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The evolutionary interrelationships between the archaeal organisms which comprise the class Halobacteria have proven difficult to elucidate using traditional phylogenetic tools. The class currently contains three orders. However, little is known about the family level relationships within these orders. In this work, we have completed a comprehensive comparative analysis of 129 sequenced genomes from members of the class Halobacteria in order to identify shared molecular characteristics, in the forms of conserved signature insertions/deletions (CSIs) and conserved signature proteins (CSPs), which can provide reliable evidence, independent of phylogenetic trees, that the species from the groups in which they are found are specifically related to each other due to common ancestry. Here we present 20 CSIs and 31 CSPs which are unique characteristics of infra-order level groups of genera within the class Halobacteria. We also present 40 CSIs and 234 CSPs which are characteristic of Haloarcula, Halococcus, Haloferax, or Halorubrum. Importantly, the CSIs and CSPs identified here provide evidence that the order Haloferacales contains two main groups, one consisting of Haloferax and related genera supported by four CSIs and five CSPs and the other consisting of Halorubrum and related genera supported by four CSPs. We have also identified molecular characteristics that suggest that the polyphyletic order Halobacteriales contains at least two large monophyletic clusters of organisms in addition to the polyphyletic members of the order, one cluster consisting of Haloarcula and related genera supported by ten CSIs and nineteen CSPs and the other group consisting of the members of the genus Halococcus supported by nine CSIs and 23 CSPs. We have also produced a highly robust phylogenetic tree based on the concatenated sequences of 766 proteins which provide additional support for the relationships identified by the CSIs and CSPs. On the basis of the phylogenetic analyses and the identified conserved molecular characteristics presented here, we propose a division of the order Haloferacales into two families, an emended family Haloferacaceae and Halorubraceae fam. nov. and a division of the order Halobacteriales into three families, an emended family Halobacteriaceae, Haloarculaceae fam. nov., and Halococcaceae fam. nov.

Microorganisms are known to play fundamental roles in the biogeochemical cycling of carbon in the coastal environments. To get to know the composition and ecological roles of the archaeal communities within the sediments of the Pearl River Estuary, Southern China, the diversity and vertical distribution of archaea in a sediment core was reported based on the 16S rRNA and mcrA genes for the first time. Quantitative PCR analysis revealed that archaea were present at 10(6)-10(7) 16S rRNA gene copies/g (wet weight) in the sediment core, and the proportion of mcrA versus 16S rRNA gene copies varied from 11 to 45%. 16S rRNA gene libraries were constructed and analyzed for the top layer (0-6 cm), middle layer (18-24 cm), sulfate-methane transition zone (SMTZ, 32-42 cm), and bottom layer (44-50 cm) sediments. The results indicated that Miscellaneous Crenarchaeotal Group (MCG) was the main component in the sediments. The MCG archaea could be further divided into six subgroups: MCG-A, B, C, D, E, and F. On the other hand, mcrA sequences from methanogens related to the order Methanomicrobiales and ANME-2 methanotrophs were detected in all sediment layers. Taken together, our data revealed a largely unknown archaeal community in which MCG dominated within the Pearl River estuarine sediments, while methanogens and methane-oxidizing archaea putatively involving in methane metabolism, were also found in the community. This is the first important step towards elucidating the biogeochemical roles of these archaea in the Pearl River Estuary.

The diversity of archaebacteria associated with anaerobic rumen protozoan Entodinium caudatum in long term in vitro culture was investigated by denaturing gradient gel electrophoresis (DGGE) analysis of hypervariable V3 region of archaebacterial 16S rRNA gene. PCR was accomplished directly from DNA extracted from a single protozoal cell and from total community genomic DNA and the obtained fingerprints were compared. The analysis indicated the presence of a solitary intensive band present in Entodinium caudatum single cell DNA, which had no counterparts in the profile from total DNA. The identity of archaebacterium represented by this band was determined by sequence analysis which showed that the sequence fell to the cluster of ciliate symbiotic methanogens identified recently by 16S gene library approach.

The prokaryotic diversity of culturable thermophilic communities of deep-sea hydrothermal chimneys was analysed using a continuous enrichment culture performed in a gas-lift bioreactor, and compared to classical batch enrichment cultures in vials. Cultures were conducted at 60 degrees C and pH 6.5 using a complex medium containing carbohydrates, peptides and sulphur, and inoculated with a sample of a hydrothermal black chimney collected at the Rainbow field, Mid-Atlantic Ridge, at 2,275 m depth. To assess the relevance of both culture methods, bacterial and archaeal diversity was studied using cloning and sequencing, DGGE, and whole-cell hybridisation of 16S rRNA genes. Sequences of heterotrophic microorganisms belonging to the genera Marinitoga, Thermosipho, Caminicella (Bacteria) and Thermococcus (Archaea) were obtained from both batch and continuous enrichment cultures while sequences of the autotrophic bacterial genera Deferribacter and Thermodesulfatator were only detected in the continuous bioreactor culture. It is presumed that over time constant metabolite exchanges will have occurred in the continuous enrichment culture enabling the development of a more diverse prokaryotic community. In particular, CO(2) and H(2) produced by the heterotrophic population would support the growth of autotrophic populations. Therefore, continuous enrichment culture is a useful technique to grow over time environmentally representative microbial communities and obtain insights into prokaryotic species interactions that play a crucial role in deep hydrothermal environments.

Surveys of Champagne Pool, one of New Zealand's largest terrestrial hot springs and rich in arsenic ions and compounds, have been restricted to geological and geochemical descriptions, and a few microbiological studies applying culture-independent methods. In the current investigation, a combination of culture and culture-independent approaches were chosen to determine microbial density and diversity in Champagne Pool. Recovered total DNA and adenosine 5'-triphosphate (ATP) content of spring water revealed relatively low values compared to other geothermal springs within New Zealand and are in good agreement with low cell numbers of 5.6 +/- 0.5 x 10(6) cells/ml obtained for Champagne Pool water samples by 4',6-diamidino-2-phenylindole (DAPI) staining. Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rRNA (small-subunit ribosomal nucleic acid) gene clone library analyses of environmental DNA indicated the abundance of Sulfurihydrogenibium, Sulfolobus, and Thermofilum-like populations in Champagne Pool. From these results, media were selected to target the enrichment of hydrogen-oxidizing and sulfur-dependent microorganisms. Three isolates were successfully obtained having 16S rRNA gene sequences with similarities of approximately 98% to Thermoanaerobacter tengcongensis, 94% to Sulfurihydrogenibium azorense, and 99% to Thermococcus waiotapuensis, respectively.

Eleven 16S rRNA gene clone libraries including 34 archaeal and 72 bacterial phylotypes were constructed from total 708 clones of hydrothermal vent prokaryotes trapped by 0.2- and 0.1-microm-pore-size filters. Crenarchaeota and Proteobacteria phylotypes dominated the archaeal and bacterial populations, respectively. Novel unaffiliated phylotypes occurred only in the 0.1-microm-trapped populations.

A hyperthermophilic anaerobic archeon, strain HT3, was isolated from hydrothermal hot spring in Northeast Algeria. The strain is a regular coccus, highly motile, obligatory anaerobic, heterotrophic. It utilizes proteinaceous complex media (peptone, tryptone or yeast extract). Sulfur is reduced to Hydrogen sulfide and enhances growth. It shares with other Pyrococcus species the heterotrophic mode of nutrition, the hyperthermophily, the ability to utilize amino acids as sole carbon and nitrogen sources and the ether lipid composition. The optimal growth occurs at 80-85 degrees C, pH 7.5 and 1.5% NaCl. The G + C content was 43 mol%. Considering its morphology, physiological properties, nutritional features and phylogenetic analyses based on 16S rRNA gene sequencing, this strain is described as a new terrestrial isolate pertaining to the genus Pyrococcus.

A new species of Archaea was isolated from an industrial mineral sulphide bioleach heap. Strain BH2, a non-motile pleomorphic coccus, was capable of chemomixotrophic growth on ferrous sulphate and yeast extract. Growth was not supported in the absence of yeast extract. Phylogenetic analysis based on the 16S rRNA gene showed that strain BH2 was most closely related to the species Ferroplasma acidiphilum; however, it showed only 95% sequence similarity with this species. Strain BH2 had a temperature optimum of 53.6 degrees C and a temperature range for growth between 22 and 63 degrees C. Thus, it is the first moderately thermophilic member of the genus Ferroplasma. The optimum pH for the growth of the strain occurred between pH 1.0 and 1.2 and the lowest pH at which growth was observed was 0.4. Based on 16S rRNA gene sequence analysis and other physiological characteristics, strain BH2 constitutes a new species within the genus Ferroplasma. The name Ferroplasma cupricumulans is proposed for the new species and strain BH2 (DSM 16651) is proposed as the type strain.

Archaeal microbial communities present in municipal solid waste landfill leachates were characterized using a 16S rDNA approach. Phylogenetic affiliations of 239 partial length 16S rDNA sequences were determined. Sequences belonging to the order Methanosarcinales were dominant in the clone library and 65% of the clones belonged to the strictly acetoclastic methanogenic family Methanosaetaceae. Sequences affiliated to the metabolically versatile family Methanosarcinaceae represented 18% of the retrieved sequences. Members of the hydrogenotrophic order Methanomicrobiales were also recovered in limited numbers, especially sequences affiliated to the genera Methanoculleus and Methanofollis. Eleven euryarchaeal and thirteen crenarchaeal sequences (i.e. 10%) were distantly related to any hitherto cultivated microorganisms, showing that archaeal diversity within the investigated samples was limited. Lab-scale incubations were performed with leachates mixed with several methanogenic precursors (acetate, hydrogen, formate, methanol, methylamine). Microbial populations were followed using group specific 16S rRNA targeted fluorescent oligonucleotidic probes. During the incubations with acetate, acetoclastic methanogenesis was rapidly induced and led to the dominance of archaea hybridizing with probe MS1414 which indicates their affiliation to the family Methanosarcinaceae. Hydrogen and formate addition induced an important acetate synthesis resulting from the onset of homoacetogenic metabolism. In these incubations, species belonging to the family Methanosarcinaceae (hybridizing with probe MS1414) and the order Methanomicrobiales (hybridizing with probe EURY496) were dominant. Homoacetogenesis was also recorded for incubations with methanol and methylamines. In the methanol experiment, acetoclastic methanogenesis took place and archaea hybridizing with probe MS821 (specific for Methanosarcina spp.) were observed to be the dominant population. These results confirm that acetoclastic methanogenesis performed by the members of the order Methanosarcinales is predominant over the hydrogenotrophic and methylotrophic pathways in landfill leachates.

The microbial diversity in maritime meltwater pond sediments from Bratina Island, Ross Sea, Antarctica was investigated by 16S rDNA-dependent molecular phylogeny. Investigations of the vertical distribution, phylogenetic composition, and spatial variability of Bacteria and Archaea in the sediment were carried out. Results revealed the presence of a highly diverse bacterial population and a significantly depth-related composition. Assessment of 173 partial 16S rDNA clones analyzed by amplified rDNA restriction analysis (ARDRA) using tetrameric restriction enzymes (HinP1I 5'G/CGC3'and Msp I. 5'C/CGG3', BioLabs) revealed 153 different bacterial OTUs (operational taxonomic units). However, only seven archaeal OTUs were detected, indicating low archaeal diversity. Based on ARDRA results, 30 bacterial clones were selected for sequencing and the sequenced clones fell into seven major lineages of the domain Bacteria; the alpha, gamma, and delta subdivisions of Proteobacteria, the Cytophaga-Flavobacterium-Bacteroides, the Spirochaetaceae, and the Actinobacteria. All of the archaeal clones sequenced belonged to the group Crenarchaeota and phylogenetic analysis revealed close relationships with members of the deep-branching Group 1 Marine Archaea.

The prokaryotic diversity of aerobic and anaerobic bacterial isolates and of bacterial and archaeal 16S rDNA clones was determined for a microbial mat sample from the moated region of Lake Fryxell, McMurdo Dry Valleys, Antarctica. Among the anaerobic bacteria, members of Clostridium estertheticum and some other psychrotolerant strains dominated whereas methanogens and other Archaea were lacking. Isolates highly related to Flavobacterium hibernum, Janthiniobacterium lividum, and Arthrobacter flavus were among the aerobic bacteria most frequently isolated. Assessment of more than 350 partial 16S rDNA clone sequences of libraries generated by Bacteria- and Archaea-specific PCR primers revealed a rich spectrum of bacterial diversity but only two different archaeal clone sequences. Among the Bacteria, representative sequences belonged to the class Proteobacteria, order Verrucomicrobiales, class Actinobacteria, Clostridium/Bacillus subphylum of Gram-positives, and the Cytophaga-Flavobacterium-Bacteroides phylum. The clones formed about 70 higher taxonomy groups (<98% sequence similarity) and 133 potential species, i.e., groups of clones sharing greater than 98% similarity. Only rarely were clone sequences found to be highly related to Lake Fryxell isolates and to strains of described species. Subsequent analysis of ten sequencing batches of 36 individual clones indicated that the diversity might be still higher than had been assessed.

In March 1996, a survey of hydrothermal sites on the island of Montserrat was carried out. Six sites (Galway's Soufrière. Gages Upper and Lower Soufrières, Hot Water Pond, Hot River, and Tar River Soufrière) were mapped and sampled for chemical, ATP, and microbial analyses. The hydrothermal Soufrière sites on the slopes of the active Chances Peak volcano exhibited temperatures up to almost 100 degrees C and were generally either mildly acidic at pH 5-7 or strongly acidic at pH 1.5-3, but with some hot streams and pools of low redox potential at pH 7-8. Hot Water Pond sites, comprising a series of heated pools near the western shoreline of the island. were neutral and saline, consistent with subsurface heating of entrained seawater. Biological activity shown by ATP analyses was greatest in near-neutral pH samples and generally decreased as acidity increased. A variety of heterotrophic and chemolithotrophic thermophilic organisms were isolated or observed in enrichment cultures. Most of the bacteria that were obtained in pure culture were familiar acidophiles and neutrophiles, but novel, iron-oxidizing species of Sulfobacillus were revealed. These species included the first mesophilic iron-oxidizing Sulfobacillus strains to be isolated and a strain with a higher maximum growth temperature (65 degrees C) than the previously described moderately thermophilic Sulfobacillus species.

DNA was extracted from water and sediment samples taken from acidic, geothermal pools on the Caribbean island of Montserrat. 16S rRNA genes were amplified by PCR, cloned, sequenced, and examined to indicate some of the organisms that might be significant components of the in situ microbiota. A clone bank representing the lowest temperature pool that was sampled (33 degrees C) was dominated by genes corresponding to two types of acidophiles: Acidiphilium-like mesophilic heterotrophs and thermotolerant Acidithiobacillus caldus. Three clone types with origins in low- and moderate- (48 degrees C) temperature pools corresponded to bacteria that could be involved in metabolism of sulfur compounds: the aerobic A. caldus and putative anaerobic, moderately thermophilic, sulfur-reducing bacteria (from an undescribed genus and from the Desulfurella group). A higher-temperature sample indicated the presence of a Ferroplasma-like organism, distinct from the other strains of these recently recognized acidophilic, iron-oxidizing members of the Euryarchaeota. Acidophilic Archaea from undescribed genera related to Sulfolobus and Acidianus were predicted to dominate the indigenous acidophilic archaeal population at the highest temperatures.

From a shallow marine hydrothermal system at Vulcano (Italy), a new hyperthermophilic member of the Archaea was isolated. The cells are coccoid - shaped and possess up to five flagella. They grow between 56 degrees and 93 degrees C (optimum 85 degrees C) and pH 5.0-9.5 (optimum 9.0). The organism is strictly anaerobic and grows heterotrophically on defined amino acids and complex organic substrates such as casamino acids, yeast extract, peptone, meat extract, tryptone, and casein. Polysulfide and elemental sulfur are reduced to H2S. In the absence of polysulfide or elemental sulfur, the isolate grows at a significantly reduced rate. Growth is not influenced by the presence of H2. DNA- DNA hybridization and 16S rRNA partial sequences indicated that the new isolate belongs to the genus Thermococcus, and represents a new species, Thermococcus acidaminovorans. The type stain is isolate AEDII10 (DSM 11906).