from various non-Streptomycetes [5]. However, even more GBL
synthases and repressors have been identified in genome sequence
data [2, 6], suggesting the presence of more GBLs yet to be discov-
ered. Moreover, recent publications [7, 8] have shown that GBLs
have cross-species activities and signals from oneStreptomycesspecies
can induce antibiotic production in another one; consequently, their
potential role in synthetic biology has been proposed [2].
Butyrolactones are very potent signaling molecules, andStrep-
tomycesspecies produce them in low amounts. Therefore, complete
purification and structural determination (e.g., by NMR) are not
the first choice when attempting to isolate GBLs for bioactivity
assays, as this may require culture volumes of up to 300 l [9].
Instead, we here describe a rapid method for GBL extraction
from solid medium and a small-scale GBL extraction from
liquid medium, which provide sufficient amounts of GBLs for
sensitive bioassays and allow partial structural characterization and
quantification using relatively small quantities of crude extract
using current chromatography and mass spectrometry techniques
[10, 11].
We then describe two bioassays that are used for GBL detec-
tion: the traditional antibiotic bioassay [12] and the kanamycin
bioassay [13]. The antibiotic bioassay is based on the observation
of precocious antibiotic production as a response to the exogenous
addition of GBLs. InS. coelicolorM145, the antibiotic bioassay
measures the enhanced production of pigmented antibiotics prodi-
giosins (RED: red) and actinorhodin (Act: blue) [9, 12]. Other
antibiotic bioassays have been described for other strains, such as
Streptomyces griseus[14], which involve extraction of antibiotic in a
disk and evaluation of antibiotic activity measuring growth inhibi-
tion of aBacillus subtilisindicator strain.
An alternative to the antibiotic bioassay is the kanamycin bio-
assay, developed by Hsiao et al. [13]. This method is based on the
repression ofcpkOpromoter activity by the GBL receptor ScbR
[15]. In the presence of GBLs, this repression is released, allowing
transcription of thecpkOgene, a master regulator of thecpkcluster
[15, 16].
Based on this concept, plasmid pTE1062 (Fig.1), an analogue
of plasmid pTE134 [13], was built and integrated into strain LW16
(M145scbR/A::aac3(IV)), which does not produce butyrolac-
tones, to generate the reporter strain LW18. The pTE1062 plasmid
contains thescbRgene, regulated by its own promoter region, and
theneogene, regulated by thecpkOpromoter. Thus, the reporter
strain is sensitive to kanamycin unless GBLs are present in the
medium. Interestingly, this system allows for detection of GBLs
other than the ones fromS. coelicolor[13], even though a higher
concentration of these GBLs is required.118 Marc Biarnes-Carrera et al.