The lantibiotic nisin of Lactococcus lactis is matured from a ribosomally synthesized prepeptide by post-translational modification. Genetic and biochemical evidence suggests that genes nisB and nisC of the nisin gene cluster encode proteins necessary for prenisin modification. Inactivation of both genes resulted in complete loss of nisin production. The preparation of membrane vesicles revealed that NisB and NisC are attached to the cellular membrane, and co-immunoprecipitation experiments showed that they are associated with each other. By using the yeast two-hybrid system, which is a highly sensitive method to unravel protein-protein interactions, we could show that the nisin prepeptide physically interacts with the NisC protein, suggesting that NisC contains a binding site for prenisin. This was also confirmed by co-immunoprecipitation of the NisC protein and the NisA prepeptide by antibodies directed against the leader sequence of the nisin prepeptide. The two-hybrid analysis also confirmed the interaction between NisB and NisC as well as the interaction between NisC and the NisT ABC transporter. A minor interaction was also indicated between prenisin and the NisB protein. Furthermore, the two-hybrid investigations also revealed that at least two molecules of NisC and two molecules of NisT are part of the modification and transport complex. Our results suggest that lantibiotic maturation and secretion occur at a membrane-associated multimeric lanthionine synthetase complex consisting of proteins NisB, NisC, and the ABC transporter molecules NisT.

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