Pseudomonas aeruginosa uses the two major quorum-sensing (QS) regulatory systems las and rhl to modulate the expression of many of its virulence factors. The las system is considered to stand at the top of the QS hierarchy. However, some virulence factors such as pyocyanin have been reported to still be produced in lasR mutants under certain conditions. Interestingly, such mutants arise spontaneously under various conditions, including in the airways of cystic fibrosis patients. Using transcriptional lacZ reporters, LC/MS quantification and phenotypic assays, we have investigated the regulation of QS-controlled factors by the las system. Our results show that activity of the rhl system is only delayed in a lasR mutant, thus allowing the expression of multiple virulence determinants such as pyocyanin, rhamnolipids and C 4 -homoserine lactone (HSL) during the late stationary phase. Moreover, at this stage, RhlR is able to overcome the absence of the las system by activating specific LasR-controlled functions, including production of 3-oxo-C 12 -HSL and Pseudomonas quinolone signal (PQS). P. aeruginosa is thus able to circumvent the deficiency of one of its QS systems by allowing the other to take over. This work demonstrates that the QS hierarchy is more complex than the model simply presenting the las system above the rhl system.

4-Hydroxy-2-alkylquinolines (HAQs), especially 3,4-dihydroxy-2-heptylquinoline (Pseudomonas quinolone signal) and its precursor, 4-hydroxy-2-heptylquinoline, are attracting much attention, mainly because of their role as signaling molecules in Pseudomonas aeruginosa. The pqsABCDE operon is centrally involved in their biosynthesis. The presence of a homologous operon in Burkholderia pseudomallei and B. thailandensis was recently reported. Thus, we have investigated the abilities of 11 Burkholderia species to produce HAQ-like molecules by liquid chromatography/mass spectrometry. We have identified 29 different HAQ derivatives produced by the only three Burkholderia species where a pqsABCDE homologue was found among available sequenced Burkholderia species genomes, including B. ambifaria, a member of the Burkholderia cepacia complex. In contrast with those of P. aeruginosa, Burkholderia HAQs typically bear a methyl group, hence their designation as 4-hydroxy-3-methyl-2-alkylquinolines (HMAQs). We identified three families of HMAQs with a saturated or unsaturated alkyl chain at the 2 position, in contrast with the 1 position of P. aeruginosa, including one with an N-oxide group. Furthermore, the operon in these species contains two more genes downstream of the pqsE homologue, resulting in the hmqABCDEFG operon. While the inactivation of hmqA inhibits the production of HMAQs, the methylation of the quinoline ring requires a putative methyltransferase encoded by hmqG. Interestingly, hmqA or hmqG mutations increase the production of acyl homoserine lactones and, consequently, phenotypes under the control of quorum sensing in B. ambifaria: antifungal activity, siderophore production, and proteolytic activity. These results indicate that only HAQs bearing a methyl group (HMAQs) are involved in quorum-sensing regulation.

A significant number of environmental microorganisms can cause serious, even fatal, acute and chronic infections in humans. The severity and outcome of each type of infection depends on the expression of specific bacterial phenotypes controlled by complex regulatory networks that sense and respond to the host environment. Although bacterial signals that contribute to a successful acute infection have been identified in a number of pathogens, the signals that mediate the onset and establishment of chronic infections have yet to be discovered. We identified a volatile, low molecular weight molecule, 2-amino acetophenone (2-AA), produced by the opportunistic human pathogen Pseudomonas aeruginosa that reduces bacterial virulence in vivo in flies and in an acute mouse infection model. 2-AA modulates the activity of the virulence regulator MvfR (multiple virulence factor regulator) via a negative feedback loop and it promotes the emergence of P. aeruginosa phenotypes that likely promote chronic lung infections, including accumulation of lasR mutants, long-term survival at stationary phase, and persistence in a Drosophila infection model. We report for the first time the existence of a quorum sensing (QS) regulated volatile molecule that induces bistability phenotype by stochastically silencing acute virulence functions in P. aeruginosa. We propose that 2-AA mediates changes in a subpopulation of cells that facilitate the exploitation of dynamic host environments and promote gene expression changes that favor chronic infections.

Groups of pathogenic bacteria employ diffusible signals to regulate their virulence in a concerted manner. Pseudomonas aeruginosa uses 4-hydroxy-2-alkylquinolines (HAQs), including HHQ and PQS, as unique signals. We demonstrate that octanoic acid is directly incorporated into HHQ. This finding rules out the long-standing hypothesis that 3-ketofatty acids are the precursors of HAQs. We found that HAQ biosynthesis, which requires the PqsABCD enzymes, proceeds by a two-step pathway: [1] PqsD mediates the synthesis of 2-aminobenzoylacetate (2-ABA) from anthraniloyl-CoA and malonyl-CoA, then [2] the decarboxylating coupling of 2-ABA to an octanoate group linked to PqsC produces HHQ, the direct precursor of PQS (Pseudomonas Quinolone Signal). PqsB is tightly associated with PqsC and required for the second step. This finding uncovers promising targets for the development of specific antivirulence drugs to combat this opportunistic pathogen.