ABSTRACT: Sodium chloride is an important ionic-osmotic stressor in bulk and rhizosphere soils. Members of the alphaproteobacterial genus Methylocystis are known to have a low salt tolerance (≤ 1.0% NaCl),despite the fact that these methane oxidizers are soil inhabitants. We therefore tested various amino acids and other well-known osmolytes for their potential to act as a compatible solute or osmoprotectant under otherwise growth-inhibitory NaCl conditions. Besides ornithine, the addition of 10 mM asparagine to the medium had the greatest stress relief effect under severe salinity (1.50% NaCl), leading to a partial growth recovery of strain SC2. The analysis of its exo-metabolome revealed that asparagine was taken up quantitatively. This resulted in an intracellularconcentration of 264 ± 57 mM asparagine, with a certain portion hydrolyzed to aspartate (4.20 ± 1.41 mM). In addition to general and oxidative stress responses, the uptake of asparagine induced 34 major proteome rearrangements related to the KEGG level 2 categories energy metabolism, amino acid metabolism, and cell growth and death. In particular, various proteins involved in cell division (e.g., ChpT, CtrA, PleC, FtsA, FtsH1) and peptidoglycan synthesis showed a positive expression response. Asparagine-derived 13C-carbon was incorporated into nearly all amino acids. Both the exo-metabolome and the 13C-labeling degree suggests that in addition to aspartate, the amino acids glutamate, glycine, serine, and alanine, but also pyruvate and malate, were most crucially involved in the osmoprotective effect of asparagine, with glutamate being a major hub between the central carbon and amino acid pathways. In summary, asparagine induced significant proteome rearrangements, leading to major changes in the flux of central metabolites and free amino acids. In consequence, asparagine acted, in part, as a carbon and nitrogen source for the growth recovery of strain SC2 under severe salinity (1.50% NaCl).
Timo Glatter 
