SummaryN-acetylmuramyl-L-alanine amidases are widely distributed among bacteria. However, in Escherichia coli, only one periplasmic amidase has been described until now, which is suggested to play a role in murein recycling. Here, we report that three amidases, named AmiA, B and C, exist in E. coli and that they are involved in splitting of the murein septum during cell division. Moreover, the amidases were shown to act as powerful autolytic enzymes in the presence of antibiotics. Deletion mutants in amiA, B and C were growing in long chains of unseparated cells and displayed a tolerant response to the normally lytic combination of aztreonam and bulgecin. Isolated murein sacculi of these chain-forming mutants showed rings of thickened murein at the site of blocked septation. In vitro, these murein ring structures were digested more slowly by muramidases than the surrounding murein. In contrast, when treated with the amidase AmiC or the endopeptidase MepA, the rings disappeared, and gaps developed at these sites in the murein sacculi. These results are taken as evidence that highly stressed murein crossbridges are concentrated at the site of blocked cell division, which, when cleaved, result in cracking of the sacculus at this site. As amidase deletion mutants accumulate trimeric and tetrameric cross-links in their murein, it is suggested that these structures mark the division site before cleavage of the septum.

The multiplicity of murein hydrolases found in most bacteria presents an obstacle to demonstrating the necessity of these potentially autolytic enzymes. Therefore, Escherichia coli mutants with deletions in multiple murein hydrolases, including lytic transglycosylases, amidases, and DD-endopeptidases, were constructed. Even a mutant from which seven different hydrolases were deleted was viable and grew at a normal rate. However, penicillin-induced lysis was retarded. Most of the mutants were affected in septum cleavage, which resulted in the formation of chains of cells. All three enzymes were shown to be capable of splitting the septum. Failure to cleave the septum resulted in an increase in outer membrane permeability, and thus the murein hydrolase mutants did not grow on MacConkey agar plates. In addition, the hydrolase mutants not only could be lysed by lysozyme in the absence of EDTA but also were sensitive to high-molecular-weight antibiotics, such as vancomycin and bacitracin, which are normally ineffective against E. coli.

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 ...

Epicidin 280 is a novel type A lantibiotic produced byStaphylococcus epidermidis BN 280. During C18reverse-phase high-performance liquid chromatography two epicidin 280 peaks were obtained; the two compounds had molecular masses of 3,133 ± 1.5 and 3,136 ± 1.5 Da, comparable antibiotic activities, and identical amino acid compositions. Amino acid sequence analysis revealed that epicidin 280 exhibits 75% similarity to Pep5. The strains that produce epicidin 280 and Pep5 exhibit cross-immunity, indicating that the immunity peptides cross-function in antagonization of both lantibiotics. The complete epicidin 280 gene cluster was cloned and was found to comprise at least five open reading frames (eciI, eciA, eciP,eciB, and eciC, in that order). The proteins encoded by these open reading frames exhibit significant sequence similarity to the biosynthetic proteins of the Pep5 operon ofStaphylococcus epidermidis 5. A gene for an ABC transporter, which is present in the Pep5 gene cluster but is necessary only for high yields (G. Bierbaum, M. Reis, C. Szekat, and H.-G. Sahl, Appl. Environ. Microbiol. 60:4332–4338, 1994), was not detected. Instead, upstream of the immunity gene eciI we found an open reading frame, eciO, which could code for a novel lantibiotic modification enzyme involved in reduction of an N-terminally located oxopropionyl residue. Epicidin 280 produced by the heterologous host Staphylococcus carnosus TM 300 after introduction of eciIAPBC (i.e., no eciO was present) behaved homogeneously during reverse-phase chromatography.

Pep5 is a 34-amino-acid antimicrobial peptide, produced by Staphylococcus epidermidis 5, that contains the thioether amino acids lanthionine and methyllanthionine, which form three intramolecular ring structures. In addition, two didehydrobutyrines are present in the central part of the lantibiotic and an oxobutyryl residue is located at the N terminus. All rare amino acids are introduced by posttranslational modifications of a ribosomally made precursor peptide. To elucidate the function of the modified residues for the antimicrobial action of Pep5, mutant peptides, in which single modified residues had been eliminated, were produced by site-directed mutagenesis. All of these peptides showed a reduced antimicrobial activity. In addition, those peptides from which the ring structures had been deleted became susceptible to proteolytic digest. This demonstrates that the ring structures serve as stabilizers of conformations essential for activity, e.g., amphiphilicity, as well as for protecting Pep5 against proteases of the producing strains. In addition, residues that could serve as precursors of new modified amino acids in lantibiotics were introduced into the Pep5 precursor peptide. This way, a novel methyllanthionine and a didehydroalanine were inserted into the flexible central part of Pep5, demonstrating that biosynthesis of modified amino acids is feasible by protein engineering and use of the lantibiotic modification system.