Unexpected Diversity and Complexity of the Guerrero Negro Hypersaline Microbial Mat

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 (≥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 O2 and H2S. 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.

Microbial mats are benthic aquatic ecosystems fueled by light energy and composed of microbial cells attached to extracellular polymeric material and mineralized scaffolds in visible millimeter scale layers (12). Unlike stromatolites, which have a biotic mechanism for calcification (49), microbial mats become layered because of occasional sedimentation and regrowth. Microbial mats and stromatolites are found in the fossil record dating back 3.4 billion years (60) and are thought to have significantly influenced the composition of the atmosphere with production of O2, H2, and CH4 (21). Ancient and modern mats share properties inherent to their structure. For instance, different wavelengths of light penetrate differentially, gas exchange with the atmosphere occurs at the surface, an organic carbon-based matrix provides a scaffold for growth, and sedimentation occasionally buries the surface, which is then overgrown, leading to layering. Comparisons of biosignatures in modern and fossilized mats seek to describe ancient biogeochemical cycles and the microbial activities of ancient communities (34, 57, 58).

The microbial mats within the hypersaline lagoons of the Exportadora de Sal SA saltern in Guerrero Negro, Baja California Sur, Mexico, cover an extensive area of artificial shallow lagoons protected from tidal disturbance by levees. Biogeochemical studies of these mats have shown that oxygen and light, as well as photosynthetic capacity, are rapidly depleted with depth. Degradation of organic matter occurs largely by two processes, fermentation and substrate oxidation through sulfate reduction (12). Both processes take on unusual characteristics in the mats. Fermentation contributes to molecular hydrogen release into the overlying water column, even in the presence of oxygen, such that bubbles of mixed oxygen and hydrogen gases form on the surface of the mat (21). In addition, the highest rates of sulfate reduction occur in the upper, oxygen-rich layers of the mat (5). Known sulfate-reducing bacteria of the delta group of proteobacteria occupy the anoxic zone, however, which suggests that novel groups of sulfate-reducing bacteria reduce sulfate in the mats aerobically (51).

Despite the intriguing biogeochemistry of hypersaline microbial mats and their importance as model systems for studies of the early Earth (21), the composition of the microbiota has not been surveyed comprehensively by culture-independent molecular methods. Classic microbiological studies and limited molecular studies have shown that cyanobacteria dominate the surface layers and revealed five of the other main bacterial phyla (phylogenetic divisions Chloroflexi, Spirochaetes, Proteobacteria, Bacteroidetes, and Firmicutes) and thus indicated a relatively simple community with little deep evolutionary diversity (10, 39-42, 51, 57). The biological simplicity of the mats was the basis for their recommended use as model systems for metagenomic analyses (4). The dominance of cyanobacteria and the biological simplicity of the community have not been verified by culture-independent methods, however.

The aim of this study was a more comprehensive description of the microbial diversity within the mats and how that diversity is distributed in relation to depth and chemical gradients characteristic of the depth profile. We studied a limited area of one mat intensively. In situ gradients of O2 and H2S concentrations and pH, measured on a micrometer scale with microelectrodes, provided a backdrop of vertical chemical gradients onto which we mapped the biological data. ATP concentrations were measured to provide an estimate of living biomass distribution throughout the mat. Pigment concentrations measured by high-pressure liquid chromatography (HPLC) offered a view of the distribution of oxygenic chlorophyll a (Chl a)-containing cyanobacteria in relation to bacteriochlorophyll (BChl)-bearing anoxygenic photosynthetic bacteria. A survey of rRNA genes provided a culture-independent assessment of dominant organisms. For each of 10 layers that divided the mat into a millimeter-centimeter scale depth profile, the composition and diversity of communities were determined by sequence analysis of 16S rRNA genes generated by PCR with universal and bacterium-specific primers from community genomic DNA. We used RNA extraction and quantitative hybridization with group-specific probes as a PCR-independent verification of the abundance of the dominant group identified by sequence analysis and of the cyanobacteria. In addition, we visualized the morphologies and associations of these bacteria by fluorescence in situ hybridization (FISH) with tyramide signal amplification to overcome the intrinsic fluorescence of the mat. These studies collectively revealed unexpected diversity, complexity, and structure within the mat.