Cheng Xi, He Fuming, Sun Ping, Chen Qianming
The Affiliated Hospital of Stomatology, School of Stomatology, Zhejiang University School of Medicine, and Key Laboratory of Oral Biomedical Research of Zhejiang Province, Hangzhou, 310006, Zhejiang, China.
AMB Express. 2021 Mar 6;11(1):39. doi: 10.1186/s13568-021-01199-4.
Acid resistance is critical for the survival of bacteria in the dental caries oral micro-environment. However, there are few acid-resistant genes of microbiomes obtained through traditional molecular biology experimental techniques. This study aims to try macrogenomics technologies to efficiently identify acid-resistant genes in oral microbes of patients with dental caries. Total DNA was extracted from oral microbiota obtained from thirty dental caries patients and subjected to high-throughput sequencing. This data was used to build a metagenomic library, which was compared to the sequences of two Streptococcus mutant known acid-resistant genes, danK and uvrA, using a BLAST search. A total of 19 and 35 unknown gene sequences showed similarities with S. mutans uvrA and dnaK in the metagenomic library, respectively. Two unknown genes, mo-dnaK and mo-uvrA, were selected for primer design and bioinformatic analysis based on their sequences. Bioinformatics analysis predicted them encoding of a human heat-shock protein (HSP) 70 and an ATP-dependent DNA repair enzyme, respectively, closely related with the acid resistance mechanism. After cloning, these genes were transferred into competent Escherichia coli for acid resistance experiments. E. coli transformed with both genes demonstrated acid resistance, while the survival rate of E. coli transformed with mo-uvrA was significantly higher in an acidic environment (pH = 3). Through this experiment we found that identify unknown acid-resistant genes in oral microbes of patients with caries by establishing a metagenomic library is very efficient. Our results provide an insight into the mechanisms and pathogenesis of dental caries for their treatment without affecting oral probiotics.
耐酸性对于细菌在龋病口腔微环境中的存活至关重要。然而，通过传统分子生物学实验技术获得的微生物耐酸性基因很少。本研究旨在尝试宏基因组学技术，以高效鉴定龋病患者口腔微生物中的耐酸性基因。从30名龋病患者的口腔微生物群中提取总DNA，并进行高通量测序。利用这些数据构建宏基因组文库，并使用BLAST搜索将其与两种变形链球菌已知的耐酸性基因danK和uvrA的序列进行比较。在宏基因组文库中，分别有19个和35个未知基因序列与变形链球菌uvrA和dnaK具有相似性。基于其序列选择了两个未知基因mo-dnaK和mo-uvrA进行引物设计和生物信息学分析。生物信息学分析预测它们分别编码一种人类热休克蛋白（HSP）70和一种ATP依赖性DNA修复酶，与耐酸性机制密切相关。克隆后，将这些基因转入感受态大肠杆菌进行耐酸性实验。转入这两个基因的大肠杆菌均表现出耐酸性，而转入mo-uvrA的大肠杆菌在酸性环境（pH = 3）中的存活率显著更高。通过本实验我们发现，通过建立宏基因组文库鉴定龋病患者口腔微生物中的未知耐酸性基因非常有效。我们的结果为龋病的治疗机制和发病机制提供了见解，且不会影响口腔益生菌。
