloadaptation at a per residue level. Our analysis of an extensive set of NMR observables, determined over several proteins, allowed us to disentangle the synergistic contributions of protein haloadaptation: cation exclusion and electrostatic repulsion between negatively charged residues destabilize the denatured state ensemble while cumulative weak cation-protein interactions stabilize the folded conformations.

Microbial weeds in hypersaline habitats: the enigma of the weed-like Haloferax mediterranei.

Heterotrophic prokaryotic communities that inhabit saltern crystallizer ponds are typically dominated by two species, the archaeon Haloquadratum walsbyi and the bacterium Salinibacter ruber, regardless of location. These organisms behave as 'microbial weeds' as defined by Cray et al. (Microb Biotechnol 6: 453-492, 2013) that possess the biological traits required to dominate the microbiology of these open habitats. Here, we discuss the enigma of the less abundant Haloferax mediterranei, an archaeon that grows faster than any other, comparable extreme halophile. It has a wide window for salt tolerance, can grow on simple as well as on complex substrates and degrade polymeric substances, has different modes of anaerobic growth, can accumulate storage polymers, produces gas vesicles, and excretes halocins capable of killing other Archaea. Therefore, Hfx. mediterranei is apparently more qualified as a 'microbial weed' than Haloquadratum and Salinibacter. However, the former differs because it produces carotenoid pigments only in the lower salinity range and lacks energy-generating retinal-based, light-driven ion pumps such as bacteriorhodopsin and halorhodopsin. We discuss these observations in relation to microbial weed biology in, and the open-habitat ecology of, hypersaline systems.

Trehalose/2-sulfotrehalose biosynthesis and glycine-betaine uptake are widely spread mechanisms for osmoadaptation in the Halobacteriales.

We investigated the mechanisms of osmoadaptation in the order Halobacteriales, with special emphasis on Haladaptatus paucihalophilus, known for its ability to survive in low salinities. H. paucihalophilus genome contained genes for trehalose synthesis (trehalose-6-phosphate synthase/trehalose-6-phosphatase (OtsAB pathway) and trehalose glycosyl-transferring synthase pathway), as well as for glycine betaine uptake (BCCT family of secondary transporters and QAT family of ABC transporters). H. paucihalophilus cells synthesized and accumulated ∼1.97-3.72 µmol per mg protein of trehalose in a defined medium, with its levels decreasing with increasing salinities. When exogenously supplied, glycine betaine accumulated intracellularly with its levels increasing at higher salinities. RT-PCR analysis strongly suggested that H. paucihalophilus utilizes the OtsAB pathway for trehalose synthesis. Out of 83 Halobacteriales genomes publicly available, genes encoding the OtsAB pathway and glycine betaine BCCT family transporters were identified in 38 and 60 genomes, respectively. Trehalose (or its sulfonated derivative) production and glycine betaine uptake, or lack thereof, were experimentally verified in 17 different Halobacteriales species. Phylogenetic analysis suggested that trehalose synthesis is an ancestral trait within the Halobacteriales, with its absence in specific lineages reflecting the occurrence of gene loss events during Halobacteriales evolution. Analysis of multiple culture-independent survey data sets demonstrated the preference of trehalose-producing genera to saline and low salinity habitats, and the dominance of genera lacking trehalose production capabilities in permanently hypersaline habitats. This study demonstrates that, contrary to current assumptions, compatible solutes production and uptake represent a common mechanism of osmoadaptation within the Halobacteriales.

Cellular and organellar membrane-associated proteins in haloarchaea: perspectives on the physiological significance and biotechnological applications.

Halophilic archaea (haloarchaea) inhabit hypersaline environments, tolerating extreme salinity, low oxygen and nutrient availability, and in some cases, high pH (soda lakes) and irradiation (saltern ponds). Membrane-associated proteins of haloarchaea, such as surface layer (S-layer) proteins, transporters, retinal proteins, and internal organellar membrane proteins including intracellular gas vesicle proteins and those associated with polyhydroxyalkanoate (PHA) granules, contribute greatly to their environmental adaptations. This review focuses on these haloarchaeal cellular and organellar membrane-associated proteins, and provides insight into their physiological significance and biotechnological potential.

Temperature and pH optima of extremely halophilic archaea: a mini-review.

Archaeal microorganisms that grow optimally at Na(+) concentrations of 1.7 M, or the equivalent of 10% (w/v) NaCl, and greater are considered to be extreme halophiles. This review encompasses extremely halophilic archaea and their growth characteristics with respect to the correlation between the extent of alkaline pH and elevated temperature optima and the extent of salt tolerance. The focus is on poly-extremophiles, i.e., taxa growing optimally at a Na(+) concentration at or above 1.7 M (approximately 10% w/v NaCl); alkaline pH, at or above 8.5; and elevated temperature optima, at or above 50°C. So far, only a very few extreme halophiles that are able to grow optimally under alkaline conditions as well as at elevated temperatures have been isolated. The distribution of extremely halophilic archaea growing optimally at 3.4 M Na(+) (approximately 20% w/v NaCl) is bifurcated with respect to pH optima, either they are neutrophilic, with a pH(opt) of approximately 7, or strongly alkaliphilic, with pH(opt) at or above 8.5. Amongst these extreme halophiles which have elevated pH optima, only four taxa have an optimum temperature above 50°C: Haloarcula quadrata (52°C), Haloferax elongans (53°C), Haloferax mediterranei (51°C) and Natronolimnobius 'aegyptiacus' (55°C).

Exploring research frontiers in microbiology: recent advances in halophilic and thermophilic extremophiles.

Extremophilic prokaryotes inhabit ecosystems that are, from a human perspective, extreme, and life in these environments requires far-reaching cellular adaptations. Here, we will describe, for two examples (Thermus thermophilus, Halobacillus halophilus), how thermophilic or halophilic bacteria adapt to their environment; we will describe the molecular basis of sensing and responding to hypersalinity and we will analyze the impact and basis of natural competence for survival in hot environments.

Recombinant expression in moderate halophiles.

A novel expression of recombinant proteins was developed using moderate halophiles that accumulate osmolytes and hence provide cytoplasmic environments where osmolyte-driven folding can take place. Promoters and selection marker were developed for high expression of foreign proteins. Examples are given for expression of bacterial nucleoside diphosphate kinase and human serine racemase.

Molecular signature of hypersaline adaptation: insights from genome and proteome composition of halophilic prokaryotes.

BACKGROUND: Halophilic prokaryotes are adapted to thrive in extreme conditions of salinity. Identification and analysis of distinct macromolecular characteristics of halophiles provide insight into the factors responsible for their adaptation to high-salt environments. The current report presents an extensive and systematic comparative analysis of genome and proteome composition of halophilic and non-halophilic microorganisms, with a view to identify such macromolecular signatures of haloadaptation. RESULTS: Comparative analysis of the genomes and proteomes of halophiles and non-halophiles reveals some common trends in halophiles that transcend the boundary of phylogenetic relationship and the genomic GC-content of the species. At the protein level, halophilic species are characterized by low hydrophobicity, over-representation of acidic residues, especially Asp, under-representation of Cys, lower propensities for helix formation and higher propensities for coil structure. At the DNA level, the dinucleotide abundance profiles of halophilic genomes bear some common characteristics, which are quite distinct from those of non-halophiles, and hence may be regarded as specific genomic signatures for salt-adaptation. The synonymous codon usage in halophiles also exhibits similar patterns regardless of their long-term evolutionary history. CONCLUSION: The generality of molecular signatures for environmental adaptation of extreme salt-loving organisms, demonstrated in the present study, advocates the convergent evolution of halophilic species towards specific genome and amino acid composition, irrespective of their varying GC-bias and widely disparate taxonomic positions. The adapted features of halophiles seem to be related to physical principles governing DNA and protein stability, in response to the extreme environmental conditions under which they thrive.

The tatC gene cluster is essential for viability in halophilic archaea.

In prokaryotes the twin-arginine translocase (Tat) is a unique transport system for the export of folded proteins. The Tat pathway is usually involved in the export of a small proportion of extracytoplasmic proteins. An exception is found in halophilic archaea, in which the majority of secretory proteins have been predicted to be Tat-dependent. All haloarchaea analysed to date contain two genes encoding homologues of the Tat-component TatC. In all of these cases both genes are located adjacently on the chromosome, indicating that they form a functional unit. We show that this gene cluster is essential for viability in haloarchaea, which is in complete contrast to all other prokaryotes that have been tested thus far.

Organic solvent tolerance of halophilic archaea, Haloarcula strains: effects of NaCl concentration on the tolerance and polar lipid composition.

Strains of halophilic archaea, Haloarcula vallismortis and two Haloarcula strains OHF-1 and OHF-2, showed high tolerance to organic solvents at high media NaCl concentrations. For example, the lowest log Pow of the solvent which allowed growth (log Pow is the common logarithm of the partition coefficient of a given solvent in a mixture of n-octanol and water) for H. vallismortis was 5.1 at 20% NaCl and 4.4 at 30% NaCl. The solvent tolerance of Haloarcula argentinensis, on the other hand, was not affected by the NaCl concentration. Cells of strains OHF-1 and OHF-2 were of triangular or irregular morphology but became spherical in cultures in NaCl media overlaid with cyclohexane (log Pow=3.4), but returned to the triangular shape when the organic solvent evaporated from the medium. When cells of strains OHF-1, OHF-2, and H. argentinensis were grown in NaCl media in the presence of n-decane, they contained less phosphatidylglycerol and more phosphatidylglycerosulfate and phosphatidylglycerophosphate methyl ester than when grown without added n-decane. When the solvent was removed from the media after cultivation, the levels of these compounds returned to their initial ones.

Identification and distribution of new insertion sequences in the genome of the extremely halotolerant and alkaliphilic Oceanobacillus iheyensis HTE831.

Six kinds of new insertion sequences (ISs), IS667 to IS672, a group II intron (Oi.Int), and an incomplete transposon (Tn852loi) were identified in the 3,630,528-bp genome of the extremely halotolerant and alkaliphilic Oceanobacillus iheyensis HTE831. Of 19 ISs identified in the HTE831 genome, 7 were truncated, indicating the occurrence of internal rearrangement of the genome. All ISs except IS669 generated a 4- to 8-bp duplication of the target site sequence, and these ISs carried 23- to 28-bp inverted repeats (IRs). Sequence analysis revealed that four ISs (IS669, IS670, IS671, and IS672) were newly identified as belonging to separate IS families (IS200/IS605, IS30, IS5, and IS3, respectively). IS667 and IS668 were also characterized as new members of the ISL3 family. Tn8521oi, which belongs to the Tn3 family as a new member, generated a 5-bp duplication of the target site sequence and carried complete 38-bp IRs. Of the eight protein-coding sequences (CDSs) identified in Tn8521oi, three CDSs (OB481, OB482, and OB483) formed a ger gene cluster, and two other paralogous gene clusters were found in the HTE831 genome. Most of the ISs and the group II intron widely distributed throughout the genome were inserted in noncoding regions, while two ISs (IS667-08 and IS668-02) and Oi.Int-04 were inserted in the coding regions.

Cultivation of Walsby's square haloarchaeon.

The square haloarchaea of Walsby (SHOW group) dominate hypersaline microbial communities but have not been cultured since their discovery 25 years ago. We show that natural water dilution cultures can be used to isolate members of this group and, once in pure culture, they can be grown in standard halobacterial media. Cells display a square morphology and contain gas vesicles and poly-beta-hydroxybutyrate (PHB) granules. The 16S rRNA gene sequence was >99% identical to other SHOW group sequences. They prefer high salinities (23-30%), and can grow with a doubling time of 1-2 days in rich media. The ability to culture SHOW group organisms makes it possible to study, in a comprehensive way, the microbial ecology of salt lakes.

Crowding in extremophiles: linkage between solvation and weak protein-protein interactions, stability and dynamics, provides insight into molecular adaptation.

The study of the molecular adaptation of microorganisms to extreme environments (solvent, temperature, etc.) has provided tools to investigate the complex relationships between protein-solvent and protein-protein interactions, protein stability and protein dynamics, and how they are modulated by the crowded environment of the cell. We have evaluated protein-solvent and protein-protein interactions by solution experiments (analytical ultracentrifugation, small angle neutron and X-ray scattering, density) and crystallography, and protein dynamics by energy resolved neutron scattering. This review concerns work from our laboratory on (i) proteins from extreme halophilic Archaea, and (ii) psychrophile, mesophile, thermophile and hyperthermophile bacterial cells.

Transport of compatible solutes in extremophiles.

Salt-tolerant as well as moderately halophilic and halophilic organisms have to maintain their turgor. One strategy is to accumulate small organic compounds, compatible solutes, by de novo synthesis or uptake. From a bioenergetic point of view, uptake is preferred over biosynthesis. The transport systems catalyzing uptake of compatible solutes are of primary or secondary nature and coupled to ATP hydrolysis or ion (H+, Na+) symport. Expression of the transporter genes as well as the activity of the transporters is regulated by salinity/osmolarity and one of the key questions is how salinity or osmolarity is sensed and the signal transmitted as far as to gene expression and transporter activation. Recent studies shed light on the nature and the activation mechanisms of solute transporters in extremophiles, and this review summarizes current knowledge on the structure, function and osmo- or salt-regulation of transporters for compatible solutes in extremophiles.