Isolation and characterization of a moderately thermophilic nitrite-oxidizing bacterium from a geothermal spring

Geothermal environments are a suitable habitat for nitrifying microorganisms. Conventional and molecular techniques indicated that chemolithoautotrophic nitrite-oxidizing bacteria affiliated with the genus Nitrospira are widespread in environments with elevated temperatures up to 55 °C in Asia, Europe, and Australia. However, until now, no thermophilic pure cultures of Nitrospira were available, and the physiology of these bacteria was mostly uncharacterized. Here, we report on the isolation and characterization of a novel thermophilic Nitrospira strain from a microbial mat of the terrestrial geothermal spring Gorjachinsk (pH 8.6; temperature 48 °C) from the Baikal rift zone (Russia). Based on phenotypic properties, chemotaxonomic data, and 16S rRNA gene phylogeny, the isolate was assigned to the genus Nitrospira as a representative of a novel species, for which the name Nitrospira calida is proposed. A highly similar 16S rRNA gene sequence (99.6% similarity) was detected in a Garga spring enrichment grown at 46 °C, whereas three further thermophilic Nitrospira enrichments from the Garga spring and from a Kamchatka Peninsula (Russia) terrestrial hot spring could be clearly distinguished from N. calida (93.6-96.1% 16S rRNA gene sequence similarity). The findings confirmed that Nitrospira drive nitrite oxidation in moderate thermophilic habitats and also indicated an unexpected diversity of heat-adapted Nitrospira in geothermal hot springs.     

Phylogenetic analysis of several Thermus strains from Rehai of Tengchong, Yunnan, China

Several Thermus strains were isolated from 10 hot springs of the Rehai geothermal area in Tengchong, Yunnan province. The diversity of Thermus strains was examined by sequencing the 16S rRNA genes and comparing their sequences. Phylogenetic analysis showed that the 16S rDNA sequences from the Rehai geothermal isolates form four branches in the phylogenetic tree and had greater than 95.9% similarity in the phylogroup. Secondary structure comparison also indicated that the 16S rRNA from the Rehai geothermal isolates have unique secondary structure characteristics in helix 6, helix 9, and helix 10 (reference to Escherichia coli). This research is the first attempt to reveal the diversity of Thermus strains that are distributed in the Rehai geothermal area.     

Phylogenetic analysis of the bacterial community in a geothermal spring,Rupi Basin,Bulgaria

Hot environments are between the supporting life extreme niches that appear to have maintained some degree of pristine quality and of special biotechnological interest. Knowledge on biodiversity in terrestrial hot springs is still scanty and has not been compared in the light of the specificity of those extreme ecological niches. Study on diversity of thermophilic bacteria inhabiting a hot spring located in Rupi Basin (RB), South-West Bulgaria, revealed a high phylogenetic richness in it (genotypic diversity is 0.37). A total of 120 clones were examined, and grouped in 28 phylogenetic types by their RFLP profile. 16S rRNA gene analysis allowed the identification of nine divisions from the domain Bacteria and one Candidate division. Ten of the retrieved bacterial sequences representing one third of the sequence types showed less than 97% similarity to the closest neighbor and were referred as new sequences. Four of them were distantly related to validly described bacteria (showed ≤90% similarity) suggesting new taxons on at least genus level. Comparison of biodiversity in the spring from Rupi Basin, Bulgaria with that described from other terrestrial hot springs revealed that Proteobacteria, Hydrogenobacter/Aquifex and Thermus are common bacterial groups for terrestrial hot springs. Simultaneously, specific bacterial taxons were observed in different springs.     

Analysis of the diversity of aerobic,thermophilic endospore-forming bacteria in two Algerian hot springs using cultural and non-cultural methods

Terrestrial hot environments are important resources for isolation of thermophilic microorganisms. Few studies have been made on microbial diversity of Algerian geothermal sites. This paper reports the diversity of thermophilic, aerobic endospore-forming bacteria from water and sediment samples taken from Hammam Ouled Ali and Hammam Debagh, two hot springs with a wide range of temperatures and a very rich mineral composition, located in the region of Guelma, north-east of Algeria using culture-dependent and culture-independent approaches Sequences of the V4 region of the 16S rRNA gene from environmental DNA extracted from sediment samples were analyzed and a set of isolates from water and sediment have been characterized by phenotypic and molecular methods. Phylogenetic surveys using environmental DNA sequences indicated that three families dominated the two hot springs: Planococcaceae, Bacillaceae, and Paenibacillaceae. Phenotypic characterization revealed the morphological, biochemical, and physiological properties of these microorganisms, all of which exhibited a range of common extracellular enzymatic activities. Amplified ribosomal DNA restriction analysis (ARDRA) was used to cluster isolates into different phylotypic groups and phylogenetic analysis of 16S rRNA gene sequences of selected isolates showed that all were closely related to four genera of thermophilic Bacilli: Bacillus, Anoxybacillus, Geobacillus, and Brevibacillus. Our results provide important insights into the microbial ecology of Guelma hot springs. They showed that the phylogenetic diversity of bacterial communities within the two studied hot springs was mostly aerobic, with the presence of taxonomic groups of great biotechnological interest. Bioprospection of thermozymes and other biomolecules within these communities will probably provide a data basis for their industrial exploitation.     

Distribution and Diversity of Aerobic Carbon Monoxide-Oxidizing Bacteria in Geothermal Springs of China,the Philippines,and the United States

Accumulating genomic evidence suggests that a variety of thermophilic bacteria contain cox operons and may be capable of aerobic carbon monoxide (CO) oxidation. However, little is known about the distribution and diversity of the cox-encoding (COXE) bacteria in natural geothermal environments. In this study, we examined coxL gene (encoding the large subunit of carbon monoxide dehydrogenase: CoxL) sequences retrieved from the sediments of 25 geothermal sites located in the Qinghai-Tibetan Plateau (QTP) and Yunnan Province (YP) of China, the Bacon-Manito Geothermal Production Field (BGPF) of the Philippines, and the Great Basin of the United States (USGB). Temperature and pH ranges of the studied hot springs were 22.1 to 90.8°C and 2.7 to 9.4, respectively. Phylogenetic analyses showed that most CoxL sequences were closely related to the classes Actinobacteria, Deinococci, Ktedonobacteria, Thermomicrobia, and Clostridia, and hot springs from different regions hosted different COXE communities. In addition, these hot springs harbored some COXE bacteria that were phylogenetically distinct from those inhabiting nongeothermal ecosystems. This study revealed no significant correlation between temperature or pH and the composition or diversity of COXE communities at the global scale. However, within a given region, temperature was correlated with the COXE bacterial community composition.     

分子生物学技术在热泉地质微生物学研究中的应用

陆地热泉是一类典型的极端生命-环境互作的地质系统,是我们认识生命与环境协同演化的天然实验室。然而,受限于有限的研究手段,热泉中仍存在大量未解密的微生物"暗物质"。这种困境随着技术的革新得到了改善,尤其是近几年来基于组学、探针和同位素标记的多元化检测手段在嗜热微生物多样性的挖掘、新物种和新代谢途径的发现以及嗜热微生物对元素地球化学循环的调控和响应等方面取得了一系列令人瞩目的研究成果,使得热泉极端微生物和地质环境内在联系的研究也成为地质微生物学研究的热点。立足于前人研究成果,本文将简述常用于热泉地质微生物学研究的分子生物学手段的发展,重点总结其在挖掘热泉微生物多样性和热泉微生物的环境功能研究中的应用及进展,最后对未来研究方向提出展望。     

Hydrogen production potential of fermentative microorganisms from the Sargasso Sea

Mounting evidence suggests that ammonia-oxidizing archaea (AOA) may play important roles in nitrogen cycling in geothermal environments. In this study, the diversity, distribution and ecological significance of AOA in terrestrial hot springs in Kamchatka (Far East Russia) were explored using amoA genes complemented by analysis of glycerol dialkyl glycerol tetraethers (GDGTs) of archaea. PCR amplification of functional genes (amoA) from AOA and ammonia-oxidizing bacteria (AOB) was performed on microbial mats/streamers and sediments collected from three hot springs (42°C to 87°C and pH 5.5-7.0). No amoA genes of AOB were detected. The amoA genes of AOA formed three distinct phylogenetic clusters with Cluster 3 representing the majority (～59%) of OTUs. Some of the sequences from Cluster 3 were closely related to those from acidic soil environments, which is consistent with the predominance of low pH (<7.0) in these hot springs. Species richness (estimated by Chao1) was more frequently higher at temperatures below 75°C than above it, indicating that AOA may be favored in the moderately high temperature environments. Quantitative PCR of 16S rRNA genes showed that crenarchaeota counted for up to 80% of total archaea. S-LIBSHUFF separated all samples into two phylogenetic groups. The profiles of GDGTs were well separated among the studied springs, suggesting a spatial patterning of archaeal lipid biomarkers. However, this patterning did not correlate significantly with variation in archaeal amoA, suggesting that AOA are not the predominant archaeal group in these springs producing the observed GDGTs.     

Hot crenarchaeal viruses reveal deep evolutionary connections

The discovery of archaeal viruses provides insights into the fundamental biochemistry and evolution of the Archaea. Recent studies have identified a wide diversity of archaeal viruses within the hot springs of Yellowstone National Park and other high-temperature environments worldwide. These viruses are often morphologically unique and code for genes with little similarity to other known genes in the biosphere, a characteristic that has complicated efforts to trace their evolutionary history. Comparative genomics combined with structural analysis indicate that spindle-shaped virus lineages might be unique to the Archaea, whereas other icosahedral viruses might share a common lineage with viruses of Bacteria and Eukarya. These studies provide insights into the evolutionary history of viruses in all three domains of life.     

Distribution and phylogenetic diversity of the subsurface microbial community in a Japanese epithermal gold mine

Distribution and phylogenetic diversity of microbial communities in hot, deep underground environments in the Hishikari epithermal gold mine, southern part of Kyushu, Japan, were evaluated using molecular phylogenetic analyses. Samples included drilled cores such as andesitic volcanic rock (0.95-1.78 Ma) and the oceanic sedimentary basement rock of Shimanto-Supergroup (100 Ma), as well as geothermal hot aquifer waters directly collected from two different sites: AW-site (71.5 degrees C, pH 6.19) and XW-site (85.0 degrees C, pH 6.80) at a depth of 350 mbls (meters below land surface). Based on PCR-amplified 16S rRNA gene clone analysis, the microbial communities in the drilled cores and the hot aquifer water from the XW-site consisted largely of the 16S rRNA gene sequences, closely related to the sequences often found in marine environments, while the aquifer water from the AW-site contained 16S rRNA gene sequences representing members of Aquificales, thermophilic methanotrophs within the gamma-subdivision of the Proteobacteria and uncultivated strains within the beta-subdivision of Proteobacteria. The cultivable microbial community detected by enrichment cultivation analysis largely matched that detected by the culture-independent molecular analysis.     

Diversity of thermophilic fungi in Tengchong Rehai national park revealed by ITS nucleotide sequence analyses

The geothermal sites near neutral and alkalescent thermal springs in Tengchong Rehai National Park were examined through cultivation-dependent approach to determine the diversity of thermophilic fungi in these environments. Here, we collected soils samples in this area, plated on agar media conducive for fungal growth, obtained pure cultures, and then employed the method of internal transcribed spacer (ITS) sequencing combined with morphological analysis for identification of thermophilic fungi to the species level. In total, 102 strains were isolated and identified as Rhizomucor miehei, Chaetomium sp., Talaromyces thermophilus, Talaromyces byssochlamydoides, Thermoascus aurantiacus Miehe var. levisporus, Thermomyces lanuginosus, Scytalidium thermophilum, Malbranchea flava, Myceliophthora sp. 1, Myceliophthora sp. 2, Myceliophthora sp. 3, and Coprinopsis sp. Two species, T. lanuginosus and S. thermophilum were the dominant species, representing 34.78% and 28.26% of the sample, respectively. Our results indicated a greater diversity of thermophilic fungi in neutral and alkaline geothermal sites than acidic sites around hot springs reported in previous studies. Most of our strains thrived at alkaline growth conditions.     

云南干热环境可培养高温放线菌多样性及产纤维素酶活性评价

[目的]探索云南东川干热河谷、元谋土林以及昆明周边高温堆肥、热泉等环境可培养高温放线菌的多样性及其产纤维素酶的潜力.[方法]利用稀释涂布平板法从采集于上述环境的样品中分离得到菌株500余株,通过形态去重复后对300余株进行16SrRNA基因测序分析,并对获得的菌株利用刚果红染色的方法进行纤维素酶活性初步筛选.[结果]分离到的菌株共分布于放线菌纲下9个亚目15个科33个属,其中候选新属2个、候选新种3个.451株菌的纤维素酶筛选结果显示57％具有纤维素酶活性,其中链霉菌、小单孢菌、野野村氏菌在纤维素酶活性菌株中占较大比例.[结论]云南干热环境下蕴含着丰富的放线菌资源,纤维素酶初步筛选显示出了良好的降解活性,为下一步的深入研究提供良好的菌源.     

Four newly isolated fuselloviruses from extreme geothermal environments reveal unusual morphologies and a possible interviral recombination mechanism

Spindle-shaped virus-like particles are abundant in extreme geothermal environments, from which five spindle-shaped viral species have been isolated to date. They infect members of the hyperthermophilic archaeal genus Sulfolobus , and constitute the Fuselloviridae , a family of double-stranded DNA viruses. Here we present four new members of this family, all from terrestrial acidic hot springs. Two of the new viruses exhibit a novel morphotype for their proposed attachment structures, and specific features of their genome sequences strongly suggest the identity of the host-attachment protein. All fuselloviral genomes are highly conserved at the nucleotide level, although the regions of conservation differ between virus-pairs, consistent with a high frequency of homologous recombination having occurred between them. We propose a fuselloviral specific mechanism for interviral recombination, and show that the spacers of the Sulfolobus CRISPR antiviral system are not biased to the highly similar regions of the fusellovirus genomes.     

Identification of novel positive-strand RNA viruses by metagenomic analysis of archaea-dominated Yellowstone hot springs

There are no known RNA viruses that infect Archaea. Filling this gap in our knowledge of viruses will enhance our understanding of the relationships between RNA viruses from the three domains of cellular life and, in particular, could shed light on the origin of the enormous diversity of RNA viruses infecting eukaryotes. We describe here the identification of novel RNA viral genome segments from high-temperature acidic hot springs in Yellowstone National Park in the United States. These hot springs harbor low-complexity cellular communities dominated by several species of hyperthermophilic Archaea. A viral metagenomics approach was taken to assemble segments of these RNA virus genomes from viral populations isolated directly from hot spring samples. Analysis of these RNA metagenomes demonstrated unique gene content that is not generally related to known RNA viruses of Bacteria and Eukarya. However, genes for RNA-dependent RNA polymerase (RdRp), a hallmark of positive-strand RNA viruses, were identified in two contigs. One of these contigs is approximately 5,600 nucleotides in length and encodes a polyprotein that also contains a region homologous to the capsid protein of nodaviruses, tetraviruses, and birnaviruses. Phylogenetic analyses of the RdRps encoded in these contigs indicate that the putative archaeal viruses form a unique group that is distinct from the RdRps of RNA viruses of Eukarya and Bacteria. Collectively, our findings suggest the existence of novel positive-strand RNA viruses that probably replicate in hyperthermophilic archaeal hosts and are highly divergent from RNA viruses that infect eukaryotes and even more distant from known bacterial RNA viruses. These positive-strand RNA viruses might be direct ancestors of RNA viruses of eukaryotes.     

Bacterial Diversity and Functional Activity of Microbial Communities in Hot Springs of the Baikal Rift Zone

In this study the bacterial diversity of thermophilic microbial mats (40 to 65°C) in three alkaline hot springs of the Baikal Rift Zone (BRZ) was determined through pyrosequencing of 16S rRNA gene libraries. Significant diversity of bacterial species was found in the biomats of the hot springs with total number of detected phylotypes of 607. The highest share of the microbial community was represented by the phyla Chloroflexi (Seya Spring, 76.4%), Deinococcus-Thermus (Alla Spring, 45.1%), Nitrospira (Alla Spring, 36.1%), Cyanobacteria (Tsenkher Spring, 33.1%), and Proteobacteria (Tsenkher Spring, 22.6%), but their ratio varied significantly in different springs. A comparison of the biodiversity and composition of microbial communities between hot springs showed a decrease in biodiversity with increasing temperature. A large number of sequences showed a low degree of similarity with cultivated representatives in public databases. Microbial communities showed intensive rates of production and destruction of organic compounds, as revealed by the quantitative assessment of their functional activity.     

Metagenomic Investigation of Bacterial and Archaeal Diversity of Hammam Essalihine Hot Spring from Khenchela,Algeria

Abstract

Hot springs are natural environments where hot groundwater comes out from the earth. Exploring the microbial diversity present in hot springs is important first to determine the microorganisms able to proliferate there and to understand their role in biogeochemical cycles. In Algeria, research concerning microbial populations in those ecosystems is limited. This study describes bacterial and archaeal diversity of the ‘Hammam Essalihine’ hot spring in Khenchela province in north-east Algeria using a culture-independent approach. This is the first microbial diversity investigation in the ‘Hammam Essalihine’ hot spring using next-generation sequencing techniques to assess the species classification of thermophilic microorganisms. Genomic DNA was extracted from water samples and the V4–V5 region of 16S rRNA gene were amplified, sequenced, and analyzed. The average temperature of water varies from 68 to 70?°C. High-throughput sequencing analysis revealed the presence of 21 bacterial phyla, including an unknown phylum and distributed across 42 families and 39 genera. The majority of the sequences were observed to belong to the kingdom Bacteria. The bacterial community from this hot spring is dominated by Proteobacteria (41.52%), Chloroflexi (7.62%), and Bacteroidetes (7.62%), whereas the community of Archaea is scarcely present in the study site and the two identified operational taxonomic units (OTUs) are far from what is known in the GenBank database. The study shows several uncharacterized sequences, indicating that the water of ‘Hammam Essalihine’ hot spring contains undescribed microorganisms. This study is thought to add to the understanding of thermophile diversity and ecology of ‘Hammam Essalihine’ hot spring.     

腾冲两热泉泉古菌多样性及系统发育的初步分析

通过构建16S rRNA基因片段的克隆文库对腾冲热海两温泉中泉古菌的多样性和系统发育关系进行了初步的研究.一共得到18个泉古茵克隆序列,可分为12个OTUs,两温泉的克隆序列与已知GenBank上关系最近序列的平均相似性较低,无名泉为92.56%,热爆区为93%.从基于16S rRNA基因片段序列构建的系统发育树来看,74℃的无名泉样点中既有属于超高温环境类群的泉古菌,同时又有属于和常温环境较接近的泉古菌;45℃的热爆区样点的泉古茵,相对来说则更接近于常温类群.本次研究表明,腾冲热泉与世界其它同类热泉之间的泉古茵类群存在着一定的差异;而且两实验样点代表了超高温和高温环境泉古菌逐渐向常温过度的两个重要环境.     

Linkage of high rates of sulfate reduction in Yellowstone hot springs to unique sequence types in the dissimilatory sulfate respiration pathway

Diversity, habitat range, and activities of sulfate-reducing prokaryotes within hot springs in Yellowstone National Park were characterized using endogenous activity measurements, molecular characterization, and enrichment. Five major phylogenetic groups were identified using PCR amplification of the dissimilatory sulfite reductase genes (dsrAB) from springs demonstrating significant sulfate reduction rates, including a warm, acidic (pH 2.5) stream and several nearly neutral hot springs with temperatures reaching 89 degrees C. Three of these sequence groups were unrelated to named lineages, suggesting that the diversity and habitat range of sulfate-reducing prokaryotes exceeds that now represented in culture.     

Life in Hot Spring Microbial Mats Located in the Trans-Mexican Volcanic Belt: A 16S/18S rRNA Gene and Metagenomic Analysis

The geothermal system of the Araró region, located in the Trans-Mexican Volcanic Belt of México, hosts various hot springs with unique physicochemical characteristics, including temperatures ranging from 45°C to 78°C. The microbial diversity in these hot springs has been explored only by culture-dependent surveys. In this study, we performed metagenomic Illumina MiSeq, and 16S and 18S rRNA pyrosequencing analysis of the microbial life are residing in the microbial mats of the springs called “Tina–Bonita”. Our results show the presence of 186 operational taxonomic units, 99.7% of which belong to bacteria, 0.27% to eukaryotes, and 0.03% to archaea. The most abundant bacterial divisions are the Proteobacteria, Chloroflexi, and Cyanobacteria, which include 105 genera. The ecological indexes indicate that the microbial mats have moderate microbial diversity. An abundant group of genes that participate in photosynthesis, including photosynthetic electron transport, as well as photosystems I and II, were detected. Another cluster of genes was found that participates in sulfur, nitrogen, and methane metabolism. Finally, this phylogenetic and metagenomic analysis revealed an unexpected taxonomic and genetic diversity, expanding our knowledge of microbial life under specific extreme conditions.     

Metagenomic analyses of novel viruses and plasmids from a cultured environmental sample of hyperthermophilic neutrophiles

Two novel viral genomes and four plasmids were assembled from an environmental sample collected from a hot spring at Yellowstone National Park, USA, and maintained anaerobically in a bioreactor at 85°C and pH 6. The double‐stranded DNA viral genomes are linear (22.7 kb) and circular (17.7 kb), and derive apparently from archaeal viruses HAV1 and HAV2. Genomic DNA was obtained from samples enriched in filamentous and tadpole‐shaped virus‐like particles respectively. They yielded few significant matches in public sequence databases reinforcing, further, the wide diversity of archaeal viruses. Several variants of HAV1 exhibit major genomic alterations, presumed to arise from viral adaptation to different hosts. They include insertions up to 350 bp, deletions up to 1.5 kb, and genes with extensively altered sequences. Some result from recombination events occurring at low complexity direct repeats distributed along the genome. In addition, a 33.8 kb archaeal plasmid pHA1 was characterized, encoding a possible conjugative apparatus, as well as three cryptic plasmids of thermophilic bacterial origin, pHB1 of 2.1 kb and two closely related variants pHB2a and pHB2b, of 5.2 and 4.8 kb respectively. Strategies are considered for assembling genomes of smaller genetic elements from complex environmental samples, and for establishing possible host identities on the basis of sequence similarity to host CRISPR immune systems.