Community phylogenetic analysis of moderately thermophilic cyanobacterial mats from China, the Philippines and Thailand

Most community molecular studies of thermophilic cyanobacterial mats to date have focused on Synechococcus occurring at temperatures of ~50–65°C. These reveal that molecular diversity exceeds that indicated by morphology, and that phylogeographic lineages exist. The moderately thermophilic and generally filamentous cyanobacterial mat communities occurring at lower temperatures have not previously been investigated at the community molecular level. Here we report community diversity in mats of 42–53°C recovered from previously unstudied geothermal locations. Separation of 16S rRNA gene-defined genotypes from community DNA was achieved by DGGE. Genotypic diversity was greater than morphotype diversity in all mats sampled, although genotypes generally corresponded to observed morphotypes. Thirty-six sequences were recovered from DGGE bands. Phylogenetic analyses revealed these to form novel thermophilic lineages distinct from their mesophilic counterparts, within Calothrix, Cyanothece, Fischerella, Phormidium, Pleurocapsa, Oscillatoria and Synechococcus. Where filamentous cyanobacterial sequences belonging to the same genus were recovered from the same site, these were generally closely affiliated. Location-specific sequences were observed for some genotypes recovered from geochemically similar yet spatially separated sites, thus providing evidence for phylogeographic lineages that evolve in isolation. Other genotypes were more closely affiliated to geographically remote counterparts from similar habitats suggesting that adaptation to certain niches is also important.     

Phylogenetic Evidence for the Existence of Novel Thermophilic Bacteria in Hot Spring Sulfur-Turf Microbial Mats in Japan

So-called sulfur-turf microbial mats, which are macroscopic white filaments or bundles consisting of large sausage-shaped bacteria and elemental sulfur particles, occur in sulfide-containing hot springs in Japan. However, no thermophiles from sulfur-turf mats have yet been isolated as cultivable strains. This study was undertaken to determine the phylogenetic positions of the sausage-shaped bacteria in sulfur-turf mats by direct cloning and sequencing of 16S rRNA genes amplified from the bulk DNAs of the mats. Common clones with 16S rDNA sequences with similarity levels of 94.8 to 99% were isolated from sulfur-turf mat samples from two geographically remote hot springs. Phylogenetic analysis showed that the phylotypes of the common clones formed a major cluster with members of the Aquifex-Hydrogenobacter complex, which represents the most deeply branching lineage of the domain bacteria. Furthermore, the bacteria of the sulfur-turf mat phylotypes formed a clade distinguishable from that of other members of the Aquifex-Hydrogenobacter complex at the order or subclass level. In situ hybridization with clone-specific probes for 16S rRNA revealed that the common phylotype of sulfur-turf mat bacteria is that of the predominant sausage-shaped bacteria.Microbial mats develop in a wide variety of aquatic environments, including geothermal hot springs and hydrothermal vents. There are several types of thermophilic microbial mats, e.g., those of cyanobacteria, anoxygenic phototrophic bacteria, and chemotrophic sulfur bacteria, which differ according to the physical and chemical conditions they favor and other environmental factors (10, 38). These microbial mats in thermal habitats have been studied extensively as a peculiar microbial community of the ecosystem, in relation to the phylogeny and evolution of thermophilic prokaryotes, or as a source of new functional enzymes.So-called sulfur-turf microbial mats are macroscopic bundles of white filaments consisting of colorless sulfur bacteria and elemental sulfur particles that form in shallow streams of sulfide-containing high-temperature hot springs. Since first reported by Miyoshi in 1897 (33), this kind of microbial mat has been recorded for several geographically remote hot springs in Japan, although there have been only scattered reports of sulfur-turf microbial mats or chemotrophic sulfur streamers in geothermal springs in other countries (9, 13, 14). The sulfur-turf mats generally develop within a temperature range of 45 to 73°C, within a pH range of 6 to 9, and at discrete sulfide-oxygen interfaces in geothermal springs. These characteristics suggest that the major constituents of the sulfur-turf prokaryotic community are (hyper)thermophilic, neutrophilic, microaerophilic, and chemolithotrophic bacteria. Early studies of these sulfur-turf mats distinguished microscopically three morphotypes of bacteria, two of which were tentatively named Thiovibrio miyoshi and Thiothrix miyoshi (15). Moreover, in situ ecophysiological and microscopic studies have shown that one of these bacteria, the large sausage-shaped “Thiovibrio miyoshi,” predominates in sulfur-turf mats and oxidizes environmental sulfide to elemental sulfur and then to sulfate via thiosulfate (27–31). So far, however, it has not been possible to isolate and cultivate any thermophilic prokaryotes from the sulfur-turf mats predominated by these sausage-shaped bacteria with artificial media, and no attempt has been made to clarify their taxonomic and phylogenetic positions.Determination of 16S rRNA genes is a useful research strategy for identifying uncultivated prokaryotes and is now commonly performed in ecological studies. This technique, involving PCR amplification of 16S rRNA genes or synthesis of cDNAs from bulk 16S rRNAs of natural mixed microbial populations, has been used successfully for the phylogenetic characterization of prokaryotes in hydrothermal environments (6, 7, 34, 40, 41, 47, 48). In the present study, this approach was applied to characterize the sausage-shaped bacteria in sulfur-turf mats without isolating and cultivating them. Here we report that sulfur-turf mats contain novel thermophilic bacteria belonging to the earliest-branching lineage of the domain bacteria.     

Alkane-1,2-diol-based glycosides and fatty glycosides and wax esters in Roseiflexus castenholzii and hot spring microbial mats

The lipid composition of Roseiflexus castenholzii, a thermophilic filamentous phototrophic bacterium related to uncultivated filamentous phototrophic bacteria that predominate in hot spring microbial mats, is reported. R. castenholzii lipid extracts were dominated by components characterized by alkane-1-ol-2-alkanoate moieties glycosidically bonded to a C(6) sugar. Similar fatty glycosides, with an additional fatty acid esterified, were detected by HPLC-MS. R. castenholzii also produces a suite of wax esters ranging from 37 to 40 carbon atoms in length. In lipid extracts from two nonsulfidic hot spring microbial mats, similar alkane-1,2-diol-based lipids were detected in minor amounts. R. castenholzii lipids are compared to lipids of mats and other thermophilic mat isolates.     

Differential microbial communities in hot spring mats from Western Thailand

The microbial communities of freshwater hot spring mats from Boekleung (Western Thailand) were studied. Temperatures ranged from over 50 up to 57°C. Green-, red-, and yellow colored mat layers were analyzed. In order to detect the major components of the microbial communities constituting the mat as well as the microorganisms showing significant metabolic activity, samples were analyzed using DNA- and RNA-based molecular techniques, respectively. Microbial community fingerprints, performed by denaturing gradient gel electrophoresis (DGGE), revealed clear differences among mat layers. Thermophilic phototrophic microorganisms, Cyanobacteria and Chloroflexi, constituted the major groups in these communities (on average 65 and 51% from DNA and RNA analyses, respectively). Other bacteria detected in the mat were Bacteroidetes, members of the Candidate Division OP10, Actinobacteria, and Planctomycetes. Differently colored mat layers showed characteristic bacterial communities and the major components of the metabolically active fraction of these communities have been identified.     

Anoxygenic phototrophic bacteria from microbial communities of Goryachinsk thermal spring (Baikal Area,Russia)

Species composition of anoxygenic phototrophic bacteria in microbial mats of the Goryachinsk thermal spring was investigated along the temperature gradient. The spring belonging to nitrogenous alkaline hydrotherms is located at the shore of Lake Baikal 188 km north-east from Ulan-Ude. The water is of the sulfate-sodium type, contains trace amounts of sulfide, and salinity does not exceed 0.64 g/L, pH 9.5. The temperature at the outlet of the spring may reach 54°C. The cultures of filamentous anoxygenic phototrophic bacteria, nonsulfur and sulfur purple bacteria, and aerobic anoxygenic phototrophic bacteria were identified using the pufLM molecular marker. The fmoA marker was used for identification of green sulfur bacteria. Filamentous cyanobacteria predominated in the mats, with anoxygenic phototrophs comprising a minor component of the phototrophic communities. Thermophilic bacteria Chloroflexus aurantiacus were detected in the samples from both the thermophilic and mesophilic mats. Cultures of nonsulfur purple bacteria similar to Blastochloris sulfoviridis and Rhodomicrobium vannielii were isolated from the mats developed at high (50.6–49.4°C) and low temperatures (45–20°C). Purple sulfur bacteria Allochromatium sp. and Thiocapsa sp., as well as green sulfur bacteria Chlorobium sp., were revealed in low-temperature mats. Truly thermophilic purple and green sulfur bacteria were not found in the spring. Anoxygenic phototrophic bacteria found in the spring were typical of the sulfur communities, for which the sulfur cycle is mandatory. The presence of aerobic bacteriochlorophyll a-containing bacteria identified as Agrobacterium (Rhizobium) tumifaciens in the mesophilic (20°C) mat is of interest.     

Bacterial community shift along a subsurface geothermal water stream in a Japanese gold mine

Change of bacterial community occurring along a hot water stream in the Hishikari gold mine, Japan, was investigated by applying a combination of various culture-independent techniques. The stream, which is derived from a subsurface anaerobic aquifer containing plentiful CO₂, CH₄, H₂, and NH₄⁺, emerges in a mine tunnel 320 m below the surface providing nutrients for a lush microbial community that extends to a distance of approximately 7 m in the absence of sunlight-irradiation. Over this distance, the temperature decreases from 69°C to 55°C, and the oxidation-reduction potential increases from –130 mV to +59 mV. In the hot upper reaches of the stream, the dominant phylotypes were: 1) a deeply branching lineage of thermophilic methane-oxidizing -Proteobacteria, and 2) a thermophilic hydrogen- and sulfur-oxidizing Sulfurihydrogenibium sp. In contrast, the prevailing phylotypes in the middle and lower parts of the stream were closely related to ammonia-oxidizing Nitrosomonas and nitrite-oxidizing Nitrospira spp.. Changes in the microbial metabolic potential estimated by competitive PCR analysis of genes encoding the enzymes, particulate methane monooxygenase (pmoA), ammonia monooxygenase (amoA), and putative nitrite oxidoreductase (norB), also substantiated the community shift indicated by 16S rRNA gene analysis. The diversity of putative norB lineages was assessed for the first time in the hot water environment. Estimation of dominant phylotypes by whole-cell fluorescent in situ hybridization and changes in inorganic nitrogen compounds such as decreasing ammonium and increasing nitrite and nitrate in the mat-interstitial water along the stream were consistent with the observed transition of the bacterial community structure in the stream.     

Physiological Ecology of a Gliding Bacterium Containing Bacteriochlorophyll a

A filamentous, gliding, thermophilic bacterium, found growing abundantly as a surface mat in a limited number of alkaline hot springs in Oregon, is described and designated F-1. The bacteria were studied in the field and in coculture with an aerobic chemoheterotroph. The bacteria are phototrophic and contain bacteriochlorophyll a and several carotenoid pigments. Unlike the other gliding phototrophic bacteria, members of the family Chloroflexaceae, F-1 does not contain chlorosomes or bacteriochlorophyll c or d. The light-dependent uptake of simple organic compounds (acetate and glucose) was demonstrated in field populations. Near-infrared radiation sustained this uptake, which occurred equally well under aerobic or anaerobic conditions and was insensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea. The bacteria formed conspicuous dominant mats from about 35 to 56°C, and they covered mats of cyanobacteria in the spring, summer, and autumn months. It appears that they depend on high light intensities to maintain a dense population.     

Probing the archaeal diversity of a mixed thermophilic bioleaching culture by TGGE and FISH

The archaeal community present in a sample of Mixed Thermophilic Culture-B (MTC-B) from a laboratory-scale thermophilic bioleaching reactor was investigated by temperature gradient gel electrophoresis (TGGE) and fluorescence in situ hybridisation (FISH). Both techniques were specifically adapted for use on native state bioleaching samples, with a view to establishing convenient means for monitoring culture composition. Using the TGGE protocol developed, the relative species composition of the thermophilic bioleaching sample was analysed, and included four phylotypes belonging to the Sulfolobales, which were related to Stygiolobus azoricus, Metallosphaera sp. J1, Acidianus infernus and Sulfurisphaera ohwakuensis. However, the St. azoricus-like phylotype was difficult to resolve and some micro-heterogeneity was observed within this phylotype. Specific FISH probes were designed to qualitatively assess the presence of the phylotypes in MTC-B. The sample was dominated by Sf. ohwakuensis-like Archaea. In addition, the St. azoricus-like, Metallosphaera species-like and Acidianus species-like cells appeared in similar low abundance in the community. Most strikingly, FISH identified Sulfolobus shibatae-like cells present in low numbers in the sample even though these were not detected by PCR-dependent TGGE. These results highlight the importance of using more than one molecular technique when investigating the archaeal diversity of complex bioleaching reactor samples.     

Microbial mats in tidal channels at San Carlos,Baja California Sur,Mexico

Microbial communities of stratified phototrophic bacteria in laminated intertidal sediments north of Estuary El Puente, near San Carlos, Baja California Sur, Mexico,‐were studied. This study describes the macroscopic and microscopic characteristics of the mats, including their annual growth. The mats were located in and along meandering mangrove‐lined tidal channels. Their thickness ranged from 0.5 to 25 cm. Square‐meter areas of polygonal mats were detected in several ponds infiltrated by sea water. The principal microbial community of the upper surface of various morphotypes of microbial mats was identified as cyanobacteria belonging to the genera Microcoleus, Lyngbya, Phormidium, and Oscillatoria. Other cyanobacte‐rial genera such as Pseudanabaena, Spirulina, Synechococcus, and Gloeocapsa, as well as many unidentified diatoms, were also present but at lower population densities. The second inward reddish layers of the microbial mats contained similar cyano‐bacterial genera plus anoxygenic phototrophic bacteria belonging to the genera Chloroflexus, Thiocapsa, Chromatium, Prosthecochloris, Rhodopseudomonas, and Chlorobium, as well as several unidentified bacteria. In situ measurements on the growth of the mats, from intermittent tide sites, showed an annual buildup of two layers: green and reddish. These layers corresponded to a vertical growth of 1.4 ± 0.27 mm/year. Permanently submerged mats did not show vertical growth during the same period of time.     

Benthic phototrophic community from Kiran soda lake,south-eastern Siberia

Phototrophic bacterial mats from Kiran soda lake (south-eastern Siberia) were studied using integrated approach including analysis of the ion composition of water, pigments composition, bacterial diversity and the vertical distribution of phototrophic microorganisms in the mats. Bacterial diversity was investigated using microscopic examination, 16S rRNA gene Illumina sequencing and culturing methods. The mats were formed as a result of decomposition of sedimented planktonic microorganisms, among which cyanobacteria of the genus Arthrospira predominated. Cyanobacteria were the largest part of phototrophs in the mats, but anoxygenic phototrophs were significant fraction. The prevailing species of the anoxygenic phototrophic bacteria are typical for soda lakes. The mats harbored aerobic anoxygenic phototrophic bacteria, purple sulfur and non-sulfur bacteria, as well as new filamentous phototrophic Chloroflexi. New strains of Thiocapsa sp. Kir-1, Ectothiorhodospira sp. Kir-2 and Kir-4, Thiorhodospira sp. Kir-3 and novel phototrophic Chloroflexi bacterium Kir15-3F were isolated and identified.

     

Biogeochemical processes in the algal-bacterial mats of the Urinskii alkaline hot spring

The structure and production characteristics of microbial communities from the Urinskii alkaline hot spring (Buryat Republic, Russia) have been investigated. A distinctive characteristic of this hot spring is the lack of sulfide in the issuing water. The water temperature near the spring vents ranged from 69 to 38.5°C and pH values ranged from 8.8 to 9.2. The total mineralization of water was less than 0.1 g/liter. Temperature has a profound effect on the species composition and biogeochemical processes occurring in the algal-bacterial mats of the Urinskii hot spring. The maximum diversity of the phototrophic community was observed at the temperatures 40 and 46°C. A total of 12 species of cyanobacteria, 4 species of diatoms, and one species of thermophilic anoxygenic phototrophic bacteria, Chloroflexus aurantiacus, have been isolated from mat samples. At temperatures above 40°C, the filamentous cyanobacterium Phormidium laminosum was predominant; its cell number and biomass concentration comprised 95.1 and 63.9%, respectively. At lower temperatures, the biomass concentrations of the cyanobacterium Oscillatoria limosa and diatoms increased (50.2 and 36.4%, respectively). The cyanobacterium Mastigocladus laminosus, which is normally found in neutral or slightly acidic hydrothermal systems, was detected in microbial communities. As the diatom concentration increases, so does the dry matter concentration in mats, while the content of organic matter decreases. The concentrations of proteins and carbohydrates reached their maximum levels at 45–50°C. The maximum average rate of oxygenic photosynthesis [2.1 g C/(m² day)], chlorophyll a content (343.4 mg/m²), and cell number of phototrophic microorganisms were observed at temperatures from 45 to 50°C. The peak mass of bacterial mats (56.75 g/m²) occurred at a temperature of 65–60°C. The maximum biomass concentration of phototrophs (414.63 × 10^?6 g/ml) and the peak rate of anoxygenic photosynthesis [0.42 g C/(m² day)] were observed at a temperature of 35–40°C.     

Dominant phylotypes in the 16S rRNA gene clone libraries from bacterial mats of the Uzon caldera (Kamchatka,Russia) hydrothermal springs

In situ analysis of the 16S rRNA genes from bacterial mats of five hydrothermal springs (36–58°C) in the Uzon caldera (Kamchatka, Russia) was carried out using clone libraries. Eight clone libraries contained 18 dominant phylotypes (over 4–5%). In most clone libraries, the phylotype of the green sulfur bacterium Chlorobaculum sp. was among the dominant ones. The phylotypes of the green nonsulfur bacteria Chloroflexus and Roseiflexus and of purple nonsulfur bacteria Rhodoblastus, Rhodopseudomonas, and Rhodoferax were also among the dominant ones. Cyanobacteria were represented by one dominant phylotype in a single spring. Among nonphototrophic bacteria, the dominant phylotypes belonged to Sulfyrihydrogenibium sp., Geothrix sp., Acidobacterium sp., Meiothermus sp., Thiomonas sp., Thiofaba sp., and Spirochaeta sp. Three phylotypes were not identified at the genus level. Most genera of phototrophic and nonphototrophic organisms corresponding to the phylotypes from Uzon hydrotherms have been previously revealed in the hydrotherms of volcanically active regions of America, Asia, and Europe. These results indicate predominance of bacterial mats carrying out anaerobic photosynthesis in the hydrotherms of the Uzon caldera.     

Molecular diversity of arbuscular mycorrhizal fungi in onion roots from organic and conventional farming systems in the Netherlands

Diversity and colonization levels of naturally occurring arbuscular mycorrhizal fungi (AMF) in onion roots were studied to compare organic and conventional farming systems in the Netherlands. In 2004, 20 onion fields were sampled in a balanced survey between farming systems and between two regions, namely, Zeeland and Flevoland. In 2005, nine conventional and ten organic fields were additionally surveyed in Flevoland. AMF phylotypes were identified by rDNA sequencing. All plants were colonized, with 60% for arbuscular colonization and 84% for hyphal colonization as grand means. In Zeeland, onion roots from organic fields had higher fractional colonization levels than those from conventional fields. Onion yields in conventional farming were positively correlated with colonization level. Overall, 14 AMF phylotypes were identified. The number of phylotypes per field ranged from one to six. Two phylotypes associated with the Glomus mosseae–coronatum and the G. caledonium–geosporum species complexes were the most abundant, whereas other phylotypes were infrequently found. Organic and conventional farming systems had similar number of phylotypes per field and Shannon diversity indices. A few organic and conventional fields had larger number of phylotypes, including phylotypes associated with the genera Glomus-B, Archaeospora, and Paraglomus. This suggests that farming systems as such did not influence AMF diversity, but rather specific environmental conditions or agricultural practices. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Authors Guillermo A. Galván and István Parádi contributed equally to this research and share first co-authorship.     

Heterotrophic dinitrogen fixation (acetylene reduction) in phosphate-fertilised Microcoleus chthonoplastes microbial mat from the hypersaline inland lake ‘la Salada de Chiprana’ (NE Spain)

Microcoleus chthonoplastes dominated microbial mats are conspicuous along the shallow littoral zone in Lake Chiprana, a hypersaline lake located in the Ebro river basin in north-eastern Spain. Pigment data show that these mats included diatom species and anoxygenic phototrophs, Chloroflexus-type bacteria and purple bacteria. In situ, these mats showed low rates of dinitrogen fixation (acetylene reduction). Acetylene reduction was stimulated about 30-fold in excised mats after moderate phosphate fertilisation during 2 weeks incubation in a mesocosm. Pigment analyses showed that this treatment had little impact on the phototrophic community structure, except that it induced a decrease of Chloroflexus-type bacteria. The use of metabolic inhibitors indicated that methanogenic archaea and aerobic heterotrophic bacteria were the major dinitrogen fixers in this system. This is in agreement with the fact that the mat-building cyanobacterium M. chthonoplastes lacks the dinitrogenase reductase nifH gene and with the fact that acetylene reduction rates were strongly stimulated by additions of H₂/CO₂, methanol, fructose and sucrose, but not by lactate, acetate, formate and glucose. No significant differences where found for acetylene reduction rates when comparing light and dark incubations of these microbial mats. However, acetylene reduction rates were enhanced in the light when the near infrared (NIR) light was filtered out, which arrested anoxygenic photosynthesis. We suggest, therefore, that the chemoheterotrophic dinitrogen fixing bacteria were in competition with anoxygenic phototrophic bacteria for organic substrates, while the latter did not contribute to dinitrogen fixation in the mat.     

Diversity and Function of Chloroflexus-Like Bacteria in a Hypersaline Microbial Mat: Phylogenetic Characterization and Impact on Aerobic Respiration

We studied the diversity of Chloroflexus-like bacteria (CLB) in a hypersaline phototrophic microbial mat and assayed their near-infrared (NIR) light-dependent oxygen respiration rates. PCR with primers that were reported to specifically target the 16S rRNA gene from members of the phylum Chloroflexi resulted in the recovery of 49 sequences and 16 phylotypes (sequences of the same phylotype share more than 96% similarity), and 10 of the sequences (four phylotypes) appeared to be related to filamentous anoxygenic phototrophic members of the family Chloroflexaceae. Photopigment analysis revealed the presence of bacteriochlorophyll c (BChlc), BChld, and γ-carotene, pigments known to be produced by phototrophic CLB. Oxygen microsensor measurements for intact mats revealed a NIR (710 to 770 nm) light-dependent decrease in aerobic respiration, a phenomenon that we also observed in an axenic culture of Chloroflexus aurantiacus. The metabolic ability of phototrophic CLB to switch from anoxygenic photosynthesis under NIR illumination to aerobic respiration under non-NIR illumination was further used to estimate the contribution of these organisms to mat community respiration. Steady-state oxygen profiles under dark conditions and in the presence of visible (VIS) light (400 to 700 nm), NIR light (710 to 770 nm), and VIS light plus NIR light were compared. NIR light illumination led to a substantial increase in the oxygen concentration in the mat. The observed impact on oxygen dynamics shows that CLB play a significant role in the cycling of carbon in this hypersaline microbial mat ecosystem. This study further demonstrates that the method applied, a combination of microsensor techniques and VIS and NIR illumination, allows rapid establishment of the presence and significance of CLB in environmental samples.     

Enrichment of anaerobic heterotrophic thermophiles from four Azorean hot springs revealed different community composition and genera abundances using recalcitrant substrates

DGGE analysis combined with a metagenomic approach was used to get insights into heterotrophic anoxic enrichment cultures of four hot springs of Vale das Furnas, Portugal, using the recalcitrant substrate spent coffee ground (SCG). Parallel enrichment cultures were performed using the major components of spent coffee ground, namely arabinogalactan, galactomannan, cellulose, and proteins. DGGE revealed that heterotrophic thermophilic bacteria are highly abundant in the hydrothermal springs and significant differences in community composition depending on the substrate were observed. DNA, isolated from enrichment cultures of different locations that were grown on the same substrate were pooled, and the respective metagenomes were analyzed. Results indicated that cultures grown on recalcitrant substrate SCG consists of a totally different thermophilic community, dominated by Dictyoglomus. Enrichments with galactomannan and arabinogalactan were dominated by Thermodesulfovibrio, while cultures with casein and cellulose were dominated by Thermus. This study indicates the high potential of thermophilic bacteria degrading recalcitrant substrate such as SCG and furthermore how the accessibility to complex polymers shapes the bacterial community.

     

Diversity of phototrophic bacteria in microbial mats from Arctic hot springs (Greenland)

We investigated the genotypic diversity of oxygenic and anoxygenic phototrophic microorganisms in microbial mat samples collected from three hot spring localities on the east coast of Greenland. These hot springs harbour unique Arctic microbial ecosystems that have never been studied in detail before. Specific oligonucleotide primers for cyanobacteria, purple sulfur bacteria, green sulfur bacteria and Choroflexus/Roseiflexus-like green non-sulfur bacteria were used for the selective amplification of 16S rRNA gene fragments. Amplification products were separated by denaturing gradient gel electrophoresis (DGGE) and sequenced. In addition, several cyanobacteria were isolated from the mat samples, and classified morphologically and by 16S rRNA-based methods. The cyanobacterial 16S rRNA sequences obtained from DGGE represented a diverse, polyphyletic collection of cyanobacteria. The microbial mat communities were dominated by heterocystous and non-heterocystous filamentous cyanobacteria. Our results indicate that the cyanobacterial community composition in the samples were different for each sampling site. Different layers of the same heterogeneous mat often contained distinct and different communities of cyanobacteria. We observed a relationship between the cyanobacterial community composition and the in situ temperatures of different mat parts. The Greenland mats exhibited a low diversity of anoxygenic phototrophs as compared with other hot spring mats which is possibly related to the photochemical conditions within the mats resulting from the Arctic light regime.     

Molecular Community Analysis of Arbuscular Mycorrhizal Fungi in Roots of Geothermal Soils in Yellowstone National Park (USA)

To better understand adaptation of plants and their mycorrhizae to extreme environmental conditions, we analyzed the composition of communities of arbuscular mycorrhizal fungi (AMF) in roots from geothermal sites in Yellowstone National Park (YNP), USA. Arbuscular mycorrhizal fungi were identified using molecular methods including seven specific primer pairs for regions of the ribosomal DNA that amplify different subgroups of AMF. Roots of Dichanthelium lanuginosum, a grass only occurring in geothermal areas, were sampled along with thermal and nonthermal Agrostis scabra and control plants growing outside the thermally influenced sites. In addition, root samples of Agrostis stolonifera from geothermal areas of Iceland were analyzed to identify possible common mycosymbionts between these geographically isolated locations. In YNP, 16 ribosomal DNA phylotypes belonging to the genera Archaeospora, Glomus, Paraglomus, Scutellospora, and Acaulospora were detected. Eight of these phylotypes could be assigned to known morphospecies, two others have been reported previously in molecular studies from different environments, and six were new to science. The most diverse and abundant lineage was Glomus group A, with the most frequent phylotype corresponding to Glomus intraradices. Five of the seven phylotypes detected in a preliminary sampling in a geothermal area in Iceland were also found in YNP. Nonthermal vegetation was dominated by a high diversity of Glomus group A phylotypes while nonthermal plants were not. Using multivariate analyses, a subset of three phylotypes were determined to be associated with geothermal conditions in the field sites analyzed. In conclusion, AMF communities in geothermal soils are distinct in their composition, including both unique phylotypes and generalist fungi that occur across a broad range of environmental conditions. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Enrichment of previously uncultured bacteria from natural complex communities by adhesion to solid surfaces

The adhesion to inert solid surfaces was explored as a novel approach for the enrichment of previously uncultured bacteria from natural microbial communities. Enrichments on solid steel, glass and synthetic polymeric surfaces were established using samples from five freshwater lakes, a marine microbial mat and an alpine soil, and were subsequently analysed by molecular fingerprinting and sequencing of their 16S rRNA gene fragments. The majority of the enriched phylotypes grouped with the Alphaproteobacteria, Betaproteobacteria or Bacteroidetes and in several cases were related to typical biofilm‐forming species and genera. Most enrichments were most closely related to previously uncultured phylotypes and none had previously been cultivated from the original environments even when applying improved high throughput liquid cultivation techniques. Of the 13 phylotypes enriched from freshwater samples, seven were previously unknown, three matched so‐far uncultured environmental clones, and three were identical to previously cultivated bacteria. Of the 17 phylotypes recovered from soil, 12 were previously unknown with five of these phylotypes representing novel genera, whereas five phylotypes were identical to previously cultured soil bacteria. The feasibility of the biofilm‐enrichment approach was exemplified by the successful isolation of a not‐yet cultured Betaproteobacterium that constituted a discernible component of the alpine soil microbial community in situ and exhibited only 93% similarity to its closest cultured relative. Based on these results, cultivation on solid surfaces represents a promising approach to recover isolates that have so far escaped cultivation as suspended cultures in liquid media.