## 3 Materials and Methods
### Detection of (+)-Ectoine in Fermentation Broths  
**Sample collection and bacteria isolation. ** Marine-derived _Streptomyces_ were isolated from three different invertebrates and sediments collected at several locations in the Adriatic Sea, Croatia (Table S2). Briefly, samples were collected in sterile bags which were kept on ice and processed immediately upon arrival to the laboratory. Marine invertebrates were washed several times in sterile seawater and approximately two grams of sponge tissue and tunicate or mussel digestive system were macerated and incubated in 10 volumes of sterile seawater. Sea sediments (5 g) were transferred into 5 mL of sterile seawater and vigorously shaken for about 10 min. The supernatants were 10-fold serially diluted (\(10^{-2}\), \(10^{-3}\), \(10^{-4}\), \(10^{-5}\)) and 100 \(\mu\)L of each dilution was plated on ISP2 plates supplemented with cycloheximide (100 \(\mu\)g/mL) and nalidixic acid (25 \(\mu\)g/mL). Plates were incubated at 12 \({}^{\circ}\)C for two to three weeks, and the appearance and growth of colonies was monitored regularly. **Selection and molecular identification of salt-tolerant _Streptomyces_. ** Colonies that morphologically resemble streptomyces (velvety surface, ingrowth into agar medium, production of diffuse pigment) were selected, and a pure culture was obtained from a mixed population using the streak-plate method. The obtained isolates were further tested for salt tolerance by inoculation on ISP2 plates containing 3, 6 or 9% (\(w/v\)) NaCl. Plates were incubated at 30 \({}^{\circ}\)C for five days and inspected for growth. Strains tolerant to 9% (\(w/v\)) NaCl were inoculated in nutrient broth (Biolab Diagnostics Laboratory Inc., Budapest, Hungary) and incubated in a heated shaker (200 rpm) at 30 \({}^{\circ}\)C for three days. Cells were harvested by centrifugation (4 \({}^{\circ}\)C, 6000\(\times\)\(g\), 10 min) and total genomic DNA was extracted with Duseasy Blood and Tissue Kit (Qiagen, Venlo, The Netherlands). PCR amplification of the 16S rDNA gene sequence was performed using the universal electrical primers 27F (5\({}^{\prime}\)-GAGTTTGATCCTGGCTCAG-3\({}^{\prime}\)) and 1492R (5\({}^{\prime}\)-GGTTACCTTGTTACGATT-3\({}^{\prime}\)) [34]. Amplified PCR products were visualized by agarose gel electrophoresis, purified with QI-Aquick PCR Purification Kit (Qiagen, The Netherlands) and sequenced using 27F primer at Macrogen Europe, Amsterdam, The Netherlands. The obtained sequences were analyzed using NCBI BLAST tool ([[http://www.ncbi.nlm.nih.gov/BLAST](http://www.ncbi.nlm.nih.gov/BLAST)]([http://www.ncbi.nlm.nih.gov/BLAST](http://www.ncbi.nlm.nih.gov/BLAST)), accessed on 31 August 2024). **Growth conditions for bacterial isolates and the HPLC analysis of (+)-ectoine. ** Salt-tolerant marine _Streptomyces_ strains (\(n=11\)) and reference strain _Streptomyces_ _ecolicor_ A3(2) were inoculated in 10 mL of minimal liquid medium [35] supplemented with the indicated NaCl concentrations. The bacteria were first kept at room temperature for 24 h without shaking, then transferred to a shaker (30 \({}^{\circ}\)C, 200 rpm) and grown for 48 h. The bacterial cells were pelleted by centrifugation (4 \({}^{\circ}\)C, 6000\(\times\)\(g\), 10 min), and the supernatant (fermentation broth) was stored at \(-\)20 \({}^{\circ}\)C until analysis. A quantitative analysis of ectoine in samples was performed on an HPLC Shimadzu LC.2010 HTC (Kyoto, Japan) with DAD detector (wavelengths 210 and 220 nm) and a Synergi Polar-RP 80 A column (150 mm \(\times\) 4.6 mm i.d., 4 \(\upmu\)m, Phenomenex, Torrance, CA, USA). The supernatants were filtered through PTFE filters (0.20 \(\upmu\)m, 0.013 mm) without dilution and further processing. A solvent gradient was used for HPLC analysis: 0\(\rightarrow\)7 min 100% H\({}_{2}\)O; 7\(\rightarrow\)20 min 100%H\({}_{2}\)O\(\rightarrow\)100%ACN; 20\(\rightarrow\)25 min 100%ACN; 25\(\rightarrow\)25.01 min 100%ACN\(\rightarrow\)100%H\({}_{2}\)O; 25.01\(\rightarrow\)30 min 100% H\({}_{2}\)O.