Strain-dependent diversity in the Pseudomonas aeruginosa quorum-sensing regulon.

Quorum sensing allows bacteria to sense and respond to changes in population density. Acyl-homoserine lactones serve as quorum-sensing signals for many Proteobacteria, and acyl-homoserine lactone signaling is known to control cooperative activities. Quorum-controlled activities vary from one species to another. Quorum-sensing controls a constellation of genes in the opportunistic pathogen Pseudomonas aeruginosa, which thrives in a number of habitats ranging from soil and water to animal hosts. We hypothesized that there would be significant variation in quorum-sensing regulons among strains of P. aeruginosa isolated from different habitats and that differences in the quorum-sensing regulons might reveal insights about the ecology of P. aeruginosa. As a test of our hypothesis we used RNA-seq to identify quorum-controlled genes in seven P. aeruginosa isolates of diverse origins. Although our approach certainly overlooks some quorum-sensing-regulated genes we found a shared set of genes, i.e., a core quorum-controlled gene set, and we identified distinct, strain-variable sets of quorum-controlled genes, i.e., accessory genes. Some quorum-controlled genes in some strains were not present in the genomes of other strains. We detected a correlation between traits encoded by some genes in the strain-variable subsets of the quorum regulons and the ecology of the isolates. These findings indicate a role for quorum sensing in extension of the range of habitats in which a species can thrive. This study also provides a framework for understanding the molecular mechanisms by which quorum-sensing systems operate, the evolutionary pressures by which they are maintained, and their importance in disparate ecological contexts.

LuxR homolog-independent gene regulation by acyl-homoserine lactones in Pseudomonas aeruginosa.

Pseudomonas aeruginosa quorum control of gene expression involves three LuxR-type signal receptors LasR, RhlR, and QscR that respond to the LasI- and RhlI-generated acyl-homoserine lactone (acyl-HSL) signals 3OC12-HSL and C4-HSL. We found that a LasR-RhlR-QscR triple mutant responds to acyl-HSLs by regulating at least 37 genes. LuxR homolog-independent activation of the representative genes antA and catB also occurs in the wild type. Expression of antA was influenced the most by C10-HSL and to a lesser extent by other acyl-HSLs, including the P. aeruginosa 3OC12-HSL and C4-HSL signals. The ant and cat operons encode enzymes for the degradation of anthranilate to tricarboxylic acid cycle intermediates. Our results indicate that LuxR homolog-independent acyl-HSL control of the ant and cat operons occurs via regulation of antR, which codes for the transcriptional activator of the ant operon. Although P. aeruginosa has multiple pathways for anthranilate synthesis, one pathway-the kynurenine pathway for tryptophan degradation-is required for acyl-HSL activation of the ant operon. The kynurenine pathway is also the critical source of anthranilate for energy metabolism via the antABC gene products, as well as the source of anthranilate for synthesis of the P. aeruginosa quinolone signal. Our discovery of LuxR homolog-independent responses to acyl-HSLs provides insight into acyl-HSL signaling.

An evolving perspective on the Pseudomonas aeruginosa orphan quorum sensing regulator QscR.

Many Proteobacteria govern responses to changes in cell density by using acyl-homoserine lactone (AHL) quorum-sensing (QS) signaling. Similar to the LuxI-LuxR system described in Vibrio fischeri, a minimal AHL QS circuit comprises a pair of genes, a luxI-type synthase gene encoding an enzyme that synthesizes an AHL and a luxR-type AHL-responsive transcription regulator gene. In most bacteria that utilize AHL QS, cognate luxI and luxR homologs are found in proximity to each other on the chromosome. However, a number of recent reports have identified luxR homologs that are not linked to luxI homologs; in some cases luxR homologs have been identified in bacteria that have no luxI homologs. A luxR homolog without a linked luxI homologs is termed an orphan or solo. One of the first reports of an orphan was on QscR in Pseudomonas aeruginosa. The qscR gene was revealed by whole genome sequencing and has been studied in some detail. P. aeruginosa encodes two AHL synthases and three AHL responsive receptors, LasI-LasR form a cognate synthase-receptor pair as do RhlI-RhlR. QscR lacks a linked synthase and responds to the LasI-generated AHL. QS regulation of gene expression in P. aeruginosa employs multiple signals and occurs in the context of other interconnected regulatory circuits that control diverse physiological functions. QscR affects virulence of P. aeruginosa, and although it shows sensitivity to the LasI-generated AHL, 3-oxo-dodecanoylhomoserine lactone, it's specificity is relaxed compared to LasR and can respond equally well to several AHLs. QscR controls a set of genes that overlaps the set regulated by LasR. QscR is comparatively easy to purify and study in vitro, and has become a model for understanding the biochemistry of LuxR homologs. In fact there is a crystal structure of QscR bound to the LasI-generated AHL. Here, we review the current state of research concerning QscR and highlight recent advances in our understanding of its structure and biochemistry.

Bacterial quorum sensing and metabolic incentives to cooperate.

The opportunistic pathogen Pseudomonas aeruginosa uses a cell-cell communication system termed "quorum sensing" to control production of public goods, extracellular products that can be used by any community member. Not all individuals respond to quorum-sensing signals and synthesize public goods. Such social cheaters enjoy the benefits of the products secreted by cooperators. There are some P. aeruginosa cellular enzymes controlled by quorum sensing, and we show that quorum sensing-controlled expression of such private goods can put a metabolic constraint on social cheating and prevent a tragedy of the commons. Metabolic constraint of social cheating provides an explanation for private-goods regulation by a cooperative system and has general implications for population biology, infection control, and stabilization of quorum-sensing circuits in synthetic biology.

The influence of human respiratory epithelia on Pseudomonas aeruginosa gene expression.

The opportunistic pathogen Pseudomonas aeruginosa can cause acute or chronic infections in humans. Little is known about the initial adaptation of P. aeruginosa to host tissues and the factors that determine whether a P. aeruginosa-epithelial cell interaction will manifest as an acute or a chronic infection. To gain insights into the initial phases of P. aeruginosa infections and to identify P. aeruginosa genes regulated in response to respiratory epithelia, we exposed P. aeruginosa to cultured primary differentiated human airway epithelia. We used a P. aeruginosa strain that causes acute damage to the epithelia and a mutant with defects in Type III secretion and in rhamnolipid synthesis. The mutant did not cause rapid damage to epithelia as did the wildtype. We compared the transcriptomes of the P. aeruginosa wildtype and the mutant to each other and to P. aeruginosa grown under other conditions, and we discovered overlapping sets of differentially expressed genes in the wildtype and mutant exposed to epithelia. A recent study reported that exposure of P. aeruginosa to epithelia is characterized by a repression of the bacterial iron-responsive genes. These findings were suggestive of ample iron availability during infection. In contrast, we found that P. aeruginosa shows an iron-starvation response upon exposure to epithelial cells. This observation highlights the importance of the iron starvation response in both acute and chronic infections and suggests opportunities for therapy.

Critical nucleotides in the interaction of CatR with the pheBA promoter: conservation of the CatR-mediated regulation mechanisms between the pheBA and catBCA operons.

The promoter of the plasmid-borne pheBA genes encoding enzymes for phenol degradation resembles the catBCA promoter and is activated by CatR, the regulator of the chromosomally encoded catechol-degradative catBCA genes in Pseudomonas putida. In this study, site-directed mutagenesis of the pheBA promoter region was performed. The interrupted inverted repeat sequence of the CatR recognition binding site (RBS) of the pheBA promoter is highly homologous to that of the catBCA promoter. However, the RBS was shown not to be the sole important feature for high-affinity binding of CatR to this site. Mutagenesis of the activation binding site (ABS) of CatR, which overlaps the -35 hexamer sequence TTGGAT of the promoter, revealed that the two G nucleotides in this sequence are important for promoter activity but not for CatR binding. All other substitutions made in the ABS negatively affected both the promoter activity and CatR binding. The spacer sequence of the pheBA and catBCA promoters between the -10 and -35 hexamers is 19 bp, which is longer than optimal. However, reducing the spacer region of the pheBA promoter was not sufficient for CatR-independent promoter activation. An internal binding site (IBS) for CatR is located downstream of the transcriptional start site of the catBCA genes and it negatively regulates the operon. A similar IBS was identified in the case of the pheBA operon and tested for its functionality. The results indicate a conservation of CatR-mediated regulation mechanisms between the pheBA promoter and the catBCA promoter. This universal mechanism of CatR-mediated transcriptional activation could be of great importance in enabling catechol-degrading bacteria to expand their substrate range via horizontal transfer of the phenol degradative genes.