Downloaded from www.microbiologyresearch.org by IP: 54.237.57.119 On: Sat, 02 Apr 2016 12:35:15 Expression of biofilm-associated genes of Streptococcus mutans in response to glucose and sucrose Moshe Shemesh, Avshalom Tam and Doron Steinberg Correspondence Doron Steinberg dorons@cc.huji.ac.il Institute of Dental Sciences, Faculty of Dental Medicine, Hebrew University-Hadassah, POB 12272, Jerusalem 91120, Israel Received 2 January 2007 Accepted 29 June 2007 Streptococcus mutans is known as a primary pathogen of dental caries, one of the most common human infectious diseases. Exopolysaccharide synthesis, adherence to tooth surface and biofilm formation are important physiological and virulence factors of S. mutans. In vitro comparative gene expression analysis was carried out to differentiate 10 selected genes known to be mostly involved in S. mutans biofilm formation by comparing the expression under biofilm and planktonic environments. Real-time RT-PCR analyses indicated that all of the genes tested were upregulated in the biofilm compared to cells grown in planktonic conditions. The influence of simple dietary carbohydrates on gene expression in S. mutans biofilm was tested also. Among the tested genes, in the biofilm phase, the greatest induction was observed for gtf and ftf, which are genes encoding the extracellular polysaccharide-producing enzymes. Biofilm formation was accompanied by a 22- fold induction in the abundance of mRNA encoding glucosyltransferase B (GTFB) and a 14.8 - fold increase in mRNA encoding GTFC. Levels of mRNA encoding fructosyltransferase were induced approximately 11.8-fold in biofilm-derived cells. Another notable finding of this study suggests that glucose affects the expression of S. mutans GS5 biofilm genes. In spite of a significant upregulation in biofilm-associated gene expression in the presence of sucrose, the presence of glucose with sucrose reduced expression of most tested genes. Differential analysis of the transcripts from S. mutans, grown in media with various nutrient contents, revealed significant shifts in the expression of the genes involved in biofilm formation. The results presented here provide new insights at the molecular level regarding gene expression in this bacterium when grown under biofilm conditions, allowing a better understanding of the mechanism of biofilm formation by S. mutans. INTRODUCTION Streptococcus mutans is a bacterium that has evolved to depend on a biofilm lifestyle for survival and persistence in its natural ecosystem. S. mutans is assembled as communities attached to dental surfaces and forms matrix-embedded biofilms (Marsh, 2005). Such biological organization provides a sheltered microenvironment for the immobilized bacteria (Bowden & Hamilton, 1998; Hall-Stoodley et al., 2004). Adhesion is the initial step in the formation of biofilm communities. As a primary bacterial agent of dental caries, the mechanisms by which S. mutans adheres to tooth surfaces are important potential targets for anti-cariogenic intervention. Sucrose-dependent mechanisms of adherence, as mediated by extracellular enzymes [glucosyltransferases (GTFs) and fructosyltrans- ferases (FTFs)] and glucan-binding proteins (GBPs), have well-established roles in the virulence of S. mutans (Kuramitsu, 1993, 2001; Steinberg, 2000; Banas & Vickerman, 2003). Sucrose-independent mechanisms can also foster microbial colonization by providing binding sites for bacteria (Lee et al., 1989; Shemesh & Steinberg, 2006). Beyond initial adherence, it appears that a variety of genes are required for the adaptation of S. mutans and other oral streptococci in biofilms. Cells existing in the biofilm have phenotypic characteristics, which are distinct from those of their planktonic counterparts, probably accompanied with significant changes in the patterns of gene expression (Costerton, 1987; Whiteley et al., 2001). These include genes associated with intercellular communication systems and environmental sensing systems, regulators of carbohy- drate metabolism, and adhesion-promoting genes (Lemos & Burne, 2002; Shemesh et al., 2007; Senadheera et al., 2005). Among the genes selected for the study were brpA Abbreviations: CLSM, confocal laser scanning microscope; C t , threshold cycle; FTF, fructosyltransferase; GBP, glucan-binding protein; GTF, glucosyltransferase. A figure showing depth analysis of live/dead stained biofilms is available as supplementary material with the online version of this paper. Journal of Medical Microbiology (2007), 56, 1528–1535 DOI 10.1099/jmm.0.47146-0 1528 47146 G 2007 SGM Printed in Great Britain