Osmoadaptative Strategy and Its Molecular Signature in Obligately Halophilic Heterotrophic Protists

Abstract

Halophilic microbes living in hypersaline environments must counteract the detrimental effects of low water activity and salt interference. Some halophilic prokaryotes equilibrate their intracellular osmotic strength with the extracellular milieu by importing inorganic solutes, mainly potassium. These “salt-in” organisms characteristically have proteins that are highly enriched with acidic and hydrophilic residues. In contrast, “salt-out” halophiles accumulate large amounts of organic solutes like amino acids, sugars and polyols, and lack a strong signature of halophilicity in the amino acid composition of cytoplasmic proteins. Studies to date have examined halophilic prokaryotes, yeasts, or algae, thus virtually nothing is known about the molecular adaptations of the other eukaryotic microbes, that is, heterotrophic protists (protozoa), that also thrive in hypersaline habitats. We conducted transcriptomic investigations to unravel the molecular adaptations of two obligately halophilic protists, Halocafeteria seosinensis and Pharyngomonas kirbyi. Their predicted cytoplasmic proteomes showed increased hydrophilicity compared with marine protists. Furthermore, analysis of reconstructed ancestral sequences suggested that, relative to mesophiles, proteins in halophilic protists have undergone fewer substitutions from hydrophilic to hydrophobic residues since divergence from their closest relatives. These results suggest that these halophilic protists have a higher intracellular salt content than marine protists. However, absence of the acidic signature of salt-in microbes suggests that Haloc. seosinensis and P. kirbyi utilize organic osmolytes to maintain osmotic equilibrium. We detected increased expression of enzymes involved in synthesis and transport of organic osmolytes, namely hydroxyectoine and myo-inositol, at maximal salt concentration for growth in Haloc. seosinensis, suggesting possible candidates for these inferred organic osmolytes.

A fundamental difference between macrobiota and microbial eukaryotes: protistan plankton has a species maximum in the freshwater‐marine transition zone of the Baltic Sea

“…The physiological adaptations, which govern the distribution of different taxon groups in different salinity regimes are largely unknown. First molecular studies show versatile cellular responses of different protists to salinity shifts (Harding et al, 2016;Skarlato et al, 2018;Weinisch et al, 2018a;Weinisch et al, 2018b). One observed strategy is the exclusion of salt ions from the cytoplasm while acquiring or synthesizing concentrations of organic compatible solutes.…”

Recent Advances in Halophilic Protozoa Research

“…First, we detected a similar amino acid bias in their predicted cytoplasmic proteomes, which leads to increased hydrophilicity compared to proteomes from marine protists (Harding et al. ). Since the hydrophobic effect increases with salinity, proteins exposed to high salt require fewer hydrophobic interactions to avoid over‐rigid conformations and protein–protein aggregation (Elcock and McCammon ).…”

“…However, this hydrophilic signature was not coupled to an acidic signature, as commonly detected in prokaryotic halophiles that accumulate high levels of inorganic osmolytes like potassium ions (Harding et al. ). We interpreted this observation as indirect evidence for a significant contribution by organic solutes to achieving osmotic equilibrium with the extracellular solvent, and speculated that these protozoa are fundamentally “salt‐out” halophiles.…”

“…Concordantly, at high salt, H. seosinensis up‐regulates genes putatively involved in organic osmolyte synthesis and transport (Harding et al. ). For example H. seosinensis expressed all genes required to synthesise of ectoine and hydroxyectoine (two compounds commonly used as osmolytes by bacteria), and the gene coding for ectoine hydroxylase, which converts ectoine to hydroxyectoine, was some 200‐fold over‐expressed at 30% salt compared to 15% salt.…”

Mitochondrial Genome Evolution and a Novel RNA Editing System in Deep-Branching Heteroloboseids

“…Pharyngomonas kirbyi strain AS12B was grown at 12.5% salt at 37 °C as described in Harding et al (2016). Total RNA was isolated from BB2 cells harvested using TRIzol (Rio et al 2010) following the manufacturer’s instructions (Ambion), and treated with Turbo DNAse (Ambion) to remove residual DNA.…”