Scl1 expression in L. lactis promotes biofilm formation. L. lactis was transformed with the plasmid construct pSL230 to express Scl1.41 surface protein or with pJRS525 vector. (a) PCR analysis of L. lactis transformants using scl1.41-gene-specific primers; lanes: (1) MG1363 wild-type (WT) cells; (2) MG1363::pJRS525 vector-only control; (3) MG1363::pSL230 transformant; (4) control pSL230 plasmid DNA. (b) Scl1.41 expression by western blot analysis of cell-wall extracts prepared from transformed L. lactis and control GAS strains using anti- P176 (rScl1.41) antibodies; lanes: (1) purified recombinant P176 protein (truncated Scl1.41); (2) MG1363 WT strain; (3) MG1363::pJRS525 vector; (4) MG1363::pSL230 transformant; (5) MGAS6183 (M41) control. (c) Analysis of Sc1.41 expression by flow cytometry with anti-P176 (rScl1.41) rabbit polyclonal antibodies on the surface of MGAS1363 WT strain (black trace), MGAS1363::pJRS525 vector-only control (green trace) and MG1363:pSL230 transformant (red trace). (d) Crystal violet staining of 24 h biofilms formed by L. lactis WT strain, MG1363::pJRS525 vector-only control or MG1363::pSL230 transformant (top) with visual representation of the corresponding wells (bottom). Statistical significance is denoted as **P ≤ 0.001. (e) CLSM analysis of 24 h biofilms from same experiment shown in (d). Images are X-Y orthogonal Z-stack views representative of ten images within a single experiment. Average vertical biofilm thickness is indicated in micrometers (top right).