Molecular characterization of the diversity and distribution of a thermal spring microbial community by using rRNA and metabolic genes - PubMed (original) (raw)

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

Justine R Hall et al. Appl Environ Microbiol. 2008 Aug.

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.

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Figures

FIG. 1.

Panoramic view of Coffee Pots Hot Spring (Mirror Plateau, northeastern quadrant of Yellowstone National Park). Image is a compilation of overlapping individual digital photographs with brightness uniformly increased in Adobe Photoshop 10. Sample sites are indicated by triangles with pH in parentheses. The arrow indicates the source of the spring.

FIG. 2.

Energy yield of metabolic reactions common to hydrothermal systems as a function of H2 and O2 concentrations reported for other Yellowstone springs (3, 57, 59). Forty-one reactions were evaluated using the analytical results for Coffee Pots (Table 3). Reactions were evaluated for a suite of six models using a range of H2 and O2 concentrations reported for Yellowstone hot springs: models 1 to 4 examine low (4.1 × 10−6 ppm), medium (2.05 × 10−5 ppm), medium-high (2.05 × 10−4 ppm), and high (6.67 × 10−4 ppm) H2 concentrations, respectively, with an O2 concentration of 0.1 ppm; and models 5, 3, and 6 examine a range of O2 concentrations (0.01, 0.1, and 0.5 ppm, respectively) at a fixed H2 concentration of 2.05 × 10−4 ppm. The chemical reactions are displayed and ranked in the order obtained from Obsidian Pool in a previous study (57) of decreasing energy released (per mole of electrons transferred in the reaction).

FIG. 3.

(A) Phylogenetic analysis of 16S rRNA gene sequences obtained from both clone libraries for sample COF_65.7. (B) Phylogenetic tree of the Toll clone and a subset of sequences from the tree in panel A. Both trees are based on 1,293 nucleotides of the 16S rRNA gene and are rooted with Methanocaldococcus jannaschii. Bold names indicate sequences obtained in this study. Single-letter designations and the Toll clone represent sequences from the first clone library, and alphanumeric names represent sequences from the second clone library. The number of clones found for each phylotype is given in parentheses.

FIG. 4.

16S rRNA secondary structure for the Toll sequence. Capital letters are bases conserved between A. pyrophilus and Toll. Regions V1 to V9 are hypervariable regions as determined by Ashelford et al. (5); approximate nucleotide positions are given in parentheses. Regions T1 to T9 are established tertiary interactions. Numbering is unique to this structure.

FIG. 5.

Phylogenetic analysis of the nifH gene sequences obtained from COF_39.3 and COF_65.7. Bold text indicates sequences obtained in this study. The tree was based on 113 amino acid residues and was constructed with neighbor-joining analysis using a heuristic tree search with TBR branch swapping. The tree was rooted with the C. tepidum bchL gene. Bootstrap values are based on 100 resamplings.

FIG. 6.

Phylogenetic analysis of the aclB gene sequences obtained from the seven sample sites. Bold text indicates sequences obtained in this study. The tree was based on 107 amino acid residues and was constructed with neighbor-joining analysis using a heuristic tree search with TBR branch swapping. The tree was rooted with C. limicola. Bootstrap values are based on 1,000 resamplings.

FIG. 7.

Gene copy numbers for 16S rRNA and aclB gene assays for each sampling location. Increasing distance from the source waters of the spring correlates to decreasing temperature. Gene copy numbers were determined using assay-specific standard curves and normalized by calculating the number of gene copies per microliter of total extracted DNA in the reaction. Error bars represent the standard deviation of three replicates.

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Molecular characterization of the diversity and distribution of a thermal spring microbial community by using rRNA and metabolic genes - PubMed (original) (raw)