| In a mechanism called quorum sensing (QS), Gram-negative bacteria use signaling molecules such as autoinducer-1 (acylated homoserine lactone/AHL) and autoinducer-2 (4,5-dihydroxy-2,3-pentanedione derivative) to regulate bacterial gene expression in a population density-dependent manner. Plants have been shown to produce QS mimic compounds that interfere with these bacterial signals, presumably as a mechanism to prevent or alter their niche. Since the first QS mimics were actually discovered from seaweeds, we explored the possibility that QS mimics may also be produced by the unicellular green algae, Chlamydomonas reinhardtii, Chlorella fusca, and Chlorella vulgaris. As detailed in chapter 2, using solvent partitioning or solid phase extraction methods coupled with HPLC fractionation and bacterial reporter strain bioassays, we have demonstrated the presence of various AHL mimic compounds in culture filtrates of the algae cell filtrates.;The chapter 3 details the development of a fluorescence resonance energy transfer (FRET)-based biosensor for the detection of the boron derivative of the autoinducer-2 class of bacterial QS signals (BAI-2) and potential mimics. This biosensor construct contains a bacterial periplasmic binding protein, LuxP, fused at its N-terminus to a cyan fluorescent protein (CFP) and at its C-terminus to a yellow fluorescent protein (YFP). We have demonstrated that in the presence of synthetic BAI-2, LuxP protein binds BAI-2 and changes its conformation. This ligand induced conformation change was monitored as a decrease in fluorescence ratio between YFP and CFP. Importantly, site-directed mutagenesis of the ligand binding blocked the BAI-2-dependent change in FRET, indicating that the observed change in FRET ratio (YFP/CFP) was BAI-2 specific. For the first time, the apparent binding affinity of the ligand BAI-2 for the LuxP receptor protein has been determined (270 nM) and is consistent with sub-micromolar range for similar class of proteins.;Extending our FRET studies, we also generated a biosensor for use in quantification of bioavailable heavy metals in aqueous environments. Similar to the QS-FRET biosensor, the heavy metal biosensor (CMY) has an N-terminal CFP, an intervening heavy metal-binding, metallothionein II (MTII) receptor protein fused to a C-terminal YFP. (Abstract shortened by UMI.). |
