Identifying SloR Binding Partners in Streptococcus mutans, Poster 38

Streptococcus mutans, a gram-positive oral pathogen, is the main causative agent of caries on the human dentition. Its success as an oral pathogen is due to its obligate biofilm lifestyle and ability to withstand oxidative... [ view full abstract ]

Streptococcus mutans, a gram-positive oral pathogen, is the main causative agent of caries on the human dentition. Its success as an oral pathogen is due to its obligate biofilm lifestyle and ability to withstand oxidative stress that can derive from Fenton chemistry. A 25-kDa metalloregulatory protein in S. mutans, called SloR, maintains essential metal ion homeostasis by modulating expression of the sloABC metal ion transport operon along with other virulence genes that can promote cariogenesis. The goal of this study is to identify potential SloR binding partners that facilitate SloR’s role as a master regulator of essential metal ion uptake and virulence gene expression in S. mutans. We used a Bacterial Adenylate Cyclase Two Hybrid (BACTH) system to reveal putative SloR binding partners.  We cloned the S. mutans sloR gene into a pKT25 vector and transformed it into an adenylate cyclase-deficient E. coli host that had been previously transformed with a S. mutans genomic library (cloned on plasmid pUT18). The resulting co-transformants were grown on LB/X-Gal/IPTG or maltose MacConkey plates supplemented with antibiotics, and colonies with a blue or red phenotype, respectively, were considered positive for SloR-protein interactions in E. coli.  Recombinant pUT18 plasmids were then isolated by mini prep (Qiagen) and the cloned inserts were sequenced. We identified many different S. mutans proteins as putative SloR binding partners, among which are proteins involved in ABC-type transport, two component signal transduction, and central metabolism.  Co-immunoprecipitation experiments are underway in an effort to validate the SloR-protein interactions predicted in BACTH.