Phenotype differences of the four strains

Four streaked strains of WT, ΔdgcA, OdgcA, and CdgcA were respectively picked up from plates and inoculated into the M210 medium (containing antibiotic if needed), which were then grown for three days with shaking at 28 °C. Each culture was diluted to a starting OD₆₀₀ of approximately 0.01 in 100 ml of fresh M210 medium containing the required antibiotic, which was then incubated at 28 °C with shaking for about 44 h to reach the late logarithmic phase. This activated culture was diluted to an OD₆₀₀ of 0.05 and prepared for the following phenotypic assays.

Growth curve was measured by taking aliquots of culture at approximately 4 h intervals until 73 h, and the OD₆₀₀ values of each strain were recorded and plotted versus time⁹. For colony morphology observation, cultures were initiated at an OD₆₀₀ of 0.05 and 3 μl of each culture was dropped onto the M210 plates. Cells were grown at 28 °C and monitored after three days.

After growing at 28 °C for 44 h, half cultures were collected by centrifugation at 10,000 × g for 10 min for measuring the EPS production as reported^1,5,11. 10 ml cultures were transferred to a 15 ml round bottom glass tube to determine the autoaggregation ability as previously described^31,57. These tubes were allowed to sit without agitation at room temperature for 0, 1, 2, 3 and 12 h respectively. The OD₆₀₀ of the supernatant in the static tube culture was determined to monitor relative autoaggregation activity, which was calculated using the OD difference value relative to the densities at the initial experiment⁵⁷.

Swimming motility assays were investigated on the semi-solid M210 medium plates with 0.3% agar as described¹⁵. Aliquots (3 μl) of each diluted culture were spotted onto the plates, which were photographed after incubating at 28 °C for three and four days, respectively^3,50. The experiments were repeated at least three times and a representative result was chosen for display. Biofilm formation assay was carried out by incubating the diluted culture for five days at 28 °C under static conditions. Non-adherent bacteria and medium were discarded and biofilm was washed with deionized water. The attached biofilm was stained with 0.1% (w/v) crystal violet for 30 min at room temperature⁵⁸, thoroughly washed with water for 3 times and air-dried. The bound dye was solubilized in 90% ethanol and quantified by measuring absorption at 590 nm⁹. All the phenotypic assays were performed in triplicate.