Abstract
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 β-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.
Highlights
LuxI-type synthases, and detected by LuxR-type regulator proteins [8]
The link between LasR and oxidative stress response was supported by Northern hybridization analysis that revealed that the RNA level of lasI, under LasR control, was reduced after cells were treated with H2O2
We investigated the impact of Cys79 mutagenesis on ligand binding domain (LBD) structure by measuring the thermal stability of LBD mutants C79S (Tm ϭ 55.1 °C) and C79A (Tm ϭ 55.7 °C), finding that replacement of Cys79 does lower thermal stability for these mutants expressed in the presence of native 3O-C12-HSL compared with wild-type LBD (Tm ϭ 59.4 °C)
Summary
Expression, and Purification of LasR, LBD, and DBD—The P. aeruginosa PA01 lasR gene was amplified by PCR from a lasR-containing pDONR201 vector obtained from the Harvard Medical School PlasmID Repository. Fractions containing the desired protein as judged by SDS-PAGE were pooled and dialyzed against dialysis buffer (50 mM sodium phosphate (pH 7.0), 200 mM NaCl, and 10% (v/v) glycerol, with 1 mM DTT added for LasR and DBD purification). LBD expressed in the presence of non-native AHLs was diluted in non-reducing SDS sample buffer and resolved on a 12% polyacrylamide gel. Serial dilutions of LasR, LasR mutants, and DBD were incubated with 100 nM lasB OP1 for 30 min at 25 °C before binding reactions were applied to a nondenaturing 6% polyacrylamide DNA retardation gel (Invitrogen) with 0.5ϫ Tris borate-EDTA as the electrophoresis running buffer. Digested proteins were subjected to LC-MS, with ions of interest selected and subjected to collision-induced dissociation (MS/MS) to confirm peptide identity
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