produced by the opportunistic pathogenPseudomonas aeruginosa
[9],N-3-oxo-octanoyl HSL (3-o-C8 HSL) produced byAgrobac-
terium tumefaciens[10], and N-hexanoyl HSL (C6-HSL) pro-
duced by Chromobacterium violaceum[11]. AHL-based QS is
reviewed in [7, 12].
AHLs are associated with a number of important microbial
activities including virulence gene regulation [13, 14], antibiotic
resistance [15, 16], and aspects of biofilm formation inP. aerugi-
nosa[17, 18]. Quorum interference by brominated furanones and
other compounds [19–21] as well as AHL-degrading enzymes has
shown promise as a novel antibacterial treatment strategy due to its
effectiveness against highly resistant biofilm populations [22, 23].
As a result, there is considerable interest in the identification of QS
systems and QS inhibitors (QSIs). There are a number of chemical
approaches including chromatography and mass spectrometry that
can be used to characterize AHLs (e.g., [24–27]); however, the
equipment and expertise needed for these approaches can be quite
significant. In contrast, QS bioassays (reviewed in [28]) are rela-
tively inexpensive and thus allow screening for QSIs and AHLs in
regions of the world including places with high biodiversity, such as
the tropics, and limited financial resources.
Bioassay organisms for QS have a transcriptional response reg-
ulator (luxRhomologue in the case of AHLs) coupled to a reporter
gene allowing a readily observable phenotype. In the case of the
widely usedA. tumefaciensAHL bioassays [29, 30], thetraRgene
(luxRhomologue) is coupled tolacZ. In this fashion, AHLs can be
detected on the basis ofβ-galactosidase activity often using 5-
bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal). The
normal phenotype associated with QS inA. tumefaciensis gene
conjugation [29], which is readily visualized. In contrast toA.
tumefaciens, several organisms, including Serratia marscesens
[31],Pseudomonas aureofaciens[32], andC. violaceum[11, 33]
naturally produce pigmented compounds in response to QS. We
shall focus on the use ofA. tumefaciensandC. violaceumin this
chapter.1.2 Agrobacterium
tumefaciens
as a Biosensor
A. tumefaciensis a Gram-negative opportunistic plant pathogen
that causes crown gall formation on plants through the transmis-
sion of DNA fragments into the nuclei of infected plants [29].
Conjugation-based gene transfer is under the regulation of 3-o-
C8 HSL-dependent QS. The genes responsible for synthesis and
response regulation of this signal molecule aretraIand traR,
respectively, which are homologues ofluxIandluxR[34]. During
their investigations of QS-related functions ofA. tumefaciens, S.C.
Winans, C. Fuqua, J. Zhu, and colleagues developed several
reporter strains, notably A136 (pCF218)(pCF372) (also referred
in literature as WCF47(pCF218)(pCF372)) [29, 35], NTL
(pCF218)(pCF372) [36, 37], and KYC55 (pJZ372)(pJZ384)Bioassays of Quorum and Biofilm Dispersion Signals 5