Abstract
Transcription of the genes for a fructan hydrolase (fruA) and a fructose/mannose sugar:phosphotransferase permease (levDEFG) in Streptococcus mutans is activated by a four-component regulatory system consisting of a histidine kinase (LevS), a response regulator (LevR) and two carbohydrate-binding proteins (LevQT). The expression of the fruA and levD operons was at baseline in a levQ mutant and substantially decreased in a levT null mutant, with lower expression with the cognate inducers fructose or mannose, but slightly higher expression in glucose or galactose. A strain expressing levQ with two point mutations (E170A/F292S) did not require inducers to activate gene expression and displayed altered levD expression when growing on various carbohydrates, including cellobiose. Linker-scanning (LS) mutagenesis was used to generate three libraries of mutants of levQ, levS and levT that displayed various levels of altered substrate specificity and of fruA/levD gene expression. The data support that LevQ and LevT are intimately involved in the sensing of carbohydrate signals, and that LevQ appears to be required for the integrity of the signal transduction complex, apparently by interacting with the sensor kinase LevS.
Highlights
As the major etiological agent of human tooth decay, Streptococcus mutans is well-adapted to growth in oral biofilms, where the intermittent nature of human feeding presents the organisms with a ‘‘feast or famine’’ existence [1]
To test whether the sensor kinase and carbohydrate binding proteins could be exported from the cell, portions of the genes encoding the N-terminal segments of LevQ, LevT or LevS were fused to the DSPNuc sequence (Figure S4), which encodes a nuclease derived from Staphylococcus aureus that lacks its export signal [15]
The reason for nuclease activity detected in the supernatant fluid of these samples was likely due to auto-cleavage of the fusion proteins caused by an internal peptide sequence of Nuc, which can result in release to the culture supernatant of mature NucA from cell surface [15]
Summary
As the major etiological agent of human tooth decay, Streptococcus mutans is well-adapted to growth in oral biofilms, where the intermittent nature of human feeding presents the organisms with a ‘‘feast or famine’’ existence [1]. S. mutans extracts energy from a spectrum of carbohydrates almost exclusively through glycolysis, releasing lactic and other organic acids that are responsible for demineralization of the tooth. The organism secretes a fructosyltransferase (ftf) enzyme that converts sucrose into a fructose homopolymer (fructan) that accumulates rapidly in oral biofilms [2] and functions as an extracellular storage compound [3]. These fructans can be hydrolyzed into free fructose by the action of a secreted exo-bD-fructosidase enzyme encoded by the fruA gene [4,5], which is inducible and under the control of catabolite repression. A gene for a second predicted b-fructosidase enzyme (FruB) is co-transcribed with fruA, but the growth characteristics and fructosidase activity of a fruA mutant do not differ from those of a fruAB deletion mutant
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