Journal: Applied and Environmental Microbiology

Article Title: SMU_1361c regulates the oxidative stress response of Streptococcus mutans

doi: 10.1128/aem.01871-23

Figure Lengend Snippet: Effect of smu_1361c on the growth, biofilm formation, and oxidative tolerance of S. mutans. (A) Growth characteristics of UA159, UA159/pDL278, UA159/pDL278-1361c, and UA159 Δ1361c. Bacteria were grown aerobically at 37°C, and OD600nm was monitored at 30-min intervals for 12 h. (B) The biofilm biomass was determined using crystal violet staining assay when bacteria were cultured in BHIS under aerobic conditions for 6, 12, and 24 h, respectively. The exponential cultures of UA159, UA159/pDL278, UA159/pDL278-1361c, and UA159 Δ1361c were exposed to oxidative stress with 0.2% (66.05 mM) hydrogen peroxide for 0, 20, 40, and 60 min, respectively, which were serially diluted and cultured on the BHI agar plates. The representative pictures of the hydrogen peroxide challenge are shown (C), colony-forming units were counted, and percent survivals of different strains were calculated relative to the control sample UA159 (D). The plates were overlaid with soft agar containing UA159, UA159/pDL278, UA159/pDL278-1361c, and UA159 Δ1361c and challenged with filter discs soaked with 0, 20, 40, and 60 mM hydrogen peroxide. The representative plates are shown to demonstrate the differences in inhibition zones (E), which are measured at three different positions and averaged to serve as a single data point, and the ratio of inhibition zone to disc diameter was calculated (F). Each experiment was repeated at least thrice. Results are presented as mean ± SD (*P < 0.05 or ***P < 0.001).

Article Snippet: TABLE 1 Strains or plasmids Description Source S. mutans UA159 Wild-type strain ATCC 700610 UA159/pDL278 UA159 pDL278; Spe r This study UA159/pDL278 -1361c UA159/pDL278 -1361c; Spe r This study UA159 Δ 1361c UA159 Δ 1361c ; Em s ; p -Cl-Phe r This study S. gordonii S. gordonii Wild-type strain DL1 S. sanguinis S. sanguinis Wild-type strain SK36 E. coli DH5α F- φ80dlacZΔM15 Δ(lacZYA-argF)U169 deoR recA1 endA1 hsdR17 Laboratory stock (rk−, mk+) phoA supE44 λ- thi-1 gyrA96 relA1 BL21(DE3) F-ompT hsdS B(rB-mB-)dcm gal (DE3) Novagen Plasmids pET28a Kan r expression vector with the 6His-tag coding sequence Novagen pET 1361c pET derivative for expression 6His-1361c This study pDL278 E. coli-Streptococcus shuttle vector (Spe r ) ( 52 ) pDL278 -1361c pDL278 derivative for overexpression of smu_1361c in S. mutans This study Open in a separate window Bacterial strains and plasmids used in this study. . Construction of overexpression strains The genes of smu_1361c and ldh promoter regions were amplified from S. mutans genomic DNA by polymerase chain reaction.

Techniques: Bacteria, Staining, Cell Culture, Inhibition

Journal: Applied and Environmental Microbiology

Article Title: SMU_1361c regulates the oxidative stress response of Streptococcus mutans

doi: 10.1128/aem.01871-23

Figure Lengend Snippet: Transcriptomics analysis of UA159/pDL278 and UA159/pDL278-1361c strains. (A) Functional classification of differentially expressed genes (DEGs) was performed based on the clusters of orthologous groups type. (B) The volcano plot illustrates DEGs between UA159/pDL278 and UA159/pDL278-1361c. Functional annotation and enrichment analysis of DEGs were performed in the Kyoto Encyclopedia of Genes and Genomes (KEGG, C) and Gene Ontology (GO, D) databases. Downregulated genes are shown in green. (E) Genetic organization of gene clusters that were significantly downregulated in UA159/pDL278-1361c.

Article Snippet: TABLE 1 Strains or plasmids Description Source S. mutans UA159 Wild-type strain ATCC 700610 UA159/pDL278 UA159 pDL278; Spe r This study UA159/pDL278 -1361c UA159/pDL278 -1361c; Spe r This study UA159 Δ 1361c UA159 Δ 1361c ; Em s ; p -Cl-Phe r This study S. gordonii S. gordonii Wild-type strain DL1 S. sanguinis S. sanguinis Wild-type strain SK36 E. coli DH5α F- φ80dlacZΔM15 Δ(lacZYA-argF)U169 deoR recA1 endA1 hsdR17 Laboratory stock (rk−, mk+) phoA supE44 λ- thi-1 gyrA96 relA1 BL21(DE3) F-ompT hsdS B(rB-mB-)dcm gal (DE3) Novagen Plasmids pET28a Kan r expression vector with the 6His-tag coding sequence Novagen pET 1361c pET derivative for expression 6His-1361c This study pDL278 E. coli-Streptococcus shuttle vector (Spe r ) ( 52 ) pDL278 -1361c pDL278 derivative for overexpression of smu_1361c in S. mutans This study Open in a separate window Bacterial strains and plasmids used in this study. . Construction of overexpression strains The genes of smu_1361c and ldh promoter regions were amplified from S. mutans genomic DNA by polymerase chain reaction.

Techniques: Functional Assay

Journal: Applied and Environmental Microbiology

Article Title: SMU_1361c regulates the oxidative stress response of Streptococcus mutans

doi: 10.1128/aem.01871-23

Figure Lengend Snippet: Bacterial strains and plasmids used in this study

Article Snippet: TABLE 1 Strains or plasmids Description Source S. mutans UA159 Wild-type strain ATCC 700610 UA159/pDL278 UA159 pDL278; Spe r This study UA159/pDL278 -1361c UA159/pDL278 -1361c; Spe r This study UA159 Δ 1361c UA159 Δ 1361c ; Em s ; p -Cl-Phe r This study S. gordonii S. gordonii Wild-type strain DL1 S. sanguinis S. sanguinis Wild-type strain SK36 E. coli DH5α F- φ80dlacZΔM15 Δ(lacZYA-argF)U169 deoR recA1 endA1 hsdR17 Laboratory stock (rk−, mk+) phoA supE44 λ- thi-1 gyrA96 relA1 BL21(DE3) F-ompT hsdS B(rB-mB-)dcm gal (DE3) Novagen Plasmids pET28a Kan r expression vector with the 6His-tag coding sequence Novagen pET 1361c pET derivative for expression 6His-1361c This study pDL278 E. coli-Streptococcus shuttle vector (Spe r ) ( 52 ) pDL278 -1361c pDL278 derivative for overexpression of smu_1361c in S. mutans This study Open in a separate window Bacterial strains and plasmids used in this study. . Construction of overexpression strains The genes of smu_1361c and ldh promoter regions were amplified from S. mutans genomic DNA by polymerase chain reaction.

Techniques: Expressing, Plasmid Preparation, Sequencing, Over Expression