Spherical particles of halophilic archaea correlate with exposure to low water activity - implications for microbial survival in fluid inclusions of ancient halite

Viable extremely halophilic archaea (haloarchaea) have been isolated from million‐year‐old salt deposits around the world; however, an explanation of their supposed longevity remains a fundamental challenge. Recently small roundish particles in fluid inclusions of 22 000‐ to 34 000‐year‐old halite were identified as haloarchaea capable of proliferation (Schubert BA, Lowenstein TK, Timofeeff MN, Parker MA, 2010, Environmental Microbiology, 12, 440–454). Searching for a method to produce such particles in the laboratory, we exposed rod‐shaped cells of Halobacterium species to reduced external water activity (a_w). Gradual formation of spheres of about 0.4 μm diameter occurred in 4 m NaCl buffer of a_w ≤ 0.75, but exposure to buffered 4 m LiCl (a_w ≤ 0.73) split cells into spheres within seconds, with concomitant release of several proteins. From one rod, three or four spheres emerged, which re‐grew to normal rods in nutrient media. Biochemical properties of rods and spheres were similar, except for a markedly reduced ATP content (about 50‐fold) and an increased lag phase of spheres, as is known from dormant bacteria. The presence of viable particles of similar sizes in ancient fluid inclusions suggested that spheres might represent dormant states of haloarchaea. The easy production of spheres by lowering a_w should facilitate their investigation and could help to understand the mechanisms for microbial survival over geological times.     

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     

Selective enrichment,isolation and molecular detection of <Emphasis Type="Italic">Salinibacter</Emphasis> and related extremely halophilic <Emphasis Type="Italic">Bacteria</Emphasis> from hypersaline environments

Salinibacter is a genus of red, extremely halophilic Bacteria. Thus far the genus is represented by a single species, Salinibacter ruber, strains of which have been isolated from saltern crystallizer ponds in Spain and on the Balearic Islands. Both with respect to its growth conditions and its physiology, Salinibacter resembles the halophilic Archaea of the order Halobacteriales. We have designed selective enrichment and isolation techniques to obtain Salinibacter and related red extremely halophilic Bacteria from different hypersaline environments, based on their resistance to anisomycin and bacitracin, two antibiotics that are potent inhibitors of the halophilic Archaea. Using direct plating on media containing bacitracin, we found Salinibacter-like organisms in numbers between 1.4×10³ and 1.4×10⁶ml⁻¹ in brines collected from the crystallizer ponds of the salterns in Eilat, Israel, being equivalent to 1.8–18% of the total colony counts obtained on identical media without bacitracin. A number of strains from Eilat were subjected to a preliminary characterization, and they proved similar to the type strain of S. ruber. We also report here the isolation and molecular detection of Salinibacter-like organisms from an evaporite crust on the bottom of salt pools at the Badwater site in Death Valley, CA. These isolates and environmental 16S rRNA gene sequences differ in a number of properties from S. ruber, and they may represent a new species of Salinibacter or a new related genus. Guest Editor: John M. Melack Saline Waters and their Biota     

Impact of paleoclimate on the distribution of microbial communities in the subsurface sediment of the Dead Sea

A long sedimentary core has been recently retrieved from the Dead Sea Basin (DSB) within the framework of the ICDP‐sponsored Dead Sea Deep Drilling Project. Contrasting climatic intervals were evident by distinctive lithological facies such as laminated aragonitic muds and evaporites. A geomicrobiological investigation was conducted in representative sediments of this core. To identify the microbial assemblages present in the sediments and their evolution with changing depositional environments through time, the diversity of the 16S rRNA gene was analyzed in gypsum, aragonitic laminae, and halite samples. The subsurface microbial community was largely dominated by the Euryarchaeota phylum (Archaea). Within the latter, Halobacteriaceae members were ubiquitous, probably favored by their ‘high salt‐in’ osmotic adaptation which also makes them one of the rare inhabitants of the modern Dead Sea. Bacterial community members were scarce, emphasizing that the ‘low salt‐in’ strategy is less suitable in this environment. Substantial differences in assemblages are observed between aragonitic sediments and gypsum–halite ones, independently of the depth and salinity. The aragonite sample, deposited during humid periods when the lake was stratified, consists mostly of the archaeal MSBL1 and bacterial KB1 Candidate Divisions. This consortium probably relies on compatible solutes supplied from the lake by halotolerant species present in these more favorable periods. In contrast, members of the Halobacteriaceae were the sole habitants of the gypsum–halite sediments which result from a holomictic lake. Although the biomass is low, these variations in the observed subsurface microbial populations appear to be controlled by biological conditions in the water column at the time of sedimentation, and subsequently by the presence or absence of stratification and dilution in the lake. As the latter are controlled by climatic changes, our data suggest a relationship between local lacustrine subsurface microbial assemblages and large‐scale climatic variations over the Dead Sea Basin.     

Archaeal diversity along a subterranean salt core from the Salar Grande (Chile)

The Salar Grande in the Coastal Range of Northern Chile is a fossil evaporitic basin filled with almost pure halite (95% NaCl average). It is assumed that the basin has not received input of brines since the Pliocene (5.3 to 1.8 million years). Below 1 m the halite has remained undissolved since this time, whereas the upper layer has been dissolved and recrystallized by dripping fogs and occasional rainfall. We compared the archaeal community at different depths using both nested PCR and cultivation. The upper 10 cm of halite crust contained diverse haloarchaeal species, including several from new genera, but their provenance is unknown. For samples deeper in the core, a new and rigorous procedure for chemically sterilizing the surface of single halite crystals was developed. These halite crystals contained only species of the genus Halobacterium (Hbt.). Halobacterium salinarum-like sequences were detected by PCR, and evidence that they were from ancient DNA include: comparison with numerous negative controls; detection of 16S rRNA sequence differences in non-conserved regions, indicating genuine evolutionary mutations rather than PCR-cloning artefacts; independent isolation of Hbt. salinarum from ancient halite; and diverse mechanisms possessed by this species for minimizing radiation damage and thus enhancing its potential for long-term survival. Haloarchaea related to Hbt. noricense were obtained from enrichment cultures from ≈ 0.4 and 15.4 m depth. We investigated Hbt. noricense strain A1 and found that when trapped inside halite crystals its recovery was as rapid after 27 months of entombment as at day 0, faring much better than other extreme halophiles. A biogeographical investigation showed that Hbt. noricense-like organisms were: commonly found in surface-sterilized ancient halite, associated with salt mines, in halite crusts, and, despite a much more intense search, only rarely detected in surface environments. We conclude that some Halobacterium species are specialists at long-term survival in halite.     

A New Halophilic Heterolobosean Flagellate,Aurem hypersalina gen. n. et sp. n., Closely Related to the Pleurostomum‐Tulamoeba Clade: Implications for Adaptive Radiation of Halophilic Eukaryotes

Halophilic protozoa are independently scattered across the molecular phylogeny of eukaryotes; most of which are assigned to Heterolobosea. Here, we isolated a biflagellate from a hypersaline water of 342‰ salinity. This isolate shared several morphological features with typical halophilic heterolobosean flagellates. In addition, molecular phylogenetic trees of the 18S rRNA gene sequences clearly indicated flagellate is a heterolobosean species closely related to the halophilic Tulamoebidae. However, the flagellate was not accommodated to any described genus. Cells were ovoid‐shaped, and no amoebae were observed. The two unequal flagella beat heterodynamically. An ear‐like bulge at the margin of a cytostomal groove was observed. Flagellates could grow at 100–200‰ salinity, suggesting an obligately halophilic species. Currently, it appears that the new halophilic Aurem hypersalina forms a strong clade with Tulamoebidae, and is sister to the Tulamoebidae, indicating that this new clade is composed almost entirely of obligate halophilic taxa. Thus, A. hypersalina and the Tulamoebidae clade currently represent a unique adaptive radiation of halophilic eukaryotes.     

Enzyme Characteristics of Extremely Halophilic Archaeal Community in Tuzkoy Salt Mine,Turkey

Tuzkoy is a large and unpolluted salt mine in Central Anatolia, Turkey. High quality salt in this mine may be supplied to the table salt market after a simple process instead of complicated refining. However, the extremely halophilic microbial content of the salt has not yet been investigated which may result in detrimental effects in industrial processes such the food and leather industries, if unprocessed salt is directly used. Therefore, to identify and characterize the microbial contaminants in Tuzkoy salt, chemical and microbial analyses were conducted on salt crystal samples collected from three different locations of the mine. Generally 10⁵–10⁶ colony forming units of extremely halophilic microorganisms were detected per gram of salt samples. Twelve colonies were selected randomly for further characterization. Phenotypic characterization, lipid analysis, antibiotic susceptibility tests and positive PCR amplification results with Archaea-specific primers confirmed that all strains were halophilic Archaea belong to family Halobacteriaceae. According to denaturing gradient gel electrophoresis (DGGE) results, 10 comparatively different strains were selected for DNA sequencing. DNA sequences and phylogenetic analyses revealed that the isolated strains were mainly species of the genera Halobacterium, Haloarcula, Natrinema and Halorubrum. In addition enzymatic activity tests were also conducted to evaluate the salt quality for industrial applications. Results of gelatinase, caseinase, amylase, cellulase and lipase activity tests revealed that the isolated strains produced hydrolytic enzymes, which could cause deterioration in salt-treated food and hide. It may be pointed out that cellulase activity in halophilic Archaea has not been reported previously. Moreover, β-galactosidase activity has been reported in some Haloferax and Halorubrum species but not in the genus Halobacterium. This revised version was published online in July 2006 with corrections to the Cover Date.     

Culturable prokaryotic diversity of deep,gas hydrate sediments: first use of a continuous high‐pressure,anaerobic, enrichment and isolation system for subseafloor sediments (DeepIsoBUG)

Deep subseafloor sediments may contain depressurization‐sensitive, anaerobic, piezophilic prokaryotes. To test this we developed the DeepIsoBUG system, which when coupled with the HYACINTH pressure‐retaining drilling and core storage system and the PRESS core cutting and processing system, enables deep sediments to be handled without depressurization (up to 25 MPa) and anaerobic prokaryotic enrichments and isolation to be conducted up to 100 MPa. Here, we describe the system and its first use with subsurface gas hydrate sediments from the Indian Continental Shelf, Cascadia Margin and Gulf of Mexico. Generally, highest cell concentrations in enrichments occurred close to in situ pressures (14 MPa) in a variety of media, although growth continued up to at least 80 MPa. Predominant sequences in enrichments were Carnobacterium, Clostridium, Marinilactibacillus and Pseudomonas, plus Acetobacterium and Bacteroidetes in Indian samples, largely independent of media and pressures. Related 16S rRNA gene sequences for all of these Bacteria have been detected in deep, subsurface environments, although isolated strains were piezotolerant, being able to grow at atmospheric pressure. Only the Clostridium and Acetobacterium were obligate anaerobes. No Archaea were enriched. It may be that these sediment samples were not deep enough (total depth 1126–1527 m) to obtain obligate piezophiles.     

Microbial Diversity in Maras Salterns, a Hypersaline Environment in the Peruvian Andes

Maras salterns are located 3,380 m above sea level in the Peruvian Andes. These salterns consist of more than 3,000 little ponds which are not interconnected and act as crystallizers where salt precipitates. These ponds are fed by hypersaline spring water rich in sodium and chloride. The microbiota inhabiting these salterns was examined by fluorescence in situ hybridization (FISH), 16S rRNA gene clone library analysis, and cultivation techniques. The total counts per milliliter in the ponds were around 2 × 10⁶ to 3 × 10⁶ cells/ml, while the spring water contained less than 100 cells/ml and did not yield any detectable FISH signal. The microbiota inhabiting the ponds was dominated (80 to 86% of the total counts) by Archaea, while Bacteria accounted for 10 to 13% of the 4′,6′-diamidino-2-phenylindole (DAPI) counts. A total of 239 16S rRNA gene clones were analyzed (132 Archaea clones and 107 Bacteria clones). According to the clone libraries, the archaeal assemblage was dominated by microorganisms related to the cosmopolitan square archaeon “Haloquadra walsbyi,” although a substantial number of the sequences in the libraries (31% of the 16S rRNA gene archaeal clones) were related to Halobacterium sp., which is not normally found in clone libraries from solar salterns. All the bacterial clones were closely related to each other and to the γ-proteobacterium “Pseudomonas halophila” DSM 3050. FISH analysis with a probe specific for this bacterial assemblage revealed that it accounted for 69 to 76% of the total bacterial counts detected with a Bacteria-specific probe. When pond water was used to inoculate solid media containing 25% total salts, both extremely halophilic Archaea and Bacteria were isolated. Archaeal isolates were not related to the isolates in clone libraries, although several bacterial isolates were very closely related to the “P. halophila” cluster found in the libraries. As observed for other hypersaline environments, extremely halophilic bacteria that had ecological relevance seemed to be easier to culture than their archaeal counterparts.     

Description of a New Freshwater Ciliate Epistylis wuhanensis n. sp. (Ciliophora,Peritrichia) from China,with a Focus on Phylogenetic Relationships within Family Epistylididae

Two populations of Epistylis wuhanensis n. sp., a new freshwater peritrich ciliate, were isolated from different freshwater ponds located in Hubei, China. Their morphological characteristics were investigated using live observation, protargol impregnation, and scanning electron microscopy (SEM). Specimens from the two populations showed identical arrangement of the infraciliature and identical small subunit ribosomal RNA (SSU rRNA) gene and ITS1‐5.8S‐ITS2 sequences. The zooids present bell‐shaped and 90–175 × 27–54 μm in vivo. Macronucleus is variable in shape and located in the middle of cell. Pellicle is usually smooth with 139–154 and 97–105 striations above and below the trochal band, respectively. SSU rRNA gene and ITS1‐5.8S‐ITS2 sequences of E. wuhanensis n. sp. did not match any available sequences in GenBank. Phylogenetically, E. wuhanensis n. sp. clusters with the other Epistylis within the family Epistylididae, but is distinct from the major clades of Epistylis. Above all, the morphological characteristics and molecular analyses support that the present Epistylis is a new species. Expanded phylogenetic analyses of sessilids based on both SSU rRNA gene sequences and ITS1‐5.8S‐ITS2 sequences reveal that the genus Epistylis consists of Epistylis morphospecies and taxonomic revision of the genus is needed.     

Reconstruction of Late Cretaceous coastal paleotemperature from halite deposits of the Late Cretaceous Nongbok Formation (Khorat Plateau,Laos)

Cretaceous evaporites of the Maha Sarakhan Formation in Thailand (e.g., the Nongbok Formation, Laos) have been studied for almost a century as the huge potash deposits in the world. The consistently high local paleotemperatures should lead to huge salt deposits during the evaporation process. Primary fluid inclusions in halite can provide surface brine water temperatures directly and quantitatively. Until now, there have been no data published from paleotemperature of primary fluid inclusions of Cretaceous halite. The non-marine halite from the Cretaceous Nongbok Formation (Laos) precipitated from shallow brine waters with temperatures of 17.7–42.3 °C.     

Application of rpoB sequence similarity analysis, REP-PCR and BOX-PCR for the differentiation of species within the genus Geobacillus

Aim:  To investigate the applicability of rpoB gene, which encodes the β subunit of RNA polymerase, to be used as an alternative to 16S rRNA for sequence similarity analysis in the thermophilic genus Geobacillus. Rapid and reproducible repetitive extragenic palindromic fingerprinting techniques (REP‐ and BOX‐polymerase chain reaction) were also used. Methods and Results:  rpoB DNA (458 bp) were amplified from 21 Geobacillus‐ and Bacillus type strains, producing different BOX‐ and REP‐PCR profiles, in addition to 11 thermophilic isolates of Geobacillus and Bacillus species from a Santorini volcano habitat. The sequences and the phylogenetic tree of rpoB were compared with those obtained from 16S rRNA gene analysis. The results demonstrated between 90–100% (16S rRNA) and 74–100% (rpoB) similarity among examined bacteria. Conclusion:  BOX‐ and REP‐PCR can be applied for molecular typing within Geobacillus genus. rpoB sequence similarity analysis permits a more accurate discrimination of the species within the Geobacillus genus than the more commonly used 16S rRNA. Significance and Impact of the Study:  The obtained results suggested that rpoB sequence similarity analysis is a powerful tool for discrimination between species within the ecologically and industrially important strains of Geobacillus genus.     

青藏高原察尔汗盐湖地区可培养中度嗜盐菌的群落结构与多样性

【目的】研究青海察尔汗盐湖地区的可培养中度嗜盐菌的群落结构及多样性。【方法】采用多种选择性培养基进行中度嗜盐菌的分离、培养;通过16S r RNA基因序列扩增、测定,根据序列信息,进行系统进化树构建、群落结构组成分析及多样性指数计算。【结果】从察尔汗盐湖卤水及湖泥中分离到中度嗜盐菌421株,合并重复菌株后共83株中度嗜盐菌。菌株16S rRNA基因序列信息显示,4株中度嗜盐菌为潜在的新分类单元。83株嗜盐细菌分布于3个门的6个科16个属。其中,Bacillus属、Oceanobacillus属和Halomonas属为优势属。多样性结果显示,水样中的菌株多样性高于泥样,而泥样中的菌株优势度高于水样。【结论】察尔汗盐湖中度嗜盐菌具有丰富的遗传多样性,种群种类丰富,优势菌群集中,该盐湖地区存在可分离培养的中度嗜盐菌的疑似新物种。     

Microbial diversity and activity through a permafrost/ground ice core profile from the Canadian high Arctic

Culture‐dependent and culture‐independent methods were used in an investigation of the microbial diversity in a permafrost/massive ground ice core from the Canadian high Arctic. Denaturing gradient gel electrophoresis as well as Bacteria and Archaea 16S rRNA gene clone libraries showed differences in the composition of the microbial communities in the distinct core horizons. Microbial diversity was similar in the active layer (surface) soil, permafrost table and permafrost horizons while the ground ice microbial community showed low diversity. Bacteria and Archaea sequences related to the Actinobacteria (54%) and Crenarchaeota (100%) respectively were predominant in the active layer while the majority of sequences in the permafrost were related to the Proteobacteria (57%) and Euryarchaeota (76%). The most abundant phyla in the ground ice clone libraries were the Firmicutes (59%) and Crenarchaeota (82%). Isolates from the permafrost were both less abundant and diverse than in the active layer soil, while no culturable cells were recovered from the ground ice. Mineralization of [1‐¹⁴C] acetic acid and [2‐¹⁴C] glucose was used to detect microbial activity in the different horizons in the core. Mineralization was detected at near ambient permafrost temperatures (?15°C), indicating that permafrost may harbour an active microbial population, while the low microbial diversity, abundance and activity in ground ice suggests a less hospitable microbial habitat.     

Morphology and Phylogeny of Two New Pleurostomatid Ciliates,Epiphyllum shenzhenense n. sp. and Loxophyllum spirellum n. sp. (Protozoa,Ciliophora) from A Mangrove Wetland,South China

ABSTRACT. The morphology, infraciliature, and small subunit (SSU) rRNA gene sequences of two new pleurostomatid ciliates, Epiphyllum shenzhenense n. sp. and Loxophyllum spirellum n. sp., isolated from a mangrove wetland near Shenzhen, South China, were investigated. Epiphyllum shenzhenense n. sp. is morphologically characterized by leaf‐shaped cell about 150 × 35 μm in vivo, usually with four contractile vacuoles, 20–29 right kineties and 10–26 left kineties, ca. four macronuclear nodules, and two types of extrusomes (i.e. short spindle‐shaped and long bar‐shaped). As a new species, L. spirellum n. sp. is distinguished from its congeners by its posterior dorsal margin twisted onto the left side, the distribution of extrusomes (evenly arranged along the oral slit, the posterior end, and clustered to 7–13 warts on dorsal margin), the subterminally positioned contractile vacuole, the number of kineties (8–10 on right side, 4–5 on left side), and its genetic distance from congeners. Phylogenetic trees based on the SSU rRNA gene sequence for both organisms were constructed, which indicate that Epiphyllum is a distinct genus and occupies a basal position in the Pleurostomatida clade; L. spirellum n. sp. falls well into the Loxophyllum clade, which has a close relationship with Litonotus and Spiroloxophyllum.     

A New Heterolobosean Amoeboflagellate,Tetramitus dokdoensis n. sp., Isolated from a Freshwater Pond on Dokdo Island in the East Sea,Korea

The genus Tetramitus is a representative amoeboflagellate group within the Heterolobosea, and currently contains over a dozen species. Here, a new heterolobosean amoeboflagellate was isolated from a freshwater pond on Dokdo Island, Korea. The amoebae have eruptive pseudopodia, no uroidal filament, and a nucleus with a central nucleolus. The length and width of the amoebae are 15.5–28.0 μm and 5.4–12.6 μm, respectively. The flagellates are conical, with 4 flagella of equal length (~10 μm). There is a discrete rostrum in the subapical region of the flagellate form. The cyst has thin endo‐ and ectocyst layers and no cyst pores. The amoeba shows slow movement at 37 °C, but does not move at 42 °C under a light microscope. Phylogenies of the 18S rRNA gene and the ITS1‐5.8S rRNA gene‐ITS2 sequence show that the strain belongs to a subclade of Tetramitus that includes Tetramitus rostratus, Tetramitus waccamawensis and Tetramitus entericus, amongst others. Nonetheless, the strain is distinct from other species in both molecular phylogenetic trees. Thus the strain isolated from the Dokdo Island is proposed as a novel species, Tetramitus dokdoensis n. sp.