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

Article abstract of DOI:10.1074/jbc.M117.806240

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 genomewide 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 LCFAinduced 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.

Full text of DOI:10.1074/jbc.M117.806240

JBC Papers in Press. Published on October 17, 2017 as Manuscript M117.806240
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M117.806240
Role of ubiquinone in LCFA metabolism
A genome-wide screen in Escherichia coli reveals that ubiquinone is a key antioxidant for
metabolism of long chain fatty acids
1
#
1#
2
1
1†
Shashank Agrawal , Kanchan Jaswal , Anthony L Shiver , Himanshi Balecha , Tapas Patra and
1
Rachna Chaba *
1
Department of Biological Sciences, Indian Institute of Science Education and Research (IISER) Mohali,
2
SAS Nagar, Punjab 140306, India; Department of Bioengineering, Stanford University, Stanford,
California 94305, USA
Running title: Role of ubiquinone in LCFA metabolism
#
S.A. and K.J. contributed equally to this work.
*
To whom correspondence should be addressed:
Rachna Chaba, Department of Biological Sciences, Indian Institute of Science Education and Research
(
IISER) Mohali, SAS Nagar, Punjab 140306, India.
Email: rachnachaba@iisermohali.ac.in; rachnachaba@gmail.com
Keywords: fatty acid metabolism, long chain fatty acids, respiratory chain, electron transport, quinone,
oxidative stress, non-fermentable carbon sources, ubiI, ubiK, bacterial genetics
ABSTRACT
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.
Long chain fatty acids (LCFAs) are used as a rich
source of metabolic energy by several bacteria
including important pathogens. Since 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
amongst 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 to 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
INTRODUCTION
Long chain fatty acids (LCFAs) are
carboxylic acids with an unbranched aliphatic
chain of 12-20 carbon atoms. Several bacterial
pathogens such as Mycobacterium tuberculosis,
Pseudomonas aeruginosa, and Salmonella
typhimurium metabolize LCFAs derived from host
tissues, which enable their survival in harsh
environments and contribute to their virulence (1-
3). Much of our understanding of LCFA
metabolism is obtained from studies in E. coli.
LCFA metabolism is mediated by proteins
(
ROS) 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
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1
Copyright 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Role of ubiquinone in LCFA metabolism
encoded by the fad (fatty acid degradation) genes
that transport LCFAs inside the cell and
subsequently degrade it to acetyl-CoA via the β-
oxidation pathway. Acetyl-CoA feeds into the
tricarboxylic acid (TCA) and glyoxylate cycles to
generate metabolic intermediates for growth.
Reduced cofactors generated during β-oxidation
and TCA cycle are oxidized by the electron
transport chain (ETC) resulting in production of
ATP. The fad genes are controlled at a
transcriptional level by three regulatory systems: i)
positive regulation by the global cyclic AMP
receptor protein-cyclic AMP complex (catabolite
repression), ii) negative regulation by the
transcriptional regulator, FadR, repression of
which is relieved by binding to acyl-CoA, a
metabolic intermediate in LCFA degradation, and
iii) negative regulation by the ArcA-ArcB two-
component system [reviewed in (4,5)].
carbon sources there could be a difference in the
requirement of ETC components. Hence, the
requirement of individual components of the ETC
in LCFA metabolism should be investigated.
In addition, a recent study has shown that
LCFAs induce oxidative stress in E. coli (13),
however, the mechanism by which bacteria
combat this stress is not known. The enzymatic
scavengers (peroxidases, catalases and superoxide
dismutases) are known to be the major oxidative
stress combat players in bacteria (14). On the other
hand, the role of the ETC component, ubiquinone,
as an antioxidant in bacteria is underappreciated.
There is only one report in E. coli that suggested
ubiquinone to function as an antioxidant based on
oxidative stress phenotypes of mutants defective in
ubiquinone biosynthesis (15). But, how
ubiquinone counteracts ROS, what is the
physiological condition under which ubiquinone
plays a predominant role, and what is the relative
contribution of ubiquinone to the overall oxidative
stress response remains to be assessed. The
remarkable use of LCFAs by pathogens such as
Growth on LCFAs requires the presence
of either oxygen as terminal electron acceptor
(
aerobic metabolism) or alternative electron
acceptor such as nitrate (anaerobic metabolism).
Thus, LCFAs are non-fermentable carbon sources,
which unlike glucose, a fermentable carbon
source, require optimal functioning of ETC (6,7).
Components of ETC are located in the inner
membrane of E. coli. During aerobic metabolism
Mycobacterium
tuberculosis,
Pseudomonas
aeruginosa, and Salmonella typhimurium (1-3)
warrants a detailed investigation of the strategies
employed by bacteria to mitigate LCFA-mediated
oxidative stress.
NADH and FADH
2
are oxidized by NADH
With the motive to fill in the above-
dehydrogenases (Ndh and Nuo) and succinate
dehydrogenase (Sdh), respectively, and the
electrons are transferred to the lipid-soluble
electron carrier, ubiquinone. The reduced form of
ubiquinone, ubiquinol, in turn donates electrons to
the terminal oxidases, cytochrome bo (Cyo) and
cytochrome bd (Cyd), which further transfer
electrons to molecular oxygen reducing it to water
molecule. During the flow of electrons through
ETC, Nuo, Cyo and Cyd generate proton motive
force that further drives ATP synthesis through
ATP synthase (8,9). Consistent with the increased
requirement of ETC on non-fermentable carbon
sources, in contrast to growth on glucose, mutants
of several ETC components exhibit severe growth
defect on succinate, acetate, lactate and malate (9-
mentioned knowledge gaps in LCFA metabolism,
using the Keio deletion library of E. coli (16), we
performed a quantitative genetic screen on oleate
(C18:1 cis-9) and compared our LCFA dataset
with recent high-throughput genetic screens of
additional carbon sources (17). This comparative
analysis revealed that amongst all aerobic ETC
components only genes involved in the
biosynthesis of ubiquinone are more important for
growth in LCFAs when compared to other non-
fermentable carbon sources. Our detailed studies
show that of the various oxidative stress combat
players in E. coli, ubiquinone is the key player that
mitigates elevated levels of ROS generated by
LCFA degradation, however, its role as an
antioxidant cannot be described solely by its
known electron carrier function in the ETC.
1
2). However, the importance of ETC components
for growth on LCFAs has not been examined.
Different non-fermentable carbon sources enter
into the TCA cycle at varied steps and are
theoretically expected to generate different amount
of reduced cofactors, thus even amongst these
RESULTS
A
chemical-genomic screen reveals the
pathways used by E. coli to aerobically
metabolize long chain fatty acids
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2

Role of ubiquinone in LCFA metabolism
We tested the ability of 3994 mutants from the
Keio deletion library to grow on oleate as the sole
carbon source. To measure fitness defects during
growth on oleate, we pinned the Keio library onto
M9 minimal agar plates containing either oleate or
glucose as the sole carbon source. Since oleate
was solubilized in the detergent Brij-58, the
growth medium containing glucose was also
supplemented with Brij-58. We took images of the
plates at a single time point and quantified colony
size using image analysis software (18). Replicate
colony size measurements were reproducible in
the screen (Fig. 1A, R=0.86). We then assigned a
fitness-score to mutants in the oleate condition by
calculating the statistical significance of the
difference in colony size between oleate and the
glucose control using an established analysis
pathways being critical for generation of reduced
cofactors and metabolites for growth on non-
fermentable carbon sources (4,5). Furthermore,
oleate, acetate and succinate utilization exhibited
enrichment in multiple pathways for electron
transfer activity (FDR q-value <5%), underscoring
the importance of ETC for energy generation
during growth on non-fermentable carbon sources.
We also performed GSEA analysis for protein
complexes with 3 or more proteins (20,22). Oleate,
acetate and succinate utilization were enriched for
ETC complexes, NADH dehydrogenase I (Nuo)
(FDR q-value <1%) and succinate dehydrogenase
(FDR q-value <7%) (Dataset S1C). The enriched
metabolic pathways and complexes for oleate
utilization are depicted in Fig. 1B.
Although glycerol is considered to be a
pipeline
(19)
with
slight
modifications
non-fermentable carbon source, this substrate did
not show a significant enrichment of any of the
ETC pathways or complexes in our GSEA
analysis. We speculate that since there is ATP
generation (by substrate-level phosphorylation)
after the entry of glycerol in the later part of
glycolysis (23), this ATP might allow for growth
on glycerol. Moreover, the fermentative utilization
of glycerol has also been suggested (24). Thus, for
detailed studies on the requirement of ETC
components we further focused on oleate, acetate
and succinate. To circumvent problems of high-
throughput screens (incorrect strains, suppressors
(
Experimental Procedures). To facilitate the
comparison of oleate to other carbon sources, we
reanalyzed data from a previous screen (17),
comparing both fermentable (glucosamine, N-
acetyl glucosamine and maltose) and non-
fermentable (acetate, succinate and glycerol)
carbon sources to glucose as a control. The fitness-
scores we report thus represent the statistical
significance of a change in colony size on a
particular carbon source as compared to growth on
glucose with positive and negative fitness-scores
representing increased and decreased colony size,
respectively. A full list of fitness scores of Keio
library strains in different carbon sources
and cross-contamination), before
a
detailed
follow-up analysis of ETC mutants, we verified
phenotypes using transductants. Further, since
oxygen availability to cells in a colony varies with
the size of colony (25), in our subsequent
experiments we also validated the requirement of
ETC components in aerobic metabolism by
assessing their phenotypes in liquid medium.
(
normalized to a glucose control) is available in
Dataset S1A.
The screen identified a large number of
mutants with severe growth defects on oleate. To
better understand the physiological basis for these
growth defects, we performed a global analysis
using Gene Set Enrichment Analysis (GSEA) and
gold-standard biological pathways (20-22), to find
pathways that play a significant role in growth on
oleate (Dataset S1B). Importantly, global analysis
highlighted the β-oxidation pathway (FDR q-
value: 1.1%), critical for the utilization of LCFAs
as an energy source, as a unique signature of
growth on oleate. This result emphasizes the
robustness of our high-throughput genetic screen
and of our statistical approach. In addition, oleate,
Requirement of ubiquinone does not correlate
with the energy yield of non-fermentable
carbon sources
A comparison of our LCFA dataset with GSEA
analysis for additional carbon sources clearly
showed that ETC is critical for growth of E. coli
on non-fermentable carbon sources. However,
there was a difference in the requirement of
various ETC components. Of the two NADH
dehydrogenases involved in ETC, Ndh is known to
be more important during aerobic respiration
whereas Nuo is essential for dimethyl sulfoxide
acetate and succinate shared
enrichment in the TCA and glyoxylate cycles
a
significant
(
FDR q-value <5%), a result consistent with these
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3

Role of ubiquinone in LCFA metabolism
and fumarate (anaerobic) respiration (26,27).
However, we noted that for aerobic metabolism of
oleate, acetate, and succinate, Nuo was important
whereas Ndh was dispensable (Fig. 1C). Early
studies had reported the growth defect of nuo
mutants in acetate, however they did not assess the
relative requirement of Ndh and Nuo (10,28). We
validated our results by comparing the growth
profile of Δnuo (the entire nuo operon is deleted)
and Δndh in liquid medium; only Δnuo showed
significant growth defect in oleate, acetate, and
succinate (Fig. 2). We suggest that since Nuo
couples NADH oxidation to proton translocation
(30,31) (Fig. 1C). We thus constructed several
transductants of all five ubi mutants and assessed
their growth at a candidate level (Fig. S1). The
phenotype of all ubi mutants corroborated with
their known phenotype in succinate: ubiE, ubiF
and ubiH mutants exhibited no growth (30,32),
ubiX mutant showed growth defect (33) and ubiI
mutant displayed wild-type growth (30,31). In
acetate, all ubi mutants had a phenotype similar to
that in succinate. In oleate, ubi mutants either did
not grow at all (ubiE, ubiF and ubiH) or showed
significant growth defect (ubiI and ubiX) (Fig. S1).
Upon relating the growth profile of ubi deletion
strains observed in our study with their ubiquinone
levels reported in the literature we find that
mutants with no detectable ubiquinone (ubiE, ubiF
and ubiH) (30,32) do not grow in any of the three
non-fermentable carbon sources, whereas a ubiI
mutant which produces reduced levels of
ubiquinone (30,31) exhibits growth defect only in
oleate. UbiI catalyzes C5-hydroxylation of the 3-
octaprenylphenol intermediate in the ubiquinone
biosynthesis pathway and the residual level of
ubiquinone in a ΔubiI strain is attributed to the
suboptimal C5-hydroxylase activity of a C6-
monooxygenase, UbiF (31).
+
-
+
-
(
H /e =2) whereas Ndh does not (H /e =0) (8,26),
it is likely that growth on non-fermentable carbon
sources is dependent on Nuo for ATP production.
Further, amongst oleate, acetate, and succinate,
Nuo was more significantly required for growth in
acetate (Fig. 1C and 2) that has the worst net ATP
yield of these three carbon sources (4). Whereas
Δnuo did not grow in acetate, the strain exhibited
an extended lag in oleate and succinate, and then
grew at the same rate and to the same yield as
wild-type (WT) (Fig. 2). To rule out the possibility
that Δnuo strain was picking up suppressors in
oleate and succinate, we sub-cultured cells from
stationary phase. We again observed extended lag
in succinate and oleate. Similar to Nuo, Cyo which
is the major terminal oxidase during aerobic
metabolism and also generates proton motive force
For the last several decades, the increased
requirement of ubiquinone for energy generation
during growth with succinate compared to glucose
has been the rationale for identifying genes
involved in ubiquinone biosynthesis (34,35).
Despite this, it was surprising that a recent study
showed that ΔubiI, where ubiquinone level is
reduced to only ~10-15% of WT level, exhibited
normal growth in succinate (30,31). Data from
genetic screens and candidate studies in solid
media reinforce that even ~10-15% ubiquinone is
sufficient for growth in succinate, whereas, this
ubiquinone level is suboptimal for growth in
LCFAs.
+
-
in the ETC (H /e =2) (8), was maximally required
in acetate (Fig. 1C). Collectively, our results
suggest that the quantitative contribution of Nuo
and Cyo to growth on non-fermentable carbon
sources is inversely correlated with the energy
yield of these substrates.
In contrast to Nuo and Cyo, we observed
that the requirement of ubiquinone was more in
oleate compared to other non-fermentable carbon
sources (Fig. 1C). In E. coli, the biosynthesis of
ubiquinone is known to involve at least twelve ubi
genes (9,29) (Fig. 1B). The data for only seven ubi
deletion strains were available from previous
genetic screens for comparison with our oleate
dataset (17) (Fig. 1C). In independent studies the
growth phenotype of null mutants of five ubi
genes (ubiE, ubiF, ubiH, ubiI and ubiX) have been
reported in succinate (30-33). In our analysis, only
the phenotype of ubiF, ubiH and ubiI mutants
agreed with their known phenotypes in succinate
We investigated further the differential
requirement of ubiquinone on non-fermentable
carbon sources. We compared the growth profile
of ΔubiH and ΔubiI strains in liquid media.
Whereas ΔubiH showed a growth reduction on
glucose, the strain did not grow at all with oleate,
acetate and succinate (Fig. 3A). On the other hand,
ΔubiI showed a significant growth defect only in
oleate (Fig. 3A); the growth defect was
complemented by ubiI cloned on plasmid (Fig.
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4

Role of ubiquinone in LCFA metabolism
S2). Taken together, our analysis of the growth
phenotypes of ETC mutants on various carbon
sources highlights a specific role of ubiquinone in
LCFA metabolism.
change was comparable with a previous report
where ~2-fold higher ROS levels were observed in
WT cells grown in minimal medium supplemented
with oleate compared to cells grown in glucose
supplemented medium (13). Moreover, ROS
levels further increased in ΔubiI in all media
conditions with maximal ROS levels again in TB-
Ole grown cells (Fig. 3D). Brij-58 alone did not
result in increased ROS. Importantly, when
ubiquinone-8 was supplied exogenously, ROS
levels decreased by ~15-25% in WT grown in TB-
Ole and ΔubiI grown either in TB or TB-Ole (Fig.
Ubiquinone is maximally required in oleate to
mitigate elevated levels of ROS
E. coli grown in oleate is reported to accumulate
higher levels of ROS compared to cultures grown
in glucose (13). In a separate study, ubiquinone
was suggested to function as an antioxidant in E.
coli (15). A ubiCA knockout which produces no
detectable ubiquinone (36) was shown to exhibit
several oxidative stress phenotypes in LB:
3
E), reiterating that ubiquinone relieves oxidative
stress.
accumulation of superoxide and
H
2
O
2
in
NBT enters inside the cells and has been
membranes, hypersensitivity to oxidative stress
inducing agents, and upregulation of catalase (15).
In the present work, whereas ubi deletion strains
that produce no detectable ubiquinone did not
grow at all in oleate, acetate and succinate, ubiI
mutant that produces decreased levels of
ubiquinone showed significant growth defect only
in oleate (Fig. 1C and 3A; Fig. S1). Collectively,
these observations led us to argue that of the above
three non-fermentable carbon sources, E. coli
generates highest ROS levels when cultured in
oleate and the increased requirement of
ubiquinone in oleate is to counteract elevated
levels of ROS.
extensively used in E. coli and other gram-
negative bacteria to measure intracellular
superoxide (40-43). We independently validated
that the reduction of NBT reports on intracellular
superoxide levels by showing that overexpressing
superoxide dismutase, SodA, from plasmid
decreases NBT signal in all tested media
conditions (TB, TB-Brij and TB-Ole; Fig. S3A
and B). We further confirmed our results from
NBT assay by using
a
fluorescent dye,
dihydroethidium (DHE) that also detects
superoxide (Fig. S3C) (44). Experiments involving
measurement of ROS levels were carried out in
TB to support the growth of ubiI knockout, which
shows growth defect in minimal medium
containing oleate as the sole carbon source (Fig.
S1; Fig. 3A). Furthermore, since TB causes mild
catabolite repression (45), supplemented carbon
sources are expected to be co-utilized with carbon
components of TB. As a representative, the
increase in biomass and transcript levels of fadL
(outer membrane transporter for LCFAs) and fadE
(acyl-CoA dehydrogenase involved in β-oxidation
of LCFAs) in WT grown in TB-Ole confirmed the
co-utilization of oleate with TB (46,47) (Fig. S4A
and B). Since maximum difference in ROS levels
was observed in the stationary phase (time point
T4 in Fig. S4A) (Fig. S4C), for single time point
experiments cultures were sampled in this growth
phase.
Consistent with the above proposal,
glutathione and thiourea, widely used chemical
antioxidants in E. coli (37-39), partially recovered
the growth defect of ΔubiI in oleate (Fig. 3B and
C). We did not observe a complete recovery since
another factor responsible for the poor growth of
ΔubiI in oleate would be reduced energy
generation due to lowering of ETC function.
Interestingly, WT strain also grew better in the
presence of glutathione and thiourea consistent
with previous report that cells suffer oxidative
stress during growth in oleate (13) (Fig. 3B and
C). Further, we tested whether E. coli indeed
generates highest ROS levels when cultured in
oleate in comparison to acetate and succinate. For
this, we measured intracellular ROS levels in WT
and ΔubiI cultured in Tryptone Broth (TB)
supplemented with various carbon sources by a
colorimetric assay based on the reduction of
nitroblue tetrazolium (NBT) dye by superoxide.
WT had highest ROS levels in TB-Ole; ~1.5-fold
higher than basal medium (Fig. 3D). This fold
Ubiquinone is a major antioxidant during
oleate metabolism
We tested whether in addition to ubiquinone other
oxidative stress response players counteract ROS
generated by oleate. In TB medium, ROS levels
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5

Role of ubiquinone in LCFA metabolism
were ~1.3 to 1.7 fold higher when strains lacked
either the ubi genes or other players [(alkyl
hydroperoxide reductase subunit- AhpC, oxidative
stress regulator- SoxR, superoxide dismutase-
SodA, catalase- KatE, and an enzyme involved in
glutathione biosynthesis, GshB) (14,15)] (Fig.
While this manuscript was under preparation, an
uncharacterized gene, yqiC (renamed as ubiK),
was identified as a ubiquinone biosynthesis player
based on the reduction of ubiquinone levels to
~20% of WT in ΔubiK strain (29), however, the
growth of ΔubiK strain on non-fermentable carbon
sources has not been evaluated. Importantly, in our
comparative analysis, we observed that similar to
ΔubiI, ΔubiK showed statistically significant
fitness defect only in oleate (Dataset S1, FDR
4
A). In contrast, in TB-Ole medium, ROS levels
increased only in ubi deletion strains (Fig. 4A).
We considered two possibilities for the TB-Ole
results: i) there is redundancy of enzymatic
scavengers and their regulators, and ii) as long as
ubiquinone is present, it does not allow ROS to
build-up further in TB-Ole thereby reducing
dependency on other players. Consistent with the
second possibility, the enzymatic scavengers, katG
and ahpC were induced (~2-fold) by oleate only in
a ΔubiI strain (Fig. 4B and C), and this increased
expression was reduced by ~25% upon exogenous
supplementation of ubiquinone-8 (Fig. 4D).
<
5%; Fig. 1C).
To further strengthen our proposal that
reduced levels of ubiquinone are suboptimal for
growth in oleate thus leading to oxidative stress,
we independently compared ubiquinone levels in
ΔubiI and ΔubiK, validated the growth defect of
ΔubiK in candidate studies and determined ROS
levels in ΔubiK. Q
0% in both ΔubiI and ΔubiK, and a peak
corresponding to an intermediate (elution time:
15 min) was observed in both the strains (Fig.
A; Fig. S5). Except oleate, ΔubiK did not show
8
levels were reduced to ~15-
2
Our above data suggests that ubiquinone is
a major player that mitigates LCFA-mediated
oxidative stress. We examined whether this
defense system is induced by LCFAs. E. coli
~
5
growth defect on any other non-fermentable
carbon source (Fig. 5B). The growth defect on
oleate was complemented by ubiK cloned on
plasmid (Fig. 5C), concomitant with restoration of
ubiquinone levels (Fig. S6). Further, similar to ubi
deletion strains, compared to WT in TB, ROS
levels were ~2.5-fold higher in ΔubiK in TB-Ole
ubiquinone is designated as Q
redox states in the cell, ubiquinone and ubiquinol
9). We extracted lipids from WT and several fad
knockouts, separated lipids by HPLC, and
measured total Q content (ubiquinone and
8
and exists in two
(
8
ubiquinol). Peaks for ubiquinone and ubiquinol in
the samples were assigned based on the elution
time of pure standards, and the reduction of
ubiquinone and ubiquinol peaks in ΔubiI (Fig. S5).
(
Fig. 5D).
The related phenotypes of ΔubiI and
ΔubiK prompted us to examine the phenotype of
ΔubiIΔubiK double mutant. In contrast to the
normal growth of ΔubiI and ΔubiK in LB medium,
the double mutant formed tiny colonies on LB
In WT cells, Q
TB-Ole compared to TB (Fig. 4E). Q
not increase in cells grown in TB-Brij.
Importantly, the increase in Q levels was
dependent on oleate utilization since Q levels did
8
content was ~1.8-fold higher in
8
levels did
8
(
Fig. 5E). In order to determine ubiquinone levels
8
in the ΔubiIΔubiK strain, we resorted to growing
the cultures in a rich LB-glucose medium, where
there is reduced dependency on ubiquinone for
growth (50). Interestingly, there was no detectable
ubiquinone in the ΔubiIΔubiK double mutant (Fig.
not increase in TB-Ole in fad mutants defective in
LCFA transport and β-oxidation (ΔfadL, ΔfadD
and ΔfadE) (Fig. 4E). Interestingly, the increase in
ROS levels in TB-Ole was also due to oleate
utilization; compared to TB, ROS level did not
exhibit considerable increase in TB-Ole in fad
mutants (Fig. 4F). ROS level in TB was higher in
ΔfadD strain possibly due to accumulation of
endogenous free fatty acids (48,49).
5
F; Fig. S7), suggesting redundancy in the
ubiquinone biosynthesis pathway. Importantly,
similar to single ubi deletion strains (ubiE, ubiF
and ubiH) that produce no detectable ubiquinone,
the ΔubiIΔubiK double mutant showed growth
defect in glucose but did not grow at all with
oleate, acetate and succinate (Fig. 5G). Taken
together, our data convincingly establishes that
there is a differential requirement of ubiquinone
ΔubiIΔubiK double mutant produces no
detectable ubiquinone
ꢀ
6

Role of ubiquinone in LCFA metabolism
amongst
non-fermentable
carbon
sources,
disruption of Cyo complex would be expected to
result in a more compromised growth on carbon
sources such as acetate that have poor net ATP
yield.
requirement being maximal in oleate to alleviate
oxidative stress.
DISCUSSION
Quantitative contribution of Nuo and Cyo to
growth is inversely correlated with the energy
yield of non-fermentable carbon sources
The important functions of ETC are ATP
production and maintenance of redox balance. We
find that Nuo is the major NADH dehydrogenase
for aerobic growth on oleate, acetate and succinate
Ubiquinone relieves oxidative stress generated
by LCFAs
It was surprising that unlike other ETC
components, requirement of ubiquinone, the
electron carrier of ETC, did not correlate with the
energy yield of non-fermentable carbon sources.
Our detailed analysis shows that ubiquinone was
maximally required in oleate to mitigate elevated
levels of ROS. Compared to WT cells, ROS levels
were ~1.5-fold higher in a ΔubiI strain grown in
glucose, acetate or succinate but there was no
difference in the growth profile of WT and ΔubiI
in these carbon sources (Fig. 3A and D). The
utilization of oleate resulted in ~1.5-fold increase
in ROS levels in WT cells compared to other
carbon sources which further increased to ~2.5-
fold in a ΔubiI strain (Fig. 3D). Importantly, this
elevated level of ROS (~2.5-fold) was deleterious
as evident from the significant growth defect of
ΔubiI strain in oleate and that the growth defect
could be partially recovered by chemical
antioxidants (Fig. 3). These data clearly indicate
that in oleate utilizing cells optimum levels of
ubiquinone are required to manage ROS below a
toxic threshold. Further, we find that amongst
various oxidative stress combat players,
ubiquinone is the key antioxidant during LCFA
metabolism. This is supported by the observation
that strains deleted for oxidative stress combat
players other than ubi genes do not exhibit
increase in ROS in oleate-utilizing cells (Fig. 4A).
Moreover, whereas ubiquinone accumulates in the
presence of oleate, other players are induced only
in a mutant defective in ubiquinone biosynthesis
(
Datasets S1A and C; Fig. 1C and 2). Because nuo
mutants would be unable to oxidize NADH
efficiently this would increase NADH/NAD
+
ratios, which might further inhibit TCA and
glyoxylate cycle enzymes resulting in a decrease
in cellular metabolites (28). Besides, since Nuo is
a proton pump (H /e =2) (8), its deletion would
lead to decrease in ATP synthesis in the cell.
Therefore, in nuo mutants grown on non-
fermentable carbon sources ATP would be
produced mainly from FADH oxidation.
2
The Δnuo strain exhibited no growth in
acetate (Fig. 2). We speculate that in acetate, in
+
-
+
addition to increased NADH/NAD ratio and
decrease in cellular metabolites, nuo mutant
cannot produce energy to support growth because
only 1 molecule of ATP will be generated per
2
acetate molecule considering ATP:FADH ratio to
be 1 (8). However, 1 molecule of ATP is also
expended to activate acetate to acetyl-CoA (4),
thus in a nuo mutant there will be no net ATP
gain. On the other hand nuo mutant exhibited
extended lag in oleate and succinate (Fig. 2). We
suggest that since nuo mutant can generate energy
2
from oxidation of FADH produced during β-
oxidation of oleate and in the first step of
succinate utilization (4,51), this allows cells to re-
adjust its metabolism to maintain redox balance
resulting in same growth rate and growth yield as
WT cells. Further, unlike acetate, activation of
succinate is not required for its metabolism and
although conversion of oleate to oleoyl-CoA is
required for oleate metabolism, only 1 molecule of
ATP is consumed per 18 carbon atoms (4,51).
Reduced energy generation might also be the
reason for the significant growth defect of Δcyo
strains in acetate (Fig. 1C). Since Cyo, the major
terminal oxidase during aerobic metabolism
(Fig. 4B,
C
and E). Interestingly, oleate
degradation generates ROS and also provides a
signal for ubiquinone accumulation (Fig. 4E and
F). These results suggest a feedback loop that
prevents excessive ROS formation during growth
in LCFAs.
An earlier study has shown that
ubiquinone is present in excess over flavins and
cytochromes in the E. coli inner membrane (52).
Thus under normal conditions ubiquinone is not
limiting for its electron transfer function.
+
-
generates proton motive force (H /e =2) (8),
ꢀ
7

Role of ubiquinone in LCFA metabolism
Considering this, ~2-fold increase in ubiquinone
levels in cells utilizing oleate could bring a
significant physiological response. Few ubi genes
are regulated by the ArcA-ArcB two-component
system and catabolite repression (53-57). It will be
interesting to investigate the mechanisms that
regulate ubiquinone levels during LCFA
degradation. We suggest that ROS itself might not
be the signal for upregulation of ubiquinone, since
despite exhibiting higher level of ROS ΔfadD
strain had basal ubiquinone levels in TB medium
respiratory dehydrogenases to terminal oxidases
thereby decreasing the chance of single-electron
donation to oxygen. Second, the reduced form of
ubiquinone (ubiquinol) can scavenge ROS (15).
Fig. 6 shows the probable sites of ROS
formation during growth of E. coli in LCFAs and
the mechanisms by which ubiquinone might
counteract LCFA-induced oxidative stress.
Regarding the site of ROS formation, we speculate+
that during LCFA catabolism, high NADH/NAD
2
and FADH /FAD ratios would increase electron
(
compare Fig. 4E and F).
Succinate has been extensively used for
flow in the ETC thereby increasing the probability
of electron leakage and auto-oxidation of reduced
form of respiratory dehydrogenases resulting in
screening ubi genes (34,35). We suggest that
oleate is a better carbon source than succinate for
identifying ubi players especially ones that have
partial effect on ubiquinone levels. The unique
requirement of ubiI and ubiK for growth in oleate
validates this suggestion (Fig. 1C, 3A and 5B; Fig.
S1). UbiK is a small protein (<100 amino acids)
that belongs to BMFP (Brucella Membrane
elevated levels of ROS. Importantly,
predominant source of ROS during LCFA
degradation could be the acyl-CoA
a
dehydrogenase, FadE, which catalyzes the
oxidation of acyl-CoA to enoyl-CoA concomitant
with reduction of FAD to FADH
2
. Whether FadE
itself re-oxidizes FADH and directly transfers
2
Fusogenic
Protein)
superfamily.
In
S.
electrons to ubiquinone or requires the
involvement of an electron transfer flavoprotein
(ETF) and ETF-quinone oxidoreductase is unclear
(61). Nevertheless, the oxidation of reduced flavin
bound to FadE could result in ROS formation.
Concerning the possible mechanisms by which
ubiquinone might combat ROS, our results suggest
that its antioxidant role cannot be explained solely
by the ability to transfer electrons from NADH
and succinate dehydrogenases to terminal oxidases
since amongst all these ETC components only
ubiquinone was more important on LCFAs when
compared to other non-fermentable carbon sources
(Fig. 1C, 2, 3A and 5B; Fig. S1). It is likely that
ubiquinone also limits ROS formation at FadE by
transferring electrons from FadE to the ETC. In
addition, a recent study has demonstrated the in
vitro quinol peroxidase activity of Cyd where
quinol serves as a substrate for the peroxidase to
typhimurium, it is involved in the regulation of
flagella and fimbriae expression, motility,
colonization and virulence of the organism
(
29,58,59). E. coli UbiK physically interacts with
a non-enzymatic ubiquinone biosynthesis player,
UbiJ, and is proposed to be an assembly factor for
additional Ubi proteins (29). Our results that a
double mutant of ubiK and an enzyme involved in
ubiquinone biosynthesis, ubiI, shows synthetic
sick/lethal phenotype with no detectable
ubiquinone provides a strong genetic evidence of
the interaction between UbiK and other Ubi
proteins (Fig. 5E, F and G; Fig. S7).
Proposed mechanisms by which ubiquinone
might counteract LCFA-mediated oxidative
stress
Several mechanisms have been suggested for ROS
formation that includes extraction of electrons
from reduced metal centers of certain enzymes by
molecular
oxidation-reduction cycles of ETC promoting the
reaction of free electrons with O and auto-
oxidation of flavoproteins (14,15,60). Soballe and
Poole first demonstrated the role of ubiquinone in
counteracting ROS in bacteria and proposed two
mechanisms to explain its antioxidant function.
First, ubiquinone limits ROS formation due to its
ability to rapidly transfer electrons from upstream
detoxify
H
2
O
2
(62). Thus, during LCFA
catabolism, besides decreasing ROS formation
owing to its electron shuttling role in ETC,
ubiquinone might promote the peroxidase activity
of terminal oxidase to detoxify ROS. Since ETC is
one of the sites for ROS formation, it might be
advantageous for the cell to have antioxidant in the
membrane to detoxify ROS locally. Ubiquinone in
conjunction with Cyd might fulfill this role.
O
2
,
leakage of electrons during
2
Several bacterial pathogens use LCFAs
derived from host tissues as their nutrient source
ꢀ
8

Role of ubiquinone in LCFA metabolism
(
1-3). It will be interesting to examine whether
were grown for defined time periods. For the
ubiquinone participates in managing LCFA-
mediated oxidative stress in these pathogens. In
fact ubiB, ubiE, ubiJ, and ubiK mutants of S.
typhimurium are impaired for intracellular
proliferation in macrophages (29,63). Since S.
typhimurium utilizes fatty acids in macrophages
during chronic infection (2), it is possible that
ubiquinone is required by this intracellular
pathogen to combat oxidative stress generated by
LCFAs.
detection of Q in ΔubiIΔubiK double mutant,
strains were grown in LB supplemented with 0.2%
glucose.
8
Library screening and data processing
The chemical genomics screen was performed
using the same methodology as reported
previously with slight modifications (17). Briefly,
the Keio deletion library was arrayed in 1536-
format and pinned onto plates containing minimal
medium agar supplemented either with oleate or
glucose with Brij-58, using a Singer Rotor robot.
Plates were incubated at 37°C for 21 hours for
glucose with Brij-58 and 42 hours for oleate. Time
points were chosen such that fitness differences
were apparent but growth had not saturated.
Pictures of the plates were taken using a
EXPERIMENTAL PROCEDURES
Strains and plasmids
Chemical genomics screen used the Keio deletion
library derived from BW25113 (16). Majority of
the follow-up experiments were conducted in
BW25113
background,
and
either
both
independent clones from the library and/or fresh
transductants were analyzed to rule out genetic
errors. Strains, plasmids, and primers used for
plasmid construction are listed in Table S1.
Canon G10 digital camera. Colony size was
quantified from plate images using the HT Colony
Grid Analyzer software package (18). Colony
sizes were filtered and normalized using
established methods for chemical genomics in E.
coli K-12 (19). To account for potential effects of
Brij-58 on growth, fitness scores for the oleate
condition were generated by directly comparing
colony size between oleate and glucose with Brij-
58 control using the same statistical test as the S-
score (18).
Media composition and growth conditions
Media had the following composition: M9
minimal medium- 5.3 g/L Na
KH PO , 0.5 g/L NaCl, 1g/L NH
MgSO , 2 mg/L Biotin, 2 mg/L Nicotinamide, 0.2
2
HPO
4
, 3 g/L
2
4
4
Cl, 1.2 g/L
4
mg/L Riboflavin, and 2 mg/L Thiamine; Lysogeny
Broth (LB)- 5 g/L yeast extract, 10 g/L Bacto
Tryptone, and 5 g/L NaCl; and Tryptone Broth
To facilitate comparison of the chemical
genomic profile of oleate to other carbon sources,
raw data from minimal media conditions of a
previous large-scale chemical genomic screen (17)
were reanalyzed using the same workflow above
and M9 minimal with 0.2% glucose as a
normalization. Finally, fitness score distributions
for each condition were scaled to have a standard
inter-quartile range of 1.35 (17) to minimize bias
in any downstream analysis from conditions with a
large variance. The MATLAB code and raw data
for this analysis are available online at
https://github.com/AnthonyShiverMicrobes/fitness
_lcfa.
(
TB)- 10 g/L Bacto Tryptone, and 5 g/L NaCl.
Unless otherwise specified, when required, media
were supplemented with one of the following
carbon sources at a final concentration of 5 mM:
glucose or sodium salt of acetate, succinate, or
oleate. Stock of oleate (50 mM) was prepared in
5
1
.0% Brij-58 (64). Media were solidified using
.5% (w/v) bacto agar. For chemical genomics
screen, minimal medium was supplemented either
with 5 mM oleate or 0.2% glucose with 0.5% Brij-
5
8. To facilitate comparison of our screen on
oleate with published screens on other carbon
sources (17), same concentration of glucose was
used in our control condition.
Growth curves
Cultures were incubated at 37°C. For
Overnight cultures grown in LB were pelleted,
washed and re-suspended in M9 minimal medium.
Cells were re-inoculated in 200 µl M9 minimal
medium containing the desired carbon source to a
starting OD450 of ~0.03, in 96-well plates, using a
robotic liquid handling system (Tecan). When
experiments in liquid medium, unless indicated
otherwise, primary cultures were grown in 3 ml
TB, which were further re-inoculated either in TB
or TB supplemented with desired carbon source to
an initial OD600 of ~0.01. These secondary cultures
ꢀ
9

Role of ubiquinone in LCFA metabolism
required, antibiotic, glutathione, thiourea or urea at
a desired concentration was added to the medium.
Plates were incubated in a shaker at 37°C, and
OD450 of the cultures was measured at designated
β-galactosidase assay
β-gal assays were performed in MC4100
background. Secondary cultures (15 ml) were
grown in 125 ml flasks for 14-16 hours. Cells were
pelleted, washed atleast 4 times with Z-buffer and
diluted to OD450 ~0.5. Several washings of the
culture were critical to remove Brij-58 since the
detergent interferes with β-gal assay. katG and
ahpC promoter activity was measured by
monitoring β-gal expression from single-copy
katG-lacZ and ahpC-lacZ transcriptional fusion,
respectively as described (65).
time
intervals
(Tecan
Infinite
M200
monochromator). The incubator shaker and
microplate reader were integrated with the liquid
handling system, and the transfer of plates
between shaker and reader was automated.
Dilution spotting
Overnight cultures grown in LB were pelleted,
washed and re-suspended in M9 minimal medium.
Several dilutions of cultures were spotted on M9
minimal medium containing the desired carbon
source. Antibiotic was added whenever required.
Plates were incubated and imaged at various time
Preparation of ubiquinol standard
Ubiquinol-8 was prepared by reduction of
ubiquinone-8 (Avantis Polar Lipids) following the
procedure used for reduction of ubiquinone-10
(66). Briefly, 19 ml hexane was added to 1 ml
ubiquinone solution (1 mg/ml in hexane). 1 ml
methanol and 200 mg sodium borohydride were
added (solution was covered to avoid light), stirred
and kept for 5 min. 2 ml water was added and
mixed thoroughly to dissolve sodium borohydride.
The mix was centrifuged at 2050 X g for 5 min.
Colorless organic supernatant was transferred to a
fresh tube and stored at -20°C. The conversion of
yellow colored ubiquinone-8 to colorless
ubiquinol-8 indicated complete reduction.
+
intervals using the Gel Doc XR imaging system
from BioRad. A representative image where
growth differences were apparent is shown in the
figures.
Nitroblue tetrazolium (NBT) reduction assay
Secondary cultures (3 ml) were grown in culture
tubes (27 ml capacity) for 16 hours. ROS levels
were determined by NBT reduction assay
following the protocol described in (42) with
slight modifications. Cells were pelleted and
washed with Hanks’ balanced salt solution
(
HBSS). Washing was required to remove growth
Quinone extraction and detection by HPLC-
Photodiode Array (PDA) detection analysis
Quinones were extracted using the protocol
described in (31) with slight modifications. 15 ml
secondary cultures were grown in 125 ml flasks
9
medium to avoid interference of Brij-58. 10 cells
were re-suspended in 0.2 ml HBSS and were split
in two aliquots: 0.5 ml NBT (1 mg/ml) was added
to one aliquot and the other aliquot was left
untreated. Both aliquots were incubated at 37°C
for 30 min. 0.1 ml of 0.1 M HCl was added and
samples were centrifuged at 18,400 X g for 15
min. Supernatant was discarded, and pellet was
treated with 0.4 ml dimethyl sulfoxide (DMSO) to
dissolve reduced NBT (formazan blue), followed
by addition of 0.8 ml HBSS. Formazan blue was
quantified at 575 nm. To determine the absorbance
corresponding to formazan blue, absorbance of
aliquot without NBT was deducted from
absorbance obtained for NBT treated sample. For
experiments where ROS levels were determined in
different phases of growth, 15 ml secondary
cultures were grown in 125 ml flasks. Independent
cultures (from the same primary culture) were set-
up for each time point.
1
0
for 16 hours. Equal number of cells (~3 x 10
cells) for all cultures were pelleted and the pellet
mass was determined. Pellets were re-suspended
in 100 µl of 0.15 M KCl and to the re-suspension
200 µl glass beads (acid washed ≤ 106 µm,
Sigma), 600 µl methanol and 12 µg ubiquinone-10
standard (Sigma) in hexane (used as internal
control for extraction efficiency) were added.
Samples were vortexed for 15 min, and 400 µl
hexane was added. Samples were again vortexed
for 3 min and then centrifuged at 3380 X g for 1
min. Upper hexane layer was transferred to fresh
micro-centrifuge tube. 100 µl of this hexane layer
was completely dried under vacuum and re-
suspended in 100 µl of mobile phase, an isocratic
solution constituted by using 40% ethanol, 40%
acetonitrile, and 20% of a mix of 90% isopropyl
ꢀ
10

Role of ubiquinone in LCFA metabolism
alcohol and 10% lithium perchlorate (1 M). Lipid
extracts were separated by reverse-phase HPLC
with a C18 column (Waters Sunfire 5 µm column,
ubiquinol-8 was 290 nm. Peaks for ubiquinone-8
and ubiquinol-8 in the effluent were assigned
based on the elution time of pure standards, and
reduction of ubiquinone-8 and ubiquinol-8 peaks
4
.6 X 250 mm) at a flow rate of 1 ml/min using
mobile phase. Quinones were detected using a
Photodiode array detector. In order to detect total
in ΔubiI strain. For each sample, Q peak area per
unit mass was calculated, and to account for
8
Q
8
(ubiquinone-8 + ubiquinol-8) in samples, λmax
difference in extraction efficiency between
of ubiquinone-8 and ubiquinol-8 standards was
determined by scanning from 240 nm to 399 nm.
samples, Q
by ubiquinone-10 peak area.
8
peak area per unit mass was divided
λ
max of ubiquinone-8 was 275 nm, and that of
ACKNOWLEDGMENTS
We acknowledge Carol Gross for access to the robotics facility for high-throughput genetic screens. We
thank Gisela Storz for strains GS022 and MC4100 ahpC-lacZ, and plasmid pAQ6, and Frédéric Barras
for strain BEFB05. We especially thank Patricia Kiley for valuable suggestions and critical reading of the
manuscript. We thank members of the Chaba lab for discussions. SA and TP acknowledge fellowship
support from IISER-Mohali for doctoral and postdoctoral work, respectively. KJ and HB are recipients of
DST-Inspire fellowship for doctoral and undergraduate studies, respectively. This work was supported by
start-up funds from IISER-Mohali to RC and was partly funded by CSIR (project no. 37(1623)/14/EMR-
II), Govt. of India to RC.
CONFLICT OF INTEREST
The authors declare that they have no conflict of interest with the contents of this article.
AUTHOR CONTRIBUTIONS
RC, SA, and KJ designed the study. RC performed the high-throughput genetic screen. ALS performed
analysis of the high-throughput data. SA, KJ and HB performed follow-up experiments. TP standardized
initial experiments. RC, KJ, SA, ALS and HB wrote the manuscript. All authors reviewed and edited the
manuscript. RC supervised the project.
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FOOTNOTES
†
Present address: Department of Signal Transduction and Biogenic Amines, Chittaranjan National Cancer
Institute, Kolkata, West Bengal 700026, India.
ABBREVIATIONS
LCFAs, long chain fatty acids; ETC, electron transport chain; ROS, reactive oxygen species; fad, fatty
acid degradation; TCA, tricarboxylic acid; GSEA, gene set enrichment analysis; TB, tryptone broth;
NBT, nitroblue tetrazolium
FIGURE LEGENDS
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 FADH
been speculated that FadE itself might oxidize FADH to FAD by transferring electrons from its
dehydrogenase domain to the ETC (61). However, there is no experimental evidence for the same. (C)
2
during β-oxidation. It has
2
ꢀ
16

Role of ubiquinone in LCFA metabolism
Fitness-scores were calculated for oleate and other carbon sources as compared to glucose control.
Fitness-scores of components of the β-oxidation pathway and ETC are shown.
FIGURE 2. Amongst 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
SD 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 1mM glutathione (GSH) or 1mM thiourea,
and OD450 was measured. 1mM urea was included as control for thiourea. For Figs 3A, B and C
experiments were done 3 times; each experiment had 3 technical replicates. A representative dataset, with
average and SD 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 (± SD) 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 (± SD) of 3
independent experiments. *P<0.05, **P<0.005 and 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 (±
SD) 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
(
2 2
C) (lacZ placed under the control of H O 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 (± SD) 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 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 (± SD) 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
and NS: not significant (unpaired two-tailed Student’s t-test). (E) Ubiquinone accumulates in WT cells in
response to oleate utilization. Total Q
grown either in TB or TB-Ole was determined. Q
8
level in lipid extracts from WT and various fad deletion strains
levels were normalized to the Q level of WT in TB
8
8
and represent average (± SD) of at least 4 independent experiments. (F) Oleate transport and degradation
is responsible for increase in ROS levels in WT grown in TB-Ole. WT and various fad deletion strains
ꢀ
17

Role of ubiquinone in LCFA metabolism
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 (± SD) of 3 independent experiments.
FIGURE 5. ΔubiIΔubiK double mutant produces no detectable ubiquinone. (A) ΔubiI and ΔubiK
have similar Q
determined. Q
8
content. Total Q
levels were normalized to the Q
8
level in lipid extracts from WT, ΔubiI and ΔubiK cells grown in TB was
level of WT in TB and represent average (± SD) of 3
8
8
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-
5
8. 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 (±
SD) 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 Q
ΔubiIΔubiK cells grown in LB-Glucose was determined. Q
8
level in lipid extracts from WT, ΔubiI, ΔubiK and
levels were normalized to the Q level of WT
8
8
in LB-Glucose and represent average (± SD) 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 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 FADH
2
/FAD ratios during β-oxidation and TCA cycle increase
electron flow in the ETC thereby increasing electron leakage and auto-oxidation of 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 FADH . Ubiquinone limits ROS formation by
2
rapidly transferring electrons from upstream dehydrogenases to terminal oxidases (Cyo and Cyd) thus
preventing electron leakage and auto-oxidation of reduced form of dehydrogenases. In addition, the
-
quinol peroxidase activity of Cyd will detoxify H
2
O
2
. 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 H
2 2
O by Cyd). Abbreviations: LCFA, long chain fatty acid; TCA,
tricarboxylic acid; Oaa, oxaloacetate; Cit, citrate; Isocit, isocitrate; α-KG, α-ketoglutarate; Suc-CoA,
-
2
succinyl-CoA; Suc, succinate; Fum, fumarate; Mal, malate; Glo, Glyoxylate; O , superoxide; Sdh,
succinate dehydrogenase; Q
8
, ubiquinone-8; Q
8
H
2
, ubiquinol-8; Cyd, cytochrome bd; Cyo, cytochrome
bo.
ꢀ
18

A genome-wide screen in Escherichia coli reveals that ubiquinone is a key antioxidant
for metabolism of long chain fatty acids
Shashank Agrawal, Kanchan Jaswal, Anthony L. Shiver, Himanshi Balecha, Tapas Patra
and Rachna Chaba
J. Biol. Chem. published online October 17, 2017
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