Another approach to identify QS-controlled genes is
performing “signal add-back” experiments. In this method, sam-
ples from two conditions are compared: (1) for the reference con-
dition, the signal-negative strain is grown, and (2) for the
experimental condition, the signal-negative mutant is grown in
the presence of exogenous signal to induce QS. This approach is
advantageous when dealing with an organism that has multiple QS
systems, and you want to examine each QS system individually
without making a slew of individual mutants. Please confirm that
you are adding the correct signal at an appropriate concentration
[see[3, 4] for specific examples of experimental design using “signal
add-back” experiments].
Finally, QS regulons may be identified with an alternative
approach by using QS signal interference, also called quorum
quenching. This method is useful for organisms that are not-
genetically tractable or for situations in which one does not want
to generate QS mutants. QS signal interference has been described
for a handful of organisms and must be evaluated on a case-by-case
basis. For example, AHL-mediated QS systems are candidates for
QS quenching by an AHL-degrading enzyme AiiA [5, 6], and
Staphylococcusspecies that use Agr-mediated QS are candidates for
QS interference by using non-cognate signals [7, 8]. QS interfer-
ence can be achieved in two ways: (1) by adding purified QS
quencher to the growth medium, or (2) by genetically engineering
your organism to produce the QS quencher. This approach has
limitations. Specifically, a quorum quencher may not be available
for your organism, and/or addition of the quorum quencher may
produce artifacts and identify factors that are not truly QS-
controlled.
Fig. 1Overview of conditions to compare to identify QS-controlled factors or
genes
RNAseq of QS Regulons 179