FIG. 2.

Well diffusion assay, with the L. lactis indicator strain HP, to test the inhibitory activity of 50-μl volumes of cell-free supernatant from a number of strains. (A) The zone size produced by MG1614(pMRC01) supernatant is identical to that of MG1363(pMRC01), demonstrating that plasmid transfer did not impact lacticin 3157 production. (B) Deletion of ltnA1A2 eliminates inhibitory activity. (C) Supernatant from MG1363(pOM31), which produces LtnA1 but not LtnA2, does not produce zones [a phenotype that cannot be complemented by supernatant from MG1614(pMRC01ΔltnA1A2)]. However, supernatant from MG1363(pOM31) and MG1363(pMRC01ΔltnA1) combined by either diffusion from two wells (each containing 50 μl of supernatant) or the mixing of the two supernatants in one well (25 μl of each supernatant) results in activity, demonstrating that LtnA2 is being produced by the latter and thus that the SOEing deletion of ltnA1 is not polar.