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

142j 7 Quorum Sensing in Bacteria: Potential in Plant Health Protection
anactivatingAHL(OHHLorHHL)isincludedintotheassaymedium[26].Inthiscase,a
white halo on a purple background constitutes a positive result. However, this bioreportercannotdetectanyofthe3-hydroxy-derivativesandlackssensitivitytomostofthe3oxo
derivatives [26,41]. However, this biosensor is activated by cyclic dipeptides [33].
Similarly, another strain that is used as bioreporter based on AHL-induced bacterial
swarming is S. liquefaciens MGI. The S. liquefaciem MG44 strain is a SWrI::T45
mutant of MGI that cannot synthesize BHL or HHL and therefore requires an
exogenous supply for serrawetin synthesis and swarming [136,137]. Many Gramnegative
bacteria that employ QS systems produced multiple AHL molecules, for
example Rhizobium elti produces at least seven AHLs [44]. It is presumed that these
additional signals may be due to the presence of multiple QS systems or may be the
products of a single AHL synthase.
Similarly, some bacteria may produce signals that are not detected by one of the
reporters or they may produce molecules at levels below the threshold of sensitivity
of the reporter [40]. Therefore, combinations of different bioreporters have been
used to detect AHL-like activities.
7.5.2
Chemical Characterization of Signal Molecules
Autoinducers can be separated and purified by preparative reverse-phase high-pressure
liquid chromatography. Normally 4–6 l of bacterial culture supernatant grown
in chemically defined medium is extracted with dichloromethane, ethyl acetate or
chloroform. The extract is evaporated on a rotary evaporator. The residue is then
applied to a C18-reverse-phase semi-preparation column and eluted with methanol
gradient or an isocratic mobile phase of acetonitrile : water [31]. Fractions can be
employed by using bioreporters and active fractions can be rechromatographed.
Once a single active peak has been obtained, it can be further analyzed by analytical
HPLC or subjected to identification techniques (Figure 7.3).
Partial characterization of autoinducers is normally carried out by TLC on
C18-reversed-phase plates with the sample (supernatant or extracts) and with different
standards and after chromatography overlaid with a soft agar suspension of the
indicators [26,40]. Using C. violaceum biosensor strain, N-acyl-homoserine lactones
and cyclic peptides are detected [33].
TLC provides preliminary information on both the number and the structural
groups of the compounds present in the supernatant or the extract fractions. The Rfs
value can be compared with known standards.
Not only AHL detection but also QS inhibition by various compounds could be
easily detected when TLC is coupled with other QSI indicator strains. Using A.
tumefaciens bioreporter and CV026, bioassays are useful and rapid to test a large
number of different microbial isolates [41,47]. Characterization can also be carried
out by analytical HPLC using C18-reversed-phase column.
Further identification of QS molecules have been carried out using spectroscopic
techniques, such as mass spectrometry (MS), nuclear magnetic resonance spectroscopy
(NMR) and infrared spectroscopy (IR).