(Received for publication, January 25, 1996)
From the
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.
Nisin is a ribosomally synthesized peptide antibiotic containing the unusual amino acids lanthionine, dehydrobutyrine, and dehydroalanine(1, 2) . It belongs to a class of peptide antibiotics that is referred to as lantibiotics because of their characteristic thioether bridges consisting of meso-lanthionine and 3-methyl-lanthionine. Nisin occurs naturally in dairy products (3) and is used as a food preservative because it exhibits high levels of antimicrobial activity against several pathogenic Gram-positive bacteria, such as staphylococci, streptococci, and clostridia(4) . The bactericidal action of nisin and other lantibiotics is mainly caused by pore formation in the cytoplasmic membrane(5, 6, 7) . Nisin, like the other lantibiotics described so far is ribosomally synthesized. The primary transcript of the nisin structural gene nisA encodes a 57-amino acid prepeptide, which consists of a N-terminal leader sequence followed by the C-terminal propeptide from which the lantibiotic is matured(1, 2) .
Based on the results of Ingram (8) the following model was proposed for the formation of the unusual amino acids. First, a dehydratase reaction occurs at serine and threonine residues, resulting in amino acids dehydroalanine and dehydrobutyrine, respectively. Thereafter, sulfur from neighboring cysteine residues is added to the double bonds, resulting in meso-lanthionine and 3-methyl-lanthionine, respectively. After the isolation of the first lantibiotic structural gene (epiA for epidermin) it was stated that maturation reactions occur at the prepeptide(9) . This hypothesis was supported by the isolation of prepeptides containing dehydroalanine(10) .
The genes for the biosynthesis of nisin are located on a 70-kilobase pair conjugative transposon(11) , which also contains the genetic information for sucrose metabolism. Several genes encoding proteins that are involved in the biosynthesis, secretion, and immunity of different lantibiotics have been characterized (for reviews see Refs. 12, 13, and 51). Proteins encoded by the genes nisB, nisT, nisC, nisI, nisP, nisR, nisK, nisF, nisE, and nisG have been found to be homologous to respective gene products of the subtilin(14, 15, 16, 17, 18) , epidermin(19, 20, 21, 22) , gallidermin (23) or Pep5 (24, 25) gene clusters. Gene deletion experiments of the genes spaB, spaC, spaT, spaR, and spaK in Bacillus subtilis have proven that they are essential for subtilin biosynthesis(15, 17, 18) . Like epiP, nisP codes for a subtilisin-like serine protease that is involved in processing of the post-translational modified prenisin(22, 26, 27, 28) . For nisI, nisF, nisE, and nisG we recently demonstrated an involvement in the self-protection mechanism of the producer against nisin(29) . Similar results have also been found for the respective genes of the subtilin-producing strain B. subtilis(16) .
Many lantibiotic-producing strains have three genes in common considered as lanB, lanC, and lanT. With respect to nisin biosynthesis, the nisT gene encodes a protein of 600 amino acid residues with an predicted molecular mass of 69 kDa. Its gene product shares strong homology with several ATP-dependent transport proteins having two ATP-binding sites and a very hydrophobic region at the N terminus with six potential membrane-spanning domains(26) . The NisT protein is expected to be necessary for the secretion of the modified nisin peptide. Proteins encoded by genes nisB and nisC share no homologies with other known proteins in the data bases except similar gene products of other lantibiotic-producing strains. As the functions of all other gene products found in lantibiotic gene clusters became obvious by their similarities to previously described proteins and by biochemical experiments, lanB and lanC most likely encode the proteins catalyzing the modification reactions.
Here we report on genetic and biochemical experiments proving the existence of a membrane-bound maturation complex, which we name lanthionine synthetase. These results were obtained by two independent experimental approaches. To prove any interactions between the nisin-prepeptide and its possible maturation proteins we used the yeast two-hybrid system, which is a highly sensitive method to unravel protein-protein interactions(30, 31) . This test system detects the functional reconstitution of GAL4, a transcriptional activator from yeast. The interaction of two hybrid proteins, one bearing the GAL4 DNA binding domain and the other fused to the transcriptional activation domain of GAL4, creates a functional activator by bringing the activation domain into close proximity with the DNA binding domain. This results in transcriptional activation of a lacZ reporter gene containing upstream GAL4 binding sites. Independently, co-immunoprecipitation experiments also confirmed the physical interactions indicated by the two-hybrid system. Our results suggest that this multimeric protein complex consists of NisB and at least two NisC subunits for the modification of the prepeptide and in analogy with other bacterial ABC transporters two NisT transporter subunits (32) .
Figure 1: Inactivation of genes nisB, nisC, and nisT.A, bioassay for nisin production. Strains were streaked onto plates containing M. luteus as a test organism. The zone of growth inhibition around the cells of the wild-type strain L. lactis KS100 indicates nisin production, whereas no growth inhibition can be observed around cells of the nonproducing strain MG1614 and mutants of KS100 where the gene nisB, nisC, or nisT has been inactivated, respectively. B, immunoblot analysis of NisB. Lane 1, molecular mass standards (kDa); lanes 2, 3, and 4 (from left to right), protein extracts of L. lactis wild-type cells (KS100), nonproducing cells (MG1614), and nisB disruption mutant. C, immunoblot analysis of NisC. Lane 1, molecular mass standards (kDa); lanes 2, 3, and 4 (from left to right), protein extracts of L. lactis wild-type cells (KS100), nonproducing cells (MG1614), and nisC disruption mutant.
Figure 2:
Membrane localization of NisC. Protein
extracts were separated by SDS gel electrophoresis and detected by
Western blot analysis. Lane 1, molecular mass standards (kDa);
crude extracts of nisin producing cells L. lactis KS100 (lane 2) were centrifuged at 1,000 g for 60
min at 4 °C (lane 3, supernatant; lane 4,
sediment). The supernatant was further centrifuged at 48,000
g for 30 min at 4 °C (lane 5, sediment; lane
6, supernatant). The final vesicle fraction (sediment) was washed
twice with the same centrifugation parameters, and the NisC protein was
still associated with the membrane fractions (lanes 7 and 8, sediments after each centrifugation
step).
Figure 3: A, organization of the genes nisA, nisB, nisT, nisC, and corresponding protein fragments used in the two-hybrid assays. The genes encoding proteins NisA, NisB, NisT, and NisC are indicated by arrows, and the lengths of protein fragments encoded by plasmids derived from pGBT9 and pGAD424 are shown below. B, composition of the suggested lanthionine synthetase complex derived from the data obtained in the two-hybrid assays. Lengths of bars correspond to the sizes of interacting fragments indicated by the number of amino acids.
In further experiments the NisA protein
was divided in the leader peptide and the propeptide region, and both
parts were fused to the respective GAL4 domains. These constructs were
also assayed for interaction with NisC, but no interaction of NisC with
either part of NisA could be observed, indicating that the complete
NisA protein is necessary for an interaction with NisC. The two-hybrid
analysis system also allowed us to test for self-interactions of
proteins involved in nisin maturation and transport by fusing the gene
of interest to the DNA binding and activation domain as well. When
yeast cells were cotransformed with plasmids carrying fusions of the nisC gene with the GAL4 activation domain and the
DNA-binding domain we observed -galactosidase activity, which
suggests that the protein complex that mediates the maturation of
prenisin contains more than one NisC protein. The same result (blue
coloration of the yeast cells) was also observed when plasmids carrying
fusions of the entire nisT gene with both parts of GAL4 were cotransformed. This finding suggests that at least two NisT
molecules are part of the supposed complex, which is in accordance with
the general view that bacterial ABC transporters occur as
dimers(46) . The results of the two-hybird investigations
suggested that all proteins involved in nisin maturation are associated
in a multimeric complex consisting of proteins NisB, NisC, and NisT (Fig. 3B).
Figure 4: Co-immunoprecipitation experiments. A, co-immunoprecipitation of NisB and NisC analyzed with NisB-specific antibodies. After precipitation, protein complexes were separated on 7.5% SDS-polyacrylamide gels. The NisB cross-reacting band is marked. Lane 1, molecular mass standards (kDa). Lane 2, NisB antibody-protein complex precipitated with protein A of S. aureus. Lane 3, NisC antibody-protein complex precipitated with protein A of S. aureus. Lanes 2 and 3 show the result of the co-immunoprecipitation experiment carried out with nisin-producing L. lactis KS100 cells. Lanes 4 and 5, control experiments with L. lactis MG1614 lacking the genes for nisin biosynthesis. Lane 6, vesicle fraction of L. lactis KS100 as positive control. Additional bands in lanes 2, 3, 4, and 5 are due to unspecific cross-reactions of the used antibodies with protein A of S. aureus. B, co-immunoprecipitation of NisB and NisC analyzed with NisC-specific antibodies. Lanes 1-6 are identical to lanes 1-6 in Fig. 4A. The NisC protein is marked. C, co-immunoprecipitation of NisC and NisA analyzed with NisC-specific antibodies. Lane 1, molecular mass standards (kDa). Lane 2, NisC antibody-protein complex precipitated with protein A of S. aureus. Lane 3, NisA antibody-protein complex precipitated with protein A of S. aureus. Lanes 2 and 3 show the result of the co-immunoprecipitation experiment carried out with L. lactis KS100 cells. Lanes 4 and 5 show control experiments with vesicles of L. lactis MG1614 with NisC-specific antibodies (lane 4) and NisA-specific antibodies (lane 5). Lane 6, vesicle fraction of L. lactis KS100 as positive control. Additional bands in lanes 2, 3, 4, and 5 are due to unspecific cross-reactions of the used antibodies with protein A of S. aureus. NisC protein is marked by an asterisk. D, co-immunoprecipitation of NisB and NisA analyzed with NisA-specific antibodies. After precipitation protein complexes were separated on 15% Tricine-polyacrylamide gels. Lane 1, molecular mass standards (kDa). Lane 2, NisB antibody-protein complex precipitated with protein A of S. aureus. Lane 3, NisA antibody-protein complex precipitated with protein A of S. aureus. Lanes 4 and 5 show control experiments with vesicles of L. lactis MG1614 with NisC-specific antibodies (lane 4) and NisA-specific antibodies (lane 5). Additional bands in lanes 2, 3, 4, and 5 are due to unspecific cross-reactions of the used antibodies with protein A of S. aureus.
Since the isolation of lantibiotic structural genes, which proved that lantibiotics are encoded by distinct genes, several genes involved in lantibiotic biosynthesis have been identified flanking the structural genes. The genes found near the structural genes of different producers show strong similarities indicating their similar function in lantibiotic maturation, secretion, processing, immunity, and the regulation of biosynthesis. The lantibiotics are considered to be formed by posttranslational modifications that convert the ribosomally synthesized prepeptides into peptides that contain the characteristic ring structure of lantibiotics. Two reactions have been proposed for the maturation of lantibiotics, dehydration of serine and threonine residues in the propeptide region and the addition of sulfur from neighboring cysteine residues to the resulting double bounds.
Our experiments revealed that the genes nisB and nisC encode two proteins of 117.5 and 47 kDa that are both associated with the cellular membrane. Inactivation of the genes by insertion of antibiotic resistance markers completely abolished nisin production in both cases, demonstrating their involvement in the biosynthesis of nisin. Furthermore, the results of the two-hybrid assay and co-immunoprecipitation experiments indicated that these proteins are attached to each other. In addition to this we could also demonstrate that the NisB protein as well as NisC interact with NisA. Therefore, we assume that proteins NisB and NisC form a complex that mediates the maturation of the nisin prepeptide. Since the proteins encoded by genes nisB and nisC share no homologies with other known proteins in the data bases except products of similar genes found in the gene clusters of different lantibiotic producers, we suppose that they might catalyze reactions that are specific for lantibiotic maturation.
The NisA antibody we used in the co-immunoprecipitation experiments is directed against the leader sequence of the prepeptide. Co-immunoprecipitation of NisB was impossible with the prepeptide antibody, which is directed against the leader sequence of NisA. However, when the cell extracts were first incubated with antibodies directed against NisB, the prepeptide could be co-immunoprecipitated. Co-immunoprecipitation in only one direction indicated that the nisin-prepeptide is not accessible to its leader-directed antibody in the NisB-NisA complex, suggesting that the leader peptide region of the prepeptide is involved in NisB binding. The finding that the complex consisting of NisA and NisC could be precipitated by the antibody directed against the NisA leader sequence indicates that in the complex the leader peptide is still accessible and not completely covered by NisC and only temporary covered by NisB. Possibly, the leader peptide is necessary for the recognition of NisA by the NisB protein, and the binding of the NisC protein takes place in the propeptide region.
Another interesting result of our experiments is that the protein complex mediating maturation of nisin is attached to the ABC transporter NisT, which has been implicated in the translocation process of the modified precursor peptide. Inactivation of nisT by insertion of the erythromycin resistance marker abolished nisin production, indicating that the gene product of nisT is essential for nisin biosynthesis. Results obtained from the two-hybrid assay revealed an interaction between NisC and the C terminus of NisT. Furthermore, we observed in the two-hybrid assay that NisT interacts with NisT, indicating that the ABC transporter consists of at least two nisT gene products. Since this interaction could only be observed when the entire nisT gene was fused with the respective GAL4 gene fragments, the N terminus of NisT seems to be important for the dimerization. The same result was observed with NisC, which means that at least two molecules of NisC are part of the protein complex.
Taking together our recent results we suggest the following maturation pathway of the nisin prepeptide. The translation product of the nisin structural gene, the nisin prepeptide, is matured at a protein complex consisting of proteins NisB and NisC, which is membrane-associated. Since the modification complex is directly attached to the ABC transporter consisting of NisT proteins, we assume that the fully modified peptide is subsequently translocated over the cytoplasmic membrane. Following translocation the leader peptide is removed by the specific extracellular protease NisP, which is bound to the cell surface(28) , and active nisin is released.
Recently it has been demonstrated for Pep5 that incompletely modified Pep5 precursor molecules could be isolated when the pepC gene was truncated, leaving only 167 amino acids of the PepC protein behind(25) . It was proposed that dehydratization is carried out by PepB and that PepC binds to the dehydrated prepeptides and catalyzes thioether formation. Our results of the two-hybrid assay demonstrated that the NisC protein binds the nisin prepeptide within the yeast cells. This strongly suggests that NisC is able to bind the unmodified prepeptide. Since mutants in the nisB gene as well as spaB mutants of B. subtilis do not produce nisin or subtilin(18) , we think that in addition to the LanC proteins, LanB is also an essential component of the lanthionine synthetase complex.
It has been reported that the gene clusters for cytolysin and lacticin 481 production contain genes that encode proteins CylM and LctM(47, 48) , whose C-terminal domains exhibit strong similarities with LanC proteins, whereas no similarity with LanB proteins was observed(12) . Interestingly the lack of LanB-homologous proteins is correlated with differences in the leader peptides. The structural genes for these lantibiotics encode prepeptides whose leader peptides differ from whose encoded by the nisin, epidermin, subtilin, and Pep5 gene clusters(12) . The fact that organisms that produce lantibiotics with class two leader sequences like lacticin 481 and cytolysin contain only proteins with similarities to LanC (12) is in accordance with our results suggesting that LanB may mainly interact with the lantibiotic leader and that the catalytic subunit of the lanthionine synthetase complex for modification of the prepeptides is located within the LanC proteins. LanB proteins might be necessary for the recognition of the prepeptides, stabilization of the complex, and maintenance of a conformation of the prepeptides that allows the modification reactions to proceed. In cytolysin- or lacticin 481-producing cells, the function of the LanB proteins might be provided by the N-terminal domain of the CylM and LctM proteins, respectively.
We propose the existence of a lanthionine synthetase complex of at least 350 kDa consisting of NisB, at least two molecules of NisC, and a NisT dimer (Fig. 5). Due to unsuccessful attempts to isolate the complex by the native blue gel method (50) we propose that NisC is only loosely attached to the NisT transporter molecules. However, the genetic and biochemical data gave convincing evidence that lantibiotic prepeptides are matured at a multimeric lanthionine synthetase complex that catalyzes the dehydration of amino acid residues and the subsequent thioether formation between the dehydrated residues and neighboring cysteine residues within the lantibiotic prepeptides.
Figure 5: Model of the suggested lanthionine synthetase complex. The NisT ABC transporter is integrated as a dimer in the cellular membrane and linked to NisB via a NisC dimer.