Cyanobacterial Ecotypes in Different Optical Microenvironments of a 68°C Hot Spring Mat Community Revealed by 16S-23S rRNA Internal Transcribed Spacer Region Variation†

We examined the population of unicellular cyanobacteria (Synechococcus) in the upper 3-mm vertical interval of a 68°C region of a microbial mat in a hot spring effluent channel (Yellowstone National Park, Wyoming). Fluorescence microscopy and microsensor measurements of O2 and oxygenic photosynthesis demonstrated the existence of physiologically distinct Synechococcus populations at different depths along a light gradient quantified by scalar irradiance microprobes. Molecular methods were used to evaluate whether physiologically distinct populations could be correlated with genetically distinct populations over the vertical interval. We were unable to identify patterns in genetic variation in Synechococcus 16S rRNA sequences that correlate with different vertically distributed populations. However, patterns of variation at the internal transcribed spacer locus separating 16S and 23S rRNA genes suggested the existence of closely related but genetically distinct populations corresponding to different functional populations occurring at different depths. Developments in genetic-sequence-based identification methods and microenvironmental-analysis techniques have significantly enhanced the ability to describe the ecology of bacterial populations in situ (2). Our research has focused on the ecology of phototrophic bacteria in alkaline hot spring microbial mats, where cellular morphologies are of limited use in distinguishing populations. The original descriptions of these mats were relatively simple: a green surface layer containing unicellular cyanobacteria (Synechococcus) overlying an orange layer containing green nonsulfur (Chloroflexus-like) bacteria. Cultivation-independent genetic-sequence-based analysis of 16S rRNA gene segments has been used to sample the diversity and distribution of Synechococcus populations in these communities (33). These studies have shown that numerous genetically distinct Synechococcus populations appear to be adapted to and organized along temperature-defined niches due to thermal gradients in hot spring effluent channels (7, 9). These results were consistent with earlier studies of cultivated Synechococcus that were not genetically characterized (23), as well as recent studies in which isolates were genetically characterized (16). In nearly all instances, two or three Synechococcus sequences were detected at a given temperature by 16S rRNA analysis (7–9, 25). We investigated the possibility that multiple 16S rRNA sequences at the same temperature site represented Synechococcus populations that coexist by adapting to different light environments within the mat. We analyzed Synechococcus populations over the vertical interval at a 60°C site (25). Here, the degree of 16S rRNA sequence variation between populations