Investigating a Potential Manganese Efflux Transporter in Streptococcus mutans

Objectives: Streptococcus mutans is a successful dental pathogen owing, in part, to its ability to colonize and persist on the dentition where it regulates essential metal ion transport. Work in the Spatafora laboratory centers on a 25kDa SloR metalloregulatory protein that regulates transcription of the sloABC operon which encodes a transport system that imports essential metal ions, particularly Mn²⁺. We propose that S. mutans achieves intracellular Mn²⁺ homeostasis when metal ion uptake is in equilibrium with metal ion export, although very little is known about the latter in this and other important bacterial pathogens.
Methods: In the present study, we applied a bioinformatics approach to identify a novel gene, called SMU_1176, that may contribute to metal ion efflux in S. mutans. We went on to perform cation sensitivity and growth determination assays, as well as bacterial spot inoculations with the S. mutans UA159 wild-type strain and its isogenic SMU_1176 insertion-deletion mutant, called GMS3000, to reveal the metal ion specificity of the SMU_1176 gene product. We also quantified ⁵⁴Mn transport in uptake assays and metal ion accumulation in ICP-MS experiments to reveal the directionality of Mn²⁺ transport in the UA159 and GMS3000 strains.
Results: The experimental results support SMU_1176 as a Mn²⁺-specific efflux protein given the significantly heightened sensitivity of GMS3000 to MnSO₄ at concentrations ranging from 10mM to 1M, a phenotype that was rescued in the GMS3001 complemented strain. Accumulation of ⁵⁴Mn in the GMS3000 cell pellet versus in the supernatant of its UA159 progenitor implicates the SMU_1176 gene product in Mn²⁺ export, as does the metal ion insensitivity of S. mutans GMS284, a Mn²⁺ import mutant, and the accumulation of ⁵⁴Mn in GMS284 supernatants.
Conclusions: The SMU_1176 gene product is involved in Mn²⁺ efflux, and so contributes to S. mutans metal ion homeostasis.