Project description:CodY and GlnR are two major transcriptional regulators in nitrogen metabolism in Gram-positive bacteria. CodY regulates genes involved in the adaptive response to poor growth conditions, especially to nutrient limitation. GlnR controls nitrogen utilization according to the availability of nitrogen source. In this study we used microarray to investigate the regulatory roles that CodY and GlnR play in nitrogen metabolism in Streptococcus mutans.
Project description:CodY and GlnR are two major transcriptional regulators in nitrogen metabolism in Gram-positive bacteria. CodY regulates genes involved in the adaptive response to poor growth conditions, especially to nutrient limitation. GlnR controls nitrogen utilization according to the availability of nitrogen source. In this study we used microarray to investigate the regulatory roles that CodY and GlnR play in nitrogen metabolism in Streptococcus mutans. Streptococcus mutans UA159 wild-type cells, ΔcodY, ΔglnR, and ΔcodYglnR strains were grown in a chemically-defined medium until the mid-log phase. The nitrogen source was 1% tryptone. Twenty millimolar sodium thiosulfate was added to the medium to support cysteine biosynthesis. The transcriptional profile of the whole genome was examined with microarray.
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
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 For microarray analysis, cells were grown in complete FMC to an optical density at 600 nm (OD600) of 0.3 collected by centrifugation, quick frozen in a dry-ice/ethanol bath, and stored at - 80â??C for RNA isolation. RNA was isolated from exponential phase (OD600 0.3), as described previously. All samples were digested with DNase I (Ambion, Austin, TX) and purified using the RNeasy mini kit column (Qiagen, Inc., Chatsworth, CA). RNA concentrations were determined spectrophotometrically in triplicate and one µg of RNA was run in a formaldehyde gel to verify RNA quality. Reverse transcription (RT) reaction was performed with 10 mg of RNA following the protocol provided by TIGR with minor modifications. Transcriptome analysis was performed using the S. mutans UA159 microarrays provided by The Institute for Genomic Research (TIGR). The microarrays consisted of 1,948 70-mer oligonucleotides representing 1,960 open reading frames printed four times on the surface of each microarray slide. Additional details regarding the arrays are available at http://pfgrc.tigr.org/descriptions/S_mutans.shtml. A reference RNA prepared from a large-scale culture of S. mutans UA159 cells that had been grown in BHI broth to an OD600 = 0.5 was used in every experiment. cDNA labeling was performed according to TIGR protocols with minor modifications as described elsewhere. Four cDNA samples originating from four independent cultures of UA159 and JLcodY strains were hybridized to the arrays along with the reference cDNA, generating a total of 16 slides. Hybridizations were performed in a Maui hybridization chamber (BioMicro Systems, Salt Lake City, UT). Additional details regarding array protocols are available at http://pfgrc.tigr.org/protocols/protocols.shtml. Data from all individual experiments were analyzed using Spotfinder software and normalized using Midas according to TIGR specifications (http://www.tigr.org/software/). Statistical analysis was carried out using BRB Arrays Tools (http://linus.nci.nih.gov/BRB-ArrayTools.html) with a P-value of 0.001.
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.