Short-Term Stable Isotope Probing of Proteins Reveals Taxa Incorporating Inorganic Carbon in a Hot Spring Microbial...

The upper green layer of the chlorophototrophic microbial mats associated with the alkaline siliceous hot springs of Yellowstone National Park consists of oxygenic cyanobacteria (Synechococcus spp.), anoxygenic Roseiflexus spp., and several other anoxygenic chlorophototrophs. Synechococcus spp. are believed to be the main fixers of inorganic carbon (C_i), but some evidence suggests that Roseiflexus spp. also contribute to inorganic carbon fixation during low-light, anoxic morning periods. Contributions of other phototrophic taxa have not been investigated. In order to follow the pathway of C_i incorporation into different taxa, mat samples were incubated with [¹³C]bicarbonate for 3 h during the early-morning, low-light anoxic period. Extracted proteins were treated with trypsin and analyzed by mass spectrometry, leading to peptide identifications and peptide isotopic profile signatures containing evidence of ¹³C label incorporation. A total of 25,483 peptides, corresponding to 7,221 proteins, were identified from spectral features and associated with mat taxa by comparison to metagenomic assembly sequences. A total of 1,417 peptides, derived from 720 proteins, were detectably labeled with ¹³C. Most ¹³C-labeled peptides were derived from proteins of Synechococcus spp. and Roseiflexus spp. Chaperones and proteins of carbohydrate metabolism were most abundantly labeled. Proteins involved in photosynthesis, C_i fixation, and N₂ fixation were also labeled in Synechococcus spp. Importantly, most proteins of the 3-hydroxypropionate bi-cycle for C_i fixation in Roseiflexus spp. were labeled, establishing that members of this taxocene contribute to C_i fixation. Other taxa showed much lower [¹³C]bicarbonate incorporation.IMPORTANCE Yellowstone hot spring mats have been studied as natural models for understanding microbial community ecology and as modern analogs of stromatolites, the earliest community fossils on Earth. Stable-isotope probing of proteins (Pro-SIP) permitted short-term interrogation of the taxa that are involved in the important process of light-driven C_i fixation in this highly active community and will be useful in linking other metabolic processes to mat taxa. Here, evidence is presented that Roseiflexus spp., which use the 3-hydroxypropionate bi-cycle, are active in C_i fixation. Because this pathway imparts a lower degree of selection of isotopically heavy C_i than does the Calvin-Benson-Bassham cycle, the results suggest a mechanism to explain why the natural abundance of ¹³C in mat biomass is greater than expected if only the latter pathway were involved. Understanding how mat community members influence the ¹³C/¹²C ratios of mat biomass will help geochemists interpret the ¹³C/¹²C ratios of organic carbon in the fossil record.