A genome-wide screen in Escherichia coli reveals that ubiquinone is a key antioxidant for metabolism of long-chain fatty acids

Abstract

Long-chain fatty acids (LCFAs) are used as a rich source of metabolic energy by several bacteria including important pathogens. Because LCFAs also induce oxidative stress, which may be detrimental to bacterial growth, it is imperative to understand the strategies employed by bacteria to counteract such stresses. Here, we performed a genetic screen in Escherichia coli on the LCFA, oleate, and compared our results with published genome-wide screens of multiple non-fermentable carbon sources. This large-scale analysis revealed that among components of the aerobic electron transport chain (ETC), only genes involved in the biosynthesis of ubiquinone, an electron carrier in the ETC, are highly required for growth in LCFAs when compared with other carbon sources. Using genetic and biochemical approaches, we show that this increased requirement of ubiquinone is to mitigate elevated levels of reactive oxygen species generated by LCFA degradation. Intriguingly, we find that unlike other ETC components whose requirement for growth is inversely correlated with the energy yield of non-fermentable carbon sources, the requirement of ubiquinone correlates with oxidative stress. Our results therefore suggest that a mechanism in addition to the known electron carrier function of ubiquinone is required to explain its antioxidant role in LCFA metabolism. Importantly, among the various oxidative stress combat players in E. coli, ubiquinone acts as the cell's first line of defense against LCFA-induced oxidative stress. Taken together, our results emphasize that ubiquinone is a key antioxidant during LCFA metabolism and therefore provides a rationale for investigating its role in LCFA-utilizing pathogenic bacteria.

Keywords: bacterial genetics; electron transport; fatty acid metabolism; long chain fatty acids; non-fermentable carbon sources; oxidative stress; quinone; respiratory chain; ubiI; ubiK.

Figure 1.

Growth of the Keio deletion library on oleate reveals pathways and ETC components critical for the utilization of LCFAs. A, colony sizes of individual mutants are normalized to the plate average and biological replicates (n > 3) are plotted for the two conditions tested: oleate and glucose with Brij-58. Points are colored by the logarithm of local density in the plot. Normalized colony sizes from biological replicates are highly correlated (R = 0.86, Pearson's correlation). B, a schematic of the metabolic route of LCFAs highlighting pathways and complexes significantly enriched in oleate. p- and q- represent nominal p value and FDR q value, respectively, obtained from GSEA analysis of pathways and complexes in E. coli. *FadE is a flavoprotein that reduces FAD to FADH₂ during β-oxidation. It has been speculated that FadE itself might oxidize FADH₂ to FAD by transferring electrons from its dehydrogenase domain to the ETC (61). However, there is no experimental evidence for the same. C, fitness scores were calculated for oleate and other carbon sources as compared with glucose control. Fitness scores of components of the β-oxidation pathway and ETC are shown.

Figure 2.

Among NADH dehydrogenases, only Nuo complex is required for growth on non-fermentable carbon sources. WT, Δnuo, and Δndh strains were grown in minimal medium containing one of the carbon sources, and OD₄₅₀ was measured. Each medium condition had Brij-58. The experiment was done 4 times; each experiment had 2 technical replicates. A representative dataset with average and S.D. of technical replicates is shown.

Figure 3.

The increased requirement of ubiquinone for growth in oleate is to mitigate elevated levels of ROS. A, ΔubiI shows significant growth defect in liquid medium containing oleate as the sole carbon source. WT, ΔubiH, and ΔubiI strains were grown in minimal medium containing one of the carbon sources, and OD₄₅₀ was measured. Each medium condition had Brij-58. B and C, the growth defect of ΔubiI in oleate is partially recovered by glutathione (B) and thiourea (C). WT and ΔubiI were grown in minimal medium containing oleate with or without 1 mm glutathione (GSH) or 1 mm thiourea, and OD₄₅₀ was measured. 1 mm urea was included as control for thiourea. For A–C experiments were done 3 times; each experiment had 3 technical replicates. A representative dataset, with average and S.D. of technical replicates, is shown. D, WT and ΔubiI strains exhibit maximum ROS levels in TB-Ole. WT and ΔubiI were grown either in TB or TB supplemented with carbon sources or Brij-58: glucose (TB-Glu), acetate (TB-Ace), succinate (TB-Suc), oleate (TB-Ole), and Brij-58 (TB-Brij). ROS levels were determined by NBT assay. Data were normalized to the ROS level of WT in TB and represent average (± S.D.) of 5 independent experiments. E, supplementation of ubiquinone-8 suppresses ROS levels. WT and ΔubiI were grown either in TB or TB-Ole. Media contained either 20 μm ubiquinone-8 or 0.1% ethanol (solvent for ubiquinone-8). ROS levels were determined by NBT assay. Data were normalized to the ROS level of WT in TB containing 0.1% ethanol and represent average (± S.D.) of 3 independent experiments. *, p < 0.05; **, p < 0.005; NS, not significant (unpaired two-tailed Student's t test).

Figure 4.

Ubiquinone is a major player that counteracts oxidative stress generated by oleate degradation. A, in the presence of oleate, ROS levels increase only in strains defective in ubiquinone biosynthesis. WT and various deletion strains were grown either in TB or TB-Ole. ROS levels were determined by NBT assay. Data were normalized to the ROS level of WT in TB and represent average (± S.D.) of 3 independent experiments. B and C, enzymatic scavengers are induced by oleate when ubiquinone levels are low. WT, ΔubiI, and ΔfadLΔubiI strains carrying either katG-lacZ (B) or ahpC-lacZ (C) (lacZ placed under the control of H₂O₂ responsive katG or ahpC promoter) in MC4100 background, were grown in TB, TB-Brij, or TB-Ole, and β-gal activity was determined. Data were normalized to the β-gal activity of WT in TB and represent average (± S.D.) of at least 4 independent experiments. The average β-gal activity of WT carrying katG-lacZ in TB was 166 ± 45 Miller units, and that of WT carrying ahpC-lacZ in TB was 38 ± 15 Miller units. D, supplementation of ubiquinone-8 suppresses the induction of katG-lacZ in ΔubiI grown in TB-Ole. WT and ΔubiI strains carrying katG-lacZ fusion in the MC4100 background were grown either in TB-Brij or TB-Ole. Media contained either 20 μm ubiquinone-8 or 0.1% ethanol. β-Gal activity was determined. Data were normalized to the β-gal activity of WT in TB-Brij with 0.1% ethanol and represent average (± S.D.) of 3 independent experiments. The average β-gal activity of WT in TB-Brij with 0.1% ethanol across 3 experiments was 257 ± 88 Miller units. **, p = 0.0058; NS, not significant (unpaired two-tailed Student's t test). E, ubiquinone accumulates in WT cells in response to oleate utilization. The total Q₈ level in lipid extracts from WT and various fad deletion strains grown either in TB or TB-Ole was determined. Q₈ levels were normalized to the Q₈ level of WT in TB and represent average (± S.D.) of at least 4 independent experiments. F, oleate transport and degradation are responsible for an increase in ROS levels in WT grown in TB-Ole. WT and various fad deletion strains were grown either in TB or TB-Ole. ROS levels were determined by NBT assay. Data were normalized to the ROS level of WT in TB and represent average (± S.D.) of 3 independent experiments.

Figure 5.

ΔubiIΔubiK double mutant produces no detectable ubiquinone. A, ΔubiI and ΔubiK have similar Q₈ content. Total Q₈ level in lipid extracts from WT, ΔubiI, and ΔubiK cells grown in TB was determined. Q₈ levels were normalized to the Q₈ level of WT in TB and represent average (± S.D.) of 3 independent experiments. B, ΔubiK shows significant growth defect in oleate. Dilutions of the cultures were spotted on minimal medium containing one of the carbon sources. Each medium condition had Brij-58. ΔfadL was used as a control. The experiment was repeated 3 times. A representative dataset is shown. C, ubiK cloned on plasmid complements the growth defect of ΔubiK in oleate. Dilutions of WT and ΔubiK carrying either empty plasmid (pACYC184) or pACYC184 with ubiK (pSA4) were spotted on minimal medium containing oleate as the sole carbon source. ΔfadL transformed with pACYC184 was used as a control. The experiment was repeated 2 times. A representative dataset is shown. D, ΔubiK strain has increased ROS levels. WT and ΔubiK were grown either in TB or TB-Ole, and ROS levels were determined by NBT assay. Data were normalized to the ROS level of WT in TB and represent average (± S.D.) of 3 independent experiments. E, ΔubiIΔubiK shows a synthetically sick phenotype in LB. WT, ΔubiI, ΔubiK, and ΔubiIΔubiK strains were streaked on LB and incubated overnight. F, ubiquinone is not detected in the ΔubiIΔubiK double mutant. Total Q₈ level in lipid extracts from WT, ΔubiI, ΔubiK, and ΔubiIΔubiK cells grown in LB-glucose was determined. Q₈ levels were normalized to the Q₈ level of WT in LB-glucose and represent the average (± S.D.) of 3 independent experiments. G, ΔubiIΔubiK shows a synthetic lethal phenotype in non-fermentable carbon sources. Dilutions of the cultures were spotted on minimal medium containing one of the carbon sources. Each medium condition had Brij-58. ΔfadL was used as a control. The experiment was repeated 2 times. A representative dataset is shown.

Figure 6.

Probable sites of ROS formation during LCFA degradation and the mechanisms employed by ubiquinone to mitigate LCFA-induced oxidative stress. Exogenous LCFAs are transported inside the cell by an outer membrane protein, FadL. Subsequently, the inner membrane-associated acyl-CoA synthetase, FadD, extracts LCFAs from the inner membrane concomitant with esterification to acyl-CoA. Acyl-CoAs are degraded to acetyl-CoA via the β-oxidation pathway mediated by enzymatic activities of FadE, FadB, and FadA. Acetyl-CoA feeds into the TCA cycle for further metabolism. High NADH/NAD⁺ and FADH₂/FAD ratios during β-oxidation and TCA cycle increase the electron flow in the ETC thereby increasing electron leakage and autoxidation of the reduced form of NADH dehydrogenase resulting in ROS formation. In addition, a predominant source of ROS could be the acyl-CoA dehydrogenase, FadE, which reduces FAD to FADH₂. Ubiquinone limits ROS formation by rapidly transferring electrons from upstream dehydrogenases to terminal oxidases (Cyo and Cyd) thus preventing electron leakage and autoxidation of the reduced form of dehydrogenases. In addition, the quinol peroxidase activity of Cyd will detoxify H₂O₂. Arrows with e⁻ labeled on the line show the direction of electron transfer. Dotted arrows indicate reactions for which either the components involved are not known (oxidation of FadE and electron transfer from FadE to the ETC) or the mechanisms are not established in vivo (detoxification of H₂O₂ by Cyd). Abbreviations: Oaa, oxaloacetate; Cit, citrate; Isocit, isocitrate; α-KG, α-ketoglutarate; Suc-CoA, succinyl-CoA; Suc, succinate; Fum, fumarate; Mal, malate; Glo, glyoxylate; O₂⁻, superoxide; Sdh, succinate dehydrogenase; Q₈, ubiquinone-8; Q₈H₂, ubiquinol-8; Cyd, cytochrome bd; Cyo, cytochrome bo.