Protein and CPS expression

All bacterial cultures described in this work were grown at 37 °C and with shaking at 220 rpm, unless noted otherwise. Working concentrations: ampicillin 100 mg l⁻¹, kanamycin 50 mg l⁻¹, streptomycin 50 mg l⁻¹ and chloramphenicol 25 mg l⁻¹. Appropriate plasmids were transformed into C43 cells (for protein purification) or C43ΔCPS1 cells (for in vivo encapsulation, MINFLUX and CPS purification) for overnight growth in the presence of suitable antibiotics. All collected cell pellets in this study were flash-frozen in liquid nitrogen and stored at −80 °C for further use.

For western blotting: For expression testing, an overnight starter culture of cells expressing all PmCPS components was used to inoculate 1 l of LB medium supplemented with ampicillin and chloramphenicol. At an optical density at 600 nm (OD₆₀₀) of 0.6, protein expression was induced with 100 mg l⁻¹ of IPTG. Growth was continued for another 3–4 h, after which cells were collected (4,500 rpm for 20 min) and flash-frozen in liquid nitrogen. This cell pellet was used to prepare inverted membrane vesicles, as described previously⁴⁴. The inverted membrane vesicles were then run on a 12.5% polyacrylamide gel and analysed using the western blot technique detecting the engineered affinity tags, as described previously⁴⁵.

For in vivo encapsulation assays and spheroplasting: 20 ml LB culture was inoculated with a single stab of the appropriate transformants and grown in the presence of appropriate antibiotics and 100 mg l⁻¹ IPTG. Growth was carried out for 6–8 h, after which cells were collected. Spheroplasts were prepared as described previously⁴⁶. After removal of the outer membrane, spheroplasts were resuspended in PBS supplemented with 200 mM sucrose, and used for in vivo encapsulation assays.

For purification of CPS: 8× 1 l of 2× LB supplemented with appropriate antibiotics were inoculated from an overnight starter culture. At an OD₆₀₀ of 0.6, the medium was cooled to 20 °C, and then cells were induced using 100 mg l⁻¹ of IPTG, grown overnight and collected.

For purification of the CBM70, SNAP–CBM70 and KpsMT–KpsE proteins: 6× 1 l of 2× LB supplemented with appropriate antibiotics were inoculated from an overnight starter culture. At an OD₆₀₀ of 0.6, protein expression was induced using 200 mg l⁻¹ of IPTG, and cells were grown for another 3–4 h at 37 °C, after which they were collected.