Purification of the hydrophilic antibiotics epidermin, gallidermin and nikkomycin Z by preparative reversed-phase HPLC

Fiedler, H.-P.; Hörner, Thomas; Decker, Heinrich

doi:10.1007/bf02268154

Cited by 8 publications

(6 citation statements)

References 9 publications

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“…Gallidermin and pregallidermin analytics. Detection of gallidermin and pregallidermin was performed with a protocol adapted from Fiedler et al (12). Culture supernatants were centrifuged at 16,000 ϫ g at 4°C for 10 min, filtered through a 0.2-m cellulose-acetate membrane filter (Carl Roth GmbH, Karlsruhe, DE), and separated on a Prontosil Eurobond C 18 5.0-m column with a guard column (Bischoff, Leonberg, Germany) operated in a La Chrom highperformance liquid chromatography (HPLC) system (Merck, Germany) equipped with an L-7100 pump, L-7200 autosampler, L-7455 diode array detector, and L-7490 refractive index detector.…”

Section: Methodsmentioning

confidence: 99%

“…The transfer buffer was composed of 25.5 mM Tris, 192 mM glycine, and 0.1% SDS in 20% (vol/vol) methanol. Silver staining of the membranes was performed as described elsewhere (12).…”

Section: Methodsmentioning

confidence: 99%

“…The isolation of gallidermin and pregallidermin was performed with a protocol adapted from Fiedler et al (12). Gallidermin or pregallidermin was adsorbed from the cell-free fermentation broth onto XAD-7 (4%, vol/vol; Fluka, Buchs, Switzerland) over 90 min.…”

Section: Methodsmentioning

confidence: 99%

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Circumventing the Effect of Product Toxicity: Development of a Novel Two-Stage Production Process for the Lantibiotic Gallidermin

Valsesia

Medaglia

Held

et al. 2007

Appl Environ Microbiol

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Lantibiotics such as gallidermin are lanthionine-containing polypeptide antibiotics produced by grampositive bacteria that might become relevant for the treatment of various infectious diseases. So far, selftoxicity has prevented the isolation of efficient overproducing strains, thus hampering their thorough investigation and preventing their exploitation in fields other than the food area. We wanted to investigate the effect of lantibiotic precursor peptides on the producing strains in order to evaluate novel strategies for the overproduction of these promising peptides. In this study, gallidermin was chosen as a representative example of the type A lantibiotics. A Staphylococcus gallinarum Tü3928 mutant, whose gene for the extracellular pregallidermin protease GdmP was replaced by a kanamycin-resistance gene, was constructed. Mass spectrometry (MS) analysis indicated that this mutant produced fully posttranslationally modified gallidermin precursors with truncated versions of the leader peptide, but not the entire leader as predicted from the gdmA sequence. In filter-on-plate assays, these truncated pregallidermins showed no toxicity against Staphylococcus gallinarum Tü3928 up to a concentration of 8 g/liter (corresponding to approximately 2.35 mM), while gallidermin produced clear inhibitory zones at concentrations as low as 0.25 g/liter (0.12 mM). We showed that the lack of toxicity is due entirely to the presence of the truncated leader, since MS as well as bioassay analysis showed that the peptides resulting from tryptic cleavage of pregallidermins and gallidermin produced by S. gallinarum Tü3928 had identical masses and approximately the same specific activity. This demonstrates that even a shortened leader sequence is sufficient to prevent the toxicity of mature gallidermin. In nonoptimized fermentations, the gdmP mutant produced pregallidermin to a 50%-higher molar titer, suggesting that the absence of self-toxicity has a beneficial effect on gallidermin production and giving a first confirmation of the suitability of the overproduction strategy.Antibiotic resistance has spread dramatically among pathogens throughout the last two decades. Of particular concern is the increasing emergence of multiresistant pathogens in health care settings. Nowadays, about 70% of nosocomial infections in the United States are resistant to at least one antibiotic (35), and in 2002, the first clinical isolate of Staphylococcus aureus with high-level resistance to vancomycin, a drug of last resort, was isolated (8). As the number of resistant pathogens continues to grow, the number of new antibiotics to fight them is declining steeply (37). Therefore, the development of new antimicrobial agents should be encouraged.Type A lantibiotics are cationic, amphiphilic peptides that are produced by a wide range of gram-positive bacteria and show bactericidal activity against other gram-positive bacteria. They show a dual mode of action at nanomolar concentrations which involves pore formation and inhibition of peptidoglycan biosynth...

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Section: Methodsmentioning

confidence: 99%

“…The transfer buffer was composed of 25.5 mM Tris, 192 mM glycine, and 0.1% SDS in 20% (vol/vol) methanol. Silver staining of the membranes was performed as described elsewhere (12).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Circumventing the Effect of Product Toxicity: Development of a Novel Two-Stage Production Process for the Lantibiotic Gallidermin

Valsesia

Medaglia

Held

et al. 2007

Appl Environ Microbiol

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“…Epidermin production was detected as a zone of growth inhibition of M . luteus around the colonies or was determined by HPLC analysis [18].…”

Section: Epidermin Production Bioassaymentioning

confidence: 99%

Genetic analysis of epidermin biosynthetic genes and epidermin‐negative mutants of Staphylococcus epidermidis

Augustin

Rosenstein

Wieland

et al. 1992

European Journal of Biochemistry

246

184

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Epidermin is produced by Staphylococcus epidermidis Tu3298 which harbors the 54-kb plasmid, pTu32. The plasmid contains not only the epidermin structural gene epiA, but also a flanking DNA region which is necessary for epidermin biosynthesis. The DNA sequence of this region revealed, in addition to epiA, five additional open reading frames, epiB, C , D , Q and P [Schnell, N., Engelke, G., Augustin J., Rosenstein, R., Ungermann, V., Gotz, F. & Entian, K.-D. (1992) Eur. J. Biochem. 204, 57 -681. Wc isolated a number of stable mutants from strain Tii3298 which are unable to produce biologically active epidermin. Complementation studies using the newly constructed staphylococcal plasmid vectors pT18lmcs and pCUl led to their classification as epiA, epiB, e p i c or epiD mutants. Furthermore, evidence is presented that epiB lacks its own promoter and is co-transcribed from the epiA promoter. There is evidence that e p i c and D possess their own promoters. Although epiQ and epiP mutants were not isolated, it could be shown by heterologous gene expression in S . carnosus and S. xylosus that the corresponding DNA region is involved in epidermin biosynthesis. We can not exclude the possibility that, in addition to the four open reading frames, epiA, B, C, D , and the DNA region comprising epiQ and P, host-encoded functions are necessary for epidermin production. Thus, the genetic information for epidermin biosynthesis in S. carnosus and S. xylosus is located on an 8-kb DNA fragment of pTii32. A further characterization of the two epiA mutants revealed that in both mutants, the preepidermin nucleotide sequence was changed. In one mutant, the mutation led to a substitution of Ser3 by Asn; in the other of GlylO by Glu.So far, three staphylococcal strains have been shown to produce a lantibiotic: Staphylococcus epidermidis strain 5 produces Pep5 [l], S. gullinurum Tii3928 produces gallidermin [2] and S. epidermidis Tii3298 produces epidermin [3]. Epidermin is a 22-amino-acid peptide which exhibits antibiotic activity against many gram-positive bacteria. Chemical sequence determination revealed that epidermin is distinguished by the occurrence of unusual amino acids such as meso-lanthionine, 3-methyl lanthionine and the newly described S-(Zaminovinyl)-D-cysteine. The a$-unsaturated amino acid, dehydrobutyrine, is situated at the tryptic cleavage site [3]. Epidermin production in a fermentor has been previously optimized in S. epidermidis Tu3298 ; growth and epidermin production are correlated [4]. Epidermin is ribosomally synthesized and the epidermin structural gene, epiA, resides on the 54-kb plasmid, pTii32, of S. epidermidis Tii3298 [5]. Sequence analysis revealed that epiA encodes a 52-amino-acid prepeptide which is modified and processed to the mature tetracyclic lantibiotic. For a short review on the structure and function of staphylococcal lantibiotics, see Since the biosynthesis of epidermin and other lantibiotics appears to be rather complex, we expect that there are several genes involved in epidermin biosynthesis...

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“…Epidermin, which is produced and secreted into the medium by Staphylococcus epidermidis Tu3298, can be purified by reversed-phase high-performance liquid chromatography (12). The optimization of fermentative production leads to a large increase in the epidermin yield (14).…”

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confidence: 99%

Purification and characterization of EpiD, a flavoprotein involved in the biosynthesis of the lantibiotic epidermin

et al. 1992

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The plasmid-encoded epidermin biosynthesis gene, epiD, of Staphylococcus epidermidis Tü3298 was expressed in Escherichia coli by using both the malE fusion system and the T7 RNA polymerase-promoter system. EpiD was identified by Western blotting (immunoblotting) with anti-maltose-binding protein (MBP)-EpiD antiserum. EpiD and the MBP-EpiD fusion protein, which were mainly present in the soluble protein fraction, were purified from the respective E. coli clones. Purified EpiD showed the typical absorption spectrum of an oxidized flavoprotein with maxima at 274, 382, and 453 nm. The coenzyme released from EpiD by heat treatment was identified as flavin mononucleotide. S. epidermidis Tü3298/EMS11, containing a mutation within epiD, was unable to synthesize active epidermin. This mutated gene, epiD*, was cloned in E. coli and expressed as an MBP-EpiD* fusion protein. DNA sequencing of epiD* identified a point mutation that led to replacement of Gly-93 with Asp. Unlike MBP-EpiD, the fusion protein MBP-EpiD* could not bind flavin mononucleotide. We propose that EpiD catalyzes the removal of two reducing equivalents from the cysteine residue of the C-terminal meso-lanthionine to form a --C==C-- double bond and is therefore involved in formation of the unusual S-[(Z)-2-aminovinyl[-D-cysteine structure in epidermin.

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Purification of the hydrophilic antibiotics epidermin, gallidermin and nikkomycin Z by preparative reversed-phase HPLC

Cited by 8 publications

References 9 publications

Circumventing the Effect of Product Toxicity: Development of a Novel Two-Stage Production Process for the Lantibiotic Gallidermin

Circumventing the Effect of Product Toxicity: Development of a Novel Two-Stage Production Process for the Lantibiotic Gallidermin

Genetic analysis of epidermin biosynthetic genes and epidermin‐negative mutants of Staphylococcus epidermidis

Purification and characterization of EpiD, a flavoprotein involved in the biosynthesis of the lantibiotic epidermin

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