ABSTRACT: Toxin-antitoxin (TA) systems are ubiquitous throughout bacterial and archaeal genomes. TA systems consist of a stable toxin that inhibits growth and a labile antitoxin that prevents toxicity of the toxin. Here we made an artificial TA system (arT/arA) and performed a DNA microarray study for overproduction of the toxin. arT was overexpressed in Escherichia coli BW25113 and compared to the empty vector.
Project description:MqsR/MqsA is a well-characterized toxin/antitoxin system with several regulatory roles. Deletion of mqsRA reduced growth with deoxycholate stress. Here we performed a microarray to determine the expression profile with and without mqsRA under deoxycholate stress to determine transcriptional regulation. Escherichia coli K-12 BW25113 with and without mqsRA was exposed to deoxycholate stress.
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 °C, adjusted the turbidity to 1, and exposed to 20 μg/mL ampicillin with 1 mM IPTG for 1 h. Cells were isolated by centrifuging at 0°C, and RNALater® 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 45°C with 60 rpm for 16 hours. The probe array was washed, stained using Affymetrix Genechip Fluidics Station 450 and the software GenomeChipOperating Software (GCOS)”, 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.
Project description:Quorum sensing controls the expression of multiple virulence factors. PA14 genes lasR and rhlR are necessary for quorum sensing via homoserine lactones. A PA14 lasR rhlR deficient mutant exhibits a reduced oxidative stress response. Here we conducted a microarray to determine oxidative stress response gene regulation mediated by the homoserine lactone quorum sensing circuits. A PA14 lasR rhlR deficient mutant was compared to the wild-type with and without H2O2 stress.
Project description:Persister cells are a sub-population of all bacterial cultures which exhibit a non-inheritable, multi-drug tolerance when subjected to lethal antibiotic challenge. These persisters arise as a result of metabolic dormancy, and can resume growth subsequent to antibiotic challenge, leading to recalcitrance of bacterial infections. Overproduction of DosP, an oxygen sensing protein with phosphodiesterase activity, increases bacterial persistence. Here we performed a microarray to determine the expression profile induced by DosP as a means to elucidate mechanisms of persister cell formation. dosP was overexpressed in Escherichia coli K-12 BW25113 and compared to the empty vector.
Project description:The previously uncharacterized proteins HigB (unannotated) and HigA (PA4674) of Pseudomonas aeruginosa PA14 were found to form a type II TA system in which antitoxin HigA masks the RNase activity of toxin HigB through direct binding. To determine the physiological role of HigB/HigA in P. aeruginosa, a whole-transcriptome experiment was performed for the higA antitoxin deletion mutant of the PA14 strain compared to the wild-type PA14 strain. The rationale was that for the strain that lacks the antitoxin, the effect of the toxin could be discerned due to enhanced activity of the toxin. Furthermore, toxin HigB reduces production of the virulence factors pyochelin, pyocyanin, swarming, and biofilm formation. Overall design: The higA mutant of Pseudomonas aeruginosa PA14 compared to the P. aeruginosa PA14 wild-type
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. Overall design: Strains: E. coli K-12 MG1655 and E. coli K-12 LVM100 delta 5 TA mutant Medium: LB Cells: biofilm cells on glass wool Time: 15 h Temperature: 37oC
Project description:Pathogenic biofilms have been associated with persistent infections due to high resistance to antimicrobial agents while commensal biofilms often fortify host immune system. Hence, controlling biofilm formation of both pathogenic bacteria and commensal bacteria is important in bacteria-related diseases. We investigated the effect of plant flavonoids on biofilm formation of both enterohemorrhagic Escherichia coli O157:H7 and three commensal E. coli K-12 strains. Phloretin abundant in apples markedly reduced E. coli O157:H7 biofilm formation without affecting the growth of planktonic cells while phloretin did not harm commensal E. coli K-12 biofilms. Also, phloretin reduced E. coli O157:H7 attachment to human colon epithelial cells. Global transcriptome analyses revealed that phloretin repressed toxin genes (hlyE and stx2), autoinducer-2 importer genes (lsrACDBF), a curli gene (csgA), and a dozens of prophage genes in E. coli O157:H7 cells. Electron microscopy confirmed that phroretin reduced the curli production in E. coli O157:H7. In addition, phloretin suppressed TNF-α-induced inflammatory response in vitro using human colonic epithelial cells. Moreover, in the trinitrobenzene sulfonic acid (TNBS)-induced rat colitis model, phloretin significantly ameliorated colon inflammation and body weight loss. Taken together, our results suggest that phloretin may act as an inhibitor of E. coli O157:H7 biofilm formation as well as anti-inflammatory agent on inflammatory bowel diseases while leaving beneficial commensal E. coli biofilm intact. For the microarray experiments, E. coli O157:H7 EDL933 was inoculated in 25 ml of LB in 250 ml flasks with overnight cultures that were diluted at 1:100. Cells were shaken at 100 rpm and 37°C for 7 hrs. Cells were immediately chilled with dry ice and 95% ethanol (to prevent RNA degradation) for 30 sec before centrifugation in 50 ml centrifuge tubes at 13,000 g for 2 min; cell pellets were frozen immediately with dry ice and stored -80°C. RNA was isolated using Qiagen RNeasy mini Kit (Valencia, CA, USA). To eliminate DNA contamination, Qiagen RNase-free DNase I was used to digest DNA. RNA quality was assessed by Agilent 2100 bioanalyser using the RNA 6000 Nano Chip (Agilent Technologies, Amstelveen, The Netherlands), and quantity was determined by ND-1000 Spectrophotometer (NanoDrop Technologies, Inc., DE, USA).
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
Project description:An antivirulence approach targets bacterial virulence rather than cell viability in the antibiotic approach that can readily lead to drug resistance. Opportunistic human pathogen Pseudomonas aeruginosa produces a variety of virulence factors, and biofilm cells of this bacterium are much more resistant to antibiotics than planktonic cells. To identify novel inorganic antivirulence compounds, the dual screenings of thirty-six metal ions were performed to identify that zinc ions and ZnO nanoparticle inhibited the pyocyanin production and biofilm formation in P. aeruginosa without affecting the growth of planktonic cells. Moreover, zinc ion and ZnO nanoparticle markedly reduced the production of 2-heptyl-3-hydroxy-4(1H)-quinolone and siderophore pyochelin, while increased the production of another sideropore pyoverdine and swarming motility. Further, zinc ion and ZnO nanoparticle clearly suppressed hemolytic activity in P. aeruginosa. Transcriptome analyses showed that ZnO nanoparticle induced zinc cation efflux pump czc operon, porin genes (oprD and opdT), and Pseudomonas type III repressor A ptrA, while repressed pyocyanin-related phz operon, which partially explains the phenotypic changes. Overall, ZnO nanoparticle is a potential candidate for use in an antivirulence approach against persistent P. aeruginosa infection. P. aeruginosa Genechip Genome Array (Affymetrix, P/N 900339) was used in order to study the cells after the addition of ZnO nanoparticles. DNA microarray analysis with one biological replicate was performed with an Affymetrix system. P. aeruginosa was inoculated in 25 ml of LB medium in 250 ml shaker flasks with overnight cultures (1 : 100 dilution). Cells were cultured for 5 h with shaking at 250 rpm with and without ZnO nanoparticles (1 mM). Before sample collection, RNase inhibitor (RNAlater, Ambion, TX, USA) was added, and the cells were immediately chilled with dry ice and 95% ethanol (to prevent RNA degradation) for 30 s before centrifugation at 16,000 g for 2 min. The cell pellets were immediately frozen with dry ice and stored at –80°C. Total RNA was isolated using a Qiagen RNeasy mini Kit (Valencia, CA, USA).