Cloning of a Streptococcus sobrinus gtf gene that encodes a glucosyltransferase which produces a high-molecular-weight water-soluble glucan.
The gtf gene coding for glucosyltransferase (GTF), which produces a water-soluble glucan, was cloned from Streptococcus sobrinus OMZ176 (serotype d) into plasmid vector pBR322. This gene was expressed in Escherichia coli, and the product was purified to near homogeneity. The antigenicity of recombinant GTF (rGTF) was examined with the antisera raised against purified GTF P1, P2, P3, and P4 obtained from S. sobrinus AHT (serotype g). The rGTF reacted only with anti-GTF P1 serum in a Western blot (immunoblot) analysis. The rGTF closely resembled GTF P1 in its molecular mass, Km value for sucrose, optimal pH, primer dependency, and immunological properties. The high-molecular-weight, water-soluble glucan produced by the rGTF also resembled that of GTF P1, which is the most efficient primer donor for primer-dependent, water-insoluble glucan synthesis. Properties of the rGTF were also compared with those of rGTFS, which was purified from E. coli carrying the gtfS gene isolated from Streptococcus downei (previously S. sobrinus serotype h) MFe28. Both rGTF and rGTFS synthesized water-soluble glucan from sucrose without primer dextran, but their characteristics in Km values for sucrose, optimal pHs, and polymer sizes of the glucan were different. Furthermore, the gtf gene did not hybridize with the gtfS gene in a Southern blot analysis. These results showed that rGTF is similar to S. sobrinus AHT GTF P1 but distinct from rGTFS that has been previously purified from E. coli carrying the gtfS gene.
Hanada
N
N
Department of Preventive Dentistry, Iwate Medical University School of Dentistry, Morioka, Japan.
Yamashita
Y
Y
Shibata
Y
Y
Sato
S
S
Katayama
T
T
Takehara
T
T
Inoue
M
M
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D005936:Glucans / Q000096:biosynthesis*; D005964:Glucosyltransferases / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000096:biosynthesis; D012689:Sequence Homology, Nucleic Acid
D005936:Glucans; D011994:Recombinant Proteins; D005964:Glucosyltransferases