Regulation of urease gene expression by Streptococcus salivarius growing in biofilms

The metabolism of urea by urease enzymes of oral bacteria profoundly influe nces oral biofilm pH homeostasis and oral microbial ecology. The purpose of this study was to gain insight into the regulation of expression of the lo w pH-inducible urease genes in populations of Streptococcus salivarius grow ing in vitro in biofilms and to explore whether urease regulation or the le vels of urease expression in biofilm cells differed significantly from plan ktonic cells. Two strains of S, salivarius harbouring urease promoter fusio ns to a chloramphenicol acetyltransferase (cat) gene were used: PurelCAT, c ontaining a fusion to the full-length, pH-sensitive promoter; or Purel Delt a 100CAT, a constitutively derepressed deletion derivative of the urease ge ne promoter. The strains were grown in a Rototorque biofilm reactor in a tr yptone-yeast extract-sucrose medium with or without pH control. Both CAT an d urease activities in biofilms were measured at 'quasisteady state' and af ter a 25mM glucose pulse. The results showed that CAT expression in PurelCA T was repressed at relatively neutral pH values, and that expression could be induced by acidic pH after carbohydrate challenge, Biofilms of PurelCAT grown at low pH, without buffering, had about 20-fold higher CAT levels, an d only a modest further induction could be elicited with carbohydrate pulsi ng, The levels of CAT in biofilms of PurelCAT grown in buffered medium were slightly higher than those reported for planktonic cells cultured at pH 7. 0, and the levels of CAT in PurelCAT growing at low pH or after induction w ere similar to those reported for fully induced planktonic cells. CAT activ ity in Purel Delta 100CAT was constitutively high, regardless of growth con ditions, Interestingly, urease activity detected in biofilms of the parent strain, S, salivarius 57,1, could be as much as 130-fold higher than that r eported for fluid chemostat cultures grown under similar conditions. The hi gher level of urease activity in biofilms was probably caused by the accumu lation of the stable urease enzyme within biofilm cells, low pH microenviro nments and the growth phase of populations of cells in the biofilm. The abi lity of S, salivarius biofilm cells to upregulate urease expression in resp onse to pH gradients and to accumulate greater quantities of urease enzyme when growing in bioiflms may have a significant impact on oral biofilm pH h omeostasis and microbial ecology in vivo. Additionally, S. salivarius carry ing the pH-sensitive urease gene promoter fused to an appropriate reporter gene may be a useful biological probe for sensing biofilm pH in situ.