Cloning, sequencing and overexpression of the mannitol-specific enzyme-III-encoding gene of Staphylococcus carnosus

Roland Fischer, Reinhard Eisermann, Bernd Reiche, Wolfgang Hengstenberg

Research output: Contribution to journalArticlepeer-review

22 Scopus citations

Abstract

The gene which encodes the mannitol-specific enzyme III (EIIPmtl) of the phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus carnosus, has been cloned. Genomic libraries of S. carnosus DNA were constructed using the expression vector pUC19 and EIIImtl-producing clones were identified using rabbit polyclonal antiserum. A 700-bp DdeI fragment, containing the complete gene encoding EIIImtl, was sequenced by the dideoxy chain-termination technique. Upstream from the ORF for EIIImtl one can find a sequence analogous to that of the Escherichia coli promoter. This region acts as a strong promoter when subcloned into the promoter test vector M 13HDL17. EIIImtl was overproduced using the inducible T7 polymerase system and purified to homogeneity. Amino acid sequence comparison confirmed a 38% similarity to the hydrophilic enzyme-III-like portion of enzyme IImtl of E. coli. There is also a 36% similarity to the N terminus of the fructose-specific phospho-carrier protein from E. coli.

Original languageEnglish
Pages (from-to)249-257
Number of pages9
JournalGene
Volume82
Issue number2
DOIs
StatePublished - 30 Oct 1989
Externally publishedYes

Keywords

  • Recombinant DNA
  • antibody screening
  • dideoxy sequencing
  • enzyme purification
  • genomic libraries
  • mannitol metabolism
  • phosphoenolpyruvate-dependent phosphotransferase system

Fingerprint

Dive into the research topics of 'Cloning, sequencing and overexpression of the mannitol-specific enzyme-III-encoding gene of Staphylococcus carnosus'. Together they form a unique fingerprint.

Cite this