Metagenomic Evaluation of Bacterial and Archaeal Diversity in the Geothermal Hot Springs of Manikaran, India

Bhatia, Sonu; Pathak, Ashish; Green, Stefan J.; Joshi, Amit; Chauhan, Ashvini

doi:10.1128/genomea.01544-14

Cited by 26 publications

(11 citation statements)

References 11 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Thus, our protocol was optimized to capture the highest diversity of diazotrophs while minimizing the costs of next-generation sequencing through multiplexing. Two different barcoding approaches were investigated for sequencing on an Illumina MiSeq platform, one whereby the 5= ends of the nifH primers are barcoded, and another whereby common sequences are synthesized onto the 5= ends of the nifH primers, and then the common sequence is used to attach the amplicon to a set of barcodes and adapter sequences developed by Fluidigm (31). The 5= approach was used to identify the best-performing primer sets.…”

Section: Discussionmentioning

confidence: 99%

Diazotroph Community Characterization via a High-Throughput nifH Amplicon Sequencing and Analysis Pipeline

Gaby

Rishishwar

Valderrama-Aguirre

et al. 2018

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

The dinitrogenase reductase gene () is the most widely established molecular marker for the study of nitrogen-fixing prokaryotes in nature. A large number of PCR primer sets have been developed for amplification, and the effective deployment of these approaches should be guided by a rapid, easy-to-use analysis protocol. Bioinformatic analysis of marker gene sequences also requires considerable expertise. In this study, we advance the state of the art for analysis by evaluating primer set performance, developing an improved amplicon sequencing workflow, and implementing a user-friendly bioinformatics pipeline. The developed amplicon sequencing workflow is a three-stage PCR-based approach that uses established technologies for incorporating sample-specific barcode sequences and sequencing adapters. Based on our primer evaluation, we recommend the Ando primer set be used with a modified annealing temperature of 58°C, as this approach captured the largest diversity of templates, including paralog cluster IV/V sequences. To improve sequence analysis, we developed a computational pipeline which infers taxonomy and optionally filters out paralog sequences. In addition, we employed an empirical model to derive optimal operational taxonomic unit (OTU) cutoffs for the gene at the species, genus, and family levels. A comprehensive workflow script named TaxADivA (TAXonomy Assignment and DIVersity Assessment) is provided to ease processing and analysis of amplicons. Our approach is then validated through characterization of diazotroph communities across environmental gradients in beach sands impacted by the Deepwater Horizon oil spill in the Gulf of Mexico, in a peat moss-dominated wetland, and in various plant compartments of a sugarcane field. Nitrogen availability often limits ecosystem productivity, and nitrogen fixation, exclusive to prokaryotes, comprises a major source of nitrogen input that sustains food webs. The gene, which codes for the iron protein of the nitrogenase enzyme, is the most widely established molecular marker for the study of nitrogen-fixing microorganisms (diazotrophs) in nature. In this study, a flexible sequencing/analysis pipeline, named TaxADivA, was developed for amplicons produced by Illumina paired-end sequencing, and it enables an inference of taxonomy, performs clustering, and produces output in formats that may be used by programs that facilitate data exploration and analysis. Diazotroph diversity and community composition are linked to ecosystem functioning, and our results advance the phylogenetic characterization of diazotroph communities by providing empirically derived similarity cutoffs for species, genus, and family levels. The utility of our pipeline is validated for diazotroph communities in a variety of ecosystems, including contaminated beach sands, peatland ecosystems, living plant tissues, and rhizosphere soil.

show abstract

Section: Discussionmentioning

confidence: 99%

Diazotroph Community Characterization via a High-Throughput nifH Amplicon Sequencing and Analysis Pipeline

Gaby

Rishishwar

Valderrama-Aguirre

et al. 2018

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

show abstract

“…Simultaneously, the microbial community and geochemical parameters from Manikaran hot spring, India (north western Himalayas) (Bhatia et al 2015; Chandrasekharam et al 2005) and Sungai Klah (SK) alkaline hot spring, Malaysia (Chan et al 2015) were taken into account to overcome the problem of small sample size in the present study for two component analysis. PCA method showed that the community composition was significantly (p < 0.05) linked to temperature, dissolved SiO 2 , elemental S, total sulphide, calcium etc.…”

Section: Resultsmentioning

confidence: 99%

Bacterial and archeal community composition in hot springs from Indo-Burma region, North-east India

et al. 2016

View full text Add to dashboard Cite

Bacterial and archaeal diversity of two alkaline Indian hot springs, Jakrem (Meghalaya) and Yumthang (Sikkim), were studied. Thirteen major bacterial phyla were identified of which Firmicutes, Chloroflexi and Thermi were dominant in Jakrem and Proteobacteria in Yumthang. The dominant genera were Clostridium, Chloroflexus and Meiothermus at Jakrem (water temperature 46 °C, pH 9) and Thiobacillus, Sulfuritalea at Yumthang (water temperature 39 °C, pH 8) hot springs. The four Euryarchaeota taxa that were observed in both the hot springs were Methanoculleus, Methanosaeta, Methanosarcina and Methanocorposculum. Elstera litoralis, Thiovirga sp., Turneriella sp. were observed for the first time in association with hot springs along with Tepidibacter sp., Ignavibacterium sp., Teribacillus sp. and Dechloromonas sp. Individual bacterial phyla were found to be specifically correlated with certain physico-chemical factors such as temperature, dissolved SiO2, elemental S, total sulphide, calcium concentrations in hot spring water. Bacterial reads involved in sulfur cycle were identified in both16S rRNA gene library and sulfur metabolism may play key physiological functions in this hot spring. Members within Desulfobacterales and Thermodesulfovibrionaceae were identified and hypothesized their role in regulating sulfur cycle. The presence of many taxonomically unsolved sequences in the 16S rRNA gene tag datasets from these hot springs could be a sign of novel microbe richness in these less known hot water bodies of Northeastern India.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-016-0284-y) contains supplementary material, which is available to authorized users.

show abstract

“…The high abundance of P. aerophilum in this environment is an interesting finding as it is a unique hyperthermophilic archaeal species reported extensively for its nitrate reducing properties with the help of a molybdenum-associated nitrate reductase which is distinct from other mesophilic nitrate reductases (Afshar et al, 2001). P. aerophilum also found to be abundant in Manikaran hot spring in India (96°C), is also known to grow anaerobically by dissimilatory nitrate reduction, which is an exclusive catabolic feature of this species (Afshar et al, 2001; Bhatia et al, 2015). Fervidobacterium thermophilum and Fervidobacterium pennivorans are among the other thermophilic eubacteria observed to be abundant in Tattapani, and are reported to produce thermostable cellulases and keratinases (fervidolysin) respectively, which have potential application in the conversion of biomass to biofuels at high temperature and other biotechnological processes (Kim et al, 2004; Wang et al, 2010).…”

Section: Discussionmentioning

confidence: 99%

“…Many studies have also revealed the presence of genes encoding for enzymes of biotechnological interest, such as hydrolases, xylanases, proteases, galactosidases, and lipases (Ferrandi et al, 2015; Littlechild, 2015). Some studies focusing on the metagenomic analysis of hot springs located in India have reported Bacillus licheniformis (Mangrola et al, 2015), Bacillus megaterium, Bacillus sporothermodurans, Hydrogenobacter sp., Thermus thermophilus, Thermus brockianus (Bhatia et al, 2015), Clostridium bifermentans, Clostridium lituseburense (Ghelani et al, 2015), Opitutus terrae, Rhodococcus erythropolis , and Cellovibrio mixtus (Mehetre et al, 2016) as major bacterial genera. Genes for stress responses and metabolism of aromatic and other organic compounds have been identified by preliminary functional analysis of these sites (Mangrola et al, 2015; Mehetre et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

Metagenomic Analysis of Hot Springs in Central India Reveals Hydrocarbon Degrading Thermophiles and Pathways Essential for Survival in Extreme Environments

et al. 2017

View full text Add to dashboard Cite

Extreme ecosystems such as hot springs are of great interest as a source of novel extremophilic species, enzymes, metabolic functions for survival and biotechnological products. India harbors hundreds of hot springs, the majority of which are not yet explored and require comprehensive studies to unravel their unknown and untapped phylogenetic and functional diversity. The aim of this study was to perform a large-scale metagenomic analysis of three major hot springs located in central India namely, Badi Anhoni, Chhoti Anhoni, and Tattapani at two geographically distinct regions (Anhoni and Tattapani), to uncover the resident microbial community and their metabolic traits. Samples were collected from seven distinct sites of the three hot spring locations with temperature ranging from 43.5 to 98°C. The 16S rRNA gene amplicon sequencing of V3 hypervariable region and shotgun metagenome sequencing uncovered a unique taxonomic and metabolic diversity of the resident thermophilic microbial community in these hot springs. Genes associated with hydrocarbon degradation pathways, such as benzoate, xylene, toluene, and benzene were observed to be abundant in the Anhoni hot springs (43.5–55°C), dominated by Pseudomonas stutzeri and Acidovorax sp., suggesting the presence of chemoorganotrophic thermophilic community with the ability to utilize complex hydrocarbons as a source of energy. A high abundance of genes belonging to methane metabolism pathway was observed at Chhoti Anhoni hot spring, where methane is reported to constitute >80% of all the emitted gases, which was marked by the high abundance of Methylococcus capsulatus. The Tattapani hot spring, with a high-temperature range (61.5–98°C), displayed a lower microbial diversity and was primarily dominated by a nitrate-reducing archaeal species Pyrobaculum aerophilum. A higher abundance of cell metabolism pathways essential for the microbial survival in extreme conditions was observed at Tattapani. Taken together, the results of this study reveal a novel consortium of microbes, genes, and pathways associated with the hot spring environment.

show abstract

Metagenomic Evaluation of Bacterial and Archaeal Diversity in the Geothermal Hot Springs of Manikaran, India

Cited by 26 publications

References 11 publications

Diazotroph Community Characterization via a High-Throughput nifH Amplicon Sequencing and Analysis Pipeline

Diazotroph Community Characterization via a High-Throughput nifH Amplicon Sequencing and Analysis Pipeline

Bacterial and archeal community composition in hot springs from Indo-Burma region, North-east India

Metagenomic Analysis of Hot Springs in Central India Reveals Hydrocarbon Degrading Thermophiles and Pathways Essential for Survival in Extreme Environments

Contact Info

Product

Resources

About