Ti s l100

Electron microscopy was performed based on previous methods [53 (link)]. For microscopy with SEM (S-3400N, Hitachi, Tokyo, Japan), B. cereus MB1 and M. luteus were cultured in LB medium to logarithmic phase, and the cells were washed and resuspended in PBS to 1 × 10⁶ CFU/mL. The cells were pretreated with 1 × MBC rCrus1 or PBS at 37 °C for 2 h. After treatment, the cells were fixed with 5% glutaraldehyde in PBS (pH 7.4) for 2 h and dehydrated in a series of increased concentration of ethanol (30, 50, 70, 80, 90 and 100%) for 10 min at 4 °C. The cells were treated with isoamyl acetate for 10 min, critical point-dried (Hitachi-HCP, Hitachi, Japan), sputter-coated with platinum (MC1000, Hitachi, Japan) and examined with a SEM. For microscopy with TEM (HT7700, Hitachi, Tokyo, Japan), B. cereus and M. luteus were pretreated with rCrus1 or PBS as above. TEM microscopy was performed as previously reported [54 (link)]. For the PI assay, B. cereus MB1 and M. luteus were pretreated with rCrus1 or PBS as above. The sample was stained with a PI staining kit (BestBio, Shanghai, China) following the manufacturer’s instruction. The cells were observed with a fluorescence microscope (TiS/L100, Nikon, Tokyo, Japan).

For morphological observation, P18 was cultured in Marine 2216E medium at 28°C to an OD₆₀₀ of 0.8. The culture was centrifuged at 5,000 rpm for 10 min, and the bacterial pellet was re-suspended in ddH₂O. For spore observation, P18 was cultured at 28°C in Marine 2216E medium containing 5mg/L MnSO₄ for 3 days with shaking (180 rpm/min) (Osadchaia et al., 1997 (link)); the culture was then centrifuged and re-suspended in ddH₂O. After fixation with glutaraldehyde and dehydration with acetone, the vegetative cells and spores of P18 were observed with a transmission electron microscope (Hitachi, JEM-2100, Japan). The spores were also stained with a Spore Staining Kit (HaiBo, China) and observed with a light microscope (Ti-S/L100, Nikon, Japan). The temperature range of growth was determined by culturing P18 at 4, 16, 28, 37, and 50°C in Marine 2216E medium for 36 h to 7 days. The pH range was determined by culturing P18 in Marine 2216E medium of different pH (pH 5–10, with an increment of 1 pH unit). The NaCl range was determined by culturing P18 in 2216E medium with different salinities (NaCl 0–8%, w/v, with an increment of 1%). The motility of P18 was determined as reported previously (Gu et al., 2019 (link)).

To observe the morphology of MB1 on the solid medium, MB1 was streaked in 2216EA plate and incubated at 28 °C overnight. The scanning electron microscope (SEM) and transmission electron microscope (TEM) observations were performed as previously reported [22 ]. Sporulation was induced by continuous culturing of MB1 in 2216E medium supplemented with 5 mg/L MnSO₄ at 28 °C for more than 72 h. The spores were stained using a spore staining kit (Solarbio, Beijing, China), followed by observation with a microscope (Ti-S/L100, Nikon, Tokyo, Japan). MB1 growth at different temperatures, pH, and NaCl was examined as reported previously [23 (link)]. For the haemolysis assay, MB1 was cultured in marine 2216E medium to an OD₆₀₀ of 0.8 and centrifuged at 8000 g. The bacterial cells and supernatant were collected after centrifugation. The cells were resuspended in PBS to 10⁸ CFU/mL. Ten microlitres of cell suspension, supernatant, and 2% TritonX-100 were each dropped onto filter discs in a 2216EA plate containing 5% sheep blood. The plate was incubated at 28 °C for 24 h and observed for haemolysis. For swimming analysis, 10 μl of the above MB1 suspension was dropped onto a 2216EA plate containing 0.3% (w/v) agar. The plate was incubated at 28 °C for 24 h and observed for bacterial motility.

Scanning electron microscopy and energy-dispersive X-ray mapping analysis images were obtained using a JEOL JSM 6335F instrument, at an acceleration voltage of 10 kV and 22 kV, respectively. High resolution transmission electron microscopy images were obtained using a JEOL JEM 3000 F microscope at an acceleration voltage of 300 kV. An inverted optical microscope (Nikon Eclipse Instrument Inc. Ti-S/L100), coupled with 20× objective was used to track the speed of the micromotors. Fluorescence images were obtained using an Epi-fluorescence attachment with a UV-2E/C (DAPI) filter cube. UV-VIS experiments were carried out using a Perkin-Elmer Lambda 20 spectrophotometer. Fluorescence spectra were recorded at 25 °C with a PerkinElmer LS-50B luminescence spectrophotometer equipped with a Xe flash lamp. The excitation and emission slit widths were 5 nm and scan speed was 1000 nm min^–1.

B. cereus MB1 and V. harveyi were cultured in corresponding media to OD₆₀₀ of 0.8. The bacteria were resuspended in PBS to 1 × 10⁶ CFU/mL. Crus2 or Crus2DC (1 × MBC) was added to the bacteria, and the mixture was incubated at 28 °C for 2 h. The cells were stained with a PI Staining Kit (BestBio, Shanghai, China) and examined using a fluorescence microscope (TiS/L100, Nikon, Tokyo, Japan). For electron microscopy, the above treated bacterial samples were processed as previously described [22 (link)] and observed with scanning electron microscopy (S-3400N, Hitachi, Tokyo, Japan) and transmission microscopy (HT7700, Hitachi, Tokyo, Japan).