Search

Quorum sensing is a chemical communication process that bacteria use to coordinate group behaviors. Pseudomonas aeruginosa, an opportunistic pathogen, employs multiple quorum-sensing systems to control behaviors including virulence factor production and biofilm formation. One P. aeruginosa quorum-sensing receptor, called RhlR, binds the cognate autoinducer N-butryl-homoserine lactone (C4HSL), and the RhlR:C4HSL complex activates transcription of target quorum-sensing genes. Here, we use a genetic screen to identify RhlR mutants that function independently of the autoinducer. The RhlR Y64F W68F V133F triple mutant, which we call RhlR*, exhibits ligand-independent activity in vitro and in vivo. RhlR* can drive wildtype biofilm formation and infection in a nematode animal model. The ability of RhlR* to properly regulate quorum-sensing-controlled genes in vivo depends on the quorum-sensing regulator RsaL keeping RhlR* activity in check. RhlR is known to function together with PqsE to control production of the virulence factor called pyocyanin. Likewise, RhlR* requires PqsE for pyocyanin production in planktonic cultures, however, PqsE is dispensable for RhlR*-driven pyocyanin production on surfaces. Finally, wildtype RhlR protein is not sufficiently stabilized by C4HSL to allow purification. However, wildtype RhlR can be stabilized by the synthetic ligand mBTL (meta-bromo-thiolactone) and RhlR* is stable without a ligand. These features enabled purification of the RhlR:mBTL complex and of RhlR* for in vitro examination of their biochemical activities. To our knowledge, this work reports the first RhlR protein purification., Competing Interests: No. The authors have declared that no competing interests exist.

Bacterial quorum sensing systems regulate the production of an ample variety of bioactive extracellular compounds that are involved in interspecies microbial interactions and in the interplay between the microbes and their hosts. The development of new approaches for enabling chemical detection of such cellular activities is important in order to gain new insight into their function and biological significance. In recent years, surface-enhanced Raman scattering (SERS) spectroscopy has emerged as an ultrasensitive analytical tool employing rationally designed plasmonic nanostructured substrates. This review highlights recent advances of SERS spectroscopy for label-free detection and imaging of quorum sensing-regulated processes in the human opportunistic pathogen Pseudomonas aeruginosa . We also briefly describe the challenges and limitations of the technique and conclude with a summary of future prospects for the field.

The opportunistic pathogen Pseudomonas aeruginosa produces colorful redox-active metabolites called phenazines, which underpin biofilm development, virulence, and clinical outcomes. Although phenazines exist in many forms, the best studied is pyocyanin. Here, we describe pyocyanin demethylase (PodA), a hitherto uncharacterized protein that oxidizes the pyocyanin methyl group to formaldehyde and reduces the pyrazine ring via an unusual tautomerizing demethylation reaction. Treatment with PodA disrupts P. aeruginosa biofilm formation similarly to DNase, suggesting interference with the pyocyanin-dependent release of extracellular DNA into the matrix. PodA-dependent pyocyanin demethylation also restricts established biofilm aggregate populations experiencing anoxic conditions. Together, these results show that modulating extracellular redox-active metabolites can influence the fitness of a biofilm-forming microorganism., (Copyright © 2017, American Association for the Advancement of Science.)

The human pathogen Pseudomonas aeruginosa produces pyocyanin, a blue-pigmented phenazine derivative, which is known to play a role in virulence. Pyocyanin is produced from chorismic acid via the phenazine pathway, nine proteins encoded by a gene cluster. Phenazine-1-carboxylic acid, the initial phenazine formed, is converted to pyocyanin in two steps that are catalyzed by the enzymes PhzM and PhzS. PhzM is an adenosylmethionine dependent methyltransferase, and PhzS is a flavin dependent hydroxylase. It has been shown that PhzM is only active in the physical presence of PhzS, suggesting that a protein-protein interaction is involved in pyocyanin formation. Such a complex would prevent the release of 5-methyl-phenazine-1-carboxylate, the putative intermediate, and an apparently unstable compound. Here, we describe the three-dimensional structure of PhzS, solved by single anomalous dispersion, at a resolution of 2.4 A. The structure reveals that PhzS is a member of the family of aromatic hydroxylases characterized by p-hydroxybenzoate hydroxylase. The flavin cofactor of PhzS is in the solvent exposed out orientation typically seen in unliganded aromatic hydroxylases. The PhzS flavin, however, appears to be held in a strained conformation by a combination of stacking interactions and hydrogen bonds. The structure suggests that access to the active site is gained via a tunnel on the opposite side of the protein from where the flavin is exposed. The C-terminal 23 residues are disordered as no electron density is present for these atoms. The probable location of the C-terminus, near the substrate access tunnel, suggests that it may be involved in substrate binding as has been shown for another structural homologue, RebC. This region also may be an element of a PhzM-PhzS interface. Aromatic hydroxylases have been shown to catalyze electrophilic substitution reactions on activated substrates. The putative PhzS substrate, however, is electron deficient and unlikely to act as a nucleophile, suggesting that PhzS may use a different mechanism than its structural relatives.

The blue chloroform-soluble bacterial metabolite pyocyanin (1-hydroxy-5-methyl-phenazine) contributes to the survival and virulence of Pseudomonas aeruginosa, an important Gram-negative opportunistic pathogen of humans and animals. Little is known about the two enzymes, designated PhzM and PhzS, that function in the synthesis of pyocyanin from phenazine-1-carboxylic acid. In this study, the FAD-dependent monooxygenase PhzS was purified and crystallized from lithium sulfate/ammonium sulfate/sodium citrate pH 5.5. Native crystals belong to space group C2, with unit-cell parameters a = 144.2, b = 96.2, c = 71.7 A, alpha = gamma = 90, beta = 110.5 degrees. They contain two monomers of PhzS in the asymmetric unit and diffract to a resolution of 2.4 A. Seleno-L-methionine-labelled PhzS also crystallizes in space group C2, but the unit-cell parameters change to a = 70.6, b = 76.2, c = 80.2 A, alpha = gamma = 90, beta = 110.5 degrees and the diffraction limit is 2.7 A.

Pyocyanin, phenazine-1-carboxylic acid and more than 70 related compounds collectively known as phenazines are produced by various species of Pseudomonas, including the fluorescent pseudomonad P. aeruginosa, a Gram-negative opportunistic pathogen in humans and animals. P. aeruginosa synthesizes a characteristic blue water-soluble compound called pyocyanin (1-hydroxy-5-methyl-phenazine). Two enzymes designated PhzM and PhzS are involved in the terminal steps of its synthesis and very little is known about these enzymes. In this study, PhzM, a dimeric S-adenosylmethionine-dependent methyltransferase, was purified and crystallized from PEG 3350/sodium cacodylate/sodium citrate pH 6.5. The crystals belong to space group P1, with unit-cell parameters a = 46.1, b = 61.8, c = 69.6 A, alpha = 96.3, beta = 106.6, gamma = 106.9 degrees . They contain one dimer in the asymmetric unit and diffract to a resolution of 1.8 A. Anomalous data to 2.3 A resolution have been collected from seleno-L-methionine-labelled PhzM.