were first identified, thep-coumaroyl- and isovaleryl-linked AHLs
(Fig. 1), they were not detected by the existing repertoire of
bioassay reporters [6, 9]. There are also examples of non-HSL
compounds activating reporter strains [18, 19], which could con-
found results. Additionally, bioassays are sensitive to inhibition by
other compounds sometimes present in sample extracts (e.g., we
had difficulties detecting AHLs in cystic fibrosis patient sputum
extracts using bioassays, but not using the radiolabel assay
described here [15]).
Mass spectrometry (MS) analyses are essential to QS research
(seeChapter4), especially with regard to structural elucidation of
undefined AHL compounds, and can be quantitative when an
internal deuterated AHL standard is included [14]. However, not
all laboratories have ready access to a mass spectrometer and many
of the MS techniques rely on the presence of a fragment ion at (m/
z+ H) 102 (corresponding to aminobutyrolactone), which is
observed in most, but not all [9], AHLs. Recently ELISA techni-
ques (seeChapter5) have been used to detect acyl-HSLs, but
require custom monoclonal antibodies and cannot differentiate
between closed HSL ring compounds (acyl-HSL, active QS signal)
and lactonized ones (acyl-homoserine, inactive QS signal) [13].
The^14 C-radiolabel assay detailed here is similar to that first
described by Eberhard et al. [20]. AHL-synthesizing bacteria are
fed radiolabeled L-[1-^14 C]-methionine (^14 C-met), which is trans-
ported into the cell, and a portion is converted into SAM via
enzymes like theEscherichia coliMetK SAM synthetase. The^14 C-
SAM is used by LuxI-type synthases as the substrate for the con-
served HSL ring in the AHL QS signal (diagrammed in Fig.2).(^14) C-AHLs are solvent extracted from cell-free supernatants, sepa-
rated by HPLC, and radioactivity in the HPLC fractions is
measured by liquid scintillation counting. Because all LuxI-
synthesized QS signals utilize SAM as the HSL ring substrate, all
HSL-type signals (including novel AHL structures) are detected by
the radiolabel technique so long as the bacterium tested is capable
of assimilating exogenous^14 C-met. The presence of a single radi-
olabeled carbon in the HSL ring, regardless of side-chain moiety,
allows for easy comparison of relative HSL compounds in a single
sample (Fig.3 illustrates the detection of both major and minor
AHL compounds present inPseudomonascultures). It is possible
that some bacteria could incorporate^14 C-met into other non-
AHL, extracellular, solvent-extractable compounds; however, we
have not found this to be the case for the dozens ofProteobacteria
species we have tested using the radiolabel assay.
Although some labs prefer to avoid using radioactivity due to
licensing and disposal cost issues, the radiolabel protocol for moni-
toring AHL production is a powerful technique that shows no bias
with regard to AHL side chain and is faster and more sensitive than
many traditional bioassays. Variations of this protocol have been
38 Amy L. Schaefer et al.