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Repurposing the Streptococcus mutans CRISPR-Cas9 system to understand essential gene function

ABSTRACT: RNA-seq was utilized to compare the transcriptomes of S. mutans strains that had or hadn't experienced CRISPR interference of a green fluorescent protein gene

ORGANISM(S): Streptococcus mutans

PROVIDER: GSE141863 | GEO | 2019/12/12

REPOSITORIES: GEO

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Repurposing the Streptococcus mutans CRISPR-Cas9 System to Understand Essential Gene Function.

Project description:A recent genome-wide screen identified ~300 essential or growth-supporting genes in the dental caries pathogen Streptococcus mutans. To be able to study these genes, we built a CRISPR interference tool around the Cas9 nuclease (Cas9Smu) encoded in the S. mutans UA159 genome. Using a xylose-inducible dead Cas9Smu with a constitutively active single-guide RNA (sgRNA), we observed titratable repression of GFP fluorescence that compared favorably to that of Streptococcus pyogenes dCas9 (Cas9Spy). We then investigated sgRNA specificity and proto-spacer adjacent motif (PAM) requirements. Interference by sgRNAs did not occur with double or triple base-pair mutations, or if single base-pair mutations were in the 3' end of the sgRNA. Bioinformatic analysis of >450 S. mutans genomes allied with in vivo assays revealed a similar PAM recognition sequence as Cas9Spy. Next, we created a comprehensive library of sgRNA plasmids that were directed at essential and growth-supporting genes. We discovered growth defects for 77% of the CRISPRi strains expressing sgRNAs. Phenotypes of CRISPRi strains, across several biological pathways, were assessed using fluorescence microscopy. A variety of cell structure anomalies were observed, including segregational instability of the chromosome, enlarged cells, and ovococci-to-rod shape transitions. CRISPRi was also employed to observe how silencing of cell wall glycopolysaccharide biosynthesis (rhamnose-glucose polysaccharide, RGP) affected both cell division and pathogenesis in a wax worm model. The CRISPRi tool and sgRNA library are valuable resources for characterizing essential genes in S. mutans, some of which could prove to be promising therapeutic targets.

| S-EPMC7082069 | biostudies-literature

Repurposing the Streptococcus mutans CRISPR-Cas9 system to understand essential gene function

Project description:Repurposing the Streptococcus mutans CRISPR-Cas9 system to understand essential gene function

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Transcriptome analysis of Streptococcus mutans biofilms in response to cranberry proanthocyanidins

Project description:The influence of cranberry proanthocyanidins on the transcriptomic responses of Streptococcus mutans during biofilm formation was investigated. Treatment regimens simulating topical exposures experienced clinically (twice-daily, 60 s each) were used over saliva-coated hydroxyapatite biofilm model. Cranberry proanthocyanidins (1.5 mg/ml) in 15% ethanol was used to treat the biofilms. Four biological replicates each for the treatment and vehicle control were used for RNA extraction and microarray.

2012-08-22 | E-GEOD-40172 | biostudies-arrayexpress

Copper ions inhibit Streptococcus mutans-Veillonella parvula dual biofilm by activating Streptococcus-mutans reactive nitrogen species

Project description:To investigate the inhibition mechanism of copper ions on S. mutans-V. parvula dual biofilm.

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Streptococcus mutans UA159 control vs. Streptococcus mutans UA159 supplemented with 20 uM 4,5-dihydroxy-2,3-pentadione (DPD) at 3 different pH values

Project description:Transcriptional profiling of early logarithmic phase culture (O.D=0.2-0.3) of Streptococcus mutans UA159 comparing control of untreated Streptococcus mutans UA159 bacteria with Streptococcus mutans UA159 bacteria spplemented with 20µM synthetic DPD (pre-AI-2) which regulates gene expression via AI-2 quorum sensing system.Three compairisons were performed at pHs of 7,6 and 5.

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Streptococcus mutans UA159 vs. clp mutant strains

Project description:Transcriptional profiling to investigate the roles of ClpP and ClpX of S. mutans

2011-06-10 | GSE29871 | GEO

Streptococcus mutans UA159 vs. spx mutant strains

Project description:Transcriptional profiling to investigate the regulatory roles of SpxA and SpxB of S. mutans.

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glucose amendment of Streptococcus mutans biofilm

Project description:In this experiment we collected small molecule data that represent excreted molecules by Streptococcus mutans growing as a biofilm. The S. mutans biofilms were established and incubated in anaerobic conditions. Samples were collected before and after a drastic pH drop due to glucose amendments. Control samples are included in this folder that represent molecules that were extracted from sterilized growth media only. These peaks should be subtracted from the biofilm samples prior to analyses.

2014-04-10 | MSV000078578 | MassIVE

Transcriptome analysis of Streptococcus mutans ΔSMU.1147 strain

Project description:RNA-seq was performed to examine the overall gene expression in a S. mutans ΔSMU.1147 strain and to identify potential genes that can mainly contribute to the changes of gene expression in S. mutans. Using RNA-seq technique, the differentially expressed genes that are implicated in phenotypic changes of the ΔSMU.1147 strain were studied.

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Streptococcus mutans UA159 vs. clp mutant strains

Project description:Transcriptional profiling to investigate the roles of ClpP and ClpX of S. mutans RNA was extracted from four replicate samples of each strain of interest and labeled with Cy3. For each replicate, labeled RNA was hybridized to slides along with Cy5-labeled reference RNA, extracted from S. mutans UA159 cells grown to mid-log.

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