| Fluorine is ubiquitous and the most active non-metal element in nature. It is widely used in oral hygiene products and municipal water supplies due to its strong anticaries function. Because of its widespread application and its toxic effects,organism must have evolved strategies to overcome this toxicity. However, the mechanisms of fluoride resistance are incompletely understood.A fluoride-resistant strain, Enterobacter cloacae FRM, was identified by our laboratory, which could grow well at a fluoride concentration of 4,000 mg/L.According to comparative genomics, transcriptome under fluoride stress, the genomic island GI3 was found to be important for fluoride resistance. The purpose of this study was to determine the genes on genomic island GI3 which are responsible for high fluoride resistance and elucidate the mechanism of fluoride resistance.We demenstrated that six genes on GI3, ppaC, uspA, eno, gpmA, crcB, and orf5249,which encode a fluoride transporter, fluoride-inhibited enzymes, and a universal stress protein, reside in an operon and are transcribed into two mRNAs activated by fluoride with a fluoride riboswitch,by using RT-PCR and quantitative RT-PCR. To clarify the functions of these genes, we established the genetic knockout systerm in E. cloacae FRM, based on the CRISPR-Cas9 method. The results of knockout and complementation experiments indicated that these genes work together to provide high fluoride resistance to E. cloacae FRM. This study clarified the resistance mechanism of this high fluoride-resistant organism and provided a deeper understanding of the regulation mechanisms of microbial resistance to fluoride. This study can also serve as theoretical basis for further application of fluoride-resistant microorganism, such as development of more effective oral hygiene products and microbial treatment of fluoride pollution. |
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