Antimicrobial activity of nisin variants produced in L. lactis. Samples were tested for growth inhibition of the nisin-sensitive indicator strain L. lactis NZ9000 pNZnisPT pIL253. The producer strains lack NisP, so that produced nisin variants remain inactive since the leader peptide stays attached. Upon addition of cell lysate or purified peptide samples to the NisP-expressing indicator strain, active nisin can be liberated. (A) Control samples: 30 μg pure nisin was used as positive control and pure CDM medium as negative control. To exclude antimicrobial activity resulting from PylRS–tRNAPyl expression or addition of 5 mM BocK to the medium, the corresponding supernatant samples were tested separately and showed no antimicrobial activity. (B) Comparison of antimicrobial activity of WT nisin and nisin(BocK) variants produced in presence of different BocK concentrations. The rising BocK concentrations did not affect the antimicrobial activity of nisin WT. In the absence of BocK, expression of nisA(I4amber) or nisA(K12amber) did not result in antimicrobial activity. Only upon addition of up to 5 mM BocK, antimicrobial activity was observed, with 2 mM as optimal concentration.