Physiological Nitrogen Isotopic Responses in Paracoccus denitrificans
The natural abundances of stable isotopes in our environment serve as intrinsic tracers that are incorporated into biological compounds and create unique isotopic signatures that record molecular history. Use of stable isotopes in the investigation of biochemical, geological, and forensic applications has become increasingly popular in the past two decades, in large part due to the recent development of requisite instrumentation capable of measuring the subtle variations of naturally occurring isotope ratios. Progress towards a universal model describing the behavior of isotopes in physiology has seen little consolidation due to the empirical nature of isotopic response. Elucidation of underlying mechanisms requires tightly controlled experiments with careful isotopic characterization of specific physiological states. We exploit the environmentally-relevant and nutritionally versatile bacteria, Paracoccus denitrificans, to assess the fractionation of nitrogen in the metabolism of amino acids as a means to deduce physiological state. The manipulation of respiratory state and osmotic stress yields distinctive distributions of relative isotopic enrichment which is indicative of changes in metabolic pathways. We report the first in vivo position-specific measurement of the poly-nitrogenous amino acid, Lysine, and note a dramatic deviation in isotope ratios between sidechain and peptide nitrogen, which is correlated with respiratory state. In aerobic cells, the lysine intramolecular delta-delta-15N was negligible, but in anaerobic cells it was a remarkable delta-delta-15N = +11.0 permil, driven predominantly by enrichment at the peptide N. With regard to osmotic stress, we report a linear response with salt concentration in nitrogen isotope ratios of several amino acids, indicating an isotopic enrichment in amino acids which exhibit known osmoprotective properties. Bulk 15N/14N increased by delta-delta-15N = +5.603 ? 0.79 permil from 1% to 1.75% NaCl and significant linear trends of delta-delta-15N values with salt concentration between 1% and 1.75% (p<0.05) were found for five amino acids, Ala (delta-delta-15N = 4.66 ? 0.23 permil), Gly (delta-delta-15N = 4.03 ? 0.84 permil), Val (delta-delta-15N = 3.59 ? 0.32 permil), Pro (delta-delta-15N = 6.13 ? 0.76 permil), and Asx (delta-delta-15N = 5.27 ? 0.56 permil). These findings illustrate the sensitivity of isotope ratios in amino acids to physiological state and establish a premise for diagnostic techniques to assess metabolism.
Nitrogen; Isotopic Fractionation; Isotope Ratio Mass Spectrometry; Intramolecular; Position-Specific; Paracoccus denitrificans
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