Fig 1.
Extracted Ion chromatographs of AHL standards.
The ion counts of each pseudo-molecular ion [M + H]+ are shown against retention time. Retention time axis is broken to expand the scale after five minutes, where most AHLs elute. Range for concentration of AHLs was between 0.04 μM to 3.70 μM. Compound structures are shown in S1 Table.
Table 1.
Limits of detection and retention times of AHL standards.*
Fig 2.
Characteristic MS/MS fragmentation of AHLs.
A) Characteristic fragments originating from the lactone ring. These fragments were used to identify AHLs in bacterial samples. Labeled masses for carbon (13C) and nitrogen (15N) isotopes are indicated in parenthesis. B) Characteristic fragmentation observed when no substitution is present at the third carbon of the acyl chain. C) Characteristic fragmentation observed when a 3-oxo substitution is present. In B) and C), black wavy lines indicate the bonds that are broken during fragmentation, the measured fragment is highlighted in red. D) Fragmentation spectra of selected AHL standards. Blue lines indicate peaks for characteristic fragments of the lactone ring.
Fig 3.
Relative abundance of characteristic lactone ring fragments obtained from MS/MS spectra of AHL strandards.
Table 2.
AHL detection in bacterial samples.*
Fig 4.
3-oxo-C7-HL detection in P. stewartii.
(A) Fragmentation spectra when bacteria are grown in medium with no labeled components, medium with 100% uniformly labeled glucose (13C labeling), and medium with labeled ammonium (15N labeling). Characteristic lactone ring fragments are highlighted in blue and parent ion is highlighted in red. (B) Structure of 3-oxo-C7-HL and its 12C, 13C, and 15N masses. (C) Structures and masses of fragments of 3-oxo-C7-HL in non-labeled, 13C-labeled, and 15N-labeled media. Similar results were observed for E. carotovora.
Fig 5.
(A) AHL standards were added at 1 μM concentration to exponentially growing cultures and AHL degradation was measured after 24 hours. The amount of AHL degradation at 24 hours is shown as: +++, >90% degradation; ++ 60–90% degradation; +, 30–60% degradation; -, less than 30% degradation. (B) Dynamic AHL degradation in selected bacteria. AHLs (10 μM each) were added to exponentially growing cultures and AHL degradation was monitored over time. Data represents the average of three biological replicates.