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Molecular characterization of the diversity and distribution of a thermal spring microbial community by using rRNA and metabolic genes.

ABSTRACT: The diversity and distribution of a bacterial community from Coffee Pots Hot Spring, a thermal spring in Yellowstone National Park with a temperature range of 39.3 to 74.1 degrees C and pH range of 5.75 to 6.91, were investigated by sequencing cloned PCR products and quantitative PCR (qPCR) of 16S rRNA and metabolic genes. The spring was inhabited by three Aquificae genera--Thermocrinis, Hydrogenobaculum, and Sulfurihydrogenibium--and members of the Alpha-, Beta-, and Gammaproteobacteria, Firmicutes, Acidobacteria, Deinococcus-Thermus, and candidate division OP5. The in situ chemical affinities were calculated for 41 potential metabolic reactions using measured environmental parameters and a range of hydrogen and oxygen concentrations. Reactions that use oxygen, ferric iron, sulfur, and nitrate as electron acceptors were predicted to be the most energetically favorable, while reactions using sulfate were expected to be less favorable. Samples were screened for genes used in ammonia oxidation (amoA, bacterial gene only), the reductive tricarboxylic acid (rTCA) cycle (aclB), the Calvin cycle (cbbM), sulfate reduction (dsrAB), nitrogen fixation (nifH), nitrite reduction (nirK), and sulfide oxidation (soxEF1) by PCR. Genes for carbon fixation by the rTCA cycle and nitrogen fixation were detected. All aclB sequences were phylogenetically related and spatially correlated to Sulfurihydrogenibium 16S rRNA gene sequences using qPCR (R(2) = 0.99). This result supports the recent finding of citrate cleavage by enzymes other than ATP citrate lyase in the rTCA cycle of the Aquificaceae family. We briefly consider potential biochemical mechanisms that may allow Sulfurihydrogenibium and Thermocrinis to codominate some hydrothermal environments.

SUBMITTER: Hall JR

PROVIDER: S-EPMC2519350 | biostudies-literature | 2008 Aug

REPOSITORIES: biostudies-literature

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Molecular characterization of the diversity and distribution of a thermal spring microbial community by using rRNA and metabolic genes.

Hall Justine R JR Mitchell Kendra R KR Jackson-Weaver Olan O Kooser Ara S AS Cron Brandi R BR Crossey Laura J LJ Takacs-Vesbach Cristina D CD

Applied and environmental microbiology 20080606 15

The diversity and distribution of a bacterial community from Coffee Pots Hot Spring, a thermal spring in Yellowstone National Park with a temperature range of 39.3 to 74.1 degrees C and pH range of 5.75 to 6.91, were investigated by sequencing cloned PCR products and quantitative PCR (qPCR) of 16S rRNA and metabolic genes. The spring was inhabited by three Aquificae genera--Thermocrinis, Hydrogenobaculum, and Sulfurihydrogenibium--and members of the Alpha-, Beta-, and Gammaproteobacteria, Firmic ...[more]

PMID: 18539788

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Project description:BackgroundWater springs provide important ecosystem services including drinking water supply, recreation, and balneotherapy, but their microbial communities remain largely unknown. In this study, we characterized the spring water microbiome of Comano Terme (Italy) at four sampling points of the thermal spa, including natural (spring and well) and human-built (storage tank, bathtubs) environments. We integrated large-scale culturing and metagenomic approaches, with the aim of comprehensively determining the spring water taxonomic composition and functional potential.ResultsThe groundwater feeding the spring hosted the most atypical microbiome, including many taxa known to be recalcitrant to cultivation. The core microbiome included the orders Sphingomonadales, Rhizobiales, and Caulobacterales, and the families Bradyrhizobiaceae and Moraxellaceae. A comparative genomic analysis of 72 isolates and 30 metagenome-assembled genomes (MAGs) revealed that most isolates and MAGs belonged to new species or higher taxonomic ranks widely distributed in the microbial tree of life. Average nucleotide identity (ANI) values calculated for each isolated or assembled genome showed that 10 genomes belonged to known bacterial species (> 95% ANI), 36 genomes (including 1 MAG) had ANI values ranging 85-92.5% and could be assigned as undescribed species belonging to known genera, while the remaining 55 genomes had lower ANI values (< 85%). A number of functional features were significantly over- or underrepresented in genomes derived from the four sampling sites. Functional specialization was found between sites, with for example methanogenesis being unique to groundwater whereas methanotrophy was found in all samples.ConclusionsCurrent knowledge on aquatic microbiomes is essentially based on surface or human-associated environments. We started uncovering the spring water microbiome, highlighting an unexpected diversity that should be further investigated. This study confirms that groundwater environments host highly adapted, stable microbial communities composed of many unknown taxa, even among the culturable fraction.

Project description:As new sequencing technologies become cheaper and older ones disappear, laboratories switch vendors and platforms. Validating the new setups is a crucial part of conducting rigorous scientific research. Here we report on the reliability and biases of performing bacterial 16S rRNA gene amplicon paired-end sequencing on the MiSeq Illumina platform. We designed a protocol using 50 barcode pairs to run samples in parallel and coded a pipeline to process the data. Sequencing the same sediment sample in 248 replicates as well as 70 samples from alkaline soda lakes, we evaluated the performance of the method with regards to estimates of alpha and beta diversity. Using different purification and DNA quantification procedures we always found up to 5-fold differences in the yield of sequences between individually barcodes samples. Using either a one-step or a two-step PCR preparation resulted in significantly different estimates in both alpha and beta diversity. Comparing with a previous method based on 454 pyrosequencing, we found that our Illumina protocol performed in a similar manner - with the exception for evenness estimates where correspondence between the methods was low. We further quantified the data loss at every processing step eventually accumulating to 50% of the raw reads. When evaluating different OTU clustering methods, we observed a stark contrast between the results of QIIME with default settings and the more recent UPARSE algorithm when it comes to the number of OTUs generated. Still, overall trends in alpha and beta diversity corresponded highly using both clustering methods. Our procedure performed well considering the precisions of alpha and beta diversity estimates, with insignificant effects of individual barcodes. Comparative analyses suggest that 454 and Illumina sequence data can be combined if the same PCR protocol and bioinformatic workflows are used for describing patterns in richness, beta-diversity and taxonomic composition.

Dataset Information

Molecular characterization of the diversity and distribution of a thermal spring microbial community by using rRNA and metabolic genes.

Publications

Molecular characterization of the diversity and distribution of a thermal spring microbial community by using rRNA and metabolic genes.

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