Casamino acids

P. aeruginosa PAO1 was propagated on trypticase soy agar (TSA) for plate-based assays or in trypticase soy broth (TSB) for liquid culture. M9 growth media supplemented with 0.4% (w/v) glucose and 0.4% (wt/v) casamino acids was used for biofilm formation experiments. Culture media (TSB, TSA, M9 salts and casamino acids) were obtained from Difco/Becton Dickinson (Franklin Lakes, NJ, USA) and all other reagents (phosphate buffered saline, glucose, ethanol and crystal violet) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Corning 35–1172 flat-bottomed polystyrene 96-well plates were used for biofilm formation experiments and optical density measurements were performed in a Tecan M-200 (Durham, NC, USA) plate reader. Optical micrographs of biofilms were obtained using a Nikon Eclipse 80i microscope.
A microplate based assay, modified from Junker et al.[32] (link) was used to screen compounds for QSI. Briefly, P. aeruginosa PAO1 was grown in TSB for 18 h at 37°C with rotary shaking at 225 rpm. The culture was then centrifuged at 14,000 rpm and rinsed with phosphate buffered saline (PBS, pH 7.4) three times, then was re-suspended in M9 minimal growth media to approximately 1×10⁷ cfu/ml (determined by OD and plate count assay). Test compounds were dissolved in DMSO and were added to sterile distilled water to achieve concentrations ranging from 0.1–10 mM while keeping DMSO at a maximum of 1% (v/v). P. aeruginosa inocula (360 µl) were then pre-mixed with 40 µl of the test compound solutions to achieve final compound concentrations ranging from 0.01–1 mM. An aliquot (100 µl) of this cell/compound mixture was then added to three separate wells in a 96-well microplate for replicate testing. For control wells (no inhibitor), dilute DMSO was added to the inocula instead of test compounds, to a final concentration of 1% (v/v). Optical density (OD_600nm) measurements were performed immediately after inoculation and after 24 h incubation at 37°C (without shaking) to monitor planktonic cell growth. To determine the amount of biofilm formation, supernatant from the microplate wells was gently removed and the wells were washed twice with 150 µl of PBS using a multichannel pipette. The remaining biofilm was then stained using 100 µl of a 0.2% (w/v) crystal violet solution for 15 min at room temperature. The crystal violet was then removed from the wells, the wells were rinsed four times with PBS, and then 100 µl of 95% ethanol was added to extract the crystal violet solution from the biofilm. The OD_600nm of the extracted crystal violet was then measured, yielding a measure of biofilm formation (relative to the control). For optical imaging, crystal violet stained biofilms were washed with distilled water and no ethanol extraction was performed.
In addition to crystal violet based quantification of biofilm biomass, cell viability within biofilms exposed to inhibitor compounds was determined using the formazan dye-based MTT assay (Cell Proliferation Kit I, Roche Diagnostics, Mannheim, Germany). This assay has previously been described for determination of biofilm cell viability [43] (link)–[45] (link). Briefly, biofilms were grown in 96 well microplates for 24 h as described above, in the presence and absence of inhibitor compounds. After this initial inoculation period, planktonic cells were removed and the remaining biofilm was gently rinsed three times with 100 µl of PBS. After rinsing, 100 µl of PBS and 10 µl of the MTT labeling reagent were added and the suspension was incubated for 4 h at 37°C, followed by addition of 100 µl of solubilization solution. Plates were then incubated for 24 h at 37°C and absorbance measurements were taken using a Tecan M-200 plate reader at 560 nm (peak absorbance for the formazan dye breakdown product) and at 700 nm (reference wavelength for the intact dye).

Bacteria were precultured in eight media treatments (Jensen’s, 0.25 g/L glucose, 0.5 g/L glucose, 1 g/L glucose, 5 g/L casamino acids, 10 g/L casamino acids, 20 g/L casamino acids, and 2 g/L glucose 20 g/L casamino acids). Fresh aliquots of amended media were then inoculated with precultured cells at a 10% dilution in triplicate. Cultures were then loaded onto a 96-well plate and the plate was loaded into a BioTek Synergy H1 microplate reader where cultures were kept at 25 °C and continually shaken. OD600 was measured at 30-min intervals over 5 days.

A. vinelandii was grown in 5 mL of each media treatment (Jensen’s, 0.25 g/L glucose, 0.5 g/L glucose, 1 g/L glucose, 5 g/L casamino acids, 10 g/L casamino acids, 20 g/L casamino acids, and 2 g/L glucose 20 g/L casamino acids) for 3 days at 25 °C. Cells were washed, harvested and diluted to 10% before being added to fresh aliquots of the amended media treatments in 30 mL serum bottles. Serum bottles were sealed to be gas-tight and then flushed with argon for 10 min. The serum bottle headspace was injected with 10% vol oxygen and 10% vol acetylene. Nitrogen fixation activity was then measured using the acetylene reduction assay method based on acetylene reduction into ethylene by nitrogenase⁴⁴ . Ethylene concentrations were measured over a 5-day time series using gas chromatography.