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 ...

Ethyl glucuronide (EtG) is a non-volatile, water-soluble, direct metabolite of ethanol that can be detected in body fluids and hair. We investigated urine and serum samples from three patient groups: (1) 33 in-patients in acute alcohol withdrawal; (2) 30 detoxified in-patients (treated for at least 4 weeks) from a 'motivation station'; and (3) 43 neuro-rehabilitation patients (non-alcoholics; most of them suffering from stroke, traumatic brain injury, Parkinson's disease etc.) using gas chromatography/mass spectrometry (GC/MS) with deuterium-labelled EtG as the internal standard and additionally in the second group of patients using liquid chromatography (LC/MS-MS). We found no correlation between the concentration of EtG in urine at hospitalization and the blood-ethanol concentration (r = 0.17), the time frame of detection (r = 0.5) or the total amount of clomethiazole required for the treatment of withdrawal symptoms (r = 0.28). In four out of 30 in-patients from the 'motivation station'--where neither clinical impression nor routine laboratory findings gave indications of relapse--concentrations of EtG in urine ranged between 4.2 and 196.6 mg/l. EtG concentrations in urine of between 2.89 and 23.49 mg/l were found in seven out of 43 neuro-rehabilitation patients using GC/MS. The GC/MS and the LC/MS-MS results showed a correlation of 0.98 with Pearson's correlation test and 1.0 with Spearman's correlation test. We suggest that EtG is a marker of alcohol consumption that can be detected for an extended time period after the complete elimination of alcohol from the body. When used as a relapse marker with a specific time frame of detection intermediate between short- and long-term markers, EtG fills a clinically as well as forensically important gap. Its specificity and sensitivity exceed those of all other known ethanol markers.

No abstract

Strains of Stenotrophomonasmaltophilia R3089, isolated from the rhizosphere of rape plants (Brassica napus L.), produced a novel antifungal compound,namedmaltophilin, which inhibited the growth of various saprophytic, human-pathogenic and phytopathogenic fungi but was inactive against Gram-positive and Gram-negative bacteria. Maltophilin is a novel macrocyclic lactam antibiotic with a molecular massof 510mu.The compound wasisolated from the culture nitrate by ethyl acetate extraction and gel filtration on Sephadex LH20and purified by preparative HPLC on reversed phase. The structure of maltophilin was elucidated by electrospray mass spectrometry and XHand 13C NMRspectroscopy.

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.