For Congo red binding and fluorescence assays –

For CR assays, the Ec Bcs total membrane complex (TMC; including inner and outer membrane components) constituting the functional cellulose synthase machinery was expressed using all three plasmids (pETDuet_Ec_Bcs_AB-AdrA, pACYCDuet_Ec_Bcs_CZFG and pCDFDuet_Ec_Bcs_R_Q_E-HA and the Ec Bcs_inner membrane complex (IMC) was expressed using the pACYCDuet_Ec_Bcs_FG construct instead of pACYCDuet_Ec_Bcs_CZFG, along with the other two constructs (pETDuet_Ec_Bcs_AB-AdrA and pCDFDuet_Ec_Bcs_R_Q_E-HA). Different versions of BcsZ or the BcsG mutant in the pACYCDuet_Ec_Bcs_CZFG plasmid generated in this study (as described above in construct design) were also expressed like the TMC complex using the two other pET and pCDFDuet_Ec_Bcs plasmids.