SMU_1361c regulates the oxidative stress response of Streptococcus mutans

Dental caries is the most common chronic infectious diseases around the world and disproportionately affects the marginalized socioeconomic group. Streptococcus mutans, considered a primary etiological agent of caries, must depend on the coordinated physiological response to tolerate the oxidative stress generated by commensal species within dental plaque, which is a critical aspect of its pathogenicity. Here, we identified and characterized a novel TetR family regulator SMU_1361c, encoded by the TnSmu2 operon, which appears to be acquired by the bacteria via horizontal genes transfer. Surprisingly, smu_1361c functions as a transcriptional repressor to regulate gene expression outside its operon, involved in the oxidative stress response of S. mutans. The smu_1361c overexpression strain UA159/pDL278-1361c was more susceptible to oxidative stress and less competitive against hydrogen peroxide generated by commensal species Streptococcus gordonii and Streptococcus sanguinis. Transcriptomics analysis revealed that smu_1361c overexpression resulted in the significant downregulation of 22 genes mainly belonging to three gene clusters responsible for the oxidative stress response. The conversed DNA binding motif of SMU_1361c was determined by electrophoretic mobility shift and DNase I footprinting assay with purified SMU_1361c protein, therefore, smu_1361c is directly involved in gene transcription related to the oxidative stress response. Crucially, our finding provides a new understanding of how S. mutans deals with the oxidative stress that is required for pathogenesis and will facilitate the development of new and improved therapeutic approaches for dental caries.


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3 8 its competitiveness with commensal species that occupy the same ecological niche three-species biofilm communities (Fig 2A). Quantitative analysis of FISH-labeled biofilms showed that the overexpression strain UA159/pDL278-1361c was less 1 4 5 competitive than the UA159/pDL278 strain against S. sanguinis and S. gordonii ( Fig 2B). To determine whether the less competitiveness of UA159/pDL278-1361c is caused UA159/pDL278-1361c, which was also recently reported to be involved in the oxidative 1 7 9 stress response of S. mutans [34]. Significantly, these results indicate that smu_1361c 1 8 0 may regulate their gene expression by affecting the promoter of these gene clusters ( Fig   1  8  1 3). His-SMU_1361c protein using EMSA, and the smu_136c ORF fragment (116 bp) was 1 9 1 used to exclude the nonspecific bindings of SMU_1361c to DNA substrates ( Fig 4A).
The mobility shifts were only observed when SMU_1361c was incubated with the 1 9 3 smu_137 promoter, and no mobility shifts appeared when 1361c was incubated with the 1 9 4 smu_136c ORF fragment ( Fig 4A). These results indicate that SMU_1361c can directly 1 9 5 bind to the promoters of smu_137-smu_141. Identifying the specific binding motif of SMU_1361c The DNase I footprinting assay was used to demonstrate the specific binding motif of 1 9 9 SMU_1361c to the putative smu_137 promoter region. After the smu_137 promoter 2 0 0 region was exposed to DNase I, the fragment containing 2 0 1 5′-CATATTATAACATAAAATG-3′ with high AT content was gradually protected with 2 0 2 the increasing SMU_1361c concentrations ( Fig 4B). The results showed that the specific  Oral biofilms are microbial communities with a high density and diversity of bacterial tooth surface before S. mutans [37,38]. They create oxidative stress conditions by 2 1 1 producing hydrogen peroxide, which gives them the ability to compete with S. mutans for 2 1 2 the same ecological niche [39,40]. Therefore, the ability to tolerate oxidative stress is repressor that manipulates numerous related virulence genes in S. mutans by directly 2 1 8 binding to their specific promoter regions.

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Since S. mutans lacks catalase, alternative strategies must be used to cope with the 2 2 0 oxidative stress challenges imposed by the competing species and host [41,42]. In this 2 2 1 study, we found that the overexpression strain UA159/pDL278-1361c was more sensitive 2 2 2 to hydrogen peroxide stress than the UA159/pDL278 strain (Fig 1). In addition, the and S. sanguinis in the well-characterized interspecies interaction three-species biofilms 2 2 5 model, signifying that the ability to tolerate oxidative stress is impaired (Fig 2). This 2 2 6 inhibition phenotype was lost when catalase was added to the three-species biofilms, 2 2 7 further approving that this inhibition of overexpression strain UA159/pDL278-1361c was smu_137-smu_141 is closely associated with the oxidative adaptive adaptability of S.
The TetR family, a family of transcriptional regulators, is well represented and 2 3 5 widely distributed amid bacteria with a DNA-binding motif, whose function is usually 2 3 6 that of repressors [43,44]. In this study, the expression levels of the three operons homeostasis.

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According to the transcriptomics analysis in this study, smu_1361c does not show a 2 4 6 regulatory relationship with its neighboring genes; however, it is involved in the 2 4 7 regulation of genes outside its operon. The EMSA results support a direct mechanism for was identified by DNase I footprinting assays, and the results showed that a palindromic 2 5 0 sequence with high AT content was protected by SMU_1361c, suggesting that In summary, SMU_1361c, a novel TetR family transcription factor, acts as a 2 5 5 repressor to impair the ability of S. mutans to survive and compete with commensal involved in the oxidative stress response by binding to their specific promoter regions.

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Thus, an improved understanding of the mechanism by which SMU_1361c regulates the All strains, plasmids, and primers used in this study are listed in the S1 and S2 Tables. S. The genes of smu_1361c and ldh promoter regions were amplified from S. mutans 2 7 9 genomic DNA by polymerase chain reaction (PCR). All the primers were designed using Construction of markerless in-frame deletion strains 2 9 0 The markerless in-frame deletion strain UA159 Δ 1361c was made using a two-step and dnF/dnR primers and the selection cassette IFDC2 (positive for erythromycin and 2 9 5 negative for p-Cl-phe) was amplified by the ldhF/ermR primers, respectively. Then, the 2 9 6 fragments containing the overlapping regions were ligated using the overlap extension 2 9 7 PCR with upF/dnR primers, which were transformed into S. mutans UA159 and selected  Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to 3 0 7 quantify the expression of the smu_1361c gene in overexpression and deletion strains. Bacterial cells were cultured to the mid-logarithmic phase (OD 600nm =0.5), harvested by  Primer3 online tool (http://simgene.com/Primer3) was used to design the specific primer The hydrogen peroxide sensitivity assay was performed as follows: overnight cultures of 3 1 9 S. mutans and its derivatives were washed and adjusted to the OD 600nm of 0.5. Four plates of the test strains followed by 24 h of incubation at 37°C. Inhibition zones were inhibition zone to disc diameter was calculated. Overnight cultures of S. mutans and its derivatives, S. gordonii, and S. sanguinis were 3 3 0 harvested until OD 600nm to 0.5, and simultaneously inoculated (inoculum ratio of S. Biofilm images were captured using an Olympus FV3000 confocal laser scanning  For RNA-sequencing, the subcultured UA159/pDL278 and UA159/pDL278-1361c