D-100. The Expression of Streptococcus mutans vicRKX Operon Is Influenced by the LiaFSR Three-Component Regulatory System, The Alternative Sigma Factor, ComX, and the Competence-Signal Peptide

Y. D. N. Tremblay, Y-H. Li, S. A. Halperin, S. F. Lee;
Dalhousie Univ., Halifax, NS, CANADA.

As an inhabitant of the human oral cavity, Streptococcus mutans faces frequent environmental changes. Two-component-regulatory systems (TCSs) play a critical role in responding to these changes and the S. mutans genome encodes 12 TCSs. TCSs such as ComDE and LiaRS (Hk11-Rr11) have been linked to acid stress response and biofilm formation. Another TCS, VicRK, has been shown to be essential for growth. We have previously shown that that the expression of the vicRKX operon is influenced by culture pH and growth phase but not by the carbohydrate source available to S. mutans. The objective of this study was to identify the proteins that either directly or indirectly control the expression of the vicRKX operon. In our previous study, the promoter of the vicRKX operon was fused to a promoterless lacZ reporter gene and introduced in S. mutans strain UA159. In this work, we proceeded to inactivate the liaF, liaS and LiaR genes in the strain carrying the reporter gene construct. All strains were grown to a mid-exponential phase in tryptone-vitamin medium supplemented with glucose (TVG) at pH 6.0 or in HEPES-buffered-TVG (HTVG) at pH 7.8 and harvested by centrifugation. The cells were lysed and the hydrolysis of 2-nitrophenyl-β-D-galactopyranoside in the cell lysates was measured to assess the PvicR-dependent expression of lacZ. The deletion of liaS induced an approximately 100% increase in the expression of LacZ, regardless of the growth media. The liaF mutant showed 50% up-regulation of lacZ only in HTVG whereas the liaR mutant displayed a 12% down-regulation of lacZ only in TVG. The impact of the competence-signal peptide (CSP) and ComX on the expression of LacZ was also examined. In these cases, both the parent and the comX mutant were grown to early-exponential phase in TVG or HTVG, CSP was then added to the cultures and after an hour cells, were harvested. An approximate 35% decrease in LacZ activity was only observed when CSP was added to HTVG culture in both the parent strain and comX mutant. The comX mutant displayed 12% up-regulation of lacZ only in TVG. In conclusion, the LiaFSR regulatory system, ComX and the CSP have an impact on the regulation of the vicRKX operon.