Expression of the gtfI gene from Streptococcus sobrinus in Streptococcus anginosus using integration-mediated transformation system.
We have constructed a Streptococcus anginosus transformant expressing the gtfI gene from Streptococcus sobrinus, using a previously developed integration-mediated transformation system to introduce foreign genes onto the oral streptococcal chromosome, and attempted to evaluate the gene expression. In this system, one cloning plasmid and three pACYC184 derivatives, anchor, heterodimer, and integration plasmids were used for the construction of a series of integrants via homologous recombination. A portion of S. sobrinus gtfI gene devoid of approximately 1 kb of the 5'-region derived from pMD39 was cloned into the integration plasmid and introduced onto the S. anginosus chromosome. Next, the polymerase chain reaction product corresponding to 2.0 kb of the 5'-region of the gtfI gene from S. sobrinus chromosome was further cloned into the cloning plasmid, and the intact gtfI gene was reconstructed following integration. The final S. anginosus integrant successfully secreted the enzymatically active gtfI gene products and extracellular enzyme was characterized. This enzyme produced water-insoluble glucans and glucan-forming activity was stimulated by the addition of dextranT10. When this integrant was grown in Todd-Hewitt broth supplemented with sucrose, the integrant adhered to the glass surface in vitro and this integrant exhibited the different colony morphology on Mitis-Salivarius agar plates compared to S. sobrinus and S. anginosus. These observations strongly suggest that the construction of S. anginosus integrant expressing S. sobrinus gtfI gene using this transformation system may be an effective means of analysis of cariogenic biofilm formation.
Shinozaki-Kuwahara
Noriko
N
Department of Microbiology, Nihon University School of Dentistry at Matsudo, Matsudo, Chiba 271-8587, Japan. nshino@mascat.nihon-u.ac.jp
Shiroza
Teruaki
T
Hayakawa
Mitsuo
M
Abiko
Yoshimitsu
Y
Fukushima
Kazuo
K
D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D018441:Biofilms; D003001:Cloning, Molecular / Q000379:methods*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics; D018014:Gene Transfer Techniques; D005964:Glucosyltransferases / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D034366:Streptococcus anginosus / Q000235:genetics*; D016986:Streptococcus sobrinus / Q000235:genetics*
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D011994:Recombinant Proteins; C067118:GTF-I protein, Streptococcus; D005964:Glucosyltransferases