Coomassie brilliant blue

Three microliters of overnight culture (from a single colony grown in 5 mL BHI broth) from all strains were dropped on span agar plates (H. Carroux, Germany) with or without sodium chloride (5%) and congo red solution [0.5% congo red (Sigma-Aldrich, Taufkirchen, Germany) and 0.25% coomassie-brilliant-blue (Carl Roth, Karlsruhe, Germany) diluted in ethanol]. Plates were incubated for 5 days at 28°C (Romling, 2005 (link); Richter, 2011 ). Following an initial comprehensive screening to detect differences between WT and PCV strains regarding their cellulose and/or curli fimbriae production, follow-up runs were performed on plates containing and lacking sodium chloride for all strains (Table 2). These follow-up runs were repeated six times. Reference strains (AAEC189, Blomfield et al., 1991 (link), IMT26949, and W3110 Hayashi et al., 2006 (link)) were included in all runs for all plates and at all temperatures.

Proteins were mixed in 200 μL of binding buffer (10 mM HEPES/NaOH, 10 mM MgCl₂, 300 mM NaCl, 0.1% Triton X-100, pH 7.5). The protein concentrations were 1.5 or 2.0 μM for PBP1A or PBP1B, 3 μM for PgpB-ht and 5 μM for ht-BacA. Samples were incubated at ambient temperature for 10 min to allow possible complexes to form. If cross-linking was required, 0.2% (w/v) formaldehyde (Millipore Sigma) was added followed by incubation at 37 °C for 10 min. Excess cross-linking was blocked by addition of 100 mM Tris/HCl, pH 7.5. Complexes were pulled down by O/N incubation at 4 °C with 100 µL of washed and equilibrated Ni-NTA superflow beads (QIAGEN, Hilden, Germany). Beads were washed 5–8 times with 1.5 mL of wash buffer (10 mM HEPES/NaOH, 10 mM MgCl₂, 500 mM NaCl, 50 mM imidazole, 0.05% Triton X-100, pH 7.5). For samples without cross-linker a lower imidazole concentration of 20 mM was used in the wash buffer. Retained proteins were eluted by boiling the beads in SDS-PAGE loading buffer; beads were then removed, and samples resolved by SDS-PAGE. Gels were stained with Coomassie brilliant blue (Carl Roth, Germany).

This method was adapted from (Egan et al., 2015 (link)). Proteins (1 μM) were mixed in 200 μl binding buffer (10 mM Hepes/NaOH, 10 mM MgCl₂, 150 mM NaCl, 0.05% Triton X-100, pH 7.5). Samples were applied to 100 μl of washed and equilibrated Ni-NTA superflow beads (Qiagen, The Netherlands) and incubated overnight at 4˚C with gentle mixing. The beads were then washed with wash buffer (10 mM Hepes/NaOH, 10 mM MgCl₂, 500 mM NaCl, 50 mM imidazole, 0.05% Triton X-100, pH 7.5) and boiled in SDS–PAGE loading buffer. Beads were pelleted by centrifugation and samples analysed by SDS–PAGE. Gels were stained with Coomassie brilliant blue (Roth, Germany).