The Pseudomonas aeruginosa oxidative stress regulator OxyR influences production of pyocyanin and rhamnolipids: protective role of pyocyanin

Abstract

The LysR-type transcriptional regulator (LTTR) OxyR orchestrates the defence of the opportunistic pathogen Pseudomonas aeruginosa against reactive oxygen species. In previous work we also demonstrated that OxyR is needed for the utilization of the ferrisiderophore pyoverdine, stressing the importance of this regulator. Here, we show that an oxyR mutant is unable to swarm on agar plates, probably as a consequence of absence of production of rhamnolipid surfactant molecules. Another obvious phenotypic change was the increased production of the phenazine redox-active molecule pyocyanin in the oxyR mutant. As already described, the oxyR mutant could not grow in LB medium, unless high numbers of cells (>108 ml−1) were inoculated. However, its growth in Pseudomonas P agar (King's A), a medium inducing pyocyanin production, was like that of the wild-type, suggesting a protective action of this redox-active phenazine compound. This was confirmed by the restoration of the capacity to grow in LB medium upon addition of pure pyocyanin. Although both rhamnolipid and pyocyanin production are controlled by quorum sensing, no obvious changes were observed in the production of N-acylhomoserine lactones or the Pseudomonas quinolone signal (PQS). Complementation of rhamnolipid production and motility, and restoration of normal pyocyanin levels, was only possible when the oxyR gene was in single copy, while pyocyanin levels were increased when oxyR was present in a multicopy vector. Conversely, plating efficiency was increased only when the oxyR gene was present in multicopy, but not when in single copy in the chromosome, due to lower expression of oxyR compared with the wild-type, suggesting that some phenotypes are differently affected in function to the levels of OxyR molecules in the cell. Analysis of transcripts of oxidative stress-response enzymes showed a strong decrease of katB, ahpC and ahpB expression in the oxyR mutant grown in LB, but this was not the case when the mutant was grown on P agar, suggesting that the OxyR dependency for the transcription of these genes is not total.

A Survival Strategy for Pseudomonas aeruginosa That Uses Exopolysaccharides To Sequester and Store Iron To Stimulate Psl-Dependent Biofilm Formation

“…Swarming assay. A swarming motility assay was performed as previously described by using nutrient broth with 0.5% Difco Bacto agar (42). After inoculation, swarming plates were incubated for another 12 to 24 h at 37°C until swarming zones formed.…”

Pseudomonas aeruginosa AlgR Phosphorylation Modulates Rhamnolipid Production and Motility

“…Rhamnolipid assays. Rhamnolipid production was determined using three different assays: (i) an orcinol assay, (ii) thin-layer chromatography (TLC), and (iii) a hemolysis assay, as described previously (28,(40)(41)(42)(43). To quantify the amount of rhamnolipid produced by each strain, the orcinol rhamnolipid assay was used (28,42,44).…”

“…Total-rhamnolipid values were normalized to total-protein values of cell pellets measured by a Bradford assay. TLC was performed as described previously (43). Briefly, samples were extracted as described for the orcinol assay.…”

MexT Functions as a Redox-Responsive Regulator Modulating Disulfide Stress Resistance in Pseudomonas aeruginosa

“…Several LTTRs are known to activate gene expression as higher oligomeric complexes, which form on recognition of their cognate signals (2,53,55,57). Studies on the classical LysR regulator OxyR, which forms oligomeric complexes when oxidized, suggest that a subset of LTTRs are activated via a redox-sensing mechanism (2,69,70,72). MexT is quiescent in wild-type P. aeruginosa cells, suggesting that the MexT coinducer is absent or present at low concentrations under standard laboratory growth conditions (60,67).…”