Niso4

Droplets containing 100 μl of diluted (1 mg/ml) pH7.5–8.0 His-tagged recombinant N92 amelogenin were placed on carbon coated copper TEM grids (Ted Pella, Redding, CA) and incubated in a moisturized container at 37°C for 2 h. Thereafter, TEM grids were quickly rinsed with DDW, immersed into 100 μl of freshly prepared 1% NiSO₄ (Sigma, St. Louis, MO) solution for 30 min, quickly rinsed with DDW again, air dried, and analyzed using a JEOL 1220EX transmission electron microscope at the UIC Research Resources Center (Chicago, IL).

Chemicals and solvents (acrylamide, N,N′-methylenebisacrylamide, DTT, urea, glycerol, Hepes, isopropyl-β-d-thiogalactopyranoside, imidazole, Tris, NaCl, KCl, NaOH, EDTA, HCl, SDS, Coomassie Brilliant Blue G-250 and divalent metal salts CoCl₂, MgCl₂, MnCl₂, ZnCl₂, and NiSO₄) were purchased from Sigma-Aldrich and Panreac Química SLU and used without further purification. dNTPs were bought from Biosan. All the solutions were prepared using double-distilled water.

To assess the metal resistance of the produced biofilms, Pb²⁺, Zn²⁺, Cd²⁺, Cu²⁺, and Ni²⁺ salts were generated from Pb(NO₃)₂, ZnSO₄, CdSO₄, CuSO₄, and NiSO₄ salts (Sigma-Aldrich, St. Louis, MO, USA). No more than 60 min before usage, working solutions were produced in TSB medium from stock solutions. Based on the previous research (Basak et al., 2014 (link)) and the preliminary test, a concentration range of 100,000–781 μg/ml was chosen (Buzejić et al., 2016 (link)).

The ability of the ureA transcriptional fusions to drive expression of GFP was assessed visually utilizing an Olympus BX61 fluorescent microscope, as well as using flow cytometry as previously described (Carpenter et al., 2007 (link)). Briefly, strains containing the ureA promoter fusions were grown overnight in liquid cultures containing varying NiSO₄ concentrations (0, 0.5, 1.0, 10 μM) (Sigma). Following overnight growth, 0.5–1.5 ml of each culture was pelleted and resuspended in 1–2 ml of sterile 1× phosphate-buffered saline depending on the density of the culture. Bacterial clumps and culture debris were subsequently removed by passing the resuspended culture through a 1.2-μm Acrodisc PSF syringe filter (Pall). Flow cytometry analysis for the ureA fusion constructs was performed using either a Beckman Coulter Epics XL-MCL flow cytometer with a laser setting of 750 V or a BD SLR II flow cytometer. 20,000 events were collected for each assay. WinList 3D, version 6.0 (Verity Software House) and FlowJo, version X (FLOWJO, LLC) were used to analyze the flow cytometry data. These experiments were performed 3–5 times for each strain-reporter plasmid combination.

HEPES, iodoacetamide, Zincon sodium salt, NaCl, hydrogen peroxide, ZnCl₂, KCl, NiSO₄, gallium (99.9995%), NaN₃, HNO₃ 70%, methanol, acetic acid, and ethanol were purchased from Sigma Aldrich (Merck Life Science UK Ltd., Gillingham, UK); Tris hydrochloride (TRIS/HCl) from MP Biomedicals (Fisher Scientific UK, Loughborough Leicester, UK); 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB; Ellman’s reagent) from Invitrogen (Thermo Fisher Scientific UK, Ashford, UK); ≥18.2 MΩ cm ultrapure water was obtained from an ELGA LabWater UK system (High Wycombe, UK).