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Metabolomics analysis of multiple metabolic functions in the YjgF/YER057c/UK114 (Rid) protein family GCMS (part 2)

ABSTRACT: Metabolomics analysis of wild-type S. enterica, single RidA knock-out and triple Rid (ridA Rid2 Rid7) knock-out S.enterica cells

ORGANISM(S): Salmonella Escherichia Coli Salmonella Enterica Salmonella Enterica;escherichia Coli E. Coli

TISSUE(S): Bacterial Cells

SUBMITTER: Mona ElBadawi-Sidhu

PROVIDER: ST000118 | MetabolomicsWorkbench | Thu Nov 06 00:00:00 GMT 2014

REPOSITORIES: MetabolomicsWorkbench

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Project description:Purpose: Members of the broadly distributed Rid/YER057c/UK114 protein family have imine/enamine deaminase activity, notably on a 2-aminoacrylate (2AA). Strains of Salmonella enterica, and other organisms lacking RidA, have diverse growth phenotypes, attributed to the accumulation of 2AA. The goal of this study was to use transcriptional differences between S. enterica wild-type and ridA strains to explore the breadth of the cellular consequences that resulted from accumulation of 2AA. Methods: S. enterica LT2 mRNA profiles of wild-type (WT) and ridA mutant cells were generated by Next Generation Sequencing, in biological quadruplicate, using Illumina Nextseq (150 cycles) Mid Output Flowcell in the paired end mode with a read length of 75bp. The sequence reads that passed quality filters were analyzed by DEseq to call for statistically significant (FDR < 0.05) differential gene expression. qRT–PCR experiemnts were performed using SYBR Green assays to corroborate the differential expression for 9 of the genes identified as differentially expressed. Results: Pair-wise comparison of gene expression identified 413 genes with statistically significant changes (186 higher expression, and 227 lower expression) in transcript abundance between ridA mutant and wild-type strains (FDR < 0.05). Of these loci,when genes with no known function (STMxxxx and yxxX locus tags--120 genes) and genes showing less than a two-fold difference in expression were excluded, 84 genes remained which showed differential expression between strains. RNA-seq data had a linear relationship with qRT–PCR for four genes (napF, metH, dadX, and fliI) belonging to the 84 gene dataset, one gene (thiF) with an FDR < 0.05, but failing the two-fold cutoff threshold and one gene (sdaC) that did not meet the statistical cutoff (FDR > 005). Pearson's correlation coefficient (R2) for the relationship between RNAseq and qRT-PCR data was measured at 0.96. Analysis of the differentially expressed genes uncovered several areas of metabolism and physiology which may be affected by RidA elimination, and subsequent 2-aminoacrylate accumulation. Conclusions: Our study represents the first detailed analysis on the transcriptional effect RidA elimination has in S. enterica LT2, using biologic replicates, and generated by RNA-seq technology. Our results uncovered a motility effect associated with the accumulation of 2-aminoacrylate in a ridA mutant as well as identified additional areas of metabolism impacted by the metabolic stress associated with 2AA accumulation. These data also emphasized the value of integrating global approaches with biochemical genetic approaches to understand the complex system of microbial metabolism.

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Metabolomics analysis of multiple metabolic functions in the YjgF/YER057c/UK114 (Rid) protein family GCMS (part 2)

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