Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

E.coli MG1655M-NM-^T6/pCA24N-dinJ treated with erythromycin vs. MG1655M-NM-^T6/pCA24N

ABSTRACT: Antitoxins are becoming recognized as proteins that regulate more than their own synthesis; for example, we found previously that antitoxin MqsA represses the gene encoding the stationary phase sigma factor RpoS. Here, we investigated the physiological role of antitoxin DinJ of the DinJ/YafQ toxin/antitoxin system and found DinJ also affects the general stress response by decreasing RpoS levels. Corroborating the reduced RpoS levels upon producing DinJ, catalase activity, cell adhesins, and cyclic diguanylate decreased while swimming increased. Using a transcriptome search and DNA-binding assays, we determined that the mechanism by which DinJ reduces RpoS is by repressing cspE which encodes cold-shock protein CspE that inhibits translation of rpoS mRNA. Hence, DinJ influences the general stress response indirectly by regulating cspE. strain: E.coli MG1655M-NM-^T6/pCA24N-dinJ vs MG1655M-NM-^T6/pCA24N treatment:erythromycin Medium:LB low salt Time:10 min Temp: 37M-BM-0C

ORGANISM(S): Escherichia coli

SUBMITTER: Thomas Wood

PROVIDER: E-GEOD-30692 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Antitoxin DinJ influences the general stress response through transcript stabilizer CspE.

Hu Ying Y Benedik Michael J MJ Wood Thomas K TK

Environmental microbiology 20111026 3

Antitoxins are becoming recognized as proteins that regulate more than their own synthesis; for example, we found previously that antitoxin MqsA of the Escherichia coli toxin/antitoxin (TA) pair MqsR/MqsA directly represses the gene encoding the stationary-phase sigma factor RpoS. Here, we investigated the physiological role of antitoxin DinJ of the YafQ/DinJ TA pair and found DinJ also affects the general stress response by decreasing RpoS levels. Corroborating the reduced RpoS levels upon prod ...[more]

PMID: 22026739

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Project description:Acetylation of lysine residues is conserved in all three kingdoms; however, its role in prokaryotes is unknown. Here we demonstrate that acetylation enables the reference bacterium Escherichia coli to withstand environmental stress. Specifically, the bacterium reaches higher cell densities and becomes more resistant to heat and oxidative stress when its proteins are acetylated, as shown by deletion of the gene encoding acetyltransferase YfiQ and the gene encoding deacetylase CobB, as well as by overproducing YfiQ and CobB. Furthermore, we show that the increase in oxidative stress resistance with acetylation is due to the induction of catalase activity through enhanced katG expression. We also found that two-component system proteins CpxA, PhoP, UvrY, and BasR are associated with cell catalase activity and may be responsible as the connection between bacterial acetylation and the stress response. This is the first demonstration of a specific environmental role of acetylation in prokaryotes. Overnight cultures of cobB/pCA24N-cobB and cobB/pCA24N were cultured to a turbidity of 0.05 at 600 nm and grown 2 h. Then, 0.1 mM IPTG was added for another 4 h to induce cobB expression, and then the cells were exposed to 20 mM H2O2 for 10 min. Cell pellets were collected and resuspended in RNAlater (Ambion Inc., Austin, TX), and total RNA was isolated using the RNeasy Mini Kit (Qiagen Inc., Valencia, CA). The E. coli GeneChip Genome 2.0 array (Affymetrix, P/N 900551) was used, and cDNA synthesis, fragmentation, and hybridizations were performed as described previously. If the gene with the larger transcription rate did not have a consistent transcription rate based on the 11-15 probe pairs (P-value less than 0.05), these genes were discarded. A gene was considered differentially expressed when the P-value for comparing two chips was lower than 0.05 (to assure that the change in gene expression was statistically significant and that false positives arise less than 5%) and if their fold change is higher than the standard deviation for the whole genome.

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.

Dataset Information

E.coli MG1655M-NM-^T6/pCA24N-dinJ treated with erythromycin vs. MG1655M-NM-^T6/pCA24N

Publications

Antitoxin DinJ influences the general stress response through transcript stabilizer CspE.

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