Pep5 is a tricyclic peptide antibiotic which contains the unusual amino acids dehydrobutyrine, lanthionine and 3-methyllanthionine. It is matured from a 60-amino-acid precursor peptide (pre-Pep5) deduced from the sequence of the structural genepepA. To study the biosynthesis of Pep5 we tried to isolate the primary translation product. We identified a peptide in crude extracts of the Pep5-producing Staphylococcus epidermidis strain using antibodies raised against a synthetic 26-residue peptide representing the leader peptide region of pre-Pep5. The putative precursor was purified by reversed-phase HPLC. The isolated peptide did not react with antibodies directed against a C-terminal fragment of mature Pep5 containing two sulfide bridges. Neither lanthionine nor 3-methyllanthionine was detected in amino acid analysis of the isolated precursor. Its amino acid sequence was identical with the sequence predicted from pepA, but Edman degradation stopped at the first threonine residue of the prolantibiotic region indicating a posttranslational modification at this position. The molecular mass of the isolated peptide was 6575.4 1.7 Da, determined by ion-spray mass spectrometry. This is in agreement with a molecule being dehydrated at the four threonine and the two serine residues in the propeptide region; such a peptide has a calculated molecular mass of 6576.7 Da. The results strongly suggest that maturation of the lantibiotic Pep5 is initiated by selective dehydration of hydroxyamino acids in the propeptide region of the primary translation product and that thioether ring formation is not closely linked to dehydration.Pep5 is the largest member of a group of lanthioninecontaining peptide antibiotics recently named lantibiotics. Other prominent lantibiotics are subtilin [l], epidermin [2], gallidermin [3] and the widely used nisin [4]. They are exclusively produced by Gram-positive bacteria and their action spectrum is restricted to the same group of prokaryotes. The mode of bactericidal action of the above mentioned lantibiotics is characterized by disruption of the energized state of the cytoplasmic membrane with subsequent equilibration of vital ion gradients of the cell [5-71. In planar membranes they form voltage-dependent, short-lived pores of up to 2 nm in size [8 -101. Additionally, Pep5 and nisin were shown to induce autolysis of staphylococci by activation of cell-wall-hydrolysing enzymes [ l l , 121.Elucidation of the structure of Pep5 [13a, b] and subsequent DNA-hybridization experiments with a specific oligonucleotide led to the detection of the structural genepepA [14]. pepA shares the general features of lantibiotic structural genes sequenced so far . It codes for a prepeptide which is, in this case, 60 amino acids long, possessing a typical proteolytic cleavage site (Fig. 1). The 26-residue leader peptide region is followed by a 34-residue propeptide, which is modified posttranslationally to mature Pep5. For biosynthesis of lanthionine and 3-methyllanthionine the serines and threonines of the propeptide...

A strain of a gram-positive, coccoid, yellow-pigmented bacterium was isolated from human blood. The bacterium was aerobic, non-encapsulated and non-motile. Phenotypically, the bacterium closely resembled Kytococcus sedentarius, but could be distinguished from this species by physiological tests and chemotaxonomic investigations. The peptidoglycan type is L-Lys-Glu2, variation A4alpha. The predominant menaquinones are MK-8 and MK-7. The major cellular fatty acids are iso-C17:1, iso-C17:0, iso-C15:0 and anteiso-C17:0. The strain contains catalase and does not produce acid from carbohydrates. The ability to hydrolyse Tween 80 and the lack of alpha-glucosidase activity are the most characteristic features. The results of comparative 16S rDNA analysis revealed that the strain represents a novel species within the genus Kytococcus, for which the name Kytococcus schroeteri sp. nov. is proposed. The type strain is strain Muenster 2000T (= DSM 13884T = CCM 4918T).

Protein A from Staphylococcus aureus, characterized by high affinity binding properties for IgG-type antibodies, was labeled with peroxidase to form a stable immuno-histological tracer molecule of comparatively low molecular weight. It has been used for demonstration of antibodies against tissue antigens, in direct and indirect techniques, and the findings were consistent with those in routine immunofluorescence and in staining with peroxidase-coupled anti-IgG. In comparison, the lowest unspecific tissue adsorption and staining was obtained with Protein A-peroxidase in buffer containing glucose and mannose. The immunohistological preparations were mounted and stored for documentation without apparent loss of staining.

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