The ecology of the extremely halophilic archaea

Abstract: The extremely halophilic archaea (family Halobacteriaceae) are the dominant heterotrophic organisms in hypersaline environments in which salt concentrations exceed 250–300 g l⁻¹. During the last decades our knowledge on the taxonomy, physiology and biochemistry of the Halobacterium group has greatly increased. However, our understanding of the ecology of the halophilic archaea lags far behind the progess made in the study of other aspects of their biology. A few hypersaline environments, such as the Dead Sea and solar salterns, have been studied more in depth, using techniques such as lipid analysis to obtain information on the types of organisms present and measurement of uptake of labeled substrates to quantify the dynamics of bacterial processes. The results of these studies, in combination with the information obtained from laboratory studies of representative isolates of the Halobacteriaceae, enable the beginning of an understanding of the functioning of the halophilic archaea in nature.     

Lipid composition of halophilic species of Dunaliella from the dead sea

The lipid patterns of six halophilic Dunaliella species from the Dead Sea (C₉, D_11a, D_11b, D₁₃, F_20a and D. viridis) were found to be generally similar to those of halotolerant Dunaliella species previously examined, except for the presence in the halphilic Dunaliella of small to trace amounts of one or more (up to five) unidentified glycolipids. The lipids of two of the halophilic algae, species C₉ and D_11b, were studied in detail and were found to be similar in composition. Glycolipids were the major group (55.0 and 53.2 mol % for C₉ and D_11b, respectively), followed by neutral lipids (mainly triacyglycerols: 21.3 mol %; 24.6 mol %), whereas phospholipids were a much smaller fraction (6.5 mol %; 5.8 mol %). Monogalactosyldiacylglycerol was the largest component (22.0 mol %; 24.3 mol %) but digalactosyldiacylglycerol (18.7 mol %; 14.9 mol %) and sulfoquinovosyldiacylglycerol (14.3 mol %; 14.0 mol %) were also present in high concentrations. All phospholipids were present at low concentrations: phosphatidylglycerol (4.4 mol %; 3.1 mol %); phosphatidylethanolamine (1.1 mol %; 0.7 mol %); phosphatidylcholine (0.9 mol %; 1.9 mol %); and phosphatidylinositol (traces). Diacylglyceryl-O-(N,N,N-trimethyl)homoserine was present in C₉ and D_11b (3.3 mol %; 9.3 mol %) and in all the other species examined. Fatty acid composition of the individual lipid components of C₉ and D_11b showed characteristic differences between glycolipids and phospholipids, in a similar pattern for both algae. The major fatty acids detected in all species examined were -linolenic, linoleic, palmitic, oleic and a polyunsaturated sixteen carbon acid.     

Dynamics of a bloom of halophilic archaea in the Dead Sea

After a period of more than ten years in which bacterial and algal community sizes were extremely small, a dense bloom of halophilic archaea developed in the upper 5–10 m of the Dead Sea water column in the summer of 1992. The development of this bloom followed a dilution of the upper water layer by winter rainfloods, which enabled the development of a short-lived dense bloom of the unicellular green alga Dunaliella parva. The dense archaeal community (up to 3.5 × 10⁷ cells m1^–1 in June 1992) imparted a red coloration to the Dead Sea, due to its high content of bacterioruberin. Bacteriorhodopsin was not detected. High levels of potential heterotrophic activity were associated with the bloom, as measured by the incorporation of labeled organic substrates. After the decline of the algal bloom, archaeal numbers in the lake decreased only little, and most of the community was still present at the end of 1993. The amount of carotenoid pigment per cell, however, decreased 2–3-fold between June 1992 and August 1993. No new algal and archaeal blooms developed after the winter floods of 1992–1993, in spite of the fact that salinity values in the surface layer were sufficiently low to support a new algal bloom. A remnant of the 1992 Dunaliella bloom maintained itself at the lower end of the pycnocline at depths between 7 and 13 m (September 1992–August 1993). Its photosynthetic activity was small, and very little stimulation of archaeal growth and activity was associated with this algal community.     

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.     

Metabolism of halophilic archaea

In spite of their common hypersaline environment, halophilic archaea are surprisingly different in their nutritional demands and metabolic pathways. The metabolic diversity of halophilic archaea was investigated at the genomic level through systematic metabolic reconstruction and comparative analysis of four completely sequenced species: Halobacterium salinarum, Haloarcula marismortui, Haloquadratum walsbyi, and the haloalkaliphile Natronomonas pharaonis. The comparative study reveals different sets of enzyme genes amongst halophilic archaea, e.g. in glycerol degradation, pentose metabolism, and folate synthesis. The carefully assessed metabolic data represent a reliable resource for future system biology approaches as it also links to current experimental data on (halo)archaea from the literature. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

The use of protein synthesis inhibitors in the estimation of the contribution of halophilic archaebacteria to bacterial activity in hypersaline environments

Abstract Antibiotics affecting protein synthesis were used to differentiate between the activity of different groups of organisms (halophilic archaebacteria, eubacteria and eukaryotes) in water samples from hypersaline ecosystems. Anisomycin (inhibiting both archaebacterial halophiles and eukaryotes) can be used to quantitate the contribution of the archaebacterial halophiles to amino acid incorporation by the microbial community, when cycloheximide (inhibiting eukaryotic protein synthesis, but not affecting halobacteria) is used as a control. Both in saltern ponds at salinities above 300 g/1 and in Dead Sea surface water more than 95% of the amino acid incorporation activity was abolished by anisomycin, but not by cycloheximide. Inhibition by anisomycin was well correlated with inhibition by low concentrations of bile salts, which specifically affect bacteria of the Halobacterium group. Chloramphenicol (an inhibitor of eubacterial protein synthesis) quantitatively inhibited amino acid incorporation in saltern brines of relatively low salinity, but also caused significant (28–42%) inhibition at high salinities. Erythromycin was also found valuable in the estimation of activities of the different bacterial groups.     

Uptake of zinc and copper by halophilic bacteria isolated from the dead sea shore,Jordan

Ten Gram-positive and gram-negative bacterial cultures were recovered from nine water, mud, and soil samples collected from the Dead Sea shore at Suwaymah. All bacterial cultures were able to grow at 10% NaCl and at 45°C. They were able to grow in nutrient media supplemented with 1250 ppm of Zn. Most of them, except cultures 2 and 8, were able to grow in nutrient medium supplemented with 1000 ppm of Cu. After 2 wk of incubation of these 10 cultures at different concentrations (5, 25, 100, and 500 ppm), stock solutions of both Zn and Cu elements, the maximum absorption using atomic absorption spectrometry for Zn was achieved by culture 7 at 11.2%, 1.0%, 38.4%, and 84.54%, respectively, from the previous stock solutions, whereas the maximum absorption of the same concentration of Cu was achieved by culture 3 at 6.2%, 55.56%, 85.66%, and 90.82%, respectively, of the different concentrations. After 3 wk of incubation, the estimated absorption for Zn was achieved by cultures 2, 9, and 10 at 19.2%, 16.68%, 42.92%, and 76.5%, 18.2%, 21.56%, 32.22%, and 77.43%, and 20.8%, 23.52%, 32.22%, and 82.84% of the previous stocks. The maximum absorption of the same concentration of Cu was achieved by culture 3 at 32.6%, 49.88%, 90.44%, and 91.86%, respectively. The accumulation of the absorbed metals was found to be maximum in the protoplast of all cultures. The accumulation at the cell wall was maximum for cultures 2 and 6 for Zn and Cu, respectively, and between the cell wall and the plasma membrane, it was maximum for cultures 2 and 8 for Zn and Cu, respectively.     

Diversity of extremely halophilic bacteria

In this review, the history of the classification of the family Halobacteriaceae, the extremely halophilic aerobic Archaea, is reviewed with some emphasis on the recently described new genera Halobaculum, Halorubrum, Natrialba, Natronomonas, and "Haloterrigena." Speculation is made about the evolutionary relationship between members of the Halobacteriaceae and the extremely halophilic, anaerobic methanogens of the genera Methanohalobium and Methanohalophilus. Efforts to find missing links between the two groups are also reviewed. Received: January 22, 1998 / Accepted: February 16, 1998     

NADP-dependent isocitrate dehydrogenase from the halophilic archaeon Haloferax volcanii: cloning,sequence determination and overexpression in Escherichia coli

A gene encoding NADP-dependent Ds-threo-isocitrate dehydrogenase was isolated from Haloferax volcanii genomic DNA by using a combination of polymerase chain reaction and screening of a lambda EMBL3 library. Analysis of the nucleotide sequence revealed an open reading frame of 1260 bp encoding a protein of 419 amino acids with 45837 Da molecular mass. This sequence is highly similar to previously sequenced isocitrate dehydrogenases. In the alignment of the amino acid sequences with those from several archaeal and mesophilic NADP-dependent isocitrate dehydrogenases, the residues involved in dinucleotide binding and isocitrate binding are well conserved. We have developed methods for the expression in Escherichia coli and purification of the enzyme from H. volcanii. This expression was carried out in E. coli as inclusion bodies using the cytoplasmic expression vector pET3a. The enzyme was refolded by solubilisation in 8 M urea followed by dilution into a buffer containing EDTA, MgCl(2) and 3 M NaCl. Maximal activity was obtained after several hours incubation at room temperature.     

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.     

Availability, uptake and turnover of glycerol in hypersaline environments

Abstract A sensitive assay for glycerol and other polyols was developed, based on periodate oxidation to formaldehyde, followed by a colorimetric assay with 3-methyl-2-benzothiazolone hydrazone. Apparent glycerol concentrations thus measured in saltern crystallizer ponds were around 20–36 μM, while in the Dead Sea, during a Dunaliella bloom, values were up to 27 μM. However, these values probably overestimate the glycerol concentrations present, as shown by labeled glycerol uptake experiments. Values of [K + S_n] (natural concentration + affinity constant) in saltern ponds were as low as 0.76–1.4 μM, with V_max values of 193–303 nmol 1⁻¹h⁻¹, and turnover times between 2.6–7.2 h at 35°C. Similar measurements in the Dead Sea were: [K + S_n] 0.07–1.41 μM, V_max values 160–426 nmol 1⁻¹h⁻¹, and turnover times in the range of 0.45–3.3 h.     

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.     

Bacteriorhodopsin in a bloom of halobacteria in the Dead Sea

A dense bloom of red halobacteria developed in the Dead Sea in the summer 1980, bacterial densities of up to 1.9 x10⁷ cells ml^-1 were observed. The population consisted of two types: pleomorphic, cup-shaped cells and rod-shaped cells. A high content of bacteriorhodopsin was found in the bloom (up to 0.4 nmol per mg protein). The rod-shaped Halobacterium was isolated and was shown to contain bacteriorhodopsin.Abbreviations 20 specific gravity at 20°C - Tris Tris-(hydroxymethyl)-aminomethane     

Characterization of extremely halophilic Archaea isolated from the Ayvalik Saltern,Turkey

Seven extremely halophilic strains were isolated from the Ayvalik Saltern in the north-eastern part of Turkey. Chemical analyses of the brine and salt samples were performed to measure their salt content, hardness and pH. Isolated strains were tested for their antibiotic sensitivities; cell and colony morphologies; hydrolysis of casein, starch, gelatin, Tween 20 and Tween 80; and oxidase and catalase activity. All strains were found to belong to the domain Archaea. Characterization of polar lipids by thin layer chromatography indicated that all isolates contained phytanyl diether derivatives of phosphatidylglycerol (PG), the methyl ester of phosphatidyl glycerophosphate (PGP-Me), and phosphatidylglycerosulphate (PGS). Four isolates had triglycosyl diether (TGD-2) as glycolipid, and the other three contained a sulphated diglycosyl diether instead. All isolates were examined for the presence of plasmids by agarose gel electrophoresis. Four strains were found to harbour plasmids ranging in size from 13.8 to 15.3 kbp. Correlation between the protein profiles in SDS–PAGE and the phenotypic properties of the strains was poor. The data presented here provide the first published account of the microbiota of the Ayvalik saltern, which provides a large part of the salt produced in Turkey.     

新疆两盐湖可培养嗜盐古菌多样性研究

从新疆地区艾比盐湖和艾丁盐湖卤水及泥土样品中分离到86株嗜盐古菌。16S rRNA基因序列分析结果表明,分离自艾比湖的嗜盐古菌分别属于Haloarcula、Halobacterium、Halorubrum、Haloterrigena、Natrinema和Natronorubrum6个属的11个分类单元,而分离自艾丁湖的嗜盐古菌分别属于Haloarcula、Halobiforma、Halorubrum、Haloterrigena、Natrialba、Natrinema6个属的8个分类单元,这一结果表明艾比湖可培养嗜盐古菌生物多样性稍高于艾丁湖。基于16S rRNA基因序列的系统发育分析表明代表菌株ABH15应为Natronorubrum属的中性嗜盐古菌新种,代表菌株ABH07、ABH12、ABH17、ABH19、ABH51和AD30可能是Halobacterium、Halorubrum、Haloterrigena、Haloarcula的新成员。     

Occurrence of virus-like particles in the Dead Sea

Electron-microscopic examination of water samples from the hypersaline Dead Sea showed the presence of high numbers of virus-like particles. Between 0.9 and 7.3 × 10⁷ virus-like particles ml⁻¹ were enumerated in October 1994 in the upper 20 m of the water column during the decline of a bloom of halophilic Archaea. Virus-like particles outnumbered bacteria by a factor of 0.9–9.5 (average 4.4). A variety of viral morphologies were detected, the most often encountered being spindle-shaped, followed by polyhedral and tailed phages. In addition, other types of particles were frequently found, such as unidentified algal scales, and virus-sized star-shaped particles. Water samples collected during 1995 contained low numbers of both bacteria and virus-like particles (1.9–2.6 × 10⁶ and 0.8–4.6 × 10⁷ ml⁻¹ in April 1995), with viral numbers sharply declining afterwards (less than 10⁴ ml⁻¹ in November 1995–January 1996). It is suggested that viruses may play a major role in the decline of halophilic archaeal communities in the Dead Sea, an environment in which protozoa and other predators are absent. Received: February 5, 1997 / Accepted: May 24, 1997     

Uptake and turnover of acetate in hypersaline environments

Abstract: Acetate uptake and turnover rates were determined for the heterotrophic community in hypersaline environments (saltern crystallizer ponds, the Dead Sea) dominated by halpphilic Archaea. Acetate was formed from glycerol, which is potentially the major available carbon source for natural communities of halophilic Archaea. Values of [ K _t+ S _n] (the sum of the substrate affinity and the substrate concentration present in situ) for acetate measured in saltern crystallizer ponds were around 4.5–11.5 μM, while in the Dead Sea during a Dunaliella bloom values up to 12.8 μM were found. Maximal theoretical rates ( V _max) of acetate uptake in saltern crystallizer ponds were 12–56 nmol l⁻¹ h⁻¹, with estimated turnover times for acetate ( T _t) between 127–730 h at 35°C. V _max values measured in the Dead Sea were between 0.8 and 12.8 nmol l⁻¹ h⁻¹, with turnover times in the range of 320–2190 h. V _max values for acetate were much lower than those for glycerol. Comparisons with pure cultures of halophilic Archaea grown under different conditions showed that the natural communities were not adapted for preferential use of acetate. Both in natural brines and in pure cultures of halophilic Archaea, acetate incorporation rates rapidly decreased above the optimum pH value, probably since acetate enters the cell only in its unionized form. The low affinity for acetate, together with low potential utilization rates result in the long acetate turnover times, which explains the accumulation of acetate observed when low concentrations of glycerol are supplied as a nutrient to natural communities of halophilic Archaea.     

Characterization of a novel plasmid from extremely halophilic Archaea: nucleotide sequence and function analysis

We determined the complete nucleotide sequence of the 16341 bp plasmid pHH205 of the extremely halophilic archaeon Halobacterium salinarum J7. The plasmid has a G+C content of 61.1%. A number of direct and inverted repeat sequences were found in pHH205, while no insertion sequences were found. Thirty-eight large open reading frames (ORFs) were identified in both strands, and most of them had no significant similarities to known proteins. A putative protein encoded by ORF31 showed 20-41% homology to some hypothetical proteins, which are annotated in several archaeal genome databases as predicted nucleic acid-binding proteins containing PIN domain. Sequence analysis using the GC skew procedure predicted a possible origin of replication. A 4.8 kb PvuII-SnaBI fragment containing both this region and ORF31 was shown to be able to restore replicate of pWL102, a replicon-deficient plasmid in Haloferax volcanii and in H. salinarum R1. Several methods failed to completely cure H. salinarum J7 of pHH205, suggesting that the plasmid probably played an important role in the growth and metabolism of the host. Our work describes a novel haloarchaeal replicon, which may be useful in the construction of cloning and shuttle vectors.     

Characterization of the glyceraldehyde 3-phosphate dehydrogenase from the extremely halophilic archaebacterium Haloarcula vallismortis

Glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) from the extremely halophilic archaebacterium Haloarcula vallismortis has been purified in a four step procedure to electrophoretic homogeneity. The enzyme is a tetramer with a relative molecular mass of 160000. It is strictly NAD⁺-dependent and exhibits its highest activity in 2 mol/l KCl at 45°C. Amino acid analysis and isoelectric focusing indicate an excess of acidic amino acids. Two parts of the primary sequence are reported. These peptides have been compared with glyceraldehyde 3-phosphate dehydrogenases from other archaebacteria, eubacteria and eucaryotes. The peptides show a high grade of similarity to glyceraldehyde 3-phosphate dehydrogenase from eucaryotes.Abbreviations BCA bicinchoninic acid - CTAB cetyltrimethyl ammonium bromide - DTE dithioerythritol - DTT dithiothreitol - GAP glyccraldehyde 3-phosphate - GAPDH glyceraldehyde 3-phosphate dehydrogenase