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).

No abstract

Endocytic recycling is a complex itinerary, critical for many cellular processes. Membrane tubulation is a hallmark of recycling endosomes (REs), mediated by KIF13A, a kinesin-3 family motor. Understanding the regulatory mechanism of KIF13A in RE tubulation and cargo recycling is of fundamental importance but is overlooked. Here, we report a unique mechanism of KIF13A dimerization modulated by Rab22A, a small guanosine triphosphatase, during RE tubulation. A conserved proline between neck coil–coiled-coil (NC-CC1) domains of KIF13A creates steric hindrance, rendering the motors as inactive monomers. Rab22A plays an unusual role by binding to NC-CC1 domains of KIF13A, relieving proline-mediated inhibition and facilitating motor dimerization. As a result, KIF13A motors produce balanced motility and force against multiple dyneins in a molecular tug-of-war to regulate RE tubulation and homeostasis. Together, our findings demonstrate that KIF13A motors are tuned at a single-molecule level to function as weak dimers on the cellular cargo.

Reaction based rapid detection of trace quantities of sulfite.

Enhanced emission and selective binding with albumins.