Involvement of an Inducible Fructose Phosphotransferase Operon in Streptococcus gordonii Biofilm Formation

Abstract

Oral streptococci, such as Streptococcus gordonii, are the predominant early colonizers that initiate biofilm formation on tooth surfaces. Investigation of an S. gordonii::Tn917-lac biofilm-defective mutant isolated by using an in vitro biofilm formation assay showed that the transposon insertion is near the 3 end of an open reading frame (ORF) encoding a protein homologous to Streptococcus mutans FruK. Three genes, fruR, fruK, and fruI, were predicted to encode polypeptides that are part of the fructose phosphotransferase system (PTS) in S. gordonii. These proteins, FruR, FruK, and FruI, are homologous to proteins encoded by the inducible fruRKI operon of S. mutans. In S. mutans, FruR is a transcriptional repressor, FruK is a fructose-1-phosphate kinase, and FruI is the fructose-specific enzyme II (fructose permease) of the phosphoenolpyruvate-dependent sugar PTS. Reverse transcription-PCR confirmed that fruR, fruK, and fruI are cotranscribed as an operon in S. gordonii, and the transposon insertion in S. gordonii fruK::Tn917-lac resulted in a nonpolar mutation. Nonpolar inactivation of either fruK or fruI generated by allelic replacement resulted in a biofilm-defective phenotype, whereas a nonpolar mutant with an inactivated fruR gene retained the ability to form a biofilm. Expression of fruK, as measured by the ␤-galactosidase activity of the fruK::Tn917-lac mutant, was observed to be growth phase dependent and was enhanced when the mutant was grown in media with high levels of fructose, sucrose, xylitol, and human serum, indicating that the fructose PTS operon was fructose and xylitol inducible, similar to the S. mutans fructose PTS. The induction by fructose was inhibited by the presence of glucose, indicating that glucose is able to catabolite repress fruK expression. Nonpolar inactivation of the fruR gene in the fruK::Tn917-lac mutant resulted in a greater increase in ␤-galactosidase activity when the organism was grown in media supplemented with fructose, confirming that fruR is a transcriptional repressor of the fructose PTS operon. These results suggest that the regulation of fructose transport and metabolism in S. gordonii is intricately tied to carbon catabolite control and the ability to form biofilms. Carbon catabolite control, which modulates carbon flux in response to environmental nutritional levels, appears to be important in the regulation of bacterial biofilms.The process of bacterial accumulation and proliferation after initial bacterial adhesion leads to the formation of persistent, complex, organized sessile communities on oral surfaces. The multistep process of oral biofilm formation is a complex developmental process initiated by attachment to saliva-conditioned oral surfaces of primary colonizers, such as viridans streptococci (including Streptococcus gordonii), which constitute a majority of the cultivable bacteria found in dental plaque (21). Subsequent accumulation and growth of attached bacteria result in microcolonies that increase in size and eventually form biofilms. Fully develo...

Involvement of Streptococcus gordonii Beta-Glucoside Metabolism Systems in Adhesion, Biofilm Formation, and In Vivo Gene Expression

“…As expected, mutants with mutations in several bfb genes showed reduced biofilm formation on plastic in vitro. Likewise, a fructose PTS operon has been recently linked to S. gordonii biofilm formation (20). The fourth putative regulon, gom, is for glycoprotein-derived oligosaccharide metabolism.…”

“…While genes for these adhesin proteins may contribute to colonization in vivo, none are well established at this time. Recently described genes associated with S. gordonii biofilm formation encode putative proteins involved in signal transduction, peptidoglycan biosynthesis, manganese homeostasis, and fructose PTS (18)(19)(20). Furthermore, S. gordonii cells forming biofilms in vivo on heart valves (15) or in vitro on sHA (17) induced genes associated with beta-glucoside metabolism at unexpected frequencies.…”

StreptococcusAdherence and Colonization

“…Thus, mutations in genes encoding components of quorumsensing systems, e.g., comCDE (354); TCSS, e.g., vicRK (543); GBPs (370); and metal ion permeases (503) can impair S. mutans biofilm formation. Optimal biofilm formation by S. gordonii and S. sanguinis also requires multiple gene products including sortase (428) and proteins involved with ␤-glucoside metabolism and carbon catabolite control (296,364), nucleotide biosynthesis (181), oxidative stress (322,363), and environmental sensing and signaling (193). The growth of nonmitis-group streptococci as biofilms is only just beginning to be studied, but the complexities of M serotypes, capsular serotypes, and the expression of pilus types and antigenically variable surface proteins will probably make interpretations of the molecular pathways involved extremely difficult.…”

Probiotics and the Reduction of Dental Caries Risk

“…The fact that sugars are readily metabolized by oral bacteria, leading to the production of organic acids and extra cellular polysaccharides such as glucan and fructan was shown repeatedly in clinical studies (Zero, 2004;Touger et al, 2003). Numerous studies have established the role www.intechopen.com of sugars in caries etiology and the importance of sugars as the principal dietary substrate that drives the caries process (Caglar et al, 2005;Touger et al, 2003;Loo et al, 2003).…”