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. 2015 Apr 9;10(4):e0122630.
doi: 10.1371/journal.pone.0122630. eCollection 2015.

Identification and functional analysis of genome mutations in a fluoride-resistant Streptococcus mutans strain

Ying Liao  1 , Jianwei Chen  2 , Bernd Willem Brandt  3 , Yuanfang Zhu  2 , Jiyao Li  4 , Cor van Loveren  3 , Dong Mei Deng  3
Affiliations

Identification and functional analysis of genome mutations in a fluoride-resistant Streptococcus mutans strain

Ying Liao et al. PLoS One. .

Abstract

It is known that fluoride-resistant microorganisms are different from fluoride-sensitive ones in growth, adherence and metabolic activity. It was hypothesized that these phenotypic differences were due to stable genotypic changes in the fluoride-resistant strains. However, until now, no studies have reported these genotypic changes. The aim of this study is to identify such changes in a fluoride-resistant Streptococcus mutans strain (C180-2FR) using whole-genome shotgun (WGS) sequencing and to examine the potential function of the identified mutations by comparing gene expression between the fluoride-sensitive (C180-2) and C180-2FR strains. We performed 50 bp paired-end Illumina shotgun sequencing for both strains. Through extensive bioinformatic analysis, we were able to identify 8 single nucleotide polymorphisms (SNPs) in the genome of C180-2FR, which were further confirmed by Sanger sequencing. Expression of the genes containing or in proximity to the SNPs in C180-2 and C180-2FR was then quantified by real-time PCR. A gene cluster containing genes coding for fluoride antiporters was up-regulated 10-fold in C180-2FR when compared to that in C180-2, independent of growth phase. Two SNPs are located in this gene cluster, one in its promoter region and the other in its protein-coding region. In addition, one gene, which codes for a putative glycerol uptake facilitator protein, was found to be down-regulated by 60% in C180-2FR at an early growth phase. The promoter region of this gene contained a SNP. No difference in expression was found for the other SNP-containing genes. In summary, using WGS sequencing, we were able to uncover genetic changes in the genome of a fluoride-resistant strain. These findings can provide new insights into the mechanism of microbial fluoride resistance.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Colonies of S. mutans C180-2 and C180-2FR.
S. mutans C180-2 and C180-2FR were grown anaerobically on BHI agar or TYCSB agar for 3 days. The images of the colonies on the agar plates were taken with an Anxion stereo-microscope with 50x magnification.
Fig 2
Fig 2. Gene organization at two intergenic regions in S. mutans C180-2 and C180-2FR.
A. Orientation of the genes up- and down-stream of the intergenic region 1 (Inter-1). The sequences of Inter-1 in C180-2, C180-2FR, S. mutans UA159, S. mutans LJ23, S. mutans NN2025 and S. mutans GS5 are given in the blue bar. The red letter indicates the SNP; the purple box indicates the TATAAT box; the red box indicates the transcription start site of the operon; B. Orientation of the genes up- and down-stream of the intergenic region 2 (Inter-2). In both A and B, the red lines indicate the location of the SNPs.
Fig 3
Fig 3. Comparison of gene expression between S. mutans C180-2 and C180-2FR.
S. mutans C180-2 and C180-2FR at early exponential phase (A), late exponential phase (B) and stationary phase (C). Overall expression of each selected gene in C180-2FR relative to that in C180-2 is presented as average fold-change ± SD. This experiment was repeated 3 times. All tested genes are categorized into three groups based on the type of relative fold changes. The significance level (α) was set at 0.005 (after Bonferroni correction). ** indicates p < 0.005. *** indicates p < 0.0005.
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Grants and funding

This research was supported by the National Natural Science Foundation of China (No. 81371135) and "Twelfth Five-Year" National Science and Technology Support Project (20911BAZ03171). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.