FIG. 4.

Influence of quorum sensing on biofilm formation by P. putida PCL1445 in PVC microtiter wells. Cells of PCL1445 and its quorum-sensing mutant derivatives were incubated in microtiter plates in M63 medium, and their biofilm formation was quantified over time using the crystal violet staining procedure. Cells attached to the microtiter wells were stained with crystal violet and washed, and the crystal violet in the biofilm was dissolved in ethanol, after which the OD₅₉₅ was measured. To determine surface tension-reducing activity in the well, 25 μl of culture was pipetted as a droplet on parafilm and allowed to dry. The diameter of the dried droplet correlates with surface tension reduction. A surfactant activity index based on the droplet diameter and ranging from 0 to 4 was used to quantify surface tension reduction. All experiments were performed in triplicate. (A) Time course of biofilm formation of PCL1445 (•), PCL1633 (psoA) (□), PCL1637 (ppuI) (▪), PCL1638 (ppuR) (○), and PCL1639 (rsaL) (Δ). As a negative control, uninoculated M63 medium was used (▴). (B) Biosurfactant activity present in the titer well during biofilm formation by bacterial cultures shown in panel A, as determined by the drop-collapsing assay. (C) Biofilm formation by PCL1445 and PCL1636 (ppuI) measured after 24 h of incubation. Bars: a, M63 medium without bacteria; b, PCL1445; c, PCL1445 containing pME6010; d, PCL1436 (psoA); e, PCL1636 (ppuI); f, PCL1636 (ppuI) harboring pMP5548 (ppuI); g, PCL1639 (psoA/ppuI); h, M63 plus 3-oxo-C12-AHL (5 μM); i, PCL1636 (ppuI) plus pure acetonitrile (control); j, PCL1636 (ppuI) plus 3-oxo-C12-AHL (5 μM). Standard deviations are based on the mean values of triplicate cultures. (D) Biosurfactant activity as determined by the drop-collapsing assay of bacterial cultures shown in the biofilm assay in panel C. Lanes: a, M63 medium without bacteria; b, PCL1445; c, PCL1445 containing pME6010; d, PCL1436 (psoA); e, PCL1636 (ppuI); f, PCL1636 (ppuI) harboring pMP5548 (ppuI); g, PCL1639 (psoA/ppuI); h, M63 plus 3-oxo-C12-AHL (5 μM); i, PCL1636 (ppuI) plus pure acetonitrile (control); j, PCL1636 (ppuI) plus 3-oxo-C12-AHL (5 μM). (E) An index ranging from 0 to 4 was used for the detection of biosurfactant production by bacterial cells in the biofilm assay. Shown are dried droplets of 25-μl culture supernatants with increased diameter due to decreased surface tension caused by increased biosurfactant activity.