Genus Haloferax Research Articles

Halophilic archaea isolated from man-made young (200 years) salt lakes in Slănic, Prahova, Romania

Abstract We isolated a number of extremely halophilic archaea from four salt lakes (Red Bath, Green Bath, Shepherd Bath and Bride Cave) located in Slanic Prahova, Romania. The characterization of 12 representative isolates by polyphasic approach revealed that 11 strains were members of the genus Haloferax and only one was a member of the genus Haloarcula. The 11 Haloferax isolates possessed sulfated diglycosylarchaeol-1 as the major membrane glycolipid, and G+C contents of total DNA were 63.4–65.8 mol%. The predominant isolation of Haloferax species from the lakes may suggest that the underground salt deposit possesses Haloferax species as the major biota of ancient origin. To the best of the authors’ knowledge, this is the first paper on the survey of halophilic archaea of man-made young salt lakes.

Haloferax elongans sp. nov. and Haloferax mucosum sp. nov., isolated from microbial mats from Hamelin Pool, Shark Bay, Australia

Extremely halophilic archaea were cultivated from smooth and pustular microbial mats collected from Hamelin Pool, Shark Bay, Western Australia. On the basis of morphology, two phenotypes were present and 16S rRNA gene sequence analysis indicated that all strains were most closely related to members of the genus Haloferax (98.1-99.4 % similarity). One representative strain from each phenotype was selected for further taxonomic characterization. Strain SA5T, isolated from the smooth mat, formed small ( approximately 1 mm diameter), red, translucent colonies on agar medium and strain PA12T, isolated from the pustular mat, formed large (3-5 mm diameter), pink, mucoid, domed colonies. Both strains grew in media with 1.7-5.1 M NaCl, required at least 0.2 M Mg2+ for growth and had pH optima of 7.4. The 16S rRNA gene similarity between strains SA5T and PA12T was 97.1 %. Physiological properties, G+C content and polar lipid composition supported placement of both strains in the genus Haloferax. Phenotypic analysis indicated that the two strains were distinct from each other and from all other members of the genus. This was confirmed by the low DNA-DNA relatedness between strains SA5T and PA12T (18-30 %) and between both strains and all other recognized Haloferax species. Two novel species of the genus Haloferax are proposed to accommodate these novel isolates, Haloferax elongans sp. nov. (type strain SA5T=JCM 14791T=ATCC BAA-1513T=UNSW 104100T) and Haloferax mucosum sp. nov. (type strain PA12T=JCM 14792T=ATCC BAA-1512T=UNSW 104200T).

Haloferax larsenii sp. nov., an extremely halophilic archaeon from a solar saltern

Three strains of Gram-negative, aerobic, neutrophilic, extremely halophilic archaea, designated ZJ206(T), ZJ203 and ZJ204, were isolated from a solar saltern in Zhe-Jiang Province, China. Phenotypically and on the basis of 16S rRNA gene sequences, the strains were very similar. Comparative 16S rRNA gene analysis revealed 96.4-97.4 % sequence similarity to members of the genus Haloferax. The major polar lipids were C(20)C(20) derivatives of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, diglycosyl glycerol diether and sulfated diglycosyl diether. The DNA G+C content of strain ZJ206(T) was 62.2 mol%. The results of DNA-DNA hybridizations and physiological and biochemical tests allowed genotypic and phenotypic differentiation of the isolates from closely related species. Therefore the isolates should be classified as members of a novel species, for which the name Haloferax larsenii sp. nov. is proposed. The type strain is ZJ206(T) (=CGMCC 1.5347(T)=JCM 13917(T)).

Haloferax prahovense sp. nov., an extremely halophilic archaeon isolated from a Romanian salt lake

A novel halophilic archaeon, strain TL6T, was isolated from Telega Lake, a hypersaline environment in Prahova county, Romania. Strain TL6T was able to grow in media with a salt concentration of between 2.5 and 5.2 M, with optimum growth at a concentration of 3.5 M. The novel strain was able to grow at concentrations of 1 M MgCl2 or less, with an optimum of 0.4 M Mg2+. Growth of the novel strain occurred between pH 6.0 and 8.5, with an optimum of pH 7.0-7.5. The G+C content of the total DNA was 63.7 mol%. The 16S rRNA gene sequence of the novel strain was most closely related to species of the genus Haloferax (97.3-99.3 % sequence similarity). The lipid profile of the novel strain corresponded to that of other species belonging to the genus Haloferax. A comparative analysis of the phenotypic properties and DNA-DNA hybridization between the novel strain and other species of the genus Haloferax strongly supported the conclusion that strain TL6T represents a novel species within this genus, for which the name Haloferax prahovense sp. nov., is proposed. The type strain is TL6T (=JCM 13924T=DSM 18310T).

Haloferax sulfurifontis sp. nov., a halophilic archaeon isolated from a sulfide- and sulfur-rich spring

A pleomorphic, extremely halophilic archaeon (strain M6(T)) was isolated from a sulfide- and sulfur-rich spring in south-western Oklahoma (USA). It formed small (0.8-1.0 mm), salmon pink, elevated colonies on agar medium. The strain grew in a wide range of NaCl concentrations (6 % to saturation) and required at least 1 mM Mg(2+) for growth. Strain M6(T) was able to reduce sulfur to sulfide anaerobically. 16S rRNA gene sequence analysis indicated that strain M6(T) belongs to the family Halobacteriaceae, genus Haloferax; it showed 96.7-98.0 % similarity to other members of the genus with validly published names and 89 % similarity to Halogeometricum borinquense, its closest relative outside the genus Haloferax. Polar lipid analysis and DNA G+C content further supported placement of strain M6(T) in the genus Haloferax. DNA-DNA hybridization values, as well as biochemical and physiological characterization, allowed strain M6(T) to be differentiated from other members of the genus Haloferax. A novel species, Haloferax sulfurifontis sp. nov., is therefore proposed to accommodate the strain. The type strain is M6(T) (=JCM 12327(T)=CCM 7217(T)=DSM 16227(T)=CIP 108334(T)).

Haloferax alexandrinus sp. nov., an extremely halophilic canthaxanthin-producing archaeon from a solar saltern in Alexandria (Egypt).

An extremely halophilic red micro-organism designated strain TM(T) was isolated from a solar saltern in Alexandria, Egypt. The micro-organism stains gram-negative, is very pleomorphic, non-motile and strictly aerobic and requires at least 10 g NaCl l(-1) for growth. The growth optimum is 250 g NaCl l(-1). Growth is also observed over a wide range of MgSO4 concentrations (10-40 g l(-1)). Aerobic reduction of nitrate without gas production was detected. Cells grew aerobically in a minimal salts medium containing ammonium chloride and glucose. Strain TM(T) produced acid from fructose, glucose, rhamnose, maltose and glycerol. The G+C content of the DNA was 59.5+/-0.3 mol %. On the basis of polar lipid analysis, the isolate belonged to the genus Haloferax. Analysis of the 16S rDNA sequence showed the highest similarity (>99%) to be to the type strain Haloferax volcanii. Although the spectrum of antibiotic susceptibility was similar to that of validly described species of the genus Haloferax, the strain could be distinguished from them by its different response to josamycin and rifampicin. Strain TM(T) is unique within the genus Haloferax in producing canthaxanthin. Comparative analysis of phenotypic properties and DNA-DNA hybridization between strain TM(T) and Haloferax species supported the conclusion that TM(T) is a novel species within this genus, for which the name Haloferax alexandrinus sp. nov. is proposed. The type strain is TM(T) (= JCM 10717T = IFO 16590T).

Lipids of Haloferax alexandrinus strain TM T: an extremely halophilic canthaxanthin-producing archaeon

The core lipids, polar and non polar lipids, of the novel canthaxanthin-producing archaeon, Haloferax alexandrinus strain TM T, were investigated using thin-layer chromatography (TLC), spectrophotometry, electron ionization-mass spectroscopy (EI-MS), fast atom bombardment mass spectrometry (FAB-MS), nuclear magnetic response spectrometry ( 13C-NMR) and high performance liquid chromatography (HPLC). This archaeon appeared to contain diphytanyl diether lipids (C 20-C 20) as core lipids. The major phospholipids were found to be phosphatidylglycero-phosphate-methyl ester (PGP-Me) and phosphatidylglycerol (PG), but no phosphatidylglycero-sulfate was detected. The strain contained two glycolipids, sulfated diglycosyl diether (S-DGD-1) and unsulfated diglycosyl diether (DGD). Analysis of the non polar lipids revealed the presence of β-carotene, 3-hydroxyechinenone, γ-carotene, cis-astaxanthin, lycopene, trisanhydro-bacterioruberin,monanhydro-bacterioruberin, bacterioruberin isomer, bacterioruberin and canthaxanthin. Although the polar lipids profile of Hfx. alexandrinus strain TM T was similar to those of other species of the genus Haloferax, the non polar lipid (carotenoids) profile was markedly different. Further experiments on the influence of NaCl concentration on the lipids composition of Hfx. alexandrinus strain TM T demonstrated the dependence of the proportion of each of bacterioruberin, β-carotene and canthaxanthin on the NaCl concentration in the growth media.

The ftsZ gene of Haloferax mediterranei: sequence, conserved gene order, and visualization of the FtsZ ring

We sequenced the ftsZ gene region of the halophilic archaeon Haloferax mediterranei and mapped the transcription start sites for the ftsZ gene. The gene encoded a 363-amino-acid long FtsZ protein with a predicted molecular mass of 38 kDa and an isoelectric point of 4.2. A high level of similarity to the FtsZ protein of Haloferax volcanii was apparent, with 97 and 90% identity at the amino acid and nucleotide levels, respectively. Structural conservation at the protein level was shown by visualization of the FtsZ ring structure in H. mediterranei cells using an antiserum raised against FtsZ of H. volcanii. FtsZ rings were observed in cells in different stages of division, including cells with pleomorphic shapes and cells that appeared to be undergoing asymmetric division. Cells were also observed that displayed constriction-like invaginations in the absence of an FtsZ ring, indicating that morphological data are not sufficient to determine whether pleomorphic Haloferax cells are undergoing cell division. Both the upstream and downstream gene order in the ftsZ region was found to be conserved within the genus Haloferax. Furthermore, the downstream gene order, which includes the secE and nusG genes, is conserved in almost all euryarchaea sequenced to date. The secE and nusG genes are likely to be transcriptionally and translationally coupled in Haloferax, and this co-expression may have been a selective force that has contributed to keeping the gene cluster intact.

Halogeometricum borinquense gen. nov., sp. nov., a novel halophilic archaeon from Puerto Rico.

A novel extremely halophilic archaeon was isolated from the solar salterns of Cabo Rojo, Puerto Rico. The organism is very pleomorphic, motile and requires at least 8% (w/v) NaCl to grow. Polar lipid composition revealed the presence of a novel non-sulfate-containing glycolipid and the absence of the glycerol diether analogue of phosphatidylglycerosulfate. The G + C content of the DNA is 59 mol%. On the basis of 16S rRNA sequence data, the new isolate cannot be classified in one of the recognized genera, but occupies a position that is distantly related to the genus Haloferax. All these features justify the creation of a new genus and a new species for the family Halobacteriaceae, order Halobacteriales. The name Halogeometricum borinquense gen. nov., sp. nov. is proposed. The type strain is ATCC 700274T.

Halobaculum gomorrense gen. nov., sp. nov., a Novel Extremely Halophilic Archaeon from the Dead Sea

A novel extremely halophilic archaeon was isolated from the Dead Sea. This isolate is rod shaped and, like Halobacterium sodomense, requires a relatively low level of sodium ions for growth and a very high level of magnesium; optimal growth occurs in the presence of 0.6 to 1.0 M Mg2+. The new strain resembles members of the Halobacterium saccharovorum-Halobacterium sodomense-Halobacterium trapanicum group in many physiological properties. However, the polar lipid composition of this organism is characteristic of representatives of the genus Haloferax; a sulfated diglycosyl diether is present, and the glycerol diether analog of phosphatidylglycerosulfate is absent. The G+C content of the DNA is 70 mol%. We found that on the basis of 16S rRNA sequence data our new isolate occupies a position intermediate between the position of the Halobacterium saccharovorum group and the position of the genus Haloferax and is sufficiently different from the previously described members of the Halobacteriaceae to justify classification in a new species and a new genus. We propose the name Halobaculum gomorrense gen. nov., sp. nov. for this organism; the type strain is strain DSM 9297.

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.

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

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.

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

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.

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

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.

DNA-rRNA hybridization studies onHalococcus saccharolyticusand other halobacteria

Several representative extremely halophilic archaea (formerly archaebacteria) were studied with respect to their genetic relatedness by using a DNA-rRNA hybridization approach. Our results confirm the phylogenetic relatedness of Halococcus morrhuae and Halococcus saccharolyticus but support their separation as two different taxa, at least at species level. In addition, this phylogenetic relatedness was significantly lower than that between species belonging to the genera Haloferax and Haloarcula.

A plasmid vector with a selectable marker for halophilic archaebacteria

A mutant resistant to the gyrase inhibitor novobiocin was selected from a halophilic archaebacterium belonging to the genus Haloferax. Chromosomal DNA from this mutant was able to transform wild-type cells to novobiocin resistance, and these transformants formed visible colonies in 3 to 4 days on selective plates. The resistance gene was isolated on a 6.7-kilobase DNA KpnI fragment, which was inserted into a cryptic multicopy plasmid (pHK2) derived from the same host strain. The recombinant plasmid transformed wild-type cells at a high efficiency (greater than 10(6)/micrograms), was stably maintained, and could readily be reisolated from transformants. It could also transform Halobacterium volcanii and appears to be a useful system for genetic analysis in halophilic archaebacteria.

The glycolipid ofHalobacterium saccharovorum

Abstract The polar lipids of the halophilic archaebacterium Halobacterium saccharovorum have been analyzed by spectroscopic methods, including 13 C NMR to establish structural detail. C 20 , C 20 diether forms of phosphatidyl glycerol (PG), phosphatidyl glycerol phosphate (PGP) and phosphatidyl glycerol sulphate (PGS) are the major polar lipids, together with the sulphated diglycosyl diether, 2, 3-di-O-phytanyl-1-O-[mannopyrsanosyl-(6-sulphate)-α- d -1 → 2-glucopyranosyl-α- d ]-sn-glycerol (SDGD-1) characteristics of members of the genus Haloferax .