Uptake and Metabolism of N -Acetylglucosamine and Glucosamine by Streptococcus mutans

Abstract

Glucosamine and N-acetylglucosamine are among the most abundant sugars on the planet, and their introduction into the oral cavity via the diet and host secretions, and through bacterial biosynthesis, provides oral biofilm bacteria with a source of carbon, nitrogen, and energy. In this study, we demonstrated that the dental caries pathogen Streptococcus mutans possesses an inducible system for the metabolism of N-acetylglucosamine and glucosamine. These amino sugars are transported by the phosphoenolpyruvate:sugar phosphotransferase system (PTS), with the glucose/mannose enzyme II permease encoded by manLMN playing a dominant role. Additionally, a previously uncharacterized gene product encoded downstream of the manLMN operon, ManO, was shown to influence the efficiency of uptake and growth on N-acetylglucosamine and, to a lesser extent, glucosamine. A transcriptional regulator, designated NagR, was able to bind the promoter regions in vitro, and repress the expression in vivo, of the nagA and nagB genes, encoding N-acetylglucosamine-6-phosphate deacetylase and glucosamine-6-phosphate deaminase, respectively. The binding activity of NagR could be inhibited by glucosamine-6-phosphate in vitro. Importantly, in contrast to the case with certain other Firmicutes, the gene for de novo synthesis of glucosamine-6-phosphate in S. mutans, glmS, was also shown to be regulated by NagR, and NagR could bind the glmS promoter region in vitro. Finally, metabolism of these amino sugars by S. mutans resulted in the production of significant quantities of ammonia, which can neutralize cytoplasmic pH and increase acid tolerance, thus contributing to enhanced persistence and pathogenic potential.

NagR Differentially Regulates the Expression of the glmS and nagAB Genes Required for Amino Sugar Metabolism by Streptococcus mutans

“…2), the expression of nagA and especially nagB was higher in cells growing on GlcN than on glucose, whereas glmS expression was much higher in glucose-grown cells than in GlcN-grown cells, confirming previous results (3,8). However, in the nagR deletion mutant, glmS expression was greatly elevated regardless of the carbohydrate source, nagB expression was actually higher in glucose-grown cells than in GlcNgrown cells of the mutant, and nagA expression was elevated in cells lacking NagR compared with the wild-type strain grown on glucose.…”

“…In particular, we recently reported that the loss of an apparent homolog of NagR in S. mutans led to the derepression not only of nagA and nagB but also of glmS (3). Moreover, (i) the glmS gene of S. mutans lacks the ribozyme sequences conserved in many Gram-positive bacteria, (ii) dre sites could be identified upstream of the glmS coding sequence, and (iii) purified NagR of S. mutans could bind to the glmS promoter region in vitro (3). Here, we report a more detailed characterization of NagR with respect to its interaction with dre sites and the mechanisms by which it exerts control over the genes needed for the biosynthesis and catabolism of GlcN and GlcNAc.…”

“…Recently, it was shown that the PTS was responsible for GlcN and GlcNAc internalization by the dental caries pathogen S. mutans, and that the loss of NagB or GlmS significantly affected the expression of virulence attributes in this organism (3,8). Importantly, catabolism of GlcNAc or GlcN also yields ammonia, which can increase ⌬pH (i.e., the difference in pH inside and outside the cell) and be utilized for the production of amino acids and other essential compounds (3).…”

“…We recently showed that the human dental pathogen Streptococcus mutans internalizes GlcN and GlcNAc via the phosphoenolpyruvate (PEP): sugar phosphotransferase system (PTS), which consists of a general enzyme I (EI) and a phospho-carrier protein (HPr) that transfer the phosphoryl group from PEP to a variety of substratespecific EII enzymes responsible for the concurrent phosphorylation and internalization of their cognate sugars (1). S. mutans transports GlcN and GlcNAc primarily via the EII Man permease, a fructose:mannose-type EII that is also the dominant transporter for glucose, mannose, and galactose (2)(3)(4). In fact, the PTS is the primary route for amino sugar uptake for most bacteria, providing cells with internalized GlcN-6-phosphate (GlcN-6-P) or GlcNAc-6-phosphate (GlcNAc-6-P) (5)(6)(7).…”

Sucrose- and Fructose-Specific Effects on the Transcriptome of Streptococcus mutans, as Determined by RNA Sequencing

“…Similarly, all point mutations (in gtfD, ftf, and fruB) in strain MMZ1170 were confirmed by allele-specific PCR followed by sequencing. Lastly, a PCR product containing a manL::Sp replacement marker (conferring spectinomycin resistance) (29) was introduced into strain MMZ1009 via transformation, resulting in strain MMZ1196 (gtfABCD ftf fruAB manL).…”