employlacZas a reporter gene and require supplementation of
X-gal to the medium. A comparison of the sensitivity of theC.
violaceumCVO26 assay to the aforementionedA. tumefaciens
bioassays is shown in Fig.4. Here, the CVO26 assay readily
shows the presence of C6-HSL, whereas this AHL is weakly
detected by strain KYC55 (PJZ372)(PJZ384)(PJZ410) and
not detected by strain A136 (PCF218)(PCF372) (Fig.3a). In
contrast, the twoA. tumefaciensbiosensors readily detect their
cognate AHL (3-o-C8-HSL), whereas CVO26 does not.- Pigmentation inhibition (Fig.3d) can give a useful indication
of potential QS inhibition. However, the CviR transcriptional
activator (LuxR homologue) inC. violaceumcan be blocked by
other AHLs (Fig.4c)[33] and so identification of inhibitory
substances should not be made on the basis of aC. violaceum
bioassay alone. We recommend that potential QS inhibitors be
screened for AHLs with a broad-range bioassay system such as
theA. tumefaciensreporter strains mentioned in Subheading
2.2(above). - If using a solvent to extract AHLs (typically acidified ethyl
acetate) or potential QS-inhibiting compound (usually an
organic solvent such as acetone), test the solvent on the bioas-
say strain to ensure that any pigmentation or viability alteration
due to the solvent is measured. As a safety matter, wear gloves
(we recommend nitrile gloves) and perform any solvent-based
studies in a fume hood.
Acknowledgements
We are grateful to Clay Fuqua and Steve Winans for providing these
strains and introducing us to quorum signaling. We are also grateful
to Karin Sauer for introducing us to biofilm dispersion. Work in
RJCM’s laboratory is supported by grants from NASA
(NNX17AC79G), the Texas State University Research Enhance-
ment Program, and the Biology Department at Texas State
University.References
- Chu W, Vattem DA, Maitin V, Barnes MB,
McLean RJC (2011) Bioassay of quorum sens-
ing compounds usingAgrobacterium tumefa-
ciens and Chromobacterium violaceum. In:
Rumbaugh K (ed) Quorum sensing methods
and protocols. Springer, New York, NY, pp
3–19 - Nealson KH, Platt T, Hastings JW (1970) Cel-
lular control of the synthesis and activity of the
bacterial luminescent system. J Bacteriol
104:313–322- Ng WL, Bassler BL (2009) Bacterial quorum-
sensing network architectures. Annu Rev
Genet 43:197–222 - Whiteley M, Lee KM, Greenberg EP (1999)
Identification of genes controlled by quorum
sensing inPseudomonas aeruginosa. Proc Natl
Acad Sci U S A 96:13904–13909
Bioassays of Quorum and Biofilm Dispersion Signals 21