Fig 1.
Synthesis and morphological analysis of ZnO NPs.
A. Scanning electron microscope image of ZnO NPs used in this study; white bar: 100 nm. B. Size distribution of ZnO NPs. C. X-ray diffraction patterns of ZnO NPs synthesized by the sol-gel method.
Fig 2.
Effects of various ZnO-NP concentrations on the growth of Bacillus subtilis.
A. Growth analysis curves were measured by monitoring the optical density (OD) at 600 nm. B. Antibacterial activity of ZnO NPs on B. subtilis cells; ZnO-NP concentrations are shown as -￭-: 0 ppm, -●-:5 ppm -▲-: 10 ppm, -▼-: 25 ppm, -◆-: 50 ppm, -◀-: 100 ppm, and -▶-: 200 ppm.
Fig 3.
Localization of FtsZ in wild-type cells grown under concentrations of 0, 50, and 100 ppm ZnO NPs in LB at 37°C.
FtsZ was stained green; cell membranes were stained red; and DNA was stained blue. The closed yellow arrows indicate medial FtsZ rings.
Fig 4.
Flow cytometry and fluorescent micrograph analysis of RedoxSensor activity in B. subtilis.
Wild-type bacteria were grown for 3 hrs at ZnO-NP concentrations of 0 ppm, 10 ppm, 25 ppm, 50 ppm, 100 ppm. Unstained samples and PBS buffer alone were used as controls. The X axis indicates RedoxSensor or PI fluorescence intensity (arbitrary units: au), and the Y axis indicates cell counts. A. Flow cytometry analysis of Redoxsensor activity, and B. Flow cytometry analysis of PI activity. C. Fluorescent micrographs of B. subtilis cells indicate RedoxSensor activity or PI fluorescence after incubation with different concentrations of ZnO-NPs for 3 h. Redoxsensor activity presents a false green color. PI presents a false red color. Scale bar: 10 μm.
Fig 5.
Phag-GFP at varying concentrations of ZnO NPs.
Fluorescent micrographs of B. subtilis cells show the expression of Phag-GFP after cultivation with ZnO-NP concentrations of 0, 5, 10, 25, 50, and 100 ppm for 3 h. GFP reporter expression presents a false green color. DAPI presents a false blue color. Scale bar: 10 μm.
Fig 6.
Flow cytometry analysis of Phag-GFP at various concentrations of ZnO NPs.
Wild-type bacteria were grown at different ZnO-NP concentrations for 3 h. A: PBS, B: unstained cells, C: 0 ppm, D: 10 ppm, E: 25 ppm, F: 50 ppm, and G: 100 ppm. The X axis indicates GFP fluorescence intensity (arbitrary units: au), and the Y axis indicates cell counts.
Fig 7.
ZnO NPs affect biofilm formation.
A. The pellicle column depicts microtiter wells (6-well plate) in which cells were grown in biofilm medium with various concentrations of ZnO NPs at 25°C for 3 days (scale bar: 2 cm). Bacterial wild-type (3610) and mutant strains are indicated as follows: sinR (DS92), epsA-O (DS696), sfp (DS3629), tasA (DS3630), and sinR epsA-O (HS222). B. Images of a 12-well microtiter dish containing ethanol-precipitated supernatant from the indicated strain, following treatment with different concentrations of ZnO NPs. C. The supernatants of the indicated strains were treated with proteinase K, DNase, and RNase, precipitated with ethanol, and resolved through SDS-PAGE on a 12% gel, after which staining with Stains-All was performed. D. FT-IR spectra analysis of EPS from Bacillus cells treated with ZnO NPs. Wild type bacteria were grown at ZnO NP concentrations of 0, 5, 10, 25, and 50 ppm, and untreated eps mutant cells were grown to serve as a negative control.
Fig 8.
XANES and EXAFS spectra for B. subtilis cells treated with ZnO NPs.
A. ZnO K-edge XANES spectra of silver standards and B. subtilis cells treated with 100 ppm of ZnO-NPs. B. ZnO K-edge EXAFS spectra of ZnO standards and B. subtilis cells treated with 100 ppm of ZnO-NPs. The best-fitting EXAFS spectra are indicated by the colored symbol lines. (Zn-Zn standard: red; ZnO standard: blue; Bacillus subtilis cells treated with 100 ppm of ZnO-NPs: black).