Abstract
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.
Highlights
Dental caries, one of the most common oral diseases, is capable of destroying the hard tissues of teeth, and acts as an initial stimulus for multiple systemic diseases
Probiotics account for a large proportion of human oral microbiota: they are able to inhibit oral pathogenic microbiota, assist in the food metabolism, and regulate the immune system (Selwitz et al 2007)
The ability to accurately eliminate the pathogenic microbiotas of caries without decreasing the levels of oral probiotics is one of the main goals of scholars in this field
Summary
One of the most common oral diseases, is capable of destroying the hard tissues of teeth, and acts as an initial stimulus for multiple systemic diseases. Extensive research was considered on the acid-producing and acid-resistant properties of Streptococcus mutans, which resulted in the identification of several acid-resistant genes, including ffh, uvrA, and dnaK (Jin et al 2011). These achievements can be attributed to the effective breeding of S. mutans in laboratories. Except for Streptococcus mutans, we have limited knowledge of the acid resistance and regulatory genes of caries pathogens, especially in terms of non-cultivable oral microbiotas. This forms the basis for our experiments, with the aim to identify unknown acidresistant genes using metagenomics analysis
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