The autotransporter esterase EstA of Pseudomonas aeruginosa is required for rhamnolipid production, cell motility, and biofilm formation

Abstract

Pseudomonas aeruginosa PAO1 produces the biodetergent rhamnolipid and secretes it into the extracellular environment. The role of rhamnolipids in the life cycle and pathogenicity of P. aeruginosa has not been completely understood, but they are known to affect outer membrane composition, cell motility, and biofilm formation. This report is focused on the influence of the outer membrane-bound esterase EstA of P. aeruginosa PAO1 on rhamnolipid production. EstA is an autotransporter protein which exposes its catalytically active esterase domain on the cell surface. Here we report that the overexpression of EstA in the wild-type background of P. aeruginosa PAO1 results in an increased production of rhamnolipids whereas an estA deletion mutant produced only marginal amounts of rhamnolipids. Also the known rhamnolipid-dependent cellular motility and biofilm formation were affected. Although only a dependence of swarming motility on rhamnolipids has been known so far, the other kinds of motility displayed by P. aeruginosa PAO1, swimming and twitching, were also affected by an estA mutation. In order to demonstrate that EstA enzyme activity is responsible for these effects, inactive variant EstA* was constructed by replacement of the active serine by alanine. None of the mutant phenotypes could be complemented by expression of EstA*, demonstrating that the phenotypes affected by the estA mutation depend on the enzymatically active protein.

FIG. 1.

Amounts of rhamnolipids in culture supernatants of different P. aeruginosa PAO1 strains. The amounts of rhamnolipids in culture supernatants were determined by an indirect assay (orcinol test). Wild-type (wt) P. aeruginosa PAO1, the estA mutant, and mutants overexpressing estA from plasmid pBBX+ and pBBXmut (inactive variant) were compared, and the rhlA mutant and strains containing the empty vector pBBR1MCS (V) served as controls. Culture supernatants were collected after 48 h of growth. The results were plotted into two diagrams because of the significant differences in the rhamnolipid amounts. Standard deviations were derived from three independent experiments.

FIG. 2.

TLC separation of extracellular and cell-bound rhamnolipids from P. aeruginosa. P. aeruginosa PAO1 (wild type [wt]) and the estA-negative mutant (estA-) were examined for rhamnolipid production. Rhamnolipids were extracted from cultures which were grown for 48 h in PPGAS medium. The rhamnolipid standard (RL) was purified from P. aeruginosa. Rhamnolipids were separated by TLC and stained with a reagent containing orcinol and sulfuric acid in ethanol.

FIG. 3.

Agrobacterium tumefaciens-based bioassay for autoinducer (AI) detection in culture supernatants of P. aeruginosa. Tested were wild-type (WT) P. aeruginosa PAO1 and the estA-negative mutant for the presence of AIs, and an AI-deficient mutant (rhlRI and lasRI deficient) served as a negative control. Shown is the A. tumefaciens-based bioassay, where the formation of blue halos confirmed the presence of AI molecules.

FIG. 4.

Swimming (A), swarming (B), and twitching (C) motility of P. aeruginosa PAO1. Wild-type P. aeruginosa (wt) and estA-negative mutant (estA-) strains were analyzed on agar plates. In addition the estA-negative mutant was transformed with different plasmids, i.e., the empty vector (v) as a negative control, the estA-carrying plasmid pBBX+, and a mutated version of pBBX coding for an inactive esterase (pBBXmut). The agar plates contained different agar concentrations to establish swimming (0.3%) and swarming (0.5%) motility and were incubated overnight at room temperature (swimming) or at 30°C (swarming plus twitching). (D) Motility of the P. aeruginosa estA mutant with the addition of exogenous rhamnolipids (RLs) and the wild type was monitored. Extracted RL was added to the agar plates at a concentration of 0.4 g/liter, which represents the wild-type level of RLs.

FIG. 5.

Biofilm confocal laser scanning micrographs of different P. aeruginosa strains. P. aeruginosa PAO1, the estA mutant, and mutants overexpressing estA from plasmids pBBX+ and pBBXmut (inactive variant) were compared, and the strain containing the empty vector pBBR1MCS (V) served as a control. Biofilms were grown in a flow cell, and section pictures were taken after 24 and 72 h of growth with ×630 magnification.