Cloning, sequencing and overexpression of the mannitol-specific enzyme-III-encoding gene of Staphylococcus carnosus.
The gene which encodes the mannitol-specific enzyme III (EIIImtl) 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 Dde I 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 M13HDL17. 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.
Fischer
R
R
Ruhr-Universität Bochum, F.R.G.
Eisermann
R
R
Reiche
B
B
Hengstenberg
W
W
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000096:biosynthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity; D013210:Staphylococcus / Q000235:genetics*
D011993:Recombinant Fusion Proteins; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C061852:phosphoenolpyruvate-mannitol-specific enzyme III