Quorum Sensing

4. The concentration of DSF and BDSF in the extract is calculated
   based on the peak area and the standard curve as shown in
   Fig.5.

4 Notes

1. In this protocol, the DSF-overproducing strainΔrpfCwas used
   as an example. This protocol is also applicable for extraction of
   DSF family signals from the cell cultures of other bacterial
   species. The QS signals are usually biosynthesized in a growth
   phase-dependent manner. As a result, to detect DSF family
   signals in cell cultures, it is essential to collect cell cultures in
   the right growth stage (usually early stationary phase). There-
   fore, it is advised to do pilot experiments to determine the
   optimal growth stage for the extraction of DSF family signals.


2. As mass spectrometry is a very sensitive technique, a DSF-
   deficient strain is recommended to be included as a negative
   control for DSF extraction and quantification in each individual
   experiment. For example, the DSF-deficient mutantΔrpfFof
   Xcc[10] could be used as a control.


3. DSF and BDSF signal molecules can be purchased from Sigma-
   Aldrich.

Fig. 2Typical MS spectrum of BDSF. (a) Ion chromatograms of BDSF in the

culture supernatant ofΔrpfCstrain (20 times concentrated). (b) MS analysis

showing an exact molecular weight of 198.1543 Dalton for the detected BDSF

signal

102 Lian Zhou et al.