Ectoine

The strains and plasmids constructed and used in this study are listed in Table 1. All restriction digest enzymes, DNA polymerase and DNA ligase were obtained from Takara Bio Inc. (Dalian, China). Oligonucleotides were synthesized by GENEWIZ Bio Inc. (Suzhou, China). Ectoine was purchased from Sigma-Aldrich (Shanghai, China).

Ethanol (ETH) (96%) was purchased from POCH (Polskie Odczynniki Chemiczne). Ectoine (ECT) of 99% purity produced by Halomonas elongata was purchased from Sigma-Aldrich. Two concentrations: 20 and 60 g/L of ETH alone were selected on the basis of our previous toxicity trials and the toxicological data (Dom et al. 2012 (link)). Exposure of daphnids to these two different concentrations of the toxicant alone allowed us to observe different levels of toxic changes after 24 h. After determination of toxic concentrations of ETH alone, we selected appropriate concentrations of ECT to be used in each combination at which we expected possible protective effects. We used the non-toxic concentrations with documented protective effects of ECT from our previous experiments (Bownik et al. 2014 (link), 2015 (link)). In each treatment, the experimental animals were exposed to ETH alone at concentrations of 20 and 60 g/L and the following combinations of ETH and various concentrations of ECT: (a) 20 g/L ETH + 5 mg/L ECT, (b) 20 g/L ETH + 10 mg/L ECT, (c) 20 g/L ETH + 25 mg/L ECT, (d) 60 g/L ETH + 5 mg/L ECT, (e) 60 g/L ETH + 10 mg/L ECT, and (f) 60 g/L ETH + 25 mg/L ECT. Each solution was renewed after 24 h. The unexposed animals in medium only were treated as the control.

Monomethyl auristatin F (MMAF) was sourced from BrightGene Bio-Medical Technology (China) and the compound BG-modified to generate BG–linker–AURIF by Professor Roger Hunter’s organic synthesis group, Department of Chemistry, University of Cape Town, South Africa. Detailed information about the synthesis, spectroscopic, and analytical data for the BG–linker–AURIF product is provided in Huysamen et al. (2023 (link)) (supplementary information). The purified recombinant fusion protein was incubated for 4 h at room temperature, with a threefold molar excess of BG–linker–AURIF [initially in lyophilized form, BG–linker–AURIF was solubilized in 100% (v/v) dimethyl sulfoxide (DMSO) (Sigma-Aldrich, South Africa), 1 M ectoine (a protein-stabilizing compatible solute (Lippert and Galinski 1992 ) (Sigma-Aldrich, South Africa) in 1 × PBS and 1 mM DTT]. The unconjugated BG–linker–AURIF was removed using 10 K-sized Amicon filters (Sigma-Aldrich, South Africa) according to the manufacturer’s instructions. Since the resulting product cannot be directly visualized, saturation of the binding domain of SNAP-tag with BG–linker–AURIF was ascertained by post-incubation (double conjugation) with a twofold molar excess of BG-Alexa Fluor 488 for 1 h at 37 °C. Next, SDS-PAGE analysis was conducted, and visualization of any potential fluorescence signal was carried out as described previously.

The total osmolyte content was quantified by liquid chromatography mass spectrometry (LC-MS) on extracted samples using an Agilent 6540 UHD Accurate Mass QToF LCMS with 1290 HPLC System. Sample injection volumes were 1 µL, auto-loaded and run on an Agilent InfinityLab Poroshell 120 HILIC-Z column, (2.1 × 100 mm) (1.9 µm × 120 Å pore size) with a pre-programmed ID tag. Separation solvents were solvent A (20 mM NH₄HCO₂ in 100% ddH₂O, pH adjusted to 3.0 with HCOOH) and solvent B (20 mM NH₄HCO₂ in ddH₂O and 1:10 acetonitrile solution, pH adjusted to 3.0 with HCOOH). Electrospray ionisation mass spectrometry (ESI-MS) source settings are listed in Table 2. Aqueous external standards of betaine, proline, ectoine and hydroxyectoine (Sigma-Aldrich), dissolved in ddH₂O and serial diluted to 0.36 µg mL⁻¹ were used to calibrate the data. Acquired data were processed by MassHunter Qualitative Analysis B.05.00. Results were calculated using ratios of the peak area of the analytes to their standards. Results from the analysis were standardised and recorded as a response factor via the following equation.
Response factor (RF) = ((PA_s/PA_x)/T_c) × 10⁹ whereby PA_s is the peak area of sample, PA_x is peak area of known standard signal and T_c is the total cell count of the sample. Significance of differences between the samples were determined using two-sample t-tests.

The adaptation of L. ferriphilum DSM 14647 was performed in growth medium (see above) with increasing NaCl concentrations (50-100-120-150-180 mM) and supplementation with 1 mM ectoine as a compatible solute (Sigma-Aldrich). The adaptation was performed sequentially and with 3 passages per salt concentration. Cultures were maintained until the late exponential phase and used to inoculate fresh NaCl and ectoine-containing medium (10% v/v) and generate a new culture. After the 180 mM NaCl-adapted culture had been obtained, the compatible solute was gradually (1–0.5–0 mM) removed from the medium. Adapted cells were constantly grown in the presence of 180 mM NaCl.

Antibiotic broth medium No. 3 (complex medium) was purchased from Oxoid LTD (Hampshire, Great Britain). Pyruvic acid, sucrose, KH₂PO₄, KOH, and (NH₄)₂SO₄ were purchased from Roth (Karlsruhe, Germany). D-glucose, MgSO₄·7 H₂O, and FeSO₄·7 H₂O were obtained from Merck (Darmstadt, Germany). Trehalose and NaCl were purchased from Fluka (Buchs, Schwitzerland). Ectoine (≥99%) for lactate deydrogenase stress experiments was purchased from bitop AG (Witten, Germany). HydroxyEctoine (≥99%) for lactate dehydrogenase (LDH) stress experiments was isolated from H. elongata strain DSM 2581^T in our laboratories. Freeze-dried LDH (rabbit muscle) and nicotinamide adenine dinucleotide were purchased from Sigma (Steinheim, Germany). Phosphate buffered saline (PBS) was purchased from AppliChem (Darmstadt, Germany). Ectoine (≥99.0%) and hydroxyEctoine (≥98%) for spin lable and Fourier transform infrared (FTIR) experiments were purchased from Sigma (USA). Perdeuterated spin probe Tempone-d₁₆ (4-Oxo-2,2,6,6-tetramethylpiperidine-d₁₆-1-oxyl; Figure 7 inset) was a kind gift of Prof. I. Grigoriev (Institute of Organic Chemistry of the Russian Academy of Sciences, Novosibirsk, Russia).

Adonitol, betaine anhydrous, chlorocholine chloride, choline chloride, D–maltose monohydrate, D–arabinose, D–arabitol, D–glucose, D–mannitol, D–mannose, D–sorbitol, D–threitol, D–xylose, diethylene glycol, ectoine, ethyleneglycol, glycine, L–alanine, L–arabinose, L–arabitol, L–arginine, L–lysine, L–proline, maltose, myo–inositol, sarcosine, taurine, TMAO, xylitol and β–alanine were purchased from Sigma–Aldrich. D–galactose was purchased from Frutarum. D–raffinose and trehalose were purchased from Acros. Glycerol was purchased from Gadot.