Physiological response to silver toxicity in the extremely halophilic archaeon Halomicrobium mukohataei.

Adaptive strategies responsible for heavy metal tolerance were explored in the extremely halophilic archaeon Halomicrobium mukohataei DSM 12286. The tested strain was seemingly able to overcome silver-induced oxidative stress (assessed by malondialdehyde quantification, catalase assay and total antioxidant capacity measurement) mainly through non-enzymatic antioxidants. Energy dispersive spectrometry analysis illustrated the presence of colloidal silver in Hmc. mukohataei cultures exposed to AgNO3. Bright-field and transmission electron microscopy images, as well as dynamic light scattering analysis, demonstrated the presence of intracellular nanoparticles, mostly spherical, within a size range of 20-100 nm. As determined by the zeta potential measurement, the biosynthesized nanoparticles were highly stable, with a negative surface charge. Our research is a first attempt in the systematic study of the oxidative stress and intracellular silver nanoparticle accumulation, generated by exposure to silver ions, in members of Halobacteria class, thus broadening our knowledge on mechanisms supporting heavy metal tolerance of microbial cells living under saline conditions.

Species Widely Distributed in Halophilic Archaea Exhibit Opsin-Mediated Inhibition of Bacterioruberin Biosynthesis.

Halophilic Archaea are a distinctive pink color due to a carotenoid pigment called bacterioruberin. To sense or utilize light, many halophilic Archaea also produce rhodopsins, complexes of opsin proteins with a retinal prosthetic group. Both bacterioruberin and retinal are synthesized from isoprenoid precursors, with lycopene as the last shared intermediate. We previously described a regulatory mechanism by which Halobacterium salinarum bacterioopsin and Haloarcula vallismortis cruxopsin inhibit bacterioruberin synthesis catalyzed by lycopene elongase. In this work, we found that opsins in all three major Halobacteria clades inhibit bacterioruberin synthesis, suggesting that this regulatory mechanism existed in the common Halobacteria ancestor. Halophilic Archaea, which are generally heterotrophic and aerobic, likely evolved from an autotrophic, anaerobic methanogenic ancestor by acquiring many genes from Bacteria via lateral gene transfer. These bacterial "imports" include genes encoding opsins and lycopene elongases. To determine if opsins from Bacteria inhibit bacterioruberin synthesis, we tested bacterial opsins and found that an opsin from Curtobacterium, in the Actinobacteria phylum, inhibits bacterioruberin synthesis catalyzed by its own lycopene elongase, as well as that catalyzed by several archaeal enzymes. We also determined that the lycopene elongase from Halococcus salifodinae, a species from a family of Halobacteria lacking opsin homologs, retained the capacity to be inhibited by opsins. Together, our results indicate that opsin-mediated inhibition of bacterioruberin biosynthesis is a widely distributed mechanism found in both Archaea and Bacteria, possibly predating the divergence of the two domains. Further analysis may provide insight into the acquisition and evolution of the genes and their host species.IMPORTANCE All organisms use a variety of mechanisms to allocate limited resources to match their needs in their current environment. Here, we explore how halophilic microbes use a novel mechanism to allow efficient production of rhodopsin, a complex of an opsin protein and a retinal prosthetic group. We previously demonstrated that Halobacterium salinarum bacterioopsin directs available resources toward retinal by inhibiting synthesis of bacterioruberin, a molecule that shares precursors with retinal. In this work, we show that this mechanism can be carried out by proteins from halophilic Archaea that are not closely related to H. salinarum and those in at least one species of Bacteria Therefore, opsin-mediated inhibition of bacterioruberin synthesis may be a highly conserved, ancient regulatory mechanism.

Metabolomic characterization of halophilic bacterial isolates reveals strains synthesizing rare diaminoacids under salt stress.

Metabolomics-based approaches to study stress responses in bacteria have received much attention in recent years. In the present study, a metabolomic analysis of the representative halophilic bacterial isolates (Halomonas hydrothermalis VITP9, Bacillus aquimaris VITP4, Planococcus maritimus VITP21 and Virgibacillus dokdonensis VITP14) from a saltern region in India was performed using nuclear magnetic resonance spectroscopy. Chemometric analysis of (1)H NMR spectra revealed salt-dependent increase in the levels of metabolites, mainly from the aspartate and glutamate family, that are directed from the glycolytic pathway, pentose phosphate pathway and citric acid cycle. The composition of the metabolites was found to be different with respect to the species and the type of growth medium. Analysis of the two dimensional NMR data revealed accumulation of two rare diaminoacids, Nε-acetyl-α-lysine and Nδ-acetylornithine (by VITP21 and VITP4 strains respectively) apart from other well known solutes such as ectoine, proline, glutamate and glycine betaine. Metabolite profiles of strains capable of synthesizing Nε-acetyl-α-lysine and Nδ-acetylornithine suggested their biosynthesis from lysine and ornithine using aspartate and glutamate as their precursors, respectively. Further, the cells in moderate salinity (5% w/v NaCl) showed an increase in growth rate along with increase in the levels of nucleotides, whereas at higher salinity (10% w/v NaCl), the levels of aromatic and hydrophobic metabolites dropped, accompanied with a decrease in growth rate, rightly suggesting that at any salt-stress condition provided, cellular homeostasis was favored over growth.

A novel halophilic lipase, LipBL, showing high efficiency in the production of eicosapentaenoic acid (EPA).

BACKGROUND: Among extremophiles, halophiles are defined as microorganisms adapted to live and thrive in diverse extreme saline environments. These extremophilic microorganisms constitute the source of a number of hydrolases with great biotechnological applications. The interest to use extremozymes from halophiles in industrial applications is their resistance to organic solvents and extreme temperatures. Marinobacter lipolyticus SM19 is a moderately halophilic bacterium, isolated previously from a saline habitat in South Spain, showing lipolytic activity. METHODS AND FINDINGS: A lipolytic enzyme from the halophilic bacterium Marinobacter lipolyticus SM19 was isolated. This enzyme, designated LipBL, was expressed in Escherichia coli. LipBL is a protein of 404 amino acids with a molecular mass of 45.3 kDa and high identity to class C ß-lactamases. LipBL was purified and biochemically characterized. The temperature for its maximal activity was 80°C and the pH optimum determined at 25°C was 7.0, showing optimal activity without sodium chloride, while maintaining 20% activity in a wide range of NaCl concentrations. This enzyme exhibited high activity against short-medium length acyl chain substrates, although it also hydrolyzes olive oil and fish oil. The fish oil hydrolysis using LipBL results in an enrichment of free eicosapentaenoic acid (EPA), but not docosahexaenoic acid (DHA), relative to its levels present in fish oil. For improving the stability and to be used in industrial processes LipBL was immobilized in different supports. The immobilized derivatives CNBr-activated Sepharose were highly selective towards the release of EPA versus DHA. The enzyme is also active towards different chiral and prochiral esters. Exposure of LipBL to buffer-solvent mixtures showed that the enzyme had remarkable activity and stability in all organic solvents tested. CONCLUSIONS: In this study we isolated, purified, biochemically characterized and immobilized a lipolytic enzyme from a halophilic bacterium M. lipolyticus, which constitutes an enzyme with excellent properties to be used in the food industry, in the enrichment in omega-3 PUFAs.

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.

Extreme halophilic enzymes in organic solvents.

The use of halophilic extremozymes in organic media has been limited by the lack of enzymological studies in these media. To explore the behaviour of these extremozymes in organic media, different approaches have been adopted, including the dispersal of the lyophilised enzyme or the use of reverse micelles. The use of reverse micelles in maintaining high activities of halophilic extremozymes under unfavourable conditions could open new fields of application such as the use of these enzymes as biocatalysts in organic media.

[Adaptation strategies of halophilic microorganisms and Debaryomyces hansenii (halophilic yeast)]. / Estrategias de adaptación de microorganismos halófilos y Debaryomyces hansenii (Levadura halófila).

The term halophile is used for all those organisms belonging to hypersaline habitats; they constitute an interesting class of organisms able to compete successfully in salt water and to resist its denaturing effects. A wide diversity of microorganisms, prokaryotic and eukaryotic belong to this category. Halophile organisms have strategies allowing them not only to withstand osmotic stress, but also to function better in the presence of salt, in spite of maintaining high intracellular concentrations of salt, partly due to the synthesis of compatible solutes that allow them to balance their osmotic pressure. We describe the characteristics of some halophile organisms and D. hansenii (halophile yeast), that allow them to resist high concentrations of salt. The interest to know the great diversity microorganisms living in hypersaline habitats is growing, and has begun to be the center of recent investigations, since halophile organisms produce an wide variety of biomolecules that can be used for different applications. In this review we describe some mechanisms with which some halophile organisms count to resist the high concentration of salts, mainly NaCl.

Optimization of the production of a bacteriocin from Haloferax mediterranei Xia3.

The optimal conditions for the production of the halocin H1, a 31 kDa bacteriocin-like molecule produced by the extreme halophilic Archaea Haloferax mediterranei Xia3 active against Gram-negative haloarchaea, was characterized. The physico-chemical conditions required for the optimal production of halocin H1 are similar to those found in the habitat in which the microorganism was isolated: 20% salt concentration and temperature range between 37 and 42 degrees C. Optimal antimicrobial activity was obtained using 0.5% of N-Z amine E as nutrient.

Evidence for farnesol-mediated isoprenoid synthesis regulation in a halophilic archaeon, Haloferax volcanii.

Farnesol strongly inhibited growth of a halophilic archaeon, Haloferax volcanii, with an IC50 value of only 2 microM (0.4 microgram/ml) in rich medium and 50 nM (0.01 microgram/ml) in minimal medium without lysis. Other isoprenoid alcohols such as isopentenol, dimethylallyl alcohol, geraniol, and geranylgeraniol at 500 microM did not affect its growth. Mevalonate, which is the precursor of all isoprenoid membrane lipids in archaea, led to recovery of the growth inhibition of H. volcanii, but acetate had no such effect. Farnesol inhibited incorporation of acetate, but not mevalonate, into the lipid fraction. These results suggest that farnesol inhibited the biosynthetic pathway from acetate (acetyl-CoA) to mevalonate. Farnesol is known to be derived from the important intermediate of isoprenoids, farnesyl diphosphate (FPP), and found in neutral lipid fraction from this archaeon. Moreover, the cell-free extracts from H. volcanii could phosphorylate farnesol with ATP to generate farnesyl monophosphate and FPP. We conclude that farnesol-mediated isoprenoid synthesis regulation system by controlling farnesol concentration is present in H. volcanii.

Glucose transport of Haloferax volcanii requires the Na(+)-electrochemical potential gradient and inhibitors for the mammalian glucose transporter inhibit the transport.

The uptake of glucose and its non-metabolizing analogues by Haloferax volcanii, one of the glucose-utilizing Halobacteria, was examined using intact cells and envelope vesicles. Results obtained were: (1) The transport system is inducible. (2) The uptake requires the gradient of Na(+)-electrochemical potential. (3) Inhibitors for mammalian glucose transport also have an effect on this system, implying that the transporters resemble each other. (4) It is suggested that the mobility of the transporter is regulated by the membrane energization.