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16S rRNA-based stable isotope probing identified active xylose- and glucose-fermenting Bacteria and active Archaea, including methanogens, in anoxic slurries of material obtained from a moderately acidic, CH(4)-emitting fen. Xylose and glucose were converted to fatty acids, CO(2), H(2), and CH(4) under moderately acidic, anoxic conditions, indicating that the fen harbors moderately acid-tolerant xylose- and glucose-using fermenters, as well as moderately acid-tolerant methanogens. Organisms of the families Acidaminococcaceae, Aeromonadaceae, Clostridiaceae, Enterobacteriaceae, and Pseudomonadaceae and the order Actinomycetales, including hitherto unknown organisms, utilized xylose- or glucose-derived carbon, suggesting that highly diverse facultative aerobes and obligate anaerobes contribute to the flow of carbon in the fen under anoxic conditions. Uncultured Euryarchaeota (i.e., Methanosarcinaceae and Methanobacteriaceae) and Crenarchaeota species were identified by 16S rRNA analysis of anoxic slurries, demonstrating that the acidic fen harbors novel methanogens and Crenarchaeota organisms capable of anaerobiosis. Fermentation-derived molecules are conceived to be the primary drivers of methanogenesis when electron acceptors other than CO(2) are absent, and the collective findings of this study indicate that fen soils harbor diverse, acid-tolerant, and novel xylose-utilizing as well as glucose-utilizing facultative aerobes and obligate anaerobes that form trophic links to novel moderately acid-tolerant methanogens.

Nitrification plays an important role in marine biogeochemistry, yet efforts to link this process to the microorganisms that mediate it are surprisingly limited. In particular, ammonia oxidation is the first and rate-limiting step of nitrification, yet ammonia oxidation rates and the abundance of ammonia-oxidizing bacteria (AOB) have rarely been measured in tandem. Ammonia oxidation rates have not been directly quantified in conjunction with ammonia-oxidizing archaea (AOA), although mounting evidence indicates that marine Crenarchaeota are capable of ammonia oxidation, and they are among the most abundant microbial groups in the ocean. Here, we have directly quantified ammonia oxidation rates by 15N labeling, and AOA and AOB abundances by quantitative PCR analysis of ammonia monooxygenase subunit A (amoA) genes, in the Gulf of California. Based on markedly different archaeal amoA sequence types in the upper water column (60 m) and oxygen minimum zone (OMZ; 450 m), novel amoA PCR primers were designed to specifically target and quantify 'shallow' (group A) and 'deep' (group B) clades. These primers recovered extensive variability with depth. Within the OMZ, AOA were most abundant where nitrification may be coupled to denitrification. In the upper water column, group A tracked variations in nitrogen biogeochemistry with depth and between basins, whereas AOB were present in relatively low numbers or undetectable. Overall, 15NH4+ oxidation rates were remarkably well correlated with AOA group A amoA gene copies (r2=0.90, P<0.001), and with 16S rRNA gene copies from marine Crenarchaeota (r2=0.85, P<0.005). These findings represent compelling evidence for an archaeal role in oceanic nitrification.

The extraction of nucleic acids from a given environment marks a crucial and essential starting point in any molecular investigation. Members of Halococcus spp. are known for their rigid cell walls, and are thus difficult to lyse and could potentially be overlooked in an environment. Furthermore, the lack of a suitable lysis method hinders subsequent molecular analysis. The effects of six different DNA extraction methods were tested on Halococcus hamelinensis, Halococcus saccharolyticus and Halobacterium salinarum NRC-1 as well as on an organic rich, highly carbonated sediment from stromatolites spiked with Halococcus hamelinensis. The methods tested were based on physical disruption (boiling and freeze/thawing), chemical lysis (Triton X-100, potassium ethyl xanthogenate (XS) buffer and CTAB) and on enzymatic lysis (lysozyme). Results showed that boiling and freeze/thawing had little effect on the lysis of both Halococcus strains. Methods based on chemical lysis (Triton X-100, XS-buffer, and CTAB) showed the best results, however, Triton X-100 treatment failed to produce visible DNA fragments. Using a combination of bead beating, chemical lysis with lysozyme, and thermal shock, lysis of cells was achieved however DNA was badly sheared. Lysis of cells and DNA extraction of samples from spiked sediment proved to be difficult, with the XS-buffer method indicating the best results. This study provides an evaluation of six commonly used methods of cell lysis and DNA extraction of Halococcus spp., and the suitability of the resulting DNA for molecular analysis.

The genome of the hyperthermophile archaeon Pyrococcus furiosus encodes two transcription factor B (TFB) paralogs, one of which (TFB1) was previously characterized in transcription initiation. The second TFB (TFB2) is unusual in that it lacks recognizable homology to the archaeal TFB/eukaryotic TFIIB B-finger motif. TFB2 functions poorly in promoter-dependent transcription initiation, but photochemical cross-linking experiments indicated that the orientation and occupancy of transcription complexes formed with TFB2 at the strong gdh promoter are similar to the orientation and occupancy of transcription complexes formed with TFB1. Initiation complexes formed by TFB2 display a promoter opening defect that can be bypassed with a preformed transcription bubble, suggesting a mechanism to explain the low TFB2 transcription activity. Domain swaps between TFB1 and TFB2 showed that the low activity of TFB2 is determined mainly by its N terminus. The low activity of TFB2 in promoter opening and transcription can be partially relieved by transcription factor E (TFE). The results indicate that the TFB N-terminal region, containing conserved Zn ribbon and B-finger motifs, is important in promoter opening and that TFE can compensate for defects in the N terminus through enhancement of promoter opening.

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.

Methyl coenzyme-M reductase A (mcrA) clone libraries were generated from microbial DNA extracted from the rumen of cattle fed a roughage diet with and without supplementation of the antimethanogenic compound bromochloromethane. Bromochloromethane reduced total methane emissions by c. 30%, with a resultant increase in propionate and branched chain fatty acids. The mcrA clone libraries revealed that Methanobrevibacter spp. were the dominant species identified. A decrease in the incidence of Methanobrevibacter spp. from the clone library generated from bromochloromethane treatment was observed. In addition, a more diverse methanogenic population with representatives from Methanococcales, Methanomicrobiales and Methanosacinales orders was observed for the bromochloromethane library. Sequence data generated from these libraries aided in the design of an mcrA-targeted quantitative PCR (qPCR) assay. The reduction in methane production by bromochloromethane was associated with an average decrease of 34% in the number of methanogenic Archaea when monitored with this qPCR assay. Dissociation curve analysis of mcrA amplicons showed a clear difference in melting temperatures for Methanobrevibacter spp. (80-82 degrees C) and all other methanongens (84-86 degrees C). A decrease in the intensity of the Methanobrevibacter spp. specific peak and an increase for the other peak in the bromochloromethane-treated animals corresponded with the changes within the clone libraries.

The diversity of methanogenic archaea in enrichment cultures established from the sediments of Lonar Lake (India), a soda lake having pH approximately 10, was investigated using 16S rDNA molecular phylogenetic approach. Methanogenic enrichment cultures were developed in a medium that simulated conditions of soda lake with three different substrates viz., H(2):CO(2), sodium acetate, and trimethylamine (TMA), at alkaline pH. Archaeal 16S rRNA clone libraries were generated from enrichment cultures and 13 RFLP groups were obtained. Representative sequence analysis of each RFLP group indicated that the majority of the 16S rRNA gene sequences were phylogenetically affiliated with uncultured Archaea. Some of the groups may belong to new archaeal genera or families. Three RFLP groups were related to Methanoculleus sp, while two related to Methanocalculus sp. 16S rRNA gene sequences found in Lonar Lake were different from sequences reported from other soda lakes and more similar to those of oil reservoirs, palm oil waste treatment digesters, and paddy fields. In culture-based studies, three isolates were obtained. Two of these were related to Methanoculleus sp. IIE1 and one to Methanocalculus sp. 01F97C. These results clearly show that the Lonar Lake ecosystem harbors unexplored methanogens.

The anaerobic free-living ciliate, Trimyema compressum, is known to harbor both methanogenic archaeal and bacterial symbionts in the cytoplasm. To clarify their phylogenetic belongings, a full-cycle rRNA approach was applied to this symbiosis. Phylogenetic analysis showed that the methanogenic symbiont was related to Methanobrevibacter arboriphilicus, which was distantly related to symbionts found in other Trimyema species. This result suggested that Trimyema species do not require very specific methanogenic symbionts, and symbiont replacement could have occurred in the history of Trimyema species. On the other hand, the bacterial symbiont was located near the lineage of the family Syntrophomonadaceae in the phylum Firmicutes. The sequence similarity between the bacterial symbiont and the nearest species was 85%, indicating that bacterial symbionts may be specific to the Trimyema species. The elimination of bacterial symbionts from the ciliate cell by antibiotic treatment resulted in considerably decreased host growth. However, it was not restored by stigmasterol addition (<2 microg ml(-1)), which was different from the previous report that showed that the symbiont-free strain required exogenous sterols for growth. In addition, the decline of host growth was not accompanied by host metabolism shift toward the formation of more reduced products, which suggested that the contribution of bacterial symbionts to the host ciliate was not a dispose of excessive reducing equivalent arising from the host's fermentative metabolism as methanogenic symbionts do. This study showed that bacterial symbionts make a significant contribution to the host ciliate by an unknown function and suggested that interactions between bacterial symbionts and T. compressum are more complicated than hitherto proposed.

A hyperthermophilic, sulfur-reducing, organo-heterotrophic archaeon, strain OGL-20P(T), was isolated from 'black smoker' chimney material from the Rainbow hydrothermal vent site on the Mid-Atlantic Ridge (36.2 degrees N, 33.9 degrees W). The cells of strain OGL-20P(T) have an irregular coccoid shape and are motile with a single flagellum. Growth was observed within a pH range of 5.0-8.5 (optimum pH 7.0), an NaCl concentration range of 1-5 % (w/v) (optimum 3 %) and a temperature range of 55-94 degrees C (optimum 83-85 degrees C). The novel isolate is strictly anaerobic and obligately dependent upon elemental sulfur as an electron acceptor, but it does not reduce sulfate, sulfite, thiosulfate, Fe(III) or nitrate. Proteolysis products (peptone, bacto-tryptone, Casamino acids and yeast extract) are utilized as substrates during sulfur reduction. Strain OGL-20P(T) is resistant to ampicillin, chloramphenicol, kanamycin and gentamicin, but sensitive to tetracycline and rifampicin. The G+C content of the DNA is 52.9 mol%. The 16S rRNA gene sequence analysis revealed that strain OGL-20P(T) is closely related to Thermococcus coalescens and related species, but no significant homology by DNA-DNA hybridization was observed between those species and the new isolate. On the basis of physiological and molecular properties of the new isolate, we conclude that strain OGL-20P(T) represents a new separate species within the genus Thermococcus, for which we propose the name Thermococcus thioreducens sp. nov. The type strain is OGL-20P(T) (=JCM 12859(T)=DSM 14981(T)=ATCC BAA-394(T)).

A novel, extremely thermoacidophilic, obligately chemolithotrophic archaeon (strain JP7(T)) was isolated from a solfatara on Lihir Island, Papua New Guinea. Cells of this organism were non-motile, Gram-negative staining, irregular-shaped cocci, 0.5-1.5 microm in size, that grew aerobically by oxidation of sulfur, Fe(2+) or mineral sulfides. Cells grew anaerobically using Fe(3+) as a terminal electron acceptor and H(2)S as an electron donor but did not oxidize hydrogen with elemental sulfur as electron acceptor. Strain JP7(T) grew optimally at 74 degrees C (temperature range 45-83 degrees C) and pH 0.8-1.4 (pH range 0.35-3.0). On the basis of 16S rRNA gene sequence similarity, strain JP7(T) was shown to belong to the Sulfolobaceae, being most closely related to the type strains of Acidianus ambivalens (93.7 %) and Acidianus infernus (93.6 %). Cell-membrane lipid structure, DNA base composition and 16S rRNA gene sequence similarity data support the placement of this strain in the genus Acidianus. Differences in aerobic and anaerobic metabolism, temperature and pH range for growth, and 16S rRNA gene sequence differentiate strain JP7(T) from recognized species of the genus Acidianus, and an emendation of the description of the genus is proposed. Strain JP7(T) is considered to represent a novel species of the genus Acidianus, for which the name Acidianus sulfidivorans sp. nov. is proposed. The type strain is JP7(T) (=DSM 18786(T)=JCM 13667(T)).

The last decade has been marked by a large number of studies focused on understanding the distribution of microorganisms in volcanic environments. These studies are motivated by the desire to elucidate how the geochemically extreme conditions of such environments can influence microbial diversity both on the surface and in the subsurface of the Earth. The exploration of microbial community diversity has generally not relied on culture-dependent methods, but has been carried out using environmental DNA extraction. Because of the large diversity of chemically and physically complex samples, extracting DNA from volcanic environments is technically challenging. In view of the emerging literature, and our own experience in the optimisation of methods for DNA extraction from volcanic materials, it is timely to provide a methodological comparison. This review highlights and discusses new insights and methods published on DNA extraction methods from volcanic samples, considering the different volcanic environments. A description of a recent method for DNA extraction from basalt and obsidian glass rock samples from Iceland is included. Finally, we discuss these approaches in the wider context of modern work to understand the microbial diversity of volcanic environments.

An extremely halophilic archaeon belonging to the order Halobacteriales was isolated from Fuente de Piedra salt lake, Spain. This strain, designated FP1(T), was a pleomorphic coccoid, neutrophilic and required at least 15 % (w/v) NaCl for growth. Strain FP1(T) grew at 37-60 degrees C, with optimal growth at 50 degrees C. Mg(2+) was not required, but growth was observed with up to 10 % (w/v) MgSO(4). Polar lipid analysis revealed the presence of mannose-6-sulfate(1-2)-glucose glycerol diether as a major glycolipid. Both C(20)C(20) and C(20)C(25) core lipids were present. The genomic DNA G+C content was 62.0 mol%. Phylogenetic analysis based on comparison of 16S rRNA gene sequences demonstrated that the isolate was most closely related to species of the genus Haloterrigena. DNA-DNA reassociation values between strain FP1(T) and the most closely related species of the genus Haloterrigena (Haloterrigena thermotolerans, Haloterrigena saccharevitans and Haloterrigena limicola) were lower than 29 %. It is therefore considered that strain FP1(T) represents a novel species of the genus Haloterrigena, for which the name Haloterrigena hispanica sp. nov. is proposed. The type strain is FP1(T) (=DSM 18328(T)=ATCC BAA-1310(T)).

Strain SH-6(T) was isolated from the sediment of Lake Shangmatala, a saline lake in Inner Mongolia (China). Cells were pleomorphic. The organism was neutrophilic and required at least 2.5 M (15 %) NaCl, but not MgCl(2), for growth; optimal growth occurred at 4.3 M (25 %) NaCl. The G+C content of its DNA was 63.1 mol%. 16S rRNA gene sequence analysis revealed that strain SH-6(T) is a member of the family Halobacteriaceae, but there was a low level of similarity with other members of this family. Highest sequence similarity (94.6 %) was obtained with the 16S rRNA genes of the type strains of Natronolimnobius innermongolicus and Natronolimnobius baerhuensis. Polar lipid analyses revealed that strain SH-6(T) contains phosphatidylglycerol and phosphatidylglyceromethylphosphate, derived from both C(20)C(20) and C(20)C(25) glycerol diethers together with the glycolipid S(2)-DGD-1. On the basis of the data obtained, the new isolate could not be classified in any recognized genus. Strain SH-6(T) is thus considered to represent a novel species in a new genus within the family Halobacteriaceae, order Halobacteriales, for which the name Halopiger xanaduensis gen. nov., sp. nov. is proposed. The type strain of Halopiger xanaduensis is SH-6(T) (=CECT 7173(T)=CGMCC 1.6379(T)=JCM 14033(T)).

Background: The cell cycle of all organisms includes mass increase by a factor of two, replication of the genetic material, segregation of the genome to different parts of the cell, and cell division into two daughter cells. It is tightly regulated and typically includes cell cycle-specific oscillations of the levels of transcripts, proteins, protein modifications, and signaling molecules. Until now cell cycle-specific transcriptome changes have been described for four eukaryotic species ranging from yeast to human, but only for two prokaryotic species. Similarly, oscillations of small signaling molecules have been identified in very few eukaryotic species, but not in any prokaryote., Results: A synchronization procedure for the archaeon Halobacterium salinarum was optimized, so that nearly 100% of all cells divide in a time interval that is 1/4th of the generation time of exponentially growing cells. The method was used to characterize cell cycle-dependent transcriptome changes using a genome-wide DNA microarray. The transcript levels of 87 genes were found to be cell cycle-regulated, corresponding to 3% of all genes. They could be clustered into seven groups with different transcript level profiles. Cluster-specific sequence motifs were detected around the start of the genes that are predicted to be involved in cell cycle-specific transcriptional regulation. Notably, many cell cycle genes that have oscillating transcript levels in eukaryotes are not regulated on the transcriptional level in H. salinarum. Synchronized cultures were also used to identify putative small signaling molecules. H. salinarum was found to contain a basal cAMP concentration of 200 microM, considerably higher than that of yeast. The cAMP concentration is shortly induced directly prior to and after cell division, and thus cAMP probably is an important signal for cell cycle progression., Conclusion: The analysis of cell cycle-specific transcriptome changes of H. salinarum allowed to identify a strategy of transcript level regulation that is different from all previously characterized species. The transcript levels of only 3% of all genes are regulated, a fraction that is considerably lower than has been reported for four eukaryotic species (6%-28%) and for the bacterium C. crescentus (19%). It was shown that cAMP is present in significant concentrations in an archaeon, and the phylogenetic profile of the adenylate cyclase indicates that this signaling molecule is widely distributed in archaea. The occurrence of cell cycle-dependent oscillations of the cAMP concentration in an archaeon and in several eukaryotic species indicates that cAMP level changes might be a phylogenetically old signal for cell cycle progression.

A Gram-negative, aerobic, neutrophilic and extremely halophilic archaeon (strain AJ201(T)), isolated from Ayakekum salt lake on the Qinghai-Tibet Plateau, was investigated by a polyphasic approach. The DNA G+C content of strain AJ201(T) was 65.7 mol%. The major polar lipid profile and phylogenetic analysis based on 16S rRNA gene sequences supported the allocation of the strain to the genus Halorubrum. The results of DNA-DNA hybridizations and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain AJ201(T) from closely related species. Therefore, strain AJ201(T) represents a novel species of the genus Halorubrum, for which the name Halorubrum arcis sp. nov. is proposed. The type strain is strain AJ201(T) (=CGMCC 1.5343(T)=JCM 13916(T)).

A Gram-negative, pleomorphic, extremely halophilic archaeon, designated strain XH-70(T), was isolated from the saline Lake Xilinhot, in Inner Mongolia, China. It formed small (0.9-1.5 mm), red-pigmented, elevated colonies on agar medium. The strain required at least 2.5 M NaCl and 5 mM Mg(2+) for growth. The 16S rRNA gene sequence analysis indicated that strain XH-70(T) belongs to the family Halobacteriaceae, showing 99.5 % similarity to the type strain of Halovivax asiaticus and 94.7 and 94.6 % similarity, respectively, to the type strains of Natronococcus amylolyticus and Natronococcus occultus. Polar lipid analysis supported the placement of strain XH-70(T) in the genus Halovivax. DNA-DNA hybridization studies (32 % with Halovivax asiaticus CGMCC 1.4248(T)), as well as biochemical and physiological characterization, allowed strain XH-70(T) to be differentiated from Halovivax asiaticus. A novel species, Halovivax ruber sp. nov., is therefore proposed to accommodate this strain. The type strain is XH-70(T) (=CGMCC 1.6204(T)=DSM 18193(T)=JCM 13892(T)).

Group 1 Crenarchaeota are an abundant component of soil microbial communities. A number of distinct lineages have been discovered, and the Group 1.1b lineage is present in most soil types. Others, such as the Group 1.1c lineage, may be restricted to specific soil types, such as acidic forest soils. To determine the effects of afforestation on the Archaea of moorland soils, archaeal community structure was examined across three parallel 180 m transects, running from open heather moorland into a Scots pine forest. Communities were characterized using a combination of cloning and denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA reverse transcriptase-polymerase chain reaction products. Although forest soils selected for a reproducibly distinct crenarchaeal community structure from moorland soils, both habitats contained similar populations, indicated by comigrating DGGE bands. Archaeal communities in soils of both ecosystems were dominated by Group 1.1c crenarchaea, and reproducible differences in community structure within this lineage were observed between forest and moorland soils. The findings indicate that the afforestation of moorland soils can lead to changes in crenarchaeal community structure with a potential impact on ecosystem function.

This study provides data on the diversities of bacterial and archaeal communities in an active methane seep at the Kazan mud volcano in the deep Eastern Mediterranean sea. Layers of varying depths in the Kazan sediments were investigated in terms of (1) chemical parameters and (2) DNA-based microbial population structures. The latter was accomplished by analyzing the sequences of directly amplified 16S rRNA genes, resulting in the phylogenetic analysis of the prokaryotic communities. Sequences of organisms potentially associated with processes such as anaerobic methane oxidation and sulfate reduction were thus identified. Overall, the sediment layers revealed the presence of sequences of quite diverse bacterial and archaeal communities, which varied considerably with depth. Dominant types revealed in these communities are known as key organisms involved in the following processes: (1) anaerobic methane oxidation and sulfate reduction, (2) sulfide oxidation, and (3) a range of (aerobic) heterotrophic processes. In the communities in the lowest sediment layer sampled (22-34 cm), sulfate-reducing bacteria and archaea of the ANME-2 cluster (likely involved in anaerobic methane oxidation) were prevalent, whereas heterotrophic organisms abounded in the top sediment layer (0-6 cm). Communities in the middle layer (6-22 cm) contained organisms that could be linked to either of the aforementioned processes. We discuss how these phylogeny (sequence)-based findings can support the ongoing molecular work aimed at unraveling both the functioning and the functional diversities of the communities under study.

We in this study describe a new method for genomic studies of individual uncultured prokaryotic organisms, which was used for the isolation and partial genome sequencing of a soil archaeon. The diversity of Archaea in a soil sample was mapped by generating a clone library using group-specific primers in combination with a terminal restriction fragment length polymorphism profile. Intact cells were extracted from the environmental sample, and fluorescent in situ hybridization probing with Cy3-labeled probes designed from the clone library was subsequently used to detect the organisms of interest. Single cells with a bright fluorescent signal were isolated using a micromanipulator and the genome of the single isolated cells served as a template for multiple displacement amplification (MDA) using the Phi29 DNA polymerase. The generated MDA product was afterwards used for 16S rRNA gene sequence analysis and shotgun-cloned for additional genomic analysis. Sequence analysis showed >99% 16S rRNA gene homology to soil crenarchaeotal clone SCA1170 and shotgun fragments had the closest match to a crenarchaeotal BAC clone previously retrieved from a soil sample. The system was validated using Methanothermobacter thermoautotrophicus as single-cell test organism, and the validation setup produced 100% sequence homology to the ten tested regions of the genome of this organism.

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).

Strains C23T and HBSQ001 were isolated from solar salterns and are novel square-shaped, aerobic, extremely halophilic members of the domain Archaea and family Halobacteriaceae. Cells stained Gram-negative and grew optimally in media containing 18 % salts at around neutral pH. Mg2+ is not required. The DNA G+C content of both isolates was 46.9 mol% and DNA-DNA cross-hybridization showed a relatedness of 80 %. Their 16S rRNA gene sequences showed only 2 nucleotide differences (99.9 % identity) and phylogenetic tree reconstructions with other recognized members of the Halobacteriaceae indicated that they formed a distinct clade, with the closest relative being Halogeometricum borinquense PR 3T (91.2 % sequence identity). The major polar glycolipid of both isolates was the sulfated diglycosyl diether lipid S-DGD-1. Electron cryomicrosopy of whole cells revealed similar internal structures, such as gas vesicles and polyhydroxyalkanoate granules, but the cell wall of isolate HBSQ001 displayed a more complex S-layer compared with that of isolate C23T. The phenotypic characterization and phylogenetic data support the placement of isolates C23T and HBSQ001 in a novel species in a new genus within the Halobacteriaceae, for which we propose the name Haloquadratum walsbyi gen. nov., sp. nov. The type strain of Haloquadratum walsbyi is C23T (=JCM 12705T=DSM 16854T).

Permafrost environments in the Arctic are characterized by extreme environmental conditions that demand a specific resistance from microorganisms to enable them to survive. In order to understand the carbon dynamics in the climate-sensitive Arctic permafrost environments, the activity and diversity of methanogenic communities were studied in three different permafrost soils of the Siberian Laptev Sea coast. The effect of temperature and the availability of methanogenic substrates on CH4 production was analysed. In addition, the diversity of methanogens was analysed by PCR with specific methanogenic primers and by denaturing gradient gel electrophoresis (DGGE) followed by sequencing of DGGE bands reamplified from the gel. Our results demonstrated methanogenesis with a distinct vertical profile in each investigated permafrost soil. The soils on Samoylov Island showed at least two optima of CH4 production activity, which indicated a shift in the methanogenic community from mesophilic to psychrotolerant methanogens with increasing soil depth. Furthermore, it was shown that CH4 production in permafrost soils is substrate-limited, although these soils are characterized by the accumulation of organic matter. Sequence analyses revealed a distinct diversity of methanogenic archaea affiliated to Methanomicrobiaceae, Methanosarcinaceae and Methanosaetaceae. However, a relationship between the activity and diversity of methanogens in permafrost soils could not be shown.

To examine the diversity and structure of archaeal communities in the Yangtze River estuarine region of East China Sea (ECS), the 16S rRNA gene clone libraries of two typical sites were constructed with the archaea-specific primers. In total, 71 clones randomly selected were screened by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis and 21 clones with unique RFLP pattern were sequenced. All the sequences are clustered into the two groups of Marine Group I (MGI) and Marine Group II (MGII) which are affiliated with the phyla Crenarchaeota and Euryarchaeota, respectively. MGI clones dominate both libraries with 20 MGI sequences obtained. The majority of sequences are closely related to uncultured marine archaea except for two sequences of which the nearest neighbor is a newly identified isolate of nitrifying marine archaeon Nitrosopumilus maritimus (98% identity). The results indicate that ECS coastal waters are inhabited by archaeal community with low dominance and high diversity corresponding to the complex estuarine environments, suggesting that archaea may perform an important role in the estuarine ecosystem.

A motile, pleomorphic, red-pigmented archaeon, strain EJ-52T, was isolated from water from Lake Ejinor, a saline lake in Inner Mongolia, China. Analysis of the almost-complete 16S rRNA gene sequence showed that the isolate was phylogenetically related to species of the genus Halorubrum, being most closely related to Halorubrum saccharovorum ATCC 29252T (96.1% sequence similarity), Halorubrum lacusprofundi JCM 8891T (95.9%), Halorubrum tibetense AS 1.3239T (95.2%), Halorubrum alcaliphilum AS 1.3528T (95.2%) and Halorubrum vacuolatum JCM 9060T (95.1%). The polar lipids of strain EJ-52T were C20C20 derivatives of phosphatidylglycerol phosphate and phosphatidylglycerol phosphate methyl ester and a sulfated diglycosyl diether. Strain EJ-52T requires at least 2.5 M NaCl for growth and grows optimally at 3.4 M NaCl. The strain grows at 25-50 degrees C, with optimal growth occurring at 35-45 degrees C. Mg2+ is not required. The DNA G+C content is 64.2 mol%. On the basis of the data obtained in this study, strain EJ52T represents a novel species, for which the name Halorubrum orientale sp. nov. is proposed. The type strain is EJ-52T (=CECT 7145T=JCM 13889T=CGMCC 1.6295T).

A Gram-negative, non-motile, neutrophilic, pleomorphic and extremely halophilic archaeon, strain EJ-57T, was isolated from saline Lake Ejinor in Inner Mongolia, China. Strain EJ-57T was able to grow at 25-50 degrees C, required at least 1.8 M NaCl for growth (optimum at 3.4 M NaCl) and grew over a pH range from 6.0 to 8.5 (optimum at pH 7.0). Hypotonic treatment with less than 1.5 M NaCl caused cell lysis. Analysis of the almost complete 16S rRNA gene sequence indicated that the isolate represented a member of the genus Natrinema in the family Halobacteriaceae. Strain EJ-57T was most closely related to Natrinema versiforme JCM 10478T (96.2% sequence similarity), Natrinema pallidum NCIMB 777T (95.9% sequence similarity), Natrinema altunense JCM 12890T (95.8% sequence similarity) and Natrinema pellirubrum NCIMB 786T (95.5 % sequence similarity). However, DNA-DNA hybridization experiments showed that strain EJ-57T was not related to these species, with levels of DNA-DNA relatedness equal to or below 39%. The major polar lipids of the isolate were C20C20 and C20C25 derivatives of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and the disulfated glycolipid S2-DGA-1. The G+C content of the genomic DNA was 64.7 mol%. Comparative analysis of phenotypic characteristics between strain EJ-57T and recognized Natrinema species supported the conclusion that EJ-57T represents a novel species within this genus, for which the name Natrinema ejinorense sp. nov. is proposed. The type strain is EJ-57T (=CECT 7144T=JCM 13890T=CGMCC 1.6202T).

Aims: To determine carriage rates and densities of methanogens and sulfate-reducing bacteria in adults and children using molecular methods, and to also determine if a reciprocal relationship exists between these organisms., Methods and Results: Real-time PCR was used to detect and quantify methanogens and sulfate-reducing bacteria. Real-time PCR was more sensitive than breath methane measurements. Real-time PCR assays were applied to faecal DNA samples collected from 40 children and 12 adults. Methanogens were present in 25% of the children and 42% of the adults studied, and sulfate-reducing bacteria were detected in 15% of the children and 58% of the adults. High levels of sulfate-reducing bacteria were found in two methanogenic adults., Conclusions: Carriage rates and densities of methanogens and sulfate-reducing bacteria are greater in adults than in children. Competition does not necessarily lead to the predominance of one group in the faecal microflora., Significance and Impact of the Study: This study describes sensitive, molecular assays that could be used to monitor these organisms in gastrointestinal disease. Therapeutic exclusion of one group from the bowel would not necessarily lead to the expansion of the other, as there does not appear to be a reciprocal relationship between these groups.

Two extremely halophilic archaea, strains 9-3(T) and 31-hong(T), were isolated from Aibi salt lake and Aiding salt lake in Xin-Jiang, China. Their morphology, physiology, biochemical features, polar lipid compositions and 16S rRNA gene sequences were characterized in order to elucidate their taxonomic status. The results from this study indicated that strains 9-3(T) and 31-hong(T) are members of the genus Halorubrum. Their physiological properties and polar lipid compositions are clearly different from those of the currently described species of Halorubrum. DNA-DNA relatedness values for strain 9-3(T) with respect to its closely related neighbours Halorubrum saccharovorum JCM 8865(T) and Halorubrum lacusprofundi JCM 8891(T) were 51.6 and 25.1 %, respectively, DNA-DNA relatedness values for strain 31-hong(T) with respect to its closely related neighbours Hrr. saccharovorum JCM 8865(T) and Hrr. lacusprofundi JCM 8891(T) were 29.4 and 44.9 %, respectively, and DNA-DNA relatedness between strains 9-3(T) and 31-hong(T) was 54 %. Thus, two novel species of the genus Halorubrum are proposed, Halorubrum lipolyticum sp. nov. (type strain 9-3(T)=CGMCC 1.5332(T)=JCM 13559(T)) and Halorubrum aidingense sp. nov. (type strain 31-hong(T)=CGMCC 1.2670(T)=JCM 13560(T)).

Strain XH-48(T) was isolated from the sediment of Lake Xilinhot, a saline lake in Inner Mongolia (China). The organism is pleomorphic, neutrophilic and requires at least 2.5 M (15 %) NaCl, but not MgCl(2), for growth; it exhibits optimal growth at 3.4 M (20 %) NaCl. The G+C content of its DNA is 61 mol%. 16S rRNA gene sequence analysis revealed that strain XH-48(T) is a member of the family Halobacteriaceae, but there were low levels of similarity with other members of this family. The highest sequence similarity values (94.5 and 93.3 %) were obtained with the 16S rRNA genes of Natrialba aegyptiaca and Natrialba asiatica, respectively. Polar lipid analyses revealed that strain XH-48(T) contains phosphatidylglycerol and phosphatidylglyceromethylphosphate, derived from both C(20)C(20) and C(20)C(25) glycerol diethers, and two unidentified glycolipids. On the basis of the data obtained, the novel isolate cannot be classified within any recognized genus. Strain XH-48(T) should be placed within a novel genus and species within the family Halobacteriaceae, order Halobacteriales, for which the name Halostagnicola larsenii gen. nov., sp. nov. is proposed. The type strain of Halostagnicola larsenii is strain XH-48(T) (=DSM 17691(T)=CGMCC 1.5338(T)=JCM 13463(T)=CECT 7116(T)).

A novel extremely halophilic archaeon was isolated from Ezzemoul sabkha, Algeria. The strain, designated 5.1(T), was neutrophilic, motile and Gram-negative. At least 15 % (w/v) NaCl was required for growth. The isolate grew at pH 6.5-9.0, with optimum growth at pH 7.0-7.5. Mg(2+) was required for growth. Polar lipids were C(20)C(20) derivatives of phosphatidylglycerol and phosphatidylglycerol phosphate methyl ester, and phosphatidylglycerol sulfate and sulfated diglycosyl diether. The genomic DNA G+C content of strain 5.1(T) was 61.9 mol% (T(m)). Phylogenetic analysis based on comparison of 16S rRNA gene sequences revealed that strain 5.1(T) clustered with Halorubrum species. The results of DNA-DNA hybridization and biochemical tests allowed genotypic and phenotypic differentiation of strain 5.1(T) from other Halorubrum species. The name Halorubrum ezzemoulense sp. nov. (type strain 5.1(T)=CECT 7099(T)=DSM 17463(T)) is proposed.

Three strains of CO(2)-reducing methanogens were isolated from marine sediments. Strain PL-15/H(P) was isolated from marine sediments of the Lipari Islands, near Sicily and the other two strains, Nankai-2 and Nankai-3(T), were isolated from deep marine sediments of the Nankai Trough, about 50 km from the coast of Japan. Analysis of the cellular proteins and 16S rRNA gene sequences indicated that these three strains represented a single novel species that formed a deep branch of the mesophilic methanococci. Phylogenetic analysis indicated that the three strains were most closely related to Methanothermococcus okinawensis (95 % 16S rRNA gene sequence similarity). However, strains PL-15/H(P), Nankai-2 and Nankai-3(T) grew at temperatures that were more similar to those of recognized species within the genus Methanococcus. Strain Nankai-3(T) grew fastest at 46 degrees C. Results of physiological and biochemical tests allowed the genotypic and phenotypic differentiation of strains PL-15/H(P), Nankai-2 and Nankai-3(T) from closely related species. The name Methanococcus aeolicus sp. nov. is proposed, with strain Nankai-3(T) (=OCM 812(T)=DSM 17508(T)) as the type strain.

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 x 10(6) to 3 x 10(6) 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 gamma-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.

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 crenarchaeote Sulfolobus spp. is a host for a remarkably large spectrum of viruses and plasmids. The genetic elements characterized so far indicate a large degree of novelty in terms of morphology (viruses) and in terms of genome content (plasmids and viruses). The viruses and conjugative plasmids encode a great number of conserved proteins of unknown function due to the lack of sequence similarity to functionally characterized proteins. These apparently essential proteins remain to be studied and should help to understand the physiology and genetics of the respective genetic elements as well as the host. Sulfolobus is one of the best-studied archaeons and could develop into an important model organism of the crenarchaea and the archaea.

Deep-subsurface samples obtained by deep drilling are likely to be contaminated with mesophilic microorganisms in the drilling fluid, and this could affect determination of the community structure of the geothermal microflora using 16S rRNA gene clone library analysis. To eliminate possible contamination by PCR-amplified 16S rRNA genes from mesophiles, a combined thermal denaturation and enzyme digestion method, based on a strong correlation between the G+C content of the 16S rRNA gene and the optimum growth temperatures of most known prokaryotic cultures, was used prior to clone library construction. To validate this technique, hot spring fluid (76 degrees C) and river water (14 degrees C) were used to mimic a deep-subsurface sample contaminated with drilling fluid. After DNA extraction and PCR amplification of the 16S rRNA genes from individual samples separately, the amplified products from river water were observed to be denatured at 82 degrees C and completely digested by exonuclease I (Exo I), while the amplified products from hot spring fluid remained intact after denaturation at 84 degrees C and enzyme digestion with Exo I. DNAs extracted from the two samples were mixed and used as a template for amplification of the 16S rRNA genes. The amplified rRNA genes were denatured at 84 degrees C and digested with Exo I before clone library construction. The results indicated that the 16S rRNA gene sequences from the river water were almost completely eliminated, whereas those from the hot spring fluid remained.

Pyrococcus furiosus and Pyrococcus woesei grow optimally at temperatures near 100 degrees C and were isolated from the same shallow marine volcanic vent system. Hybridization of genomic DNA from P. woesei to a DNA microarray containing all 2,065 open reading frames (ORFs) annotated in the P. furiosus genome, in combination with PCR analysis, indicated that homologs of 105 ORFs present in P. furiosus are absent from the uncharacterized genome of P. woesei. Pulsed-field electrophoresis indicated that the sizes of the two genomes are comparable, and the results were consistent with the hypothesis that P. woesei lacks the 105 ORFs found in P. furiosus. The missing ORFs are present in P. furiosus mainly in clusters. These clusters include one cluster (Mal I, PF1737 to PF1751) involved in maltose metabolism and another cluster (PF0691 to PF0695) whose products are thought to remove toxic reactive nitrogen species. Accordingly, it was found that P. woesei, in contrast to P. furiosus, is unable to utilize maltose as a carbon source for growth, and the growth of P. woesei on starch was inhibited by addition of a nitric oxide generator. In P. furiosus the ORF clusters not present in P. woesei are bracketed by or are in the vicinity of insertion sequences or long clusters of tandem repeats (LCTRs). While the role of LCTRs in lateral gene transfer is not known, the Mal I cluster in P. furiosus is a composite transposon that undergoes replicative transposition. The same locus in P. woesei lacks any evidence of insertion activity, indicating that P. woesei is a sister or even the parent of P. furiosus. P. woesei may have acquired by lateral gene transfer more than 100 ORFs from other organisms living in the same thermophilic environment to produce the type strain of P. furiosus.

Human feces collected from 10 healthy teenagers was analyzed for the presence of Crenarchaeota. After a first polymerase chain reaction (PCR) with Archaea-specific primers, a nested real-time PCR was performed using Crenarchaeota-specific primers. Real-time Crenarchaeotal PCR products detected from four subjects were cloned and the sequencing revealed that most of the partial 16S rRNA gene sequences were highly similar (> or = 97% homology) to sequences affiliated to the Sulfolobus group of the Crenarchaeota phylum. Our findings suggest for the first time that Crenarchaeota might be present in the microbiota of the human digestive ecosystem in which this phylum has never been found yet.

We employed a cultivation-independent approach to examine archaeal diversity along a transient soil salinity gradient at Salt Spring in British Columbia, Canada that is routinely eroded due to heavy, recurrent rainfall. Archaeal 16S rRNA gene libraries were created using DNA extracted from three soil samples collected along this gradient. Statistical comparisons indicated similar archaeal richness across sites but, a significant shift in archaeal community composition along the salinity gradient. Seven distinct phylogenetic groups were represented in soil libraries. Haloarchaea were the most commonly sampled group. Other 16S rRNA sequences were related to uncultured Euryarchaeota and Crenarchaeota or halophilic methanogens. Haloarchaeal diversity was remarkably high in soil of elevated salinity compared with previously characterized haloarchaeal communities. Salt Spring haloarchaea were not closely related to known low-salt adapted/tolerant species, suggesting they may be frequently faced with local mortality as a result of frequent declines in soil salinity. We speculate that ecosystem disturbance -- in the form of salinity fluctuations -- is one mechanism for maintaining a diverse community of haloarchaea at Salt Spring.

Mires forming an ecohydrological gradient from nutrient-rich, groundwater-fed mesotrophic and oligotrophic fens to a nutrient-poor ombrotrophic bog were studied by comparing potential methane (CH(4)) production and methanogenic microbial communities. Methane production was measured from different depths of anoxic peat and methanogen communities were detected by detailed restriction fragment length polymorphism (RFLP) analysis of clone libraries, sequencing and phylogenetic analysis. Potential CH(4) production changed along the ecohydrological gradient with the fens displaying much higher production than the ombrotrophic bog. Methanogen diversity also decreased along the gradient. The two fens had very similar diversity of methanogenic methyl-coenzyme M reductase gene (mcrA), but in the upper layer of the bog the methanogen diversity was strikingly lower, and only one type of mcrA sequence was retrieved. It was related to the Fen cluster, a group of novel methanogenic sequences found earlier in Finnish mires. Bacterial 16S rDNA sequences from the fens fell into at least nine phyla, but only four phyla were retrieved from the bog. The most common bacterial groups were Deltaproteobacteria, Verrucomicrobia and Acidobacteria.

The active bacterial community able to utilize benzoate under denitrifying conditions was elucidated in two coastal sediments using stable-isotope probing (SIP) and nosZ gene amplification. The SIP method employed samples from Norfolk Harbor, Virginia, and a Long-Term Ecosystem Observatory (no. 15) off the coast of Tuckerton, New Jersey. The SIP method was modified by use of archaeal carrier DNA in the density gradient separation. The carrier DNA significantly reduced the incubation time necessary to detect the (13)C-labeled bacterial DNA from weeks to hours in the coastal enrichments. No denitrifier DNA was found to contaminate the archaeal (13)C-carrier when [(12)C]benzoate was used as a substrate in the sediment enrichments. Shifts in the activity of the benzoate-utilizing denitrifying population could be detected throughout a 21-day incubation. These results suggest that temporal analysis using SIP can be used to illustrate the initial biodegrader(s) in a bacterial population and to document the cross-feeding microbial community.

The methanogen community in sediment from the edge of a small brackish lake connected to the Beaulieu Estuary (Hampshire, UK) was investigated by analysis of 16S rRNA gene diversity using new methanogen-specific primers plus Archaea-specific primers. 16S rRNA gene primers previously used for polymerase chain reaction (PCR) detection of methanogenic Archaea from a variety of environments were evaluated by in silico testing. The primers displayed variable coverage of the four main orders of methanogens, highlighting the importance of this type of primer evaluation. Three PCR primer sets were designed using novel reverse primers to facilitate specific amplification of the orders Methanomicrobiales/Methanosarcinales, Methanobacteriales and Methanococcales. Diversity of the methanogen functional gene, methyl coenzyme M reductase (mcrA), was also studied. All gene libraries constructed from this sediment indicated that Methanomicrobiales and Methanosarcinales were the only methanogens detected. There was good agreement between the relative sequence abundances in the methanogen-specific 16S rRNA gene library and terminal restriction fragment length polymorphism (T-RFLP) profiling, suggesting that the population was dominated by putative H2 CO2 utilizing Methanomicrobiales, although acetate-utilizing methanogens were also present. The methanogen population analyses were in agreement with methanogenic activity measurements, which indicated that bicarbonate methanogenesis was higher than acetate methanogenesis at all depths measured and overall there was a significant difference (P = 0.001) between the rates of the two pathways. This study demonstrates the utility of new 16S rRNA gene PCR primers targeting specific methanogenic orders, and the combined results suggest that the CO2 reduction pathway dominates methanogenesis in the brackish sediment investigated.

The characterization of microbial assemblages within solid gas hydrate, especially those that may be physiologically active under in situ hydrate conditions, is essential to gain a better understanding of the effects and contributions of microbial activities in Gulf of Mexico (GoM) hydrate ecosystems. In this study, the composition of the Bacteria and Archaea communities was determined by 16S rRNA phylogenetic analyses of clone libraries derived from RNA and DNA extracted from sediment-entrained hydrate (SEH) and interior hydrate (IH). The hydrate was recovered from an exposed mound located in the northern GoM continental slope with a hydrate chipper designed for use on the manned-submersible Johnson Sea Link (water depth, 550 m). Previous geochemical analyses indicated that there was increased metabolic activity in the SEH compared to the IH layer (B. N. Orcutt, A. Boetius, S. K. Lugo, I. R. Macdonald, V. A. Samarkin, and S. Joye, Chem. Geol. 205:239-251). Phylogenetic analysis of RNA- and DNA-derived clones indicated that there was greater diversity in the SEH libraries than in the IH libraries. A majority of the clones obtained from the metabolically active fraction of the microbial community were most closely related to putative sulfate-reducing bacteria and anaerobic methane-oxidizing archaea. Several novel bacterial and archaeal phylotypes for which there were no previously identified closely related cultured isolates were detected in the RNA- and DNA-derived clone libraries. This study was the first phylogenetic analysis of the metabolically active fraction of the microbial community extant in the distinct SEH and IH layers of GoM gas hydrate.

An obligately anaerobic, hyperthermophilic, organoheterotrophic archaeon, strain Z-1312(T), was isolated from a freshwater hot spring of the Uzon caldera (Kamchatka Peninsula, Russia). The cells were regular cocci, 1-4 microm in diameter, with one long flagellum. The cell envelope was composed of a globular layer attached to the cytoplasmic membrane. The temperature range for growth was 63-89 degrees C, with an optimum between 80 and 82 degrees C. The pH range for growth at 80 degrees C was 4.8-6.8, with an optimum at pH 6.0. Strain Z-1312(T) grew by hydrolysis and/or fermentation of a wide range of polymeric and monomeric substrates, including agarose, amygdalin, arabinose, arbutin, casein hydrolysate, cellulose (filter paper, microcrystalline cellulose, carboxymethyl cellulose), dextran, dulcitol, fructose, lactose, laminarin, lichenan, maltose, pectin, peptone, ribose, starch and sucrose. No growth was detected on glucose, xylose, mannitol or sorbitol. Growth products when sucrose or starch were used as the substrate were acetate, H(2) and CO(2). Elemental sulfur, thiosulfate and nitrate added as potential electron acceptors for anaerobic respiration did not stimulate growth when tested with starch as the substrate. H(2) at 100 % in the gas phase did not inhibit growth on starch or peptone. The G+C content of the DNA was 42.5 mol%. 16S rRNA gene sequence analysis placed the isolated strain Z-1312(T) as a member of the genus Desulfurococcus, where it represented a novel species, for which the name Desulfurococcus fermentans sp. nov. (type strain Z-1312(T) = DSM 16532 (T) = VKM V-2316(T)) is proposed.

A novel extremely halophilic strain, AJ2(T), was isolated from Ayakekum salt lake located in the Altun Mountain National Nature Reserve in Xinjiang, China. This isolate was neutrophilic, motile and grew in a wide range of MgCl(2) concentrations (0.005-1.0 M). The major polar lipids of the isolate were C(20)C(20) and C(20)C(25) derivatives of phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and phosphatidylglycerol sulfate. A comprehensive 16S rRNA gene sequence analysis revealed that the isolate shared 96.6-97.7 % sequence identity with Natrinema species. The isolate, however, could be genetically differentiated from these species by DNA-DNA hybridization analysis and on the basis of its physiological properties. On the basis of the polyphasic evidence, strain AJ2(T) (= AS 1.3731(T) = JCM 12890(T)) represents the type strain of a novel species, for which the name Natrinema altunense sp. nov. is proposed.

A so far uncultured member of the Euryarchaeota was enriched from an anoxic riparian soil and phenotypically characterized using quantitative polymerase chain reaction (qPCR; "real-time PCR"). The microorganism is related to the Thermoplasmatales and belongs to Rice Cluster III (RC-III). Enrichment cultures utilized yeast extract (YE) by transiently accumulating acetate as major fermentation product, which was subsequently converted to methane. The abundance of RC-III archaea within the enrichment cultures was quantified by analysis of the terminal restriction fragment length polymorphism (T-RFLP) and by qPCR. We developed qPCR assays targeting the 16S rRNA genes (16S rDNA) specific for RC-III as well as for the Archaea in general. The enrichment cultures consisted of a mixed methanogenic community of Bacteria and Archaea, the latter consisting of up to 60% of members of RC-III. The other archaea belonged to Methanosarcinaceae, Methanomicrobiaceae and Methanobacteriaceae. The enriched RC-III archaea were represented by two sequences (LL25A, LL37A) that were highly similar to each other and to those detected in the soil inoculum (>98% similarity). However, the 16S rDNA copy numbers of RC-III archaea were about 1000-fold lower than those of Bacteria. Nevertheless, we were able to estimate growth parameters and physiological properties of one of the enriched RC-III archaea (LL25A) by measuring the increase of 16S rDNA copy numbers specific for this group under different growth conditions. The enriched RC-III archaeon grew optimally at temperatures between 20 and 30 degrees C and neutral pH using YE, meat extract, peptone or tryptone under anoxic conditions. Doubling time was approximately 3 days. No proliferation was detected on carbohydrates, amino acids, fatty acids, glycerol, alcohols, aromatic compounds, purine and pyrimidine bases or pyruvate. Various exogenous electron acceptors (e.g. ferric iron, S(0)) did not support growth on YE. Proliferation of the enriched RC-III archaeon was hardly affected by the antibiotics ampicillin, kanamycin and streptomycin. These findings suggest that the enriched archaeon is a mesophilic anaerobe, which can grow heterotrophically on peptides. Further enrichment on peptone and kanamycin eventually allowed the microscopic detection of coccoid cells stained by fluorescence in situ hybridization (FISH).

We investigated the influence of desiccation frequency, indicated by tidal position, on microbial community structure, diversity and richness of microbial mats. We independently characterized cyanobacterial, bacterial and archaeal communities, and their spatial variability for two distinct microbial mat systems: subtidal hypersaline mats and intertidal sand flat mats. Community fingerprints based on 16S rDNA were obtained via denaturing gradient gel electrophoresis using polymerase chain reaction primers specific for each group. Fingerprints for all three groups were consistently similar [> or =85% according to Weighted Pair Group with Arithmetic Mean (WPGMA) analysis] along a 1-km-long transect in subtidal mats. Here, pair-wise comparison analysis yielded minimal variation in diversity and richness for all groups. Fingerprints of three sites along an intertidal transect were heterogenous (> or =32% similarity according to WPGMA analysis) with clear shifts in community structure in all three microbial groups. Here, all groups exhibited statistically significant decreases in richness and diversity with tidal height (as desiccation frequency increases). Regression analysis yielded a strong correlation between diversity or richness estimates and position along the tidal gradient, for both Archaea and Bacteria, with Cyanobacteria exhibiting a weaker correlation. These results suggest that desiccation frequency can shape the structure of microbial mat communities, with Archea being least tolerant and Cyanobacteria most tolerant.

To get insight into the microbial community of an Upflow Anaerobic Sludge Blanket reactor treating paper mill wastewater, conventional microbiological methods were combined with 16S rRNA gene analyses. Particular attention was paid to microorganisms able to degrade propionate or butyrate in the presence or absence of sulphate. Serial enrichment dilutions allowed estimating the number of microorganisms per ml sludge that could use butyrate with or without sulphate (10(5)), propionate without sulphate (10(6)), or propionate and sulphate (10(8)). Quantitative RNA dot-blot hybridisation indicated that Archaea were two-times more abundant in the microbial community of anaerobic sludge than Bacteria. The microbial community composition was further characterised by 16S rRNA-gene-targeted Denaturing Gradient Gel Electrophoresis (DGGE) fingerprinting, and via cloning and sequencing of dominant amplicons from the bacterial and archaeal patterns. Most of the nearly full length (approximately 1.45 kb) bacterial 16S rRNA gene sequences showed less than 97% similarity to sequences present in public databases, in contrast to the archaeal clones (approximately. 1.3 kb) that were highly similar to known sequences. While Methanosaeta was found as the most abundant genus, also Crenarchaeote-relatives were identified. The microbial community was relatively stable over a period of 3 years (samples taken in July 1999, May 2001, March 2002 and June 2002) as indicated by the high similarity index calculated from DGGE profiles (81.9+/-2.7% for Bacteria and 75.1+/-3.1% for Archaea). 16S rRNA gene sequence analysis indicated the presence of unknown and yet uncultured microorganisms, but also showed that known sulphate-reducing bacteria and syntrophic fatty acid-oxidising microorganisms dominated the enrichments.

A hot spring in the solfataric field of Pisciarelli (Naples-Italy) was analysed for Archaeal diversity. Total DNA was extracted from the environment, archaeal 16S rRNA genes were amplified with Archaea specific primers, and a clone library consisting of 201 clones was established. The clones were grouped in 10 different groups each representing a specific band pattern using restriction fragment length polymorphism (RFLP). Members of all 10 groups were sequenced and phylogenetically analyzed. Surprisingly, a high abundance of clones belonging to non-thermophilic Crenarchaeal clusters were detected together with the thermophilic archaeon Acidianus infernus in this thermophilic environment. Neither Sulfolobus species nor other hyperthermophilic Crenarchaeota were detected in the clone library. The relative abundance of the sequenced clones was confirmed by terminal restriction fragment analyses. Amplification of 16S rRNA genes from Archaea transferred from the surrounding environment was considered negligible because DNA from non-thermophilic Crenarchaeota incubated under conditions similar to the solfatara could not be PCR amplified after 5 min.

Archaeal L7Ae is a multifunctional protein that binds to a distinctive K-turn motif in RNA and is found as a component in the large subunit of the ribosome, and in ribose methylation and pseudouridylation guide RNP particles. A collection of L7Ae-associated small RNAs were isolated from Sulfolobus solfataricus cell extracts and used to construct a cDNA library; 45 distinct cDNA sequences were characterized and divided into six groups. Group 1 contained six RNAs that exhibited the features characteristic of the canonical C/D box archaeal sRNAs, two RNAs that were atypical C/D box sRNAs and one RNA representative of archaeal H/ACA sRNA family. Group 2 contained 13 sense strand RNA sequences that were encoded either within, or overlapping annotated open reading frames (ORFs). Group 3 contained three sequences form intergenic regions. Group 4 contained antisense sequences from within or overlapping sense strand ORFs or antisense sequences to C/D box sRNAs. More than two-thirds of these sequences possessed K-turn motifs. Group 5 contained two sequences corresponding to internal regions of 7S RNA. Group 6 consisted of 11 sequences that were fragments from the 5' or 3' ends of 16S and 23S ribosomal RNA and from seven different tRNAs. Our data suggest that S. solfataricus contains a plethora of small RNAs. Most of these are bound directly by the L7Ae protein; the others may well be part of larger, transiently stable RNP complexes that contain the L7Ae protein as core component.

Methane production was studied in an Italian rice field over two consecutive years (1998, 1999) by measuring the rates of total and acetate-dependent methanogenesis in soil and root samples. Population dynamics of methanogens were followed by terminal restriction fragment length polymorphism and real-time PCR targeting archaeal SSU rRNA genes. Rates of total and acetate-dependent methanogenesis in soil increased during the season, reached a maximum at about 70-80 days after flooding and then decreased again. In contrast, the size of the archaeal community remained relatively constant. Therefore, the seasonal changes in the methanogenic processes were probably not caused by changes in the size of the methanogenic community but in its activity. During the 1998/1999 winter period, a slight decrease in archaeal cell numbers was found. In both years, the dominant groups were methanogens affiliated with Rice cluster I, Methanosaetaceae, Methanosarcinaceae and Methanobacteriaceae. Correspondence analysis showed, however, that the archaeal community structure was different in 1998 and 1999. Methanogens with potential acetoclastic activity made up a larger fraction of the total archaeal community in 1999 (32-53%) than in 1998 (20-32%). Furthermore, the frequency of Methanosaetaceae relative to Methanosarcinaceae was significantly higher in 1999 than in 1998. This difference could be explained by the much lower soil acetate concentrations in 1999, to which Methanosaetaceae are physiologically better adapted than Methanosarcinaceae. Over the season, however, the composition of the archaeal community remained relatively constant and thus did not reflect the observed seasonal change in CH(4) production activity. The analysis of rice root samples in 1999 showed that the archaeal community structure on the roots was similar to that in soil but with acetoclastic methanogens being relatively less common. This observation is in agreement with domination of CH(4) production by H(2)/CO(2)-dependent methanogenesis on roots. Our study provided a link between size, structure and function of the methanogenic community in an Italian rice field.

Analyses of complete genomes indicate that insertion sequences (ISs) are abundant and widespread in hyperthermophilic archaea, but few experimental studies have measured their activities in these hosts. As a way to investigate the impact of ISs on Sulfolobus genomes, we identified seven transpositionally active ISs in a widely distributed Sulfolobus species, and measured their functional properties. Six of the seven were found to be distinct from previously described ISs of Sulfolobus, and one of the six could not be assigned to any known IS family. A type II 'Miniature Inverted-repeat Transposable Element' (MITE) related to one of the ISs was also recovered. Rates of transposition of the different ISs into the pyrEF region of their host strains varied over a 250-fold range. The Sulfolobus ISs also differed with respect to target-site selectivity, although several shared an apparent preference for the pyrEF promoter region. Despite the number of distinct ISs assayed and their molecular diversity, only one demonstrated precise excision from the chromosomal target region. The fact that this IS is the only one lacking inverted repeats and target-site duplication suggests that the observed precise excision may be promoted by the IS itself. Sequence searches revealed previously unidentified partial copies of the newly identified ISs in the Sulfolobus tokodaii and Sulfolobus solfataricus genomes. The structures of these fragmentary copies suggest several distinct molecular mechanisms which, in the absence of precise excision, inactivate ISs and gradually eliminate the defective copies from Sulfolobus genomes.