Evidence for a multimeric subtilin synthetase complex

Abstract

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...

A singular enzymatic megacomplex from Bacillus subtilis

“…It will be important to determine the identity of the exporter protein(s) as well as the biochemical determinants for localization of the Pks proteins and assembly of the megacomplex. Although lantibiotic synthase factories anchored at the cytoplasmic face of bacterial membranes have been reported (26,27), our findings provide insights into quaternary organization of natural product biosynthetic factories in antibiotic producers. Finally, we highlight that the combination of the bacillaene structure and the observed synthase organization in the model organism B. subtilis holds great potential for understanding enzymatic synthesis of hybrid PK-NRP metabolites.…”

Autoregulation of subtilin biosynthesis in Bacillus subtilis: the role of the spa-box in subtilin-responsive promoters

“…The spaS gene encodes the ribosomally synthesized subtilin precursor in which the amino acid modifications are introduced enzymatically by the products of spaB and spaC. The modified precursor is then transported across the cytoplasmic membrane by the ABC transporter encoded by spaT, which has been shown to form a membrane-associated complex with the modification enzymes SpaB and SpaC [14,21]. Following transport, the N-terminal leader peptide of the modified subtilin precursor is removed by the activity of unspecific proteases secreted by B. subtilis to release the mature and active AMP [7].…”

Lantibiotic Biosynthesis: Interactions between Prelacticin 481 and Its Putative Modification Enzyme, LctM

“…LanB would be involved in the dehydration process and LanC in thioether bond formation, as shown in the cases of nisin and Pep5, respectively (10,14). It has been shown in the cases of nisin and subtilin that LanB and LanC form a lantibiotic synthetase complex also including the transmembrane ATP-binding cassette (ABC) transporter LanT and that both LanB and LanC interact directly with the lantibiotic prepeptide (11,22). In comparison, information related to the biosynthesis of other type A lantibiotics is scarce.…”

“…If the assumption that LctM modifies the propeptide part of LctA is correct, one would expect that the two molecules would make direct physical contact. We examined this hypothesis with the yeast two-hybrid system, which detects even transient protein interactions such as enzyme-substrate interactions (6) and was used to show direct contacts between prelantibiotics and LanB or LanC (11,22). lctA and lctM were amplified by PCR and cloned into pGBT9 and pGAD424 (Matchmaker GAL4 two-hybrid system; Clontech Laboratories, Palo Alto, Calif.) as indicated in Table 1, in order to produce chimeric proteins including LctA or LctM fused to one of the two separate domains of the yeast transcriptional activator GAL4: the DNA binding domain (BD) and the transcriptional activation domain (AD).…”