Abstract
N-acyl L-homoserine lactones (AHLs) constitute a predominant class of quorum-sensing signaling molecules used by Gram-negative bacteria. Here, we report a sensitive and non-targeted HPLC-MS/MS method based on parallel reaction monitoring (PRM) to identify and quantitate known, unanticipated, and novel AHLs in microbial samples. Using a hybrid quadrupole-high resolution mass analyzer, this method integrates MS scans and all-ion fragmentation MS/MS scans to allow simultaneous detection of AHL parent-ion masses and generation of full mass spectra at high resolution and high mass accuracy in a single chromatographic run. We applied this method to screen for AHL production in a variety of Gram-negative bacteria (i.e. B. cepacia, E. tarda, E. carotovora, E. herbicola, P. stewartii, P. aeruginosa, P. aureofaciens, and R. sphaeroides) and discovered that nearly all of them produce a larger set of AHLs than previously reported. Furthermore, we identified production of an uncommon AHL (i.e. 3-oxo-C7-HL) in E. carotovora and P. stewartii, whose production has only been previously observed within the genera Serratia and Yersinia. Finally, we used our method to quantitate AHL degradation in B. cepacia, E. carotovora, E. herbicola, P. stewartii, P. aeruginosa, P. aureofaciens, the non-AHL producer E. coli, and the Gram-positive bacterium B. subtilis. We found that AHL degradation ability varies widely across these microbes, of which B. subtilis and E. carotovora are the best degraders, and observed that there is a general trend for AHLs containing long acyl chains (≥10 carbons) to be degraded at faster rates than AHLs with short acyl chains (≤6 carbons).
Highlights
Quorum sensing (QS) is a widespread form of cell-to-cell communication that allows bacteria to sense their surrounding population density and coordinately regulate a range of group-level behaviors, such as production of secondary metabolites and virulence factors, bioluminescence, and biofilm formation [1]
We report a sensitive and non-targeted HPLC-MS/MS method based on parallel reaction monitoring (PRM) to identify and measure known, unanticipated, and novel acyl L-homoserine lactones (AHLs) in microbial samples
We report a non-targeted HPLC-MS/MS method for detecting and quantitating AHLs
Summary
Quorum sensing (QS) is a widespread form of cell-to-cell communication that allows bacteria to sense their surrounding population density and coordinately regulate a range of group-level behaviors, such as production of secondary metabolites and virulence factors, bioluminescence, and biofilm formation [1]. AHLs are small diffusible signaling molecules characterized by a five-membered lactone ring and linear acyl tail of variable length (typically 4 to 14 carbons), which can have an oxo or hydroxyl group at the C-3 position (S1 Table). Over two dozen such AHL have been characterized to date, and there are likely more to be discovered [7,8,9]
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