Transcription profiling of E. coli MqsR and MqsA whole DNA microarray and nickel DNA enrichment microarray
Ontology highlight
ABSTRACT:
ORGANISM(S): Escherichia coli
SUBMITTER: Younghoon Kim
PROVIDER: E-GEOD-14203 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description: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.
2009-11-27 | GSE14203 | GEO
Project description:Persisters are cells which evade stresses like antibiotics and which are characterized by reduced metabolism and a lack of genetic alterations required to achieve this state. We showed previously that MqsR and MqsA of Escherichia coli are a toxin-antitoxin pair that influence cell physiology (e.g., biofilm formation and motility) via RNase activity as well as through regulation of toxin CspD. Here, we show that deletion of the mqsRA locus decreases persister cell formation and, consistent with this result, overexpression of MqsR increases persister cell formation. Furthermore, toxins Hha, CspD, and HokA increase persister cell formation. In addition, by overproducing MqsR in a series of isogenic mutants, we show that Hha and CspD are necessary for persister cell formation via MqsR overexpression. Surprisingly, Hfq, a small RNA chaperone, decreases persistence. A whole-transcriptome study shows that Hfq induces transport-related genes (oppA, oppB, oppC, oppD, oppF, and dppA), outer membrane protein-related genes (ybfM and ybfN), toxins (hha), and proteases (clpX, clpP, and lon). Taken together, these results indicate that toxins CspD and Hha influence persister cell formation via MqsR and that Hfq plays an important role in the regulation of persister cell formation via regulation of transport or outer membrane proteins. Strains: E. coli BW25113 K-12 hfq deleted mutant vs. wild-type Medium: LB Culture: Planktonic cell grown OD=0.5, adjusted OD=1.0, and then exposed to 100 ug/mL ampicillin for 2 h.
2009-11-07 | E-GEOD-18852 | biostudies-arrayexpress
Project description:Persisters are cells which evade stresses like antibiotics and which are characterized by reduced metabolism and a lack of genetic alterations required to achieve this state. We showed previously that MqsR and MqsA of Escherichia coli are a toxin-antitoxin pair that influence cell physiology (e.g., biofilm formation and motility) via RNase activity as well as through regulation of toxin CspD. Here, we show that deletion of the mqsRA locus decreases persister cell formation and, consistent with this result, overexpression of MqsR increases persister cell formation. Furthermore, toxins Hha, CspD, and HokA increase persister cell formation. In addition, by overproducing MqsR in a series of isogenic mutants, we show that Hha and CspD are necessary for persister cell formation via MqsR overexpression. Surprisingly, Hfq, a small RNA chaperone, decreases persistence. A whole-transcriptome study shows that Hfq induces transport-related genes (oppA, oppB, oppC, oppD, oppF, and dppA), outer membrane protein-related genes (ybfM and ybfN), toxins (hha), and proteases (clpX, clpP, and lon). Taken together, these results indicate that toxins CspD and Hha influence persister cell formation via MqsR and that Hfq plays an important role in the regulation of persister cell formation via regulation of transport or outer membrane proteins.
2009-11-08 | GSE18852 | GEO
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.
2012-04-02 | E-GEOD-31054 | biostudies-arrayexpress
Project description:The role of six toxin-antitoxin (TA) systems on biofilm development was investigated (MazEF, RelBEF, ChpB, YefM-YoeB, DinJ-YafQ, and TomB-Hha). Although these TA systems were reported previously to not impact bacterial fitness, we found that biofilm formation is decreased by toxins and increased by anti-toxins, in part, through YjgK. Hence, one role of TA systems is to regulate biofilm formation. Experiment Overall Design: Strains: E. coli K-12 MG1655 and E. coli K-12 LVM100 delta 5 TA mutant Experiment Overall Design: Medium: LB Experiment Overall Design: Cells: biofilm cells on glass wool Experiment Overall Design: Time: 15 h Experiment Overall Design: Temperature: 37oC
2009-02-09 | E-GEOD-11932 | biostudies-arrayexpress
Project description:The role of six toxin-antitoxin (TA) systems on biofilm development was investigated (MazEF, RelBEF, ChpB, YefM-YoeB, DinJ-YafQ, and TomB-Hha). Although these TA systems were reported previously to not impact bacterial fitness, we found that biofilm formation is decreased by toxins and increased by anti-toxins, in part, through YjgK. Hence, one role of TA systems is to regulate biofilm formation. Experiment Overall Design: Strains: E. coli K-12 BW25113 wild-type and E. coli BW25113 yjgK deleted mutant Experiment Overall Design: Medium: LB Experiment Overall Design: Cells: biofilm cells on glass wool Experiment Overall Design: Time: 8 h Experiment Overall Design: Temperature: 37oC
2009-02-09 | E-GEOD-12701 | biostudies-arrayexpress
Project description:E. coli K-12 BW25113 mutant strain yncC expression in biofilm cells relative to E. coli wild-type strain expression in biofilm cells. All samples were cultured in LB with glasswool at 37C for 15 hours and E. coli K-12 MG1655 mutant yncC colony cells vs wild type colony cells in LB plates 15h 37C. Quorum-sensing signal autoinducer 2 (AI-2) stimulates Escherichia coli biofilm formation through the motility regulator MqsR that induces expression of the putative transcription factor encoded by yncC. Here we show YncC increases biofilm formation by decreasing mucoidy (corroborated by decreased exopolysaccharide production and increased sensitivity to bacteriophage P1 infection). Differential gene expression and gel shift assays demonstrated that YncC is a repressor of the predicted periplasmic protein-encoding gene ybiM which was corroborated by the isogenic yncC ybiM double mutation which repressed the yncC phenotypes (biofilm formation, mucoidy, and bacteriophage resistance). Through nickel-enrichment microarrays and additional gel shift assays, we found that the putative transcription factor B3023 (directly upstream of mqsR) binds the yncC promoter. Overexpressing MqsR, AI-2 import regulators LsrR/LsrK, and AI-2 exporter TqsA induced yncC transcription whereas the AI-2 synthase LuxS and B3023 repressed yncC. MqsR has a toxic effect on E. coli bacterial growth which is partially reduced by the b3023 mutation. Therefore, AI-2 quorum-sensing control of biofilm formation is mediated through regulator MqsR that induces expression of the transcription factor YncC which serves to inhibit the expression of periplasmic YbiM; this inhibition of YbiM prevents it from overexpressing exopolysaccharide (causing mucoidy) and prevents YbiM from inhibiting biofilm formation. Experiment Overall Design: Strains: E. coli K-12 BW25113 wild-type, mutant yncC Experiment Overall Design: and E. coli K-12 MG1655 wild-type, mutant yncC Experiment Overall Design: Medium: LB Experiment Overall Design: Biofilm grown on glasswool or colony cells growth in LB plates Experiment Overall Design: Time: 15 hours Experiment Overall Design: Temperature: 37C Experiment Overall Design: Cell type: biofilm or colony Experiment Overall Design: For the biofilm arrays in BW25113 background: Experiment Overall Design: Overnight cultures (16 h, 2.5 mL) of wild type E. coli BW25113 and BW25113 yncC in LB and LB with kanamycin (50 μg/mL), respectively, were used to inoculate 250 mL LB with 10 g of glass wool (Corning Glass Works, Corning, NY) for forming biofilm. After incubating at 37°C for 15 h with shaking (250 rpm), biofilm cells were prepared by rinsing and sonicating the glass wool in sterile 0.85% NaCl solution at 0°C as described before. The total RNA was isolated from biofilm cells as described previously. Experiment Overall Design: The E. coli Genechip antisense genome array (P/N 900381, Affymetrix, Santa Clara, CA) containing probes for more than 4200 open reading frames (ORFs) was used to analyze the complete E. coli transcriptome as described previously. Hybridizations were performed for 16 h and the total cell intensity was scaled automatically in the software to an average value of 630. The Gene Expression Technical Manual (Affymetrix) was followed for the procedures of DNA microarrays, and the GeneChip operating software (Affymetrix) was applied to analyze data of DNA microarrays. The data quality was assessed following the manufacturer's guidelines (GeneChip Expression Analysis: Data Analysis Fundamentals; Affymetrix) and also was based on the expected signals of E. coli BW25113 and the yncC mutant genotypes (e.g., signals of the deleted genes, araA and rhaA, were low for both BW25113 and BW25113 yncC, while the signal of yncC was low for the yncC mutant). A gene was identified as differentially-expressed when the P value based on the False Discovery Rate Method was less than 0.05 and the expression ratio was greater than threefold since the standard deviation for the expression ratio for all of genes was 2. Experiment Overall Design: for the colony arrays in MG1655 background: Experiment Overall Design: For the agar biofilm, fresh single colonies of wild-type E. coli MG1655 and MG1655 yncC were re-streaked on LB agar plates, incubated, and about 0.05 g of the colony cultures on LB plate were quickly transferred to 2-mL collection tubes. The total RNA was isolated from these colony cells as described previously. The E. coli GeneChip Genome 2.0 Array (Affymetrix, P/N 900551, Santa Clara, CA) containing 10,208 probe sets for open reading frames, rRNA, tRNA, and intergenic regions for four E. coli strains: MG1655, CFT073, O157:H7-Sakai, and O157:H7-EDL933, was used to analyze the complete E. coli transcriptome. A gene was identified as differentially-expressed when the P value based on the False Discovery Rate Method (Benjamini & Hochberg, 1995) was less than 0.05 and the expression ratio was greater than threefold since the standard deviation for the expression ratio for all of MG1655 genes (except the deleted yncC gene) was 2.
2008-04-07 | E-GEOD-8706 | biostudies-arrayexpress
Project description:Bacterial toxin-antitoxin systems are thought to help bacteria to reduce their metabolism in various stressful conditions. Many toxins of the bacterial toxin-antitoxin systems are ribonucleases. Such toxins have been mostly viewed as degraders of mRNA, however recently it was demonstrated that they are also capable of cleaving non-coding RNA. MazF toxin is also hypothesized to reprogram E. colis translational machinery. Current cDNA libraries are part of a project which aims to identify the major RNA cleavage sites (in mRNA, rRNA and regulatory non-coding RNA) of MazF and MqsR toxins in E. coli. We wanted to elaborate on the roles of MazF and MqsR in E. coli: is MazF really involved in reprogramming the translational machinery or is MazF, along with MqsR, just a robust cleaver of RNA? We extracted total RNA from cultures where the expression of MazF or MqsR was induced for 2h and from cultures that had been recovering from toxin production for 30 minutes; RNA from log phase culture was used as the control. We used strand specific random primed paired end RNA sequencing data to locate the major cleavage sites. To map the cleavage sites, we counted 5’ end stacks of forward reads in each genomic position and compared them with total coverage. MazF and MqsR cleave at specific recognition sequences, ˇACA and GˇC respectively, which allowed us to eliminate the false positives. One of our initial aims was also to look for irregular RNA ligation events following toxin expression, thus we used long, 300 base reads in sequencing.
2018-10-11 | E-MTAB-6993 | biostudies-arrayexpress
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.
2012-04-03 | GSE31054 | GEO
Project description:The features of Mycoplasma in human organ such lung and urinary tract are enigmatic. Here, the role of M. hominis in regard to biofilm formation of uropathogenic Escherichia coli (UPEC) strain CFT073 was investigated. Although M. hominis were inferred to not impact on UPEC bacterial fitness including growth and productions of signaling molecules as autoinducer-2 (AI-2) and indole, we found that the presence of M. hominis dramatically decreased biofilm formation of UPEC CFT073 as well as slightly repressed attachment and cytotoxicity of that. Importantly, this activity was observed on UPEC strain specifically, not enterohemorrhagic E. coli (EHEC) strain that exists on intestine. Whole-transcriptome profiling and quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed PhoPQ system and anti-termination protein (encoded by ybcQ) participates on the reduction of biofilm formation by M. hominis (corroborated by qRT-PCR). Furthermore, collaborating with previous report that toxin-antitoxin (TA) system involved in biofilm formation, M. hominis increased on the transcriptions of toxin genes including hha (toxin gene in Hha-TomB TA system) and pasT (toxin part in PasT-PasI TA system). Hence, we propose that one possible role of M. hominis is to influence bacterial biofilm formation in urinary tract. Only fourteen genes were induced (2.5-fold) by the presence of M. hominis in Uropathogenic Escherichia coli (UPEC) biofilm cells. Among upregulated genes, ybcQ (encodes anti-termination protein Q homolog) and phoP/phoQ (encode DNA-binding response regulators in two-component regulatory system), were induced by the presence of M. hominis. Two-condition experiment, UPEC CFT073 alone vs. UPEC CFT073 with Mycoplasma hominis PG21 (10^5 ccu/ml). For preparing the total RNA, UPEC CFT073 cells were grown at 37°C in biofilm cells on glass wool with or without M. hominis for 24 h.
2016-01-08 | E-GEOD-43376 | biostudies-arrayexpress