Expression, purification, and characterization of EpiC, an enzyme involved in the biosynthesis of the lantibiotic epidermin, and sequence analysis of Staphylococcus epidermidis epiC mutants

@article{Kupke1996ExpressionPA,
  title={Expression, purification, and characterization of EpiC, an enzyme involved in the biosynthesis of the lantibiotic epidermin, and sequence analysis of Staphylococcus epidermidis epiC mutants},
  author={Thomas Kupke and F. Gotz},
  journal={Journal of Bacteriology},
  year={1996},
  volume={178},
  pages={1335 - 1340}
}
The plasmid-encoded epidermin biosynthetic gene epiC of Staphylococcus epidermidis Tü3298 was expressed in Escherichia coli by using the T7 RNA polymerase-promoter system, and the gene product EpiC was identified by Western blotting (immunoblotting) with an anti-EpiC-peptide antiserum. EpiC was a hydrophobic but soluble protein. EpiC was purified by hydrophobic-interaction chromatography. The determined amino-terminal amino acid sequence was M I N I N N I .... The electrophoretic migration… 
Inducible production and cellular location of the epidermin biosynthetic enzyme EpiB using an improved staphylococcal expression system.
TLDR
In order to obtain high-level production of EpiB, an improved staphylococcal expression vector based on the xylose-inducible xylA promoter of Staphylitis xylosus was constructed and the new plasmid pTX15 mediated a considerably higher expression level after induction and a lower background expression level in the uninduced state than the previously described vector pCX15.
In vivo reaction of affinity-tag-labelled epidermin precursor peptide with flavoenzyme EpiD.
The Staphylococcus epidermidis genes encoding the His-tag-labelled epidermin precursor peptide EpiA and the flavoenzyme EpiD or the mutant protein EpiD-G93D, which lacks the coenzyme, were
Secretion of the lantibiotics epidermin and gallidermin: sequence analysis of the genes gdmT and gdmH, their influence on epidermin production and their regulation by EpiQ
TLDR
Describing two additional genes, epiH and epiT, as well as the homologous gallidermin genes gdmH and gdmT, resulted in a strong increase (seven- to tenfold) in the production level, which is very likely to be due to increased efficiency of epidermin secretion.
Organization and expression of a gene cluster involved in the biosynthesis of the lantibiotic lactocin S
TLDR
It is proposed that a palindromie sequence constitutes the 3′ terminus of both RNAI and RNAII and has a dual regulatory function in controlling the expression of las operon, acting both as a barrier to 3′–5′ exonuclease degradation of the lasA-specific transcript(s), and as a “leaky” transcriptional terminator which limits theexpression of down-stream genes.
The genetics of lantibiotic biosynthesis
TLDR
A number of genes involved in the biosynthesis of these highly modified peptides have been identified, including genes encoding the precursor peptide, enzymes responsible for specific amino acid modifications, proteases able to remove the leader peptide and regulatory proteins controlling lantibiotic biosynthesis.
Lantibiotic production is a burden for the producing staphylococci
TLDR
The authors' observations explain why antibiotic-producing strains occur as a minority on their skin and other ecological niches, but retain corresponding antibiotic resistance.
Lantibiotics: biosynthesis and biological activities of uniquely modified peptides from gram-positive bacteria.
TLDR
The fundamental aspects of the biosynthetic machinery, which include information for the antibiotic prepeptide, the modification enzymes and accessory functions such as dedicated proteases and ABC transporters as well as immunity factors and regulatory proteins, are discussed along with the biotechnological potential of the peptides and of the enzymes, which could be used for construction of novel, peptide-based biomedical effector molecules.
The biology of lantibiotics from the lacticin 481 group is coming of age.
TLDR
This group differs in many aspects from the best known lantibiotic group (nisin group), but shares properties with less-studied groups such as the mersacidin, cytolysin and lactocin S groups.
Purification of Mutacin III from Group IIIStreptococcus mutans UA787 and Genetic Analyses of Mutacin III Biosynthesis Genes
TLDR
The cloned and sequenced the mutacin III biosynthesis gene locus from a group III strain of S. mutans, UA787, consistent with the supposition that mutac in III has posttranslational modifications similar to those of the lantibiotic epidermin.
...
...

References

SHOWING 1-10 OF 61 REFERENCES
Purification and characterization of EpiD, a flavoprotein involved in the biosynthesis of the lantibiotic epidermin
TLDR
It is proposed 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.
Analysis of genes involved in the biosynthesis of lantibiotic epidermin.
TLDR
Heterologous epidermin synthesis in the non-producing organism Staphylococcus carnosus finally proved that these reading frames are necessary for epidermine biosynthesis.
Nucleotide sequence of the lantibiotic Pep5 biosynthetic gene cluster and functional analysis of PepP and PepC. Evidence for a role of PepC in thioether formation.
TLDR
It is demonstrated that PepC is a thioether-forming protein and strongly suggest that PepB is responsible for dehydration of serine and threonine in the cells without excretion of processed peptide.
Genetic analysis of epidermin biosynthetic genes and epidermin-negative mutants of Staphylococcus epidermidis.
TLDR
It 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.
Analysis of genes involved in biosynthesis of the lantibiotic subtilin
TLDR
The results clearly proved that reading frames spaB and spaC are essential for subtilin biosynthesis whereas spaT mutants are probably deficient in subtil in transport.
Biosynthesis of the lantibiotic Pep5. Isolation and characterization of a prepeptide containing dehydroamino acids.
TLDR
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.
Identification of serine-143 as the most likely precursor of dehydroalanine in the active site of histidine ammonia-lyase. A study of the overexpressed enzyme by site-directed mutagenesis.
TLDR
Serine-143 is the most probable precursor of the active-site dehydroalanine in the active site of histidase, and its role in the biosynthesis of active Histidine ammonia-lyase is discussed.
Cloning, nucleotide sequence, and expression of the Escherichia coli gene encoding carnitine dehydratase
TLDR
Northern (RNA) analysis showed that caiB is likely to be cotranscribed with at least one other gene, which could be the gene encoding a 47-kDa protein, which was overexpressed upstream of caoB.
Purification and characterization of EpiA, the peptide substrate for post-translational modifications involved in epidermin biosynthesis.
For the investigation of enzymes involved in epidermin biosynthesis it is necessary to produce sufficient amounts of preepidermin (EpiA) as a substrate and to design EpiA detection systems.
Prepeptide sequence of epidermin, a ribosomally synthesized antibiotic with four sulphide-rings
TLDR
It is proposed that the N-terminus (–30 to –1) plays a cooperative role during modification reactions and prevents toxicity of the mature epidermin to the producing strain before the antibiotic is cleaved off and secreted.
...
...