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 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, 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.

Metabolism of long-chain fatty acids affects disulfide bond formation inEscherichia coliand activates envelope stress response pathways as a combat strategy

“…We observed ∼4 fold induction of fadE in cells grown in TBK-Ole in comparison to TBK-Brij (Fig 1F). This data is consistent with our previous observation of increased fadE transcript levels in oleate [23] and collectively suggests an increased flow of electrons from FadE to the ETC during LCFA metabolism.…”

“…For this, WT BW25113 was grown in buffered tryptone broth (TBK) supplemented either with oleate (TBK-Ole) or with the detergent Brij-58 used for solubilizing oleate (TBK-Brij) (Fig 1B), and steady-state levels of NADH and NAD + were measured in exponential phase (time point T1, Fig 1B). These and subsequent experiments were carried out in TBK-Ole to support the growth of control fad knockouts, which otherwise do not grow in minimal medium containing oleate as a sole carbon source [23]. Moreover, because tryptone causes mild catabolite repression [24], oleate is co-utilized with carbon components of tryptone [23].…”

“…These and subsequent experiments were carried out in TBK-Ole to support the growth of control fad knockouts, which otherwise do not grow in minimal medium containing oleate as a sole carbon source [23]. Moreover, because tryptone causes mild catabolite repression [24], oleate is co-utilized with carbon components of tryptone [23]. We observed ∼2.5-fold increase in NADH in WT cells grown in TBK-Ole compared to TBK-Brij; however, there was no significant change in NAD + levels.…”

“…As expected, the activity of NADH and succinate dehydrogenases was considerably lower in Δ nuoK ( nuoK codes for a subunit of NADH dehydrogenase I) and Δ sdhB ( sdhB codes for a subunit of succinate dehydrogenase) strains, respectively, in both TBK-Brij and TBK-Ole (Figs 1D and E). We previously reported an increase in transcript levels of an acyl-CoA dehydrogenase, FadE, in E. coli cultured in TB medium supplemented with oleate (compared to TB-Brij control) [23]. FadE catalyzes the oxidation of acyl-CoA (an intermediate in LCFA metabolism) concomitant with reduction of FAD to FADH 2 and has also been speculated to re-oxidize FADH 2 to FAD by transferring electrons from its dehydrogenase domain to the ETC (Fig 1A) [25].…”

Contextual flexibility inPseudomonas aeruginosacentral carbon metabolism during growth in single carbon sources

“…Acetate and glycerol have different entry points into P. aeruginosa central carbon metabolism, and are also thought to have distinct effects on redox metabolism [10–13]. We therefore anticipated a carbon source-specific impact on the expression of enzymes (and corresponding fluxes) involved in the relevant pathways.…”