Quorum Sensing

Allow the MgSO 4 to sediment at the bottom of the beaker and

then filter.

6. Remove the solvent by rotary evaporation at room
   temperature.


7. Resuspend the remaining brown oil in 5 ml of 100% methanol
   and transfer to three 2-ml plastic tubes. Dry the solution using
   a speedy vacuum concentrator and resuspend in a total volume
   of 50–150μl of 100% methanol per each tube. Make sure that
   the entire remaining brown oil dissolves, by gentle vortexing
   for 5–10 s. Pool all the samples.

3.3 Detection
of Butyrolactones:
Antibiotic Bioassay

1. Prepare plates of SMMS (25 ml/100 mm diameter plate).


2. From the M145 spore stock (seeSubheading2.3,item 2), add
   108 spores of M145/plate, diluted in H 2 O to 100μl final
   volume, and spread evenly over the plate using sterile cotton
   buds (seeNote 7).


3. Under a sterile laminar flow hood, allow plates to dry for 3 min
   at room temperature, keeping the lid open.


4. Add 2–3μl of methanol extract to the dried plates by careful-
   ly pipetting a drop on the center of the plate and let dry for
   1 min. If more sample needs to be added, because of the
   low expected concentration of GBL, repeat the operation. Do
   not add more than 3μl of sample at once (seeNotes 8and 9 ).
   When necessary (e.g., when comparing the relative bioactivity
   of different GBLs or serial dilutions of methanol extract),
   multiple samples can be tested on the same plate, ensuring
   that enough space is left between them (around 5 cm) so that
   there is no interaction between different diffusing GBL
   samples.


5. Incubate at 30C for 16–18 h. Evaluate precocious antibiotic
   production. This can be seen as a red halo surrounding the area
   where the sample was applied (Fig.2), due to the induced
   production of prodigiosins. Record results using a conven-
   tional flatbed scanner or camera.


6. If no antibiotic production was detected after 18 h, check plates
   every 2–3 h to monitor precocious antibiotic production, until
   the onset of prodigiosin production, which would result in all
   the plate acquiring a red color. Use a conventional scanner to
   record results on final plates as soon as precocious antibiotic
   production is evident (Fig.2).


7. The concentration of GBLs can be approximately determined
   by interpolating the diameter of the red antibiotic halo of the
   sample to a calibration curve obtained by performing the bio-
   assay using standards of known concentration (e.g., purified
   SCB1).

Butyrolactones inStreptomyces 123