Membrane vesicle formation as a multiple-stress response mechanism enhances Pseudomonas putida DOT-T1E cell surface hydrophobicity and biofilm formation

Abstract

Among the adaptive responses of bacteria to rapid changes in environmental conditions, those of the cell envelope are known to be the most crucial. Therefore, several mechanisms with which bacteria change their cell surface and membranes in the presence of different environmental stresses have been elucidated. Among these mechanisms, the release of outer membrane vesicles (MV) in Gram-negative bacteria has attracted particular research interest because of its involvement in pathogenic processes, such as that of Pseudomonas aeruginosa biofilm formation in cystic fibrosis lungs. In this study, we investigated the role of MV formation as an adaptive response of Pseudomonas putida DOT-T1E to several environmental stress factors and correlated it to the formation of biofilms. In the presence of toxic concentrations of long-chain alcohols, under osmotic stress caused by NaCl, in the presence of EDTA, and after heat shock, cells of this strain released MV within 10 min in the presence of a stressor. The MV formed showed similar size and charge properties, as well as comparable compositions of proteins and fatty acids. MV release caused a significant increase in cell surface hydrophobicity, and an enhanced tendency to form biofilms was demonstrated in this study. Therefore, the release of MV as a stress response could be put in a physiological context.

Fig 1

Representative cryo-transmission electron microscopy images of MV formed by P. putida DOT-T1E after incubation with 1-octanol (A), NaCl (B), heat shock (C), and EDTA (D). Bars, 200 nm.

Fig 2

Comparative overview of SDS-PAGE results of the isolated MV. Lane 1, standard; lane 2, 1-octanol-induced MV; lane 3, heat shock-induced MV; lane 4, NaCl-induced MV; lane 5, EDTA-induced MV.

Fig 3

Effects of 1-octanol (A), heat shock (B), NaCl (C), and EDTA (D) on water contact angles of growing cells of P. putida DOT-T1E. Error bars show standard deviations.

Fig 4

Microscopic images of biofilms formed by P. putida on polystyrene microtiter wells after incubation with HgCl₂ (A), 1-octanol (B), heat shock (C), NaCl (D), and EDTA (E).

Fig 5

Quantification of biofilm formation of P. putida after incubation with different stressors. Error bars show standard deviations.

Fig 6

Possible sequence of biofilm formation in P. putida after exposure to a stressor.