Nucleotide Sequence of the Lantibiotic Pep5 Biosynthetic Gene Cluster and Functional Analysis of PepP and PepC. Evidence for a Role of PepC in Thioether Formation

Abstract

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 ...

Organization and expression of a gene cluster involved in the biosynthesis of the lantibiotic lactocin S

“…The C-termini of these proteins, however, show considerable sequence similarity to another class of proteins associated with lantibiotic expression, namely Pep C, EpiC, NisC and SpaC. Where the issue have been addressed Klein et al 1992;Segarra 1992;Meyer et al 1995), it has been shown that the proteins carrying the C-domain are necessary for normal synthesis of lantibiotics. This apparent indispensability, and the fact that similar proteins have not been identified outside a lantibiotic context, suggests that LasM and the other C-domain proteins have important and specific functions in lantibiotic biosynthesis, and, in a recently published report, evidence for involvement of PepC in thioether bridge formation has been provided .…”

“…Two glycine residues in the C-domain (residues 8 and 9 in motifs C2 and C4, respectively) have recently been shown to be essential for EpiC function (Kupke and Götz 1996). B Schematic comparison of LasP to the primary sequences of the leader peptidases involved in the processing of Pep5 (PepP; Meyer et al 1995), CylL S/L (CylA; Segarra 1992; Gilmore et al 1994), epidermin (EpiP;Schnell et al 1992), and nisin (NisP;van der Meer et al 1993) prepeptides. The Bacillus subtilis major intracellular serine protease ISSP1 (Koide et al 1986) is included as an example of a non-exported proteinase.…”

Molecular nature of a novel bacterial toxin: the cytolysin of Enterococcus faecalis

“…The shaded C-terminal portions of the peptides are present in the active cytolysin. Residues that are known or thought to be post-translationally modified by CylM are indicated by arrows cluster from Staphylococcus epidermidis, was reported to be a thioether-forming protein, since a pepC mutant produced peptides with modified Ser and Thr residues but did not contain Lan or MeLan [33]. It was, therefore suggested that PepB mediates the dehydration of Ser and Thr residues and PepC is responsible for the formation of the thioether-bridge in Lan and MeLan.…”

Posttranslationally modified bacteriocins—the lantibiotics

“…Both steps are catalyzed by specific enzymes; in producers of the class I lantibiotics, the dehydration reaction is likely performed by the LanB proteins, while LanC is responsible for thioether formation. 22 The LanB proteins are of about 1000 amino acids in size and most likely associated with the cell membrane. [23][24][25] LanC proteins are smaller and consist of about 400 amino acids.…”

“…The location of the respective protease determines the timing of processing; it can take place before or after export from the cell through the dedicated LanT transport proteins. 22,37,38 In almost every gene cluster of the class I lantibiotics a gene encoding such a protein with significant sequence similarities to the group A (i.e., encoded by one gene) ATP-binding-cassette (ABC)-transport protein superfamily was found. 5 These transport proteins of 500 -600 amino acids can be functionally divided into two domains.…”