Altering translation allows E. coli to overcome G-quadruplex stabilizers

Cueny, Rachel 1 ; Voter, Andrew1; McKenzie, Aidan1; Morgenstern, Marcel1; Myers, Kevin1; Place, Michael1; Peters, Jason1; Coon, Joshua1; Keck, James1

Published Feb 27, 2025 on Dryad. https://doi.org/10.5061/dryad.zcrjdfnn9

Abstract

G-quadruplex (G4) structures can form in guanine-rich DNA or RNA and have been found to modulate cellular processes including replication, transcription, and translation. Many studies on the cellular roles of G4s have focused on eukaryotic systems, with far fewer probing bacterial G4s. Using a chemical-genetic approach, we identified genes in Escherichia coli that are important for growth in G4-stabilizing conditions. Reducing levels of elongation factor Tu or slowing translation elongation with chloramphenicol suppress the effects of G4 stabilization. In contrast, reducing the expression of certain translation termination or ribosome recycling proteins is detrimental to growth in G4-stabilizing conditions. Proteomic and transcriptomic analyses demonstrate that ribosome assembly factors and other proteins involved in translation are less abundant in G4-stabilizing conditions. Our results suggest that RNA G4s can present barriers to E. coli growth and reducing the rate of translation can compensate for G4-related stress.

https://doi.org/10.5061/dryad.zcrjdfnn9

Description of the data and file structure

The data included in this Dryad submission was collected in order to understand how the model organism* Escherichia coli* overcomes stabilized G-quadruplexes. This work involved a multi-omics approach to studying how the G-quadruplex stabilizers NMM and Braco-19 impact growth, gene importance, and mRNA/proteomic abundance in G-quadruplex stabilizing conditions.

Files and variables

File: Western_total_protein_stain_1.tif

Description: Total protein staining for western blots assessing EF-Tu abundance in ∆tolC, ∆tolC tufA::kan, and ∆tolC tufB::kan cells. Order of gel is ∆tolC, ∆tolC tufA::kan, and ∆tolC tufB::kan at three different dilutions added into the gel. This is the total protein stain image used in the figure of the manuscript.

File: Western_Blot_1_from_Figure.tif

Description: Western blot assessing EF-Tu abundance in ∆tolC, ∆tolC tufA::kan, and ∆tolC tufB::kan cells. Order of gel is ∆tolC, ∆tolC tufA::kan, and ∆tolC tufB::kan at three different dilutions added into the blot. This is the blot used in the figure of the manuscript.

File: Western_Blot_2.tif

File: Western_Blot_3.tif

File: Western_total_protein_stain_3.tif

File: Western_total_protein_stain_2.tif

File: Growth_Curves.txt

Description: Growth curve carried out on a plate reader to assess MG1655, ∆tolC, ∆tolC tufA::kan, and tolC tufB::kan growth in the presence and absence of NMM. This txt file contains the OD600 values for each growth condition and replicate at 10 minute intervals. This is the data shown in Figure 2 of the manuscript for the growth curves.

File: Proteomics_Data.xlsx

Description: This file includes the proteomics dataset collected from the ∆tolC cells and ∆tolC tufA::kan cells each grown in the presence and absence of NMM. This dataset includes information for each detected protein (the LFQ values indicating the detection for each protein) as well as the unique peptides and sequence coverage for each protein in each growth condition/replicate. The highlighted values in this sheet indicate imputed values from the proteomics analysis.

File: Proteomics_Comparison.xlsx

Description: This spreadsheet has Log2 comparisons of the proteomics data between the ∆tolC cells and ∆tolC tufA::kan cells each grown in the presence and absence of NMM. This spreadsheet also has p-values and q-values to determine significance of differences between data.

File: RNA_seq_raw_counts.csv

Description: This file contains the raw counts for mRNA corresponding to a different gene in* E. coli*. This is listed under each replicate for ∆tolC and ∆tolC tufA::kan each grown in the presence and absence of NMM.

File: RNA_seq_TPM_values.csv

Description: This file contains the transcripts per million for mRNA corresponding to a different gene in* E. coli*. This is listed under each replicate for ∆tolC and ∆tolC tufA::kan each grown in the presence and absence of NMM. This is data normalized for comparison between datasets and replicates.

File: Consolidated_polysome_data.xlsx

Description: This files includes all of the data obtained from the Gradient station for detecting A260 values from the sucrose gradients for ∆tolC and ∆tolC tufA::kan grown in the presence and absence of NMM. For each replicate, the corresponding position of the piston as well as the absorbance values are reported.

File: Polysome_traces_AUC.xlsx

Description: This file includes the area under the curve (percentage) corresponding for each replicate (∆tolC ± NMM and ∆tolC tufA::kan ± NMM) for the 30S, 50S, monosome, and polysome abundance in the sample.

File: TNseq_original.xlsx

Description: This sheet includes the data from the original Tn-seq screen (in a tolC+ background) in the presence and absence of NMM. This includes the reads and hits in each gene as well as the weighted reads in NMM or in the control condition.

File: TNseq_passage_1.xlsx

Description: This is the subsequent Tn-seq screen (Fig 1) of the manuscript that contains the information for the Tn-seq screen done in a ∆tolC background in the presence and absence of NMM. This includes the reads and hits in each gene as well as the weighted reads in NMM or in the control condition. This is the first passage on NMM plates or control plates.

File: TNseq_passage_2.xlsx

Description: This is the subsequent Tn-seq screen of the manuscript that contains the information for the Tn-seq screen done in a ∆tolC background in the presence and absence of NMM. This includes the reads and hits in each gene as well as the weighted reads in NMM or in the control condition. This is the second passage on NMM plates or control plates.

File: EFFLUX_230811_lb_1_PUB_416.tif

Description: This contains a spot plate on LB for the following strains (in order): MG1655, ∆tolC, arcA::kan, acrB::kan, macA::kan, macB::kan. This is a replicate for the plate shown in Supp Fig 1 of the manuscript.

File: EFFLUX_230811_lb_2_PUB_416.tif

File: EFFLUX_230811_lb_5nmm_2_PUB_416.tif

Description: This contains a spot plate on LB with 5 µM NMM for the following strains (in order): MG1655, ∆tolC, arcA::kan, acrB::kan, macA::kan, macB::kan. This is a replicate for the plate shown in Supp Fig 1 of the manuscript.

File: EFFLUX_230811_lb_5nmm_1_PUB_416.tif

File: EFFLUX_230811_lb_5nmm_in_Figure_PUB_416.tif

Description: This contains a spot plate on LB with 5 µM NMM for the following strains (in order): MG1655, ∆tolC, arcA::kan, acrB::kan, macA::kan, macB::kan. This is shown in Supp Fig 1 of the manuscript.

File: EFFLUX_230811_lb_10nmm_1_PUB_416.tif

Description: This contains a spot plate on LB with 10 µM NMM for the following strains (in order): MG1655, ∆tolC, arcA::kan, acrB::kan, macA::kan, macB::kan. This is a replicate for the plate shown in Supp Fig 1 of the manuscript.

File: EFFLUX_230811_lb_10nmm_2_PUB_416.tif

File: EFFLUX_230811_lb_in_Figure_PUB_416.tif

Description: This contains a spot plate on LB for the following strains (in order): MG1655, ∆tolC, arcA::kan, acrB::kan, macA::kan, macB::kan. This is shown in Supp Fig 1 of the manuscript.

File: EFFLUX_230811_lb_10nmm_in_Figure_PUB_416.tif

Description: This contains a spot plate on LB with 10 µM NMM for the following strains (in order): MG1655, ∆tolC, arcA::kan, acrB::kan, macA::kan, macB::kan. This is shown in Supp Fig 1 of the manuscript.

File: MG1655_tufA_tufB_021420_lb_1_pub.tif

Description: This contains a spot plate on LB for the following strains (in order): MG1655, recA::kan, tufA::kan, and tufB::kan. Only MG1655, tufA::kan, and tufB::kan were assessed in the paper, with recA::kan being a control as a strain known to be sensitive to NMM treatment.

File: MG1655_tufA_tufB_used_in_figure_S2_021420_lb_pub.tif

File: MG1655_tufA_tufB_021420_lb_2_pub.tif

File: MG1655_tufA_tufB_021420_nmm_1_pub.tif

Description: This contains a spot plate on LB with 30 µM NMM for the following strains (in order): MG1655, recA::kan, tufA::kan, and tufB::kan. Only MG1655, tufA::kan, and tufB::kan were assessed in the paper, with recA::kan being a control as a strain known to be sensitive to NMM treatment.

File: MG1655_tufA_tufB021420_nmm_2_pub.tif

File: MG1655_tufA_tufB_used_in_figure_S2_021420_nmm_pub.tif

File: dtolC_tufA_tufB_062620_5um_nmm_1_pub.tif

Description: This contains a spot plate on LB with 5 µM NMM for the following strains (in order): ∆tolC, tufA::kan, and tufB::kan.

File: dtolC_tufA_tufB_062620_5um_nmm_2pub.tif

Description: This contains a spot plate on LB with 5 µM NMM for the following strains (in order): ∆tolC, tufA::kan, and tufB::kan.

File: dtolC_tufA_tufB_062620_lb_1_pub.tif

Description: This contains a spot plate on LB for the following strains (in order): ∆tolC, tufA::kan, and tufB::kan.

File: dtolC_tufA_tufB_062620_lb_2_pub.tif

Description: This contains a spot plate on LB for the following strains (in order): ∆tolC, tufA::kan, and tufB::kan.

File: dtolC_tufA_tufB_used_in_figure_2_062620_5um_nmm_pub.tif

Description: This contains a spot plate on LB with 5 µM NMM for the following strains (in order): ∆tolC, tufA::kan, and tufB::kan. This was used in the figure of the paper.

File: dtolC_tufA_tufB_used_in_figure_2_062620_lb_pub.tif

Description: This contains a spot plate on LB for the following strains (in order): ∆tolC, tufA::kan, and tufB::kan. This was used in the figure of the paper.

File: BRACO19_dtolC_tufA_tufB_031321_braco_1_pub.tif

Description: This includes spot plates on LB with 60 µM Braco-19 and the following strains: ∆tolC, ∆tolC recA::kan, ∆tolC tufA::kan, ∆tolC tufB::kan, and ∆tolC ypjD::kan. recA::kan and ypjD::kan were strains not included in the paper so are not shown.

File: BRACO19_dtolC_tufA_tufB031321_braco_2_pub.tif

File: BRACO19_dtolC_tufA_tufB031321_lb_1_pub.tif

Description: This includes spot plates on LB and the following strains: ∆tolC, ∆tolC recA::kan, ∆tolC tufA::kan, ∆tolC tufB::kan, and ∆tolC ypjD::kan. recA::kan and ypjD::kan were strains not included in the paper so are not shown.

File: BRACO19_dtolC_tufA_tufB031321_lb_2_pub.tif

File: BRACO19_dtolC_tufA_tufBfor_figure2_031321_braco_pub.tif

File: BRACO19_dtolC_tufA_tufBfor_figure2_031321_lb_pub.tif

File: PROTEOMICS_231130_nmm_1_PUB_416.tif

Description: These spot plates included LB supplemented with 3 µM NMM and the following strains: ∆tolC, ∆tolC rimP::kan, ∆tolC rplI::kan, ∆tolC rsfS::kan, and ∆tolC deaD::kan.

File: PROTEOMICS_231130_lb_2_PUB_416.tif

Description: These spot plates included LB and the following strains: ∆tolC, ∆tolC rimP::kan, ∆tolC rplI::kan, ∆tolC rsfS::kan, and ∆tolC deaD::kan.

File: PROTEOMICS_for_figure4_231130_lb_PUB_416.tif

Description: These spot plates included LB and the following strains: ∆tolC, ∆tolC rimP::kan, ∆tolC rplI::kan, ∆tolC rsfS::kan, and ∆tolC deaD::kan. This was used in the figure.

File: PROTEOMICS_231130_lb_1_PUB_416.tif

Description: These spot plates included LB and the following strains: ∆tolC, ∆tolC rimP::kan, ∆tolC rplI::kan, ∆tolC rsfS::kan, and ∆tolC deaD::kan.

File: PROTEOMICS_231130_nmm_2_PUB_416.tif

Description: These spot plates included LB supplemented with 3 µM NMM and the following strains: ∆tolC, ∆tolC rimP::kan, ∆tolC rplI::kan, ∆tolC rsfS::kan, and ∆tolC deaD::kan.

File: PROTEOMICS_for_figure4_231130_nmm_PUB_416.tif

Description: These spot plates included LB supplemented with 3 µM NMM and the following strains: ∆tolC, ∆tolC rimP::kan, ∆tolC rplI::kan, ∆tolC rsfS::kan, and ∆tolC deaD::kan. This was used in the figure.

File: CRISPRI_tolC_NMM_240113_iptg_2_PUB_416.tif

Description: These spot plates include LB supplemented with 10 µM IPTG and the following strains: ∆tolC, ∆tolC CRISPRi aroC, ∆tolC CRISPRi prfA, ∆tolC CRISPRi fusA, ∆tolC CRISPRi prfB, ∆tolC CRISPRi frr.

File: CRISPRI_tolC_NMM_240113_iptg_1_PUB_416.tif

File: CRISPRI_tolC_NMM_240113_lb_1_PUB_416.tif

Description: These spot plates include LB and the following strains: ∆tolC, ∆tolC CRISPRi aroC, ∆tolC CRISPRi prfA, ∆tolC CRISPRi fusA, ∆tolC CRISPRi prfB, ∆tolC CRISPRi frr.

File: CRISPRI_tolC_NMM_240113_lb_2_PUB_416.tif

Description: These spot plates include LB and the following strains: ∆tolC, ∆tolC CRISPRi aroC, ∆tolC CRISPRi prfA, ∆tolC CRISPRi fusA, ∆tolC CRISPRi prfB, ∆tolC CRISPRi frr.

File: CRISPRI_tolC_NMM_240113_nmm_1_PUB_416.tif

Description: These spot plates include LB supplemented with 2 µM NMM and the following strains: ∆tolC, ∆tolC CRISPRi aroC, ∆tolC CRISPRi prfA, ∆tolC CRISPRi fusA, ∆tolC CRISPRi prfB, ∆tolC CRISPRi frr.

File: CRISPRI_tolC_NMM_240113_nmm_2_PUB_416.tif

File: CRISPRI_tolC_NMM_240113_nmm_iptg_1_PUB_416.tif

Description: These spot plates include LB supplemented with 2 µM NMM and 10 µM IPTG and the following strains: ∆tolC, ∆tolC CRISPRi aroC, ∆tolC CRISPRi prfA, ∆tolC CRISPRi fusA, ∆tolC CRISPRi prfB, ∆tolC CRISPRi frr.

File: CRISPRI_tolC_NMM_240113_nmm_iptg_2_PUB_416.tif

File: CRISPRI_tolC_NMM_used_in_figure_240113_nmm_iptg_PUB_416.tif

File: CRISPRI_tolC_NMM_used_in_figure_240113_lb_PUB_416.tif

Description: These spot plates include LB and the following strains: ∆tolC, ∆tolC CRISPRi aroC, ∆tolC CRISPRi prfA, ∆tolC CRISPRi fusA, ∆tolC CRISPRi prfB, ∆tolC CRISPRi frr. This was used in the figure of the manuscript.

File: CRISPRI_tolC_NMM_used_in_figure_240113_iptg_PUB_416.tif

File: CRISPRI_tolC_NMM_used_in_figure_240113_nmm_PUB_416.tif

File: CRISPRi_screen.pdf

Description: This is the images of the plates from the original CRISPR interference screen. This files includes strains plated on LB alone, LB + IPTG, LB + NMM, and LB with IPTG and NMM. These plates are annotated in the supplemental figure of the manuscript.

File: CRISPRi_240203_braco_1_PUB_416.tif

Description: These spot plates include LB supplemented with 75 µM Braco-19 and the following strains: ∆tolC, ∆tolC CRISPRi aroC, ∆tolC CRISPRi prfA, ∆tolC CRISPRi fusA, ∆tolC CRISPRi prfB, ∆tolC CRISPRi frr.

File: CRISPRi_240203_10_iptg_1_PUB_416.tif

File: CRISPRi_240203_lb_1_PUB_416.tif

Description: These spot plates include LB and the following strains: ∆tolC, ∆tolC CRISPRi aroC, ∆tolC CRISPRi prfA, ∆tolC CRISPRi fusA, ∆tolC CRISPRi prfB, ∆tolC CRISPRi frr.

File: CRISPRi_240203_iptg_braco_1_PUB_416.tif

Description: These spot plates include LB supplemented with 75 µM Braco-19 and 10 µM IPTG and the following strains: ∆tolC, ∆tolC CRISPRi aroC, ∆tolC CRISPRi prfA, ∆tolC CRISPRi fusA, ∆tolC CRISPRi prfB, ∆tolC CRISPRi frr.

File: CRISPRi_240211_braco_2_PUB_416.tif

File: CRISPRi_240211_braco_iptg_2_PUB_416.tif

File: CRISPRi_240211_iptg_2_PUB_416.tif

File: CRISPRi_used_in_figure_240211_braco_iptg_PUB_416.tif

File: CRISPRi_240211_lb_2_PUB_416.tif

Description: These spot plates include LB and the following strains: ∆tolC, ∆tolC CRISPRi aroC, ∆tolC CRISPRi prfA, ∆tolC CRISPRi fusA, ∆tolC CRISPRi prfB, ∆tolC CRISPRi frr.

File: CRISPRi_used_in_figure_240211_braco_PUB_416.tif

File: CRISPRi_used_in_figure_240211_lb_PUB_416.tif

File: CRISPRi_used_in_figure_240211_iptg_PUB_416.tif

File: CAM_120321_lb_1_pub.tif

Description: This spot plate includes an LB plate with ∆tolC spotted onto the plate.

File: CAM_120321_cam_nmm_1_pub.tif

Description: This spot plate includes an LB plate supplemented with 125 nM chloramphenicol and 4 µM NMM with ∆tolC spotted onto the plate.

File: CAM_120321_cam_1_pub.tif

Description: This spot plate includes an LB plate supplemented with 125 nM chloramphenicol with ∆tolC spotted onto the plate.

File: CAM_120321_nmm_1_pub.tif

Description: This spot plate includes an LB plate supplemented with 4 µM NMM with ∆tolC spotted onto the plate.

File: CAM_used_in_figure_120321_cam_nmm_pub.tif

Description: This spot plate includes an LB plate supplemented with 125 nM chloramphenicol and 4 µM NMM with ∆tolC spotted onto the plate.

File: CAM_used_in_figure_120321_cam_pub.tif

Description: This spot plate includes an LB plate supplemented with 125 nM chloramphenicol with ∆tolC spotted onto the plate. This was used in the figure.

File: CAM_used_in_figure_120321_lb_pub.tif

Description: This spot plate includes an LB plate with ∆tolC spotted onto the plate. This was used in the figure of the manuscript.

File: CAM_010622_cam_2_pub.tif

Description: This spot plate includes an LB plate supplemented with 125 nM chloramphenicol with ∆tolC spotted onto the plate.

File: CAM_010622_nmm_2_pub.tif

Description: This spot plate includes an LB plate supplemented with 4 µM NMM with ∆tolC spotted onto the plate.

File: CAM_used_in_figure_120321_nmm_pub.tif

Description: This spot plate includes an LB plate supplemented with 4 µM NMM with ∆tolC spotted onto the plate. This was used in the figure.

File: CAM_010622_nmm_cam_2_pub.tif

Description: This spot plate includes an LB plate supplemented with 125 nM chloramphenicol and 4 µM NMM with ∆tolC spotted onto the plate. This was used in the figure.

File: CAM_010622_lb_2_pub.tif

Description: This spot plate includes an LB plate with ∆tolC spotted onto the plate.

All files starting with 081420 in title:

Description: These are the minimal media spot plates for the paper.

All files starting with 20240809 in title:

Description: all of these files are the spot plates from kasugamycin treatment experiment.

All files starting with 20240819 in title:

Description: all of these files are the spot plates from the spectinomycin experiment

All files starting with initiation factor in title

Description: all of these are the spot plates from the CRISPRi initiation factor knockdown experiment.

Code/software

The files included in this submission are either .tif files of excel spreadsheets.

Strain construction

All cells used in this study are derived from an Escherichia coli MG1655 parent strain unless otherwise specified. For CRISPR interference strains, strains were a gift from Jason Peters (46). To generate E. coli knockout strains, P1 transductions were carried out using Keio collection strains as the donor strain (63,64). P1 phage lysate was grown on Keio collection donor strains, which was used to transduce the MG1655 strains or CRISPR interference strains (to make the tolC knockout of selected CRISPRi strains) which were sensitive to kanamycin. To validate strains, transductions were grown on LB plates supplemented with 50 µg/mL kanamycin and screened using colony PCR to validate proper insertion of the kanamycin resistance cassette. To remove the kanamycin resistant cassette from MG1655 tolC::kan to enable additional P1 transductions in this strain, MG1655 tolC::kan electrocompetent cells were generated and transformed with a plasmid encoding the FLP recombinase (pCP20) (65). Cells were recovered at 30 ºC and grown overnight on Super Optimal Broth (SOB) plates supplemented with 100 µg/mL ampicillin at 30 ºC. Single colonies from the plate were then grown overnight in LB at 43 ºC to promote loss of the temperature sensitive plasmid. A 10^-6 dilution of cells was grown on LB plates at 30 ºC overnight to obtain individual colonies, which were then streaked onto LB only, LB supplemented with 100 µg/mL ampicillin, and LB supplemented with 50 µg/mL kanamycin. Colonies that only grew on the LB without antibiotic plates were selected as MG1655 ∆tolC cells that were utilized for downstream applications.

Transposome preparation and transposition

Transposome preparation was carried out as previously described (66,67). Briefly, the EZ-Tn5 <DHFR-1> transposon kit (Epicentre) and the E54K/M56A/L372P Tn5 hyperactive variant transposase were used for transposon mutagenesis. The Tn5 transposon was amplified using Phusion polymerase (NEB) and oligonucleotide oAM054. Transposase purification was carried out as previously described (66,68). Transposomes were prepared by incubating 2.5 pmol Tn5 DNA with 0.5 nmol Tn5 transposase for 3 hours at ambient temperature and then dialyzed into 1x TE buffer before electroporation.

Electrocompetent E. coli cells were generated as previously described (66). Briefly, E. coli were grown at 37 ºC to an OD₆₀₀ ~0.4 and cooled at 4 ºC for an hour. Cells were centrifuged at 10,750 rcf and pellets were washed in 10% glycerol three times. Cells were then resuspended in 2 mL GYT (10% (v/v) glycerol, 0.125% (w/v) yeast extract, and 0.25% (w/v) tryptone) before flash freezing and storing electrocompetent cells at -80 ºC. Five µL of transposome was combined with 100 µL of electrocompetent cells, electroporated, and recovered in 1 mL of SOC media at 37 ºC for 1 hour. Cells were plated on SOB-agar supplemented with 10 µg/mL trimethoprim to select for cells containing transposon insertions. Transposon mutants were pooled (~200,000 colonies) from plates using 2 mL of LB to scrape colonies off plates and then stored in 50% glycerol at -80 ºC.

Selection of tolerated transposon mutations in G4 stabilizing conditions

For the pilot Tn-seq experiment (Figure S1), a Tn5 transposase generated library in MG1655 sulB103 was utilized (67). This library was diluted from a glycerol stock 1:10,000 in fresh LB and 250 µL of dilution was plated on SOC plates and SOC plates supplemented with 10 µM NMM. Plates were grown overnight at 37 ºC and there were estimated ~100,000 colonies grown on SOC alone and ~150,000 colonies grown on NMM supplemented plates. Colonies were pooled with LB and samples were diluted to an OD₆₀₀ of ~4.0 and 1 mL of concentrated cells underwent genomic DNA preparation using the Wizard Genomic DNA Purification Kit (Promega). DNA was quantified using the QuantiFluor ONE dsDNA System (Promega). Genomic DNA underwent shearing to ~200 bp fragments via sonication and the gDNA fragments were prepared for sequencing using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB). Bead-based size selection was employed to enrich for 200 bp fragments and the fragments then underwent a 17-cycle splinkerette PCR using oAM055 as the forward primer and either oAM068 (control) or oAM069 (NMM selected) as the reverse primer for barcoding and multiplexing (67). An additional bead-based size selection was used to clean up the sample before sequencing at the University of Michigan with a MiSeq platform. Primers oAM058 and oAM059 were used as unique sequencing primers for the control and NMM treated condition, respectively.

Transposon sequencing with ∆tolC cells

For the subsequent Tn-seq experiment utilizing an MG1655 ∆tolC strain, transposomes were prepared as described above and the library was prepared as before (with the exception of using 1 µg/mL trimethoprim for selection) generating ~500,000 transposon insertion mutants. MG1655 ∆tolC electrocompetent cells were generated as described above.

To select for transposon insertion mutations in control and NMM-treated conditions, libraries were grown on either SOB-agar plates or SOB-agar plates supplemented with 5 µM NMM. To ensure proper coverage after re-selection on plates, ~1.5 million colonies were collected either from the SOB-agar plates and the SOB-agar plates supplemented with NMM and split into three libraries each. Libraries were passaged a second time in the presence and absence of NMM to generate a second passage library of ~1.5 million colonies and split into three libraries each.

To prepare DNA for sequencing, libraries were prepared as described in the previous section. Each library was diluted to an OD₆₀₀ of ~4.0 and 1 mL of concentrated cells underwent genomic DNA preparation using the Wizard Genomic DNA Purification Kit (Promega). DNA was quantified using the QuantiFluor ONE dsDNA System (Promega). Genomic DNA underwent shearing to ~200 bp fragments via sonication and the gDNA fragments were prepared for sequencing using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB). Bead-based size selection was employed to enrich for 200 bp fragments and the fragments then underwent a 20-cycle splinkerette PCR using a Tn5-enrihcing forward primer (oAM55) and custom reverse primers for multiplexing (67). A final bead-based size selection was used to select for the correct length DNA. DNA was sequenced at the University of Michigan Advanced Genomics Core using a NextSeq platform (Illumina) with a custom read primer (oAM58) reading the last 10 nt of the transposon. PhiX174 DNA spike was added to the run to ensure sufficient sequence diversity on the flow cell. Then, a custom index read primer (oAM59) and standard Illumina primer were used to sequence the index reads and PhiX174, respectively.

Data analysis for Tn-seq

Tn-seq analysis was done as described previously (67). Tn-seq sequencing was trimmed with fastx_trimmer.pl version 0.0.13.2 (http://hannonlab.cshl.edu/fastx_toolkit). The default parameters were used except the first base to keep (-f flag) was edited to 10 to take out the transposon sequence. Samples were then split with fastx_barcode_splitter.pl, version 0.013.2 (http://hannonlab.cshl.edu/fastx_toolkit) using a file that contained the individual barcode sequence and the sample ID, then the barcode was removed from each read in the FASTQ file using Cutadapt version 1.13 (69). FASTQ files that were trimmed were then aligned to the E. coli K-12 MG1655 genome (NC_000913.3) using Bowtie2, version 1.2 on default parameters (70). Conditional importance or essentiality of genes was determined using TSAS, version 0.3.0 using Analysis_type2 for 2 sample analysis to compare transposon insertion profiles of NMM treated cells to cells grown without G4 stabilizer (40). The weighted reads were determined as previously described (40). The other parameters were kept at default settings.

Assessing sensitivity to G4 stabilizers using spot plates

Spot plating experiments to assess sensitivity to G4 stabilizing compounds was carried out as previously described (26). Briefly, NMM and Braco-19 were prepared by resuspending the compounds in 18 MW ultra-pure water and NMM concentration was assessed using the molar extinction coefficient 145000 M^-1 cm^-1 at 379 nm (71).

NMM and Braco-19 solutions were stored at 4 ºC. IPTG solutions were made by resuspension in 18 MW ultra-pure water and stored at -20 ºC and chloramphenicol was resuspended in ethanol and stored at -20 ºC. G4 stabilizers, IPTG, or chloramphenicol were added to LB-agar at the indicated concentrations and stored in the dark. Five mL of each E. coli strain were grown overnight and diluted in fresh LB to an OD₆₀₀ ~1. For spot plating, 10^-1 to 10^-6dilutions of strains were made in LB and 10 µL of each dilution was plated onto the LB spot plates. Spot plates were grown overnight and imaged on the Azure c600. Spot plates were done in triplicate.

CRISPR interference screen

For CRISPR interference screen, strains from the CRISPRi library were grown in plates with 200 µL of LB supplemented with 10 µg/mL chloramphenicol and 4 µL from each glycerol stock of the library. Cells were grown overnight at 37 ºC and then stored at 4 ºC overnight. The following day, plates were shaken at 37 ºC for 5 minutes and then diluted 200-fold into fresh LB and shaken for 5 minutes to mix cells. Two µL were plated onto plates with LB-agar alone or LB-agar supplemented with 15 µM NMM, 10 µM IPTG, or 10 µM IPTG and 15 µM NMM together. Plates were grown overnight at 37 ºC and imaged the following day using the Azure c600.

Assessing sensitivity to G4 stabilizers using growth curves

MG1655, ∆tolC, ∆tolC tufA::kan, and ∆tolC tufB::kan cells were grown overnight at 37 ºC in Luria Broth (LB). The next day, cells were diluted 100-fold into fresh LB and grown to an OD₆₀₀~0.2. Cells were then diluted 100-fold in a 96-well plate either in the presence or absence of NMM and OD₆₀₀of the cells was measured every ten minutes over 24 hours with continuous shaking at 37 ºC in a plate reader (BioTek Synergy H1). Growth curves were done in triplicate. The average of each growth condition was then plotted in Prism (10.2.0) with error bars representing the standard error of the mean.

Western blots

∆tolC, ∆tolC tufA::kan, and ∆tolC tufB::kan cells overnight cultures were diluted 100-fold in fresh Luria Broth (LB) and grown to an OD₆₀₀ ~0.3. One mL of cells were pelleted and resuspended in 50 µL of 1x sample buffer (0.8% SDS, 11.5% glycerol, 0.1 M Tris pH 6.8, 0.286 M bME, 0.01% bromophenol blue). Five µL of undiluted sample and 5 µL of sample at various dilutions (1:2, 1:10, or 1:15) were loaded onto a 5-15% PAGE gel (brand) and run in 1x SDS running buffer. Proteins were then transferred to a nitrocellulose membrane at 4 ºC for 1.5 hours in transfer buffer (25 mM Tris pH 8, 192 mM glycine, 0.03% SDS, 20% methanol). For total protein staining, membranes were incubated with 5 mL total protein stain (LI-COR) for 5 minutes at ambient temperature on a rocking platform before getting rinsed with wash solution (30% methanol, 6.7% acetic acid) and then imaged on the IR700 channel using the Azure c600. Following total protein stain, the membrane was blocked for 1 hour at room temperature in 5% dry milk in 1x PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, 1.47 mM KH₂PO_4,pH 7.4) and then rinsed with 1x PBS. Membrane was then incubated for 1 hour at ambient temperature with anti-EF-Tu antibody (Hycult Biotech) at 2 µg/mL and then rinsed with 1x PBS. Membrane was then incubated with peroxidase conjugated goat anti-mouse antibody (Invitrogen) at 0.1 µg/mL and then rinsed with 1x PBS. The Amersham ECL Prime Western Blotting detection kit was then used to visualize the blot on the Azure c600 using the chemiluminescence setting. Intensity of bands for total protein normalization and for EF-Tu blots was carried out using ImageJ and plotted using Prism (10.2.0). Significance was determined in Prism using the Welch’s two-tailed t-test. Western blots and total protein staining were done in triplicate.

Proteomics cell growth and cell lysis

∆tolC and ∆tolC tufA::kan cells were grown overnight and then diluted 100-fold in fresh LB and grown to an OD₆₀₀ of ~0.2. Cells were then diluted 100-fold and grown in LB ± 3.5 µM NMM. Cells were grown to an OD₆₀₀~0.2-0.4 and then 45 mL of cells were pelleted via centrifugation. The pellets were washed with 5 mL of 1x PBS to remove any residual media and then pelleted again. Cells pellets were stored at -80 ºC.

Cell lysis was initiated by resuspension in 250 µl of lysis buffer (8 M urea and 100 mM Tris pH 8, supplemented with cOmplete™ protease inhibitor cocktail (Roche) according to the manufacturer's specifications). The cell suspension was then subjected to a two-step process to complete lysis: i) sonication via a probe sonicator for 1 min at medium intensity, followed by a 1 min incubation step on ice. ii) 250 µl of glass beads (1 mm diameter) were added to each sample, and samples were subjected to 4 repetitions of the following bead-beating protocol using a Retsch MM400 oscillation mill: 4 min of milling at 30 Hz, followed by a 1 min incubation step on ice. After lysis, samples were subjected to a clarifying spin and protein concentration was determined via BCA assay (Thermo Pierce). Next, 50 µg of each sample were transferred to a new tube and diluted in lysis buffer to a concentration of 1 mg/mL. To reduce and alkylate cysteine residues, samples were adjusted to 10 mM TCEP and 40 mM chloroacetamide and incubated for 30 min at ambient temperature. Subsequently, sample were diluted in 100 mM Tris pH 8 to a urea concentration of 4 M, followed by the addition of 1 µg LysC (Wako Chemicals) and a four hour incubation at ambient temperature. For o/n tryptic digestion at ambient temperature, 50 µg of trypsin (Promega) were added after diluting samples further down to a urea concentration of 1 M. Next morning, digest was stopped by adjusting samples to 1% TFA and peptides were purified through Strata-X solid phase extraction cartridges (Phenomenex). Peptide eluates were then dried in a vacuum concentrator and afterwards resuspended in 0.2% FA to a concentration of 1 mg/ml, ready for MS analysis.

LC-MS analysis

For LC-MS analysis, the following setup was employed: a Vanquish Neo UHPLC System was coupled to an Orbitrap Astral mass spectrometer via a Nanospray Flex ionization source (all Thermo Scientific), operated at a source voltage of 2 kV. The Vanquish Neo was equipped with a 40 cm fused silica capillary column (75 μm i.d. and 360 μm o.d., Polymicro Technologies) and pulled, etched and packed in-house using 1.7 µm C18 particles (Waters) as described previously (72). Individual MS experiments were conducted by separating 1 µg of peptides at a flow rate of 300 nL/min at 55°C via a 2 h gradient (Mobile phase A: 0.2% FA, mobile phase B: 0.2% FA, 80% acetonitrile). MS experiments were conducted using a data-dependent acquisition (DDA) regime combining Orbitrap (MS1) and ion trap (MS2) scans under the following parameters: MS1 scans were recorded at a resolution of 240k, a scan range of 300-1350 m/z and a normalized AGC target of 250% with a maximum injection time of 50 ms. MS2 scan were recorded with an isolation window of 0.5 m/z, an HCD collision energy of 23%, at a “Turbo” scan rate speed, a scan range of 150-1350 m/z and a normalized AGC target of 250% with a maximum injection time of 14 ms. MIPS, charge state and dynamic exclusion filters were employed.

Proteomics data processing and analysis

LC-MS analysis resulted in 12 Thermo RAW files (2 strains x 2 growth conditions x 3 biological replicates), which were processed with MaxQuant, version 2.4.2.0 (73). MaxQuant was run using the default settings, with the following specifications and changes: i) RAW files were searched against the E.coli Uniprot reference proteome (Organism ID: 83333, downloaded in Sep 2023). All 12 files were searched together but separated into four experiments with three biological replicates per experiment. ii) Under Group-specific parameters, LFQ was enabled. Under Global parameters, Min. unique peptides was set to 1 and Match between runs as well as iBAQ were enabled. MaxQuant output files were subsequently analyzed via Perseus, version 2.0.11.0 (74). In Perseus, LFQ intensities were log-transformed, followed by a data filtering step, requiring three out of three valid LFQ intensity values for at least one of the four experiments. Next, missing values were imputed from a normal distribution using Perseus’ default settings. Differences across experiments were then assessed via a two-sided two-sample t-test. To address the multiple testing problem, a permutation-based false discovery rate calculation based on 250 randomizations was included. Our analysis yielded mean log2 LFQ intensity ratios, p-values and q-values for 2,468 protein groups.

RNA-seq sample growth and sequencing

∆tolC and ∆tolC tufA::kan cells were grown overnight, back diluted 100-fold into fresh LB, and grown to an OD₆₀₀ of ~0.2. Cells were then diluted 100-fold into 100 mL fresh LB ± 3.5 µM NMM. Cells were then grown to an OD₆₀₀ of ~0.2-0.4. Cells were harvested via centrifugation at 3214 x g at 4 ºC for 10 minutes. Pellets were transferred to 1.7 mL tubes and flash frozen in LN₂and stored at -80 ºC.

Pellets were submitted to Genewiz for RNA extraction and library preparation using their RNA-seq with rRNA depletion package. Sequencing was done using the Illumina 2x150 bp platform targeting 20 million paired-end reads per sample. Data analysis was done through Genewiz using DeSeq2 to normalize datasets and generate plots shown in Figure S7 (75). P-values were determined via the Wald test p-value and adjusted p-values were determined via the Benjamini-Hochberg adjusted p-value.

Sucrose gradients for polysome traces

∆tolC and ∆tolC tufA::kan cells were grown in 50 mL of LB overnight. Cultures were then back diluted 100-fold into fresh LB, grown to an OD₆₀₀ of ~0.2, and then diluted 100-fold into 1 L of LB ± 3.5 µM NMM. Cells were then grown to an OD₆₀₀of ~0.3-0.5. Cells were harvested via filtration with 0.45 µm filter (Whatman) and stored at -80 ºC.

Cells were then lysed via cryomilling in 1 mL of lysis buffer (20 mM Tris pH 8.0, 10 mM MgCl₂, 100 mM NH₄Cl, 5 mM CaCl₂, 100 U/mL DNase I, 1 mM chloramphenicol) at 10 s^-1 for 1 minute three times. Cells were clarified via centrifugation at 20,000 x g at 4 ºC.

For sucrose gradients, ~12.5 AU of RNA were loaded onto a 10-50% sucrose gradient (20 mM Tris pH 8.0, 15 mM MgCl₂, 100 mM NH₄Cl, 2 mM DTT) prepared using the Biocomp gradient station. Sucrose gradients were ultracentrifuged at 201,000 x g at 4 ºC for 2.5 hours at maximum acceleration and deceleration in the SW 41 Ti rotor. Sucrose gradients were then fractionated on the Biocomp Gradient Station and A₂₆₀ measurements were monitored. Sucrose gradients were done in triplicate.

To analyze the area under the curve (AUC) corresponding to the small subunit, large subunit, monosomes, and polysomes, the script from (76) was used in RStudio to quantify the fraction of the area under the curve for each component. This was done for each sucrose gradient and the AUC percentages were plotted in Prism (10.2.0). P values were determined using Welch’s two-tailed t-test.

Altering translation allows E. coli to overcome G-quadruplex stabilizers

Data files

Abstract

README: Altering translation allows E. coli to overcome chemically stabilized G-quadruplexes

Description of the data and file structure

Files and variables

File: Western_total_protein_stain_1.tif

File: Western_Blot_1_from_Figure.tif

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File: Growth_Curves.txt

File: Proteomics_Data.xlsx

File: Proteomics_Comparison.xlsx

File: RNA_seq_raw_counts.csv

File: RNA_seq_TPM_values.csv

File: Consolidated_polysome_data.xlsx

File: Polysome_traces_AUC.xlsx

File: TNseq_original.xlsx

File: TNseq_passage_1.xlsx

File: TNseq_passage_2.xlsx

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File: dtolC_tufA_tufB_used_in_figure_2_062620_5um_nmm_pub.tif

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File: CRISPRi_screen.pdf

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