Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Comparison of gene expression in Staphylococcus aureus strain SH1000 wildtype vs mepR

ABSTRACT: MepR is a substrate-responsive repressor of mepR and mepA, which encode itself and a MATE family multidrug efflux pump. Microarray analyses of Staphylococcus aureus SH1000 and its mepR-disrupted derivative revealed changes in expression of many genes in addition to mepR and mepA, notably several involved in virulence Keywords: Staphylococcus aureus, MATE efflux pump, MepR Staphylococcus aureus strains SH1000 wildtype and mepR were grown in duplicate to exponential and post-exponential phase (corresponding to an A550 nm of 0/4 and 2.0 respectively). RNA was harvested, converted to cDNA, labelled with Biotin and used to probe custom-designed Affymetrix antisense S.aureus GeneChips. Eight samples in total were prepared and analyzed.

ORGANISM(S): Staphylococcus aureus

SUBMITTER: Carmen DeMarco

PROVIDER: E-GEOD-10033 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The GntR-like protein NorG has been shown to affect Staphylococcus aureus genes involved in the resistance to quinolones and beta-lactams such as those encoding the NorB and AbcA transporters. To identify the target genes regulated by NorG, we carried out transcriptional profiling assays using S. aureus RN6390 and its isogenic norG::cat mutant. Our data showed that NorG positively affected the transcription of global regulators mgrA, arlS, and sarZ. The three putative drug efflux pump genes most positively affected by NorG were the NorB efflux pump (5.1-fold), the MmpL-like protein SACOL2566 (5.2-fold), and the BcrA-like drug transporter SACOL2525 (5.7-fold). The S. aureus predicted MmpL protein showed 53% homology with the MmpL lipid transporter of Mycobacterium tuberculosis, and the putative SACOL2525 protein showed 87% homology with the bacitracin drug transporter BcrA of Staphylococcus hominis. Two pump genes most negatively affected by NorG were NorC (4-fold) and AbcA (6-fold). Other categories of genes such as those participating in amino acid, inorganic ion, or nucleotide transporters and metabolism, were also affected by NorG. Real-time RT-PCR assays for mgrA, arlS, sarZ, norB, norC, abcA, mmpL, and bcrA-like were carried out to verify microarray data and showed the same level of up- or down regulation by NorG. The norG mutant showed a twofold increase in the resistance to norfloxacin and rhodamine, both substrates of the NorC transporter, which is consistent with the resistance phenotype conferred by overexpression of norC on a plasmid. These data indicate that NorG has broad regulatory function in S. aureus. The goal of this study was to define the transcriptional response of S. aureus norG mutant cells. To do so, commercially available S. aureus Affymetrix GeneChips (Santa Clara, CA) were used to compare the expression properties of wild type (ISP794) and isogenic norG (QT11) mutant cells. S. aureus strains ISP794 and QT11 were grown to mid exponential phase growth, lysed , total bacterial RNA was extracted, labeled and hybridized to commerically available S. aureus Affymetrix GeneChips. In total three biological replicates were compared for each strain background.

Project description:Isoprenoids are a class of ubiquitous organic molecules synthesized from the five-carbon starter unit isopentenyl pyrophosphate (IPP). Comprising more than 30,000 known natural products, isoprenoids serve various important biological functions in many organisms. In bacteria, undecaprenyl pyrophosphate is absolutely required for the formation of cell wall peptidoglycan and other cell surface structures, while ubiquinones and menaquinones, both containing an essential prenyl moiety, are key electron carriers in respiratory energy generation. There is scant knowledge on the nature and regulation of bacterial isoprenoid pathways. In order to explore the cellular responses to perturbations in the mevalonate pathway, responsible for producing the isoprenoid precursor IPP in many Gram-positive bacteria and eukaryotes, we constructed three strains of Staphylococcus aureus in which each of the mevalonate pathway genes is regulated by an IPTG inducible promoter. We used DNA microarrays to profile the transcriptional effects of downregulating the components of the mevalonate pathway in S. aureus and demonstrate that decreased expression of the mevalonate pathway leads to widespread downregulation of primary metabolism genes, an upregulation in virulence factors and cell wall biosynthetic determinants, and surprisingly little compensatory expression in other isoprenoid biosynthetic genes. We subsequently correlate these transcriptional changes with downstream metabolic consequences. We used microarrays to profile the transcriptional changes incurred when downregulating mvaS, mvaA, or mvaK in S. aureus. Keywords: stress response S. aureus mutants were generated in which mvaS, mvaA, or mvaK were placed under control of the IPTG-inducible Pspac promoter. These mutants, as well as wildtype RN4220 S. aureus, were grown with or without 1 mM IPTG, harvested in exponential phase, and their total RNA was extracted and hybridized to Affymetrix GeneChips. Experiments were done in triplicate.

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Comparison of gene expression in Staphylococcus aureus strain SH1000 wildtype vs mepR

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