Bacterial strains and primers

Cells were grown in M9 glucose medium (47.75 mM of Na 2 HPO 4 , 22.04 mM of KH 2 PO 4 , 8.56 mM of NaCl, 18.70 mM of NH 4 Cl, 2 mM of MgSO 4 , 0.1 mM of CaCl 2 , and 2 g l−1 of glucose), unless stated otherwise. Isoleucine or valine was supplemented with a final concentration of 0.15 mM, when appropriate. Primers and probes used in this study are listed in Supplementary Table 2. Uncropped and unprocessed gel images are provided in Source Data. Bacterial strains and plasmids used in this study are summarized in Supplementary Table 3.

ALE and strain isolation

Escherichia coli MS56 was grown in 50 ml of M9 glucose medium in a 250-ml Erlenmeyer flat-bottom flask at 37 °C with agitation. To support the growth of MS56 in M9 glucose medium, 0.1% LB medium was supplemented initially. The supplementation was reduced in a stepwise manner to eventually achieve supplement-free growth. Batch cultures were manually transferred to fresh medium every 12 h at an initial optical density at 600 nm (OD 600 nm ) of approximately 0.005. Number of cell divisions during ALE was calculated from final and initial cell densities according to the Eq. (1):

$${\mathrm{Number}}\;{\mathrm{of}}\;{\mathrm{generation}} = \log _2\frac{{{\mathrm{Final}}\;{\mathrm{cell}}\;{\mathrm{density}}}}{{\mathrm{Initial}\;{\mathrm{cell}}\;{\mathrm{density}}}}.$$ (1)

After ALE, single clones were isolated on M9 glucose agar medium. Because the clones showed equivalent growth rate, one of the clones was selected for further analyses and experiments (named eMS57 from ALE of MS56 and eMG1655 from ALE of MG1655).

Electron microscopy

For scanning electron microscopy (SEM), 1 ml of exponential phase culture was prefixed in 2.5% paraformaldehyde–glutaraldehyde mixture buffered with 0.1 M phosphate buffer (pH 7.2) at 4 °C for 2 h. Next, the prefixed sample was treated with 1% osmium tetroxide solution buffered with 0.1 M phosphate buffer (pH 7.2) for 1 h at room temperature (25 °C). The fixed sample was dehydrated in graded ethanol, substituted by isoamyl acetate, and critical point-dried in liquid CO 2 . The sample was finally sputter-coated with gold in a Sputter Coater SC502 (Polaron, Quorum Technologies, East Sussex, UK) to 20 nm thickness and SEM images were obtained using the FEI Quanta 250 FEG SEM (FEI, Hillsboro, OR, USA) installed at the Korea Research Institute of Bioscience and Biotechnology at a 10-kV acceleration voltage. For transmission electron microscopy, a sample fixed using the same method as used for SEM imaging was dehydrated in graded ethanol, substituted with propylene oxide, and embedded in Epon-812 resin for 36 h at 60 °C. The embedded sample was ultra-sectioned with an Ultracut E Ultramicrotome (Leica, Wetzlar, Germany) and double-stained with uranyl acetate and lead citrate. The sample was examined under a CM20 transmission electron microscope (Philips, Amsterdam, Netherlands) installed at the Korea Research Institute of Bioscience and Biotechnology at a 100-kV acceleration voltage.

Phenotype microarray

Cells were streaked on Biolog Universal Growth Agar (Biolog) plates and grown overnight at 37 °C. Then, cells were resuspended and diluted with Inoculating Fluid A (80% IF-0a GN/GP Base in sterile water; Biolog) to 42% transmittance (T) measured using a Turbidimeter (Biolog). A 42% T cell resuspension was diluted with Inoculating Fluid B (83.33% IF-0a GN/GP Base and 1.2% Biolog Redox Dye mix A in sterile water) to generate 85% T cell resuspension. For PM plate 3B and 4A, 19.8 mM of sodium succinate and 1.98 nM of ferric citrate were added to Inoculating Fluid B as carbon sources. Finally, 100 μl of the 85% T cell resuspension was inoculated onto PM plates and cellular respiration was measured using an Omnilog instrument (Biolog).

Fed-batch fermentation

Fed-batch fermentation was conducted in a 2-L stirred-tank reactor containing 1 L of LB or M9 glucose medium at 37 °C. The culture was aerated with 1 bar compressed air with a rate of 200 ml min−1 and agitated by pitched-blade impellers with speed controlled from 1000 to 1800rpm so as pO 2 was not to drop below 90% saturation. Feeding solution (50% glucose (wv−1), 23.65 mM MgSO 4 , and 8.16 mM CaCl 2 ) was added at a rate of 20 ml hr−1 to support exponential growth. Antifoam 204 (Sigma) and 2 M NaOH were added to remove excess foam and maintain pH of the medium.

Whole-genome resequencing

Genomic DNA of E. coli was isolated using a Wizard Genomic DNA Purification Kit (Promega) according to the manufacturer’s instruction. Briefly, cells were collected by centrifugation and lysed by incubating at 80 °C for 5 min with Nuclei Lysis Solution. The cell lysate was incubated at 37 °C for 15 min with 1.2 μg of RNase A (Qiagen). Protein was precipitated with Protein Precipitation Solution and genomic DNA in the supernatant was purified by isopropanol/ethanol precipitation. Finally, DNA was rehydrated by distilled water. The resequencing library was constructed from the isolated genomic DNA using the TruSeq DNA LT Sample Prep Kit (Illumina) according to the manufacturer’s instruction. Then, the library was sequenced using a MiSeq Reagent Kit v2 (Illumina) in a 50 cycle single-ended reaction on the MiSeq instrument (Illumina).

Determination of clonal lineages in the ALE population

Sequence variants were mathematically clustered according to their time-course allelic frequencies. Sequence variants with allelic frequency >0.8 once or >0.5 at more than two time points were subjected to hierarchical clustering. Clustering was performed using the SciPy clustering package44. Complete and Euclidean methods were used for linkage calculation. Variants within a Euclidean distance of 125 or less were regarded as being in a cluster. Clusters that cannot withstand together in a population (sum of allelic frequencies exceed 100%) were regarded as sub-clusters.

Pyruvate transporter assay

The pyruvate uptake function of a cell was determined with a method used in previous studies with slight modifications17,18. Escherichia coli cells were grown in LB medium at 37 °C for 8 h. The cells were washed twice with M9 pyruvate medium (M9 minimal medium supplemented with 2 g l−1of pyruvate). Then, the washed cells were grown overnight in M9 pyruvate medium at 37 °C with an initial OD 600 nm of 0.05. Overnight cultures were washed twice with M9 sorbitol medium (M9 minimal medium supplemented with 2 g l−1 of sorbitol) and inoculated in 30 ml of M9 sorbitol medium with initial OD 600 nm of 0.05. The culture was grown for one doubling (OD 600 nm of 0.1) at 37 °C. Then, each 400 μl of the grown culture was transferred into a 48-well microplate and 3-FP was added to 1 mM final concentration. The plate was incubated at 37 °C with constant double orbital shaking (5 mm of amplitude) and OD 600 nm was recorded using a Synergy H1 microplate reader (BioTek).

Intracellular and extracellular pyruvate assay

Cells were grown in M9 glucose medium at 37 °C. Then, 1 ml of culture was centrifuged briefly and the supernatant was collected for extracellular pyruvate. Intracellular pyruvate sample was prepared as described in the previous study45. Briefly, 5 ml of culture was collected and immediately quenched with 5 ml of quenching solution (40% ethanol (vv−1) 0.8% NaCl (wv−1)) pre-chilled to −35 °C. Quenched cells were collected when the temperature reached −5 °C by centrifugation at −11 °C, 3400 × g for 10 min. Then, the cell pellet was resuspended with 500 μl of methanol pre-chilled to −80 °C. The resuspension was flash frozen and thawed three times with liquid nitrogen to lyse cells. Lysed cells were centrifuged at 4 °C, 10,000 × g for 2 min and the supernatant was collected. Methanol extraction was repeated for the remaining pellet to extract pyruvate completely. The two methanol extracts were combined. Pyruvate concentration was measured using an EnzyChromTM Pyruvate Assay Kit (Bioassay Systems) according to the manufacturer’s protocol.

Construction of MS56 Δ21kb and ΔrpoS strain

Target regions were knocked out using lambda recombination of MS5646. A kanamycin resistance cassette was PCR amplified from pKD1346 using primers with homology to the target region. The DNA cassette (1 μg) was introduced to electrocompetent MS56 harboring pKD46 via electroporation on MicroPulser (Bio-Rad) with 1.8 kV in a 0.1-cm gap cuvette and a recipient was selected by incubating at 30 °C on LB agar medium containing 50 μg ml−1 of kanamycin. The electrocompetent MS56 was prepared by washing MS56 (carrying pKD46) grown to OD 600 nm of 0.4 at 30 °C in 50 ml of LB medium containing 10 mM of arabinose three times with ice-cold 10% glycerol. The knockout was confirmed by Sanger sequencing. To remove the kanamycin resistance cassette, pCP20 was introduced to the strain by electroporation. A clone carrying pCP20 was selected by incubating 30 °C on LB agar medium containing 34 μg ml−1 of chloramphenicol. A clone carrying pCP20 was then inoculated in 3 ml of liquid LB medium and incubated at 42 °C for overnight with agitation. The overnight grown culture was streaked on LB agar medium and few colonies were subjected to replica plating on LB agar plates containing ampicillin or kanamycin. A colony sensitive to both kanamycin and ampicillin is free of the plasmids and kanamycin resistance cassette and thus propagated further.

Western blot analysis

Cell lysate containing 40 μg of protein was separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Separated protein was transferred to polyvinylidene difluoride membrane using a Trans-blot Turbo Transfer System (Bio-Rad). Membranes were incubated for 2 h in blocking buffer (5% skimmed milk in Tris-buffered saline with 0.1% Tween-20; TBST) with gentle shaking at room temperature. Blocked membranes were washed three times with TBST, each for 10 min of gentle shaking at room temperature. Appropriate primary antibody (anti-RpoD mouse IgG; NeoClone; Cat. #663202; Lot. #B193929 or anti-MutS rabbit polyclonal; GeneCheck; Cat. #GC-M001; Lot. #219) diluted to 20 μg ml−1 of final concentration in dilution buffer (1% skimmed milk in TBST) was added on washed membranes. Membranes were incubated at 4 °C overnight with gentle shaking. Then, membranes were washed three times with TBST and an appropriate secondary antibody (horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG antibody (Thermo), Cat. #31430; or HRP-conjugated goat anti-rabbit IgG (Thermo), Cat. #31460) with a final concentration of 80 ng ml−1 was applied for 2 h at room temperature with gentle shaking. Bands were detected with Pierce ECL Plus Western Blotting Substrate (Thermo, Cat. #32132) using a ChemiDoc Imaging System (Bio-Rad).

Construction of the eMS57mutS+ strain

The native mutS locus was PCR amplified from MG1655 genomic DNA using MutS_Tn_F and MutS_neo_R primers. The amplified mutS DNA fragment was connected with a kanamycin resistance DNA cassette of pKD13 (PCR amplified with primers MutS_neo_F and Neo_Tn_R) using overlap extension PCR (OE-PCR) as follows: 5 ng each of MutS cassette and kanamycin cassette were first ligated by 15 cycles of the following PCR reaction: 96 °C for 30 s, 56 °C for 30 s, and 72 °C for 3 min. Ligated product was amplified in the same tube by adding two outermost primers (MutS_Tn_F and Neo_Tn_R) and 20 cycles of PCR reaction: 96 °C for 30 s, 56 °C for 30 s, and 72 °C for 4 min. Ligated product was cloned into pMOD3 with EcoRI and XbaI restriction ligation, generating pMOD3-MutS plasmid in E. coli BW25113 strain. Transposon DNA containing mosaic end (ME) sequence was PCR amplified from pMOD3-MutS using ME_plus_3′ and ME_plus_5′ primers. Then, 50 ng of transposon DNA and 1 U of EZ-Tn5 transposase (Epicentre) were mixed and incubated at room temperature for 30 min to construct a transposome complex, which was introduced into eMS57 and a transposed clone was selected by a kanamycin-selective medium. Expression of MutS was confirmed by quantitative reverse transcription-PCR (qRT-PCR) and western blotting (Supplementary Figure 5). A clone with the most similar expression level of MutS, when compared to MG1655, was chosen and termed as eMS57mutS+. The location of transposon insertion was determined from semi-random PCR using Tn_confirm_F and Random_R1 primers. The eMS57mutS+ had the mutS expression cassette inserted in putrescine symporter, PuuP (Supplementary Figure 5).

Introduction of point mutation on MS56 genome

Homologous lambda recombination was used to introduce point mutations in the MS56 genome46. The OE-PCR product of the kanamycin resistance gene cassette (amplified from pKD1346) and target gene (amplified from MS56 or eMS56) was introduced into MS56 harboring pKD4646 using electroporation as described above. Colonies grown on a kanamycin-selective medium were isolated and confirmed by Sanger sequencing. Constructed strains possessing a wild-type or mutant gene with the kanamycin cassette were compared.

TaqMan assay coupled with dPCR

The concentration of genomic DNA was measured by NanoDrop 2000 Spectrophotometer (Thermo) and diluted to a concentration of 20,000−30,000 copies ml−1. Target genes were amplified with QuantStudio 3D Digital PCR System (Thermo) as follows. Each PCR reaction was set up total 20 μl reaction volume, comprised of 10 μl of 2× QuantStudio 3D Digital PCR Master Mix (Thermo), 0.5 μl of 40× TaqMan Probe/Primer Mix (8 μM each of reporters and 36 μM each of primers, Supplementary Table 2), and 1 μl of diluted gDNA. Mixed PCR reaction was dispensed into a QuantStudio 3D Digital PCR 20 K Chip (Thermo) according to manufacturer’s instruction using a QuantStudio 3D Digital PCR Chip Loader (Thermo). After dispensing, the chip was filled with QuantStudio 3D Digital PCR Immersion Fluid (Thermo) and sealed with a QuantStudio 3D Digital PCR Chip Lid. PCR was performed by flat block thermal cycler GeneAmp PCR System 9700 (Thermo) equipped with a QuantStudio™ 3D Digital PCR Chip Adapter Kit (Thermo) and QuantStudio 3D Tilt Base (Thermo) according to the following program: 96 °C for 10 min, 56 °C for 2 min, 98 °C for 30 s, repeat the last two steps for 44 cycles, and 56 °C for 2 min for final extension. The image of the chip was read using a QuantStudio 3D Digital PCR instrument (Thermo) and analyzed by QuantStudio 3D AnalysisSuite Cloud Software (Thermo, https://apps.thermofisher.com/quantstudio3d/). Raw data were extracted and visualized in Microsoft Excel 2010 (Microsoft) and Adobe Illustrator CS6 (Adobe) without modifying data integrity.

Chromatin immunoprecipitation sequencing

Escherichia coli strains MG1655 and eMS57 were grown in 50 ml of M9 minimal medium at 37 °C with agitation. Cells were sampled at mid-log phase (OD 600 nm was approximately 0.55 and 0.50 MG1655 and eMS57, respectively). Then, 1.4 ml of 37% formaldehyde solution was added to 50 ml of culture and incubated for 25 min at room temperature with gentle shaking. Next, 2 ml of 2.5 M glycine solution was added to fixed cell culture and incubated for 5 min at room temperature with gentle shaking. The culture was washed three times with 50 ml of ice-cold TBS. After centrifugation at 4 °C, 3000 × g for 15 min, all the supernatants were removed and the cell pellet was resuspended with 1.5 ml of lysis buffer (10 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 1 mM EDTA). Forty microliters of Protease Inhibitor Cocktail (freshly prepared as per the manufacturer’s manual, Sigma) and 0.3 μl (10,500U) of Ready-Lyse Lysozyme (Epicentre) were added and incubated at 37 °C for 2 h with gentle rotation. Subsequently, 1.65 ml of IP buffer (100 mM Tris-HCl, pH 7.5, 200 mM NaCl, 2% Triton X-100, and 1 mM EDTA) was added and the sample was incubated at 4 °C with gentle rotation for 1 h. Genomic DNA was sheared by sonicating for a total of 2 min (amplitude was 13%, on sonic for 20 s, and resting for 40 s, 6 cycles) using a Sonic Dismembrator Model 500 (Fisher Scientific) equipped with micro-tip (diameter of 3 mm). A total of 700 μl homogenized sample was used for immunoprecipitation. To this, 12 μg of anti-RpoD mouse IgG (NeoClone, Cat. #663202, Lot. #B193929), anti-c-Myc mouse IgG (Santa Cruz Biotechnology; Cat. #sc-40; Lot. #B1313), or normal mouse IgG (Millipore, Cat. #12-371, Lot. #DAM1774722) was added individually and incubated overnight at 4 °C with gentle rotation. Antibody-coupled chromatin solution was added to 50 μl of Dynabeads Protein A (Life Technologies) for anti-RpoD mouse IgG sample or Dynabeads Pan Mouse IgG (Life Technologies) for anti-c-Myc mouse IgG or normal mouse IgG sample pre-washed with 1 ml of ice-cold bead washing solution (0.5% bovine serum albumin in phosphate-buffered saline (PBS)), and incubated at 4 °C for 6 h with gentle rotation. Beads were pooled down with sequential steps on a magnetic stand with washing buffer I (50 mM Tris-HCl (pH 7.5), 140 mM NaCl, 1% Triton X-100, and 1 mM EDTA) twice, washing buffer II (50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 1% Triton X-100, and 1 mM EDTA), washing buffer III (10 mM Tris-HCl, pH 8.0, 250 mM LiCl, 1% Triton X-100, and 1 mM EDTA), and TE buffer. Two hundred microliters of elution buffer (50 mM Tris-HCl, pH 8.0, 1% SDS, and 1 mM EDTA) was added to washed beads and incubated overnight at 65 °C for elution and reverse crosslinking. Beads were pooled down by magnetic stand and cleared eluate was isolated. Eluate was purified using the MinElute PCR Purification Kit (Qiagen) after treatment with 100 μg of RNase A (Qiagen) and 80 μg of Protease K (Invitrogen). Purified DNA was subjected to qPCR or ChIP-Seq library preparation. A sequencing library was prepared using a NEXTflex Illumina ChIP-Seq Library Prep Kit (Bioo Scientific) according to the manufacturer’s instruction. Constructed sequencing libraries were quantified using a Qubit dsDNA HS Assay Kit (Thermo) with a Qubit 2.0 fluorometer (Thermo). Quality of libraries was analyzed using TapeStation 2200 (Agilent) equipped with High Sensitivity D1000 Screen Tape (Agilent). The sequencing libraries were sequenced using a MiSeq Reagent Kit v2 (Illumina) with 50 cycle single-ended reaction in the MiSeq instrument (Illumina).

Heterologous expression of native and mutant σ70

Native or mutant rpoD was PCR amplified from MG1655 or eMS57 genomic DNA, respectively, using rpoD_F and rpoD_R primers. Plasmid backbone was also PCR amplified from pTrcHis2A plasmid (Invitrogen) using pTrc_inv_F and pTrc_inv_R primers. Two PCR products were combined using In-Fusion HD Cloning Kit (Takara) as per the manufacturer’s instructions. Two tandem c-Myc epitope was fused at the N terminus of rpoD by rpoD_R and pTrc_inv_F primers.

Transcriptome sequencing (RNA-Seq)

Cell culture samples (10 ml) were harvested at the mid-log growth phase (OD 600 nm ~0.55 for MG1655, ~0.50 for eMS57). Total RNA was isolated using the RNASnapTM method47. Briefly, the harvested cell pellet was resuspended with 100 μl of RNASnap solution (18 mM of EDTA, 0.025% of SDS, 1% of β-mercaptoethanol, and 95% of formamide). The resuspension was incubated at 95 °C for 7 min and centrifuged at 16,000 × g for 5 min. A clear supernatant was transferred to a new microcentrifuge tube and RNA was extracted by ethanol precipitation. Total RNA (5 μg) was treated with 2 U of DNase I (NEB) for 30 min at 37 °C to remove residual DNA contaminants. DNase I-treated RNA samples were purified with phenol–chloroform–isoamyl alcohol extraction followed by ethanol precipitation. Ribosomal RNA was removed from 2 μg of purified total RNA samples using a RiboZero rRNA (ribosomal RNA) Removal Kit (for bacteria, Illumina) according to the manufacturer’s instruction. RNA-Seq libraries were constructed from rRNA-subtracted RNA using a TruSeq Stranded mRNA LT Sample Prep Kit (Illumina) according to the manufacturer’s protocol. Constructed sequencing libraries were quantified using a Qubit dsDNA HS Assay Kit (Thermo) with a Qubit 2.0 fluorometer (Thermo). Quality of libraries was analyzed using a TapeStation 2200 (Agilent) equipped with High Sensitivity D1000 Screen Tape (Agilent). The sequencing libraries were sequenced using a MiSeq Reagent Kit v2 (Illumina) with 50 cycles single-ended reaction in the MiSeq instrument (Illumina).

qPCR and qRT-PCR

RT was performed from 1 μg of total RNA in 20 μl reaction using the SuperScript III First-Strand Synthesis System (Thermo) according to the manufacturer’s instruction. Briefly, 1 μg of DNA-subtracted RNA, 50 ng of Random Hexamer, 1 μl of 10 mM dNTP mix, and diethyl pyrocarbonate-treated water to bring reaction volume to 10 μl were mixed in RNase-free PCR tube. The mixture was incubated at 65 °C for 5 min and placed on ice immediately after incubation. Ten microliters of cDNA Synthesis Mix (2 μl of 10× RT buffer, 4 μl of 25 mM MgCl 2 , 2 μl of 0.1 M dithiothreitol, 1 μl of RNaseOUT, and 200 U of SuperScript III RT) was added and incubated at 25 °C for 10 min. Then, the mixture was incubated 50 °C for 50 min followed by 85 °C for 5 min. One microliter (2U) of E. coli RNase H was treated at 37 °C for 20 min to remove RNA. qPCR was performed in 20 μl reaction (10 μl of iQ SYBR Green Supermix (Bio-Rad), 10pmol of forward and reverse primers, 1 μl of cDNA or immunoprecipitated DNA) with the following conditions: 40 cycles of 95 °C for 10 s, 58 °C for 30 s, and 72 °C for 30 s. Reactions were monitored on a C1000 Thermal Cycler (Bio-Rad) equipped with a CFX96 Real-Time PCR Detection System (Bio-Rad). All the primers were designed by Primer-BLAST48 and there was no non-specific binding found in E. coli K-12 MG1655 genome sequence (Acc. NC_000913.3). Sequence of primers and size of amplicons are summarized in Supplementary Table 2. In ChIP-qPCR experiment, promoter region (peak region) was targeted for amplification and ΔΔCq method was used for quantification using geometric mean of four reference peaks (hpt, nrdR, yebS, and yecD) as an internal reference point49.

Differential RNA-Seq and KEGG pathway enrichment analysis

Genes with a P value lower than 0.01 (two-sided Welch’s t test) were subjected to enrichment analysis of differential expression44. The analysis was performed using ClueGO plug-in (v2.2.4) of Cytoscape (v3.3.0).

Cellular NADH/NAD+ and NADPH/NADP+ assay

NADH/NAD+ and NADPH /NADP+ ratios were determined using a NAD/NADH Quantification Kit (Sigma-Aldrich) and NADP/NADPH Quantification Kit (Sigma-Aldrich), respectively, according to the manufacturer’s instruction. Briefly, 2 × 109 and 5 × 109 cells were collected for NADH/NAD+ and NADPH/NADP+ assays, respectively. Samples were washed twice with ice-cold PBS buffer and resuspended with NAD/NADH or NADP/NADPH extraction buffer. The samples were flash frozen in liquid nitrogen and ground by a pestle and mortar. The lysates were collected and divided into two conical tubes. One was incubated at 60 °C for 30 min to deplete NAD(P)+. Then, Cycling Enzyme Mix was applied at room temperature for 5 min to convert NAD(P)+ to NAD(P)H. Absorbance at 450 nm was measured after adding NAD(P)H Developer using a Synergy H1 microplate reader (BioTek).

Cellular ATP assay

Intracellular ATP concentration was determined using an ATP Bioluminescence Assay Kit HS II (Roche) according to the manufacturer’s instruction. First, 5 × 108 cells were collected and washed twice with ice-cold PBS buffer. Then, the pellet was resuspended in 500 μl of Dilution Buffer and lysed by Cell Lysis Reagent. The samples were transferred to a Nunc F96 MicroWell Black Polystyrene Plate (Nunclon). Luminescence was measured using a Synergy H1 microplate reader immediately after adding Luciferase Reagent.

Ribosome profiling

Ribosome profiling was conducted using a method described in the previous report50 without tRNA removal step. Briefly, 50 ml of E. coli culture was collected after 5 min treatment of chloramphenicol (34 mg ml−1) at an exponential growth phase. Cells were flash frozen with 0.5 ml of lysis buffer (1% Triton X-100, 34μgml−1 chloramphenicol, 133 mM of NaCl, 4.75 mM of MgCl 2 , and 19 mM of Tris-HCl, pH 7.5) and lysed by pestle and mortar. Then, the supernatant containing 10μg of RNA treated with 2000 gel units of Micrococcal Nuclease (NEB). Polysomes were recovered from MNase-digested sample using Illustra MicroSpin S-400 HR Columns (GE Healthcare) followed by phenol:chloroform:isoamyl alcohol extraction. Ribosomal RNA was removed from 5μg of polysome-protected RNA using a RiboZero rRNA Removal Kit (Illumina) according to the manufacturer’s instruction. rRNA-subtracted RNA samples were phosphorylated by treating 10U of T4 Polynucleotide Kinase (NEB) at 37 °C for an hour and purified with RNeasy MinElute columns (Qiagen). Sequencing libraries were prepared from phosphorylated RNA samples using the NEBNext Small RNA Library Prep Set for Illumina (NEB) according to the manufacturer’s protocol. Next-generation sequencing was performed by the ChunLab (Seoul, South Korea) with high output mode using V4 sequencing-by-synthesis reagent on a HiSeq 2500 instrument.

Measurement of protein production via flow cytometry

Cells harboring high copy mRFP1 expression plasmid, BBa_J04450-pSB1C3, were grown aerobically in M9 glucose medium for 12 h at 37 °C, with 0.5 mM of isopropyl β-d-1-thiogalactopyranoside induction. Then, 1 ml of the cell culture was diluted in 9 ml of PBS and cells were dissociated using a round bottom polystyrene test tube with cell strainer snap cap (Corning). Then, samples were analyzed on an S3e Cell Sorter (Bio-Rad). A total of 100,000 events were collected and analyzed by the FlowJo software (FlowJo). Contour plots showing gating strategy are provided in Supplementary Figure 19.

Data processing

Sequencing data was processed on a CLC Genomics Workbench (CLC Bio). Raw reads were trimmed using a Trim Sequence Tool in NGS Core Tools with a quality limit of 0.05. Reads with more than two ambiguous nucleotides were discarded and the quality trimmed reads were mapped on the MG1655 reference genome (NC_000913.3) or MS56 genome sequence (http://cholab.or.kr/data/) with the following parameters: mismatch cost: 2, indel cost: 3, length and similarity fractions: 0.9. For RNA-Seq and ribosome profiling, reads were mapped strand specifically (backward). Reads mapped to the multiple genomic positions were mapped randomly for resequencing and ChIP-Seq and discarded in RNA-Seq and ribosome profiling analysis. Sequence variants were detected using a Quality-based Variant Detection Tool with the following parameters: neighborhood radius: 5; maximum gap and mismatch counts: 5; minimum neighborhood and central qualities: 30; minimum coverage: 10, minimum variant frequency: 10%, and maximum expected alleles: 4. Non-specific matches were ignored and bacterial genetic code was used. Variants in repeat region (i.e., rRNAs and transposases) were discarded. Mutation rate (mutations per genome per generation) was calculated as the number of mutation (including SNVs, MNVs, and indels) divided by cumulative generation. Binding peaks of RpoD were detected from a mapping file using the Model-based analysis of ChIP-Seq (MACS) software with shiftsize of 5051. Detected peaks were compared with RpoD binding regions in the RegulonDB database30. A promoter motif was found using the MEME Suite software (v4.11.4)52. Expression and RPF levels were normalized by reads per kilobase per million mapped reads (RPKM) metric. The mapping file was exported as BAM file format and converted to GFF file format and visualized by the SignalMap software (v2.0.0.5, Roche). TE was obtained by dividing RPF by RNA expression. In the meta-analysis of a ribosome profile, either 5′ or 3′ ends were tested to determine a position of ribosome (Supplementary Figure 17)53,54. 5′ Assignment method was used for the meta-analysis, because the method provides clearer 3-nt codon periodicity of translation than 3′ assignment.

Determination of σ70 binding peaks from ChIP-Seq

ChIP-Seq detects DNA fragments that crosslinked to the target protein. The MACS software was used to determine the binding sites of σ7051. After analysis with MACS, we obtained 1062 and 1089 raw binding sites from MG1655 and eMS57 samples. The summit of each binding signal was defined as a peak. The raw reads were further adjusted with the RegulonDB database for maximum accuracy and precision30. In adjustment, all of the raw binding sites were compared with 2055 RegulonDB binding sites. However, we had to determine a certain length threshold that determined whether two binding sites are the same. Thus, various lengths of sliding windows ranges from 10 to 150nt were examined (Supplementary Figure 8). As the length of the sliding window increases, the number of binding sites retrieved increased. The number of retrieved binding sites divided by the number of raw binding sites detected by MACS (termed retrieval rate hereafter) reached a plateau at a sliding window of 80nt or longer. Simultaneously, the number of peaks that were bound by both wild-type and mutant σ70 were compared (Supplementary Figure 8). At a sliding window of 80nt, the ratio of shared peaks to total peaks was 0.381 and reached a plateau. The ratio remained unchanged (0.407 at a sliding window of 10,000nt). A starting point of the plateau (80nt) was used as a threshold. Using the threshold, we determined a total of 421 and 418 binding sites from MG1655 and eMS57, respectively (Fig. 3a). Among them, 320 were overlapped.

Normalization of RNA expression level and RPFs

RNA expression and RPFs were normalized by the RPKM metric55. Although there remains some controversy related to RPKM transformed expression56, recent RNA-Seq analysis pipelines could not be applied on two significantly different genomes. The pipelines, such as DESeq2, assume that most of the genes are not differentially expressed57. With thorough inspection of the data, we decided to transform transcriptomic data into RPKM and genes with a wide range of expression were validated with qRT-PCR (Supplementary Figure 10). Furthermore, we believe this method is more reliable when calculating translational efficiency from RNA expression and RPFs that are expressed in the same unit.

Statistical analyses

All bacterial growth measurements were biologically triplicated and their differences were examined by two-sided t test of unequal variance (Welch’s t test). Transcriptional change according to promoter category was tested by Wilcoxon's rank-sum test (Fig. 3). Welch’s t test was used for DEG determination across biologically duplicated sequencing results. All statistical analyses were performed using the statistical analysis of SciPy package44.

Reporting summary

Further information on experimental design is available in the Nature Research Reporting Summary linked to this article.