Detection and purification of plasmids present inThermus strains from Icelandic hot springs

Summary Two procedures to detect and purify covalently closed circular plasmid DNA present inThermus strains from Icelandic hot springs are described in detail. Using these procedures, plasmid DNA was detected in IcelandicThermus strains B₁ and B₂, and 0.2–0.4 mg of pure plasmid DNA was isolated from a litre of the cultures (8.5–9.0 g wet weight). Strain B₁ harboured one plasmid species, pCLK 1; strain B₂ harboured two plasmid species, pCLK 2 and pCLK 3.

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Detección y purificación de plásmidos presentes en cepas de Thermus de manantiales calientes de Islandia

Resumen Se describen detalladamente los procedimientos para detectar y purificar DNA-plásmidos covalentemente cerrados que se hallan en cepas deThermus aisladas de manantiales calientes de Islandia. Usando la metodología descrita se detectó DNA plásmico en las cepas islandesas deThermus B₁ y B₂. Se aisló 0.2–0.4 mg de DNA plásmico puro a partir de un litro de cultivo (8.5–9.0 g de peso fresco). La cepa B₁ contenia una especie de plásmido, pCLK 1; mientras que la cepa B₂ contenia dos, pCLK 2 y pCLK 3.

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Détection et purification de plasmides présents dans des souches de Thermus provenant de sources thermales islandaises

Résumé Deux procédés pour la détection et la purification d'ADN plasmidiques circulaires de souches deThermus provenant de sources thermales islandaises sont décrits de façon détaillée. Par ces procédés, de l'ADN plasmidique a été décelé dans les souches islandaises deThermus Bl et B2, et 0.2–0.4 mg d'ADN plasmidique pur a été isolé à partir d'un litre de culture (8.5–9.0 g, poids humide). La souche Bl héberge une suele éspèce de plasmide (pCLK 1); la souche B2 héberge deux éspèces de plasmides (pCLK 2 et pCLK 3).

     

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.     

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.     

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     

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.     

Application of the filter plate microbial trap (FPMT), for cultivating thermophilic bacteria from thermal springs in Barguzin area,eastern Baikal,Russia

Hot springs are regarded as treasury of valuable thermophiles. Like other bacteria, thermophiles are not easily cultivated using conventional culture methods. We used an advanced cultivation method, the filter plate microbial trap (FPMT), to isolate bacteria from thermal springs. In total, 184 isolates were obtained from five thermal springs using the FPMT and standard agar plate method, and their 16S rRNA gene sequences were analyzed. FPMT allowed us to obtain a culture collection that was larger, richer, and more novel than that obtained by standard cultivation. Seven novel species were obtained using the FPMT technique, whereas only one was isolated using a standard cultivation. We also found clear differences in the patterns of phylogenetic diversity and physiological properties between isolates from two cultivation methods. The results have encouraged us to apply the FPMT method in other extreme environments and offer further support for fostering the development of new cultivation methods.     

Screening and identification of extracellular lipase-producing thermophilic bacteria from a Malaysian hot spring

Seven lipase-producing thermophilic bacteria (ST 1, ST 4, ST 6, ST 7, ST 8, ST 9 and ST 10) were isolated from the Setapak hot spring in Malaysia. The crude extracellular lipases recovered by ultrafiltration of cell-free culture supernatant were reacted in an olive oil mixture and their lipolytic activities were compared. Identification of the bacteria was carried out using the Biolog system and biochemical tests. Strain ST 7 that exhibited the highest lipolytic activity of 4.58 U/ml was identified as belonging to the Bacillus genus. Strain ST 6 with an activity of 3.51 U/ml, was identified as Ralstonia paucula. The lipolytic activities of strains ST 1, ST 4, ST 8, ST 9 and ST 10 were 2.39, 1.84, 2.38, 1.80 and 2.62 U/ml respectively. Strains ST 1, ST 4, and ST 10 were identified as Ralstonia paucula while strains ST 8 and ST 9 were Bacillus spp. Strains ST 7 and ST 9 were tentatively identified as Bacillus thermoglucosidasius, Bacillus stearothermophilus or Bacillus coagulans, whereas strain ST 8 was tentatively identified as Bacillus subtilis.     

Xylanases of thermophilic bacteria from Icelandic hot springs

Thermophilic, aerobic bacteria isolated from Icelandic hot springs were screened for xylanase activity. Of 97 strains tested, 14 were found to be xylanase positive. Xylanase activities up to 12 nkat/ml were produced by these strains in shake flasks on xylan medium. The xylanases of the two strains producing the highest activities (ITI 36 and ITI 283) were similar with respect to temperature and pH optima (80°C and pH 8.0). Xylanase production of strain ITI 36 was found to be induced by xylan and xylose. Xylanase activity of 24 nkat/ml was obtained with this strain in a laboratory-scale-fermentor cultivation on xylose medium. -Xylosidase activity was also detected in the culture filtrate. The thermal half-life of ITI 36 xylanase was 24 h at 70°C. The highest production of sugars from hydrolysis of beech xylan was obtained at 70°C, although xylan depolymerization was detected even up to 90°C. Correspondence to: M. Rättö     

Lipase activity of thermophilic bacteria from icelandic hot springs

Summary Several bacteria strains were choosen from pre-selected strains for further testing and characterisation. Hydrolytic activity of lipases from thermophilic bacteria was examined using olive oil as a substrate at different reaction temperatures. Alcoholytic activity was also investigated. Lipases from thermophilic bacteria have been successfully produced on a large scale. To be able to predict if these lipases can be used for transesterification reactions, these preparations need to be purified further or to be cloned.     

Thermopsis thermophila n. gen. n. sp. from hot springs in Nevada, U.S.A. (Crustacea, Ostracoda)

Thermopsis thermophila n. gen. n. sp., a new freshwater ostracod species is described from hot springs in Nevada, U.S.A. The animals were collected within a temperature range of 40–55°C. The new genus belongs to the Ostracoda Podocopida Cypridoidea Cyprididae Cypridopsinae.     

Isolation of cultivable thermophilic lactic acid bacteria from cheeses made with mesophilic starter and molecular comparison with dairy-related Lactobacillus helveticus strains

Aims:  To isolate cultivable thermophilic lactic acid bacteria from cheeses made with mesophilic starter and compare them with dairy-related Lactobacillus helveticus strains using molecular typing methods.
Methods and Results:  The number of thermophilic bacteria in seven commercial cheeses manufactured with mesophilic starters was estimated to be <10 CFU g⁻¹. Implementation of an enumeration step in the isolation method made it possible to isolate one thermophilic strain from each of five of seven cheeses. Comparing repetitive sequence PCR (rep-PCR) profiles of the isolates with dairy-related Lact. helveticus strains indicated that one isolate was a Lact. helveticus . Partial sequencing of 16S rRNA confirmed this, and the remaining four strains were identified as Lactobacillus delbrueckii , Lactobacillus fermentum and Enterococcus faecium . The rep-PCR profile of the isolated Lact. helveticus was identical to the rep-PCR profile of the Lact. helveticus adjunct culture used in the specific cheese, but their pulsed field gel electrophoresis profiles differed slightly.
Conclusion:  It was possible to isolate cultivable thermophilic bacteria from ripened cheeses manufactured with mesophilic starter and thermophilic adjunct cultures by using an enumeration step.
Significance and Impact of the Study:  Isolation of cultivable thermophilic bacteria from ripened cheeses made with mesophilic starters offers an original source for new dairy-relevant cultures.     

Activity of bacteria in water of hot springs from Southern and Central Kamchatskaya geothermal provinces, Kamchatka Peninsula, Russia

The hot-spring waters of numerous hot springs at the Kamchatka Peninsula, Russia differ in their chemical characteristics and elemental composition. Total bacterial abundance (TBA) as well as enzymatically active and respiring bacteria was enumerated in water samples collected from the Nalychevskie, Oksinskie, Apapelskie, and Dachnye hot springs. 5-Carboxyfluorescein diacetate activity was detected in all water samples and comprised 29-65% of the TBA as determined by 4',6-diamidino-2-phenylindol staining. The respiratory activity of iron-oxidizing bacteria was assayed by 5-cyano-2,3-ditolyltetrazolium chloride reduction. Respiring cells accounted for 9-14% of the TBA, indicating a positive correlation with the number of iron-oxidizing bacteria from the hot-spring biomats. Enumeration of heterotrophic bacteria revealed a high-density bacterial population only in the water of the Apapelskie hot spring, which has a temperature of 36 degrees C. Therefore, it appears that heterotrophic and iron-oxidizing bacteria inhabiting the hot-spring waters are essential for the geochemical processes occurring in hydrothermal systems.     

Microbial diversity of soil from two hot springs in Uttaranchal Himalaya

Soil samples collected from two hot springs, Soldhar and Ringigad, both located in the Garhwal region of Uttaranchal Himalaya were analysed for their physical, chemical and microbial components. The alkaline pH, total absence of carbon and nitrogen, and high temperature were features common to soil samples from both sites. The Soldhar samples contained higher amounts of Cu, Fe and Mn. Ringigad soil was devoid of Cu, but had much higher phosphate. While the optimum incubation temperature for isolating the maximum microbial counts from soil samples from the two sites was 50 degrees C, microbial growth in broth was also observed when incubated at 80 degrees C. Microscopic examination revealed three types of microbial populations, i.e., bacteria, yeast and filamentous organisms. The soil samples were found to be dominated by spore forming rods. Out of 58 aerobic isolates, 53 were gram positive bacilli. Gram positive anaerobic oval rods were also observed up to 60 degrees C. Soil dilution plates revealed the presence of antagonistic and phosphate solubilizing populations.     

Methane production from organic acid-rich wastewaters by immobilized thermophilic methane-producing bacteria

Thermophilic methane-producing bacteria isolated from a wastewater treatment facility have been immobilized in acetylcellulose filter with agar. The immobilized cells produced methane from wastewaters in rich organic acid (acetic, propionic and butyric acids) at the rate of 1.4 μmol mg protein⁻¹ h⁻¹. The optimum conditions for methane production by immobilized whole cells were 52–55°C and pH 7.0–8.0. The immobilized cells retained 80% of the initial activity after exposure to air. The immobilized thermophilic bacteria produced methane continuously over 10 days at 52°C.     

Isolation of an extremely thermophilic chemoorganotrophic anaerobe similar to Dictyoglomus thermophilum from new zealand hot springs

A strain similar to Dictyoglomus thermophilum, isolated from a New Zealand hot spring, is described. This strictly anaerobic, Gram-negative, non-motile and nonsporulating bacterium usually exists as long thin filaments of 5 to 25 m by 0.35 to 0.45 m. Rotund bodies are commonly observed. Thin sections of the cells revealed a two-layered cell wall. The optimum temperature and pH for growth was 70°C and 7.0 and 7.5 respectively. No growth was observed at 40°C and 85°C or at pH 4.5 to pH 9.0. The organism fermented glucose, maltose, mannose, xylose, lactose, cellobiose, galactose and sucrose and produced acetate as the major end-product with significant amounts of lactate, H₂ and CO₂ and only traces of ethanol. The doubling time on glucose was 10 h. The DNA base composition was 29.5% guanine plus cytosine as determined by the thermal denaturation method. Growth was inhibited by penicillin, tetracycline and chloramphenicol indicating that the organism was a eubacterium. These features are in common with the newly described species Dictyoglomus thermophilum to which the New Zealand isolate belongs.     

Fatty acids, unusual glycophospholipids and DNA analyses of thermophilic bacteria isolated from hot springs

The composition of fatty acids in 12 strains of the genera Thermus, Meiothermus, Geobacillus and Alicyclobacillus was analyzed by gas chromatography–mass spectrometry. Major FAs found in the profiles included i-15:0, i-17:0, ai-15:0, i-16:0, 16:0, ai-17:0, together with some minor components. Branched FAs were predominant, forming more than 80% of all FAs measured. Fast atom bombardment-mass spectrometry was used for analysis of unusual glycophospholipids, i.e., acylglycosylcardiolipins from genera Geobacillus and Alicyclobacillus and 1-(hydroxy(2-(O-acylglycosyl-oxy)hexadecyloxy)phosphoryloxy) hexadecan-2-yl esters of C15–C17 acids from genera Thermus and Meiothermus. Cloning and preliminary sequence analysis of 16S rDNA showed that these isolates belong to the genera Thermus, Meiothermus, Geobacillus and Alicyclobacillus.     

Isolation and characterization of highly thermophilic xylanolytic Thermus thermophilus strains from hot composts

This is the first detailed report of xylanolytic activity in Thermus strains. Two highly thermophilic xylanolytic bacteria, very closely related to non-xylanolytic T. thermophilus strains, have been isolated from the hottest zones of compost piles. Strain X6 was investigated in more detail. The growth rate (optical density monitoring) on xylan was 0.404.h-1 at 75 degrees C. Maximal growth temperature was 81 degrees C. Xylanase activity was mainly cell-bound, but was solubilized into the medium by sonication. It was induced by xylan or xylose in the culture medium. The temperature and pH optima of the xylanases were determined to be around 100 degrees C and pH 6, respectively. Xylanase activity was fairly thermostable; only 39% of activity was lost after an incubation period of 48 h at 90 degrees C in the absence of substrate. Xylanolytic T. thermophilus strains could contribute to the degradation of hemicellulose during the thermogenic phase of industrial composting.     

The isolation and initial characterization of mercury resistant chemolithotrophic thermophilic bacteria from mercury rich geothermal springs

Mercury rich geothermal springs are likely environments where mercury resistance is critical to microbial life and where microbe-mercury interactions may have evolved. Eleven facultative thermophilic and chemolithoautotrophic, thiosulfate oxidizing bacteria were isolated from thiosulfate enrichments of biofilms from mercury rich hot sulfidic springs in Mount Amiata, Italy. Some strains were highly resistant to mercury (≥200 μM HgCl₂) regardless of its presence or absence during primary enrichments, and three reduced ionic mercury to its elemental form. The gene encoding for the mercuric reductase enzyme (MerA), was amplified by PCR from seven strains. However, one highly resistant strain did not reduce mercury nor carried merA, suggesting an alternative resistance mechanism. All strains were members of the order Bacillales and were most closely related to previously described thermophiles belonging to the Firmicutes. Phylogenetic analyses clustered the MerA of the isolates in two supported novel nodes within the Firmicutes lineage and a comparison with the 16S rRNA gene tree suggested at least one case of horizontal gene transfer. Overall, the results show that the thermophilic thiosulfate oxidizing isolates were adapted to life in presence of mercury mostly, but not exclusively, by possessing MerA. These findings suggest that reduction of mercury by chemolithotrophic thermophilic bacteria may mobilize mercury from sulfur and iron deposits in geothermal environments.     

Temperature-dependent enumeration and characterization of anaerobic, thermophilic xylan-degrading bacteria present in two Icelandic hot springs

Abstract Anaerobic thermophilic xylan-degrading bacteria present in unenriched and enriched 70°C samples from two Icelandic hot springs were enumerated at 68, 78, 90 and 99°C by the use of the Most-Probable-Number method. Xylan was used as substrate. From the samples taken at 70°C and incubated at the temperatures previously described no growth was observed above 78°C. A total of ten strains were isolated and characterized from the positive MPN enrichment cultures from the MPN experiments. A higher number of different strains could be isolated in the enriched samples compared with the unenriched, control samples from the same hot spring. Introduction of xylan, i.e., in situ enrichment, into one of the hot springs changed the bacterial population, as none of the bacteria isolated from the unenriched samples were isolated from the enriched samples. All the isolated bacteria were asporogenous, non-motile and gram-negative rods. One long thin rod had morphological similarities to members of the genus Dictyoglomus and was found in both hot spring samples.     

Lysophosphatidic acid acyltransferase from the thermophilic bacterium Thermus thermophilus HB8 displays substrate promiscuity

ABSTRACT

Lysophosphatidic acid acyltransferase is a phospholipid biosynthetic enzyme that introduces a fatty acyl group into the sn-2 position of phospholipids. Its substrate selectivity is physiologically important in defining the physicochemical properties of lipid membranes and modulating membrane protein function. However, it remains unclear how these enzymes recognize various fatty acids. Successful purification of bacterial lysophosphatidic acid acyltransferases (PlsCs) was recently reported and has paved a path for the detailed analysis of their reaction mechanisms. Here, we purified and characterized PlsC from the thermophilic bacterium Thermus thermophilus HB8. This integral membrane protein remained active even after solubilization and purification and showed reactivity toward saturated, unsaturated, and methyl-branched fatty acids, although branched-chain acyl groups are the major constituent of phospholipids of this bacterium. Multiple sequence alignment revealed the N-terminal end of the enzyme to be shorter than that of PlsCs with defined substrate selectivity, suggesting that the shortened N-terminus confers substrate promiscuity.