A natural view of microbial biodiversity within hot spring cyanobacterial mat communities

Abstract

This review summarizes a decade of research in which we have used molecular methods, in conjunction with more traditional approaches, to study hot spring cyanobacterial mats as models for understanding principles of microbial community ecology. Molecular methods reveal that the composition of these communities is grossly oversimplified by microscopic and cultivation methods. For example, none of 31 unique 16S rRNA sequences detected in the Octopus Spring mat, Yellowstone National Park, matches that of any prokaryote previously cultivated from geothermal systems; 11 are contributed by genetically diverse cyanobacteria, even though a single cyanobacterial species was suspected based on morphologic and culture analysis. By studying the basis for the incongruity between culture and molecular samplings of community composition, we are beginning to cultivate isolates whose 16S rRNA sequences are readily detected. By placing the genetic diversity detected in context with the well-defined natural environmental gradients typical of hot spring mat systems, the relationship between gene and species diversity is clarified and ecological patterns of species occurrence emerge. By combining these ecological patterns with the evolutionary patterns inherently revealed by phylogenetic analysis of gene sequence data, we find that it may be possible to understand microbial biodiversity within these systems by using principles similar to those developed by evolutionary ecologists to understand biodiversity of larger species. We hope that such an approach guides microbial ecologists to a more realistic and predictive understanding of microbial species occurrence and responsiveness in both natural and disturbed habitats.

FIG. 1

Laminated cyanobacterial mat communities of alkaline siliceous hot springs in the Lower Geyser Basin, Yellowstone National Park, viewed at different scales. (A) Landscape showing green-orange mat at far edge of Octopus Spring and down effluent channel. (B) Cross section of ca. 50 to 55°C Octopus Spring cyanobacterial mat sample magnified at ca. × 1.8. (C) Phase-contrast microscopy image of homogenized 1-mm-thick upper green Octopus Spring mat layer showing the predominant cyanobacteria, sausage-shaped S. cf. lividus, embedded in a matrix of filaments, at least some of which are probably green nonsulfur bacteria such as C. aurantiacus (reprinted from reference 160). (D) Autofluorescence microscopy image of a vertical cryotome section through the upper 1-mm green layer of the 61°C Mushroom Spring mat showing banding of S. cf. lividus populations at different depths (111).

FIG. 2

Distance matrix phylogenetic trees illustrating cyanobacterial (A) and green nonsulfur bacterium-like (B) 16S rRNA sequences detected directly in the Octopus Spring cyanobacterial mat or in isolates therefrom (thick lines) relative to representative major lines of descent in each lineage (thin lines). We used the OSC1 isolate sequence (identical to that of S. cf. lividus Y-7c-s) and the C. aurantiacus J10 sequence (98.2% similar to that of C. aurantiacus Y-400-fl) because more sequence data were available. The trees were constructed with the programs DNADIST and FITCH from the Phylogenetic Inference Package (PHYLIP), version 3.57c. Trees were inferred from nucleotides which align with Escherichia coli positions 332 to 452, 480 to 507, 712 to 892, and 1140 to 1364 (A) and 256 to 446, 485 to 598, 604 to 830, 856 to 922, 933 to 939, and 954 to 966 (B), except that for sequences shown to the right of arrows, fewer nucleotides were available for analysis. In these cases, smaller trees were inferred from available data and manually added to the appropriate branch. The tree in panel A was rooted by using the 16S rRNA sequences of Thermotoga maritima, Chlorobium vibrioforme, and E. coli, while the tree in panel B was rooted by using sequences from Methanobacterium formicicum, Thermodesulfobacterium commune, Aquifex pyrophilus, T. maritima, C. vibrioforme, Agrobacterium tumefaciens, Pseudomonas testosteroni, and E. coli. Evolutionary distance is indicated by horizontal lines; each bar corresponds to 0.01 fixed point mutations per sequence position.

FIG. 3

DGGE analysis of 16S rRNA gene segments of aerobic chemoorganotrophic populations enriched from 10-fold serially diluted samples of the Octopus Spring cyanobacterial mat and present in the mat itself. Bands labeled with numbers were characterized by purification and sequencing (see reference 121).

FIG. 4

Evidence of the existence of temperature-adapted populations within hot spring cyanobacterial mats. (A) Effect of temperature on growth rate of Thermus strains ac-1, ac-2, and ac-7 isolated from a ca. 50°C Octopus Spring mat sample. Bars represent standard errors (modified from reference 87). (B) Effect of temperature on growth rate of S. cf. lividus strains isolated from the Hunter’s Springs, Oreg., mat. Bars represent ranges of replicate experiments (modified from reference 105). (C) Effect of temperature on bacterial photoautotrophy in samples of the Twin Butte Vista (Lower Geyser Basin, Yellowstone National Park) mat collected at various temperature-defined sites, as shown (redrawn from reference 9).

FIG. 5

Distribution of 16S rRNA gene segments of populations detected by DGGE in cyanobacterial mat samples collected from temperature-defined sites along the thermal gradient in the Octopus Spring effluent channel on 13 March 1995. Lanes within a single temperature interval are true replicate mat samples. Bands labeled with letters were characterized by purification and sequencing. Single letters denote homoduplex molecules from real populations, while double letters denote heteroduplex artifacts formed from the populations indicated by the two letters. Primes indicate sequences closely related to those of the same letter without primes (reprinted from reference 41).

FIG. 6

Rates of oxygenic photosynthesis determined by microelectrode analysis in a single ca. 60°C Octopus Spring mat sample incubated at different temperatures. (A) Upper mat surface. (B) 0.3 mm below the surface (modified from reference 68).

FIG. 7

Distribution of 16S rRNA gene segments of populations detected by DGGE through the upper 1-mm vertical interval of the 61°C Mushroom Spring cyanobacterial mat. Each lane represents DGGE analysis of a separate 100-μm-thick cryotome section. Bands are labeled as described in the legend to Fig. 5 (111).

FIG. 8

16S rRNA gene segments of populations detected by DGGE which recolonized the upper few millimeters of the Octopus Spring 55 to 62°C mat after removal of the upper green cyanobacterial layer. Triplicate mat samples were analyzed at each time point. Bands are labeled as described in the legend to Fig. 5. (reprinted from reference 37).