Uninoculated Nirogacestat purchase growth media were used as the negative control in all cases. Identification of transformation products Extraction and analytical methods Culture supernatants were subjected to organic extraction according to previously published procedures . Briefly, culture supernatants were extracted with an equal volume of ethyl acetate at neutral pH, the organic layer was carefully separated and the remaining aqueous phase then acidified to pH 2.0 with 5 M HCl and again extracted with an equal volume of ethyl acetate. The neutral and acidic organic layers (extracts) were pooled together, evaporated to dryness with a rotary evaporator (BUCHI-Postfach, ISRIB Flawil, Switzerland) and then dissolved
in 150 μl of ethyl acetate. The latter was then subjected to thin layer chromatography (TLC) and gas chromatography (GC) using standard procedures. The identity of transformation intermediates was ascertained by comparing the Rf and Rt values obtained from the TLC and GC analyses respectively to those of authentic standards. Uninoculated media were used as controls for abiotic transformation of test CNACs. Culture supernatants were also subjected to high performance liquid chromatography (HPLC) using a Waters 600 model (Waters, Millford USA) equipped with a Waters 996 photodiode array detector. Detection of the
transformation intermediates was carried out by scanning the samples at 210-390 nm. Sample separation was carried out using a Waters Spherisorb 5 μm C8 reverse phase column as the stationary phase and 1% glacial acetic acid in methanol and 1% glacial acetic acid in the learn more ratio 80:20 at a constant flow rate of 1.0 ml.min-1 as the mobile phase. The identity of peaks was established by comparison of UV-visible spectra and retention times (Rt) to those for the peaks obtained from standard compounds. Chemotaxis of strain SJ98 towards CNACs The chemotactic behaviour of strain SJ98 towards test CNACs was investigated qualitatively with drop plate and swarm plate assays and quantitatively with capillary assays according to procedures described earlier [9, 20, 30]. check details Competitive capillary
assays were also conducted to determine the effect of co-occurrence of potential chemotactic competitors on the chemotactic behaviour of strain SJ98 towards the CNACs. Drop plate assay Cells were grown in MM plus 10 mM glucose, MM plus the test CNAC, or MM plus both the test CNAC and 10 mM glucose. The concentration of CNACs in the growth medium was set at the optimum value (i.e., eliciting the strongest chemotactic response in the quantitative capillary assays described below). The cells were harvested at mid-log phase (OD600 ~0.35) by centrifugation at 3500 rpm for 8-10 min. Harvested cells were washed twice with phosphate buffered saline (PBS), resuspended in drop plate assay medium (MM plus 0.3% bacto agar) and poured into 96 mm petri-plates.