The biosynthetic genes of the nisin-producing strain Lactococcus lactis 6F3 are organized in an operon-like structure starting with the structural gene nisA followed by the genes nisB, nisT, and nisC, which are probably involved in chemical modification and secretion of the prepeptide (G. Engelke, Z. Gutowski-Eckel, M. Hammelmann, and K.-D. Entian, Appi. Environ. Microbiol. 58:3730-3743, 1992). Subcloning of an adjacent 5-kb downstream region revealed additional genes involved in nisin biosynthesis. The gene nisI, which encodes a lipoprotein, causes increased immunity after its transformation into nisin-sensitive L. lactis MG1614. It is followed by the gene nisP, coding for a subtilisin-like serine protease possibly involved in processing of the secreted leader peptide. Adjacent to the 3' end of nisP the genes nisR and nisK were identified, coding for a regulatory protein and a histidine kinase, showing marked similarities to members of the OmpRIEnvZ-like subgroup of two-component regulatory systems. The deduced amino acid sequences of nisR and nisK exhibit marked similarities to SpaR and SpaK, which were recently identified as the response regulator and the corresponding histidine kinase of subtilin biosynthesis. By using antibodies directed against the nisin prepeptide and the NisB protein, respectively, we could show that nisin biosynthesis is regulated by the expression of its structural and biosynthetic genes. Prenisin expression starts in the exponential growth phase and precedes that of the NisB protein by approximately 30 min. Both proteins are expressed to a maximum in the stationary growth phase.

Nisin produced by Lactococcus lactis 6F3 is used as a food preservative and is the most important member of a group of peptide-antibiotics containing lanthionine bridges (lantibiotics) (N.

1994). Transcription of the SpaC protein started in the late logarithmic growth phase, reaching a maximum in the early stationary growth phase. No SpaC was detectable in the early logarithmic growth phase. Deletions within the spaR and spaK genes, which act as a two-component regulatory system, resulted in failure to express SpaB and SpaC, indicating that these two genes are the regulatory targets. Western 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 experiments, 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.Lantibiotics such as subtilin, nisin, and epidermin are ribosomally synthesized peptide antibiotics. They contain the unusual amino acids meso-lanthionine, dehydrobutyrine, and dehydroalanine (36). The major characteristic of lantibiotics is the occurrence of the thioether amino acids meso-lanthionine and 3-methyllanthionine, which are generated during maturation of the lantibiotic prepeptides. Lantibiotics can be divided into two subgroups (19): (i) linear lantibiotics, including subtilin (16), nisin (28, 32), epidermin (1, 2), gallidermin (20), and Pep5 (33), and (ii) globular lantibiotics, including cinnamycin (RoO9-0198 or lanthiopeptin) (6, 21, 30), duramycin (15), and ancovenin (40).Epidermin and gallidermin are potentially applicable in the treatment of acne disease because of their high activity against Propionibacterium acnes. Subtilin is very similar to nisin, which is the most important member of the group of linear lantibiotics. Since the realization of the mutagenic effect of nitrite, which is used against clostridia in canned food, there has been an increasing interest in the use of nisin as a food preservative. The main difficulty in exploiting lantibiotics commercially is the low production rates. To overcome this problem, the biosynthesis of lantibiotics has to be elucidated.The structural genes of linear lantibiotics encode prepeptides which consist of an N-terminal leader sequence followed by the C-terminal propeptide from which the lantibiotics mature (36). Several genes essential for the synthesis of the lantibiotics subtilin (5, 23, 24, 25), epidermin (4, 35), and nisin (10, 38) have been identified. Sequence analysis of the DNA regions adjacent to the subtilin structural gene spaS revealed eight open reading frames (13). The genes spaB, spaT, and spaC are located upstream of spaS. Downstream to the spaS gene, spaI, spaF, spaG, spaR, and spaK were identified. All of these genes have been shown to be similar to respe...

The information responsible for biosynthesis of the lantibiotic subtilin is organized in an operon-like structure that starts with the spaB gene. The spaB gene encodes an open reading frame consisting of 1,030 amino acid residues, and it was calculated that a protein having a theoretical molecular mass of 120.5 kDa could be produced from this gene. This is consistent with the apparent molecular weight for SpaB of 115,000 which was estimated after sodium dodecyl sulfate-gel electrophoresis and identification with SpaB-specific antibodies. The SpaB protein is very similar to proteins EpiB and NisB, which were identified previously as being involved in epidermin and nisin biosynthesis. Upstream from SpaB a characteristic sigma A promotor sequence was identified. An immunoblot analysis revealed that SpaB expression was strongly regulated. No SpaB protein was detected in the early logarithmic growth phase, and maximum SpaB expression was observed in the early stationary growth phase. The expression of SpaB was strongly correlated with subtilin biosynthesis. Deletion mutations in either of two recently identified regulatory genes, spaR and spaK, which act as a "twocomponent" regulatory system necessary for growth phase-dependent induction of subtilin biosynthesis (C. Klein, C. Kaletta, and K. D. Entian, Appl. Environ. Microbiol. 59:296-303, 1993), also resulted in failure of SpaB expression. To investigate the intracellular localization of SpaB, vesicles of BaciUus subtilis were prepared. The SpaB protein cosedimented with the vesicle fraction and was released only after vigorous resuspension of the vesicles. Our results suggest that SpaB is membrane associated and that subtilin biosynthesis occurs at the cytoplasmic membrane of B. subtilis.

The Ca2+‐ATPase from sarcoplasmic reticulum can be inhibited by the Ca2+‐ and pH‐dependent reaction with ATP γP‐imidazolidate. The chemically and monofunctionally activated inhibitor introduces an intramolecular cross‐link between two neighbouring peptides of the active site. This can be followed by the reduced mobility of the ATPase upon SDS‐PAGE analysis which becomes even more pronounced after limited trypsinolysis. After cleavage of the cross‐linked ATPase molecule by cyanogen bromide and separation of the peptides a double‐peptide can be detected which upon sequencing can be identified as part of the phosphorylation and the nucleotide binding site, respectively.