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

Abstract

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 (Ci), but some evidence indicates 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 Ci 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 associated with photosynthesis, Ci fixation, and N2 fixation were alsomore » labeled in Synechococcus spp. Importantly, most proteins of the 3-hydroxypropionate bi-cycle for Ci fixation in Roseiflexus spp. were labeled, establishing that members of this taxocene contribute to Ci 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 Ci 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 Ci fixation. Because this pathway imparts a lower degree of selection of isotopically heavy Ci 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.« less

Authors:
 [1];  [2]; ORCiD logo [2];  [3]; ORCiD logo [2];  [2];  [3];  [2];  [4];  [3]
  1. Univ. of Nebraska, Lincoln, NE (United States)
  2. Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
  3. Montana State Univ., Bozeman, MT (United States)
  4. Pennsylvania State Univ., University Park, PA (United States); Montana State Univ., Bozeman, MT (United States)
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1607671
Report Number(s):
PNNL-SA-148749
Journal ID: ISSN 0099-2240
Grant/Contract Number:  
AC05-76RL01830
Resource Type:
Accepted Manuscript
Journal Name:
Applied and Environmental Microbiology
Additional Journal Information:
Journal Volume: 86; Journal Issue: 7; Journal ID: ISSN 0099-2240
Publisher:
American Society for Microbiology
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; proteomics; Yellowstone National Park; thermophile; autotrophy

Citation Formats

Steinke, Laurey A., Slysz, Gordon W., Lipton, Mary S., Klatt, Christian, Moran, James J., Romine, Margaret F., Wood, Jason, Anderson, Gordon A., Bryant, Donald A., and Ward, David M. Short-Term Stable Isotope Probing of Proteins Reveals Taxa Incorporating Inorganic Carbon in a Hot Spring Microbial Mat. United States: N. p., 2020. Web. doi:10.1128/AEM.01829-19.
Steinke, Laurey A., Slysz, Gordon W., Lipton, Mary S., Klatt, Christian, Moran, James J., Romine, Margaret F., Wood, Jason, Anderson, Gordon A., Bryant, Donald A., & Ward, David M. Short-Term Stable Isotope Probing of Proteins Reveals Taxa Incorporating Inorganic Carbon in a Hot Spring Microbial Mat. United States. doi:https://doi.org/10.1128/AEM.01829-19
Steinke, Laurey A., Slysz, Gordon W., Lipton, Mary S., Klatt, Christian, Moran, James J., Romine, Margaret F., Wood, Jason, Anderson, Gordon A., Bryant, Donald A., and Ward, David M. Wed . "Short-Term Stable Isotope Probing of Proteins Reveals Taxa Incorporating Inorganic Carbon in a Hot Spring Microbial Mat". United States. doi:https://doi.org/10.1128/AEM.01829-19. https://www.osti.gov/servlets/purl/1607671.
@article{osti_1607671,
title = {Short-Term Stable Isotope Probing of Proteins Reveals Taxa Incorporating Inorganic Carbon in a Hot Spring Microbial Mat},
author = {Steinke, Laurey A. and Slysz, Gordon W. and Lipton, Mary S. and Klatt, Christian and Moran, James J. and Romine, Margaret F. and Wood, Jason and Anderson, Gordon A. and Bryant, Donald A. and Ward, David M.},
abstractNote = {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 (Ci), but some evidence indicates 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 Ci 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 associated with photosynthesis, Ci fixation, and N2 fixation were also labeled in Synechococcus spp. Importantly, most proteins of the 3-hydroxypropionate bi-cycle for Ci fixation in Roseiflexus spp. were labeled, establishing that members of this taxocene contribute to Ci 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 Ci 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 Ci fixation. Because this pathway imparts a lower degree of selection of isotopically heavy Ci 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.},
doi = {10.1128/AEM.01829-19},
journal = {Applied and Environmental Microbiology},
number = 7,
volume = 86,
place = {United States},
year = {2020},
month = {3}
}

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