Impact of growth environment and physiological state on metal immobilization by Pseudomonas aeruginosa PAO1.

Environmental growth conditions and cell physiology have the potential to influence bacterial surface-metal interactions in both planktonic and biofilm systems. Here, Pseudomonas aeruginosa was studied to determine the influence of these factors (pH, redox potential, and active respiration) on surface electrostatics and metal immobilization. Acid-base titrations revealed a decrease in ionizable ligands at pKa 5 (putative carboxyls) in cells grown below pH 6.2 and in cells grown anaerobically relative to cells grown under oxic and circumneutral pH conditions. This observation correlates with Western immunoblotting assays that revealed a reduction in carboxylated B-band lipopolysaccharide in these cells. Furthermore, spectrophotometric analysis revealed a decrease in zinc, copper, and iron immobilization in these cells, suggesting that lipopolysaccharide modification in response to environmental stimuli influences metal binding. The effect of active versus inactive metabolism on metal adsorption was also examined using respiration inhibitors carbonyl cyanide m-chlorophenylhydrazone and sodium azide. Cells treated with these compounds bound more zinc, copper, and iron than untreated controls, suggesting proton extrusion through respiration competes with metal cations for reactive groups on the cell surface. Accumulation of gold did not show the same trend, and transmission electron microscopy studies confirmed it was not a surface-mediated process. These results suggest that variations in growth environment and cell physiology influence metal accumulation by bacterial cell surfaces and may help to explain discontinuous accumulation of metal observed throughout microbial communities.

Elucidation of functional groups on gram-positive and gram-negative bacterial surfaces using infrared spectroscopy.

Surface functional group chemistry of intact Gram-positive and Gram-negative bacterial cells and their isolated cell walls was examined as a function of pH, growth phase, and growth media (for intact cells only) using attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. Infrared spectra of aqueous model organic molecules, representatives of the common functional groups found in bacterial cell walls (i.e., hydroxyl, carboxyl, phosphoryl, and amide groups), were also examined in order to assist the interpretation of the infrared spectra of bacterial samples. The surface sensitivity of the ATR-FTIR spectroscopic technique was evaluated using diatom cells, which possess a several-nanometers-thick layer of glycoprotein on their silica shells. The ATR-FTIR spectra of bacterial surfaces exhibit carboxyl, amide, phosphate, and carbohydrate related features, and these are identical for both Gram-positive and Gram-negative cells. These results provide direct evidence to the previously held conviction that the negative charge of bacterial surfaces is derived from the deprotonation of both carboxylates and phosphates. Variation in solution pH has only a minor effect on the secondary structure of the cell wall proteins. The cell surface functional group chemistry is altered neither by the growth phase nor by the growth medium of bacteria. This study reveals the universality of the functional group chemistry of bacterial cell surfaces.