Analysis of genes involved in the biosynthesis of lantibiotic epidermin

Abstract

The structural gene of the lanthionine-containing peptide antibiotic epidermin is located on a 54-kb plasmid of Staphylococcus epidermidis [Schnelll et al. (1988) Nature 333, 276-2781. A 13.5-kb DNA region neighbouring the epidermin structural gene (@A) was subcloned and its sequencing revealed five additional open reading frames. Three of these reading frames, epiB, e p i c and epiD shared no homology with previously described proteins stored in data bases. They were located 3' adjacent to epiA. Using rpiB as a probe, a 5-kb mRNA was identified indicating that three or all four reading frames are transcribed as an operon. Additionally, a 0.3-kb mRNA specific for epiA was identified. Two open reading frames (epiP and epiQ) were located 3' to epiA, epiB, e p i c and epiD, but in the reverse orientation. The epiQ gene product shows similarity to the positive regulatory factor PhoB. This might indicate a regulatory function of epiQ in epidermin biosynthesis. The epiP gene product shows striking similarity to several serine proteases which makes epiP a likely candidate for processing the epidermin prepeptide. Heterologous epidermin synthesis in the non-producing organism Staphylococcus carnosus finally proved that these reading frames are necessary for epidermin biosynthesis.Epidermin is a 21-amino-acid peptide amide antibiotic with antimicrobial activities against Gram-positive bacteria. Some pathogenic bacteria in particular, such as Propionibacterium acnes, Staphylococci and Streptococci are highly sensitive to epidermin. It contains the unusual amino acid dehydrobutyrine and four sulfide rings consisting of two meso-kanthionine residues, one 3-methyllanthionine, and 2-aminovinyl-u-cysteine [I]. The isolation of the epidermin structural gene proved the ribosomal origin of this peptide antibiotic [2]. The primary translation product, called prepeptide, consists of 52 amino acid residues. Epidermin is derived by post-translational modification from the 22 C-terminal amino acid residues. The N-terminal 30 amino acids, which probably assume a partially amphiphilic a-helix conformation, were expected to play a cooperativc role during modification reactions [2]. A similar gene structure was confirmed for all lanthionine-containing peptide antibiotics investigated so far. These include Pep5 thesis of lantibiotics. The formation of the unusual non-proteinogenic amino acids could be explained by post-translational dehydration of peptide serine and threonine residues, with subsequent addition of cysteine sulfur to these dehydroamino acids [lo, 1 I].To elucidate the mechanism of how lantibiotics are posttranslationally modified and processed, the various enzymes involved have to be identified. Based on the prepeptide structure, our hypothesis indicates that modification reactions might occur at the entire prepeptide might occur 121. Two observations supported this view. First, in contrast to the Cterminus, the N-terminus of preepidermin contains mostly hydrophilic amino acid residues increasing propeptide solubility. Secon...

Homology modelling of the Lactococcus lactis leader peptidase NisP and its interaction with the precursor of the lantibiotic nisin

“…The general-purpose, cell-envelope proteinase of L.lactis has similar structural characteristics but is much larger (Vos et al, 1989). The catalytic domain of NisP is most similar (42% identical residues) to the putative protease EpiP encoded in the gene cluster for biosynthesis of epidermin, a related lantibiotic from Staphylococcus epidermidis (Schnell et al, 1992).…”

“…The amino acid sequences of the catalytic domain of L.lactis NisP ( Van der Meer et al, 1993) and S.epidermidis EpiP (Schnell et al, 1992) were included in the multiple sequence alignment of the catalytic domains of the entire subtilase family R.J .Siezen, manuscript in preparation); in this alignment the catalytic domain is defined as the sequence segment beginning at the predicted mature N-terminus and ending at the residue equivalent to the C-terminus of thermitase. Figure 3 shows only the alignment of NisP residues 29-377, numbered from the predicted N-terminus of the mature protease (Van der Meer et al, 1993), with EpiP residues 114--461 and with mature thermitase and subtilisin BPN'; the sequence identity of these NisP residues is 42% (146 out of 344 residues) with EpiP, 28% (76 out of 270 residues) with thermitase and 27% (74 out of 271 residues) with subtilisin BPN'.…”

Elucidation of the Primary Structure of the Lantibiotic Epilancin K7 from Staphylococcus epidermidis K7. Cloning and Characterisation of the Epilancin-K7-Encoding Gene and NMR Analysis of Mature Epilancin K7

“…These leader peptidases are involved in the processing of prePep5, prenisin and of pre-epidermin, respectively Schnell et al, 1992;van der Meer et al, 1993;Engelke et al, 1994; Meyer, C., Bierbaum, G., Heidrich, C., Siiling, J., Iglesias-Wind, M. I., Gnau, V., Kempter, C. and Sahl, H.-G., unpublished results). Therefore, the ORF downstream of the elkA gene is named elkP.…”

“…Correspondingly, the ORF upstream of the elkA gene is named elkT. Alignment of the currently known part of the ElkT sequence with the sequences of the N-terminal parts of the PepT, NisT, SpaT and EpiT' translocators Klein et al, 1992;Schnell et al, 1992;Kuipers et al, 1993a; Meyer, C., Bierbaum, G., Heidrich, C., Siiling, J., Iglesias-Wind, M. I., Gnau, V., Kempter, C. and Sahl, H.-G., unpublished results; Fig. 7) shows that the overall similarity between these hydrophobic parts of the tentative lantibiotic translocators is rather low.…”

Characterization of the nisin gene cluster nisABTCIPR of Lactococcus lactis

“…Several genes have been characterized that encode proteins involved in biosynthetic processes of different lantibiotics, e.g. epidermin (epi; Schnell et al, 1992), subtilin (spa; Banerjee and Hansen, 1988;Chung and Hansen, 1992;Klein et al, 1992;Klein et al, 1993), pep5 (pep; Reis and Sahl, 1991) and nisin (nis; Buchman et al, 1988;Steen et a)., 1991 ;Mulders et al, 1991 ;van der Meer et al, 1993). The proteins encoded by the nisB and nisC genes , and their counterparts in other lantibiotic-encoding gene clusters, might be involved in modification reactions of the prelantibiotic.…”