Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of E. coli BW25113 fhlAL/pCA24N-FhlA133 vs fhlA/pCA24N-FhlA in modified-complex 20 mM formate at 37A?C

ABSTRACT: Variant FhlA133 (Q11H, L14V, Y177F, K245R, M288K, and I342F) had eight- fold higher hydrogen production than FhlA wild-type under 30 min of anaerobic incubation in modified-complex 20 mM formate at 37ÂºC. The mechanism by which the FhlA133 mutations increase hydrogen production is by increasing the transcription of all of the genes activated by the native FhlA (FHL complex). Experiment Overall Design: Strains: E. coli BW25113 fhlA/pCA24N-FhlA wild-type and E. coli BW25113 fhlA/pCA24N-FhlA133 (Q11H, L14V, Y177F, K245R, M288K, and I342F). Experiment Overall Design: Medium: Modified-complex 20 mM formate Experiment Overall Design: 1 mL of overnight culture in modified - complex 20 mM formate + Cm 30 Âµg/mL was inoculated in 9 mL of fresh modified - complex 20 mM formate + Cm 30 Âµg/mL and incubated anaerobically at 37oC in 27 mL glass vials. Experiment Overall Design: Time: 30 minutes Experiment Overall Design: Cell type: Suspension

ORGANISM(S): Escherichia coli

SUBMITTER: Younghoon Kim

PROVIDER: E-GEOD-13902 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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 0Â°C, and RNALater bufferÂ® (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.

Dataset Information

Transcription profiling of E. coli BW25113 fhlAL/pCA24N-FhlA133 vs fhlA/pCA24N-FhlA in modified-complex 20 mM formate at 37A?C

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