Gf 75

Sample water was collected at a station in the western Black Sea located at 42° 53.8′ N 30° 40.7′ E during a cruise with the research vessel R/V Pelagia in August 2018. Water column profiling was conducted using an ultra‐clean conductivity‐temperature‐density (CTD) system, which was equipped with among others a SBE3plus thermometer, SBE4 conductivity sensor and SBE43 dissolved oxygen sensor (Sea‐Bird Electronics, Bellevue, WA). Water samples used for bottle incubations were obtained using clean 25 l bottles that were connected to N₂‐gas immediately after boarding of the sampler to minimize contact with oxygen. To analyse the total water column microbial community composition, SPM samples were collected with in situ pumps (McLane Laboratories, Falmouth, MA), by filtering through 142‐mm‐diameter 0.3 μm pore‐size glass fibre GF75 filters (Advantec MFS., Dublin, CA) and immediately stored at −80°C. While this filter size will potentially miss ultrasmall prokaryotic cells, it is in the range of filter sizes generally used to collect free‐living cells (0.2 μm) compared with the filter sized used to collect particle‐attached microbial communities (2.7 μm/30 μm; Fuchsman et al., 2011 (link), Suter et al. 2018), and therefore approximates the total microbial community residing in the water column.

Using our previously published methods^{41 (link),42 (link)}, AhEcR_S, AhEcR_R, and AhUSP-1 were translated in vitro from their corresponding cDNAs inserted in the plasmid pTnT using TNT Coupled Reticulocyte Lysate Systems (Promega Corp.), from their corresponding cDNAs inserted in the plasmid pTnT according to the vendor protocol. The details of “Cloning and sequencing of AcEcRB1 cDNAs and construction of expression plasmids” are described in Supplementary methods. In vitro translated AhEcR (_S or _R) and AhUSP-1, test compound, and PonA (25,000 dpm/tube) were mixed in the siliconized tube. After incubating the mixture at 25 °C for 60 mins, the reaction mixture was immediately filtered through a glass filter (GF-75; ADVANTEC) with the aid of a vacuum pump. Filters were washed three times with washing buffer and transferred to a vial containing 3 ml of Aquasol-2 (Perkin-Elmer) to measure the radioactivity in an Aloka LSC-6100 liquid scintillation counter (Aloka). A 1000-fold excess of unlabeled PonA was added to measure nonspecific binding, and total binding was obtained for the treatment with a carrier (DMSO or EtOH).