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

Abstract

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...

Protein engineering of lantibiotics

“…Staphylococcus epidermidis Tii32981EMS6 (Augustin et al 1992), an epiA mutant of the epidermin producer strain Staphylococcus epidermidis TiJ3298 showing an Epi-phenotype, was used to express natural and mutant gallidermin and epidermin species. The staphylococcal vector pT18 l mcs and the E. coli-Staphylococcus shuttle vector pCU1 (Augustin et al 1992) were used as expression vectors for natural and mutant gdmA and epiA genes.…”

Role for dnaK locus in tolerance of multiple stresses in Staphylococcus aureus

“…Restriction sites (underlined) were included in the primers to facilitate subcloning of the amplified fragments. The amplicon represents a fragment starting 340 nt upstream of the hrcA gene and 285 nt downstream of the prmA gene that was cloned to the KpnI and XbaI sites of a shuttle plasmid pCU1 (Augustin et al, 1992), and subsequently transferred to the dnaK mutant of S. aureus strain COL.…”

Intragenus generalized transduction in Staphylococcus spp. by a novel giant phage

“…J Uchiyama et al pCU1 and selection using chloramphenicol (20 mg ml À 1 ; Augustin et al, 1992), bacteria harboring the plasmid pCU1 were used as donor hosts. The presence of pCU1 in the donor host was confirmed by colony-direct PCR using the primers listed in Supplementary Table S3.…”