Streptococcus mutans NADH oxidase lies at the intersection of overlapping regulons controlled by oxygen and NAD+ levels

Abstract

NADH oxidase (Nox, encoded by nox) is a flavin-containing enzyme used by the oral pathogen Streptococcus mutans to reduce diatomic oxygen to water while oxidizing NADH to NAD(+). The critical nature of Nox is 2-fold: it serves to regenerate NAD(+), a carbon cycle metabolite, and to reduce intracellular oxygen, preventing formation of destructive reactive oxygen species (ROS). As oxygen and NAD(+) have been shown to modulate the activity of the global transcription factors Spx and Rex, respectively, Nox is potentially poised at a critical junction of two stress regulons. In this study, microarray data showed that either addition of oxygen or loss of nox resulted in altered expression of genes involved in energy metabolism and transport and the upregulation of genes encoding ROS-metabolizing enzymes. Loss of nox also resulted in upregulation of several genes encoding transcription factors and signaling molecules, including the redox-sensing regulator gene rex. Characterization of the nox promoter revealed that nox was regulated by oxygen, through SpxA, and by Rex. These data suggest a regulatory loop in which the roles of nox in reduction of oxygen and regeneration of NAD(+) affect the activity levels of Spx and Rex, respectively, and their regulons, which control several genes, including nox, crucial to growth of S. mutans under conditions of oxidative stress.

FIG 1

Overview of microarray analysis. The pie charts show overrepresented functional categories with altered expression in UA159 plus 8.4% O₂ compared to UA159 grown without added O₂ (A) and the Δnox strain compared to UA159 grown without added O₂ (B). The numbers next to the up arrows indicate the numbers of genes upregulated, and those next to the down arrows indicate the numbers of genes downregulated. Genes encoding hypothetical proteins were separated out to allow better visualization of genes with known functions.

FIG 2

Characterization of the nox promoter. (A) Primer extension assay for the identification of the nox transcriptional start site. Total RNA was isolated from steady-state cultures of S. mutans UA159 grown at pH values of 7 and 5 and with or without the addition of exogenous oxygen (maintained at 8.4%). Lanes with cDNA resulting from the extension of mRNA are indicated as pH 5, pH 7, or pH 7 plus 8.4% O₂. The primer was located at positions +72 to +94 bp in the nox open reading frame and was also used to generate the nucleotide ladder. (B) qRT-PCR enumerating nox transcripts. RNA from UA159 cells grown to steady state at pH 7 or pH 5 in a chemostat, with or without the addition of 8.4% oxygen. *, statistical significance between pairs, using Student's t test, with a P value of <0.05 (n = 12); ***, statistical significance between pairs, using Student's t test, with a P value of <0.0001 (n = 12). The error bars indicate standard deviations. (C) Nucleotide sequence of the intergenic region preceding nox. The site of transcription initiation (asterisk), site of translational initiation (bent arrow), ribosomal binding site (RBS), −10 region, −35 region, conserved Spx and Rex motifs, and end of rnsD (SMU.1021) are indicated.

FIG 3

Transcriptional activity of the nox promoter. CAT activity was measured using cell extracts of strains grown in batch cultures in a fermentor vessel with or without the addition of exogenous oxygen to 8.4%. The strains contained either 516 bp or 119 bp upstream of nox in front of the CAT gene in a UA159 background, as detailed in Materials and Methods. All pairs were found to be statistically significant using Student's t test with a P value of <0.05 (n = 3). The error bars indicate standard deviations.

FIG 4

nox promoter activity in Δspx strains of S. mutans. (A) Chloramphenicol acetyltransferase activity was measured using cell extracts of strains grown in batch culture. Transcription from the 119-bp promoter region preceding nox was determined in the following background strains: UA159, ΔspxA (JL13), ΔspxB (JL12), and ΔspxAB (JL21). #, statistical significance between pairs, using Student's t test, with a P value of <0.05 (n = 3); **, statistical significance between pairs, using Student's t test, with a P value of <0.01 (n = 3). The error bars indicate standard deviations. (B) qRT-PCR enumerating nox transcripts in RNA extracted from UA159 or the ΔspxA or ΔspxB strain grown to steady state in a continuous culture. *, statistical significance between pairs, using Student's t test, with a P value of <0.05 (n = 3). The error bars indicate standard deviations.

FIG 5

SpxA promotes in vitro transcription of nox. The nox promoter was incubated with B. subtilis RNAP and B. subtilis σ^A with or without SpxA or SpxB protein and DTT.

FIG 6

Inverse relationship of rex and nox transcript levels. (A) qRT-PCR enumerating rex transcripts in RNA extracted from UA159, UA159 plus 8.4% O₂, and Δnox strains grown to steady state at pH 7 in continuous culture. * and #, statistical significance between pairs, using Student's t test, with a P value of <0.05 (n = 5). The error bars indicate standard deviations. (B) nox promoter activity was quantified by CAT activity measurements as described in Materials and Methods, using cell extracts of strains grown in batch culture. Transcription from the 119-bp and 516-bp promoter regions preceding nox was determined in UA159 (parent strain) and Δrex backgrounds. * and #, statistical significance between pairs, using Student's t test, with a P value of <0.0001 (n = 5). The error bars indicate standard deviations.

FIG 7

Rex binds to the nox promoter. (A) Electrophoretic mobility shift assays were performed with radiolabeled nox promoter probe incubated with the indicated amount of purified Rex protein with or without the addition of 10 mM NAD⁺. The arrow at the left indicates migration of the free probe (FP). (B) An electrophoretic mobility shift assay was also performed to demonstrate specificity of the binding reaction. FP, indicated by the arrow, was incubated in the presence of 50 ng Rex with 10 mM NAD⁺, 10 mM NADH, and 10 mM NAD⁺ plus unlabeled cognate competitor (80 ng, or 200 times the labeled probe) or 10 mM NAD⁺ plus 2 ng poly(dG-dC). A control reaction was also performed with BSA plus 10 mM NAD⁺.

FIG 8

Proposed model for the role of Nox in the Spx and Rex regulons. Nox generates NAD⁺, which, when bound, activates the transcriptional-regulatory activity of Rex. Rex positively regulates the ROS stress genes nox, ahpC, and gor while downregulating itself and adhABCD. Rex may also regulate spxA (dashed line). Nox metabolizes oxygen, which, when abundant, leads to oxidation of SpxA, activating its transcriptional-regulatory activity. When active, SpxA positively regulates the ROS stress genes nox, dpr, tpx, trxB, ahpCF, gor, and sod, as well as the adhABCD complex.