2.1 Bacterial Cell
Cultures
- Bacterial strain: The methods presented here use therpfC
mutant (ΔrpfC) of the Gram-negative bacteriumX.campestris
pv.campestris(Xcc) strain XC1 [10]. The mutant strainΔrpfC
overproduces DSF family signals (seeNotes 1and 2 ). - NA broth: 5 g/l Peptone, 3 g/l beef extract, 10 g/l sucrose,
1 g/l yeast extract. Fill up to 1 l with bidistilled H 2 O and adjust
the pH to 6.8 with NaOH. Sterilize the medium by autoclaving
for 20 min at 115C. - NA agar: NA broth plus 15 g/l agar. Also in this case fill up to
1 l with bi-distilled H 2 O and adjust the pH to 6.8 with NaOH.
Sterilize the medium by autoclaving for 20 min at 115C. - 50 ml and 250 ml glass Erlenmeyer flasks.
- Heated/refrigerated shaker.
- 1.5 ml Cuvette.
- UV-visible spectrophotometer.
- Rifampicin stock: 50 mg/ml in ethanol.
2.2 Extraction of DSF
Family Signals
- pH test strips (pH 0.0–6.0).
- 1.5, 5, and 50 ml centrifuge tubes.
- Refrigerated centrifuge capable of 13,800gand solvent-
resistant tubes. - Ethyl acetate (ACS reagent grade, purity99.5%) containing
0.1% (v/v) acetic acid (store in the dark in volatile chemical
storage). - Rotary evaporator or centrifugal evaporator.
- Circulation cooling-water system.
- Vortex mixer.
- 0.2μm Syringe filters.
- Acetic acid (ACS reagent grade, purity99.7%).
2.3 DSF and BDSF DSF (cis-11-methy-dodecenoic acid) (HPLC grade, purity
90.0%); BDSF (cis-2-dodecenoic acid) (HPLC grade, purity
90.0%) (seeNote 3). The stock solutions (5 mM) are prepared
using methanol as solvent. The stock solution is further diluted
using methanol to get a range of diluted DSF or BDSF solutions
(1μM, 5μM, 10μM, and 50μM, respectively) for UPLC analysis.
2.4 UPLC/MS
Analysis
- 1.5 ml Tubes.
- HPLC screw-cap vials.
- Polypropylene flat-bottom inserts for HPLC screw-cap vials.
- Pipette tips (volume ranges 50–1000μl).
UPLC/MS Analysis for DSF-Family Signals 99