Outer Membrane Machinery and Alginate Synthesis Regulators Control Membrane Vesicle Production inPseudomonas aeruginosa

Abstract

The opportunistic human bacterial pathogen Pseudomonas aeruginosa produces membrane vesicles (MVs) in its surrounding environment. Several features of the P. aeruginosa MV production mechanism are still unknown. We previously observed that depletion of Opr86, which has a role in outer membrane protein (OMP) assembly, resulted in hypervesiculation. In this study, we showed that the outer membrane machinery and alginate synthesis regulatory machinery are closely related to MV production in P. aeruginosa. Depletion of Opr86 resulted in increased expression of the periplasmic serine protease MucD, suggesting that the accumulation of misfolded OMPs in the periplasm is related to MV production. Indeed, the mucD mutant showed a mucoid phenotype and the mucD mutation caused increased MV production. Strains with the gene encoding alginate synthetic regulator AlgU, MucA, or MucB deleted also caused altered MV production. Overexpression of either MucD or AlgW serine proteases resulted in decreased MV production, suggesting that proteases localized in the periplasm repress MV production in P. aeruginosa. Deletion of mucD resulted in increased MV proteins, even in strains with mutations in the Pseudomonas quinolone signal (PQS), which serves as a positive regulator of MV production. This study suggests that misfolded OMPs may be important for MV production, in addition to PQS, and that these regulators act in independent pathways.

Low concentrations of ethanol stimulate biofilm and pellicle formation in Pseudomonas aeruginosa

“…17−21) To determine the impact of ethanol on exopolysaccharide synthesis, we constructed in-frame deletion mutants, including ΔpelA, ΔpslA, and ΔalgD, which do not synthesize Pel, Psl, and alginate, respectively, by a previously described protocol, 22) and biofilm formation in these mutants was examined in LB and M63 medium (0.4% glucose and 0.2% casamino acid as carbon sources). Deletion of pslA decreased biofilm formation in the microtiter plate assay (Fig.…”

Pseudomonas Quinolone Signal Affects Membrane Vesicle Production in not only Gram-Negative but also Gram-Positive Bacteria

“…The toluene-tolerant bacterium P. putida IH-2000 releases MVs for the elimination of toluene (17). Moreover, misfolded outer membrane proteins accumulated in the periplasm are discharged through MVs in Gramnegative bacteria (29,35). Thus, MVs have a role in the elimination as well as delivery of virulence factors and cellto-cell communication.…”

“…The viability of B. subtilis in planktonic shaking cultures at the stationary phase was determined using a BacLight LIVE/DEAD bacterial viability staining kit (Molecular Probes, Eugene, OR, USA) as described previously (35). Live SYTO9-stained cells and dead propidium iodide-stained cells were counted.…”

“…Vesicle extraction was performed as described previously (35). The quantity of vesicles was determined by phospholipid assay (35).…”

“…Vesicle extraction was performed as described previously (35). The quantity of vesicles was determined by phospholipid assay (35). Vesicles were purified using a previously reported method (16).…”