Abstract
Hypersaline environments pose major challenges to their microbial residents. Microorganisms have to cope with increased osmotic pressure and low water activity and therefore require specific adaptation mechanisms. Although mechanisms have already been thoroughly investigated in the green alga Dunaliella salina and some halophilic yeasts, strategies for osmoadaptation in other protistan groups (especially heterotrophs) are neither as well known nor as deeply investigated as for their prokaryotic counterpart. This is not only due to the recent awareness of the high protistan diversity and ecological relevance in hypersaline systems, but also due to methodological shortcomings. We provide the first experimental study on haloadaptation in heterotrophic microeukaryotes, using the halophilic ciliate Schmidingerothrix salinarum as a model organism. We established three approaches to investigate fundamental adaptation strategies known from prokaryotes. First, proton nuclear magnetic resonance (1H-NMR) spectroscopy was used for the detection, identification, and quantification of intracellular compatible solutes. Second, ion-imaging with cation-specific fluorescent dyes was employed to analyze changes in the relative ion concentrations in intact cells. Third, the effect of salt concentrations on the catalytic performance of S. salinarum malate dehydrogenase (MDH) and isocitrate dehydrogenase (ICDH) was determined. 1H-NMR spectroscopy identified glycine betaine (GB) and ectoine (Ect) as the main compatible solutes in S. salinarum. Moreover, a significant positive correlation of intracellular GB and Ect concentrations and external salinity was observed. The addition of exogenous GB, Ect, and choline (Ch) stimulated the cell growth notably, indicating that S. salinarum accumulates the solutes from the external medium. Addition of external 13C2-Ch resulted in conversion to 13C2-GB, indicating biosynthesis of GB from Ch. An increase of external salinity up to 21% did not result in an increase in cytoplasmic sodium concentration in S. salinarum. This, together with the decrease in the catalytic activities of MDH and ICDH at high salt concentration, demonstrates that S. salinarum employs the salt-out strategy for haloadaptation.
Highlights
Salinity is a decisive environmental determinant of microbial community composition and dispersal, exerting a high evolutionary selection pressure [1]
We provide the first experimental evidence for the biosynthesis and accumulation of two compatible solutes that the ciliate uses to combat increasing salt concentrations
Glycine betaine, is widely distributed across kingdoms, while the other, ectoine, has to date only been identified in prokaryotes
Summary
Salinity is a decisive environmental determinant of microbial community composition and dispersal, exerting a high evolutionary selection pressure [1]. Hypertonic conditions induce an osmotic gradient, which lowers the relative water content of a cell, leaving behind a highly concentrated cytoplasm. In such a cell environment, nucleic acids, proteins, and other macromolecules denature and lose their functions [7]. Proteins form aggregates, precipitate [8], and disrupt their tertiary structure. Enzymes lose their flexibility and, their catalytic activity [9]. Flourishing communities of archaea, bacteria, and microbial eukaryotes are found in various hypersaline environments on Earth [10]
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