The LasR regulator protein functions at the top of the Pseudomonas aeruginosa quorum-sensing hierarchy and is implicated in promoting bacterial virulence. Of note is recent evidence that this transcription factor may also respond to oxidative stress. Here, all cysteines in LasR were inspected to deduce their redox sensitivity and to probe the connection between stress response and LasR activity using purified LasR and individual LasR domains. Cys 79 in the ligand binding domain of LasR appears to be important for ligand recognition and folding of this domain to potentiate DNA binding but does not seem to be sensitive to oxidative stress when bound to its native ligand. Two cysteines in the DNA binding domain of LasR do form a disulfide bond when treated with hydrogen peroxide, and formation of this Cys 201 -Cys 203 disulfide bond appears to disrupt the DNA binding activity of the transcription factor. Mutagenesis of either of these cysteines leads to expression of a protein that no longer binds DNA. A cell-based reporter assay linking LasR function with ␤-galactosidase activity gave results consistent with those obtained with purified LasR. This work provides a possible mechanism for oxidative stress response by LasR and indicates that multiple cysteines within the protein may prove to be useful targets for disabling its activity.The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic human pathogen that establishes chronic infections in immunocompromised patients, with persistent pulmonary colonization in individuals with cystic fibrosis (1-3). Like many bacteria, P. aeruginosa employs quorumsensing (QS) 4 machinery to communicate local population density through the exchange of self-produced signaling molecules (4). QS contributes to the development of acute infections by coordinating the production of multiple virulence factors, including elastase and pyocyanin, and the formation of biofilms (5-7). QS in P. aeruginosa is largely mediated by N-acyl L-homoserine lactone autoinducer molecules, biosynthesized by LuxI-type synthases, and detected by LuxR-type regulator proteins (8). The two major systems responsive to acylhomoserine lactones (AHLs) in P. aeruginosa are LasI/LasR and RhlI/RhlR. LasI and RhlI are LuxI-type synthases, whereas LasR and RhlR are LuxR-type regulatory proteins. These AHL-dependent systems are also closely connected to signaling governed by 2-alkyl-4-quinolones in P. aeruginosa (9).The regulator protein LasR, with its cognate synthase LasI, functions at the top of the P. aeruginosa quorum-sensing hierarchy (10). Like other LuxR-type proteins, this transcription factor is composed of two domains (11). The amino-terminal ligand binding domain (LBD) specifically recognizes its autoinducer, N-(3-oxo-dodecanoyl)-L-homoserine lactone (3O-C 12 -HSL), promoting protein folding and dimerization, whereas the DNA binding domain (DBD) recognizes target sites to activate downstream gene expression. There has been significant interest in understanding LasR, as its inhibition represents a...

Edited by Michael IbbaKeywords: Flavin Oxidase Non-ribosomal peptide synthesis Biosynthesis Thiazole a b s t r a c t Flavin cofactors are central to many biochemical transformations and are typically tightly bound as part of a catalytically active flavoenzyme. This work indicates that naturally occurring flavins can act as stand-alone catalysts to promote the oxidation of biosynthetically inspired heterocycles in aqueous buffers. Flavin activity was compared with that of oxidases important in non-ribosomal peptide synthesis, providing a rare direct comparison between the catalytic efficacy of flavins alone and in the context of a full flavoenzyme. This study suggests that such oxidases are likely to possess an active site base, as oxidase activity was greater than that of flavins alone, particularly for less acidic substrates. These findings offer perspective on the development of robust and catalytically effective, designed miniature flavoenzymes.