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. 2014 Feb 28;9(2):e89580.
doi: 10.1371/journal.pone.0089580. eCollection 2014.

Transcriptomic Analysis of Carboxylic Acid Challenge in Escherichia Coli: Beyond Membrane Damage

Free PMC article

Transcriptomic Analysis of Carboxylic Acid Challenge in Escherichia Coli: Beyond Membrane Damage

Liam A Royce et al. PLoS One. .
Free PMC article


Carboxylic acids are an attractive biorenewable chemical. Enormous progress has been made in engineering microbes for production of these compounds though titers remain lower than desired. Here we used transcriptome analysis of Escherichia coli during exogenous challenge with octanoic acid (C8) at pH 7.0 to probe mechanisms of toxicity. This analysis highlights the intracellular acidification and membrane damage caused by C8 challenge. Network component analysis identified transcription factors with altered activity including GadE, the activator of the glutamate-dependent acid resistance system (AR2) and Lrp, the amino acid biosynthesis regulator. The intracellular acidification was quantified during exogenous challenge, but was not observed in a carboxylic acid producing strain, though this may be due to lower titers than those used in our exogenous challenge studies. We developed a framework for predicting the proton motive force during adaptation to strong inorganic acids and carboxylic acids. This model predicts that inorganic acid challenge is mitigated by cation accumulation, but that carboxylic acid challenge inverts the proton motive force and requires anion accumulation. Utilization of native acid resistance systems was not useful in terms of supporting growth or alleviating intracellular acidification. AR2 was found to be non-functional, possibly due to membrane damage. We proposed that interaction of Lrp and C8 resulted in repression of amino acid biosynthesis. However, this hypothesis was not supported by perturbation of lrp expression or amino acid supplementation. E. coli strains were also engineered for altered cyclopropane fatty acid content in the membrane, which had a dramatic effect on membrane properties, though C8 tolerance was not increased. We conclude that achieving higher production titers requires circumventing the membrane damage. As higher titers are achieved, acidification may become problematic.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.


Figure 1
Figure 1. Octanoic acid response network.
MG1655 was challenged with sufficient octanoic acid to inhibit growth by 23% (10 mM) in MOPS minimal media at pH 7.0, 37°C, 150 rpm. This diagram shows central metabolism and highlights the regulatory effect of regulators with significantly perturbed activity during C8 challenge, as identified by network component analysis. Dashed lines indicate regulatory connections that were proposed in our previous analysis . Mechanisms for changes in transcription factor activity are discussed in the text.
Figure 2
Figure 2. Octanoic acid challenge decreases the intracellular pH.
E. coli MG1655 pJTD1 was grown to midlog in minimal media at pH(HCl). Values are the average of 4 biological replicates, with error bars indicating the standard deviation.
Figure 3
Figure 3. Supplementation with arginine or glutamate does not mitigate intracellular acidification.
Measurements of the intracellular pH of E. coli MG1655 pJTD1 during C8 challenge while grown in the presence of supplemental arginine and glutamate. The cells were incubated for 3°C in MOPS media to allow utilization of the amino acid-dependent acid resistance systems. All concentrations are 10 mM.
Figure 4
Figure 4. Supplementing glutamate increases GABA export; however, addition of C8 reduces GABA accumulation and export.
GABA measurements of MG1655 during log phase growth in MOPS with 2% dextrose at 37°C, 150 rpm. All concentrations are 10 mM. GABA: γ-amino butyric acid.
Figure 5
Figure 5. Production of carboxylic acids in strain ML103+pXZ18Z+pZS-GFP does not significantly change the intracellular pH.
Shake flasks of E. coli producing predominately C14:0 and C16:0 carboxylic acids in M9 media with 1.5% dextrose at 30°C. IPTG induces the pXZ18Z plasmid carrying a thioesterase and a β-hydroxyacyl-ACP dehydratase. The intracellular pH values are the average of four biological replicates and four technical replicates. The error bars indicate the standard deviation.
Figure 6
Figure 6. cfa mutants with altered the membrane lipid profile, S:U ratio and the average lipid length.
a: Membrane lipid profile of MG1655 and strains with altered cfa expression. Strains were incubated with 0–30 mM C8, pH = 7.0. Inset: specific growth rate in the log phase of E. coli with varying cfa expression. C16:0- palmitic acid, C16:1- palmitoleic acid, C17cyc- cyclopropane C17:0, C18:1- vaccenic acid, C18:0- stearic acid, C19cyc- cyclopropane C19:0. The complete lipid profiles are shown in Figure S2 of the Supporting Information. Membrane properties are calculated from a to obtain: b: saturated:unsaturated lipid ratio and c: average lipid length.
Figure 7
Figure 7. lrp mutations do not improve E.
coli growth rate upon addition of octanoic acid. The specific growth rate of strains with altered lrp expression during the log phase in MOPS with 2% dextrose at 37°C, 150 rpm.
Figure 8
Figure 8. Simulations of the effect of the electrochemical gradients on the proton motive force.
The appropriate range for E. coli cells is −140 to −180 mV. pH values are measurements from Figure 2 at 20 mM. Assumption 1: An average of −160 mV is used for the normal PMF. The target PMF is within the range of −140 to −180 mV. Assumption 2: Shock conditions are the instantaneous outcome of the PMF after an extreme change in the environment. Assumption 3: The cell can initiate a response that gives times to adjust the PMF to the target range. Assumption 4: Rapid addition of a strong mineral acid (e.g., HCl) adds an equal amount of anions and protons, which does not change the membrane potential. Assumption 5: C8 is at a sufficiently high concentration that it rapidly integrates into the cell membrane and releases its proton inside the cell, which is not reversible. A negative (−) PMF indicates protons diffusing into the cell, whereas a positive (+) PMF indicates protons diffusing out of the cell. A negative (−)Δψ indicates an overall negative ion gradient due to anions, whereas a positive (+)Δψ indicates an overall positive ion gradient due to cations. The mol ions is the molar ratio from the normal condition. pHi- intracellular pH; pHe- extracellular pH; formula image- ΔpH associated electrochemical force; formula image- membrane potential associated electrochemical force; PMF- proton motive force; A (red) C8 anion; A (green) another anion; C+ any cation.

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This work was supported by the National Science Foundation (NSF) Engineering Research Center for Biorenewable Chemicals (CBiRC), NSF award number EEC-0813570. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.