A novel signal transduction system and feedback loop regulate fructan hydrolase gene expression in Streptococcus mutans

Abstract

SummaryThe fruA gene of Streptococcus mutans encodes for a secreted fructan hydrolase (fructanase), an established virulence determinant required for releasing D-fructose from levan-and inulin-type fructans. Expression of fruA is under the control of carbon catabolite repression and is induced by growth in fructans. In this report, we identified an operon in S. mutans UA159 encoding a two-component system flanked by two predicted carbohydrate-binding proteins that is absolutely required for the expression of fruA. All four genes were found to be required for optimal growth of S. mutans on inulin-containing medium and for transcriptional activation of fruA. Complementation assays using a plasmid expressing the response regulator suggested that the twocomponent system works in concert with the sugarbinding proteins. This operon was also shown to activate a four-gene cluster located immediately downstream and encoding an Enzyme II (EII Lev ) for a fructose/mannose sugar : phosphotransferase enzyme, which was found to negatively regulate the expression of fruA. Using transcriptional fusions, it was found that fructose could signal induction of the fruA and levD operons through the two-component system/sugar-binding protein complex. A recombinant LevR protein was shown to bind to the promoter regions of fruA and levD in gel mobility shift assays. Thus, a 'four-component signal transduction system' activates fructan catabolism and the expression of an Enzyme II complex that functions in a feedback loop to sense the accumulation of the end-product of fructan degradation.

Deficiency of BrpB causes major defects in cell division, stress responses and biofilm formation by Streptococcus mutans

“…Briefly, the 59 and 39 regions flanking brpB were amplified by PCR using Phusion high-fidelity DNA polymerase (New England Biolabs) with the gene-specific primers shown in Table 2. Following proper restriction enzyme digestions, the flanking regions were ligated to a non-polar Kan-resistance element (Zeng et al, 2006) that was digested similarly. The resulting ligation mixtures were used to directly transform Strep.…”

Comprehensive Mutational Analysis of Sucrose-Metabolizing Pathways in Streptococcus mutans Reveals Novel Roles for the Sucrose Phosphotransferase System Permease

“…In order to understand the growth behavior of the scrA mutant on FOS, we examined the expression of the fruA gene using a fruA promoter::cat fusion (24), which was established in the wild-type and scrA mutant backgrounds. The strains were cultivated in TV medium containing 0.5% of FOS.…”

“…The studies with FOS led us to investigate in more detail how the presence of sucrose and ScrA may influence fruA expression by studying the induction of fruA by the levQRST circuit in the context of sucrose metabolism by S. mutans. We previously established the levD promoter as a useful model system for LevR-dependent activation of gene expression, particularly because its promoter region has the binding site for LevR; levD promoter activity is minimal in the absence of LevR-dependent activation; and, unlike the fruA promoter, there is no CRE for CcpA binding in the levD promoter, and therefore the results would not be confounded by the activity of an additional regulatory pathway (23,24).Since fructose is a potent signal for activation of the LevQRST pathway, a PlevD-cat fusion was introduced into the background of MMZ952 (gtfABCD ftf fruA), resulting in strain MMZ998. Similarly created was another PlevD-cat reporter strain, MMZ1002, which also lacks scrA in addition to the mutations in MMZ952.…”

“…Both types of polymers can be hydrolyzed by S. mutans by a single secreted exo-␤-D-fructosidase (FruA) (7,19,20). Expression of fruA is governed by an unusual four-component regulatory system that is composed of a conventional histidine kinase (LevS) and response regulator (LevR) and two membrane-associated carbohydrate-binding proteins (LevQ and LevT) that work in concert with LevS and LevR (21)(22)(23)(24). The same regulatory system also controls expression of the levDEFG operon that encodes the A, B, C, and D domains of a fructose/mannose-PTS EII permease (24).…”

Streptococcus mutans: a new Gram-positive paradigm?

“…Of note, elegant studies from the Burne group on carbon metabolism and carbon catabolism repression revealed a highly complex, hierarchical and efficient control of energy generation by S. mutans, and also indicated that carbon metabolism is intertwined with the expression of virulence-related traits (Abranches et al, 2003(Abranches et al, , 2008Zeng et al, 2006).…”