Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Comparison of gene expression in MGAS315 and its isogenc mtsR mutant

ABSTRACT: RATIONALE: mtsR is a regulator of genes involved in ion aquisition in S. pyogenes that has been suggested to be involved in development of invasive infections. We wanted to identify mtsR regulon. EXPERIMENT: We compared global gene expression in wild type strain MGAS315 with expression in its isogenc mtsR mutant. Experiment was performed with bacteria in exponential (EXP) and at late exponential phase (Late EXP) grown in THY medium Keywords: strain comparison 1. Three independent cultures of WT or mutant strain bacteria at exponential phase of growth (OD60=0.5, biological replicates) served as source of total RNA. RNA was transcribed into cDNA, fragmented and labeled using standard Affymetrix protocol for prokaryotic target preparation. Hybridization with custom Affymetrix array and scaning was performed using protocol recomended by manufacturer. Hybridizaton values aquired by GCOS (in CHP files) are normalized to global hybridization intesity. 2. Analogous experiment was performed with bacteria grown to OD600=1.2 representing late exponential growth phase

ORGANISM(S): Streptococcus pyogenes

SUBMITTER: Izabela Sitkiewicz

PROVIDER: E-GEOD-10252 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Decreased necrotizing fasciitis capacity caused by a single nucleotide mutation that alters a multiple gene virulence axis.

Olsen Randall J RJ Sitkiewicz Izabela I Ayeras Ara A AA Gonulal Vedia E VE Cantu Concepcion C Beres Stephen B SB Green Nicole M NM Lei Benfang B Humbird Tammy T Greaver Jamieson J Chang Ellen E Ragasa Willie P WP Montgomery Charles A CA Cartwright Joiner J McGeer Allison A Low Donald E DE Whitney Adeline R AR Cagle Philip T PT Blasdel Terry L TL DeLeo Frank R FR Musser James M JM

Proceedings of the National Academy of Sciences of the United States of America 20100104 2

Single-nucleotide changes are the most common cause of natural genetic variation among members of the same species, but there is remarkably little information bearing on how they alter bacterial virulence. We recently discovered a single-nucleotide mutation in the group A Streptococcus genome that is epidemiologically associated with decreased human necrotizing fasciitis ("flesh-eating disease"). Working from this clinical observation, we find that wild-type mtsR function is required for group A ...[more]

PMID: 20080771

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Project description:We used RNAseq to identify how membrane fatty acid changes might impact and explain the virulence defect during host infection. WT and mFabT expression was compared in THY, and in THY-Tween (as C18:1Δ9 source), which to activates WT FabT repression. RNA isolation and Illumina RNA-seq sequencing: GAS strains were cultured at 37°C in THY or THY-Tween, and cells were harvested during exponential growth (OD600 between 0.4 and 0.5). Independent triplicate cultures were prepared for each condition. For RNA preparation, 2 volumes of RNA protect (Qiagen) was added to cultures prior centrifugation (10 min 12,000 g) and total RNA was extracted after lysing bacteria by a 30 min 15 mg.ml-1 lysozyme, 300 U.ml-1 mutanolysin treatment at 20°C followed by two cycles of Fast-prep (power 6, 30 s) at 4 °C. RNA extraction (Macherey-Nagel RNA extraction kit; Germany) was done according to supplier instructions. RNA integrity was analyzed using an Agilent Bioanalyzer (Agilent Biotechnologies, Ca., USA). 23S and 16S rRNA were depleted from the samples using the MICROBExpress Bacterial mRNA enrichment kit (Invitrogen, France); depletion was controlled on Agilent Bioanalyzer (Agilent Biotechnologies). Libraries were prepared using an Illumina TS kit. Libraries were sequenced generating 10,000,000 to 20,000,000 75-bp-long reads per sample. RNA-Seq data analysis: The MGAS6180 strain sequence (NCBI), which is nearly identical to M28PF1, was used as a reference sequence to map sequencing reads using the STAR software (2.5.2b) BIOCONDA (Anaconda Inc). RNA-seq data were analyzed using the hclust function and a principal component analysis in R 3.5.1 (version 2018-07-02). For differential expression analysis, normalization and statistical analyses were performed using the SARTools package and DESeq2 p-values were calculated and adjusted for multiple testing using the false discovery rate controlling procedure. We used UpsetR to visualize set intersections in a matrix layout comprising the mFabT versus the WT strain grown in THY and in THY-Tween, and growth in THY-Tween versus THY for each strain.

Dataset Information

Comparison of gene expression in MGAS315 and its isogenc mtsR mutant

Publications

Decreased necrotizing fasciitis capacity caused by a single nucleotide mutation that alters a multiple gene virulence axis.

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