Halophilic Research Articles

Identification of extremely halophilic archaea associated with adult Artemia urmiana

The brine shrimp, Artemia is the dominant macrozooplankton present in many hypersaline environments. Artemia urmiana is the only macroscopic organism in Urmia Salt Lake (Iran), and the high salinity of the lake makes it a suitable environment for halophilic archaea too. Because of common environment for Artemia and extreme halophiles; this investigation is concentrated on studying the relationship between Artemia and halophilic archaea in Urmia Lake. In this study first the procedure of arhaea isolation was done. Then, isolated strains were sub-cultured and DNA was extracted and amplified by PCR using specific primers for amplifying archaeal 16S rRNA. The amplified archeal DNA fragments were purified, and sequenced. 16S rRNA sequences were compared to known sequences using the NCBI BLAST program. Sequences relating to Halorubrum, Haloarcula and Halobacterium species were identified in Urmia Salt Lake water and Artemia adults and the phylogenetic tree of different species was constructed. Only Halorubrum species were present in association with Artemia. They belong to Halobacteriaceae family of archeae which are isolated from different salt lakes in different parts of world and we could show their existence in adult Artemia, another organism living in hypersaline enviroments.

The potential of halophilic and halotolerant bacteria for the production of antineoplastic enzymes: L-asparaginase and L-glutaminase.

L-asparaginase and L-glutaminase can be effectively used for the treatment of patients who suffer from accute lymphoblastic leukemia and tumor cells. Microbial sources are the best source for the bulk production of these enzymes. However, their long-term administration may cause immunological responses, so screening for new enzymes with novel properties is required. Halophilic and halotolerant bacteria with novel enzymatic characteristics can be considered as a potential source for production of enzymes with different immunological properties. In this study, L-asparaginase and L-glutaminase production by halophilic bacteria isolated from Urmia salt lake was studied. Out of the 85 isolated halophilic and halotolerant bacterial strains, 16 (19 %) showed L-asparaginase activity and 3 strains (3.5 %) showed L-glutaminase activity. Strains with the highest activities were selected for further studies. Based on 16S rDNA sequence analysis, it was shown that the selected isolates for L-asparaginase and L-glutaminase production belong to the genus Bacillus and Salicola, respectively. Both enzymes were produced extracellularly. The strain with the most L-asparaginase production did not show L-glutaminase production which is medically important. The effects of key parameters including temperature, initial pH of the solution, and concentrations of glucose, asparagine or glutamine, and sodium chloride were evaluated by means of response surface methodology (RSM) to optimize enzymes production. Under the obtained optimal conditions, L-asparaginase and L-glutaminase production was increased up to 1.5 (61.7 unit/mL) and 2.6 fold (46.4 unit/mL), respectively.

L-Asparaginase Activity in Cell Lysates and Culture Media of Halophilic Bacterial Isolates.

The objective of this study was to isolate halophilic bacteria with the ability to produce intracellular or extracellular L-asparaginase. A total number of 120 halophilic bacteria were isolated from 17 different saline habitats of Iran including salt lakes, wetlands, brine springs and deserts. Among these, 68 were able to grow in the presence of 1.5 M NaCl and 52 demonstrated the ability to grow in the selection medium containing 3.5 M NaCl. None of the isolates appeared to produce appreciable amounts of extracellular L-asparaginase. Among the isolates that produced intracellular L-asparaginase, 5 moderate and 1 extreme halophiles were selected for further study based on their observed activity level. The moderately halophilic isolates were shown to belong to the genus Halomonas while the extreme halophile was identified as a member of the genus Aidingimonas.

Shelf life of packaged loins of dried salt-cured cod (Gadus morhua L.) stored at elevated temperatures

Dried salt-cured cod is a commercially important product that is mainly exported to markets in Latin America and Southern Europe. The product is usually split, and cut and packaged once it reaches the market. However, many markets do not have adequate refrigeration facilities. When stored at elevated temperatures, a red or pink discolouration can develop due to the growth of extreme halophiles, at which point the product is considered defective and cannot be sold. We have investigated the shelf life of packaged loins of dried salt-cured cod stored at 25, 30, and 35 °C and at 60 and 80% relative humidity (RH). Shelf life varied by temperature, RH, water content, and level of extreme halophiles. Product stored at 60% RH had a longer shelf life than product stored at 80% RH, and storage at 25 °C revealed a longer shelf life than storage at 30 or 35 °C. At both 60 and 80% RH, drying beyond 48% had a limited shelf life effect. As dried salt-cured fish is largely sold and consumed seasonally, its shelf life has more implications during the low season, as prolonged non-refrigerated storage may cause more product to become defective.

Metabolism and Physiology of Halophiles

Metabolism and Physiology of Halophiles

Diversification and niche adaptations of Nitrospina-like bacteria in the polyextreme interfaces of Red Sea brines.

Nitrite-oxidizing bacteria (NOB) of the genus Nitrospina have exclusively been found in marine environments. In the brine–seawater interface layer of Atlantis II Deep (Red Sea), Nitrospina-like bacteria constitute up to one-third of the bacterial 16S ribosomal RNA (rRNA) gene sequences. This is much higher compared with that reported in other marine habitats (~10% of all bacteria), and was unexpected because no NOB culture has been observed to grow above 4.0% salinity, presumably due to the low net energy gained from their metabolism that is insufficient for both growth and osmoregulation. Using phylogenetics, single-cell genomics and metagenomic fragment recruitment approaches, we document here that these Nitrospina-like bacteria, designated as Candidatus Nitromaritima RS, are not only highly diverged from the type species Nitrospina gracilis (pairwise genome identity of 69%) but are also ubiquitous in the deeper, highly saline interface layers (up to 11.2% salinity) with temperatures of up to 52 °C. Comparative pan-genome analyses revealed that less than half of the predicted proteome of Ca. Nitromaritima RS is shared with N. gracilis. Interestingly, the capacity for nitrite oxidation is also conserved in both genomes. Although both lack acidic proteomes synonymous with extreme halophiles, the pangenome of Ca. Nitromaritima RS specifically encodes enzymes with osmoregulatory and thermoprotective roles (i.e., ectoine/hydroxyectoine biosynthesis) and of thermodynamic importance (i.e., nitrate and nitrite reductases). Ca. Nitromaritima RS also possesses many hallmark traits of microaerophiles and high-affinity NOB. The abundance of the uncultured Ca. Nitromaritima lineage in marine oxyclines suggests their unrecognized ecological significance in deoxygenated areas of the global ocean.

Draft Genome Sequence of Halostagnicola sp. A56, an Extremely Halophilic Archaeon Isolated from the Andaman Islands

The first draft genome of Halostagnicola sp. A56, isolated from the Andaman Islands is reported here. The A56 genome comprises 3,178,490 bp in 26 contigs with a G+C content of 60.8%. The genome annotation revealed that A56 could have potential applications for the production of polyhydroxyalkanoate or bioplastics.

Molecular adaptation and salt stress response of Halobacterium salinarum cells revealed by neutron spectroscopy.

Halobacterium salinarum is an extreme halophile archaeon with an absolute requirement for a multimolar salt environment. It accumulates molar concentrations of KCl in the cytosol to counterbalance the external osmotic pressure imposed by the molar NaCl. As a consequence, cytosolic proteins are permanently exposed to low water activity and highly ionic conditions. In non-adapted systems, such conditions would promote protein aggregation, precipitation, and denaturation. In contrast, in vitro studies showed that proteins from extreme halophilic cells are themselves obligate halophiles. In this paper, adaptation via dynamics to low-salt stress in H. salinarum cells was measured by neutron scattering experiments coupled with microbiological characterization. The molecular dynamic properties of a proteome represent a good indicator for environmental adaptation and the neutron/microbiology approach has been shown to be well tailored to characterize these modifications. In their natural setting, halophilic organisms often have to face important variations in environmental salt concentration. The results showed deleterious effects already occur in the H. salinarum proteome, even when the external salt concentration is still relatively high, suggesting the onset of survival mechanisms quite early when the environmental salt concentration decreases.

Assemblages of endophytic fungi on Salicornia europaea disjunctively distributed in Japan: towards clarification of the ubiquity of fungal endophytes on halophytes and their ecological roles.

We studied assemblages of endophytic fungi on the halophilic plant Salicornia europaea in Japan. This plant is disjunctively distributed in the eastern Hokkaido and the Seto Inland Sea (Setouchi) regions. Various dematiaceous fungi that form dark spores and/or mycelia (and the related ascigerous stages) were common in S. europaea . Among them, Pleospora sp. (anamorph: Stemphylium ) and Alternaria alternata were the major endophytes found in the plant, followed by Alternaria phragmospora and Cladosporium cladosporioides . Fungal assemblages from Hokkaido were similar among years, as were assemblages collected in completely segregated regions in Japan, Hokkaido and Setouchi. While such dematiaceous fungi are generally known to be epiphytes living on the surface of terrestrial vascular plants, they may ubiquitously inhabit halophytes such as Salicornia .

Salinity regulation of the interaction of halovirus SNJ1 with its host and alteration of the halovirus replication strategy to adapt to the variable ecosystem.

Halovirus is a major force that affects the evolution of extreme halophiles and the biogeochemistry of hypersaline environments. However, until now, the systematic studies on the halovirus ecology and the effects of salt concentration on virus-host systems are lacking. To provide more valuable information for understanding ecological strategies of a virus-host system in the hypersaline ecosystem, we studied the interaction between halovirus SNJ1 and its host Natrinema sp.J7-2 under various NaCl concentrations. We found that the adsorption rate and lytic rate increased with salt concentration, demonstrating that a higher salt concentration promoted viral adsorption and proliferation. Contrary to the lytic rate, the lysogenic rate decreased as the salt concentration increased. Our results also demonstrated that cells incubated at a high salt concentration prior to infection increased the ability of the virus to adsorb and lyse its host cells; therefore, the physiological status of host cells also affected the virus-host interaction. In conclusion, SNJ1 acted as a predator, lysing host cells and releasing progeny viruses in hypersaline environments; in low salt environments, viruses lysogenized host cells to escape the damage from low salinity.

Annihilation of Extremely Halophilic Archaea in Hide Preservation Salt Using Alternating Electric Current

Salt contains extremely halophilic archaea and these microorganisms degrade leather quality. The aim of this study is to fi nd an effective treatment system to kill these microorganisms in salt used in hide brine curing. Ten salt samples were obtained from Tuz Lake, Turkey, and the total cell counts of extremely halophilic archaea, proteolytic and lipolytic extremely halophilic archaea were determined. Two sets of experiments were designed to detect the inactivation impact of alternating electric current on extremely halophilic archaea. In the fi rst experiment, 2 A alternating electric current was applied for 25 min to the salt samples dissolved in liquid medium. In the second experiment, 2 A alternating electric current was applied for 25 min to the isolates of proteolytic extremely halophilic archaea, lipolytic extremely halophilic archaea, both proteolytic and lipolytic extremely halophilic archaea, and a mixed culture of these isolates. The extremely halophilic archaea in salt (102–104 colony forming units (CFU) g–1) was annihilated in 1 min via alternating electric current and a 5 min treatment with the current was enough to destroy extremely halophilic archaeal isolates (106 CFU ml–1) obtained from salt samples. This electric treatment was found fairly effective to kill proteolytic and lipolytic extremely halophilic archaea in salt used for preservation of hide.

Production and Characterization of α-Amylase from an Extremely Halophilic Archaeon, Haloferax sp. HA10.

Haloarchaea are found at very high concentrations in salt-conditioned environments, hence produce enzymes which are able to catalyze reactions under harsh conditions, typical of many industrial processes. In the present study, culture conditions for extracellular amylase production from Haloarchaea isolated from a solar saltern were optimized and the purified enzyme was characterized. Haloferax sp. HA10 showed maximum amylase production at 3 M NaCl, 37 °C, pH=7 and 1% starch content. Purified α-amylase was a calcium-dependent enzyme with an estimated molecular mass of about 66 kDa and many industrially useful properties. It was found to be stable in a broad range of pH (from 5 to 9) and NaCl concentrations (from 0.5 to 3.0 M), retaining 48% activity even at 4 M. The optimal temperature for Haloferax sp. HA10 amylase activity was 55 °C (99% activity), and 57% activity was retained at 80 °C, which dropped to 44% with the increase of temperature to 90 or 100 °C. It was able to sustain various surfactants and detergents. To the best of our knowledge the detergent-stable α-amylases from halophilic archaeon have not been reported yet.

Erratum for Behera et al., Draft Genome Sequence of the Extremely Halophilic Bacterium Halomonas salina Strain CIFRI1, Isolated from the East Coast of India.

Volume 3, no. 1, [e01321-14][1], 2015. Page 1: The affiliation line should read as given above. [1]: /lookup/doi/10.1128/genomeA.01321-14

Draft Genome Sequence of the Extremely Halophilic Bacterium Halomonas salina Strain CIFRI1, Isolated from the East Coast of India.

Halomonas salina strain CIFRI1 is an extremely salt-stress-tolerant bacterium isolated from the salt crystals of the east coast of India. Here we report the annotated 3.45-Mb draft genome sequence of strain CIFRI1 having 86 contigs with 3,139 protein coding loci, including 62 RNA genes.

Structural Analysis of Halophilic Lipases Isolated from Bledug Kuwu Mud Crater, Purwodadi-Grobogan, Indonesia: A Structural Bioinformatics Study

We have cloned lipase genes isolated from six halophilic bacteria isolated from Bledug Kuwu mud crater, Purwodadi-Grobogan, Indonesia. In the present study, we carried out structural bioinformatics analysis to estimate the structural properties of the cloned gene products. In this study, crystal structures of several proteins originated from nonhalophiles, moderate and extreme halophiles were used as references. Moderate and extreme halophilic proteins were grouped into OSMOL and SALT-IN proteins, respectively, which referred to the adaptation strategy of moderate and extreme halophiles against external osmotic pressure exerted by hypersaline solution in their habitats. All structural bioinformatics analysis presented here were carried out by evaluating the relative difference of various analysed parameters between all halophiles proteins including the references and the nonhalophiles ones. Lipase samples from the six Bledug Kuwu bacterial isolates displayed different profiles in amino acid composition, secondary structures, solvent accessible surface areas, radius of gyrations and salt bridges compare to those of all the references structures including OSMOL and SALT-IN proteins. These differences may be correlated to the unique halophiles habitat of all six bacteria isolates, which is the salty mud crater situated at the mainland and far from the sea. Therefore, based on this analysis we can expect that the biochemical and structural property of those six lipase samples were unique as well.

Geological, hydrogeochemical, and microbiological characteristics of the Oil site of the Uzon caldera (Kamchatka)

This study used geological, geochemical, and microbiological data to examine the Uzon oils and conditions within the Uzon caldera. The trace-element compositions of crude oils and solutions from boreholes and hydrothermal springs were determined by ICP-MS. The majority of hydrothermal manifestations within the Uzon caldera are controlled by three trends of faults. The major fault zone, trending nearly E-W, is located between Kikhpinych and Taunshits volcanoes. It acts as a magma conduit and hosts numerous oval-shaped hydrothermal vents. The Oil site is situated on the periphery of the hottest area of highest hydrothermal activity within the Eastern thermal field. On the Eh-pH diagram, most solutions of the Uzon caldera define distinct fields and trends which correlate with the stability fields for sulfur and iron. The Oil site is characterized by very wide variations in temperature and Eh-pH parameters of pore solutions. The geochemical signatures of the solutions at this site are broadly similar to those from other areas of the Uzon caldera, but differ in their sulfide ion and sulfate ion concentrations. These differences can be explained by mixing of deep chloride-sodium hydrothermal solutions and solutions produced during surface oxidation of sulfide-containing material. With respect to the average continental crust, the Uzon oil is enriched in S, As, and Hg. The crude oil and solutions have broadly similar concentrations of B, S, Cl, As, Se, Br, Cd, I, Hg, and Pb and highly variable concentrations of Ti, V, Cr, Co, Ni, Cu, Cd, Nb, and Sn. The community structure of archaeal assemblages in springs and test pits at the Eastern thermal field was analyzed by 16S rRNA library and pyro sequencing methods. It was found that the proportion of archaea in the microbial communities of the Uzon caldera ranges from 2 to over 70% of the total sequences identified. Crenarchaeota were found in large proportions in all samples except one. The majority of the sequences in four samples were affiliated with Euryarchaeota, which comprise methanogenic archaea, extreme halophiles, and some extreme thermophiles. The results of geological, mineralogical-geochemical, microbiological and physicochemical studies of oil seeps in the Uzon caldera reveal distinctive geochemical characteristics of crude oil and the complexity of oil formation.

Isotopic labeling of proteins in Halobacterium salinarum.

It is often necessary to obtain isotopically labeled proteins containing (15)N, (13)C, or (2)H for nuclear magnetic resonance; and (2)H for small-angle neutron scattering or neutron diffraction studies. To achieve uniform isotopic labeling, protein expression is most commonly performed in Escherichia coli or yeast using labeled media. However, proteins from extreme halophiles sometimes require a cellular environment with high ionic strength and cannot be heterologously expressed in E. coli or yeast in functional form. We present here methods for the cultivation of Halobacterium salinarum in isotopically labeled rich media, using commercially available isotopically labeled hydrolysates. The methods described here are both technically simple and relatively inexpensive.

Stability of Halophilic Proteins in Hyper Saline Brine: [2Fe-2S] Ferredoxin as a Paradigm

Apart from normal or mesophilic environment, organisms are found in extreme of salinity, and other hostile environments in the earth. Extreme halophiles thrive as pure culture in their natural environments of saturated salt as other microbes can’t venture to grow. Over the evolution, these microorganism grew up with specialized transport-devices to solve the problem of osmoregulation. As a consequence whole of their biochemical machinery started functioning in this highly saline brine situation that mesophile cannot withstand. Intensive researches are thus carried out over the last fifty years to understand salt dependent properties of these proteins and enzymes. Ferredoxin is a small soluble protein that functions as electron carrier in decarboxylation reactions in cytoplasm in conjunction with an oxidoreductase. Two of its representatives from Halobacterium marismortui (HmFd) and Halobacterium salinarum (HsFd) are extensively studied. Atomic structures of HmFd and HsFd reveal that halo adaptation is largely mediated by a hyper acidic inserted domain of some 24 residues long at N-terminus region. By designed kinetics and thermodynamics experiments it was demonstrated that HsFd indeed adapted in high salt and requires ≥1.5M salt to retain its overall structural integrity. While non-specific electrostatic effect is operative at ≤0.25M salt, higher salt promote salt-bridge and hydrophobic stability. At intermediate salt where Hofmeister effects of specific ion interactions are operative, HsFd forms a hydrophobic collapsed intermediate whose structural properties differs from its native state in saturated salts. Thus intuitively, HsFd in its native state seems to entertain a post Hofmeister like effect in that wide modulation of tertiary interactions might occur.

Application of Alternating Electric Current in Leather Industry to Kill Extremely Halophilic Archaea Found in Brine Solutions

Application of Alternating Electric Current in Leather Industry to Kill Extremely Halophilic Archaea Found in Brine Solutions

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 etal. (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.