(pJZ410) [30] (listed in Table1) that have become widely used in
the detection of AHLs. These strains lack thetraIgene (AHL
synthesis), overexpress the traR gene, and employ lacZ as a
reporter gene. They require supplementation with aβ-galactosidase
substrate such as X-gal in order to visualize AHL recognition.
These strains recognize a wide range of AHLs [30, 35] with the
KYC55 (PJZ372)(PJZ384)(PJZ410) strain being able to detect a
wider range of AHLs with increased sensitivity [30]. It has been our
experience that all strains will detect their cognate AHL (3-o-C
HSL) at subpicomole concentrations, with other AHLs being
detected in the micromole to nanomole range.A. tumefaciens
bioassays have been incorporated into AHL detection by reverse-
phase thin-layer chromatography [38] and high-performance liq-
uid chromatography [24]. Alternatively, assays forβ-galactosidase
(LacZ gene product) can be employed to gain quantitative data
[30].1.3 Chromo-
bacterium violaceum
as a Biosensor
C. violaceumis a Gram-negative bacterium that produces a purple
pigment, violacein, under regulation of C6-HSL-dependent QS.
N-decanoyl homoserine lactone (C10-HSL) has also been identi-
fied as a signal inC. violaceum[39]. The C6-HSL synthesis and
response regulator genes arecviIandcviR, respectively, and are
homologues ofluxIandluxR[40]. Several strains are commonly
used for bioassays (Table1). These strains allow the direct detection
of short-chain AHLs through the induction of pigmentation in
strain CVO26 [11], which is unable to produce AHLs but is fully
capable of producing violacein in response to its cognate signal
molecule (C6-HSL) or the short-chain C4-HSL. As a positive
control for the bioassay, a C6-HSL-producing, nonpigmented
strain (ATCC 31532), is used in association with the CVO
bioassay strain [33]. Using a plate streaking protocol (Subheading
3.2.1), the pigmentation is readily visible after overnight culture
(Fig.1a) and is absent if C6 HSL is not present (Fig.1b). We have
observed that some pigmentation will occur in older (>48 h)
CVO26 cultures. AHLs can be extracted from cultures (process
described below), synthesized in the lab [26] or alternatively pur-
chased from commercial sources. Several investigators have mod-
ified theC. violaceumassay by extracting the violacein with a
solvent (typically acetone, ethanol, or butanol) and then measuring
absorption using a spectrophotometer [40]. This approach enables
to gain quantitative data from the bioassay.
AHL-based QS is very specific in that the response regulator
(cviRin the case ofC. violaceum) will only respond to the cognate
AHL (C6-HSL) or a closely related AHL such asN-butyryl HSL
(C4-HSL). Other AHLs will bind ineffectively to the CviR receptor
and competitively interfere with its ability to activate genes asso-
ciated with violacein production, which is seen as a loss in pigmen-
tation. A strategy for investigating whether quorum inhibition is6 Starla G. Thornhill and Robert J.C. McLean