Pseudomonas aeruginosa PumA acts on an endogenous phenazine to promote self-resistance

Abstract

The activities of critical metabolic and regulatory proteins can be altered by exposure to natural or synthetic redox-cycling compounds. Many bacteria, therefore, possess mechanisms to transport or transform these small molecules. The opportunistic pathogen Pseudomonas aeruginosa PA14 synthesizes phenazines, redox-active antibiotics that are toxic to other organisms but have beneficial effects for their producer. Phenazines activate the redox-sensing transcription factor SoxR and thereby induce the transcription of a small regulon, including the operon mexGHI-opmD, which encodes an efflux pump that transports phenazines, and PA14_35160 (pumA), which encodes a putative monooxygenase. Here, we provide evidence that PumA contributes to phenazine resistance and normal biofilm development, particularly during exposure to or production of strongly oxidizing N-methylated phenazines. We show that phenazine resistance depends on the presence of residues that are conserved in the active sites of other putative and characterized monooxygenases found in the antibiotic producer Streptomyces coelicolor. We also show that during biofilm growth, PumA is required for the conversion of phenazine methosulfate to unique phenazine metabolites. Finally, we compare ∆mexGHI-opmD and ∆pumA strains in assays for colony biofilm morphogenesis and SoxR activation, and find that these deletions have opposing phenotypic effects. Our results suggest that, while MexGHI-OpmD-mediated efflux has the effect of making the cellular phenazine pool more reducing, PumA acts on cellular phenazines to make the pool more oxidizing. We present a model in which these two SoxR targets function simultaneously to control the biological activity of the P. aeruginosa phenazine pool.

Phenazine oxidation by a distal electrode modulates biofilm morphogenesis

“…2 B and C. Based on these graphs, we determined the time point for the onset of wrinkling. We note the PMS-dependent delay in onset of wrinkling when no potential was applied, which is in agreement with previous studies [ 15 , 16 ] and is due to the contribution of O 2 to PMS oxidation. To assess the effect of an applied potential of +200 ​mV vs. Ag/AgCl we compared the delay in onset of wrinkling between stimulated and unstimulated colonies in the presence and absence of PMS.…”

“…Although some of these phenazines have been demonstrated to act as electron shuttles [ 8 ], only the methylated phenazines--in particular 5-Me-PCA, which has the most oxidizing redox potential--prevent wrinkle formation. We previously established that the commercially available PMS is a good surrogate for 5-Me-PCA: supplementing agar with 200 ​μM PMS does not confer toxicity and is sufficient to prevent wrinkle formation in Δ phz colony biofilms [ 15 , 16 ]. For the current study, it was important to choose a PMS concentration that (i) is low enough to allow for colony wrinkling in the absence of electrochemical stimulation but also (ii) high enough for sufficient PMS-dependent electron transfer between the electrode and the colony biofilm.…”

Resistance-Nodulation-Division Efflux Pump, LexABC, Contributes to Self-Resistance of the Phenazine Di-N-Oxide Natural Product Myxin in Lysobacter antibioticus

“…The gene LaPhzX encoding putative small monooxygenase in myxin biosynthetic gene cluster, which is similar to ActVA-Orf6, TcmH ( Zhao et al, 2016 ) and PumA ( Sporer et al, 2018 ), has not been characterized. PumA was reported to contribute to phenazine resistance and was required for the conversion of PMS to unique phenazine metabolites, but its exact function was not clear ( Sporer et al, 2018 ). We predict LaPhzX may contribute to the defense line of myxin, but further study is needed to determine its function in myxin resistance.…”

Light-mediated decreases in cyclic di-GMP levels are potentiated by pyocyanin and inhibit structure formation inPseudomonas aeruginosabiofilms

“…Transcriptional reporters of SoxR-driven mexG expression were constructed by 420 transforming plasmid pLD2726 (58) into Escherichia coli S17-1 and moved into PA14 WT and ∆phz by conjugation as described in (59). The plasmid backbone was resolved out using FLP recombinase, introduced on plasmid pFLP2, and counterselected on sucrose plates as described in (59).…”