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

Abstract

The ability of bacteria to metabolize glucosamine (GlcN) and N-acetyl-D-glucosamine (GlcNAc) is considered important for persistent colonization of the oral cavity. In the dental caries pathogen Streptococcus mutans, the NagR protein regulates the expression of glmS, which encodes a GlcN-6-P synthetase, and nagA (GlcNAc-6-P deacetylase) and nagB (GlcN-6-P deaminase), which are required for the catabolism of GlcNAc and GlcN. Two NagR-binding sites (dre) were identified in each of the promoter regions for nagB and glmS. Using promoter-reporter gene fusions, the role of each dre site was examined in the regulation of glmS and nagB promoter activities in cells grown with glucose, GlcNAc, or GlcN. A synergistic relationship between the two dre sites in the glmS promoter that required proper spacing was observed, but that was not the case for nagB. Binding of purified NagR to DNA fragments from both promoter regions, as well as to dre sites alone, was strongly influenced by particular sugar phosphates. Using a random mutagenesis approach that targeted the effector-binding domain of NagR, mutants that displayed aberrant regulation of both the glmS and nagAB genes were identified. Collectively, these findings provide evidence that NagR is essential for regulation of genes for both the synthesis and catabolism of GlcN and GlcNAc in S. mutans, and that NagR engages differently with the target promoter regions in response to specific metabolites interacting with the effector-binding domain of NagR. IMPORTANCEGlucosamine and N-acetylglucosamine are among the most abundant naturally occurring sugars on the planet, and they are catabolized by many bacterial species as sources of carbon and nitrogen. Representing a group called lactic acid bacteria (LAB), the human dental caries pathogen Streptococcus mutans is shown to differ from known paradigm organisms in that it possesses a GntR/HutC-type regulator, NagR, that is required for the regulation of both catabolism of GlcN and biosynthesis. Results reported here reveal a simple and elegant mechanism whereby NagR differentially regulates two opposing biological processes by surveying metabolic intermediates. This study provides insights that may contribute to the development of novel therapeutic tools to combat dental caries and other infectious diseases.A mino sugars, including glucosamine (GlcN) and N-acetyl-Dglucosamine (GlcNAc or NAG), are some of the most abundant carbohydrates on earth. Since amino sugars can serve as sources of carbon and nitrogen, many bacteria have the capacity to utilize them for energy production and growth. 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. mu...

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“…Similar to in S. mutans, the nagA and nagB genes are distant from one another in the genome of S. gordonii DL1, with putative promoter elements and NagRbinding sites (dre) predicted upstream of their coding sequences (see Fig. S4 in the supplemental material) (25). Different from S. mutans, though, is the fact that a three-gene locus, including another GntR-type regulator (busR) that is situated immediately downstream of nagR (24) in S. mutans, is not present anywhere in the genome of DL1.…”

“…Recombinant NagR Sg protein was used in fluorescence polarization (FP) and electrophoretic mobility shift assays (EMSA), as detailed in previous reports (24,25). For FP, a 6-carboxyfluorescein (FAM)-labeled, 45-nucleotide (nt) probe (25) that contained the consensus NagR-binding sequence (dre) deduced from a number of streptococcal genomes and that was functionally validated (25) was employed, and analysis of the FP data allowed for calculation of the equilibrium dissociation constant (K D ). EMSA used three 0.3-kbp biotin-labeled DNA fragments that contained the intergenic regions upstream of the coding sequence of nagA, nagB, and glmS in S. gordonii.…”

“…Similar to recombinant NagR from S. mutans (NagR Sm ), cleavage of the MBP fusion protein resulted in precipitation of NagR Sg . The precipitated protein was harvested by centrifugation, washed, and redissolved in a buffer that contained 300 mM NaCl, 50 mM KCl, 1 mM EDTA, 5 mM dithiothreitol, 10 mM HEPES (pH 7.9), and 10% glycerol (25).…”

“…Similar to S. mutans, computer algorithms predict at least one putative binding site (dre) for NagR upstream of ORF SGO_1757 in S. gordonii (see Fig. S4 in the supplemental material) (25). To assess the impact of the deletion of nagR on glmS transcription in S. gordonii, qRT-PCR was performed on RNA samples from the nagR-null and wild-type strains grown in Glc, GlcN, or GlcNAc.…”

“…Recently, we characterized the metabolic pathways for amino sugars in the dental pathogen S. mutans and demonstrated that regulation of the expression of the genes for NagA, NagB, and GlmS by the GntR/HutC-type regulator NagR was direct (24,25), unlike the regulatory systems for these genes in many other bacteria. We also showed that the glucose/mannose-PTS (EII Man ) is the primary transporter for GlcNAc in S. mutans, that significant amounts of ammonia are released by S. mutans when catabolizing these amino sugars, and that growth on these compounds affected acid tolerance and the acidogenic potential of the organism (24).…”