Culture conditions and chemicals
Rhodococcus opacus PWD4 (DSM 44313) (Duetz et al. 2001) was cultivated in a mineral medium as described by Hartmans et al. (1989), with 4 g/L disodium succinate as carbon and energy source. Cells were grown in 50 mL liquid cultures at 30 °C in a horizontally shaking water bath at 180 rpm. All chemicals were reagent grade and obtained from commercial sources.
The stressors were added to the cultures in the early exponential growth phase (OD560nm between 0.4 and 0.6). Cells were harvested 2 h after stressor application for fatty acid analysis and determination of water contact angle. The cell suspensions were centrifuged at 10,000g for 10 min at 4 °C. The cell pellets were frozen for fatty acid analysis or stored at 4 °C for water contact angle measurements.
Toxicity of the stressors added to cells growing in the early exponential phase was estimated by the effective concentration 50 % (EC50), i.e. the concentration that causes a 50 % inhibition of bacterial growth as described earlier by Heipieper et al. (1995). Growth inhibition caused by the toxic compounds was measured by comparing the differences in growth rate μ (h−1) between intoxicated cultures (µstressor) with that of control cultures (µcontrol). The growth inhibition of different concentrations of stressors was defined as the percentage of the growth rates of intoxicated cultures and that of control cultures without stressor addition.
Mycolic acid preparation
For the mycolic acid analysis we used a protocol modified from Sokolovská et al. (2003). Therefore, 30–50 mg of cells harvested in the late exponential growth phase were resuspended in 2 mL of minimal media. An equal volume of 25 % KOH (w/w) in 50 % ethanol–water (v/v) was added to the suspension. The mixture was then incubated for 1 h at 120 °C. Afterwards, 1.75 mL of concentrated HCl were added to the solution and the fatty acids and the mycolic acids were extracted with 3 mL dichloromethane for 3 min on a Vortexer at room temperature. This extraction procedure was done three times. The organic phases were combined and evaporated under nitrogen flux until a volume of approximately 1 mL and then transferred to a GC-vial and therein evaporated to dryness. 100 µL of BSTFA derivatization reagent and 50 µL of pyridine were added to the dried fatty acids and softly shaken. The mixture was incubated at 80 °C for 20 min. After the incubation, the mixture was evaporated under nitrogen flux. To purify the reaction product, 1 mL of benzene was added to the dried sample and evaporated under nitrogen flux. This purification step was repeated once. The purified, dried sample was taken up in 100 µL hexane and transferred into a GC-vial with micro insert. The sample was stored until analysis at −20 °C.
Gas chromatographic analysis of mycolic acids
Mycolic acids were analyzed on an Agilent 5975T GC–MS System (AgilentTechnologies, Santa Clara, CA, USA). The column used was a Zebron ZB-1MS capillary column (Phenomenex Inc, Torrance, CA, USA; length, 12 m; inner diameter, 0.20 mm; 0.33 µm film thickness), 100 % Dimethylpolysiloxane, 30 m in length, with an internal diameter of 0.18 mm and 0.18 µm of film thickness. Helium was used as mobile phase and 1 µL of sample was injected for analysis in splitless mode, with injector temperature set at 140 °C. The oven temperature program consisted of an initial steady temperature plateau at 50 °C for 1 min, followed by two consecutive ramps at 10 °C/min from 50 to 120 °C, and at 3 °C/min from 120 to 360 °C. Post run column regeneration was performed at 360 °C for 5 min.
Lipid extraction, transesterification, and fatty acid analysis
The lipids were extracted with chloroform/methanol/water as described by Bligh and Dyer (1959). Fatty acid methyl esters (FAME) were prepared by incubation for 15 min at 95 °C with boron trifluoride/methanol according to the method of Morrison and Smith (1964). FAME were extracted with hexane and analysed by GC-FID.
Analysis of fatty acid composition by GC-FID
Analysis of FAME in hexane was performed using a HP5890 GC System (Hewlett & Packard, Palo Alto, USA) equipped with a split/splitless injector and a flame ionisation detector (FID). A CP-Sil 88 capillary column (Chrompack, Middelburg, The Netherlands; length, 50 m; inner diameter, 0.25 mm; 0.25 µm film thickness) was used for the separation of the FAME. GC conditions were the following: injector temperature was held at 240 °C, detector temperature was set at 270 °C. The injection was made in splitless mode, and the carrier gas was He at a flow of 2 mL min−1. The oven temperature program was as follows: 40 °C, 2 min isothermal; 8 °C/min to 220 °C; 15 min isothermal at 220 °C. The peak areas of the FAMEs were used to determine their relative amounts. The fatty acids were identified by GC and co-injection of authentic reference compounds obtained from Supelco (Bellefonte, USA). The degree of saturation of fatty acids was defined as the ratio between the saturated fatty acids (C16:0, C17:0, C18:0) and unsaturated fatty acids (C16:1Δ9cis, C18:1Δ9cis) present in this bacterium.
Characterization of bacterial cell surface hydrophobicity
Physico-chemical cell surface properties of bacterial cells were investigated using water contact angle measurements as described by others (Van Loosdrecht et al. 1987). Bacterial lawns needed for contact angle (θw) measurements were prepared by collecting cell suspensions in 10 mM KNO3 on 0.45-μm pore-size Micropore filters (Schleicher & Schuell, Dassel, Germany), mounting the filters on glass slides, and drying them for 2 h at room temperature. Cell surface hydrophobicities were derived from θw of water drops on the bacterial lawns using a Krüss drop shape analysis system DSA 100 (Krüss GmbH, Hamburg, Germany). According to an earlier classification, cells exhibiting contact angles of θw < 20°, 20° ≤ θw ≤ 50° and θw > 50° were considered hydrophilic, intermediately hydrophilic and hydrophobic, respectively (Van Loosdrecht et al. 1987).
All experiments were carried out in triplicate. All figures show mean values with the corresponding standard deviations as error bars.