Regulation of glucosyltransferase in Streptococcus gordonii.

Streptococcus gordonii is found in dental plaque in the oral cavity, and it is also associated with infective endocarditis. It exhibits a hard colony phenotype (Spp$\sp{+}$) on media containing sucrose; however, soft colony variants (Spp$\sp{-}$) appeared at a frequency of 10$\sp{-4}$ to 10$\sp{-3}$. Selected Spp$\sp{-}$ strains were able to revert to Spp$\sp{+}$ at a similar frequency. A correlation between change in phenotype and extracellular glucosyltransferase (GTF) activity was observed. In order to determine a possible molecular basis for the phase transition, genetic analysis of the GTF locus was conducted. A structural gtf gene was cloned, and the N-terminal portion (about 1/3 of the gene) was sequenced. A region of about 1 kb displayed $>$50% amino acid identity with gtf genes identified in Streptococcus mutans and Streptococcus downei. Insertional mutagenesis of the gene resulted in a strain with an Spp$\sp{-}$ phenotype and no detectable GTF activity. The gene was designated gtfG. A regulatory gene, rgg, was identified 66-bp upstream of gtfG. When present in multiple copies on a plasmid chimera, rgg functionally complemented Spp$\sp{-}$ variants, and in an Spp$\sp{+}$ host it increased GTF expression well above (7 to 12 fold) wild type levels. Disruption of the chromosomal rgg gene in an Spp$\sp{+}$ strain resulted in an Spp$\sp{-}$ phenotype exhibiting only 3% of wild-type GTF activity. Northern analysis showed that rgg positively regulates transcription of gtfG. Nucleotide sequencing revealed two inverted repeats located before rgg and intercistronically between rgg and gtfG, both of which may play a role in the regulation of gtfG. Thus, rgg and gtfG are necessary for GTF expression and the Spp$\sp{+}$ phenotype. Comparison of chromosomal DNA from S. gordonii Spp$\sp{+}$ and Spp$\sp{-}$ phase variants by Southern blot analysis using a variety of different restriction enzymes resolved no large DNA rearrangements at the rgg/gtfG locus. Northern analysis identified a decrease in the rgg-positively-regulated gtfG message in only a subset of Spp$\sp{-}$ variants. Spp variants therefore may be generated by more than one type of genetic event, one of which involves altered levels of a positively-regulated gtfG RNA transcript.PhDMicrobiology and ImmunologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/103320/1/9308461.pdfDescription of 9308461.pdf : Restricted to UM users only