A Single Nucleotide Change in the Promoter mutp Enhances Fluoride Resistance of Streptococcus mutans

Abstract

dPreviously, we identified a single nucleotide mutation in the promoter (mutp) of the fluoride antiporter-coding genes in a naturally fluoride-resistant Streptococcus mutans strain. Here, we studied the role of this mutation in a defined genetic background. The results confirmed that this mutation alone confers fluoride resistance on S. mutans, as shown by growth and lactic acid production assays. This resistance was explained by constitutively higher mutp promoter activity and upregulation of the fluoride antiporter-coding genes. Fluoride is highly abundant in nature and is extensively used in oral hygiene products (1, 2). At high concentrations, it can be bactericidal. Due to the extensive use of fluoride, many microorganisms have developed resistance to fluoride (3). Recent studies revealed that a conserved fluoride antiporter protein (CrcB) mediates microbial fluoride resistance by exporting fluoride ions (F Ϫ ) to maintain a low F Ϫ level in the cell (4, 5). The expression of the fluoride antiporter genes can be regulated by fluoride-sensing riboswitches (4). However, these riboswitches are absent in microorganisms such as Streptococcus mutans and staphylococci (4). Little is known about the regulation of the fluoride antiporter genes in these microorganisms.In a previous study, we compared the genome sequence of a naturally selected fluoride-resistant S. mutans strain to that of a fluoride-sensitive strain using whole-genome shotgun sequencing and discovered a single nucleotide mutation (Ϫ44A¡C) located in the promoter of two tandem genes coding for the fluoride antiporters (6). The expression of these genes was 10-fold higher in the fluoride-resistant strain than in the fluoride-sensitive strain. Although this evidence indicated that this mutation is involved in the expression regulation of the fluoride antiporters, it is uncertain whether this mutation alone causes fluoride resistance, since an additional seven mutations were found in the genome comparison. Thus, we aimed to investigate the role of this single nucleotide mutation in the fluoride resistance of S. mutans in a defined genomic environment. The promoter containing this mutation is referred to as mutp.We constructed a clean mutp mutant from S. mutans strain UA159 using site-directed mutagenesis and an unlabeled gene replacement system (7). The successful introduction of the base change, Ϫ44A¡C, in mutp was confirmed by Sanger sequencing. The corresponding mutant strain was designated UF35 and char-

Fluoride resistance inStreptococcus mutans: a mini review

“…The products of these two genes share 58% amino acid identity [17]. The involvement of these two genes in fluoride resistance has been confirmed by two gene knockout studies and a gene regulation study [54,64,65]. Both gene knockout studies found that S. mutans became 100-fold more sensitive to fluoride after knocking out both eriC F copies [64,65].…”

The Fitness Cost of Fluoride Resistance for Different Streptococcus mutans Strains in Biofilms

“…As stated above, these two fluoride-resistant strains differ in the number of chromosomal mutations. Strain UF35 contains one mutation in the promoter region of the fluoride antiporter-coding genes and resists fluoride challenge through up-regulation of fluoride antiporters ( Liao et al, 2016 ). Strain UAFR was obtained by stepwise selection on agar plates containing increasing concentrations of fluoride ( Zhu et al, 2012 ).…”

“…The strains used in this study were the fluoride-sensitive S. mutans strain UA159 and two fluoride-resistant strains (UF35 and UAFR). The construction of strain UF35 was described in Liao et al (2016) . The strain UAFR (accession: NZ_CP007016.1) was kindly provided by Professor Zhimin Zhang (Jilin University, Changchun, China).…”

“…The aim of this study was to investigate the fitness of two fluoride-resistant S. mutans strains (UAFR and UF35) in biofilms, using the isogenic wild-type stain UA159 as a reference. The fluoride-resistant strain UAFR was created by culturing UA on agar plates containing increasing concentrations of fluoride ( Zhu et al, 2012 ), whereas strain UF35 was engineered by changing a single nucleotide (-44A→C) in the promoter region mutp of UA159 ( Liao et al, 2016 ). Both strains displayed a slower growth rate than the wild-type strain in suspensions.…”

Identification of an operon involved in fluoride resistance in Enterobacter cloacae FRM

“…Fluoride-resistant Streptococcus mutans , a causative agent of dental caries, which appears to lack a fluoride riboswitch 8 encodes two homologs of the EriC F protein. A single-nucleotide mutation was identified in the promoter of these two eriC F genes 9 and this mutation was found to confer fluoride resistance on S. mutans by increasing promoter activity and upregulating the expression of downstream fluoride antiporter-coding genes 10 . Moreover, these eriC F genes were confirmed to confer fluoride resistance in S. mutans 11 .…”