Thermo multiskan ex micro plate photometer

The 5 ml AgNPs solutions have been adjusted by adding the stock of AgNPs to the half-strength LB broth in order to obtain a final concentration of AgNPs of 5, 10, 20, and 30 μg/ml, respectively. The MIC of AgNPs was determined by inoculating the 100 μl of bacteria cells of Ao strain RS-2 (∼approximately 1 × 10⁸ CFU/ml) into AgNPs solution of different concentrations, while the control was only sterile ddH₂O (without AgNPs). The samples were then incubated at 30°C for 12 h at 180 rpm. The bacterial numbers in the samples were computed by reading the absorbance value at 600 nm using a ThermoMultiskan EX Microplate Photometer (Thermo Fisher Scientific, Waltham, MA, United States). With six replicates for each treatment, this experiment was repeated three times.

Biofilm formation assays were performed on T6SS mutants of the Aaa strain RS-2 in 96-well microtitre plates (Corning-Costar Corp., Corning, NY, USA) using the method of Peeters et al. [48 (link)]. Briefly, the overnight cell suspension of the Aaa wild-type strain RS-2 and mutants were re-cultured into a fresh LB broth containing appropriate antibiotics with a 1:100 dilution under shaking to mid exponential growth. Then each well was inoculated with 100 µL of approximately ~1 × 10⁸ CFU/mL (OD₆₀₀ = 0.6) bacterial suspension and incubated at 30 °C for 48 h of adhesion without agitation while twelve wells filled with sterile ddH₂O served as blanks. Culture media were then poured out and each well in the plates was washed three times with sterile ddH₂O. Following air-dried for 30 min, each well was stained with 125 µL of 0.1% (w/v) crystal Violet (CV) solution for 45 min at room temperature. The unbound crystal Violet was removed and then washed with ddH₂O. To solubilize the crystal Violet stained cells, 150 µL of 33% acetic acid was added into each well. Bacterial biofilm was quantified by measuring their optical density at 590 nm using a Thermo Multiskan EX Micro plate Photometer (Thermo Fisher Scientific Inc.). Twelve replications of each treatment were used for quantitative measurement in the three repeated experiments.

After protein purification as mentioned above, 1–2.5 μg protein was used to measure superoxide dismutase (SOD) or catalase activities. Precisely, SOD activity was determined with a SOD assay Kit-WST (Beyotime, China). The Catalase Assay Kit (Beyotime, China) was used to detect catalase activity. Briefly, for SOD activity assay, the samples were treated by WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)- 2H-tetrazolium, monosodium salt] and incubated at 37°C for 30 min. Afterward, the absorption maximum at 450 nm was measured by Thermo Multiskan EX Micro plate Photometer (Thermo Fisher Scientific, Waltham, MA, USA). SOD activity was calculated by standard curve according to the instruction of manufacturer in kit. For catalase assay, the samples were treated with 10 μl of 250 mM H₂O₂ and incubated 5 min at room temperature, and the remaining H₂O₂ (not decomposed by catalase) was coupled with a substrate to generate N-4-antipyryl-3-chloro-5-sulfonate-p-benzoquinonemonoimine, which has an absorption maximum at 520 nm and was quantified spectrophotometrically. Catalase activity was then calculated by standard curve according to the instruction of manufacturer in kit. These experiments were repeated three times independently.