Analysis of Gene Expression in Streptococcus Mutans in Biofilms in Vitro

Abstract

The purpose of this study was to develop methods for the consistent production of biofilms of S. mutans containing reporter gene fusions, and to examine the expression of genes involved in sucrose metabolism in adherent populations of this organism. Three strains of S. mutans harboring reporter gene fusions to the gene promoter regions of the gtfBC genes, ftf, and scrA were grown in a Rototorque biofilm fermenter in a tryptone-yeast extract-sucrose medium. Quasi-steady-state levels of reporter gene activity were measured after the biofilms were grown for either 48 hrs of 7 days. Also, induction of gene expression by the addition of sucrose to biofilm cells was monitored. Reporter gene activity was measurable from all gene fusion strains. This study (i) establishes the feasibility of doing detailed molecular and physiologic studies on immobilized populations of S. mutans, (ii) demonstrates that the polysaccharide synthesis machinery of S. mutans is differentially expressed in biofilms, and (iii) opens the way for a more detailed analysis of the environmental signals and signal transduction pathways governing the regulation of gene expression by S. mutans cells that are immobilized on a solid surface.

Regulation of the gtfBC and ftf genes of Streptococcus mutans in biofilms in response to pH and carbohydrate

“…The construction of these strains and their genotypic stabilities are detailed elsewhere (Burne et al, 1997 ;Hudson & Curtiss, 1990 ;Wexler et al, 1993). The stability of the gene fusions in biofilms was again confirmed in this study by quantifying biofilm bacteria on selective and non-selective media, as previously detailed (Burne et al, 1997 ;Li et al, 2000). The strains were routinely maintained on brain heart infusion (BHI) agar (Difco) supplemented with 10 µg erythromycin ml − ".…”

“…In addition, the medium contained 50 mM potassium phosphate buffer (pH 7n8) for the experiments requiring pH control, or 90 mM KCl for those not requiring pH control, to ensure that the potassium ion concentration was similar in all cases. Mono-species biofilms were developed in a modified Rototorque biofilm reactor (Burne et al, 1997) with a working volume of 0n6 l. The Rototorque is a stirred tank biofilm reactor that can be run in either batch or continuous mode. For this study, continuous culture was used by pumping medium at a fixed dilution rate and the biofilms were formed on slides that were inserted on the inner wall of the vessel.…”

“…Also, the use of gtf and ftf gene fusion strains in a continuous-flow biofilm fermenter has shown that organisms growing in thicker, mature (7-d-old) biofilms have higher levels of expression of the gtfBC genes compared with cells grown in suspension or in thinner (2-d-old) biofilms (Burne et al, 1997 ;Hudson & Curtiss, 1990 ;Kiska & Macrina, 1994 ;Wexler et al, 1993). In contrast, ftf was found to be dramatically down-regulated in 7 d biofilms compared with 2 d biofilms.…”

“…A number of studies indicate that the expression of the genes for the exopolysaccharide-synthesizing enzymes of S. mutans is dependent on environmental conditions, including growth rate, pH, carbon source, and whether the organisms are attached to the surfaces (Burne et al, 1997 ;Hudson & Curtiss, 1990 ;Kiska & Macrina, 1994 ;Wexler et al, 1993). Also, the use of gtf and ftf gene fusion strains in a continuous-flow biofilm fermenter has shown that organisms growing in thicker, mature (7-d-old) biofilms have higher levels of expression of the gtfBC genes compared with cells grown in suspension or in thinner (2-d-old) biofilms (Burne et al, 1997 ;Hudson & Curtiss, 1990 ;Kiska & Macrina, 1994 ;Wexler et al, 1993).…”

Responses of Cariogenic Streptococci to Environmental Stresses

“…In the presence of sucrose, the glucosyltranferase (GTF) enzymes, gtfB, C and D, produce water-insoluble glucan polymers that, in concert with specific glucan binding proteins (GBPs), play key roles in adhesion and accumulation of biofilms (Banas and Vickerman, 2003;Burne et al, 1997;Hudson and Curtiss, 1990;Yamashita et al, 1993a). In addition to GTFs, S. mutans makes a fructosyltransferase (FTF) enzyme that also participates in the production of extracellular polymers (Birkhed et al, 1979).…”