Characterization of the dominant halophilic archaea in a bacterial bloom in the dead sea

Abstract A mass bloom of halophilic archaea developed in the Dead Sea in the summer of 1992, with peak densities of more than 3 × 10⁷ cells/ml, imparting a red coloration to the water. Microscopical examination showed a numerical dominance of pleomorphic, flat cells. Attempts to identify the dominant type of halophilic archaea by means of growth experiments, both on agar plates and by dilution in liquid media, were unsuccessful, as viable counts obtained were two or more orders of magnitude lower than the total microscopic counts. Analysis of the polar lipids in the Dead Sea biomass during the bloom showed one major glycolipid to be present in the extracts, corresponding with the sulfated diglycosyl diether lipid (S-DGD-1) characteristic of the genus Haloferax . No indications were found for the presence of significant amounts of other glycolipids that indicate the presence of large numbers of Dead Sea archaea such as Halobacterium sodomense or Haloarcula marismortui , or Halobacterium species such as H. halobium, H. salinarium and H. saccharovorum . Thus, the numerically dominant organisms in the bloom is probably a difficult to culture, not yet isolated, representative of the genus Haloferax .     

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.     

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.     

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     

Denitrification by extremely halophilic bacteria

Abstract Extremely halophilic bacteria were isolated from widely separated sites by anaerobic enrichment in the presence of nitrate. The anaerobic growth of several of these isolates was accompanied by the production of nitrite, nitrous oxide, and dinitrogen. These results are a direct confirmation of the existence of extremely halophilic denitrifying bacteria, and suggest that such bacteria may be common inhabitants of hypersaline environments.     

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.     

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.     

Polyamine contents and amino acid decarboxylation activities of extremely halophilic archaebacteria and some eubacteria

An extremely halophilic archaebacterium Halobacterium cutirubrum was demonstrated to be devoid of any polyamine except agmatine when grown in a synthetic medium with no exogenous polyamines. Decarboxylation activities of homoarginine and canavanine as well as of arginine were shown to be present in cell lysates of 5 strains of extreme halophiles examined. H. halobium R1 was shown to have an additional pathway to synthesize agmatine from glutamic acid.     

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     

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).     

基于PHA颗粒-PhaP标签和内含肽元件的极端嗜盐古菌蛋白的表达纯化系统

【目的】建立一个基于PHA颗粒-PhaP标签的简便实用的极端嗜盐古菌蛋白表达纯化系统,并探讨嗜盐古菌内含肽在该系统优化中的应用。【方法】以嗜盐古菌-大肠杆菌穿梭载体pWL502为基本骨架,构建带有嗜盐古菌强启动子和PhaP融合标签的表达载体;在地中海富盐菌phaP缺陷株(ΔphaP)中融合表达目的基因,通过蔗糖密度梯度离心分离纯化结合于PHA颗粒上的PhaP融合蛋白;在phaP基因与多克隆位点之间引入特定的嗜盐古菌内含肽元件,尝试通过定点突变改变该内含肽的剪切活性。【结果】成功构建了以PhaP作为N端融合标签的表达载体pPM以及作为C端融合标签的载体pIP;在phaP基因簇强启动子控制下,二者均实现了目标蛋白的高效表达;通过PHA颗粒介导的蛋白分离纯化策略,实现了以PhaP为融合标签的目标蛋白的分离纯化;发现内含肽序列Hbt21在地中海富盐菌中保持了高效的剪接活性,通过定点突变其C端末位氨基酸天冬酰胺(N182)及邻位的丝氨酸(S183)失活了该内含肽的C端剪接活性。【结论】首次建立了一个基于PHA颗粒-PhaP标签的简便节约的极端嗜盐古菌蛋白表达纯化系统,并确定了嗜盐古菌型内含肽C端剪接的活性位点,为该内含肽将来应用于PhaP融合蛋白的标签去除奠定了基础。     

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 extremely halophilic archaea isolated from saline environment in different parts of Turkey

Ninety-five extremely halophilic strains were isolated from six distinct saline regions of Turkey by using complex medium containing 25% NaCl. The selected regions are Tuz Golu (salt lake), Ankara; Aci Lake, Denizli; Salda Lake, Denizli; Seyfe Lake, Kirsehir; Tuzla Lake, Kayseri; and Bolluk Lake, Konya. The isolated strains were tested for motility, Gram reaction, cell and colony morphologies, pigmentation, biochemical characteristics, and antibiotic sensitivities. According to membrane glycerol diether moieties and antibiotic susceptibilities, all isolated strains were found to belong to the domain Archaea. All isolates were examined for the presence of plasmids by agarose gel electrophoresis and it was established that most isolates contained plasmids that varied in number and whose molecular sizes ranged from 1 to 36.9 kbp. Whole-cell protein profiles from isolates were analyzed by SDS-PAGE and a similarity dendogram was constructed using the UPGMA method. Significant similarities and differences were observed among the isolates. The strains were clustered in eight groups and ten of our isolates were placed in the same group with the standard strains. The current study represents the first isolation and characterization of such a large collection of archeal strains from Turkey.     

Taxonomic analysis of extremely halophilic archaea isolated from 56-years-old dead sea brine samples

A taxonomic study comprising both phenotypic and genotypic characterization, has been carried out on a total of 158 extremely halophilic aerobic archaeal strains. These strains were isolated from enrichments prepared from Dead Sea water samples dating from 1936 that were collected by B. E. Volcani for the demonstration of microbial life in the Dead Sea. The isolates were examined for 126 morphological, physiological, biochemical and nutritional tests. Numerical analysis of the data, by using the S(J) coefficient and UPGMA clustering method, showed that the isolates clustered into six phenons. Twenty-two out of the 158 strains used in this study were characterized previously (ARAHAL et al., 1996) and were placed into five phenotypic groups. The genotypic study included both the determination of the guanineplus-cytosine content of the DNA and DNA-DNA hybridization studies. For this purpose, representative strains from the six phenons were chosen. These groups were found to represent some members of three different genera - Haloarcula (phenons A, B, and C), Haloferax (phenons D and E) and Halobacterium (phenon F) - of the family Halobacteriaceae, some of them never reported to occur in the Dead Sea, such as Haloarcula hispanica, while Haloferax volcanii (phenons D and E) was described in the Dead Sea by studies carried out several decades later than Volcani's work.     

Cytological peculiarities of some extremely halophilic soil archaeobacteria

Ultrastructure and morphogenesis of extremely halophilic neutrophilic (Halobacteriam distributum, Halococcus turkmenicus) and alkaliphilic (Natronobacterium pharaonis, Natronococcus occultus) archaeobacteria were studied. The H. distributum culture was rather polymorphous and produced cells of four types. Due to the irregular cell fission in different planes packages of various numbers of cells surrounded by a common capsule were formed. Resting forms (halocysts) with multilayer covers were present in the population. The N. pharaonis culture consisted of rod-like cells and cyst-like forms. Besides, under conditions of carbon limitation, multicellular aggregated forms were found in the culture. Encapsulated single cells and aggregated forms with a common capsule were observed in H. turkmenicus and N. occultus cultures.     

Cloning and expression of the ferredoxin gene from extremely halophilic archaeon Haloarcula japonica strain TR-1

The gene encoding a ferredoxin (Fd) from Haloarcula japonica strain TR-1 was cloned and sequenced. Sequence analysis of the cloned Ha. japonica Fd gene revealed that the structural gene consisted of an open reading frame of 387 nucleotides encoding 129 amino acids. The deduced amino acid sequence of Ha. japonica Fd showed 84 to 98% identity with corresponding sequences in other extremely halophilic archaea. The Ha. japonica Fd gene was inserted into the shuttle vector pWL102 and used to transform Ha. japonica. Ha. japonica Fd could then be produced as a fusion with HisTag (6xHis) in Ha. japonica host cells. The absorption and ESR spectra of the Fd/HisTag fusion protein revealed the presence of a [2Fe-2S] cluster which is characteristic of native Ha. japonica Fd.     

Isolation of halophilic and halotolerant bacteria from a Japanese salt field and comparison of the partial 16S rRNA gene sequence of an extremely halophilic isolate with those of other extreme halophiles

Halophilic and halotolerant bacteria were isolated from soil samples of a Japanese salt field, an environment where salt concentrations vary annually. From 1 g of each of the five samples collected, over 1×10³ bacterial colonies (colony forming units (cfu)g^-1) grew on agar medium containing 2M Na⁺. In contrast, 0–4 bacterial colonies (cfu g^-1) were observed on agar medium containing 4M Na⁺. Two of the five samples contained numerous bacteria (10²–10³ cfu g^-1) capable of growth on a 2M Na⁺ alkaline (pH=9.5) medium, while few bacterial colonies were observed from the other three samples. Only one of the five samples was shown to contain bacteria capable of growth on a 4M Na⁺ alkaline medium. Most of the bacteria isolated on 4M Na⁺ agar were eubacteria, but one extreme halophile (TR-1, already described as Haloarcula japonica JCM7785) was also isolated. The 16S rRNA sequence of TR-1 was determined and shows high homology (94.4–98.5%) to Ha. marismortui and Ha. sinaiiensis. These results suggested that: 1) environments with seasonally varying salinity can harbour halotolerants as well as halophiles and, 2) closely related halophiles can be isolated from geographically distant habitats.     

Identification and partial purification of DnaK homologue from extremely halophilic archaebacteria, Halobacterium cutirubrum

The levels of synthesis of six proteins were increased at elevated growth temperature of the extremely halophilic archaebacterium Halobacterium cutirubrum. One of these proteins, with an apparent molecular mass of 97 kDa on sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS–PAGE), bound to an ATP-agarose column in the presence of 4 M NaCl, but not in the absence of salt, indicating that this protein retained its ATP-binding activity only at high salt concentration. The NH₂-terminal sequence of this protein and the internal sequences of the tryptic peptides covering 1/3 of the total number of residues coincided with that deduced from the nucleotide sequence of the dnaK gene isolated from H. cutirubrum. The results strongly suggest that this apparent 97-kDa protein is the gene product of dnaK, although the molecular mass calculated from the nucleotide sequence is only 68,495, much smaller than the value of this protein determined by SDS–PAGE. Ferguson plot analysis indicated that this protein showed anomalous mobility on SDS–PAGE. We have purified DnaK homologue to greater than 90% homogeneity with stepwise elution from an ATP-agarose column.