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SUMMARY 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.

We compared bacterial 16S ribosomal RNA gene sequences recovered from Lake Loosdrecht, the Netherlands, to reported sequences from lakes in Alaska and New York State. In each of the three lake systems, which differ in pH and trophic state, some sequence types were found without related sequences (sequence identity < 90%) in the data sets from the other two systems. Two sequences in the Actinomycetes and Verrucomicrobia radiations were more closely related to sequences from the New York lakes data set than to any other sequence in the global databases. However, the most striking similarities were found in the subdivisions alpha and beta of the Proteobacteria. In these subdivisions three different clusters of highly related bacteria were identified (97-100% sequence identity) that were represented in all three lake regions. The clusters contained no members other than freshwater bacteria. One cluster falls within a monophyletic aquatic supergroup that apparently diverged early in evolution into an exclusive freshwater cluster and an exclusive marine cluster, the so-called SAR11 cluster. The detection of these three bacterial clades in lakes distinguished by geographic distance as well as physical and chemical diversity suggests that these organisms are dispersed globally and that they possess unique functional capabilities enabling successful competition in a wide range of freshwater environments.

To understand the composition and structure of denitrifying communities in the oxygen-deficient zone off the Pacific coast of Mexico, the molecular diversity of nir genes from sediments obtained at four stations was examined by using a PCR-based cloning approach. A total of 50 operational taxonomic units (OTUs) for nirK and 82 OTUs for nirS were obtained from all samples. Forty-four of the nirS clones and 31 of the nirK clones were sequenced; the levels of similarity of the nirS clones were 52 to 92%, and the levels of similarity of the nirS clones were 50 to 99%. The percentages of overlapping OTUs between stations were 18 to 30% for nirS and 5 to 8% for nirK. Sequence analysis revealed that 26% of the nirS clones were related to the nirS genes of Alcaligenes faecalis (80 to 94% similar) and Pseudomonas stutzeri (80 to 99%), whereas 3 to 31% of the nirK clones were closely related to the nirK genes of Pseudomonas sp. strain G-179 (98 to 99%), Bradyrhizobium japonicum (91%), Blastobacter denitrificans (83%), and Alcaligenes xylosoxidans (96%). The rest of the clones, however, were less than 80% similar to nirS and nirK sequences available in sequence databases. The results of a principalcomponent analysis (PCA) based on the percentage of OTUs and biogeochemical data indicated that the nitrate concentration and oxygen have an effect on the denitrifying communities. The communities at the stations in oxygen-deficient zones were more similar than the communities at the stations in the oxygenated zone. The denitrifying communities were more similar at the stations that were closer together and had similar nitrate levels. Also, the results of PCA based on biogeochemical properties suggest that geographic location and biogeochemical conditions, especially the nitrate and oxygen levels, appear to be the key factors that control the structure of denitrifying communities.The continental margin occupies only a fraction of the total ocean, but it is critical for carbon and nutrient cycling. This margin contributes 30 to 50% of the total marine primary productivity (28,43). The high productivity results in high carbon input into margin sediments that stimulates rapid benthic carbon and nutrient cycling (12,18). Nearly 90% of marine carbon burial (permanent sequestration) occurs in margin sediments (5,24). Recent studies have suggested that the present-day oceanic nitrogen budget is unbalanced (15), although some evidence suggests that it may be balanced (11,22). In the unbalanced view of the N budget, the rate of supply of nitrogen to the ocean is much lower than the removal rate, primarily due to denitrification (1,14,20,31). Denitrification contributes both indirectly and directly to decreasing carbon sequestration. It decreases the amount of nitrogen available to phytoplankton, thus affecting primary productivity. It also produces greenhouse gases, such as nitric (NO) and nitrous oxide (N 2 O), which contribute to global warming and the destruction of the ozone layer (16,27).The uncertainty about estimating marine denitr...

Cyanobacteria are renowned as the mediators of Earth's oxygenation. However, little is known about the cyanobacterial communities that flourished under the low-O(2) conditions that characterized most of their evolutionary history. Microbial mats in the submerged Middle Island Sinkhole of Lake Huron provide opportunities to investigate cyanobacteria under such persistent low-O(2) conditions. Here, venting groundwater rich in sulfate and low in O(2) supports a unique benthic ecosystem of purple-colored cyanobacterial mats. Beneath the mat is a layer of carbonate that is enriched in calcite and to a lesser extent dolomite. In situ benthic metabolism chambers revealed that the mats are net sinks for O(2), suggesting primary production mechanisms other than oxygenic photosynthesis. Indeed, (14)C-bicarbonate uptake studies of autotrophic production show variable contributions from oxygenic and anoxygenic photosynthesis and chemosynthesis, presumably because of supply of sulfide. These results suggest the presence of either facultatively anoxygenic cyanobacteria or a mix of oxygenic/anoxygenic types of cyanobacteria. Shotgun metagenomic sequencing revealed a remarkably low-diversity mat community dominated by just one genotype most closely related to the cyanobacterium Phormidium autumnale, for which an essentially complete genome was reconstructed. Also recovered were partial genomes from a second genotype of Phormidium and several Oscillatoria. Despite the taxonomic simplicity, diverse cyanobacterial genes putatively involved in sulfur oxidation were identified, suggesting a diversity of sulfide physiologies. The dominant Phormidium genome reflects versatile metabolism and physiology that is specialized for a communal lifestyle under fluctuating redox conditions and light availability. Overall, this study provides genomic and physiologic insights into low-O(2) cyanobacterial mat ecosystems that played crucial geobiological roles over long stretches of Earth history.

We used real-time PCR to quantify the denitrifying nitrite reductase gene (nirS), a functional gene of biogeochemical significance. The assay was tested in vitro and applied to environmental samples. The primerprobe set selected was specific for nirS sequences that corresponded approximately to the Pseudomonas stutzeri species. The assay was linear from 1 to 10 6 gene copies (r 2 ‫؍‬ 0.999). Variability at low gene concentrations did not allow detection of twofold differences in gene copy number at less than 100 copies. DNA spiking and cell-addition experiments gave predicted results, suggesting that this assay provides an accurate measure of P. stutzeri nirS abundance in environmental samples. Although P. stutzeri abundance was high in lake sediment and groundwater samples, we detected low or no abundance of this species in marine sediment samples from Puget Sound (Wash.) and from the Washington ocean margin. These results suggest that P. stutzeri may not be a dominant marine denitrifier.Denitrification is one of the important biogeochemical processes in that it is the main sink for fixed nitrogen (28). In agriculture, it accounts for 20 to 30% of fertilizer losses (11), and in marine environments it is thought to account for up to 80% of the loss of the nitrogen load to coastal areas (38). Two of its products, NO and N 2 O, are involved in global warming and the destruction of the ozone layer. Denitrification is also used in waste treatment facilities to remove excess combined nitrogen (41).More accurate understanding and modeling of denitrification should be possible if the catalyst can be quantified. We have evaluated the application of real-time PCR to the quantification of the nitrite reductase gene in Pseudomonas stutzeri. P. stutzeri is a denitrifier commonly isolated from both soil (12) and marine environments (44), and it may be of general importance in global denitrification. Nitrite reductase catalyzes a key step in the nitrogen cycle, in that the reduction of nitrite (NO 2 Ϫ ) to nitric oxide (NO) converts N to a form no longer available to most of the biota. This enzyme is found as two different variants. One contains copper and is encoded by nirK, while the other contains the hemes c and d 1 and is encoded by nirS (45). nirK is found in a wider range of physiological groups, while nirS appears to be more abundant in nature (8). P. stutzeri contains nirS (29).Several methods have been used to attempt to identify or quantify denitrifiers, including the design of specific PCR primers (6, 17) or probes (39) for genes involved in denitrification or ribosomal DNA genes (26) and immunofluorescence assays using polyclonal antibodies against specific denitrifying bacteria (44) or denitrification enzymes (8). Recently, competitive PCR and most-probable-number PCR were applied to the quantification of nirS-containing denitrifying bacteria (34).The real-time PCR technique is based on the use of the 5Ј nuclease assay, first described by Holland et al. (20) and further improved by the use of fluorescent TaqMan...

Physicochemical characterization, automated ribosomal intergenic spacer analysis (ARISA) community profiling, and 16S rRNA gene sequencing approaches were used to study bacterial communities inhabiting submerged Lake Huron sinkholes inundated with hypoxic, sulfate-rich groundwater. Photosynthetic cyanobacterial mats on the sediment surface were dominated by Phormidium autumnale, while deeper, organically rich sediments contained diverse and active bacterial communities.Groundwater intrusion is becoming recognized as an important source of nutrients, contaminants, and trace elements in aquatic ecosystems (7). Recent reports regarding marine habitats suggest that groundwater influences nitrogen inputs (29) and may have a significant impact on nutrient dynamics over seasonal (27) and longer (25) time scales. To date, investigations of groundwater effects have focused primarily on marine habitats. To better understand the impact of groundwater intrusion into freshwater habitats, we have been studying submerged sinkholes in the Laurentian Great Lakes.Sinkholes typically develop in areas of terrestrial karst when underground caverns collapse (36). We recently discovered submerged sinkholes that occur beneath the surface of Lake Huron in water up to a depth of 93 m (9, 41). These unique habitats are formed by groundwater dissolution of Paleozoic limestone and marine evaporite sediments in the Michigan Basin (16). Some sinkholes actively release cold, dense groundwater through underwater vents onto the lake floor. The venting groundwater has a lower pH (ϳ7.1), higher specific conductivity (ϳ2.3 mS ⅐ cm Ϫ1 , due to high levels of dissolved sulfate [Ͼ1,000 mg ⅐ liter Ϫ1 ], carbonate, and chloride ions), and lower concentrations of dissolved oxygen (Ͻ0.4 mg ⅐ liter Ϫ1 ) and nitrate than Lake Huron water (2,3,41,42). The intrusion of cold, hypoxic, sulfate-rich groundwater greatly alters the local lake habitat and has a significant impact on the sediment microbial community. Sediments at nearby control sites are sandy, but submerged sinkhole sediments (with carbon accounting for 5 to 35% of the sediment dry weight) are rich in organic matter originating from phytoplankton in the overlying water column

The diversity of ammonia-oxidizing bacteria in aquatic sediments was studied by retrieving ammonia monooxygenase and methane monooxygenase gene sequences. Methanotrophs dominated freshwater sediments, while ␤-proteobacterial ammonia oxidizers dominated marine sediments. These results suggest that ␥-proteobacteria such as Nitrosococcus oceani are minor members of marine sediment ammonia-oxidizing communities.