Dataset Information

E. coli MqsR and MqsA whole DNA microarray and nickel DNA enrichment microarray

ABSTRACT: Here evidence is presented that MqsR and B3021 are a novel toxin-antitoxin (TA) system related to biofilm development and quorum sensing. Using whole-transcriptome studies and nickel enrichment DNA binding microarrays coupled with cell survival studies in which MqsR was overexpressed in isogenic mutants, we identified seven genes involved in MqsR toxicity (clpX, clpP, yfjZ, cspD, relB, relE, and hokA). Quantitative real-time polymerase chain reaction (qRT-PCR) confirmed consistent induction of the seven genes by the overexpression of MqsR as well as induction of nutrient starvation related genes (cstA, rpoS, and dps). Taken together, our results indicate that MqsR toxicity is caused via CspD (a DNA replication inhibitor), other TA systems (RelE/RelB and YfjZ), HokA (a small membrane toxin peptide), and nutrient-starvation conditions induced via CstA, RpoS, and Dps. Additionally, in vivo binding results show antitoxin B3021 binds to the promoter regions of genes encoding essential proteins for stress, growth and normal physiology.

ORGANISM(S): Escherichia coli K-12 Escherichia coli

PROVIDER: GSE14203 | GEO | 2009/11/27

SECONDARY ACCESSION(S): PRJNA111327

REPOSITORIES: GEO

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Project description:Persisters are a subpopulation of metabolically-dormant cells in biofilms that are resistant to antibiotics; hence, understanding persister cell formation is important for controlling bacterial infections. Previously we discerned that MqsR and MqsA of Escherichia coli are a toxin/antitoxin pair that influence persister cell production via their regulation of Hha, CspD, and HokA. Here, to gain more insights into the origin of persisters, we used protein engineering to increase the toxicity of toxin MqsR by reasoning it would be easier to understand the effect of this toxin if it were more toxic. We found that two mutations (K3N and N31Y) increase the toxicity four fold and increase persistence 73 fold compared to native MqsR by making the protein less labile. A whole transcriptome study revealed that the MqsR variant represses acid resistance genes (gadABCEWX and hdeABD), multidrug resistance genes (mdtEF), and osmotic resistance genes (osmEY). Corroborating these microarray results, deletion of rpoS as well as the genes that the master stress response regulator RpoS controls, gadB, gadX, mdtF, and osmY, increased persister formation dramatically to the extent that nearly the whole population became persistent. Therefore, the more toxic MqsR increases persistence by decreasing the ability of the cell to respond to antibiotic stress through its RpoS-based regulation of acid resistance, multidrug resistance, and osmotic resistance systems. For the whole-transcriptome study of BW25113 mqsR/pBS(Kan)-mqsR 2-1 versus BW25113 mqsR/pBS(Kan)-mqsR, planktonic cells were grown to a turbidity of 0.5 at 600 nm in LB medium with 1 mM IPTG at 37 M-BM-0C, adjusted the turbidity to 1, and exposed to 20 M-NM-<g/mL ampicillin with 1 mM IPTG for 1 h. Cells were isolated by centrifuging at 0M-BM-0C, and RNALaterM-BM-. buffer (Ambion, Cat# AM7021) was added to stabilize RNA during the RNA preparation steps. Total RNA was isolated from cell pellets with Qiagen RNeasy mini Kit (Cat# 74104) using a bead beater. cDNA was synthesized and fragmented to obtain 50-200 base cDNA fragments. The fragmented cDNA was labelled with Biotin-ddUTP, and hybridization was performed at 45M-BM-0C with 60 rpm for 16 hours. The probe array was washed, stained using Affymetrix Genechip Fluidics Station 450 and the software GenomeChipOperating Software (GCOS)M-bM-^@M-^], and scanned using Affymetrix Genechip scanner GCS3000 7G system and the software GenomeChipOperating Software (GCOS). Data were processed with MAS 5.0 Expression Analysis Default Setting. Genes were identified as differentially expressed if the expression ratio was higher than the standard deviation: 4.0 fold (enriched and differentially degraded) cutoff for the DNA microarrays (standard deviation 1.3 fold), and if the p-value for comparing two chips was less than 0.05.