Epidermin is produced by Staphylococcus epidermidis Tu3298 which harbors the 54-kb plasmid, pTu32. The plasmid contains not only the epidermin structural gene epiA, but also a flanking DNA region which is necessary for epidermin biosynthesis. The DNA sequence of this region revealed, in addition to epiA, five additional open reading frames, epiB, C , D , Q and P [Schnell, N., Engelke, G., Augustin J., Rosenstein, R., Ungermann, V., Gotz, F. & Entian, K.-D. (1992) Eur. J. Biochem. 204, 57 -681. Wc isolated a number of stable mutants from strain Tii3298 which are unable to produce biologically active epidermin. Complementation studies using the newly constructed staphylococcal plasmid vectors pT18lmcs and pCUl led to their classification as epiA, epiB, e p i c or epiD mutants. Furthermore, evidence is presented that epiB lacks its own promoter and is co-transcribed from the epiA promoter. There is evidence that e p i c and D possess their own promoters. Although epiQ and epiP mutants were not isolated, it could be shown by heterologous gene expression in S . carnosus and S. xylosus that the corresponding DNA region is involved in epidermin biosynthesis. We can not exclude the possibility that, in addition to the four open reading frames, epiA, B, C, D , and the DNA region comprising epiQ and P, host-encoded functions are necessary for epidermin production. Thus, the genetic information for epidermin biosynthesis in S. carnosus and S. xylosus is located on an 8-kb DNA fragment of pTii32. A further characterization of the two epiA mutants revealed that in both mutants, the preepidermin nucleotide sequence was changed. In one mutant, the mutation led to a substitution of Ser3 by Asn; in the other of GlylO by Glu.So far, three staphylococcal strains have been shown to produce a lantibiotic: Staphylococcus epidermidis strain 5 produces Pep5 [l], S. gullinurum Tii3928 produces gallidermin [2] and S. epidermidis Tii3298 produces epidermin [3]. Epidermin is a 22-amino-acid peptide which exhibits antibiotic activity against many gram-positive bacteria. Chemical sequence determination revealed that epidermin is distinguished by the occurrence of unusual amino acids such as meso-lanthionine, 3-methyl lanthionine and the newly described S-(Zaminovinyl)-D-cysteine. The a$-unsaturated amino acid, dehydrobutyrine, is situated at the tryptic cleavage site [3]. Epidermin production in a fermentor has been previously optimized in S. epidermidis Tu3298 ; growth and epidermin production are correlated [4]. Epidermin is ribosomally synthesized and the epidermin structural gene, epiA, resides on the 54-kb plasmid, pTii32, of S. epidermidis Tii3298 [5]. Sequence analysis revealed that epiA encodes a 52-amino-acid prepeptide which is modified and processed to the mature tetracyclic lantibiotic. For a short review on the structure and function of staphylococcal lantibiotics, see Since the biosynthesis of epidermin and other lantibiotics appears to be rather complex, we expect that there are several genes involved in epidermin biosynthesis...

The structural gene of the lanthionine-containing peptide antibiotic epidermin is located on a 54-kb plasmid of Staphylococcus epidermidis [Schnelll et al. (1988) Nature 333, 276-2781. A 13.5-kb DNA region neighbouring the epidermin structural gene (@A) was subcloned and its sequencing revealed five additional open reading frames. Three of these reading frames, epiB, e p i c and epiD shared no homology with previously described proteins stored in data bases. They were located 3' adjacent to epiA. Using rpiB as a probe, a 5-kb mRNA was identified indicating that three or all four reading frames are transcribed as an operon. Additionally, a 0.3-kb mRNA specific for epiA was identified. Two open reading frames (epiP and epiQ) were located 3' to epiA, epiB, e p i c and epiD, but in the reverse orientation. The epiQ gene product shows similarity to the positive regulatory factor PhoB. This might indicate a regulatory function of epiQ in epidermin biosynthesis. The epiP gene product shows striking similarity to several serine proteases which makes epiP a likely candidate for processing the epidermin prepeptide. Heterologous epidermin synthesis in the non-producing organism Staphylococcus carnosus finally proved that these reading frames are necessary for epidermin biosynthesis.Epidermin is a 21-amino-acid peptide amide antibiotic with antimicrobial activities against Gram-positive bacteria. Some pathogenic bacteria in particular, such as Propionibacterium acnes, Staphylococci and Streptococci are highly sensitive to epidermin. It contains the unusual amino acid dehydrobutyrine and four sulfide rings consisting of two meso-kanthionine residues, one 3-methyllanthionine, and 2-aminovinyl-u-cysteine [I]. The isolation of the epidermin structural gene proved the ribosomal origin of this peptide antibiotic [2]. The primary translation product, called prepeptide, consists of 52 amino acid residues. Epidermin is derived by post-translational modification from the 22 C-terminal amino acid residues. The N-terminal 30 amino acids, which probably assume a partially amphiphilic a-helix conformation, were expected to play a cooperativc role during modification reactions [2]. A similar gene structure was confirmed for all lanthionine-containing peptide antibiotics investigated so far. These include Pep5 thesis of lantibiotics. The formation of the unusual non-proteinogenic amino acids could be explained by post-translational dehydration of peptide serine and threonine residues, with subsequent addition of cysteine sulfur to these dehydroamino acids [lo, 1 I].To elucidate the mechanism of how lantibiotics are posttranslationally modified and processed, the various enzymes involved have to be identified. Based on the prepeptide structure, our hypothesis indicates that modification reactions might occur at the entire prepeptide might occur 121. Two observations supported this view. First, in contrast to the Cterminus, the N-terminus of preepidermin contains mostly hydrophilic amino acid residues increasing propeptide solubility. Secon...

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.