The biosynthesis of Peps, a lanthionine-containing antimicrobial peptide, is directed by the 20-kbp plasmid pED503. We identified a 7.9-kbp DNA-fragment within this plasmid which covers the information for Peps synthesis in the homologous host Staphylococcus epidermidis S which has been cured of pED503. This fragment contained, in addition to the previously described structural gene pepA and the immunity gene pep1 [Reis, M., Eschbach-Bludau, M., Iglesias-Wind, M. I., Kupke, T. & Sahl, H . 4 . (1994) Appl. Env. Microbiol. 60, 2876-28831, a genepepTcoding for a translocator of the ABC transporter family, a gene pepP coding for a serine protease and two genes pepB and pepC coding for putative modification enzymes ; the gene arrangement is pepTIAPBC. We analyzed the biosynthetic genes with respect to their function in Peps biosynthesis. Deletion of PepT reduced Pep5 production to about lo%, indicating that it can be partially replaced by other host-encoded translocators. Inactivation of PepP by site-directed mutagenesis of the active-site His residue resulted in production of incorrectly pi-ocessed Peps fragments with strongly reduced antimicrobial activity. Deletion of pepB and pepC leads to accumulation of Pep5 prepeptide in the cells without excretion of processed peptide. A pepC-deletion clone did not excrete correctly matured Peps but it did produce fragments from which serine and threonine were absent. Only one of these fragments contained a single lanthionine residue out of three expected while the remaining, unmodified cysteine residues could be detected by reaction with Ellman's reagent. These results demonstrate that PepC is a thioether-forming protein and strongly suggest that PepB is responsible for dehydration of serine and threonine.Keywords: lantibiotics ; Peps biosynthetic gene cluster; PepC, thioether-forming enzyme; PepP, serine protease.Peps is a tricyclic peptide produced by Staphylococcus epidermidis S (Sahl and Brandis, 1981) which belongs to the family of lantibiotics, a designation introduced to characterize lanthionine containing peptides with antimicrobial activity (Schnell et al., 1988). In contrast to conventional peptide antibiotics, which are synthesized by multienzyme complexes, lantibiotics derive from gene-encoded precursor peptides. These precursors consist of a leader sequence and a propeptide part which is posttranslationally modified to give the mature lantibiotic. It was proposed that in a first modification step the serine and threonine residues of the propeptide part are dehydrated to didehydroalanine (Dha) and didehydrobutyrine (Dhb) (Schnell et al., 1988). Such dehydrated prepeptides have been isolated from S. epidermidis 5 (Weil et al., 1990). In a second step thiol groups of cysteine Note. The novel nucleotide sequence data published here have been deposited with the EMBL sequence data bank and are available under accession number 249865. The novel amino acid sequence data have also been deposited with the EMBL sequence data bank. residues react with the double bonds of ...

The biosynthesis of Peps, a lanthionine-containing antimicrobial peptide, is directed by the 20-kbp plasmid pED503. We identified a 7.9-kbp DNA-fragment within this plasmid which covers the information for Peps synthesis in the homologous host Staphylococcus epidermidis S which has been cured of pED503. This fragment contained, in addition to the previously described structural gene pepA and the immunity gene pep1 [Reis, M., Eschbach-Bludau, M., Iglesias-Wind, M. I., Kupke, T. & Sahl, H . 4 . (1994) Appl. Env. Microbiol. 60, 2876-28831, a genepepTcoding for a translocator of the ABC transporter family, a gene pepP coding for a serine protease and two genes pepB and pepC coding for putative modification enzymes ; the gene arrangement is pepTIAPBC. We analyzed the biosynthetic genes with respect to their function in Peps biosynthesis. Deletion of PepT reduced Pep5 production to about lo%, indicating that it can be partially replaced by other host-encoded translocators. Inactivation of PepP by site-directed mutagenesis of the active-site His residue resulted in production of incorrectly pi-ocessed Peps fragments with strongly reduced antimicrobial activity. Deletion of pepB and pepC leads to accumulation of Pep5 prepeptide in the cells without excretion of processed peptide. A pepC-deletion clone did not excrete correctly matured Peps but it did produce fragments from which serine and threonine were absent. Only one of these fragments contained a single lanthionine residue out of three expected while the remaining, unmodified cysteine residues could be detected by reaction with Ellman's reagent. These results demonstrate that PepC is a thioether-forming protein and strongly suggest that PepB is responsible for dehydration of serine and threonine.Keywords: lantibiotics ; Peps biosynthetic gene cluster; PepC, thioether-forming enzyme; PepP, serine protease.Peps is a tricyclic peptide produced by Staphylococcus epidermidis S (Sahl and Brandis, 1981) which belongs to the family of lantibiotics, a designation introduced to characterize lanthionine containing peptides with antimicrobial activity (Schnell et al., 1988). In contrast to conventional peptide antibiotics, which are synthesized by multienzyme complexes, lantibiotics derive from gene-encoded precursor peptides. These precursors consist of a leader sequence and a propeptide part which is posttranslationally modified to give the mature lantibiotic. It was proposed that in a first modification step the serine and threonine residues of the propeptide part are dehydrated to didehydroalanine (Dha) and didehydrobutyrine (Dhb) (Schnell et al., 1988). Such dehydrated prepeptides have been isolated from S. epidermidis 5 (Weil et al., 1990). In a second step thiol groups of cysteine Note. The novel nucleotide sequence data published here have been deposited with the EMBL sequence data bank and are available under accession number 249865. The novel amino acid sequence data have also been deposited with the EMBL sequence data bank. residues react with the double bonds of ...

The pepM gene coding for a methionine-specific aminopeptidase was cloned from Salmonella typhimurium and its nucleotide sequence determined. The gene encoded a 264 amino acid protein that was homologous to a similar protein from Escherichia coli. The sequence of an overproducer mutant allele, pepM100, contained a single base change in the likely--35 region of the pepM promoter that increased its homology to the consensus promoter sequence. A region downstream from the pepM coding sequence contained extensive inverted repeats and was homologous to sequences found elsewhere in both Salmonella and other bacterial species.

No abstract