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Project Progress

We used whole bacterial cells to address the question of whether EPS provides armor against cell lysis. A Gram negative wild-type bacterium, Pseudomonas aeruginosa PAO1, which is a robust EPS producer and its knock out mutant, Δ-psl , with defective ability to form EPS and biofilm were used to conduct cell viability studies in suspensions of oxide particles with cells (Figure 1). Wild type cell viability was greater than that of the mutant type. Furthermore, viability was oxide-dependent, increasing as γ-Al₂O₃ > amorphous SiO₂ (Figure 2). Thus, our results confirm our hypothesis that EPS acts as a shield against the toxicity of certain minerals.

We also examined the cell + oxide particle suspensions using High Resolution Transmission Electron Microscopy (HRTEM) to visualize the location of the particles with respect to the cell surface (Figure 3). The amorphous silica particles were located in the extra-cellular region of both wild type cells that possess EPS as well as washed mutant type cells that lack EPS, suggesting that the similar negative charge on both SiO₂ particles and the cell surfaces resulted in electrostatic repulsion and prevented cell-particle contact. In contrast, γ -Al₂O₃ particles of size < 80 nm were seen inside the washed mutant type cells, but were seen only in the extracellular space for the wild type cells. These results indicated that the positively-charged γ-Al₂O₃ particles were electrostatically attracted to the oppositely-charged cell surface. The particles could not penetrate in the case of the wild type due to the presence of the EPS, whereas the smaller particles < 80 nm could enter the mutant type cells that lack EPS.

The toxicity of anatse even without entering the cell was explained by the presence of photocatalytically induced reactive oxygen species (ROS) at the titania surface. ROS can oxidize organic molecules and are known to be membranolytic to cells. The reactivity of these species can be quenched, however, by specific enzymes which scavenge these free radicals. Thus, the relative viability of cells in the presence of bovine liver catalase-treated anatase was much higher than in the case of anatase alone (Figure 4).

Furthermore, we showed that EPS does not simply act as a passive barrier to nanoparticle toxicity. The surface charge of alumina particles was reversed from positive to negative by the adsorption of EPS generated by the wild-type cells, such that repulsion with the negatively-charged surfaces reduced the toxicity of alumina compared to that of mutant type cells which could not generate EPS. Most significantly, the presence of the toxic nanoparticles could induce the formation of additional EPS by the wild type cells compared to the particle-free blanks (Figure 5).

We also determined if the difference in cell wall structure of Gram negative versus Gram positive bacteria could influence the effects of EPS and nanoparticle toxicity. We used biofilm-producing wild-type, NCIB3610, and biofilm-defective mutant, yhxBΔ. We confirmed that the presence of EPS does protect Gram-positive bacteria from toxic oxides. Oxide toxicity was also specific to the mineral in the case of the Gram positive bacteria (Figure 6). Thus, our hypothesis that EPS may have evolved early in the evolution of life seems to hold across the two bacterial types. In addition, the Gram positive bacteria are slightly less susceptible to nanoparticle toxicity compared to the Gram negative bacteria. We interpret this result to indicate that the thick outer layer of peptidoglycan compared to the thin peptidoglycan layer of Gram negative bacteria provides additional protection against the toxicity of oxides (Figure 6).

Our results support our hypotheses that EPS acts an armor against mineral toxicity, cell wall structure differences also provide additional armor and that not all minerals are toxic or benign, but that toxicity depends on mineral properties such as surface charge, presence of surface free radicals and particle size.