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The microbial mats growing in the runoff channels of the hot springs of Yellowstone National Park (YNP) are a rich mix of bacterial, archaeal, and eukaryotic species. Mat samples were gathered from Octopus Hot Spring in 2005 and 2006.The samples were subjected to labeling with iTRAQ reagents followed by shotgun proteomics. Mascot was used to query an in-house YNP database derived from the microbial portion of the NCBInr. It was expected that the majority of the proteins mapping to species with high temperature optima would be associated with the sample taken at a higher temperature, while those proteins from species with lower optima would be associated with the sample from lower temperature. Although Synechococcus is the most abundant microorganism in the mat community when abundance is measured by the percentage of DNA in a metagenomics sample, more peptides were identified from Roseiflexus sp. RS-1 than from any other organism. This discrepancy is most likely due to the sample being a mix of the large red and small green layers of the mat. Synechococcus resides only in the green layer. The large number of distinct peptides from the Roseiflexus extracellular binding protein resulted in the highest Mowse score of the proteins identified. Eighty percent of the peptides associated with the extracellular binding protein were isolated from the 58°C sample, while only 20% of this protein came from the 71°C sample. This trend continues throughout the housekeeping proteins quantified from Roseiflexus, as might be expected of a thermophile with a lower temperature optimum. The remainder of the identified proteins showed the association with collection temperature that would be expected from the temperature optimums of the mciroorganisms from which they were extracted. The majority of the proteins identified in this experiment were housekeeping and structural proteins.