Systems Analyses Reveal the Resilience of Escherichia coli Physiology during Accumulation and Export of the Nonnative Organic Acid Citramalate

Growth media.Escherichia coli strains were grown in either LB (Melford, United Kingdom), SM or ML media at 37°C. ML medium (1 liter) contained MgSO₄·7H₂O (2 ml; 246.47 g liter⁻¹), salt solution (200 ml) containing K₂HPO₄ (73 g liter⁻¹), NaH₂PO₄·2H₂O (18 g liter⁻¹), (NH₄)₂SO₄ (10 g liter⁻¹), ammonium citrate (2.5 g liter⁻¹), trace elements (2 ml) containing Na₂EDTA·2H₂O (22.3 g liter⁻¹), FeCl₃ (10.03 g liter⁻¹), CaCl₂·2H₂O (0.5 g liter⁻¹), ZnSO₄·7H₂O (0.18 g liter⁻¹), CoCl₂·6H₂O (0.18 g liter⁻¹), CuSO₄·5H₂O (0.16 g liter⁻¹), and MnSO₄·H₂O (0.15 g liter⁻¹) and glucose (the concentration of which, as indicated in the text, was added from a sterile stock solution at 250 g liter⁻¹). When required, carbenicillin was added to the medium at a concentration of 50 mg liter⁻¹. Polypropylene glycol (0.1 ml liter⁻¹) was added to the medium when used in a fermentor. The SM medium (1 liter [34]) contained glycerol (30 ml; 500 g liter⁻¹), yeast extract (50 ml; 100 g liter⁻¹), MgSO₄ (2 ml; 246.47 g liter⁻¹), salt solution (200 ml) containing Na₂HPO₄·12H₂O (75.6 g liter⁻¹), KH₂PO₄ (15 g liter⁻¹), NH₄Cl (5 g liter⁻¹), NaCl (2.5 g liter¹), CaCl₂ (55 mg liter⁻¹), and SM trace element solution (100 μl) containing FeSO₄·7H₂O (80 g liter⁻¹), AlCl₃·6H₂O (10 g liter⁻¹), MnSO₄·H₂O (10 g liter⁻¹), CoCl₂ (4 g liter⁻¹), ZnSO₄·7H₂O (2 g liter⁻¹), Na₂MoO₄·2H₂O (2 g liter⁻¹), CuCl₂·2H₂O (1 g liter⁻¹), and H₃BO₃ (0.5 g liter⁻¹) dissolved in HCl (3 M). Glycerol, yeast extract, MgSO₄·7H₂O and salt solutions were prepared and autoclaved separately. Trace elements were sterilized by filtration using 0.2-μm-pore filters. All solutions were cooled before mixing.

Preparation of E. coli strains.Escherichia coli BW25113 and E. coli BW25113 ldhA were purchased from the Keio collection (35). A control plasmid (pBAD-cimA3.7_dead) expressing an inactivated CimA3.7 by virtue of a His192Ala substitution was constructed using Quickchange II XL site-directed mutagenesis kit (Agilent Technologies). The manufacturer’s protocol was followed with primers (5′-ACCTGCCGGTTAGCGTGGCCTGCCATAACGATTTCGGC-3′ and 5′-GCCGAAATCGTTATGGCAGGCCACGCTAACCGGCAGGT-3′) and pBAD-cimA3.7 (5) as the template. Electro-competent E. coli BW25113 ldhA was transformed to create strains expressing active or inactive CimA; transformants were selected on LB agar supplemented with carbenicillin (50 mg liter⁻¹). Escherichia coli BW25113 ldhA was used in the fermentation processes to minimize lactate formation based on unpublished results (J. Webb, personal communication). Other E. coli BW25113 mutants were also obtained from the Keio collection, except the mscL mscS double mutant, which was constructed using pCP20 (36) to cure the kanamycin cassette from E. coli BW25113 mscL::Kan^r, followed by deletion of mscS from the resulting strain (E. coli BW25113 mscL::FRT) by P1 transduction of the mscL mutation from E. coli BW25113 mscL::Kan^r.

Citramalate production using harvested cells.Single colonies of E. coli BW25113 ldhA expressing either active or inactive CimA3.7 were grown overnight (37°C, 250 rpm) in SM glycerol medium. Cultures were diluted to a starting OD₆₀₀ of 0.1 in SM glycerol medium, and the culture was incubated (250 rpm, 37°C) until the OD₆₀₀ reached 0.6. CimA3.7 expression was induced by the addition of l-arabinose (0.2 g liter⁻¹). Cells were harvested by centrifugation (4,000 × g, 20 min, 4°C) 4 h postinduction (OD₆₀₀ of 3 to 4) and concentrated to a dry cell weight of 15 g liter⁻¹ in SM medium without NH₄Cl or yeast extract but with glucose (20 g liter⁻¹). The cell suspensions (approximately 10 ml) were incubated in baffled flasks (250 ml, 24 h, 250 rpm, 37°C) before analysis of culture supernatants by high-pressure liquid chromatography (HPLC).

Fermentations.Inocula for fed-batch fermentations were produced by inoculating E. coli BW25113 ldhA pBAD24-cimA3.7 or E. coli BW25113 ldhA pBAD24-cimA3.7_dead into ML medium (50 ml) supplemented with glucose (10 g liter⁻¹) and carbenicillin (50 μg ml⁻¹) and incubating overnight (200 rpm, 37°C). The cultures were diluted to OD₆₀₀ of 0.1 in sterile water (50 ml) and used to inoculate ML medium (1 liter) supplemented with glucose (11.9 g liter⁻¹) and carbenicillin in a 3-liter BioFlo/CelliGen 115 bioreactor (New Brunswick Scientific). Cultures were grown at 37°C, and the pH was maintained at 7.0 ± 0.1 by the addition of NH₄OH (28 to 30%) and 2 M H₂SO₄ (2 M). The airflow rate was initially set at 1 liter min⁻¹, and the dissolved oxygen (dO₂) was maintained at 30% of saturation by automatic control of stirrer speed between 400 and 1,200 rpm and an airflow cascade between 1 and 7 liter min⁻¹. The airflow cascade was only implemented when maximum agitation was reached. Drops of polypropylene glycol (100%) were manually added to the medium when required to avoid foaming. When the glucose in the batch medium had been consumed, indicated by a sharp increase in dO₂ and confirmed using glucose test strips (117866; Merck Millipore), a feed of glucose was started containing glucose (650 g liter⁻¹), yeast extract (5.9 g liter⁻¹), MgSO₄ (7.2 g liter⁻¹), and trace elements (11.8 ml). The flow rate was adjusted manually as required to maintain a pseudoexponential growth rate of approximately 0.25 h⁻¹ and avoid the accumulation of excess glucose in the culture, using the test strips as confirmation as described above. Protein expression was induced by the addition of l-arabinose (0.02% wt/vol) when the culture OD₆₀₀ was 50.

Analytical methods.Growth was monitored by measuring OD₆₀₀. Samples were diluted in deionized water when the OD₆₀₀ was >0.8. Dry cell weight was measured by centrifuging 1-ml samples in preweighed polypropylene tubes, removing the supernatant, and drying the pellets to a constant weight. d-Glucose, (R)-citramalate, and other organic acids were quantified by HPLC using an Agilent 1200 series HPLC system equipped with both UV (215 nm) and refractive index detectors. Samples were resolved using a Rezex ROA organic acid H+ column (Phenomenex) at 55°C with 0.01 N H₂SO₄ (0.5 ml min⁻¹) as the mobile phase. Samples were prepared for HPLC analysis by centrifuging (12,000 × g, 5 min) and filtering the supernatants (0.2-μm-pore filter). Data analysis was performed with ChemStation software using calibration curves prepared using authentic standards of each compound (0.1 to 200 mM).

Transcriptomics.Fermentation samples (3 × 0.1 ml) were mixed with RNAprotect (Qiagen [0.2 ml]), vortexed, and incubated at room temperature (5 min). The mixture was then centrifuged (16,000 × g, 2.5 min, 4°C) and the supernatant discarded, and pellets were stored at −80°C. Cell pellets were thawed at room temperature and normalized in TE buffer (10 mM Tris-HCl at pH 8.0, 0.1 mM EDTA), such that 1 ml of TE buffer contained 2 OD₆₀₀ units. Aliquots (1 ml) were harvested by centrifugation (12,000 × g, 1 min), and the pellets were resuspended in 100 μl TE buffer containing lysozyme (15 mg ml⁻¹). These were incubated at room temperature (10 min) with vortexing every 2 min. Total RNA was prepared using the RNeasy RNA purification kit (Qiagen) according to the manufacturer’s protocol (including the on-column DNase treatment step). Labeled cDNA was produced using SuperScriptIII reverse transcriptase (Invitrogen) with the Cy3-dCTP included in the dNTP mixture. Labeled E. coli genomic DNA was produced using the BioPrime DNA labeling kit (Invitrogen) with Cy5-dCTP included in the dNTP mixture. Labeled genomic DNA and cDNA were combined and hybridized overnight to an oligonucleotide microarray (Agilent Technologies). Quantification of cDNA, hybridization of cDNA to microarrays, microarray processing, and microarray scanning were performed as described in the Fairplay III labeling kit (Agilent Technologies, 252009, version 1.1). Microarrays were scanned with a high-resolution microarray scanner (Agilent Technologies). Features with background intensities exceeding 10 times the array median, or with a signal/background ratio below 3 were excluded from further analysis. Background correction (37), within-array Loess normalization (38), and between-array quantile normalization were applied to the remaining features using the R statistical package LIMMA from Bioconductor (39). Moderated t statistics were calculated using gene-wise linear models with an empirical Bayes approach (40, 41). P values were adjusted for multiple testing using the Benjamini-Hochberg method (42). Transcripts exhibiting ≥2-fold change in abundance with an adjusted P value of <0.05 were deemed to be differentially regulated. The data are available in ArrayExpress. The relative activities of transcription factors were inferred using the TFInfer software package as previously described (14).

Metabolomics.Quenching of fermentation samples (0.5 ml) was performed by mixing ethanol solution (40% vol/vol) prepared in NaCl (0.8% wt/vol) at −20°C (43). The mixture was centrifuged (16,000 × g, 2.5 min, 4°C). The supernatant was discarded, and the pellets were stored (−80°C) until required. Cell pellets were resuspended to an OD₆₀₀ of 1 in chloroform-methanol (1:1, −20°C), and 1 ml was transferred to a fresh polypropylene tube. An ice-cold ball bearing was added to each tube before incubation (−80°C, 1 h). The samples were then vortexed twice (30 s) and returned to the −80°C freezer for 1 h. Milli-Q water (400 μl) was added to each sample, followed by vortex mixing (30 s). Samples were then centrifuged (4,000 × g, 1 min, 4°C). The upper layer was transferred to a fresh tube. This step was repeated, and the two organic phases were combined. Electrospray ionization time-of-flight (ESI-TOF) MS was performed on a Hybrid quadrupole time-of-flight (TOF) LC-MS-MS spectrometer (Waters, Ltd., Manchester, United Kingdom) based on the methods described by Davey et al. (44) and Walker (45). Data acquisition and processing were performed on MassLynx (version 4) to create centroid peak lists (m/z accurate to 4 decimal places versus ion counts), which were then transferred to Microsoft Excel (Microsoft Corp., USA) as text files. The mass spectrometer was operated in negative-ion mode at a rate of 1 scan per s for 6 min. Samples were loaded using a syringe pump (Razel, CT, USA) at a flow rate of 20 μl min⁻¹. A Lockspray interface was used to give an external standard and allow automated correction of mass measurements (5 ng liter⁻¹ sulfadimethoxine, giving a lock mass of 309.0653). Samples were analyzed in a randomized order to minimize effects of day-to-day machine variation. Data processing and downstream analysis were performed in R, using Bioconductor package XCMS (46). Peaks were aligned across analytical replicates and grouped into 0.2-m/z-width bins (47). Peaks were rejected if all three replicates were not present or the mass variance fell outside an acceptable range defined as a function of the m/z (formula modified from Overy et al. [48]; H. Walker, personal communication).

Proteomics. (i) Protein extraction and peptide sample preparation.Fermentation samples (500 μl) were harvested by centrifugation (16,000 × g, 2.5 min, 4°C). The supernatant was discarded and the pellet stored (−80°C). Cells were embedded in a 20% polyacrylamide gel matrix and digested and washed following the gel-aided sample preparation (GASP) protocol with three minor changes (49). First, samples were reduced using 10 mM Tris(2-carboxyethyl)phosphine (TCEP); second, they were desalted after gel extraction on Sola C₁₈ cartridges following the manufacturer’s instructions; third, after the samples were dried to near dryness, they were resuspended in 0.1% formic acid for injection into the mass spectrometry-liquid chromatography system. Injections were normalized by a quantitative colorimetric assay (Thermo) that uses a modified bicinchoninic acid (BCA) chemistry for sensitive and accurate peptide sample quantification to keep samples comparable and to optimize signal intensity.

The buffers used for the GASP method were as follows. Lysis was performed on ice (30 min) in a buffer containing urea (6 M), thiourea (1.5 M), SDS (4%), TCEP (10 mM), 20 mM Tris (20 mM, pH 8.0). Alkylation was achieved on ice (30 min) using an equal volume of monomeric acrylamide mix solution (40%, 37.5:1 acrylamide-bisacrylamide solution). Polymerization was initiated at room temperature by addition of N,N,N′,N′-tetramethylethylenediamine (TEMED [5 μl]) and ammonium persulfate (APS [10%]) followed by rapid mixing by vortexing. After polymerization was complete (∼30 min), gel plugs were cut by centrifugation through a membrane-less SpinX centrifugation filter support device (Costar) to achieve identical sizes of gel pieces. Gel pieces were fixed by addition of fixation buffer (1 ml) containing methanol-acetic acid-water (50:40:10) and overhead rotation (10 min). After brief pulse centrifugation, the supernatant was discarded and gel pieces were rehydrated with urea (6 M, 0.5 ml, 10 min). Acetonitrile (1 ml) was used to dehydrate gel pieces. Following removal of the supernatant using a gel loading pipette tip (200-μl-volume pipette tip with a long narrow end to avoid loss of gel pieces), two more rounds of urea and acetonitrile washes were performed to minimize carryover of SDS and other contaminants. After these washes, the gel pieces were pH adjusted using triethylammonium bicarbonate (TEAB [500 μl, 50 mM, pH 8.0]) and rotation (10 min). Gel pieces were dehydrated once more using acetonitrile (1 ml), supernatant was removed, and acetonitrile (0.5 ml) was used for more thorough dehydration of the gel pieces. Dried-out gel pieces were soaked with trypsin solution (1/50 enzyme/substrate ratio based on expected protein content from known cell mass in Tris buffer [20 mM, pH 8.0]). Digestion was performed overnight with shaking (1,000 rpm, 37°C). Elutions were performed using one gel volume: elution 1 with acetonitrile, 2 with formic acid (5%), and 3 with acetonitrile. Elutions 2 and 3 were combined.

(ii) Mass spectrometry analysis.Peptides (1 μg) from each label-free sample were injected and separated via 1D-ultrahigh-pressure liquid chromatography (UHPLC) using a Dionex Ultimate 3000 RSLC nanoUPLC (Thermo Fisher Scientific, Inc., Waltham, MA, USA) system, and masses were analyzed using an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Separation of peptides was performed by reverse-phase chromatography at a flow rate of 300 nl min⁻¹ on a Thermo Scientific reverse-phase nano-Easy-spray column (Thermo Scientific PepMap C₁₈, 2-μm particle size, 100-Å pore size, 75-μm inside diameter [i.d.] by 50-cm length). Peptides were loaded onto a precolumn (Thermo Scientific PepMap 100 C₁₈, 5-μm particle size, 100-Å pore size, 300-μm i.d. by 5-mm length) from the Ultimate 3000 autosampler with formic acid (0.1%) for 3 min at a flow rate of 10 μl min⁻¹. After this period, the column valve was switched to allow elution of peptides from the precolumn onto the analytical column. Solvent A was H₂O containing formic acid (0.1%), and solvent B was acetonitrile (80%), H₂O (20%), and formic acid (0.1%). The linear gradient employed was 2 to 45% B in 90 min. The total run time was 120 min, including a high-organic-wash step and reequilibration step. Peptides were transferred to the gaseous phase with positive-ion electrospray ionization at 2.1 kV. In DDA, the top 20 precursors were acquired between 360 and 1,500 m/z with a 1.5-Th (Thomson) selection window, dynamic exclusion of 35 s, normalized collision energy (NCE) of 30%, and resolutions of 70,000 for MS and 17,500 for MS2; the automatic gain control (AGC) target was 500,000. The .raw files were analyzed with MaxQuant version 1.5.5.1 using default settings. The minimal peptide length was set to 6. Trypsin/P was used as the digestion enzyme. Search criteria included propionamidation of cysteine (PAM-Cys [+71.0371 Da]) as a fixed modification and oxidation of methionine and acetyl (protein N terminus) as variable modifications. Up to two missed cleavages were allowed. The mass tolerances for the precursor were 20 and 4.5 ppm for the first and the main searches, respectively, and that for the fragment ions was 20 ppm. The files were searched against the E. coli K-12 reference UniProt fasta database (July 2017 [4,306 sequences]). The identifications were filtered to obtain a false-discovery rate (FDR) of 1% at the peptide and the protein levels. No filter was applied to the number of peptides per protein. Log₂-transformed quantification results were tested for differential abundance using moderated t statistics using the LIMMA R package. The mass spectrometry proteomics data have been deposited into the ProteomeXchange Consortium via the PRIDE partner repository (50).

Lipidomics.Fermentation samples (500 μl) were harvested by centrifugation (16,000 × g, 2.5 min, 4°C), and the supernatants were discarded. The pellet was washed twice in a salt solution consisting of (NH₄)SO₄ (2 g liter⁻¹), K₂HPO₄ (14.6 g liter⁻¹), NaH₂PO₄·2H₂O (3.6 g liter⁻¹), and (NH₄)₂citrate (0.5 g liter⁻¹). Samples were lyophilized, and 4 mg was transferred into 2-ml microcentrifuge tubes for lipid extraction. All solvents were HPLC grade from Rathburn Chemicals (Walkerburn, Scotland, United Kingdom). Lipids were extracted by adding isopropanol (400 μl) and a deuterated internal standard mix (10 μl SPLASH lipidomix, p/n 330707; Avanti Polar Lipids, AL, USA). Samples were briefly resuspended with a micropestle, and the tubes were capped and incubated (95°C, 15 min) to inactivate phospholipases. Samples were cooled (4°C) before adding hexane (600 μl), followed by brief vortex mixing and centrifuging (16,000 × g, 10 min). The supernatant was transferred to a fresh tube and aqueous Na₂SO₄ (500 μl, 6.7% wt/vol) added to promote phase separation. The tubes were briefly vortexed and centrifuged to separate the layers, and the organic (top) layer (200 μl) removed to a glass HPLC vial for LC-MS analysis of intact lipids. A separate aliquot (200 μl) was kept for transmethylation to fatty acid methyl esters (FAMEs) for analysis by GC-MS. All samples were dried on a centrifugal evaporator. Technical triplicates were extracted from each fermentor at each time point, and extraction blanks were processed in parallel to correct for any background contamination in subsequent analyses.

For GC-MS analysis, FAMEs were generated by incubating dried samples with 1 N methanolic HCl (Supelco) in sealed glass vials (85°C, 4 h). Samples were cooled before adding hexane (200 μl) and aqueous KCl (250 μl 0.9%), vortexing and centrifuging briefly, and collecting the upper phase (100 μl) for analysis. GC-MS analysis of FAMEs was performed using an Agilent 6890 gas chromatograph interfaced with a Leco Pegasus IV TOF MS (Leco, Stockport, United Kingdom) system. Data acquisition and processing was performed using ChromaTof 4.5 software (Leco). A 1-μl aliquot was injected in pulsed splitless mode onto an Rxi-5Sil MS column (30-m by 0.25-mm i.d. by 0.25-μm film thickness; Thames Restek, Saunderton, United Kingdom). FAMEs were separated in He at a constant flow of 1 ml min⁻¹ under the following conditions: initial 100°C for 2 min, linear ramp at 5°C min⁻¹ to 300°C, hold at 300°C for 2 min, and total run time of 44 min. EI spectra were obtained at −70 eV at a detector voltage of 1,790 V, at a rate 5 spectra s⁻¹ over the m/z range 50 to 450. ChromaTof was used to detect peaks at a minimum s/n of 10 and peak width of 3 s, and peak areas were reported from deconvoluted total-ion chromatograms. Peaks were identified by searching electron ionization (EI) spectra against the NIST 2014 library and comparing retention times and spectra to a bacterial FAME reference mix (Larodan, Sweden).

For LC-MS analysis, samples were reconstituted in acetonitrile-isopropanol (200 μl, 70:30 vol/vol). LC was achieved using a Waters Acquity I-Class UPLC system, fitted with an Accucore C₃₀ column (Thermo Scientific [100 mm by 2.1 mm, 2.6-μm particle size]). The column was protected with an Accucore C₃₀ guard cartridge (10 mm by 2.1 mm). Samples were chilled (10°C) in the UPLC autosampler, and a 2-μl aliquot was injected. The weak wash solvent was methanol (5% vol/vol), and the strong wash solvent was isopropanol. For sample elution, the mobile phase A was acetonitrile-water (60:40 vol/vol) plus ammonium formate (10 mM) plus formic acid (0.1% vol/vol). Mobile phase B was acetonitrile-isopropanol (10:90 vol/vol) plus ammonium formate (10 mM) plus formic acid (0.1% vol/vol). Lipids were eluted at 0.35 ml min⁻¹ and 40°C, using the following gradient: initial 1% B, linear increase to 99% B at 21 min, hold at 99% B until 24 min, return to initial conditions at 24.1 min, and hold at initial conditions until 28 min. Mass spectra were acquired using a Thermo Orbitrap Fusion Tribrid mass spectrometer, fitted with a heated electrospray ionization (HESI) ion source. The UPLC and MS were controlled by Thermo Xcalibur 4.0 software. Alternate injections were made in positive- and negative-ion acquisition modes. In positive mode, the spray voltage was set to 3,500 V, and in negative mode, it was set to 3,000 V. In both modes, nitrogen gas flows for sheath, auxiliary, and sweep gases were set to 42, 14, and 1 arbitrary unit, respectively. The ion transfer tube was set to 300°C and the HESI vaporizer to 280°C. Data were acquired in a set 1-s cycle time, with high resolution (240,000 full width at half-maximum [FWHM] at m/z 200) MS1 profile spectra acquired over the m/z 200 to 1,600 scan range, with an AGC target of 200,000 and a maximum ion injection time of 100 ms. MS1 data were internally calibrated during acquisition to <1-ppm mass error using the installed Thermo Easy IC option. Data-dependent MS2 scans were collected in parallel using a 1-m/z quadrupole isolation window and scanned at approximate unit mass resolution in centroid mode from the ion trap, alternating between higher-energy collisional dissociation (HCD) and collision-induced dissociation (CID) fragmentation modes. Only MS1 masses of >400 m/z were selected for fragmentation, with an AGC target of 10,000 and max ion time of 50 ms. HCD spectra were collected in stepped collision energy mode centered at 40% and CID spectra at a fixed 40%. Dynamic exclusion was enabled, with exclusion after n = 1 times, for 6 s.

For analysis of LC-MS-MS data, Xcalibur .raw files were converted to centroided. mzML format using the msconvert function of ProteoWizard (release 3.0.18114). Peak processing workflows were conducted in R 3.5.0. MS1 peaks were extracted using the “centWaveWithPredictedIsotopeROIs” method from the XCMS package (version 3.2.0 [46, 51]). Peaks were grouped across samples, missing peaks imputed by reintegration within group boundaries, and group median m/z values further processed using the CAMERA package (version 1.23.3 [52]), to identify isotopes and adducts. Candidate formulas were generated with adapted code from the rcdk package (version 3.4.9 [53]), with the following limits: positive mode, C10-300, H20-500, O0-20, N0-3, P0-2, Na02, RDBE −0.5-18, 2-ppm error; negative mode, C10-300, H20-500, O0-20, N0-3, P0-2, S0-2, RDBE −0.5-18, 20-ppm error. Consensus peak groups were then filtered with custom R scripts to (i) exclude any peak groups where any peak in the group was present with an area less than the mean + 3 standard deviations of the value from blank extracts, (ii) only retain the most intense monoisotopic peak identified by CAMERA, and (iii) only retain peaks with valid molecular formulas. MS1 peaks were searched against downloaded local copies of the E. coli metabolome database (ECMDB [54]) and the Lipid Maps Structure Database (LMSD [http://www.lipidmaps.org]). Consensus HCD and CID MS2 spectra were extracted with custom scripts and searched against the in silico LipidBlast (55) and LipidMatch (56) databases. Peaks were annotated following manual examination of MS2 spectra in consensus with returned database hits. All retained peaks were normalized to the SPLASH deuterated PC [15:0_18:1(d7)] internal standard and then to sample dry weight. Statistical analyses were carried out on glog normalized data (lambda value in glog transform taken to be 1/10 of nonzero minimum), using time-series ANOVA2 models from the online MetaboAnalyst resource (https://www.metaboanalyst.ca).

Citramalate synthase assay.Cell-free extracts of E. coli BW25113 ldhA pBAD24-cimA3.7, E. coli BW25113 ldhA pBAD24-cimA3.7A_dead or E. coli BW25113 ldhA were prepared from cultures (400 ml) 4 h after induction with l-arabinose (0.02 g liter⁻¹). Cells were harvested by centrifugation (8,000 × g, 10 min, 4°C) and resuspended in HEPES buffer (0.1 M, pH 7.5) containing MgCl₂ (5 mM). Cells were lysed by three passages through a French pressure cell. The resulting cell suspension was centrifuged (12,000 × g, 10 min), and the supernatant containing the soluble protein was filtered (0.2-μm-pore filter). Total protein concentration was estimated using a Bio-Rad protein assay as described by the manufacturer, and the protein content was normalized to 1 mg ml⁻¹ in HEPES buffer (0.1 M, pH 7.5) containing MgCl₂ (5 mM). CimA3.7 and CimA3.7_dead were assayed using a modified method described by Howell et al. (57). The enzyme assay mixture (1 ml) contained acetyl-CoA (1 mM), pyruvate (1 mM), cell extract (200 μl), and HEPES buffer (0.1 M, pH 7.5) containing MgCl₂ (5 mM). The reaction mixture was incubated at 37°C, and at regular intervals (10 min), samples (100 μl) were taken and mixed with a sample analysis mixture (900 μl), and the absorbance at 412 nm was measured. The sample analysis mixture (900 μl) contained 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB [0.56 mM]), Tris-HCl (78 mM, pH 8.0), and distilled water (dH₂O). Where indicated in the text, pyruvate was replaced by 2-oxobutyrate.

Data availability.Transcriptomics data are available in ArrayExpress under accession no. E-MTAB-7257. The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium database via the PRIDE partner repository under accession no. PXD013088 (50).