Pep5, a new lantibiotic: structural gene isolation and prepeptide sequence

Kaletta, Cortina; Entian, Karl‐Dieter; Kellner, Roland; Jung, Günther; Reis, Michaela; Sahl, Hans‐Georg

doi:10.1007/bf00447005

Cited by 131 publications

(66 citation statements)

References 32 publications

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“…For the isolation of epidermin, cells were grown on 3% meat extract (Lab Lemco powder, Oxoid, Wesel, FRG), 3.8% malt extract (Frankle and Eck, Stuttgart, FRG), 0.6% CaCI2 x 2 H 2 0 and 4.6% NaC1, pH 6.5. According to the transformant used, tetracycline or chloramphenicol was added.…”

Section: Epidermin Purificationmentioning

confidence: 99%

“…The clustered arrangement for all genes specific of epidermin biosynthesis may explain the occurrence of homologous lantibiotics in different Gram-positive organisms by horizontal gene transfer. Some lantibiotic structural genes, such as epiA (epidermin) [2], and p~p A (Pep 5) [ 3 ] ) are located on episomes whereas others, such as spas (subtilin) [7,341) and g h A (gallidermin) [4]) are integrated into chromosomal DNA. The nisA gene (nisin) was found to be in episomal [5] and chromosomal [6] DNA.…”

Section: Codon Bias Preferences In S Epidermidismentioning

confidence: 99%

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Analysis of genes involved in the biosynthesis of lantibiotic epidermin

Schnell¹,

Engelke²,

Augustin³

et al. 1992

European Journal of Biochemistry

153

141

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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...

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Section: Epidermin Purificationmentioning

confidence: 99%

Section: Codon Bias Preferences In S Epidermidismentioning

confidence: 99%

Analysis of genes involved in the biosynthesis of lantibiotic epidermin

Schnell¹,

Engelke²,

Augustin³

et al. 1992

European Journal of Biochemistry

153

141

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“…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 .…”

mentioning

confidence: 99%

“…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 .…”

mentioning

confidence: 99%

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Biosynthesis of the lantibiotic Pep5

Weil

Beck‐Sickinger

Metzger

et al. 1990

European Journal of Biochemistry

Self Cite

106

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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...

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Staphylococcus

Peacock

2010

Topley &Amp; Wilson's Microbiology and Microbial Infections

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Pep5, a new lantibiotic: structural gene isolation and prepeptide sequence

Cited by 131 publications

References 32 publications

Analysis of genes involved in the biosynthesis of lantibiotic epidermin

Analysis of genes involved in the biosynthesis of lantibiotic epidermin

Biosynthesis of the lantibiotic Pep5

Staphylococcus

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