Unexpected diversity and complexity of the Guerrero Negro hypersaline microbial mat - PubMed (original) (raw)

Unexpected diversity and complexity of the Guerrero Negro hypersaline microbial mat

Ruth E Ley et al. Appl Environ Microbiol. 2006 May.

Abstract

We applied nucleic acid-based molecular methods, combined with estimates of biomass (ATP), pigments, and microelectrode measurements of chemical gradients, to map microbial diversity vertically on a millimeter scale in a hypersaline microbial mat from Guerrero Negro, Baja California Sur, Mexico. To identify the constituents of the mat, small-subunit rRNA genes were amplified by PCR from community genomic DNA extracted from layers, cloned, and sequenced. Bacteria dominated the mat and displayed unexpected and unprecedented diversity. The majority (1,336) of the 1,586 bacterial 16S rRNA sequences generated were unique, representing 752 species (> or =97% rRNA sequence identity) in 42 of the main bacterial phyla, including 15 novel candidate phyla. The diversity of the mat samples differentiated according to the chemical milieu defined by concentrations of O(2) and H(2)S. Bacteria of the phylum Chloroflexi formed the majority of the biomass by percentage of bulk rRNA and of clones in rRNA gene libraries. This result contradicts the general belief that cyanobacteria dominate these communities. Although cyanobacteria constituted a large fraction of the biomass in the upper few millimeters (>80% of the total rRNA and photosynthetic pigments), Chloroflexi sequences were conspicuous throughout the mat. Filamentous Chloroflexi bacteria were identified by fluorescence in situ hybridization within the polysaccharide sheaths of the prominent cyanobacterium Microcoleus chthonoplastes, in addition to free living in the mat. The biological complexity of the mat far exceeds that observed in other polysaccharide-rich microbial ecosystems, such as the human and mouse distal guts, and suggests that positive feedbacks exist between chemical complexity and biological diversity. The sequences determined in this study have been submitted to the GenBank database and assigned accession numbers DQ 329539 to DQ 331020, and DQ 397339 to DQ 397511.

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Figures

FIG. 1.

FIG. 1.

Chemical and biochemical characteristics of the mat as a function of depth. (A) Microelectrode measurements of O2 and H2S concentrations and pH. (B) ATP concentrations. Means of three independent measurements are plotted; the bars show standard errors. (C) Pigment concentrations. For all measurements, October values are plotted; the June values were equivalent (data not shown).

FIG. 2.

FIG. 2.

Bacterial diversity within the hypersaline microbial mat. (A) Observed and predicted (Chao1, Ace1) numbers of taxa with minimum thresholds ranging from 90 to 100% ID for masked sequences. (B) Collector's curves for taxa (OTUs) with minimum thresholds of 90, 95, 97, 98, 99, and 100% ID.

FIG. 3.

FIG. 3.

Bacterial diversity in the mat. Proportions of bacterial phyla in the total data set (A), in the oxic zone (0 to 2 mm) (B), in the low-H2S zone (2 to 6 mm) (C), and in the H2S-rich zone (6 to 60 mm) (D) are shown. Others: cyanobacteria, KSB1, OP10, GN03, OP5, GN1, Firmicutes, OP11, GN04, GN05, GN09, GN10, WS1, WS2, GN2, _Deinococcus_-Thermus, GN07, Haloanaerobiales, GN06, GN11, BRC1, OP8, OS-K, GN12, GN13, GN14, actinobacteria, GN15, WS3, GN8, OP9, TM6, and VadinBE97. Abbreviations: Chloro., Chloroflexi; Cyano., Cyanobacteria; Verr., Verrucomicrobia; Planct., Planctomycetales; Spiro., Spirochaetales; Firm., Firmicutes, Bact., Bacteroidetes; Proteo., Proteobacteria. (E) Bacterial community clustering by layer studied (UPGMA tree of UniFrac metric based on 1,585 16S rRNA gene sequences). Shaded areas refer to the different chemical milieus identified by the microelectrode measurements in Fig. 1A.

FIG. 4.

FIG. 4.

Diagrammatic phylogenetic trees of microbial mat sequences and their cultured and uncultured relatives with associated GenBank accession numbers. Reference sequences of cultured representatives are shown in italics. Wedges represent groups of microbial mat sequences, and single sequences are indicated by their clone names. The length of the top and bottom edges represents the range of sequence divergence. The average depth from which sequences were obtained is indicated next to the wedge, with the total depth range in parentheses. (A) Chloroflexi sequences. Percentages indicate the fraction of Chloroflexi sequences within a given sequence cluster. “Oxic zone” indicates clusters of sequences obtained from surface layers exclusively. (B) Cyanobacterial sequences. Percentages indicate the fraction of cyanobacterial sequences within a given sequence cluster.

FIG. 5.

FIG. 5.

Depth distributions of the 10 most abundant phyla in the mat. Points indicate the percentage of sequences within each phylum (not the percentage of total sequences) obtained at each depth. The bar indicates 20% of sequences within each group. Shaded areas: see legend to Fig. 2E.

FIG. 6.

FIG. 6.

Chloroflexi bacteria and the cyanobacterium M. chthonoplastes in the mat visualized by laser confocal microscopy. (A) Chloroflexi bacteria (red, FISH Chloroflexi probe) entwined with M. chthonoplastes (green, DAPI) at a 1-mm depth. The arrow indicates the edge of the polysaccharide sheath. (B) Chloroflexi bacteria (green, Chloroflexi probe) and M. chthonoplastes (green, autofluorescence of Chl a). (C) Chloroflexi bacteria (thin filaments) and M. chthonoplastes (thick filaments), DAPI stained. (D) Chloroflexi filaments (red, Chloroflexi probe) and polysaccharide material (dull green) at a 50-mm depth. Non-Chloroflexi bacteria are visible as bright green spots (arrow 1). Arrow 2 indicates a buried M. chthonoplastes filament. Scale bars, 10 μm.

FIG. 7.

FIG. 7.

Phylogenetic structure of microbial mat (n = 1,585; this study), human colonic (n = 11,831) (15), and mouse cecal (n = 5,088) (32) 16S rRNA gene sequence data sets. Sequences were clustered into phylotypes based on percent sequence identity (OTUs with similarity thresholds ranging from 65% ID to 100% ID). The ratio of phylotypes at each threshold to the total sequence in each data set is plotted.

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References

    1. Amann, R. I., B. J. Binder, R. J. Olson, S. W. Chisolm, R. Devereux, and D. A. Stahl. 1990. Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl. Environ. Microbiol. 56:1919-1925. - PMC - PubMed
    1. Backhed, F., R. E. Ley, J. L. Sonnenburg, D. A. Peterson, and J. I. Gordon. 2005. Host-bacterial mutualism in the human intestine. Science 307:1915-1920. - PubMed
    1. Bebout, B. M., S. P. Carpenter, D. J. DesMarais, M. Discipulo, T. Embaye, F. Garcia-Pichel, T. M. Hoehler, M. Hogan, L. L. Jahnke, R. M. Keller, S. R. Miller, L. E. Prufert-Bebout, C. Raleigh, M. Rothrock, and K. Turk. 2002. Long-term manipulations of intact microbial mat communities in a greenhouse collaboratory: simulating Earth's present and past field environments. Astrobiology 2:383-402. - PubMed
    1. Buckley, M. R. 2004. The global genome question: microbes as the key to understanding evolution and ecology. American Academy of Microbiology, Washington, D.C. - PubMed
    1. Canfield, D. E., and D. J. DesMarais. 1993. Biogeochemical cycles of carbon, sulfur, and free oxygen in a microbial mat. Geochim. Cosmochim. Acta 57:3971-3984. - PubMed

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