Page 2 Plant-Bacteria Interactions Edited by Iqbal Ahmad, John ...

7.5.1
Bioassays for the Detection of Signal Molecules
7.5 Quorum-Sensing Signal Molecules in Gram-Negative Bacteriaj141
Autoinducer signal molecules are produced at very low concentrations. Many bioassays
and sensor systems have been developed that allow facile detection, characterization
and quantitative analysis of microbial acyl-HSLs [40,117,124–128]. Most of
the autoinducers are mutants that cannot synthesize their own AHL. So the wildtype
phenotype is only expressed upon the addition of exogenous AHL.
Various bioassay strains developed have reporter genes including lacZ, gfp, lux
and the production of an endogenous pigment. Some of the routinely used bioassays
and their characteristic features are presented in Table 7.2. The details of these
strains and their basis may be seen in more details from the literature published
elsewhere [17,31].
Autoinducer sensors have generally been dependent on the use of lacZ reporter
fusions in an E. coli or A. tumefaciens genetic background or on the induction or
inhibition of the purple pigment, violacein in C. violaceum [135]. The inhibition of
quorum sensing by Bacillus sp. in this system has been shown in Figure 7.2 as
demonstrated in our laboratory [161].
An A. tumefaciens based AHL sensor pDC141E33 has been developed in which
lacZ is fused to traG, that is regulated via the luxR homology TraR by incorporating
5-bromo-4-chloro-3-b-D-galactopyranoside (X-Gal) in the agar overlay. It is possible to
visualize AHLs on TLC plates or on Petri dishes. It has been found that the
pDC141E33 vector allows detection of the broadcast range of AHLs derivatives and
shows the greatest sensitivity [31]. However, this bioreporter does not detect Nbutanoyl-homoserine
lactone, even at high concentrations [40].
Another frequently used bioreporter strain is based on the induction or inhibition of
violacein production in C. violaceum. In this bacterium, production of pigment is regulated
by HHL [26]. Strain CV026 is a violacein negative miniTn5 mutants of C. violaceum
in which pigment production can be restored by incubation with exogenous AHL.
AHL compound (C10–C14) N-acyl chains are unable to induce violacein production,
butlong-chainAHLscanbedetectedbytheirabilitytoinhibitviolaceinproductionwhen
Figure 7.2 Antiquorum-sensing activity of Bacillus sp. using C. violaceum ATCC 2147 [161].