Flippity Floppity: Investigating Specific Nucleotides in Streptococcus mutans that are Essential for SloR Binding, Poster 45

Streptococcus mutans is a bacterium that lives in the mouth where it is the primary causative agent of dental cavities. A goal in the Spatafora laboratory is to understand the traits that make S. mutans a successful pathogen,... [ view full abstract ]

Streptococcus mutans is a bacterium that lives in the mouth where it is the primary causative agent of dental cavities. A goal in the Spatafora laboratory is to understand the traits that make S. mutans a successful pathogen, and so inform the development of a novel anti-cavities therapeutic. We previously identified SloR as a 25kDa metalloregulatory protein that represses multiple genes throughout the S. mutans genome that are important for disease.  Among the gene products that foster cavities development are those deriving from the sloABC operon which mediate essential Mn²⁺ transport.  Upstream of this operon and overlapping the promoter is a 22bp DNA sequence (called a SloR Recognition Element or “SRE”) to which SloR binds in a Mn²⁺-dependent manner. Recently, we characterized this SRE and expanded it to include at least 72bp on which three inverted hexameric repeats are resident. We propose that three SloR homodimers bind to this sequence to repress the downstream sloABC operon.  To address this hypothesis, I introduced site-specific mutations into the SRE using overlap extension PCR (Xie et al., 2010).  Transformants demonstrating sensitivity to erythromycin and resistance to 4-chloro-phenylalanine were selected for subsequent cell lysate PCR, which were used for nucleotide sequencing to confirm the SRE-specific mutations.  The impact of these mutations on sloABC transcription was determined in real time qRT-PCR experiments so that their effect on SloR-SRE binding could be assessed. Preliminary qRT-PCR results support a “flip-flop” model for SloR-SRE binding. A better understanding of the SloR-SRE interaction could inform therapies to reduce cavities.