The Relationship of pqs Gene Expression to Acylhomoserine Lactone Signaling in Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa has complex quorum sensing (QS) circuitry, which involves two acylhomoserine lactone (AHL) systems, the LasI AHL synthase and LasR AHL-dependent transcriptional activator system and the RhlI AHL synthase-RhlR AHL-responsive transcriptional activator. There is also a quinoline signaling system (the Pseudomonas quinolone signal, PQS, system). Although there is a core set of genes regulated by the AHL circuits, there is substantial strain-to-strain variation in the non-core QS regulated genes. Reductive evolution of the QS regulon, and variation in specific genes activated by QS, occurs in laboratory evolution experiments with the model strain PAO1. We used a transcriptomics approach to test the hypothesis that reductive evolution in the PAO1 QS regulon can in large part be explained by a simple null mutation in pqsR , the gene encoding the transcriptional activator of the pqs operon. We found that PqsR had very little influence on the AHL QS regulon. This was a surprising finding because the last gene in the PqsR-dependent pqs operon, pqsE , codes for a protein, which physically interacts with RhlR and this interaction is required for RhlR-dependent activation of some genes. We used comparative transcriptomics to examine the influence of a pqsE mutation on the QS regulon and identified only three transcripts, which were strictly dependent on PqsE. By using reporter constructs we showed that the PqsE influence on other genes was dependent on experimental conditions and we have gained some insight about those conditions. This work adds to our understanding of the plasticity of the P. aeruginosa QS regulon and to the role PqsE plays in RhlR-dependent gene activation.

Microbial Primer: LuxR-LuxI Quorum Sensing.

Quorum sensing is a term describing bacterial cell-to-cell communication systems for monitoring and responding to changes in population density. This primer serves as an introduction to the canonical LuxR-LuxI-type quorum sensing circuits common to many species of Gram-negative bacteria. Quorum sensing can synchronize behaviours across a community. Different species employ quorum sensing strategies to control specific behaviours such as bioluminescence, virulence factor production, secondary metabolite production, and biofilm formation.

Rhamnolipids produced by Pseudomonas: from molecular genetics to the market.

Rhamnolipids are biosurfactants with a wide range of industrial applications that entered into the market a decade ago. They are naturally produced by Pseudomonas aeruginosa and some Burkholderia species. Occasionally, some strains of different bacterial species, like Pseudomonas chlororaphis NRRL B-30761, which have acquired RL-producing ability by horizontal gene transfer, have been described. P. aeruginosa, the ubiquitous opportunistic pathogenic bacterium, is the best rhamnolipids producer, but Pseudomonas putida has been used as heterologous host for the production of this biosurfactant with relatively good yields. The molecular genetics of rhamnolipids production by P. aeruginosa has been widely studied not only due to the interest in developing overproducing strains, but because it is coordinately regulated with the expression of different virulence-related traits by the quorum-sensing response. Here, we highlight how the research of the molecular mechanisms involved in rhamnolipid production have impacted the development of strains that are suitable for industrial production of this biosurfactant, as well as some perspectives to improve these industrial useful strains.

The outlier Pseudomonas aeruginosa strain ATCC 9027 harbors a defective LasR quorum-sensing transcriptional regulator.

Pseudomonas aeruginosa infections represent an important health problem that has been recognized by the World Health Organization as a research priority. A complex regulatory network called the quorum sensing (QS) regulates several P. aeruginosa virulence-related traits, including production of elastase, rhamnolipids and pyocyanin. The avirulent P. aeruginosa strain ATCC 9027 belongs to clade 3, which is the more distant phylogroup in relationship to the other four clades of this species. This strain does not produce QS-regulated virulence factors such as elastase and rhamnolipids when cultured in rich LB medium. We report here that ATCC 9027 harbors a defective LasR protein, presumably due to the presence of an aspartic acid in position 196 instead of a glutamic acid which is the amino acid present in this position in functional LasR proteins of the type strains PAO1 (clade 1) and PA7 (also belonging to clade 3), among others. In addition, we report that ATCC 9027 and PA7 strains present differences compared to the PAO1 strain in lasB which encodes elastase, and in the rhlR regulatory sequences that modify las-boxes, and that these mutations have a little effect in the expression of these genes by a functional LasR transcriptional regulator.

Inactivation of the quorum-sensing transcriptional regulators LasR or RhlR does not suppress the expression of virulence factors and the virulence of Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa is an environmental bacterium but is also an opportunistic pathogen. The aim of this work is to evaluate the contribution of P. aeruginosa LasR and RhlR transcriptional regulators of the quorum-sensing response (QSR) to the production of virulence factors, and to its virulence in a mouse abscess model. The QSR is a complex regulatory network that modulates the expression of several virulence factors, including elastase, pyocyanin and rhamnolipids. LasR, when complexed with the auto-inducer 3-oxo-dodecanoyl lactone (3O-C12-HSL), produced by LasI, is at the top of the QSR regulatory cascade since it activates transcription of some genes encoding virulence factors (such as the gene coding for elastase, lasB) and also transcription of both rhlR and rhlI, encoding the synthase of the auto-inducer butanoyl-homoserine lactone (C4-HSL). In turn RhlR, coupled with C4-HSL, activates the transcription of genes encoding for the enzymes involved in pyocyanin and rhamnolipid production. Several efforts have been made to obtain inhibitors of LasR activity that would suppress the QSR. However, these attempts have used chemical compounds that might not be specific for LasR inactivation. In this work we show that individual inactivation of either lasR or rhlR did not block the QSR, nor did it impair P. aeruginosa virulence, and that even a lasR rhlR double mutant still presented residual virulence, even lacking the production of virulence factors. These results show that the inhibition of either lasR or rhlR is not a straightforward approach to blocking P. aeruginosa virulence, due to the great complexity of the QSR.

Overproduction of rhamnolipids in Pseudomonas aeruginosa PA14 by redirection of the carbon flux from polyhydroxyalkanoate synthesis and overexpression of the rhlAB-R operon.

OBJECTIVE: To construct Pseudomonas aeruginosa PA14 derivatives that overproduce rhamnolipids (RL) by blocking the synthesis of the carbon-storage polymer polyhydroxyalkanoates (PHA) and by overexpressing the rhlAB-R operon that encodes for enzymes of RL synthesis and the RhlR transcriptional regulator. RESULTS: In contrast to previous results showing that overexpression of rhlAB-R genes in two P. aeruginosa strains (PAO1 and ATCC 9027) is sufficient to overproduce RL, we show that a PA14 derivative overexpressing the rhlAB-R operon did not increase the synthesis of these biosurfactants. In addition, PA14 mutants deficient in PHA production did not overproduce RL either. However, if the rhlAB-R genes were expressed in a mutant that is completely impaired in PHA synthesis, a significant increase in RL production was observed (59%). These results show that RL production in PA14 is limited both by the availability of fatty acid precursors and by the levels of the RhlA and RhlB enzymes that are involved in the synthesis of mono-RL. CONCLUSIONS: The limitation of RL production by P. aeruginosa PA14 is multifactorial and diverse from the results obtained with other strains. Thus, the factors that limit RL production are particular to each P. aeruginosa strain, so strain-specific strategies should be developed to increase their production.