Quorum Sensing

3.4 Detection
of Butyrolactones:
Kanamycin Bioassay

The kanamycin bioassay is based on the use ofS. coelicolorLW18 or

LW94 strains [13]. Strain LW18 contains a replacement of the

GBL system inS. coelicolorby the apramycin resistance cassette

and a genomic insertion of plasmid pTE1062 (unpublished).

Therefore, this strain does not produce GBLs and will develop

kanamycin resistance in the presence of exogenous GBLs. LW94

is a derivative of LW18, with the apramycin resistance cassette

removed (unpublished).

1. Prepare one DNAgar plate with 5μg/ml kanamycin for each
   sample, one for the negative control (with methanol only), one
   for the positive control (e.g., 0.25μg of SCB1;seeNote 6), and
   one plate of DNAgar without antibiotic, which will be used as
   growth control to check the indicator strain growth.


2. Prepare a LW18 or LW94 spore solution with 2.6 106
   spores/100 μl sterile water (see Subheading2.4, item 1).
   Spread 100μl of the spore solution evenly over each plate
   using sterile cotton buds previously wetted with sterile
   deionized H 2 O to avoid the spores to stick to the dry cotton
   bud. Allow plates to dry for 3 min under a sterile laminar flow
   hood, keeping the lid open.


3. Add 2–3μl of methanol extract and let dry for 1 min (seeNotes
   8 and 9 ). In the negative control, add 2–3μl of methanol and
   let dry for 1 min, and in the positive control add 1μlofa

Fig. 2Antibiotic bioassay results in SMMS medium after incubation at 30C for up to 48 h, using two different
volumes of methanol extract containing GBL fromS. coelicolorM145 compared to a methanol control

124 Marc Biarnes-Carrera et al.