Anaerobic growth of halophilic archaeobacteria by reduction of dimethysulfoxide and trimethylamine N-oxide

Abstract Most representatives of the halophilic arachaeobacterial genera Halobacterium, Haloarcula and Haloferax tested were able to reduce dimethylsulfoxide (DMSO) to dimethylsulfide (DMS) and trimethylamine N -oxide (TMAO) to trimethylamine (TMA) under (semi)anaerobic conditions. In most cases the reduction of DMSO and TMAO was accompanied by an increase in cell yield. The ability to reduce DMSO or TMAO was not correlated to reduced DMSO or TMAO was not correlated with the ability to reduce nitrate to nitrite. Anaerobic respiration with DMSO and TMAO as electron acceptor supplies the halophilic archeobacteria with an additional mode of energy generation in the absence of molecular oxygen.     

Effects of chaotropic salts on global proteome stability in halophilic archaea: Implications for life signatures on Mars

Halophilic archaea thriving in hypersaline environments, such as salt lakes, offer models for putative life in extraterrestrial brines such as those found on Mars. However, little is known about the effect of the chaotropic salts that could be found in such brines, such as MgCl₂, CaCl₂ and (per)chlorate salts, on complex biological samples like cell lysates which could be expected to be more representative of biomarkers left behind putative extraterrestrial life forms. We used intrinsic fluorescence to study the salt dependence of proteomes extracted from five halophilic strains: Haloarcula marismortui, Halobacterium salinarum, Haloferax mediterranei, Halorubrum sodomense and Haloferax volcanii. These strains were isolated from Earth environments with different salt compositions. Among the five strains that were analysed, H. mediterranei stood out as a results of its high dependency on NaCl for its proteome stabilization. Interestingly, the results showed contrasting denaturation responses of the proteomes to chaotropic salts. In particular, the proteomes of strains that are most dependent or tolerant on MgCl₂ for growth exhibited higher tolerance towards chaotropic salts that are abundant in terrestrial and Martian brines. These experiments bridge together global protein properties and environmental adaptation and help guide the search for protein-like biomarkers in extraterrestrial briny environments.     

Utilization of protein profiles for the characterization of halophilic bacteria

Proteins from 24 halophilic bacteria, including Haloarcula marismortui, Haloarcula vallismortis, Haloferax mediteranei, Haloferax gibbonsii, Halobacterium salinarium, as well as unknown isolates from Enid, Oklahoma; Jefferson Island, Louisiana; and the Salado Formation-New Mexico, were analyzed by one-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Their protein profiles were compared, and the bacteria were grouped according to the Statistical Analysis System (SAS Institute, Cary, North Carolina) on an IBM 4316 computer. The groupings made from protein profiles showed agreement with groupings made from DNA reassociation data. The various known halophiles were easily separated into the three main halobacterial genera. The data show that one-dimensional SDS-PAGE can be easily used to rapidly screen large numbers of unknown strains to group them into related clusters. This technique offers a way to reduce the total number of halophilic isolates being studied in large taxonomic research programs.     

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).     

Phenol degradation by halophilic bacteria isolated from hypersaline environments

Phenol is a toxic aromatic compound used or produced in many industries and as a result a common component of industrial wastewaters. Phenol containing waste streams are frequently hypersaline and therefore require halophilic microorganisms for efficient biotreatment without dilution. In this study three halophilic bacteria isolated from different saline environments and identified as Halomonas organivorans, Arhodomonas aquaeolei and Modicisalibacter tunisiensis were shown to be able to grow on phenol in hypersaline media containing 100 g/L of total salts at a concentration of 3 mM (280 mg/L), well above the concentration found in most waste streams. Genes encoding the aromatic dioxygenase enzymes catechol 1,2 dioxygenase and protocatechuate 3,4-dioxygenase were present in all strains as determined by PCR amplification using primers specific for highly conserved regions of the genes. The gene for protocatechuate 3,4-dioxygenase was cloned from the isolated H. organivorans and the translated protein was evaluated by comparative protein sequence analysis with protocatechuate 3,4-dioxygenase proteins from other microorganisms. Although the analysis revealed a wide range of sequence divergence among the protocatechuate 3,4-dioxygenase family, all of the conserved domain amino acid structures identified for this enzyme family are identical or conservatively substituted in the H. organivorans enzyme.     

Production of d-lactate, acetate, and pyruvate from glycerol in communities of halophilic archaea in the Dead Sea and in saltern crystallizer ponds

Abstract When glycerol is added to cultures of halophilic archaea, especially representatives of the genera Haloferax and Haloarcula , massive amounts of acids are formed. HPLC and enzymatic analyses of supernatants of Haloferax cultures grown in the presence of glycerol showed that all produced d -lactate and acetate. Cultures of two Haloarcula species tested produced pyruvate and acetate from glycerol. In all cases only a small fraction of the added glycerol was converted to organic acids. Both lactate, pyruvate, and acetate can be used as substrates for the growth of many halophilic archaea, including those that produce them, and acid production is possibly an overflow phenomenon, due to the limited capacity of the enzymatic systems responsible for their dissimilation. To test whether lactate is formed also by natural communities of halophilic archaea at low glycerol concentrations such as may be encountered in situ, we incubated samples from the Dead Sea and from the saltern crystallizer ponds at Eilat with 1.5–3 μM [U-¹⁴C]glycerol. After depletion of the glycerol, around 10% of the label was found in lactate and acetate in both brine samples. In addition, pyruvate was formed in Dead Sea water. Upon further incubation of the Dead Sea samples after depletion of the glycerol, pyruvate disappeared rapidly, while acetate and lactate concentrations decreased only very slowly. In saltern brines the lactate formed was degraded after depletion of the glycerol, but the concentration of labelled acetate decreased only very slowly.     

Distribution of glycerol dehydrogenase and glycerol kinase activity in halophilic archaea

Abstract A constitutive NAD⁺-dependent glycerol dehydrogenase activity was detected in Halobacterium salinarium and Halobacterium cutirubrum . Optimal activity was found at 3 M KCl and pH 8–10. No glycerol dehydrogenase activity could be demonstrated in representatives of the genera Haloferax and Haloarcula , even when grown in the presence of glycerol, or in Halobacterium saccharovorum and Halobacterium sodomense . Glycerol kinase activity was shown to be present constitutively in all halophilic archaea examined. The finding that glycerol dehydrogenase is found only in part of the halophilic arachaea makes dihydroxyacetone an improbable candidate as the precursor for the glycerol moiety of halobacterial lipids.     

Taxonomy of halophilic Archaea: current status and future challenges

Several groups of Archaea, all Euryarchaeota, develop in hypersaline environments (from >10 % salt up to saturation). The cultured diversity of halophilic Archaea includes the family Halobacteriaceae of aerobic or facultative anaerobic, generally red-pigmented species (47 genera and 165 species as of February 2014) and seven representatives of four genera of methanogens, most of which obtain energy from methylated amines under anaerobic conditions. Metagenomic studies have identified an additional deep lineage of Archaea in salt lakes and ponds with brines approaching NaCl saturation. Genomic information is now available for representatives of these ‘Nanohaloarchaea’, but no members of this lineage have yet been cultured. Multilocus sequence analysis is becoming increasingly popular in taxonomic studies of the Halobacteriaceae, and such studies have demonstrated that recombination of genetic traits occurs at an extremely high frequency at least in some genera. Metagenomic studies in an Antarctic lake showed that large identical regions of up to 35 kb in length can be shared by members of different genera living together in the same environment. Such observations have important implications not only for the taxonomy of the Halobacteriaceae, but also for species concepts and questions on taxonomy and classification for prokaryotic microorganisms in general.     

Total reconstitution of active small ribosomal subunits of the extreme halophilic archaeon Haloferax mediterranei

The small ribosomal subunit of the halophilic archaeon Haloferax mediterranei has been reconstituted from its dissociated rRNA and protein components. Efficient reconstitution of particles, fully active in poly(U)-dependent polyphenylalanine synthesis, occurs after 2 h of incubation at 36°C in the presence of l.5 M of (NH₄)₂SO₄ 100 mM of MgAc₂, 20 mM Tris-HCI (pH 8.2) and 6 mM 2-mercaptoethanol. Important differences in the optimal ionic conditions for the reconstitution of the 30S and the 50S ribosomal subunits from Haloferax mediterranei have been found. K⁺ and NH₄⁺ ions have differing abilities to promote the reconstitution of the particles. The assembly of 30S ribosomal subunits of H. mediterranei has a higher tolerance to ionic strength than the assembly of the 50S subunits and it is independent of the Mg²⁺concentration present in the system.     

Phylogeny and ecophysiological features of prokaryotes isolated from temporary saline tidal pools

Although hypersaline environments have been extensively examined, only a limited number of microbial community studies have been performed in saline tide pools. We have studied a temporary salt-saturated tide pool and isolated prokaryotes from the water. Chlorinity measurements revealed that the tide pool brine could be characterized as one of the most hypersaline ecosystems on earth. Enumeration of microorganisms at different salinities showed that the tide pool was dominated by moderate halophiles. Based on 16S rRNA gene sequence analysis, the prokaryotic strains isolated were related to the bacterial genera Rhodovibrio, Halovibrio, Aquisalimonas, Bacillus and Staphylococcus and to the haloarchaeal species Haloferax alexandrinus. Four bacterial isolates were distantly related to their closest validly described species Aquisalimonas asiatica (96.5 % similarity), representing a novel phylogenetic linkage. Ecophysiological analysis also revealed distinct phenotypic profiles for the prokaryotic strains analyzed. The herbicide 2,4-dichlorophenoxyacetate could be effectively utilized by selected strains as the sole carbon source, but phenolic compounds could not be utilized by any of the halophilic isolates examined. None of the halophilic strains were able to grow without the presence of sea salt or seawater. Based on these results, we conclude that moderate halophilic bacteria rather than extremely halophilic archaea dominate in such a hypersaline environment.     

Haloferax sp. D1227, a halophilic Archaeon capable of growth on aromatic compounds

A pink-pigmented halophilic Archaeon, Strain D1227, was isolated from soil contaminated with oil brine and shown to be a member of the genus Haloferax, based on: (1) its hybridization with a 16S rRNA probe universal for the Archaea; (2) its resistance to a broad spectrum of antibiotics that affect Bacteria; (3) its requirement for at least 0.86 M NaCl and 25 mM Mg²⁺ for growth; (4) its possession of C₅₀-carotenoids characteristic of the halophilic Arachaea; (5) the thin layer chromatographic pattern of its polar lipids, which was identical to that of other species of Haloferax; and (6) its pleomorphic cell morphology. However, in contrast to the known species of Archaea, Haloferax strain D1227 was able to use aromatic substrates (e.g., benzoate, cinnamate, and phenylpropanoate) as sole carbon and energy sources for growth. Physiologically similar organisms, such as Haloferax volcanii, Haloferax mediterrani, Haloarcula vallismortis, and Haloarcula hispanica, could not grow on these aromatic substrates. When grown on ¹⁴C-benzoate, strain D1227 mineralized 70% of the substrate and assimilated 19% of the ¹⁴C-label into cell biomass. In addition to growth on aromatic substrates, D1227 was also capable of growth on a variety of carbohydrates and organic acids. Optimum growth of strain D1227 occurred at 45°C in media containing 1.7–2.6 M NaCl and 100 mM Mg²⁺. Under optimum growth conditions, the cell shape varied from that of an oblate spheroid on mineral salts medium alone, to discshaped, irregular or triangular cells on the same medium amended with yeast extract and tryptone. To our knowledge, this is the first unequivocal demonstration of the ability of an Archacon to grow by mineralization of aromatic substrates, and it adds a new dimension to our appreciation of the physiological diversity of this group of prokaryotes.Abbreviations Ha. Haloarcula - Hf. Haloferax     

Tolerance of extremely halophilic archaebacteria towards bromide

The tolerance of halophilic archaebacteria towards bromide was tested in view of the fact that bromide occurs in natural brines in concentrations of up to 66 mM. It was found that, while concentrations of up to 0.8–1M are tolerated well by all halobacterial types examined, great differences exist between species with respect to bromide tolerance. WhileHalobacterium (H. salinarium, H. halobium, andH. sodomense) andNatronobacterium species are only moderately tolerant,Haloarcula (H. vallismortis, H. marismortui), andHaloferax species (H. mediterranei, H. gibbonsii) tolerate higher concentrations.Haloferax volcanii proved extremely tolerant and showed growth in bromide media at very low chloride concentrations (below 50 mM). No correlation was found between bromide tolerance and the bromide concentration in the habitat from which the strains were isolated. Iodide proved much more toxic than bromide. Bromide-tolerant strains also proved relatively resistant to growth inhibition by iodide.     

First evidence for the salt-dependent folding and activity of an esterase from the halophilic archaea Haloarcula marismortui

A gene encoding an esterase from Haloarcula marismortui, a halophilic archaea from the Dead Sea, was cloned, expressed in Escherichia coli, and the recombinant protein (Hm EST) was biochemically characterized. The enzymatic activity of Hm EST was shown to exhibit salt dependence through salt-dependent folding. Hm EST exhibits a preference for short chain fatty acids and monoesters. It is inhibited by phenylmethylsulfonyl fluoride, diethyl-p-nitrophenyl phosphate, and 5-methoxy-3-(4-phenoxyphenyl)-3H-[1,3,4]oxadiazol-2-one, confirming the conclusion from sequence alignments that Hm EST is a serine carboxylesterase belonging to the hormone-sensitive lipase family. The activity of Hm EST is optimum in the presence of 3 M KCl and no activity was detected in the absence of salts. Far–UV circular dichroism showed that Hm EST is totally unfolded in salt-free medium and secondary structure appears in the presence of 0.25–0.5 M KCl. After salt depletion, the protein was able to recover 60% of its initial activity when 2 M KCl was added. A 3D model of Hm EST was built and its surface properties were analyzed, pointing to an enrichment in acidic residues paralleled by a depletion in basic residues. This peculiar charge repartition at the protein surface supports a better stability of the protein in a high salt environment.     

Enzyme(s) responsible for tellurite reducing activity in a moderately halophilic bacterium,Salinicoccus iranensis strain QW6

Oxyanions of tellurium, like tellurate (TeO₄ ^2?) and tellurite (TeO₃ ^2?), are highly toxic for most microorganisms. There are a few reports on the bacterial tellurite resistance mechanism(s). Salinicoccus iranensis, a Gram-positive halophilic bacterium, shows high tellurite resistance and NADH-dependent tellurite reduction activity in vitro. Since little is known regarding TeO₃ ^2? resistance mechanisms in halophilic microorganisms, here one of the enzymatic reduction activities presented in this microorganism is investigated. To enhance the enzymatic activity during purification, the effect of different parameters including time, inoculation, different pHs, different tellurite concentrations and different salts were optimized. We also examined the tellurite removal rates by diethyldithiocarbamate (DDTC) during optimization. In the culture medium the optimum conditions obtained showed that at 30 h, 2 % inoculum, pH 7.5, without tellurite and with 5 % NaCl (w/v) the highest enzyme activity and tellurite removal were observed. Results of the purification procedure done by hydroxyapatite batch-mode, ammonium sulfate precipitation, followed by phenyl-Sepharose and Sephadex G-100 column chromatography, showed that the enzyme consisted of three subunits with molecular masses of 135, 63 and 57 kDa. In addition to tellurite reduction activity, the enzyme was able to reduce nitrate too. Our study extends the knowledge regarding this process in halophilic microorganisms. Besides, this approach may suggest an application for the organism or the enzyme itself to be used for bioremediation of polluted areas with different contaminants due to its nitrate reductase activity.     

Prospects for robust biocatalysis: engineering of novel specificity in a halophilic amino acid dehydrogenase

Heat- and solvent-tolerant enzymes from halophiles, potentially important industrially, offer a robust framework for protein engineering, but few solved halophilic structures exist to guide this. Homology modelling has guided mutations in glutamate dehydrogenase (GDH) from Halobacterium salinarum to emulate conversion of a mesophilic GDH to a methionine dehydrogenase. Replacement of K89, A163 and S367 by leucine, glycine and alanine converted halophilic GDH into a dehydrogenase accepting l-methionine, l-norleucine and l-norvaline as substrates. Over-expression in the halophilic expression host Haloferax volcanii and three-step purification gave ~98 % pure protein exhibiting maximum activity at pH 10. This enzyme also showed enhanced thermostability and organic solvent tolerance even at 70 °C, offering a biocatalyst resistant to harsh industrial environments. To our knowledge, this is the first reported amino acid specificity change engineered in a halophilic enzyme, encouraging use of mesophilic models to guide engineering of novel halophilic biocatalysts for industrial application. Calibrated gel filtration experiments show that both the mutant and the wild-type enzyme are stable hexamers.     

Cloning and sequencing of ftsZ homolog from extremely halophilic archaeon Haloarcula japonica strain TR-1.

The gene encoding FtZ was cloned from triangular disc-shaped extremely halophilic archaeon Haloarcula japonica strain TR-1. Nucleotide sequencing analysis of the possible ftsZ gene revealed that the structural gene consisted of an open reading frame of 1,182 nucleotides encoding 394 amino acids. The deduced amino acid sequence of the Ha. japonica FtsZ showed high identities with those Halobacterium salinarom, Haloferax volcanii and Haloferax mediterranei FtsZs.     

Purification and characterization of a novel extracellular halophilic and organic solvent-tolerant amylopullulanase from the haloarchaeon, Halorubrum sp. strain Ha25

A halophilic archaeon, Halorubrum sp. strain Ha25, produced extracellular halophilic organic solvent-tolerant amylopullulanase. The maximum enzyme production was at high salt concentration, 3–4 M NaCl. Optimum pH and temperature for enzyme production were 7.0 and 40 °C, respectively. Molecular mass of purified enzyme was estimated to be about 140 kDa by SDS–PAGE. This enzyme was active on pullulan and starch as substrates. The apparent K _m for the enzyme activity on pullulan was 4 mg/ml and for soluble starch was 1.8 mg/ml. Optimum temperature for amylolytic and pullulytic activities was 50 °C. Optimum pH for amylolytic activity was 7 and for pullulytic activity was 7.5. This enzyme was active over a wide range of concentrations (0–4.5 M) of NaCl. The effect of organic solvents on the enzyme activities showed that this enzyme was more stable in the presence of non-polar organic solvents than polar solvents. This study is the first report on amylopullulanase production in halophilic bacteria and archaea.     

Occurrence of Methanogenic Archaea in Highly Polluted Sediments of Tropical Santos–São Vicente Estuary (São Paulo, Brazil)

Little is known about the ability of methanogens to grow and produce methane in estuarine environments. In this study, traditional methods for cultivating strictly anaerobic microorganisms were combined with Fluorescence in situ hybridization (FISH) technique to enrich and identify methanogenic Archaea cultures occurring in highly polluted sediments of tropical Santos–São Vicente Estuary (São Paulo, Brazil). Sediment samples were enriched at 30°C under strict anaerobic and halophilic conditions, using a basal medium containing 2% of sodium chloride and amended with glucose, methanol, and sodium salts of acetate, formate and lactate. High methanogenic activity was detected, as evidenced by the biogas containing 11.5 mmol of methane at 20 days of incubation time and methane yield of 0.138-mmol CH₄/g organic matter/g volatile suspense solids. Cells of methanogenic Archaea were selected by serial dilution in medium amended separately with sodium acetate, sodium formate, or methanol. FISH analysis revealed the presence of Methanobacteriaceae and Methanosarcina sp. cells.     

Halomonas nanhaiensis sp. nov., a halophilic bacterium isolated from a sediment sample from the South China Sea

A novel Gram-negative, aerobic, slightly halophilic, yellow-pigmented, oxidase-negative, Voges–Proskauer positive, non-spore-forming bacterium, designated YIM M 13059^T, was isolated from a sediment sample collected from the South China Sea at a depth of 310 m. Optimal growth was found to occur at 28–30 °C, pH 7.0 and in the presence of 3–4 % (w/v) NaCl. Cells were observed to be rod-shaped and motile by peritrichous flagella. The polar lipids of strain YIM M 13059^T were found to be diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, a ninhydrin-positive phospholipid, one glycolipid and two unknown phospholipids. The predominant respiratory quinone was determined to be Q-9. The major fatty acids were identified as C_18:1 ω7c, C_16:1 ω6c/C_16:1 ω7c, C_16:0 and C_12:0 3-OH. The genomic DNA G+C content was determined to be 54.4 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that the isolate belongs to the genus Halomonas in the family Halomonadaceae. The 16S rRNA gene sequence similarities between strain YIM M 13059 ^T and the type strains of members of the genus Halomonas were in the range 93.3–98.3 %. However, the levels of DNA–DNA relatedness values between YIM M 13059 and the type strains of the most closely related species, Halomonas zhangjiangensis, Halomonas variabilis, Halomonas neptunia, Halomonas boliviensis and Halomonas sulfadieris were 50.2 ± 0.68 %, 46.8 ± 1.9 %, 28.5 ± 0.74 %, 42.9 ± 0.55 % and 37.1 ± 0.68 %, respectively. Based on phylogenetic, chemotaxonomic and phenotypic data, the strain YIM M 13059^T is proposed to represent a novel member of the genus Halomonas, with the name Halomonas nanhaiensis sp. nov. The type strain is YIM M 13059^T (=JCM 18142^T =CCTCC AB 2012911^T).     

A potential anti-oxidant protein in a ferrous iron-oxidizing Sulfolobus species

Abstract Eight species of halophilic Archaea were tested for the presence of isocitrate lyase activity. High activities (up to 100 nmol min⁻¹ mg protein⁻¹) were detected in Haloferax mediterranei and Haloferax volcanii when grown in medium containing acetate as the principal carbon source. Little activity was found in representatives of the genera Halobacterium and Haloarcula . Isocitrate lyase from Haloferax mediterranei required high potassium chloride concentrations, optimal activity being found at 1.5–3 M potassium chloride and pH 7.0. Replacement of potassium chloride by sodium chloride resulted in much lower activities. Sulfhydryl compounds (cysteine, glutathione) were not stimulatory. In other properties (stimulation by magnesium ions, sensitivity to different inhibitors) the enzyme resembled isocitrate lyases from representatives of the Bacteria and Eucarya.