¹⁵N‐ and ²H proteomic stable isotope probing links nitrogen flow to archaeal heterotrophic activity

Abstract

Understanding how individual species contribute to nutrient transformations in a microbial community is critical to prediction of overall ecosystem function. We conducted microcosm experiments in which floating acid mine drainage (AMD) microbial biofilms were submerged - recapitulating the final stage in a natural biofilm life cycle. Biofilms were amended with either (15)NH4(+) or deuterium oxide ((2)H2O) and proteomic stable isotope probing (SIP) was used to track the extent to which different members of the community used these molecules in protein synthesis across anaerobic iron-reducing, aerobic iron-reducing and aerobic iron-oxidizing environments. Sulfobacillus spp. synthesized (15)N-enriched protein almost exclusively under iron-reducing conditions whereas the Leptospirillum spp. synthesized (15)N-enriched protein in all conditions. There were relatively few (15)N-enriched archaeal proteins, and all showed low atom% enrichment, consistent with Archaea synthesizing protein using the predominantly (14)N biomass derived from recycled biomolecules. In parallel experiments using (2)H2O, extensive archaeal protein synthesis was detected in all conditions. In contrast, the bacterial species showed little protein synthesis using (2)H2O. The nearly exclusive ability of Archaea to synthesize proteins using (2)H2O may be due to archaeal heterotrophy, whereby Archaea offset deleterious effects of (2)H by accessing (1)H generated by respiration of organic compounds.

Tracking heavy water (D 2 O) incorporation for identifying and sorting active microbial cells

“…This effect has been exploited on the bulk level to monitor lipid biosynthesis of microbes in the environment (35,36). In addition to lipid labeling, proteins of active community members will become deuterated, as demonstrated by metaproteomics (37). Here, we show that deuterium incorporation into individual active microbial cells can easily be detected within seconds by nondestructive Raman microspectroscopy and that the amount of deuterium labeling can reliably be quantified.…”

“…Raman microspectroscopy produces a chemical "fingerprint" of the abundant molecular bonds in individual microbial cells. Because bulk analyses have demonstrated significant incorporation of D into the biomass of cells growing in the presence of heavy water (35,37), we hypothesized that this incorporation might be detectable as a general metabolic activity marker in Raman spectra of single microbial cells. Indeed, Raman spectra of Escherichia coli cultivated in growth water with no, partial, or complete substitution with heavy water exhibited several distinctive shifts ( Fig.…”

“…Lipids from autotrophic and heterotrophic organisms show a predominance of water-derived protons (or D + in the presence of heavy water) (33,35,47) as protons from water are transferred to NAD(P) during its reduction, and these protons are then incorporated into lipids via the known fatty acid biosynthesis pathways (31)(32)(33). D will, of course, also be incorporated to some extent via other processes, such as amino acid and carbohydrate biosynthesis (37,48). Importantly, however, control experiments with paraformaldehyde-fixed cells demonstrated that abiotic H-D exchange reactions, such as occur in amide bonds of proteins (49) or other O-and N-bound H (32), do not contribute measurably to the C-D peak region (SI Appendix, Fig.…”

Microbial metaproteomics for characterizing the range of metabolic functions and activities of human gut microbiota

“…By quantifying abundance changes of heavy isotope-labeled proteins and light isotope-labeled proteins, protein synthesis rates and degradation rates can be determined. Furthermore, by feeding microbial communities with heavy isotope-labeled nutrients, the metabolism of those nutrients results in incorporation of those heavy isotopes into proteomes of different organisms [49,78,79]. The identification of organisms that are involved in metabolism of a specific labeled nutrient and quantification of the extent of incorporation has been automated by the development of advanced new algorithms [49].…”

Monitoring Photosynthetic Activity in Microalgal Cells by Raman Spectroscopy with Deuterium Oxide as a Tracking Probe

“…The measured difference was clear enought od iscriminate the photosynthetic activity andw as, thus, adequate as screening criteria.T he CÀDf ormation under dark conditions in autotrophic medium can be ascribed to biological metabolism,i ncluding fatty-acid biosynthesis mediated by deuterated NADPH [41][42][43] and proton-deuteriume xchange during enzymatic reactions in the glycolytic pathway. [44,45] According to aprevi- ChemBioChem 2017ChemBioChem , 18,2063ChemBioChem -2068 www.chembiochem.org ous report, [36] abiotic HÀDe xchange in ap araformaldehydefixed cell did not measurably contributet ot he CÀDs ignal region, which is consistent with the fact that the CÀDs ignals are exclusively caused by biological processes. Interestingly, the standard deviation of the CÀDf ormation ratio was remarkably largea fter 12 ho fl ight irradiation, which presumably reflects the cell-to-cell heterogeneity of the photosynthetic productivity.T hese resultss how that monitoring the CÀDs ignal intensity allows evaluation of the photosynthetic activity in E. gracilis at hourly intervals.…”