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

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

PROVIDER: PRJNA594976 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR10670100.fastq.gz	Fastqsanger.gz
	SRR10670101.fastq.gz	Fastqsanger.gz
	SRR10670102.fastq.gz	Fastqsanger.gz
	SRR10670103.fastq.gz	Fastqsanger.gz
	SRR10670104.fastq.gz	Fastqsanger.gz

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

Project description: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

2019-12-12 | GSE141863 | GEO

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

Acid-inducible genes in Streptococcus mutans: multiple two-component systems involved in acid adaptation

Project description:Global transcriptional analysis of acid-inducible genes in Streptococcus mutans: multiple two-component systems involved in acid adaptation pH is a major environmental factor that regulates gene expression in many bacteria. Streptococcus mutans in dental biofilms is regularly exposed to cycles of acidic pH during the ingestion of fermentable dietary carbohydrates. The ability of S. mutans to tolerate low pH is crucial for its virulence and the pathogenesis in dental caries. To better understand its acid tolerance mechanisms, we used DNA microarray to perform genome-wide transcriptional analysis of S. mutans in response to acidic pH. The results showed that adaptation of S. mutans to pH 5.5 for 2 hrs induced differential expression of nearly 14% of genes in the genome, including 169 up-regulated genes and 108 down-regulated genes, largely categorized into six groups. Especially, we found that the genes encoding multiple two-component systems, including CiaHR, LevSR, LiaSR, ScnKR, HK/RR07 and ComDE, were up-regulated during acid adaptation. These findings were further confirmed by real time qRT-PCR and phenotypic assays of the gene deletion mutants. The results support that the multiple two-component systems are required for S. mutans to orchestrate its signal transduction networks for optimal adaptation to acidic pH.

2009-03-06 | GSE15125 | GEO

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.

2010-07-29 | GSE23159 | GEO

The CodY Regulon in Streptoccus mutans

Project description:In this report, codY mutant strains were constructed and used to demonstrate the relationship of (p)ppGpp synthesized by RelP and RelQ with the activation of CodY. In addition, because CodY has not been studied in S. mutans, we used microarrays to demonstrate that this protein function as a global regulator of gene expression in S. mutans. Keywords: stress response, gene knockout analysis

2008-10-31 | GSE9641 | GEO

The CdaA Regulon in Streptoccus mutans

Project description:Nucleotide signaling pathways are found in all kingdoms of life and are utilized to coordinate a rapid response to changes in the environment. One more recently discovered signaling nucleotide is the secondary messenger cyclic diadenosine monophosphate (c-di-AMP), which is widely distributed among bacteria and is also found in several archaea. This cyclic nucleotide has been shown to involve in several important cellular processes, including maintenance of DNA integrity, cell wall metabolism, stress tolerance, transcription regulation and virulence. However, the mechanisms by which c-di-AMP modulates these physiological changes have remained largely unknown.In the present study, we identified and characterized a c-di-AMP synthase (CdaA) in S. mutans UA159. Furthermore, we investigated the role of CdaA in S. mutans cell physiology and global gene expression by utilizing cdaA gene in-frame deletion mutant. Our findings suggest that CdaA is an important global modulator of optimal growth and environmental adaption in this pathogen. Streptococcus mutans UA159 whole-genome arrays (8 x 15 K) were obtained from Agilent and included 1998 probes for S. mutans transcripts. For microarray analysis, S. mutans UA159 and S. mutans ?cdaA cells were routinely grown at 37°C anaerobically (90% N2, 5% CO2, 5% H2) in brain heart infusion broth (BHI; Difco, Sparks, MD, USA) to an optical density at 600 nm (OD600) of 0.5. Four RNA samples isolated from four independent cultures of UA159 and cdaA mutant strains were hybridized to the arrays and analyzed.

2015-04-06 | E-GEOD-65511 | 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.

2023-02-01 | GSE222218 | GEO

Repurposing the CRISPR-Cas9 system for targeted DNA methylation.

Project description:Epigenetic studies relied so far on correlations between epigenetic marks and gene expression pattern. Technologies developed for epigenome editing now enable direct study of functional relevance of precise epigenetic modifications and gene regulation. The reversible nature of epigenetic modifications, including DNA methylation, has been already exploited in cancer therapy for remodeling the aberrant epigenetic landscape. However, this was achieved non-selectively using epigenetic inhibitors. Epigenetic editing at specific loci represents a novel approach that might selectively and heritably alter gene expression. Here, we developed a CRISPR-Cas9-based tool for specific DNA methylation consisting of deactivated Cas9 (dCas9) nuclease and catalytic domain of the DNA methyltransferase DNMT3A targeted by co-expression of a guide RNA to any 20 bp DNA sequence followed by the NGG trinucleotide. We demonstrated targeted CpG methylation in a ∼35 bp wide region by the fusion protein. We also showed that multiple guide RNAs could target the dCas9-DNMT3A construct to multiple adjacent sites, which enabled methylation of a larger part of the promoter. DNA methylation activity was specific for the targeted region and heritable across mitotic divisions. Finally, we demonstrated that directed DNA methylation of a wider promoter region of the target loci IL6ST and BACH2 decreased their expression.

| S-EPMC4937303 | biostudies-literature

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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