To test the ability of SypA variants to induce biofilm formation despite the presence of active SypE, the sypG overexpression plasmid pEAH73 [25 (link)] was introduced into the strains by conjugation and selection for CmR. These strains were grown overnight in LBS containing Cm, then subcultured into the same medium at 28°C. After growth to mid-log phase, cultures were normalized to an OD600 of 0.2 and an aliquot of 10 μL was spotted onto LBS Cm plates. After the spots dried, the plates were inverted and incubated for 48 h at 28°C. After incubation, images of the spots were captured using a Zeiss Stemi 2000-C dissecting microscope.
To test the activity of SypA variants, we evaluated their ability to complement a ΔsypA mutant under conditions in which SypE is non-inhibitory, i.e., during overexpression of rscS from plasmid pKG11 [23 (link)]. When overproduced under these LBS conditions, RscS inhibits the kinase activity of SypE, permitting SypA to be unphosphorylated and thus active [26 (link)]. The pKG11-containing strains were grown and analyzed as described above for SypG overproduction, with the exception that the plates were incubated at 24°C.
To test the activity of SypA variants, we evaluated their ability to complement a ΔsypA mutant under conditions in which SypE is non-inhibitory, i.e., during overexpression of rscS from plasmid pKG11 [23 (link)]. When overproduced under these LBS conditions, RscS inhibits the kinase activity of SypE, permitting SypA to be unphosphorylated and thus active [26 (link)]. The pKG11-containing strains were grown and analyzed as described above for SypG overproduction, with the exception that the plates were incubated at 24°C.
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