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| Status |
Public on Oct 05, 2020 |
| Title |
The mRNA derived MalH sRNA contributes to alternative carbon source utilization by tuning maltoporin expression in E. coli |
| Organism |
Escherichia coli |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Previous high-throughput studies in Gram-negative bacteria identified a large number of 3’UTR fragments that potentially function as sRNAs. Here we extensively characterise the MalH sRNA. We show that MalH is a stable degradation intermediate derived from the 3’ end of malG, which is part of the maltose uptake operon transcript malEFG. Unlike the majority of bacterial sRNAs, MalH is transiently expressed during the transition from the exponential to the stationary growth phase, suggesting that it contributes to adaptation to changes in nutrient availability. Over-expression of MalH reduces expression of general outer membrane porins and MicA, a repressor of the high-affinity maltose/maltodextrin transporter LamB. Disrupting MalH production and function significantly reduces lamB accumulation when maltose is the only available carbon source, presumably due to the accumulation of the MicA repressor. We propose that MalH is part of a regulatory network that, during the transition phase, directly or indirectly promotes accumulation of high-affinity maltose transporters in the outer membrane by dampening competing pathways.
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| Overall design |
For pulse-overexpression studies overnight MG1655 cultures containing pBAD::sRNA and empty pBAD+1 control plasmids were inoculated in fresh LB-ampicillin medium at a starting OD600 of 0.05 and grown aerobically at 37°C to OD600 0.4. Pre-induction (0 min) and post-induction samples were harvested. For induction, cultures were supplemented with L-arabinose (Sigma, A3256) and rapidly collected by filtration and flash-frozen in liquid nitrogen at the indicated time-points. RNA was extracted from three biological replicate time-series, followed by RNA extraction using GTC-phenol. RNA-seq libraries were generated using an in-house protocol. Genomic DNA was removed by incubating 10 ug of total RNA with 2U Turbo DNase in a 50 ul final volume for 30 minutes at 37°C in the presence of 10 U superaseIn RNase Inhibitor. RNA was subsequently phenol-chloroform extracted and purified by ethanol-precipitation. Ribosomal RNA was removed with the Illumina Gram-negative Ribo-Zero rRNA Removal Kit according to the manufacturer's instructions. Successful rRNA depletion was verified on the Agilent 2100 Bioanalyzer. The RNA was fragmented for 5 min at 95°C in the presence of Superscript III buffer (Invitrogen) followed by a five-minute incubation on ice. Reverse-transcription (RT) was performed with Superscript III (Thermo Scientific) in 20 ul reactions according to manufacturer's procedures using 250 ng of ribosomal RNA depleted RNA and 2.5 uM random hexamers (PE_solexa_hexamer, 5'-CGTGTGCTCTTCCGATCTNNNNNN-3'). The RNA and free primers were degraded using 20U of Exonuclease I (NEB) and 50U RNaseIf (NEB) and the cDNA was purified with the DNA Clean & Concentrator 5 kit (Zymo Research). Ligation of the 5' P5_phospho_adapter (5'P-AGATCGGAAGAGCGTCGTGTAGGG-3SpC3) to the cDNA was performed using CircLigase II (Lucigen) for 6 hours at 60°C, followed by a 10-minute inactivation at 80°C. The cDNA was purified with the DNA Clean & Concentrator 5 kit. Half of the cDNA library was PCR amplified using Pfu polymerase (Promega) using the P5 forward PCR oligonucleotide (5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3') and barcoded BC reverse oligonucleotides (200 nM; 5'-CAAGCAGAAGACGGCATACGAGAT(BC 6nt barcode)GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3'; 95°C for 2 min, 95°C for 20s, 52°C for 30s and 72°C for 1 min, and a final extension of 72°C for 5 min. 20 cycles of amplification). The PCR products were treated with Exonuclease 1 (NEB) for 1 h at 37°C and purified by ethanol precipitation. Libraries were resolved on a 2% MetaPhor agarose gel 200-500 bp fragments were gel-extracted using the MinElute kit. All libraries were quantified on a 2100 Bionalyzer using the High-Sensitivity DNA assay (Agilent). Individual libraries were pooled in equimolar amounts. Paired-end sequencing (75 bp) was performed by Edinburgh Genomics on the Illumina HiSeq 4000 platform.
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| Contributor(s) |
Granneman S, Iosub IA |
| Citation(s) |
33043783 |
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| Submission date |
Apr 21, 2020 |
| Last update date |
May 19, 2021 |
| Contact name |
Sander Granneman |
| E-mail(s) |
[email protected]
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| Organization name |
University of Edinburgh
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| Department |
Centre for Synthetic and Systems Biology
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| Lab |
Granneman lab
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| Street address |
Mayfield Road, Kings Buildings, Waddington building, room 3.06
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| City |
Edinburgh |
| ZIP/Postal code |
EH9 3JD |
| Country |
United Kingdom |
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| Platforms (1) |
| GPL21433 |
Illumina HiSeq 4000 (Escherichia coli) |
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| Samples (6)
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| GSM3494382 |
RNA-seq over-expression empty plasmid control replicate 1 |
| GSM3494383 |
RNA-seq over-expression from empty plasmid control replicate 2 |
| GSM3494384 |
RNA-seq over-expression from empty plasmid control replicate 3 |
| GSM3494385 |
RNA-seq over-expression from MdoR sample replicate 1 |
| GSM3494386 |
RNA-seq over-expression from MdoR sample replicate 2 |
| GSM3494387 |
RNA-seq over-expression from MdoR sample replicate 3 |
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| Relations |
| BioProject |
PRJNA627225 |
| SRA |
SRP258175 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE149059_pBAD_vs_MdoR_counts_DESeq2_data.xlsx |
656.5 Kb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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