SUMMARY Quorum-sensing bacteria communicate via small molecules called autoinducers to coordinate collective behaviors. Because quorum sensing controls virulence factor expression in many clinically relevant pathogens, membrane-permeable quorum sensing antagonists that prevent population-wide expression of virulence genes offer a potential route to novel antibacterial therapeutics. Here, we report a strategy for inhibiting quorum-sensing receptors of the widespread LuxR family. Structure-function studies with natural and synthetic ligands demonstrate that the dimeric LuxR-type transcription factor CviR from Chromobacterium violaceum is potently antagonized by molecules that bind in place of the native acylated homoserine lactone autoinducer, provided that they stabilize a closed conformation. In such conformations, each of the two DNA-binding domains interacts with the ligand-binding domain of the opposing monomer. Consequently, the DNA-binding helices are held apart by ~60 Å, twice the ~30 Å separation required for operator binding. This approach may represent a general strategy for the inhibition of multi-domain proteins.

SUMMARY The Reg regulon from Rhodobacter capsulatus and Rhodobacter sphaeroides encodes proteins involved in numerous energy-generating and energy-utilizing processes such as photosynthesis, carbon fixation, nitrogen fixation, hydrogen utilization, aerobic and anaerobic respiration, denitrification, electron transport, and aerotaxis. The redox signal that is detected by the membrane-bound sensor kinase, RegB, appears to originate from the aerobic respiratory chain, given that mutations in cytochrome c oxidase result in constitutive RegB autophosphorylation. Regulation of RegB autophosphorylation also involves a redox-active cysteine that is present in the cytosolic region of RegB. Both phosphorylated and unphosphorylated forms of the cognate response regulator RegA are capable of activating or repressing a variety of genes in the regulon. Highly conserved homologues of RegB and RegA have been found in a wide number of photosynthetic and nonphotosynthetic bacteria, with evidence suggesting that RegB/RegA plays a fundamental role in the transcription of redox-regulated genes in many bacterial species.

A phytochrome-like protein called Ppr was discovered in the purple photosynthetic bacterium Rhodospirillum centenum. Ppr has a photoactive yellow protein (PYP) amino-terminal domain, a central domain with similarity to phytochrome, and a carboxyl-terminal histidine kinase domain. Reconstitution experiments demonstrate that Ppr covalently attaches the blue light-absorbing chromophore p-hydroxycinnamic acid and that it has a photocycle that is spectrally similar to, but kinetically slower than, that of PYP. Ppr also regulates chalcone synthase gene expression in response to blue light with autophosphorylation inhibited in vitro by blue light. Phylogenetic analysis demonstrates that R. centenum Ppr may be ancestral to cyanobacterial and plant phytochromes.

Summary Quorum sensing is a process of bacterial communication involving production and detection of secreted molecules called autoinducers. Gram-negative bacteria use acyl-homoserine lactone (AHL) autoinducers, which are detected by one of two receptor types. First, cytoplasmic LuxR-type receptors bind accumulated intracellular AHLs. AHL-LuxR complexes bind DNA and alter gene expression. Second, membrane-bound LuxN-type receptors bind accumulated extracellular AHLs. AHL-LuxN complexes relay information internally by phosphorylation cascades that direct gene-expression changes. Here we show that a small molecule, previously identified as an antagonist of LuxN-type receptors, is also a potent antagonist of the LuxR family, despite differences in receptor structure, localization, AHL specificity, and signaling mechanism. Derivatives were synthesized and optimized for potency, and in each case, we characterized the mode of action of antagonism. The most potent antagonist protects Caenorhabditis elegans from quorum-sensing-mediated killing by Chromobacterium violaceum, validating the notion that targeting quorum sensing has potential for antimicrobial drug development.