Project description:Jakrem hot water spring is located in the West Khasi Hill District of the state of Meghalaya, and is one of the most popular hot springs of the state. There is a populist belief among the inhabitants and people that the hot spring water has got curative properties against various skin ailments. This is the first report on V3 hyper-variable region of 16S rDNA metagenome sequence employing Illumina platform to profile the microbial community of this less known hot spring from Meghalaya, India. Metagenome comprised of 10, 74,120 raw sequences with a sequence length of 151 bp and 56.35% G + C content. Metagenome sequence information is now available at NCBI, SRA database accession no. SRP056897. A total of 8, 77, 364 pre-processed reads were clustered into 694 OTUs (operational taxonomical units) comprising of 14 bacterial phyla including unknown phylum demonstrating 49 families. Hot spring bacterial community is dominated by Firmicutes (61.60%), Chloroflexi (21.37%), Cyanobacteria (12.96%) and unclassified bacteria (1.2%) respectively.
Project description:Volcanic regions contain a variety of environments suitable for extremophiles. This study was focused on assessing and exploiting the prokaryotic diversity of two microbial communities derived from different Kamchatkian thermal springs by metagenomic approaches. Samples were taken from a thermoacidophilic spring near the Mutnovsky Volcano and from a thermophilic spring in the Uzon Caldera. Environmental DNA for metagenomic analysis was isolated from collected sediment samples by direct cell lysis. The prokaryotic community composition was examined by analysis of archaeal and bacterial 16S rRNA genes. A total number of 1235 16S rRNA gene sequences were obtained and used for taxonomic classification. Most abundant in the samples were members of Thaumarchaeota, Thermotogae, and Proteobacteria. The Mutnovsky hot spring was dominated by the Terrestrial Hot Spring Group, Kosmotoga, and Acidithiobacillus. The Uzon Caldera was dominated by uncultured members of the Miscellaneous Crenarchaeotic Group and Enterobacteriaceae. The remaining 16S rRNA gene sequences belonged to the Aquificae, Dictyoglomi, Euryarchaeota, Korarchaeota, Thermodesulfobacteria, Firmicutes, and some potential new phyla. In addition, the recovered DNA was used for generation of metagenomic libraries, which were subsequently mined for genes encoding lipolytic and proteolytic enzymes. Three novel genes conferring lipolytic and one gene conferring proteolytic activity were identified.
Project description:The spatial distribution of biodiversity (i.e., the biogeography) of the hot-spring microbiome is critical for understanding the microbial ecosystems in hot springs. We investigated the microbiome diversity scaling (changes) over space by analyzing the diversity-area relationship (DAR), which is an extension to classic SAR (species-area relationship) law in biogeography. We built DAR models for archaea and bacteria with 16S-rRNA sequencing datasets from 165 hot springs globally. From the DAR models, we sketch out the biogeographic maps of hot-spring microbiomes by constructing: (i) DAR profile-measuring the archaea or bacteria diversity scaling over space (areas); (ii) PDO (pair-wise diversity overlap or similarity) profile-estimating the PDO between two hot springs; (iii) MAD (maximal accrual diversity) profile-predicting the global MAD; (iv) LRD/LGD (ratio of local diversity to regional or global diversity) profile. We further investigated the differences between archaea and bacteria in their biogeographic maps. For example, the comparison of DAR-profile maps revealed that the archaea diversity is more heterogeneous (i.e., more diverse) or scaling faster than the bacterial diversity does in terms of species numbers (species richness), but is less heterogeneous (i.e., less diverse) or scaling slower than bacteria when the diversity (Hill numbers) were weighted in favor of more abundant dominant species. When the diversity is weighted equally in terms of species abundances, archaea, and bacteria are equally heterogeneous over space or scaling at the same rate. Finally, unified DAR models (maps) were built with the combined datasets of archaea and bacteria.
Project description:The Sungai Klah (SK) hot spring is the second hottest geothermal spring in Malaysia. This hot spring is a shallow, 150-m-long, fast-flowing stream, with temperatures varying from 50 to 110°C and a pH range of 7.0-9.0. Hidden within a wooded area, the SK hot spring is continually fed by plant litter, resulting in a relatively high degree of total organic content (TOC). In this study, a sample taken from the middle of the stream was analyzed at the 16S rRNA V3-V4 region by amplicon metagenome sequencing. Over 35 phyla were detected by analyzing the 16S rRNA data. Firmicutes and Proteobacteria represented approximately 57% of the microbiome. Approximately 70% of the detected thermophiles were strict anaerobes; however, Hydrogenobacter spp., obligate chemolithotrophic thermophiles, represented one of the major taxa. Several thermophilic photosynthetic microorganisms and acidothermophiles were also detected. Most of the phyla identified by 16S rRNA were also found using the shotgun metagenome approaches. The carbon, sulfur, and nitrogen metabolism within the SK hot spring community were evaluated by shotgun metagenome sequencing, and the data revealed diversity in terms of metabolic activity and dynamics. This hot spring has a rich diversified phylogenetic community partly due to its natural environment (plant litter, high TOC, and a shallow stream) and geochemical parameters (broad temperature and pH range). It is speculated that symbiotic relationships occur between the members of the community.
Project description:Deep-subsurface samples obtained by deep drilling are likely to be contaminated with mesophilic microorganisms in the drilling fluid, and this could affect determination of the community structure of the geothermal microflora using 16S rRNA gene clone library analysis. To eliminate possible contamination by PCR-amplified 16S rRNA genes from mesophiles, a combined thermal denaturation and enzyme digestion method, based on a strong correlation between the G+C content of the 16S rRNA gene and the optimum growth temperatures of most known prokaryotic cultures, was used prior to clone library construction. To validate this technique, hot spring fluid (76 degrees C) and river water (14 degrees C) were used to mimic a deep-subsurface sample contaminated with drilling fluid. After DNA extraction and PCR amplification of the 16S rRNA genes from individual samples separately, the amplified products from river water were observed to be denatured at 82 degrees C and completely digested by exonuclease I (Exo I), while the amplified products from hot spring fluid remained intact after denaturation at 84 degrees C and enzyme digestion with Exo I. DNAs extracted from the two samples were mixed and used as a template for amplification of the 16S rRNA genes. The amplified rRNA genes were denatured at 84 degrees C and digested with Exo I before clone library construction. The results indicated that the 16S rRNA gene sequences from the river water were almost completely eliminated, whereas those from the hot spring fluid remained.
Project description:The springs at Gypsum Hill and Colour Peak on Axel Heiberg Island in the Canadian Arctic originate from deep salt aquifers and are among the few known examples of cold springs in thick permafrost on Earth. The springs discharge cold anoxic brines (7.5 to 15.8% salts), with a mean oxidoreduction potential of -325 mV, and contain high concentrations of sulfate and sulfide. We surveyed the microbial diversity in the sediments of seven springs by denaturing gradient gel electrophoresis (DGGE) and analyzing clone libraries of 16S rRNA genes amplified with Bacteria and Archaea-specific primers. Dendrogram analysis of the DGGE banding patterns divided the springs into two clusters based on their geographic origin. Bacterial 16S rRNA clone sequences from the Gypsum Hill library (spring GH-4) were classified into seven phyla (Actinobacteria, Bacteroidetes, Firmicutes, Gemmatimonadetes, Proteobacteria, Spirochaetes, and Verrucomicrobia); Deltaproteobacteria and Gammaproteobacteria sequences represented half of the clone library. Sequences related to Proteobacteria (82%), Firmicutes (9%), and Bacteroidetes (6%) constituted 97% of the bacterial clone library from Colour Peak (spring CP-1). Most GH-4 archaeal clone sequences (79%) were related to the Crenarchaeota while half of the CP-1 sequences were related to orders Halobacteriales and Methanosarcinales of the Euryarchaeota. Sequences related to the sulfur-oxidizing bacterium Thiomicrospira psychrophila dominated both the GH-4 (19%) and CP-1 (45%) bacterial libraries, and 56 to 76% of the bacterial sequences were from potential sulfur-metabolizing bacteria. These results suggest that the utilization and cycling of sulfur compounds may play a major role in the energy production and maintenance of microbial communities in these unique, cold environments.
Project description:We have developed a ten-week curriculum for molecular biology that uses 16S ribosomal RNA genes to characterize and compare novel bacteria from hot spring communities in Yellowstone National Park. The 16S rRNA approach bypasses selective culture-based methods. Our molecular biology course offered the opportunity for students to learn broadly applicable methods while contributing to a long-term research project. Specifically, students isolated and characterized clones that contained novel 16S rRNA inserts using restriction enzyme, DNA sequencing, and computer-based phylogenetic methods. In both classes, students retrieved novel bacterial 16S rRNA genes, several of which were most similar to Green Nonsulfur bacterial isolates. During class, we evaluated student performance and mastery of skills and concepts using quizzes, formal lab notebooks, and a broad project assignment. For this report, we also assessed student performance alongside data quality and discussed the significance, our goal being to improve both research and teaching methods.
Project description:Denaturing gradient gel electrophoresis analysis of PCR-amplified 16S rRNA gene segments was used to examine the distributions of bacterial populations within a hot spring microbial mat (Octopus Spring, Yellowstone National Park). Populations at sites along the thermal gradient of the spring's effluent channel were surveyed at seasonal intervals. No shift in the thermal gradient was detected, and populations at spatially or temperature-defined sites exhibited only slight changes over the annual sampling period. A new cyanobacterial 16S rRNA sequence type was detected at temperatures from 63 to 75 degrees C. A new green nonsulfur bacterium-like sequence type was also detected at temperatures from 53 to 62 degrees C. Genetically unique though closely related cyanobacterial and green nonsulfur bacterium-like populations were successively distributed along the thermal gradient of the Octopus Spring effluent channel. At least two cyanobacterial populations were detected at each site; however, a limited ability to detect some cyanobacterial populations suggests that only dominant populations were observed.
Project description:The ability of thermophilic microorganisms and their enzymes to decompose biomass have attracted attention due to their quick reaction time, thermostability, and decreased risk of contamination. Exploitation of efficient thermostable glycoside hydrolases (GHs) could accelerate the industrialization of biofuels and biochemicals. However, the full spectrum of thermophiles and their enzymes that are important for biomass degradation at high temperatures have not yet been thoroughly studied. We examined a Malaysian Y-shaped Sungai Klah hot spring located within a wooded area. The fallen foliage that formed a thick layer of biomass bed under the heated water of the Y-shaped Sungai Klah hot spring was an ideal environment for the discovery and analysis of microbial biomass decay communities. We sequenced the hypervariable regions of bacterial and archaeal 16S rRNA genes using total community DNA extracted from the hot spring. Data suggested that 25 phyla, 58 classes, 110 orders, 171 families, and 328 genera inhabited this hot spring. Among the detected genera, members of Acidimicrobium, Aeropyrum, Caldilinea, Caldisphaera, Chloracidobacterium, Chloroflexus, Desulfurobacterium, Fervidobacterium, Geobacillus, Meiothermus, Melioribacter, Methanothermococcus, Methanotorris, Roseiflexus, Thermoanaerobacter, Thermoanaerobacterium, Thermoanaerobaculum, and Thermosipho were the main thermophiles containing various GHs that play an important role in cellulose and hemicellulose breakdown. Collectively, the results suggest that the microbial community in this hot spring represents a good source for isolating efficient biomass degrading thermophiles and thermozymes.
Project description:The phylogenetic group termed OP5 was originally discovered in the Yellowstone National Park hot spring and proposed as an uncultured phylum; the group was afterwards analyzed by applying culture-independent approaches. Recently, a novel thermophilic chemoheterotrophic filamentous bacterium was obtained from a hot spring in Japan that was enriched through various isolation procedures. Phylogenetic analyses of the isolate have revealed that it is closely related to the OP5 phylum that has mainly been constructed with the environmental clones retrieved from thermophilic and mesophilic anaerobic environments. It appears that the lineage is independent at the phylum level in the domain Bacteria. Therefore, we designed a primer set for the 16S rRNA gene to specifically target the OP5 phylum and performed quantitative field analysis by using the real-time PCR method. Thus, the 16S rRNA gene of the OP5 phylum was detected in some hot-spring samples with the relative abundance ranging from 0.2% to 1.4% of the prokaryotic organisms detected. The physiology of the above-mentioned isolate and the related environmental clones indicated that they are scavengers contributing to the sulfur cycle in nature.