A thermophilic green sulfur bacterium from New Zealand hot springs,Chlorobium tepidum sp. nov.

Thermophilic green sulfur bacteria of the genus Chlorobium were isolated from certain acidic high sulfide New Zealand hot springs. Cells were Gram-negative nonmotile rods of variable length and contained bacteriochlorophyll c and chlorosomes. Cultures of thermophilic chlorobia grew only under anaerobic, phototrophic conditions, either photoautotrophically or photoheterotrophically. The optimum growth temperature for the strains of thermophilic green sulfur bacteria isolated was 47–48°C with generation times of about 2 h being observed. The upper temperature limit for growth was about 52°C. Thiosulfate was a major electron donor for photoautotrophic growth while sulfide alone was only poorly used. N₂ fixation was observed at 48°C and cell suspensions readily reduced acetylene to ethylene. The G+C content of DNA from strains of thermophilic chlorobia was 56.5–58.2 mol% and the organisms positioned phylogenetically within the green sulfur bacterial branch of the domain Bacteria. The new phototrophs are described as a new species of the genus Chlorobium, Chlorobium tepidum.This paper is dedicated to Professor Norbert Pfennig on the occasion of his 65th birthday     

Bacterial community composition in thermophilic microbial mats from five hot springs in central Tibet

Despite detailed study of selected thermophilic taxa, overall community diversity of bacteria in thermophilic mats remains relatively poorly understood. A sequence-based survey of bacterial communities from several hot spring locations in central Tibet was undertaken. Diversity and frequency of occurrence for 140 unique 16S rRNA gene phylotypes were identified in clone libraries constructed from environmental samples. A lineage-per-time plot revealed that individual locations have evolved to support relatively large numbers of phylogenetically closely related phylotypes. Application of the F _ST statistic and P test to community data was used to demonstrate that phylogenetic divergence between locations was significant, thus emphasizing the status of hot springs as isolated habitats. Among phylotypes, only the Chlorobi were ubiquitous to all mats, other phototrophs (Cyanobacteria and Chloroflexi) occurred in most but not all samples and generally accounted for a large number of recovered phylotypes. Phylogenetic analyses of phototrophic phylotypes revealed support for location-specific lineages. The alpha, beta and gamma proteobacteria were also frequently recovered phyla, suggesting they may be abundant phylotypes in mats, a hitherto unappreciated aspect of thermophilic mat biodiversity. Samples from one location indicated that where phototrophic bacteria were rare or absent due to niche disturbance, the relative frequency of proteobacterial phylotypes increased. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Phototrophs in high iron microbial mats: microstructure of mats in iron-depositing hot springs

Chocolate Pots Hot Springs in Yellowstone National Park are high in ferrous iron, silica and bicarbonate. The springs are contributing to the active development of an iron formation. The microstructure of photosynthetic microbial mats in these springs was studied with conventional optical microscopy, confocal laser scanning microscopy and transmission electron microscopy. The dominant mats at the highest temperatures (48-54 degrees C) were composed of Synechococcus and Chloroflexus or Pseudanabaena and Mastigocladus. At lower temperatures (36-45 degrees C), a narrow Oscillatoria dominated olive green cyanobacterial mats covering most of the iron deposit. Vertically oriented cyanobacterial filaments were abundant in the top 0.5 mm of the mats. Mineral deposits accumulated beneath this surface layer. The filamentous microstructure and gliding motility may contribute to binding the iron minerals. These activities and heavy mineral encrustation of cyanobacteria may contribute to the growth of the iron deposit. Chocolate Pots Hot Springs provide a model for studying the potential role of photosynthetic prokaryotes in the origin of Precambrian iron formations.     

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.     

Alternative pathways for phosphonate metabolism in thermophilic cyanobacteria from microbial mats

Synechococcus sp. represents an ecologically diverse group of cyanobacteria found in numerous environments, including hot-spring microbial mats, where they are spatially distributed along thermal, light and oxygen gradients. These thermophiles engage in photosynthesis and aerobic respiration during the day, but switch to fermentative metabolism and nitrogen fixation at night. The genome of Synechococcus OS-B′, isolated from Octopus Spring (Yellowstone National Park) contains a phn gene cluster encoding a phosphonate (Phn) transporter and a C–P lyase. A closely related isolate, Synechococcus OS-A, lacks this cluster, but contains genes encoding putative phosphonatases (Phnases) that appear to be active only in the presence of the Phn substrate. Both isolates grow well on several different Phns as a sole phosphorus (P) source. Interestingly, Synechococcus OS-B′ can use the organic carbon backbones of Phns for heterotrophic growth in the dark, whereas in the light this strain releases organic carbon from Phn as ethane or methane (depending on the specific Phn available); Synechococcus OS-A has neither of these capabilities. These differences in metabolic strategies for assimilating the P and C of Phn by two closely related Synechococcus spp. are suggestive of niche-specific constraints in the evolution of nutrient assimilation pathways and syntrophic relationships among the microbial populations of the hot-spring mats. Thus, it is critical to evaluate levels of various P sources, including Phn, in thermally active habitats and the potential importance of these compounds in the biogeochemical cycling of P and C (some Phn compounds also contain N) in diverse terrestrial environments.     

Heliobacterium modesticaldum,sp. nov., a thermophilic heliobacterium of hot springs and volcanic soils

Enrichment cultures for heliobacteria at 50°C yielded several strains of a thermophilic heliobacterium species from Yellowstone hot spring microbial mats and volcanic soils from Iceland. The novel organisms grew optimally above 50°C, contained bacteriochlorophyll g, and lacked intracytoplasmic membranes. All isolates were strict anaerobes and grew best as photoheterotrophs, although chemotrophic dark growth on pyruvate was also possible. These thermophilic heliobacteria were diazotrophic and fixed N₂ up to their growth temperature limit of 56°C. Phylogenetic studies showed the new isolates to be specific relatives of Heliobacterium gestii and, as has been found in H. gestii, they produce heat-resistant endospores. The unique assemblage of properties found in these thermophilic heliobacteria implicate them as a new species of this group, and we describe them herein as a new species of the genus Heliobacterium, Heliobacterium modesticaldum.     

Bacterial composition of microbial mats in hot springs in Northern Patagonia: variations with seasons and temperature

Seasonal shifts in bacterial diversity of microbial mats were analyzed in three hot springs (39?C68 °C) of Patagonia, using culture-independent methods. Three major bacterial groups were detected in all springs: Phyla Cyanobacteria and Bacteroidetes, and Order Thermales. Proteobacteria, Acidobacteria and Green Non-Sulfur Bacteria were also detected in small amounts and only in some samples. Thermophilic filamentous heterocyst-containing Mastigocladus were dominant Cyanobacteria in Porcelana Hot Spring and Geyser, and Calothrix in Cahuelmó, followed by the filamentous non-heterocyst Leptolyngbya and Oscillatoria. Bacteroidetes were detected in a wide temperature range and their relative abundance increased with decreasing temperature in almost all samples. Two Meiothermus populations with different temperature optima were found. Overall, fingerprinting analysis with universal bacterial primers showed high similarities within each hot spring despite differences in temperature. On the other hand, Cahuelmó Hot Spring showed a lower resemblance among samples. Porcelana Hot Spring and Porcelana Geyser were rather similar to each other, possibly due to a common geological substrate given their geographic proximity. This was even more evident with specific cyanobacterial primers. The different geological substrate and the seawater influence in Cahuelmó might have caused the differences in the microbial community structure with the other two hot springs.     

Thermoproteus uzoniensis sp. nov., a new extremely thermophilic archaebacterium from Kamchatka continental hot springs

A new extremely thermophilic rod-shaped archaebacterium was found in the samples from hot springs and soil of the Uzon caldera (SW of Kamchatka pen.). Cells are rods from 1 to 20 m in length and 0.3 to 0.4 m in width, sometimes branching or with spherical protrusions on the ends. The cell wall consists of two layers: an internal one with distinct hexagonal structure and the outer one with less clear structure and variable thickness. Cells are non-motile and have no flagella. The new organism grows anaerobically by fermenting peptides, concurrently reducing elemental sulfur to H₂S. Fermentation products are acetate, isobutyrate, isovalerate. The G+C content of the DNA is 56.5 mol. %. A new species Thermoproteus uzoniensis is described. Type strain is isolate Z-605, DSM 5262.     

Themoanaerobacterium calidifontis sp. nov., a novel anaerobic, thermophilic, ethanol-producing bacterium from hot springs in China

A novel thermophilic Gram staining positive strain Rx1 was isolated from hot springs in Baoshan of Yunnan Province, China. The strain was characterized as a hemicellulose-decomposing obligate anaerobe bacterium that is rod-shaped (diameter: 0.5–0.7 μm; length: 2.0–6.7 μm), spore-forming, and motile. Its growth temperature range is 38–68 °C (optimum 50–55 °C) and pH range is 4.5–8.0 (optimum 7.0). The maximum tolerance concentration of NaCl was 3 %. Rx1 converted thiosulfate to elemental sulfur and reduced sulfite to hydrogen sulfide. The bacterium grew by utilizing xylan and starch, as well as a wide range of monosaccharide and polysaccharides, including glucose and xylose. The main products of fermentation were ethanol, lactate, acetate, CO₂, and H₂. The maximum xylanase activity in the culture supernatant after 30 h of incubation at 55 °C was 16.2 U/ml. Rx1 DNA G + C content was 36 mol %. 16S rRNA gene sequence analysis indicated that strain Rx1 belonged to the genus Thermoanaerobacterium of the family ‘Thermoanaerobacteriaceae’ (Firmicutes), with Thermoanaerobacterium aciditolerans 761–119 (99.2 % 16S rRNA gene sequence similarity) being its closest relative. DNA–DNA hybridization between Rx1 and T. aciditolerans 761–119 showed 36 % relatedness. Based on its physiological and biochemical tests and DNA–DNA hybridization analyses, the isolate is considered to represent a novel species in the genus Thermoanaerobacterium, for which the name Thermoanaerobacterium calidifontis sp. nov. is proposed, with the type strain is Rx1 (=JCM 18270 = CCTCC M 2011109).     

Phylogenetic diversity of thermophilic carbohydrate degrading bacilli from Bulgarian hot springs

Phylogenetic diversity of culturable bacteria from genus Bacillus and related genera, isolated from 18 Bulgarian hot springs was investigated in association with their functional diversity. Sixty-seven thermophilic and facultative thermophilic strains were isolated under aerobic conditions at 60°C. Sixty-six of them belonged to eight species in four genera from Bacillus group: Anoxybacillus, Geobacillus, Brevibacillus and Bacillus. Representatives of the genus Anoxybacillus predominated. Based on phylogenetic analysis (<97% sequence similarity) four strains belonged to groups representing potentially novel species. Producers of carbohydrases, degrading 12 from the tested 13 substrates were isolated. About half of the isolates degraded amylose by exo- or endo-mechanism of action of their enzymes. The isolates degrading hemicellulose carbohydrates like arabinan, arabinoxylan, β-glucan, galactan, galactomannan and xyloglucan were reached to. Some of the microorganisms were able to uptake microbial polysaccharides like curdlan and gellan and their enzymes were between first reported thermostable enzymes in their groups, like gellan lyase and curdlan lyase A relation between species affiliation and their functional activity was observed—all A. gonensis strains were producer of amylolytic enzymes, most of Brevibacillus ruber strains were able to grow in a minimal medium with xanthan.     

Isolation of plasmids present in thermophilic strains from hot springs in Jordan

The plasmid profile of two thermophilic bacterial strains isolated from recreation thermal springs in Jordan has been investigated. These strains are Streptococcus thermophilus and Bacillus sp1, which have been isolated from Zerka – Maeen and Himma hot springs respectively. Supercoiled and circular plasmid forms were detected, explaining the effect of DNA conformation on the mobility of the plasmid in the agarose gel electrophoresis. Two plasmids have been isolated and characterized by restriction endonucleases to facilitate their use as cloning vectors in thermophilic strains. The sizes of the plasmids were approximately 3 kb (from Streptococcus thermophilus) and 7 kb (from Bacillus spl). These plasmids were then digested with three different restriction enzymes (EcoRI, Bam HI, and HindIII), one of which was found to possess a single site for both plasmids, this enzyme is EcoRI.     

Obligately phototrophic Chloroflexus: primary production in anaerobic hot spring microbial mats

Microbial mats which lack cyanobacteria occur at 50° to 65° C in the sulfide-containing Mammoth Springs of Yellowstone National Park. The principal organisms within these mats are filamentous bacteria which resemble Chloroflexus aurantiacus. The incorporation of [¹⁴C]-HCO ₃ ^- into mat material depended upon both light and sulfide, and was not inhibited when complete natural light was replaced with far-red and infra-red radiation. [¹⁴C]-acetate was incorporated in a light-dependent reaction which was stimulated by, but did not require, sulfide. In situ experiments with microelectrodes demonstrated net sulfide uptake by the mat in the light, and net sulfide production by the mat in the dark, suggesting the operation of a sulfur cycle.Filamentous phototrophic bacteria isolated from the mat were incapable of sustained growth in the presence of O₂.Simultaneous exposure of cultures to light and O₂ caused degradation of bacteriochlorophyll c. The stimulation of light-dependent [¹⁴C]-HCO ₃ ^- -uptake by sulfide was more pronounced in these isolates than in strains of Chloroflexus aurantiacus.     

Tetramitus thermacidophilus n. sp., an amoeboflagellate from acidic hot springs

ABSTRACT. Tetramitus thermacidophilus n. sp. is a novel thermophilic and acidophilic amoeboflagellate isolated from acidic hot springs in the Caldera Uzon (Kamchatka, Russia) and in Pisciarelli Solfatara (Naples, Italy). We describe it based on physiological, morphological, and sequence data. It was grown in monoxenic culture on the archaeon Acidianus brierleyi as food. Tetramitus thermacidophilus multiplies in a pH range from 1.2 to 5 and in a temperature range from 28 °C to 54 °C. The shortest doubling time was 4.5 h at pH 3 at 45 °C. Its spindle-shaped biflagellated stage was only rarely found in culture. The amoeboid stage shows the typical locomotive form of vahlkampfiid amoebae. Sequence comparisons of the internal transcribed spacer sequences and the small subunit rRNA genes confirm that T. thermacidophilus is a novel species within the genus Tetramitus and that both isolates belong to that species.     

Spirosymplokos deltaeiberi nov. gen., nov. sp.: variable-diameter composite spirochete from microbial mats

Large (up to 100 m long), loosely coiled, free-living spirochetes with variable diameters (from 0.4 to 3 m in the same cell) were seen at least 40 times between August 1990 and January 1993. These spirochetes were observed in mud water and enrichment media from highly specific habitats in intertidal evaporite flats at three disjunct localities, one in Spain and two in Mexico. All three are sites of commerical saltworks. Associated with Microcoleus chthonoplastes, the large spirochetes from Spain display phototaxis and a composite organization. Shorter and smaller-diameter spirochetes are seen inside both healthy and spent periplasm of larger ones. Small spirochetes attached to large ones have been observed live. From two to twelve spirochete protoplasmic cylinders were seen inside a single common outer membrane. A distinctive granulated cytoplasm in which the granules are of similar diameter (20–32 nm) to that of the flagella (26 nm) was present. Granule diameters were measured in thin section and in negatively-stained whole-mount preparations. Based on their ultrastructure, large size, variable diameter, number of flagella (3 to 6), and phototactic behavior these unique spirochetes are formally named Spirosymplokos deltaeiberi. Under anoxic (or low oxygen) conditions they formed blooms in mixed culture in media selective for spirochetes. Cellobiose was the major carbon source in 80% seawater, the antibiotic rifampicin was added, mat from the original field site was present and tubes were incubated in the light at from 18–31 °C. Within 1–2 weeks populations of the large spirochete developed at 25 °C but they could not be transferred to fresh medium.     

Anoxybacillus thermarum sp. nov., a novel thermophilic bacterium isolated from thermal mud in Euganean hot springs, Abano Terme, Italy

A novel aerobe thermophilic endospore-forming bacterium designated strain AF/04^T was isolated from thermal mud located in Euganean hot springs, Abano Terme, Padova, Italy. Strain AF/04^T was Gram-positive, motile, rod-shaped, occurring in pairs, or filamentous. The isolate grew between 55 and 67°C (optimum 65°C) and at pH 6.0–7.5 (optimum pH 7.2). The strain was aerobic and grew on maltose, trehalose, and sodium acetate as sole carbon sources. The G + C content of DNA was 53.5 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain AF/04^T falls within the genus Anoxybacillus. Levels of 16S rRNA gene sequence similarity between strain AF/04^T and the type strains of recognized Anoxybacillus species ranged from 95 to 99%. Chemotaxonomic data (major isoprenoid quinone–menaquinone-7; major fatty acid iso-C15:0 and anteiso-C17:0) supported the affiliation of strain AF/04^T to the genus Anoxybacillus. Based on phenotypic and chemotaxonomic characteristics, 16S rRNA gene sequence analysis and DNA–DNA hybridization data, it was proposed that strain AF/04^T (=DSM 17141^T = ATCC BAA 1156^T) should be placed in the genus Anoxybacillus as the type strain of a novel species, Anoxybacillus thermarum sp. nov.     

Distribution of cultivated and uncultivated cyanobacteria and Chloroflexus-like bacteria in hot spring microbial mats.

Oligodeoxynucleotide hybridization probes were developed to complement specific regions of the small subunit (SSU) rRNA sequences of cultivated and uncultivated cyanobacteria and Chloroflexus-like bacteria, which inhabit hot spring microbial mats. The probes were used to investigate the natural distribution of SSU rRNAs from these species in mats of Yellowstone hot springs of different temperatures and pHs as well as changes in SSU rRNA distribution resulting from 1-week in situ shifts in temperature, pH, and light intensity. Synechococcus lividus Y-7c-s SSU rRNA was detected only in the mat of a slightly acid spring, from which it may have been initially isolated, or when samples from a more alkaline spring were incubated in the more acid spring. Chloroflexus aurantiacus Y-400-fl SSU rRNA was detected only in a high-temperature mat sample from the alkaline Octopus Spring or when lower-temperature samples from this mat were incubated at the high-temperature site. SSU rRNAs of uncultivated species were more widely distributed. Temperature distributions and responses to in situ temperature shifts suggested that some of the uncultivated cyanobacteria might be adapted to high-, moderate-, and low-temperature ranges whereas an uncultivated Chloroflexus-like bacterium appears to have broad temperature tolerance. SSU rRNAs of all uncultivated species inhabiting a 48 to 51 degrees C Octopus Spring mat site were most abundant in the upper 1 mm and were not detected below a 2.5-to 3.5-mm depth, a finding consistent with their possible phototrophic nature. However, the effects of light intensity reduction on these SSU rRNAs were variable, indicating the difficulty of demonstrating a phototrophic phenotype in light reduction experiments.     

Phototrophs in high-iron-concentration microbial mats: physiological ecology of phototrophs in an iron-depositing hot spring

At Chocolate Pots Hot Springs in Yellowstone National Park the source waters have a pH near neutral, contain high concentrations of reduced iron, and lack sulfide. An iron formation that is associated with cyanobacterial mats is actively deposited. The uptake of [(14)C]bicarbonate was used to assess the impact of ferrous iron on photosynthesis in this environment. Photoautotrophy in some of the mats was stimulated by ferrous iron (1.0 mM). Microelectrodes were used to determine the impact of photosynthetic activity on the oxygen content and the pH in the mat and sediment microenvironments. Photosynthesis increased the oxygen concentration to 200% of air saturation levels in the top millimeter of the mats. The oxygen concentration decreased with depth and in the dark. Light-dependent increases in pH were observed. The penetration of light in the mats and in the sediments was determined. Visible radiation was rapidly attenuated in the top 2 mm of the iron-rich mats. Near-infrared radiation penetrated deeper. Iron was totally oxidized in the top few millimeters, but reduced iron was detected at greater depths. By increasing the pH and the oxygen concentration in the surface sediments, the cyanobacteria could potentially increase the rate of iron oxidation in situ. This high-iron-content hot spring provides a suitable model for studying the interactions of microbial photosynthesis and iron deposition and the role of photosynthesis in microbial iron cycling. This model may help clarify the potential role of photosynthesis in the deposition of Precambrian banded iron formations.     

Speciation of sulfur from filamentous microbial mats from sulfidic cave springs using X-ray absorption near-edge spectroscopy

Most transformations within the sulfur cycle are controlled by the biosphere, and deciphering the abiotic and biotic nature and turnover of sulfur is critical to understand the geochemical and ecological changes that have occurred throughout the Earth's history. Here, synchrotron radiation-based sulfur K-edge X-ray absorption near-edge structure (XANES) spectroscopy is used to examine sulfur speciation in natural microbial mats from two aphotic (cave) settings. Habitat geochemistry, microbial community compositions, and sulfur isotope systematics were also evaluated. Microorganisms associated with sulfur metabolism dominated the mats, including members of the Epsilonproteobacteria and Gammaproteobacteria. These groups have not been examined previously by sulfur K-edge XANES. All of the mats consisted of elemental sulfur, with greater contributions of cyclo-octasulfur (S8) compared with polymeric sulfur (Smicro). While this could be a biological fingerprint for some bacteria, the signature may also indicate preferential oxidation of Smicro and S8 accumulation. Higher sulfate content correlated to less S8 in the presence of Epsilonproteobacteria. Sulfur isotope compositions confirmed that sulfur content and sulfur speciation may not correlate to microbial metabolic processes in natural samples, thereby complicating the interpretation of modern and ancient sulfur records.