EVIDENCE FOR A MULTIMERIC SUBTILIN SYNTHETASE COMPLEX

Subtilin is a lanthionine-containing peptide antibiotic (lantibiotic) produced by Bacillus subtilis. It is ribosomally synthesized as a prep eptide and modified posttranslationally. Three proteins of the suhtili n gene cluster (SpaB, SpaC, and SpaT) which are probably involved in p repeptide modification and transport have been identified genetically (C. Klein, C. Kaletta, N. Schnell, and K.-D. Entian, Appl. Environ. Mi crobiol. 58: 132-142, 1992). Immunoblot analysis revealed that product ion of SpaC is strongly regulated (Z. Gutowski-Eckel, C. Klein, K. Sie gers, K. Bohm, M. Hammelmann, and K.-D. Entian, Appl. Environ. Microbi ol. 60:1-11, 1994). Transcription of the SpaC protein started in the l ate logarithmic growth phase, reaching a maximum in the early stationa ry growth phase. No SpaC was detectable in the early logarithmic growt h phase. Deletions within the spaR and spaK genes, which act as a two- component regulatory system, resulted in failure to express SpaB and S paC, indicating that these two genes are the regulatory targets, Weste rn blot analysis of vesicle preparations of B. subtilis revealed that the SpaB, SpaT, and SpaC proteins are membrane bound, although some of the protein was also detectable in cell extracts. By using the yeast two-hybrid analysis system for protein interactions, we showed that a complex of at least two each of SpaT, SpaB, and SpaC is most probably associated with the substrate SpaS. These results were also confirmed by coimmunoprecipitation experiments. In these cosedimentation experim ents, SpaB and SpaC were coprecipitated by antisera against SpaC, SpaB , and SpaT, as well as by a monoclonal antibody against epitope-tagged SpaS, indicating that these four proteins are associated.