Characterization of the Nicotinamide Adenine Dinucleotide Biosynthesis Pathway and Regulatory Mechanisms in Streptococcus mutans

Abstract

NAD⁺ and its derivatives, which act as redox coenzymes, are crucial for cellular metabolism and energy production. Nevertheless, the processes by which Streptococcus mutans, a bacterium known for causing dental caries, synthesizes NAD⁺ are not well elucidated. Through a genome-wide screen, we identified the nicotinic acid salvage pathway and the evolutionarily incomplete PnuC-NadR pathway involved in NAD⁺ biosynthesis in S. mutans UA159. The nicotinic acid pathway is regulated by SmNiaR, a nicotinic acid-responsive transcription regulator featuring an N-terminal DNA-binding winged helix-turn-helix-like domain and a C-terminal 3-histidine domain. Notably, a single-site amino acid substitution at site K97 in SmNiaR can reverse its DNA-binding ability, an effect mediated by acetylation at this site, which impacts the intracellular production of NAD⁺ and NADH. Additionally, the deletion of niaR in S. mutans UA159 impaired bacterial proliferation, reduced acid production, and altered biofilm formation, resulting in attenuated virulence in the rat caries model. Conclusively, the regulation of NAD⁺ homeostasis via SmNiaR contributes significantly to the cariogenic virulence of S. mutans.

Keywords: Streptococcus mutans; NAD+ biosynthesis; acetylation; regulator; virulence.

FIGURE 1

NAD biogenesis and its transcriptional regulation in S. mutans containing the SmNiaR regulator. Pathway diagram showing biochemical transformations (arrows) of precursors and intermediates (shown via the same abbreviations as in the text). The NiaX and PnuC transporters take up exogenous precursors (NA, NAM, and NRam). Enzymes are shown using the respective gene names in boxes whose colors correspond to the salvage I pathway of NAD metabolism. The third pathway (salvage III pathway) of NAD biogenesis is incomplete due to the missing NadR domain (NadR^AT). Red lines indicate regulated genes (NiaR regulon members).

FIGURE 2

Characteristics of NAD⁺ biosynthesis regulatory factors. (A) Comparison of NadR domains in E. coli, S. suis , and S. mutans . (B) The sequence of the conserved NiaR‐recognizable motif in Streptococci. (C) Ribbon illustration of the modeled structure of the SmNiaR protein. (D) Structural alignment of NiaR proteins in S. mutans (SmNiaR, Chartreuse) and in Thermotoga maritima (TmNiaR, pale yellow).

FIGURE 3

Binding of SmNiaR to cognate niaX and pnuC is increased by the addition of niacin (NA). (A) Genetic context of niaR and its regulatory target genes (niaX, pnuC) and the binding sites. (B) SmNiaR binds to the pnuC probe in a dose‐dependent manner. (C) SmNiaR binds to the niaX probe in a dose‐dependent manner. (D) The addition of NA increased the binding of SmNiaR to the cognate niaX promoter. (E) The addition of NA increased the binding of SmNiaR to the pnuC promoter. A 45 bp DNA probe encompassing the niaX/pnuC gene promoter region was obtained by annealing the primers niaX‐F/R and pnuC‐F/R (Table S3). The arrow indicates the shifted/supers‐shifted DNA–protein complex. Levels of intracellular NAD⁺ (F) and NADH (G) in the WT, ΔniaR, and CΔniaR strains and their corresponding point mutants (K97Q, K97A, and K97R) were assessed to identify the role of SmNiaR and acetylation of K97 in the homeostasis of the intracellular NAD⁺ pool. All of the experiments were performed at least in triplicate, and the data are presented as the means ± SD. The p values were calculated using one‐way ANOVA along with Tukey's test. *p < 0.05, **p < 0.01, and ***p < 0.001.

FIGURE 4

Structural and acetylation characterization of SmNiaR. (A) Western blotting was used to probe the acetylation of the SmNiaR protein mutants. (B) Acetylation levels of SmNiaR mutant proteins. (C) Structural analysis of the critical amino acid residue K97 for the DNA‐binding domain through structural modeling of S. mutans NiaR. (D–F) Surrounding structural features of K97 from various angles. The recombinant SmNiaR mutant proteins were purified and analyzed by western blotting using both an anti‐acetyl‐lysine antibody (a‐acetyl) and a His‐tag antibody (A, B). Representative results from three independent experiments is shown.