Expression of green fluorescent protein in Streptococcus gordonii DL1 and its use as a species-specific marker in coadhesion with Streptococcus oralis 34 in saliva-conditioned biofilms in vitro

Streptococcus gordonii is one of the predominant streptococci in the biofil m ecology of the oral cavity, It interacts with other bacteria through rece ptor-adhesin complexes formed between cognate molecules on the surfaces of the partner cells. To study the spatial organization of S. gordonii DL1 in oral biofilms, we used green fluorescent protein (GFP) as a species-specifi c marker to identify S. gordonii In a two-species in vitro oral biofilm Boo -cell system, To drive expression of gfp, we isolated and characterized an endogenous S. gornonii promoter, PhppA, which is situated upstream of the c hromosomal hppA gene encoding an oligopeptide-binding lipoprotein. A chromo somal chloramphenicol acetyltransferase (cat) gene fusion with PhppA was co nstructed and used to demonstrate that PhppA was highly active throughout t he growth of bacteria in batch culture. A promoterless 0,8-kb gfp ('gfp) ca ssette was PCR amplified from pBJ169 and subcloned to replace the cat casse tte downstream of the S. gordonii-derived PhppA in pMH109-HPP, generating p MA1. Subsequently, the PhppA-'gfp cassette was PCR amplified from pMA1 and subcloned into pDL277 and pVA838 to generate the Escherichia coli-S, gordon ii shuttle vectors pMA2 and pMA3, respectively. Each vector was transformed into S, gordonii DL1 aerobically to ensure GFP expression, Flow cytometric analyses of aerobically grown transformant cultures were performed over a 24-h period, and results showed that GFP could be successfully expressed in S. gordonii DL1 from PhppA and that S. gordonii DL1 transformed with the P hppA-'gfp fusion plasmid stably maintained the fluorescent phenotype, Fluor escent S. gordonii DL1 transformants were used to elucidate the spatial arr angement of S, gordonii DL1 alone in biofilms or with the coadhesion partne r Streptococcus oralis 34 in two-species biofilms in a saliva-conditioned i n vitro flowcell system. These results show for the first time that GFP exp ression in oral streptococci can be used as a species-specific marker in mo del oral biofilms.