Transcription profiling of E. coli K-12 BW25113 wt and E. coli K-12 BW25113 yjgK deleted mutant on 8 h biofilm cell development
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ABSTRACT:
ORGANISM(S): Escherichia coli
SUBMITTER: Younghoon Kim
PROVIDER: E-GEOD-12701 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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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 global transcriptional regulator Hha of Escherichia coli controls hemolysin activity, biofilm formation, and virulence expressions. Earlier, we have reported that Hha represses initial biofilm formation and disperses biofilms as well as controls prophage excision in E. coli. Since biofilm dispersal is a promising area to control biofilms, here we rewired Hha to control biofilm dispersal and formation. The Hha variant Hha13D6 was obtained to have enhanced biofilm dispersal activity along with increased toxicity compared to wild-type Hha (Hha13D6 induces dispersal 60%, whereas wild-type Hha induces dispersal at early biofilms but not at mature biofilms). Toxic Hha13D6 caused cell death probably by the activation of proteases HslUV, Lon, and PrlC, and deletion of protease gene hslV with overproducing Hh13D6 repressed biofilm dispersal, indicating Hha13D6 induces biofilm dispersal through the activity of protease HslV. Furthermore, another Hha variant Hha24E9 was also obtained to decrease biofilm formation 4-fold compared to wild-type Hha by regulation of gadW, glpT, and phnF. However, the dispersal variant Hha13D6 did not decrease biofilm formation, while the biofilm variant Hha24E9 did not induce biofilm dispersal. Hence, Hha may have evolved two ways in response to environmental factors to control biofilm dispersal and formation, but both controlling mechanisms come from different regulatory systems. For the whole-transcriptome study of BW25113 hha/pCA24N-hha13D6 versus BW25113 hha/pCA24N-hha biofilm dispersal, cells were grown in 250 mL of LB glucose (0.2%) for 16 h at 125 rpm with 10 g of glass wool (Corning Glass Works, Corning, NY, USA) in 1 L Erlenmeyer flasks to form a robust biofilm (Ren et al., 2004) and incubated an additional 1 h with 1 mM IPTG to induce wild-type Hha and Hha13D6. Similarly, for the whole transcriptome study of BW25113 hha/pCA24N-hha24E9 versus BW25113 hha/pCA24N-hha biofilm formation, cells were grown in 250 mL of LB glucose (0.2%) containing 1 mM IPTG for 7 h at 250 rpm with 10 g of glass wool to form biofilms. Biofilm cells were obtained by rinsing and sonicating the glass wool in sterile 0.85% NaCl solution at 0°C, and RNALater buffer® (Applied Biosystems, Foster City, CA, USA) was added to stabilize RNA during the RNA preparation steps. Total RNA was isolated from biofilm cells using a bead beater (Biospec, Bartlesville, OK, USA). cDNA synthesis, fragmentation, and hybridizations to the E. coli GeneChip Genome 2.0 array (Affymetrix, Santa Clara, CA, USA; P/N 511302). Genes were identified as differentially expressed if the expression ratio was higher than the standard deviation: 2.0-fold (induced and repressed) cutoff for Hha13D6 DNA microarrays (standard deviation 1.3-fold) and 10.0-fold (induced) or 4.0-fold (repressed) for Hha24E9 DNA microarrays (standard deviation 4.0-fold), and if the p-value for comparing two chips was less than 0.05.
2010-10-31 | E-GEOD-21604 | 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:The global regulator, H-NS, controls genes related to stress response, biofilm formation, and virulence expression by recognizing the curved DNA and silences gene transcription acquired from lateral gene transfer. Here, we rewired H-NS to control biofilm formation using protein engineering. One H-NS variant, H-NS K57N was obtained to reduce biofilm formation 10-fold compared to H-NS wild-type. Whole-transcriptome analysis (BW25113 hha hns / pCA24N-hns K57N vs. BW25113 hha hns / pCA24N-hns) revealed that H-NS K57N represses biofilm formation through the interactinon with other nucleoid proteins, Cnu and StpA. Remarkably, H-NS K57N enhanced the excision of defective prophage Rac while H-NS wild-type represses it, and H-NS controlled only Rac excision among E. coli prophages. These results imply that the repression of Rac excision is one of the silencing manner for foreign genes by H-NS. Also, the prophage excision not only led to the change of biofilm formation but also resulted in cell lysis through the expression of toxin protein HokD with reduced viability, which are important for cell physiology in response to the change of environmental conditions. Hence, H-NS regulatory system may be evolved easily with specialized functions in terms of biofilm formation, prophage control, and cell lysis. For the whole transcriptome study of BW25113 hha hns / pCA24N-hns K57N versus BW25113 hha hns / pCA24N-hns, cells were grown in 250 mL LB containing 1 mM IPTG for 7 h with 10 g of glass wool (Corning Glass Works, Corning, N.Y.) in 1 L Erlenmeyer flasks to form a robust biofilm. Biofilm cells were obtained by rinsing and sonicating the glass wool in sterile 0.85% NaCl solution at 0°C, and RNALater buffer® (Applied Biosystems, Foster City, CA) was added for RNA stabilization and protection during the RNA preparation steps. Total RNA was isolated from biofilm cells. The E. coli GeneChip Genome 2.0 array (Affymetrix, Lot# 4059655) containing 10,208 probe sets for four E. coli strains (MG1655, CFT073, O157:H7-Sakai, and O157:H7-EDL933) was used for DNA microarray. cDNA synthesis, fragmentation, and hybridizations were performed.
2010-02-27 | E-GEOD-17853 | biostudies-arrayexpress
Project description:We have shown the quorum-sensing signals acylhomoserine lactones (AHLs), autoinducer-2 (AI-2), and indole influence the biofilm formation of Escherichia coli. Here, we investigate how the environment, i.e., temperature, affects indole and AI-2 signaling in E. coli. We show in biofilms that indole addition leads to more extensive differential gene expression at 30ï°C (186 genes) than at 37ï°C (59 genes), that indole reduces biofilm formation (without affecting growth) more significantly at 25ï°C and 30ï°C than at 37ï°C, and that the effect is associated with the quorum-sensing protein SdiA. The addition of indole at 30ï°C compared to 37ï°C most significantly repressed genes involved in uridine monophosphate (UMP) biosynthesis (carAB, pyrLBI, pyrC, pyrD, pyrF, and upp) and uracil transport (uraA). These uracil-related genes are also repressed at 30ï°C by SdiA, which confirms SdiA is involved in indole signaling. Also, compared to 37ï°C, indole more significantly decreased flagella-related qseB, flhD, and fliA promoter activity, enhanced antibiotic resistance, and inhibited cell division at 30ï°C. In contrast to indole and SdiA, the addition of (S)-4,5-dihydroxy-2,3-pentanedione (the AI-2 precursor) leads to more extensive differential gene expression at 37ï°C (63 genes) than at 30ï°C (11 genes), and, rather than repressing UMP synthesis genes, AI-2 induces them at 37ï°C (but not at 30ï°C). Also, the addition of AI-2 induces the transcription of virulence genes in enterohemorrhagic E. coli O157:H7 at 37oC but not at 30oC. Hence, cell signals cause diverse responses at different temperatures, and indole- and AI-2-based signaling are intertwined. Experiment Overall Design: We performed seven sets of microarray experiments in LB medium (Table 2): (i) biofilm cells of the BW25113 wild-type strain grown for 7 h at 30°C vs. 37°C to determine the effect of temperature on E. coli biofilm formation (initial absorbance of 0.05), (ii) biofilm cells of the tnaA mutant grown for 7 h with 1 mM indole vs. no indole at 30°C to determine the effect of temperature on indole signaling in E. coli (initial absorbance of 0.05), (iii) biofilm cells of the tnaA mutant grown for 7 h with 1 mM indole vs. no indole at 37°C to determine the effect of temperature on indole signaling in E. coli, (iv) suspension cells of the wild-type strain vs. the sdiA mutant at an absorbance of 4.0 at 600 nm at 30°C (since production of indole takes place primarily in the stationary phase) to discern the genes that SdiA controls (initial absorbance of 0.05), (v) biofilm cells of the sdiA mutant grown for 7 h with 1 mM indole vs. no indole at 30°C to determine gene expression in response to indole in the absence of SdiA, (vi) suspension cells of the luxS mutant with 100 micro M AI-2 vs. no AI-2 at 30°C for 3 h to determine the effect of temperature on AI-2 signaling in E. coli (initial absorbance of 0.5), and (vii) suspension cells of the luxS mutant with 100 micro M AI-2 vs. no AI-2 at 37°C for 3 h to determine the effect of temperature on AI-2 signaling in E. coli. Ten g of glass wool (Corning Glass Works, Corning, N.Y.) was used to form large amounts of biofilms (Ren et al., 2004a) in 250 mL LB in 1 L Erlenmeyer shake flasks. RNA was isolated from the suspension and biofilm cells as described previously (Ren et al., 2004b).
2008-06-18 | E-GEOD-9923 | biostudies-arrayexpress
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. Experiment Overall Design: Strain: E. coli K-12 BW25113 wt and mqsR deleted mutant Experiment Overall Design: Medium: LB medium Experiment Overall Design: Temperature: 37 oC Experiment Overall Design: Time: 24 h Experiment Overall Design: Cell type: Biofilm grown on glass wool and planktonic culture
2009-12-05 | E-GEOD-14203 | biostudies-arrayexpress
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
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.
2009-01-28 | GSE12701 | GEO
Project description:This Series involves two studies: 1) The gene expression of E. coli K-12 BW25113 ompA mutant strain vs. wild type strain glasswool biofilm cells and E. coli K-12 BW25113 ompA mutant vs. wild type polystyrene biofilm cells. 2) The gene expression of E. coli BW25113 ompA/pCA24N_ompA vs. ompA/pCA24N suspension cells. Strains: E. coli K-12 BW25113 wild type, ompA mutant Medium: LB Cell type: Biofilm cells grown on glasswool and polystyrene surfaces Time: 15 h Temperature: 37C Strains: BW25113 ompA/pCA24N_ompA and ompA/pCA24N Medium: LB Time: 7 h Temperature: 37C Cell type: suspension cells, induced by 0.1 mM IPTG
2010-11-03 | E-GEOD-14064 | biostudies-arrayexpress
Project description:E. coli K-12 ATCC 25404 in LB medium with 5-fluorouracil 10 uM biofilm cells relative to E. coli K-12 ATCC 25404 in LB DMF biofilm cells. The same amount of stock 5-fluoroacil stock solution (0.1% of the volume) was added as DMF into the LB DMF. Experiment Overall Design: Strain: E. coli K-12 ATCC 25404 wild-type Experiment Overall Design: Medium: LB Experiment Overall Design: Biofilm growth on glass wool Experiment Overall Design: Time: 24 h Experiment Overall Design: Cell type: biofilm Experiment Overall Design: 5-fluorouracil vs. DMF
2009-01-09 | E-GEOD-12750 | biostudies-arrayexpress