Liao Ying, Chen Jianwei, Brandt Bernd Willem, Zhu Yuanfang, Li Jiyao, van Loveren Cor, Deng Dong Mei
State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu, China; Department of Preventive Dentistry, Academic Centre for Dentistry Amsterdam, University of Amsterdam and VU University Amsterdam, Amsterdam, The Netherlands.
BGI-Shenzhen, Shenzhen, China.
PLoS One. 2015 Apr 9;10(4):e0122630. doi: 10.1371/journal.pone.0122630. eCollection 2015.
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.
已知耐氟微生物在生长、黏附及代谢活性方面与氟敏感微生物不同。据推测，这些表型差异是由于耐氟菌株中稳定的基因型变化所致。然而，截至目前，尚无研究报道这些基因型变化。本研究的目的是利用全基因组鸟枪法（WGS）测序确定耐氟变形链球菌菌株（C180 - 2FR）中的此类变化，并通过比较氟敏感菌株（C180 - 2）和C180 - 2FR菌株之间的基因表达来研究所鉴定突变的潜在功能。我们对这两种菌株都进行了50 bp双末端Illumina鸟枪法测序。通过广泛的生物信息学分析，我们在C180 - 2FR基因组中鉴定出8个单核苷酸多态性（SNP），并通过桑格测序进一步证实。然后通过实时PCR对C180 - 2和C180 - 2FR中包含SNP或与之相邻的基因的表达进行定量。与C180 - 2相比，一个包含编码氟反向转运蛋白基因的基因簇在C180 - 2FR中上调了10倍，且与生长阶段无关。两个SNP位于该基因簇中，一个在其启动子区域，另一个在其蛋白质编码区域。此外，发现一个编码假定甘油摄取促进蛋白的基因在C180 - 2FR生长早期下调了60%。该基因的启动子区域包含一个SNP。对于其他含SNP的基因，未发现表达差异。总之，利用WGS测序，我们能够揭示耐氟菌株基因组中的遗传变化。这些发现可为微生物耐氟机制提供新的见解。
