Identification of Unanticipated and Novel <i>N</i>-Acyl L-Homoserine Lactones (AHLs) Using a Sensitive Non-Targeted LC-MS/MS Method

<div><p><i>N</i>-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. <i>B</i>. <i>cepacia</i>, <i>E</i>. <i>tarda</i>, <i>E</i>. <i>carotovora</i>, <i>E</i>. <i>herbicola</i>, <i>P</i>. <i>stewartii</i>, <i>P</i>. <i>aeruginosa</i>, <i>P</i>. <i>aureofaciens</i>, and <i>R</i>. <i>sphaeroides</i>) 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 <i>E</i>. <i>carotovora</i> and <i>P</i>. <i>stewartii</i>, whose production has only been previously observed within the genera <i>Serratia</i> and <i>Yersinia</i>. Finally, we used our method to quantitate AHL degradation in <i>B</i>. <i>cepacia</i>, <i>E</i>. <i>carotovora</i>, <i>E</i>. <i>herbicola</i>, <i>P</i>. <i>stewartii</i>, <i>P</i>. <i>aeruginosa</i>, <i>P</i>. <i>aureofaciens</i>, the non-AHL producer <i>E</i>. <i>coli</i>, and the Gram-positive bacterium <i>B</i>. <i>subtilis</i>. We found that AHL degradation ability varies widely across these microbes, of which <i>B</i>. <i>subtilis</i> and <i>E</i>. <i>carotovora</i> 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).</p>