Robert A Edwards; Beltran Rodriguez-Brito; Linda Wegley; Matthew Haynes; Mya Breitbart; Dean M Peterson; Martin O Saar; Scott Alexander; E Calvin Alexander, Jr; Forest Rohwer

“ We demonstrate that completely unique microbial communities inhabit proximate environments joined by a common watercourse, and that using metagenomics we can identify the unique metabolic potentials prevalent in each environment such as their mechanisms of iron acquisition and respiration. ”

“ These subsystems show that the pyrosequencing generates sequences that represent a large swathe of central metabolism in each of the environments. Common metabolic potential that is expected to be present in sulfur-utilizing chemoautotrophs is represented in the mine libraries, including the Calvin-Benson cycle, inorganic sulfur assimilation, amino acid biosynthetic genes, and so on. The comparison of the subsystem similarities suggested the simple hypothesis that groups of genes (or subsystems) important to a particular environment will be enriched in that environment. ”

“ The water flowing away from each borehole, on the floor of the mine tunnel, is exposed to the oxygenated mine atmosphere, and transitions to a sequence of "Red" environments within a few cm of the orifices. The oxidized environments are continuously fed by anoxic water flowing from the boreholes. The water in the borehole, which yielded the Black sample, as well as a number of similar sites found throughout the mine, has a pH of 6.70 and redox potential of -142 mV. Some of the Black areas are associated with bubbling of gas. The Black sediment contained 5.8 × 105 microbes per ml. ”

“ Table 2 shows subsystems that were determined to be statistically more common, with 95% confidence, in either the Red or Black samples from the Soudan mine. The subsystems that are overrepresented in a metagenome can yield significant insights into the microbial ecology of the environment. ”

“ Nonetheless, the short fragments are sufficient to determine statistically significant differences between metagenomes that reflect the most likely biology occurring in each environment. The low cost, high yield of pyrosequencing combined with statistical analyses on the abundance of subsystems in the samples allows the rapid identification of key processes driving the metabolism of different environments. ”

“ Nitrogen assimilation, iron acquisition, and sulfur metabolism are all differentiated between these two samples from close proximity within the same mine. However, many more significant differences between the samples remain unexplained by our current knowledge of bacterial physiology and metabolism. Explaining these differences will be a grand challenge for the future. By combining pyrosequencing, subsystems analysis, comparative metagenomics, and statistics, Occam has used his razor on metagenomics. ”

“ We anticipate that pyrosequencing will be widely used to sequence environmental samples because of the speed, cost, and technical advantages. Furthermore, subsystem comparisons rapidly identify the important metabolisms employed by the microbes in different environments.
Background
Banded iron formations started appearing ~3,700 million years ago when localized sea floor cyanobacterial photosynthesis raised oxygen concentrations high enough that dissolved iron precipitated. That iron powered the industrial revolution. ”

“ Additional File 3 Table1S. Two lists (one for the Red Sample and one for the Black Sample) describing all the similarities found in the data. The table has the following columns: "Classification I" and "Classification II" are hierarchical classifications of the subsystems. "Subsystem" is the name of the subsystem [21] . "Functional Role" is the role of the protein in the subsystem to which the sequence from the Soudan Mine was similar. "Occurrence" is the number of times that a functional role is found in each sample. ”

“ Comparative metagenomics was introduced recently [4] , identifying those sets of genes that distinguish environmental samples. For example, samples from the surface of the ocean contain significantly more photosynthetic genes than soil or other samples [4] , [6] , [5] . We have used comparative metagenomics to characterize the metabolic potential of different environments, and identify those genes, pathways, and subsystems that are more common in any particular environment [7] . ”

“ To distinguish between ecologically important differences and differences caused by sampling error, a method was devised to identify those subsystems that are statistically significantly overrepresented in one sample when compared to another [7] .
Subsystems in the Red and Black Samples. The occurrence of classes of subsystems is shown as a percent of all subsystems in each sample for the Red and Black samples. Notes and abbreviations: The subsystem class "Glu, Asp" also contains Gln and Asn. The subsystem class "Lys, Thr" also contains Met and Cys. CHO: Carbohydrates; sacch: saccharides ”

“ An analysis of 160 bacterial genome sequences in the SEED database [10] , [15] with annotated 16S genes showed that about 1 in 105 bases is from a 16S gene. Based on this estimate, as a rule of thumb the Soudan samples are expected to contain approximately 3,000 bases of 16S sequence in total, or approximately 30 sequences. Twenty four sequences were found to have significant similarity (with an E value less than 1 × 10-5 and a match of 50 bp or more) to 16S rDNA from the Black sample and seventy six sequences were found to have significant similarity to 16S rDNA from the Red sample. ”

“ These two communities are fundamentally different both from each other and from the community identified in the Iron Mountain metagenom [14] . The community in the Red sample has a much higher species richness than the Black sample, and the differences between the Soudan and Iron mines reflect the iron composition (hematite versus pyrite) , temperature, and pH of the various environments [14] .
Composition of the 16S rDNA sequences from the two samples and comparison of 16S sequences from the 454 libraries and a traditional clone library. ”

“ Extracellular polysaccharides; Myco: Mycobacterial cell wall; Gm: Gram stain positive (+) or negative (-) ; Clust: clusters; RFN: Riboflavin; T: Transporters; Mot: Motility; N: Nitrogen; Resp: Respiration; e-: electron ”