Quantification of symplasmata concentration

From overnight cultures of Pe299R and mutants grown in LB medium with appropriate antibiotics, 25 μl was inoculated into 5 ml of fresh LB and incubated for 3 hours at 30 °C with shaking. Cells were harvested by centrifugation at 2,500 g for 10 min, washed twice with M9 devoid of carbon source and resuspended in the same medium to an OD₆₀₀ of 0.2. From this bacterial suspension, 400 μl was added to 20 ml of M9G in a 250 ml-flask, and incubated at 30 °C with shaking. After various periods of incubation, a sample from the culture was mixed with Indian ink (Sanford, Oak Brook, USA) in a 9:1 proportion. Symplasmata were identified as cell clusters with a clear capsule surrounding them and quantified in a hemacytometer (Sigma-Aldrich, St-Louis, USA).