AhrC Negatively Regulates Streptococcus mutans Arginine Biosynthesis

Abstract

Streptococcus mutans is a primary cariogenic pathogen in humans. Arginine metabolism is required for bacterial growth. In S. mutans, however, the involvement of transcription factors in regulating arginine metabolism is unclear. The purpose of this study was to investigate the function and mechanism of ArgR family transcription factors in S. mutans. Here, we identified an ArgR (arginine repressor) family transcription factor named AhrC, which negatively regulates arginine biosynthesis and biofilm formation in S. mutans. The ahrC in-frame deletion strain exhibited slow growth and significantly increased intracellular arginine content. The strain overexpressing ahrC showed reduced intracellular arginine content, decreased biofilm biomass, reduced production of water-insoluble exopolysaccharides (EPS), and different biofilm structures. Furthermore, global gene expression profiles revealed differential expression levels of 233 genes in the ahrC-deficient strain, among which genes related to arginine biosynthesis (argJ, argB, argC, argD, argF, argG, argH) were significantly upregulated. In the ahrC overexpression strain, there are 89 differentially expressed genes, mostly related to arginine biosynthesis. The conserved DNA patterns bound by AhrC were identified by electrophoretic mobility shift assay (EMSA) and DNase I footprinting. In addition, the analysis of β-galactosidase activity showed that AhrC acted as a negative regulator. Taken together, our findings suggest that AhrC is an important transcription factor that regulates arginine biosynthesis gene expression and biofilm formation in S. mutans. These findings add new aspects to the complexity of regulating the expression of genes involved in arginine biosynthesis and biofilm formation in S. mutans. IMPORTANCE Arginine metabolism is essential for bacterial growth. The regulation of intracellular arginine metabolism in Streptococcus mutans, one of the major pathogens of dental caries, is unclear. In this study, we found that the transcription factor AhrC can directly and negatively regulate the expression of N-acetyl-gamma-glutamyl-phosphate reductase (argC), thus regulating arginine biosynthesis in S. mutans. In addition, the ahrC overexpression strain exhibited a significant decrease in biofilm and water-insoluble extracellular polysaccharides (EPS). This study adds new support to our understanding of the regulation of intracellular arginine metabolism in S. mutans.

Keywords: Streptococcus mutans; biofilm(s); gene expression; microbial genetics; transcription factor(s).

FIG 1

The growth characteristics, the content of arginine, and biofilm formation of S. mutans. (A) Growth curves of S. mutans strains grown in BHI medium for 24 h. (B) The content of arginine in S. mutans strains. (C) The biofilm biomass of S. mutans strains grown in BHIS medium for 24 h. (D) The SEM images show the 6-h biofilms of S. mutans strains. Images were taken at ×1,000 and ×5,000 magnification. (E) Double-labeled images of 6-h S. mutans strain biofilms. Red represents the EPS (Alexa Flour 647), and green represents the bacteria (SYTO 9). Images were taken at ×60 oil magnification and analyzed with IMARIS 9.0. Representative images are shown from at least five randomly selected fields of each sample. (F) The EPS/bacteria ratio of 6-h S. mutans biofilms. The data were quantified with ImageJ. The results were the mean values of five randomly selected areas in each sample, expressed as mean ± standard deviation.

FIG 2

Transcriptomic analysis of the ΔahrC and UA159/pDL278-ahrC strains. (A) Volcano plot showing the differences in gene expression between S. mutans UA159 and the ΔahrC strain. (B) The KEGG pathway analysis and the GO enrichment analysis of DEGs in the ΔahrC strain. (C) Volcano plot showing the gene expression differences between S. mutans UA159/pDL278 and S. mutans UA159/pDL278-ahrC. (D) The KEGG pathway analysis and the GO enrichment analysis of DEGs in S. mutans UA159/pDL278-ahrC. Upregulated genes are shown in red, whereas downregulated genes are shown in green. GO, gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

FIG 3

Genes regulating the arginine biosynthesis pathway. (A) The differentially expressed arginine biosynthesis operons in the S. mutans ΔahrC strain. The genetic organization of differentially expressed gene clusters associated with arginine biosynthesis in the S. mutans ΔahrC strain. Upregulated genes are indicated in red, whereas downregulated genes are shown in green, with the fold of differential expression represented by the values in parentheses. (B) Flowchart of the arginine biosynthesis pathway in S. mutans. Genes encoding enzymes in the arginine synthesis pathway are indicated in blue.

FIG 4

Identification of conserved DNA motif bound by AhrC. (A) The result of electrophoretic mobility shift assay (EMSA) showing AhrC protein binding to the argC promoter (lanes 7 to 9). (B) DNase I footprinting experiments showing the protective effect of AhrC protein (0, 0.5, 1, and 2 μM) against 5-FAM-labeled primers of argC promoter. The sequence of the protected area is indicated in red. (C) The conserved palindromic sequences and mutated sequences of argC promoter. DNA binding activity of AhrC on argC promoter p1 (lanes 1 to 4) and argC promoter p1mut (lanes 5 to 8). (D) The prediction of AhrC binding sequence in selected gene promoters. (E) The sequence logo for AhrC binding motif was generated by the WebLogo tool.

FIG 5

AhrC served as a suppressor. Constructing a series of lacZ and promoter-lacZ coexpression plasmids to analyze the effect of AhrC on argC expression. A schematic representation of each cloned reporter box is used to produce a recombinant strain. The lacZ and ldhp-lacZ plasmids were used as controls. The recombinant strains were inoculated on BHI-agar plates containing 1 mg/mL spectinomycin and 40 μg/mL X-gal for 48 h of incubation. The results of β-galactosidase activity analysis were expressed in Miller units. The data consisted of three replicates, expressed as mean ± standard deviation.