Abstract
Fluorine is ubiquitous and the most active non-metal element in nature. While many microorganisms have developed fluoride resistance as a result of the widespread and prolonged application of oral hygiene products, the mechanisms used by these organisms to overcome fluoride toxicity are incompletely understood. In this study, a fluoride-resistant strain, Enterobacter cloacae FRM, was identified which could grow well at a fluoride concentration of 4,000 mg/L. According to comparative genomics, transcriptome under fluoride stress, and sequence analyses of two fluoride-resistant fosmid clones, the genomic island GI3 was found to be important for fluoride resistance. The result of quantitative RT-PCR indicated 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. 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 has expanded our understanding of the biological effects of fluoride.
Highlights
Fluoride is ubiquitous in nature and is commonly used as an additive in water and oral hygiene products because of its strong anticaries function[1, 2]
To determine the level of fluoride resistance of E. cloacae FRM, we examined the growth of E. cloacae FRM, as well as other seven E. cloacae strains (E. cloacae Agricultural Culture Collection of China (ACCC) 10453, ACCC 11690, ACCC0 1620, ACCC 11688, ATCC 7256, CMCC 45301, and NCTC 9394) in liquid medium with various fluoride concentrations
Our research showed that an operon consisting of six genes is involved in the fluoride resistance of E. cloacae FRM
Summary
Fluoride is ubiquitous in nature and is commonly used as an additive in water and oral hygiene products because of its strong anticaries function[1, 2]. This research group found that the conserved RNA structure identified by bioinformatics in many species of bacteria and archaea[6] functions as a fluoride-sensing riboswitch. A single-nucleotide mutation was identified in the promoter of these two eriCF genes[9] and this mutation was found to confer fluoride resistance on S. mutans by increasing promoter activity and upregulating the expression of downstream fluoride antiporter-coding genes[10]. Environmental levels of fluoride are typically found in the 10–100 μM range, this bacterium can grow at 4,000 mg/L (210 mM) fluoride Given that such high fluoride resistance is not common among fluoride-resistant microorganisms, we decided to investigate the mechanism of resistance. An operon containing six genes (ppaC, uspA, eno, gpmA, crcB, and orf5249) in the genomic island GI3 was identified and shown to be important for the survival of E. cloacae FRM in high concentration of fluoride
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