Ubiquinone Biosynthesis over the Entire O 2 Range: Characterization of a Conserved O 2 -Independent Pathway

Abstract

In order to colonize environments with large O 2 gradients or fluctuating O 2 levels, bacteria have developed metabolic responses that remain incompletely understood. Such adaptations have been recently linked to antibiotic resistance, virulence, and the capacity to develop in complex ecosystems like the microbiota. Here, we identify a novel pathway for the biosynthesis of ubiquinone, a molecule with a key role in cellular bioenergetics. We link three uncharacterized genes of Escherichia coli to this pathway and show that the pathway functions independently from O 2 . In contrast, the long-described pathway for ubiquinone biosynthesis requires O 2 as a substrate. In fact, we find that many proteobacteria are equipped with the O 2 -dependent and O 2 -independent pathways, supporting that they are able to synthesize ubiquinone over the entire O 2 range. Overall, we propose that the novel O 2 -independent pathway is part of the metabolic plasticity developed by proteobacteria to face various environmental O 2 levels.

Full text

Ubiquinone Biosynthesis over the Entire O
2
Range:
Characterization of a Conserved O
2
-Independent Pathway
Ludovic Pelosi,
a
Chau-Duy-Tam Vo,
b
Sophie Saphia Abby,
a
Laurent Loiseau,
c
Bérengère Rascalou,
a
Mahmoud Hajj Chehade,
a
Bruno Faivre,
b
Mathieu Goussé,
a
Clothilde Chenal,
a
Nadia Touati,
d
Laurent Binet,
d
David Cornu,
e
Cameron David Fyfe,
b
Marc Fontecave,
b
Frédéric Barras,
c,f
Murielle Lombard,
b
Fabien Pierrel
a
a
CNRS, CHU Grenoble Alpes, Grenoble INP, TIMC-IMAG, Université Grenoble Alpes, Grenoble, France
b
Laboratoire de Chimie des Processus Biologiques, Collège de France, CNRS UMR 8229, PSL Research University, Sorbonne Université, Paris, France
c
CNRS, Laboratoire Chimie Bactérienne, Institut Microbiologie de la Méditerranée, Aix Marseille Université, Marseille, France
d
ENSCP-Chimie ParisTech, Institut de Recherche de Chimie Paris, CNRS UMR 8247, Paris, France
e
Plateforme SICaPS, Institut de Biologie Intégrative de la Cellule (I2BC), Gif-sur-Yvette, France
f
SAMe Unit, Department of Microbiology, Institut Pasteur, Paris, France
ABSTRACT Most bacteria can generate ATP by respiratory metabolism, in which
electrons are shuttled from reduced substrates to terminal electron acceptors, via
quinone molecules like ubiquinone. Dioxygen (O
2
) is the terminal electron acceptor
of aerobic respiration and serves as a co-substrate in the biosynthesis of ubiquinone.
Here, we characterize a novel, O
2
-independent pathway for the biosynthesis of
ubiquinone. This pathway relies on three proteins, UbiT (YhbT), UbiU (YhbU), and
UbiV (YhbV). UbiT contains an SCP2 lipid-binding domain and is likely an accessory
factor of the biosynthetic pathway, while UbiU and UbiV (UbiU-UbiV) are in-
volved in hydroxylation reactions and represent a novel class of O
2
-independent
hydroxylases. We demonstrate that UbiU-UbiV form a heterodimer, wherein each
protein binds a 4Fe-4S cluster via conserved cysteines that are essential for activ-
ity. The UbiT, -U, and -V proteins are found in alpha-, beta-, and gammaproteo-
bacterial clades, including several human pathogens, supporting the widespread
distribution of a previously unrecognized capacity to synthesize ubiquinone in
the absence of O
2
. Together, the O
2
-dependent and O
2
-independent ubiquinone
biosynthesis pathways contribute to optimizing bacterial metabolism over the
entire O
2
range.
IMPORTANCE In order to colonize environments with large O
2
gradients or fluctuat-
ing O
2
levels, bacteria have developed metabolic responses that remain incom-
pletely understood. Such adaptations have been recently linked to antibiotic resis-
tance, virulence, and the capacity to develop in complex ecosystems like the
microbiota. Here, we identify a novel pathway for the biosynthesis of ubiqui-
none, a molecule with a key role in cellular bioenergetics. We link three unchar-
acterized genes of Escherichia coli to this pathway and show that the pathway
functions independently from O
2
. In contrast, the long-described pathway for
ubiquinone biosynthesis requires O
2
as a substrate. In fact, we find that many
proteobacteria are equipped with the O
2
-dependent and O
2
-independent path-
ways, supporting that they are able to synthesize ubiquinone over the entire O
2
range. Overall, we propose that the novel O
2
-independent pathway is part of the
metabolic plasticity developed by proteobacteria to face various environmental
O
2
levels.
KEYWORDS bioenergetics, facultative anaerobes, hydroxylases, iron-sulfur, oxygen,
peptidase U32, proteobacteria, quinones, respiration, ubiquinone
Citation Pelosi L, Vo C-D-T, Abby SS, Loiseau L,
Rascalou B, Hajj Chehade M, Faivre B, Goussé
M, Chenal C, Touati N, Binet L, Cornu D, Fyfe
CD, Fontecave M, Barras F, Lombard M, Pierrel
F. 2019. Ubiquinone biosynthesis over the
entire O
2
range: characterization of a
conserved O
2
-independent pathway. mBio
10:e01319-19. https://doi.org/10.1128/mBio
.01319-19.
Editor Dianne K. Newman, California Institute
of Technology
Copyright © 2019 Pelosi et al. This is an open-
access article distributed under the terms of
the Creative Commons Attribution 4.0
International license.
Address correspondence to Murielle Lombard,
murielle.lombard@college-de-france.fr,
or Fabien Pierrel,
fabien.pierrel@univ-grenoble-alpes.fr.
L.P. and C.-D.-T.V. contributed equally to the
work.
Received 21 May 2019
Accepted 7 June 2019
Published 9 July 2019
RESEARCH ARTICLE
Molecular Biology and Physiology
crossm
July/August 2019 Volume 10 Issue 4 e01319-19 ®mbio.asm.org 1

Since the oxygenation of the Earth’s atmosphere some 2.3 billion years ago, many
organisms adopted dioxygen (O
2
) as a terminal electron acceptor of their energy-
producing respiratory chains (1). Indeed, oxygenic (aerobic) respiration has an energetic
output superior to that of anaerobic respiration or fermentation, which are both
O
2
-independent processes (1). In fact, several microorganisms, including many impor-
tant human pathogens, are facultative anaerobes that are able to adopt either an
aerobic or an anaerobic lifestyle depending on the environmental conditions (2, 3). In
the laboratory, bacteria are usually cultured and studied under fully aerobic or com-
pletely anaerobic conditions (absence of O
2
)(4), whereas natural habitats cover the
entire range of O
2
concentrations (5). For instance, large O
2
gradients are typically
encountered in the human large intestine, in biofilms, or in transition zones between
oxic and anoxic environments (5). Moreover, bacteria can experience rapid transitions
between environments with vastly different O
2
contents, such as during the infection
process of enteric pathogens that progress along the gastrointestinal tract (3).
To maximize their bioenergetic capacities according to the various levels of O
2
encountered in their environment, bacteria modulate the composition of their respi-
ratory chains, notably the quinone species and the terminal reductases (3, 4, 6).
Quinones are lipophilic redox molecules that fuel electrons to terminal reductases,
which reduce O
2
whenever available, or alternative electron acceptors, for instance,
nitrate, dimethyl sulfoxide (DMSO), and trimethylamine N-oxide (7). Naphtoquinones
(menaquinone [MK] and demethylmenaquinone [DMK]) and ubiquinone (UQ) are the
two main groups of bacterial quinones. (D)MK and UQ differ by the nature of their head
group and the value of their redox midpoint potential (8). (D)MK are considered
anaerobic quinones, since they function primarily in anaerobic respiration, whereas UQ
is considered an aerobic quinone, because it supplies electrons mostly to the reduc-
tases that reduce O
2
(1, 8, 9). Accordingly, UQ is the main quinone of the facultative
anaerobe Escherichia coli under aerobic conditions, whereas the naphtoquinones are
predominant in the absence of O
2
(10, 11), with UQ nevertheless being present.
The biosynthesis of UQ requires a total of eight reactions to modify the aromatic ring
of the precursor, 4-hydroxybenzoic acid (4-HB): one prenylation, one decarboxylation,
three hydroxylation, and three methylation reactions (Fig. 1A)(12). In addition to the
enzymes that catalyze the various steps, three accessory factors, UbiB, UbiJ, and UbiK,
are also needed. UbiB has an ATPase activity (13), and we showed that UbiJ and UbiK
(14, 15) belong to a multiprotein UQ biosynthesis complex, in which the SCP2 domain
(sterol carrier protein 2) of UbiJ binds the hydrophobic UQ biosynthetic intermediates
(16). This UQ biosynthetic pathway is dependent upon O
2
, since all three hydroxylases,
UbiI, UbiH, and UbiF, use O
2
as a cosubstrate (Fig. 1A)(17, 18). We showed recently that
other hydroxylases, UbiL, UbiM, and Coq7, replace UbiI, UbiH, and UbiF in some
proteobacteria (19). The six hydroxylases have in common their dependence on O
2
and,
thus, function in UQ biosynthesis only when sufficient O
2
is available. Interestingly,
Alexander and Young established 40 years ago that E. coli was able to synthesize UQ
anaerobically (20), suggesting the existence of an O
2
-independent biosynthesis path-
way, which is still uncharacterized.
In this study, we describe the O
2
-independent UQ biosynthetic pathway in E. coli
and identify three essential components, the UbiT, UbiU, and UbiV proteins, formerly
called YhbT, YhbU, and YhbV. We show that the O
2
-independent UQ biosynthetic
pathway is widely conserved in proteobacteria. UbiT likely functions as an accessory
factor in the O
2
-independent UQ biosynthetic pathway, and we show that UbiU and
UbiV are involved in at least one O
2
-independent hydroxylation reaction. Moreover, we
demonstrate that both UbiU and UbiV bind a [4Fe-4S] cluster essential for activity,
which identifies these proteins as prototypes of a new class of O
2
-independent hy-
droxylases. Our results highlight that many proteobacterial species use two different
and complementary molecular pathways to produce UQ over the entire continuum of
environmental O
2
.
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RESULTS
4-HB is the precursor of UQ synthesized under anaerobic conditions. 4-HB is the
precursor of UQ synthesized under aerobic conditions (21). Accordingly, an E. coli ΔubiC
mutant impaired in 4-HB biosynthesis (22) is deficient in UQ
8
and is complemented by
addition of 4-HB to the growth medium (23). In order to evaluate whether 4-HB is also
the precursor of the O
2
-independent UQ biosynthetic pathway, we grew a ΔubiC strain
anaerobically. The ΔubiC strain showed a diminished level of UQ
8
, which was partially
recovered by supplementation with 4-HB (Fig. 1B). Furthermore, we grew the ΔubiC
strain in medium supplemented with
13
C
7
-4-HB and analyzed the labeling of biosyn-
thesized UQ
8
by high-performance liquid chromatography-mass spectrometry (HPLC-
MS). In cells grown under aerobic or anaerobic conditions, the labeled form of UQ
represented 98.3% and 97.3% (⫾0.2%), respectively, of the total UQ
8
pool (Fig. 1C and
D). As expected, ΔubiC cells grown anaerobically with unlabeled 4-HB did not show any
13
C
6
-UQ
8
(Fig. 1E). Together, these results establish that 4-HB is the precursor of the
O
2
-independent UQ biosynthetic pathway.
FIG 1 Aerobic and anaerobic UQ biosynthetic pathways differ only in the hydroxylation steps. (A) O
2
-dependent UQ biosynthesis pathway in E. coli. The
octaprenyl tail is represented by R on the biosynthetic intermediates, and the numbering of the aromatic carbon atoms is shown on OPP. Abbreviations used
are 4-HB for 4-hydroxybenzoic acid, OPP for octaprenylphenol, DMQ
8
for C6-demethoxy-ubiquinone 8, and UQ
8
for ubiquinone 8. (B) UQ
8
quantification of WT
and ΔubiC cells grown anaerobically in glycerol-nitrate medium supplemented with the indicated concentrations of 4-HB or left unsupplemented. Values are
means ⫾standard deviations (SD) (n⫽3to6).****,P⬍0.0001 by unpaired Student’s ttest. (C to E) Mass spectra of UQ
8
obtained by HPLC-MS analysis of lipid
extracts from cells grown with
13
C
7
-4-HB either anaerobically (C) or aerobically (D) or anaerobically with unlabeled 4-HB (E). (F) UQ
8
quantification from WT and
Δubi cells grown anaerobically in SMGN medium overnight or aerobically in LB medium until an OD of 0.8 was reached. nd, not detected under aerobic and
anaerobic conditions; nd, not detected under anaerobic conditions. Values are means ⫾SD (n⫽3 to 4). (G) HPLC-ECD analyses (mobile phase 1) of lipid extracts
from 1 mg of WT or ΔubiIHF cells grown in LB medium under air or anaerobic conditions (⫺O
2
). Chromatograms are representative of n⫽3 independent
experiments (UQ
10
used as a standard). (H) UQ biosynthesis represented with Ubi enzymes specific to the O
2
-dependent pathway (red), to the O
2
-independent
pathway (green), or common to both pathways (black). The same color code applies to the accessory factors (circled).
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2
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Ubi enzymes, except hydroxylases, are common to the aerobic and anaerobic
UQ biosynthesis pathways. The above-described result suggests that the UQ biosyn-
thetic pathways decorate 4-HB with the same chemical groups irrespective of the
presence of environmental O
2
. Thus, we evaluated whether the enzymes of the aerobic
pathway are also involved in the O
2
-independent pathway by measuring the UQ
8
content of knockout (KO) strains grown under aerobic and anaerobic conditions.
Deletion of ubiA,ubiE,orubiG abrogated UQ
8
biosynthesis under both conditions,
whereas ΔubiB,ΔubiD, and ΔubiX strains synthesized a limited amount of UQ
8
but only
under aerobic conditions (Fig. 1F). In contrast, ubiJ and ubiK had no effect on UQ
biosynthesis under anaerobic conditions (Fig. 1F).
Under aerobic conditions, the hydroxylation reactions are catalyzed by the flavin
monooxygenases (FMOs) UbiF, UbiH, and UbiI that use dioxygen as a cosubstrate (17,
18, 24)(Fig. 1A). We previously reported that cells deleted for a single O
2
-dependent
hydroxylase (ΔubiF,ΔubiI,orΔubiH cells) were deficient in UQ when cultured in the
presence of air but synthesized UQ under anaerobic conditions (17), consistent with the
existence of an alternative hydroxylation system in the O
2
-independent pathway (20).
Indeed, we confirmed that all three FMOs are dispensable for the O
2
-independent UQ
biosynthetic pathway, since a ΔubiF ΔubiI ΔubiH triple mutant was deficient for UQ
when grown in air but synthesized wild-type (WT) levels of UQ
8
under anaerobic
conditions (Fig. 1G). Together, our results demonstrate that the O
2
-dependent and
O
2
-independent UQ biosynthetic pathways share the enzymes involved in the preny-
lation (UbiA), decarboxylation (UbiX and UbiD), and methylation (UbiE and UbiG)
reactions but differ in their hydroxylases and the accessory factors UbiJ and UbiK
(Fig. 1H).
Identification of three genes required for UQ biosynthesis under anaerobic
conditions. To identify genes involved in the O
2
-independent UQ biosynthetic path-
way, we cultivated anaerobically a collection of ⬃200 E. coli strains that contained
deletions covering multiple open reading frames (ORFs) (25, 26), and we analyzed their
UQ
8
content by HPLC-electrochemical detection (HPLC-ECD). We found a complete
absence of UQ
8
in strains ME4561, ME5034, and ME4746 that carry deletions encom-
passing ubiE-ubiJ-ubiB-ubiD,ubiG, and ubiX, respectively (see Table S1 in the supple-
mental material). Several other strains had a low UQ
8
content and poor growth in
synthetic medium supplemented with glycerol and nitrate (SMGN). However, those
strains showed better growth and higher UQ
8
content in LB medium (Table S1). Thus,
we did not investigate them further, as a genetic defect affecting directly the O
2
-
independent UQ pathway was unlikely. In contrast, ME4641 showed a profound UQ
8
deficiency and robust anaerobic growth in LB and SMGN media (Table S1). Importantly,
ME4641 had a WT UQ
8
level when grown aerobically, suggesting that only the
O
2
-independent pathway was altered (Fig. 2A). ME4641 contains a deletion named
OCL30-2 that covers 9 genes, 5 of them lacking an identified function (Fig. 2B). To find
the candidate gene involved in the anaerobic biosynthesis of UQ, we obtained 8
single-gene KO strains from the Keio collection (27) and analyzed their quinone content
after anaerobic growth (Fig. 2C). The ΔyhbT and ΔyhbU strains were strongly deficient
in UQ
8
. We then transduced the ΔyhbT and ΔyhbU mutations from the Keio strains into
an MG1655 genetic background and also constructed the ΔyhbV strain, which was not
available in the Keio collection. We found that all three strains had very low levels of
UQ
8
when grown under anaerobic conditions but showed normal levels after aerobic
growth (Fig. 2D, Table 1). In addition, the mutant strains showed a 2-fold decrease in
MK
8
and a 2-fold increase in DMK
8
after anaerobic growth (Table 1). This effect might
indirectly result from the UQ
8
deficiency, as it was also observed in the ΔubiG strain
(Fig. S1A).
Deletion of yhbT,yhbU,oryhbV causes UQ
8
deficiency specifically under
anaerobic conditions. We then transformed the mutant strains with an empty vector
or a vector carrying a WT allele of the studied gene. In yhb KO strains expressing the
corresponding gene from the plasmid, we observed a complementation of the UQ
8
deficiency (Table 1) and a normalization of the levels of octaprenylphenol (OPP), an
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early UQ
8
biosynthetic intermediate (Fig. 2E). The ⬃3-fold elevation of OPP in the yhbT,
-U, and -V KO mutants suggested that the O
2
-independent UQ biosynthetic pathway
was blocked downstream of OPP in these strains. In these experiments, no cross-
complementation was observed, for example, the plasmid with yhbV had no effect on
FIG 2 yhbT,yhbU, and yhbV are essential to the anaerobic biosynthesis of UQ. (A) HPLC-ECD analysis of lipid extracts from ME4641 strain grown in SMGN either
aerobically or anaerobically (⫺O
2
). (B) Genomic region covered by the OCL30-2 deletion in the ME4641 strain. (C) HPLC-ECD analysis of lipid extracts from
knockout strains of the individual genes covered by the OCL30-2 deletion grown in SMGN anaerobically. (D) HPLC-ECD analysis of lipid extracts from ΔyhbT,
ΔyhbU, and ΔyhbV strains constructed in the MG1655 background and grown in SMGN either aerobically or anaerobically. HPLC-ECD analyses with mobile phase
2 (A, C, and D). (E) OPP content (as a percentage of the WT, mass detection M⫹NH
4
⫹
) in cells from Table 1. The Δyhb strains contain either an empty plasmid
or a plasmid carrying the indicated gene and were cultured anaerobically in SMGN containing 0.02% arabinose. Values are means ⫾SD (n⫽3to5).**,P⬍
0.01 by unpaired Student’s ttest. (F) UQ
8
content (as a percentage of the WT grown in LB medium) of cells cultured anaerobically in SM containing the indicated
carbon sources and electron acceptors. (G) Single-ion monitoring for UQ
8
(M⫹NH
4
⫹
) in HPLC-MS analysis (mobile phase 1) of lipid extracts from 1 mg of ΔubiU
or ΔubiU ΔubiH cells grown in SMGN under anaerobic conditions. (H) Single-ion monitoring in HPLC-MS analysis (mobile phase 1) of lipid extracts from 1.6 mg
of cells grown in LB medium under strict anaerobic conditions and quenched in methanol. Chromatograms are representative of n⫽3 independent
experiments (G and H). (I) UQ
8
content of cells described for panel H (quantification of the signal at 8 min with m/z 744.6). Values are means ⫾SD (n⫽3).
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2
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ΔyhbT or ΔyhbU strains, suggesting the absence of redundancy in the function of each
gene. We also measured a profound UQ
8
deficiency when the ΔyhbT,ΔyhbU, and ΔyhbV
strains were grown anaerobically in various media (glycerol plus DMSO, lactate plus
KNO
3
)(Fig. 2F), showing that the UQ
8
biosynthetic defect is not linked to a particular
carbon source or electron acceptor. Altogether, our results demonstrate that the yhbT,
yhbU, and yhbV genes are part of the O
2
-independent UQ biosynthetic pathway, so we
propose to rename them ubiT,ubiU, and ubiV, respectively.
Complete absence of UQ biosynthesis in ⌬ubiT,⌬ubiU, and ⌬ubiV mutants
grown under strict anaerobic conditions. As the ΔubiT,ΔubiU, and ΔubiV strains still
contained small amounts of UQ
8
after growth under anaerobic conditions (Table 1 and
Fig. 2F), we wondered how this UQ
8
was synthesized. To verify if the O
2
-dependent
pathway contributed to this synthesis, we inactivated the ubiH gene in the ΔubiU strain.
Sensitive HPLC-MS detection established that UQ
8
was completely absent from extracts
from the ΔubiH ΔubiU cells (Fig. 2G). This result supported that the residual UQ
8
synthesized in ΔubiU cells originated from the O
2
-dependent pathway and suggested
that our anaerobic media contain trace amounts of O
2
or that O
2
-dependent UQ
biosynthesis occurs during the handling of cells, under normal atmosphere, prior to
quinone extraction.
To eliminate the traces of O
2
, we took extra precautions in the degassing and
inoculation of our media (see Materials and Methods) and also added a reductant
(L-cysteine). In addition, we used the redox indicator resazurin to verify strict anaero-
biosis during the entire culture. We also modified our sampling procedure to rapidly
quench the anaerobic cells in ice-cold methanol in order to prevent any O
2
-dependent
UQ biosynthesis prior to quinone extraction. HPLC-MS analysis of extracts from cells
cultivated and handled under such strict anaerobic conditions showed the nearly
complete absence of UQ
8
in ΔubiT,ΔubiU, and ΔubiV strains (Fig. 2H and I). In contrast,
the UQ
8
level of the WT strain (Fig. 2I) was comparable to those we measured
previously in WT cells cultivated under suboptimal anaerobic conditions (Table 1)(14),
establishing that UQ biosynthesis occurred independently from O
2
. Together, our
results show that ΔubiT,ΔubiU, and ΔubiV cells are unable to synthesize UQ under strict
anaerobic conditions, unlike WT cells. Furthermore, our results support that the low
residual UQ content previously observed in ΔubiT,ΔubiU, and ΔubiV strains (Table 1 and
Fig. 2F) resulted from the function of the O
2
-dependent pathway.
ubiT,-U, and -V strongly cooccur exclusively in genomes with potential for UQ
biosynthesis. We investigated the distribution of ubiT,-U, and -Vin a large genome
data set of 5,750 genomes of bacteria and archaea. We found no evidence of genomes
harboring matches for more than one of the three genes of interest outside of
Alphaproteobacteria,Betaproteobacteria, and Gammaproteobacteria and three genomes
TABLE 1 Quinone content of WT and Δyhb strains cultured in SMGN either aerobically or anaerobically
a
Culture condition Strain
Quinone content
UQ
8
(pmol/mg cells) DMK
8
(247-nm peak area/mg cells) MK
8
(247-nm peak area/mg cells)
Air WT 208 ⫾9.4 0.94 ⫾0.26 0.15 ⫾0.09
Air ΔyhbT 193.9 ⫾12.4 1.34 ⫾0.05 0.13 ⫾0.03
Air ΔyhbU 233.8 ⫾16.2 0.9 ⫾0.26 0.16 ⫾0.09
Air ΔyhbV 227.9 ⫾25.1 0.93 ⫾0.15 0.13 ⫾0.04
Anaerobic WT 114.2 ⫾19.1 1.28 ⫾0.4 0.41 ⫾0.15
Anaerobic ΔyhbT 3.3 ⫾0.4 2.88 ⫾0.47 0.17 ⫾0.01
Anaerobic ΔyhbU 6.1 ⫾3.3 2.55 ⫾0.55 0.19 ⫾0.01
Anaerobic ΔyhbV 7.3 ⫾4.7 2.61 ⫾0.49 0.21 ⫾0.01
Anaerobic ΔyhbT⫹pK 2 ⫾0.4 2.46 ⫾0.33 0.16 ⫾0.04
Anaerobic ΔyhbT⫹pK-yhbT 64.6 ⫾16.1 1.77 ⫾0.29 0.3 ⫾0.04
Anaerobic ΔyhbU⫹pBAD 1 ⫾0.6 2.29 ⫾0.58 0.18 ⫾0.04
Anaerobic ΔyhbU⫹pBAD-yhbU 82 ⫾15.9 1.2 ⫾0.18 0.27 ⫾0.04
Anaerobic ΔyhbV⫹pBAD 2.2 ⫾3.5 2.14 ⫾0.15 0.23 ⫾0.01
Anaerobic ΔyhbV⫹pBAD-yhbV 120.4 ⫾18.5 1.87 ⫾0.19 0.42 ⫾0.06
a
n⫽3 to 5. For strains containing pK or pBAD vectors, SMGN was supplemented with 0.02% arabinose.
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of Acidithiobacillia (Table S2A). Interestingly, the three former classes are the only ones
known so far to be able to produce UQ (1, 8), consistent with a specific link between
UbiT, -U, and -V and UQ biosynthesis.
This was confirmed by analyzing in more detail the distribution of ubiT,-U, and -V
and three marker genes of the UQ biosynthetic pathway (ubiA,ubiE, and ubiG)in611
representative and reference genomes of alpha-, beta-, and gammaproteobacteria
(Table S2B). We chose ubiA,-G, and -E because they have a higher conservation than
other genes, like ubiC,-D, and -X (28), and because they are part of the O
2
-dependent
and O
2
-independent pathways (Fig. 1H). A total of 575 genomes had positive matches
for ubiA,-G, and -E, and 589 had positive matches for at least two of them. Regarding
the distribution of ubiT,-U, and -V, 221 genomes had matches for at least one of the
three genes, and in 210 cases (95%) the three genes were present. Importantly, all
genomes with ubiT,-U, and -Vharbored at least two of the three marker genes for the
UQ biosynthetic pathway (Table S2B). In addition, we found that 22 out of the 29
proteobacterial orders analyzed had up to 50% of genomes harboring a complete set
of the ubiT,-U, and -Vgenes (Fig. 3A), demonstrating a wide taxonomic distribution.
FIG 3 ubiT,-U, and -Voccurrence and genetic architecture in proteobacterial genomes. (A) The proportion of genomes with (green) and
without (red) all three genes, ubiTUV (left column), is indicated for each proteobacterial order known to synthesize UQ. The middle column,
labeled 3-genes loci, displays the proportion of genomes with the three genes either at a single locus (green) or at different loci (red).
The number of genomes analyzed for each order is given in the right column (Nb genomes). (B) Occurrence in the reference
proteobacterial genomes of the marker proteins (UbiA, -E, and -G), of the O
2
-dependent hydroxylases, and of the UbiT,-U, and -V proteins.
The number in boldface represents the 3 genomes (P. fulvum,M. marinus, and O. formigenes) containing exclusively the O
2
-independent
pathway. (C) The distinct genetic architectures found for ubiT,-U, and -Vin genomes where the three genes were present are displayed
as boxes with different colors. The numbers of cases corresponding to each depicted architecture are given on the right. A white box
corresponds to a gene found between the genes of interest, and a white box with dots corresponds to two to five genes between the
genes of interest.
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Overall, our analysis indicates a strong pattern of cooccurrence of ubiT,-U, and -Vand
demonstrates that they are uniquely found in genomes showing signs of UQ produc-
tion.
We found ubiA,-G, and -E and O
2
-dependent hydroxylase genes in 570 genomes, of
which 204 also contained ubiT,-U, and -V(Table S2B and Fig. 3B). Only 3 species,
Phaeospirillum fulvum,Magnetococcus marinus, and Oxalobacter formigenes, seem to
rely exclusively on the O
2
-independent pathway for UQ production, as their genomes
contain the ubiT,-U, and -Vgenes but no O
2
-dependent hydroxylases (Fig. 3B). We
noticed that the ubiT,-U, and -Vgenes are found next to other ubi genes in M. marinus
(Fig. S1B). Interestingly, P. fulvum and M. marinus were described to synthesize UQ
under microaerobic conditions (29–31), and O. formigenes has been documented as an
obligate anaerobe (32). Together, our results show that the O
2
-independent UQ bio-
synthesis pathway is widespread in alpha-, beta-, and gammaproteobacterial orders
and coexists with the O
2
-dependent pathway in 98% of the cases.
We then looked into the relative positioning of ubiT,-U, and -Vin the 210 genomes
harboring the three genes. We found 106 cases, covering alpha-, beta-, and gamma-
proteobacterial orders, where they were located next to each other (termed 3-gene
loci) and 82 cases of a 2-gene locus, with the third gene being elsewhere in the genome
(Fig. 3C). Evaluation of the genetic architecture of ubiT,-U, and -Vrevealed that ubiU
and ubiV were found exactly next to each other in 69% of the loci (in all 3-gene loci and
in 39/82 of the 2-gene loci) and that the three genes were located in three separate
parts of the genome in only 21 cases (Fig. 3C). Interestingly, as an additional support
for their involvement in the same function, we found an example of a gene fusion
between ubiT and ubiU in two genomes from Zymomonas mobilis strains (alphapro-
teobacteria), which also contain a ubiV gene directly downstream of the fused gene
(Fig. 3C).
UbiT is an SCP2 protein, and UbiU-UbiV are required for the O
2
-independent
hydroxylation of DMQ
8
.We then analyzed the sequences of the UbiT, UbiU, and UbiV
proteins. The major part of UbiT (amino acids [aa] 45 to 133, from a total of 174 aa in
the E. coli protein) corresponds to an SCP2 domain (Pfam entry PF02036) (Fig. S2). SCP2
domains typically form a hydrophobic cavity that binds various lipids (33), and our
sequence alignment indeed showed the conservation of hydrophobic amino acids at
several positions in the SCP2 domain of UbiT (Fig. S2). Recently, we reported that UbiJ
binds UQ biosynthetic intermediates in its SCP2 domain and organizes a multiprotein
complex composed of several Ubi enzymes (16). We propose that UbiT and its SCP2
domain fulfill similar functions in the O
2
-independent UQ pathway, as UbiJ is required
exclusively for the O
2
-dependent biosynthesis of UQ (Fig. 1F).
UbiU and UbiV have ⬃330 and 300 aa, respectively, and contain an uncharacterized
motif called peptidase U32 (PF01136) (Fig. S3 and S4). Since only the hydroxylation
reactions are uncharacterized in the O
2
-independent pathway (Fig. 1H), we hypothe-
sized that UbiU and UbiV function in these steps. To test our hypothesis, we developed
an in vivo assay based on the O
2
-independent conversion of labeled DMQ
8
into labeled
UQ
8
. This assay monitors the C6-hydroxylation and the subsequent O6-methylation
(Fig. 4A). ΔubiC ΔubiF cells grown aerobically with
13
C
7
-4-HB synthesized DMQ
8
, 73% of
which was labeled with
13
C
6
. Upon transfer to anaerobic conditions, the cells gradually
converted a significant part of (
13
C
6
)-DMQ
8
into (
13
C
6
)-UQ
8
(Fig. 4B). Inactivation of
either ubiU or ubiV in ΔubiC ΔubiF cells did not perturb the accumulation of (
13
C
6
)-
DMQ
8
but prevented its conversion into (
13
C
6
)-UQ
8
(Fig. 4C and D). This result dem-
onstrates that UbiU and UbiV are essential for the C6-hydroxylation reaction of the
O
2
-independent UQ biosynthetic pathway.
UbiV contains a [4Fe-4S] cluster. To gain insights into the potential presence of
cofactors in UbiU and UbiV, we attempted to characterize them biochemically. UbiU
was not soluble but we purified UbiV
6His
, which behaved as a monomer in solution
(Fig. S5A and B). UbiV was slightly pink-colored and had a UV-visible (UV-Vis) absorption
spectrum with features in the 350- to 550-nm region (Fig. 5A, dotted line), suggesting
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the presence of iron-sulfur (Fe-S) species (34–36). We indeed detected substoichiomet-
ric amounts of iron and sulfur (0.2 Fe and 0.2 S/monomer), indicating oxidative
degradation of the [Fe-S] cluster during aerobic purification, as already observed with
many other Fe-S proteins (37, 38). Consistent with this hypothesis, anaerobic reconsti-
tution of the [Fe-S] cluster yielded a UbiV protein with 3.9 iron and 3.3 sulfur/monomer
(Table 2) and with a UV-Vis spectrum characteristic of a [4Fe-4S]
2⫹
cluster (39)(Fig. 5A,
solid line) that was affected by exposure to air (Fig. S5C). The electron paramagnetic
resonance (EPR) spectrum of the cluster reduced anaerobically displayed features
characteristic of a [4Fe-4S]
1⫹
cluster in the spin state S ⫽1/2 (Fig. 5B)(38, 40). Overall,
we conclude that, under anaerobic conditions, UbiV is able to bind one air-sensitive,
redox-active [4Fe-4S] cluster.
[Fe-S] clusters are typically coordinated by cysteine residues (41, 42), and we
obtained evidence that the [4Fe-4S] cluster in UbiV is coordinated by four conserved
cysteines arranged in a CX
n
CX
12
CX
3
C motif (Fig. S4). Indeed, combinatorial elimination
of C39, C180, C193, and C197 in double, triple, and quadruple mutants resulted in
proteins incapable of binding [Fe-S] clusters in vivo, as shown by the absence of
absorption bands in the 350- to 550-nm region of their UV-Vis spectra (Fig. S5D).
Furthermore, after anaerobic reconstitution of the cluster, the Fe and S contents and
the absorbance at 410 nm were largely decreased in the double and triple mutants and
were undetectable in the quadruple mutant (Table 2 and Fig. 5C). Finally, we found that
the mutation of C180 or C193 altered the function of UbiV in vivo (Fig. 5D), suggesting
that the [4Fe-4S] cluster is important for activity.
UbiU contains a [4Fe-4S] cluster and forms a complex with UbiV. We succeeded
in purifying UbiU after coexpressing it with UbiV
6His
(Fig. S6A). The two proteins
copurified in the form of a heterodimer (Fig. S6A and B) that showed traces of [Fe-S]
clusters (Fig. 6A, dotted line), with substoichiometric amounts of iron and sulfide (0.4
Fe and 0.4 S/heterodimer). Reconstitution with iron and sulfide yielded a heterodimer
with about 8 iron and 8 sulfur (Table 2) and a UV-visible spectrum characteristic of
[4Fe-4S]
2⫹
clusters (Fig. 6A, solid line). The EPR spectrum of reduced UbiU-UbiV was
FIG 4 UbiU and UbiV are necessary for the anaerobic conversion of DMQ
8
into UQ
8
. (A) Conversion of DMQ
8
to
UQ
8
with enzymes of the O
2
-dependent and the O
2
-independent pathways, indicated above and below arrows
(numbering of carbon atoms shown on DMQ
8
and polyprenyl tail represented by R). (B) Quantification by HPLC-MS
(monitoring of Na
⫹
adducts) of unlabeled (
12
C) and labeled (
13
C
6
) DMQ
8
and UQ
8
in ΔubiC ΔubiF cells after aerobic
growth and transition to anaerobiosis. (C and D) Same as panel B but with ΔubiC ΔubiF ΔubiU cells (C) and ΔubiC
ΔubiF ΔubiV cells (D). nd, not detected. Results are representative of two independent experiments (B to D).
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also consistent with the presence of 2 different [4Fe-4S] clusters since it showed a
composite signal, which reflects the presence of two different S ⫽1/2 species (Fig. 6B).
Four strictly conserved cysteines are also found in UbiU (Fig. S3), and we hypoth-
esized that they bind the [4Fe-4S] cluster. We eliminated these cysteines in pairs and
FIG 5 UbiV binds a [4Fe-4S] cluster. (A) UV-visible absorption spectra of as-purified UbiV (dotted line, 47
␮
M) and
reconstituted holo-UbiV (solid line, 41
␮
M). The inset is an enlargement of the 300- to 700-nm region. The molar
extinction coefficient, ␧
410nm
, was determined to be 10.8 ⫾0.4 mM
⫺1
cm
⫺1
for holo-UbiV. (B) X-band EPR spectrum
of 785
␮
M dithionite-reduced holo-UbiV. Recording conditions were the following: temperature, 10K; microwave
power, 10 mW; modulation amplitude, 0.6 mT. (C) Comparative UV-visible absorption spectra of WT and different
Cys-to-Ala mutants of UbiV after [Fe-S] cluster reconstitution, with the following concentrations: 41
␮
M WT, 44
␮
M
C193A C197A, 46
␮
M C39A C193A C197A, 47
␮
M C180A C193A C197A, and 54
␮
M C39A C180A C193A C197A. (A
to C) Proteins were in 50 mM Tris-HCl, pH 8.5, 25 mM NaCl, 15% glycerol, 1 mM DTT. (D) UQ
8
quantification of ΔubiV
cells transformed with pBAD-UbiV
6His
, pBAD-UbiV
6His
C180A, pBAD-UbiV
6His
C193A, or empty pBAD and grown
overnight in anaerobic SMGN plus 0.02% arabinose. Values are means ⫾SD (n⫽4to5).*,P⬍0.05; ****,P⬍
0.0001; both by unpaired Student’s ttest.
TABLE 2 Characterization of UbiV proteins and UbiU-UbiV heterodimeric complexes
a
Proteins A
280
/A
410
Content (nmol/nmol of
protein)
Iron Sulfur
UbiV WT 5.8 3.9 ⫾0.13 3.3 ⫾0.18
UbiV C193A C197A 8.4 2.4 ⫾0.02 2.2 ⫾0.13
UbiV C39A C193A C197A 15.5 1.9 ⫾0.01 1.5 ⫾0.4
UbiV C180A C193A C197A 26.7 0.7 ⫾0.05 0.6 ⫾0.1
UbiV C39A C180A C193A C197A 57.4 0.1 ⫾0.04 0
UbiU WT-UbiV WT 4.3 7.9 ⫾0.05 7.5 ⫾0.06
UbiU C169A C176A-UbiV WT 7.3 4.7 ⫾0.0.3 5.8 ⫾0.1
UbiU C193A C232A-UbiV WT 8 3.9 ⫾0.05 5.8 ⫾0.1
a
Shown are metal content and UV-Vis properties after attempts of anaerobic reconstitution of their Fe-S
centers for both wild-type and mutant proteins.
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purified heterodimers composed of WT UbiV
6His
and mutant UbiU (Fig. S6C). After
reconstitution, UbiU C169A C176A-UbiV and UbiU C193A C232A-UbiV had about half
the iron as the WT heterodimer (Table 2), and their A
280
/A
410
ratios were also dimin-
ished about 2-fold (Fig. 6C and Table 2). Altogether, our data clearly demonstrate that
each protein of the heterodimeric UbiU-UbiV complex binds one [4Fe-4S] cluster and
that the iron-chelating cysteines in UbiU are C169, C176, C193, and C232. Finally, an in
vivo complementation assay demonstrated that C176 was important for the function of
UbiU (Fig. 6D).
Many U32 proteases display motifs of four conserved cysteines. The presence of
[Fe-S] clusters in UbiU and UbiV, two U32 protease family members, led us to evaluate
the presence of conserved Cys motifs in other U32 proteins. Kimura et al. reported a
phylogenetic tree of 3,521 peptidase U32 domains which formed 12 groups, belonging
to 10 protein families (43). We extracted and aligned the sequences of the 10 protein
families and found highly conserved 4-cysteine clusters (97 to 100% conservation) in
eight of them (Fig. 7), suggesting an important functional role for these residues. Only
families PepU32#5 and PepU32#6 had no conserved cysteines (PepU32#5) and two
mildly (60 to 80%) plus three poorly (40 to 65%) conserved cysteines (PepU32#6) in
their sequences. The cysteine motifs for each of the eight families showed a high
degree of conservation, and strikingly, most of them could even be aligned with each
FIG 6 UbiU-V complex binds two [4Fe-4S] clusters. (A) UV-visible absorption spectra of as-purified UbiU-UbiV
(dotted line, 17
␮
M) and reconstituted holo-UbiU-UbiV (solid line, 15.5
␮
M). The inset shows an enlargement of the
300- to 700-nm region. (B) X-band EPR spectrum of 339
␮
M dithionite-reduced holo-UbiU-UbiV. Recording
conditions were the following: temperature, 10K; microwave power, 2 mW; modulation amplitude, 0.6 mT. (C)
Comparative UV-visible absorption spectra of Cys-to-Ala mutants of UbiU in the UbiU-UbiV complex after metal
cluster reconstitution with the following concentrations: 15.5
␮
M WT, 16.0
␮
M UbiU C169A C176A, and 16.0
␮
M
UbiU C193A C232A. (A to C) Proteins were in 50 mM Tris-HCl, pH 8.5, 150 mM NaCl, 15% glycerol, 1 mM DTT. (D)
UQ
8
quantification of ΔubiU cells transformed with pBAD-UbiU (n⫽4), pBAD-UbiU C176A (n⫽2), or pBAD empty
vector (n⫽3) and grown overnight in anaerobic SMGN plus 0.02% arabinose. Values are means ⫾SD. **,P⬍0.01;
ns, not significant; both by unpaired Student’s ttest.
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other, with the CX
6
CX
15
CX
3/4
C patterns appearing recurrently (Fig. 7). Note that UbiV
had a slightly distinct motif, with the first cysteine occurring far upstream of the three
others and outside of the U32 domain. Overall, our data suggest that most members of
the U32 peptidase family contain a [4Fe-4S] cluster coordinated by conserved cysteines,
similar to what we demonstrated for UbiU and UbiV.
DISCUSSION
The O
2
-independent UQ biosynthesis pathway is widespread in proteobacte-
ria. The evidence for an O
2
-independent synthesis of UQ by E. coli was reported more
than forty years ago (20), yet this pathway remained uncharacterized until now.
Circumstantial evidence had been obtained that a few species, limited, to our knowl-
edge, to E. coli (20), Rhodobacter sphaeroides (44), Paracoccus denitrificans (45), and
Halorhodospira halophila (46), were able to synthesize UQ under anaerobic conditions,
as demonstrated by biochemical measurements of the quinone content of cells grown
anaerobically. Here, we demonstrate that the O
2
-dependent and O
2
-independent UQ
biosynthesis pathways differ by only three hydroxylation steps (Fig. 1), and we identify
three genes, ubiT,-U, and -V, that are essential for the O
2
-independent biosynthesis of
UQ in E. coli (Fig. 2). The facts that the UbiT, -U, and -V proteins are widespread in
alpha-, beta-, and gammaproteobacterial clades (Fig. 3) and cooccur with UbiA, -E, and
-G enzymes (Table S2B) reveal UbiT, -U, and -V as key elements of the broadly
distributed, O
2
-independent UQ pathway. Overall, our data support that many proteo-
bacteria have the previously unrecognized capacity to synthesize UQ independently
from O
2
.
Physiological possibilities offered by UQ biosynthesis over the entire O
2
range.
In our set of reference genomes, only three species (P. fulvum,M. marinus, and O.
formigenes) seem to rely exclusively on the O
2
-independent pathway for UQ production
(Table S2B). Indeed, the vast majority of proteobacteria with the O
2
-independent UQ
FIG 7 Conserved four-cysteine motifs in the U32 protease family. The conserved 4-cysteine motifs and
Pfam domains (colored boxes) found in each U32 protease family are displayed for a set of reference
sequences. These motifs were obtained by aligning the sequences listed by Kimura et al. (43). Conserved
cysteines are in red, and x6 indicates that 6 residues were found between two conserved cysteines.
Positions of the domains are displayed on the outside of the boxes for the reference sequences.
Scrambled extremities show interrupted matches for the Pfam domain. No conserved cysteines were
found for U32#5 and U32#6 (see the main text). Reference sequences were from E. coli for UbiU, UbiV,
YegQ, and RhlA (YHBU_ECOLI, YHBV_ECOLI, YEGQ_ECOLI, and YDCP_ECOLI for RlhA). For the rest of the
families, the sequence accession numbers were the following: R7JPV1_9FIRM for U32#1, R6XKQ3_9CLOT
for U32#2, S1NZZ5_9ENTE for U32#3, H1YXA1_9EURY for U32#4, H3NJ45_9LACT for U32#5, and
D5MIQ1_9BACT for U32#6.
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biosynthesis pathway also possess the O
2
-dependent hydroxylases of the aerobic
pathway (207 out of 210) (Fig. 3B). This result supports that both pathways confer
physiological advantages, allowing production of UQ over the entire spectrum of O
2
levels encountered by facultative aerobes.
So-called microaerobes, able to respire O
2
under microaerobic conditions, are very
abundant in nature (5), and E. coli is known to respire nanomolar O
2
concentrations
(47). To sustain O
2
respiration in the microaerobic range, these organisms are equipped
with high-affinity O
2
reductases (5, 47, 48). These enzymes reduce efficiently the low
levels of environmental O
2
present at the cell’s membrane, leaving the cytoplasm
devoid of any O
2
(49). Under such conditions, UQ, which is the main electron donor for
the high-affinity O
2
reductases bdI and bdII of E. coli (9), therefore must be synthesized
via the O
2
-independent pathway. Overall, we believe that the O
2
-independent UQ
biosynthesis pathway operates not only under anaerobic conditions but also under
microaerobic conditions, in which UQ is likely crucial for bacterial physiology.
The O
2
-independent UQ biosynthesis pathway may also confer a significant advan-
tage to facultative bacteria in the case of a rapid transition from an anaerobic to an
aerobic environment. Indeed, anaerobic biosynthesis of UQ will result in cellular
membranes containing UQ at the time of the transition, allowing an immediate switch
to the energetically favorable metabolism of O
2
respiration. Our identification of the
anaerobic UQ pathway provides the unique opportunity to selectively disrupt UQ
biosynthesis depending on O
2
levels and should foster new research on bacterial
physiology in the microaerobic range. Indeed, apart from E. coli, which was thoroughly
studied over the microaerobic range (4, 49, 50), details on bacterial physiology in
microaerobiosis are scarce.
ubiT,-U, and -Vmutants and pathogenicity. In addition to bioenergetics per se,
anaerobic and microaerobic respirations are thought to be important for pathogenicity
(3, 51). Interestingly, homologs of UbiT, -U, and -V have been linked to pathogenicity in
several bacterial models. Indeed, the inactivation of ubiU-ubiV homologs in Proteus
mirabilis leads to a decreased infection of the urinary tract of mice (52) and to a
diminished virulence of Yersinia ruckeri (53), a pathogen that develops in the gut of fish,
an environment with a notoriously low O
2
content. Furthermore, inactivation of PA3911
(54)(ubiT) and PA3912-PA3913 (55)(ubiU-ubiV)inPseudomonas aeruginosa abolished
nitrate respiration, the main anaerobic metabolism used by the bacterium in the lungs
of cystic fibrosis patients (56, 57). Based on our results showing that the deletion of
ubiT,ubiU,orubiV abrogates the O
2
-independent biosynthesis of UQ in E. coli,we
suggest that the attenuation of the mutants discussed above results from their UQ
deficiency under microaerobic/anaerobic conditions.
Proposed roles for UbiT, UbiU, and UbiV. UbiT possesses an SCP2 domain, similar
to that of UbiJ, which we recently demonstrated to be an accessory factor that binds
the hydrophobic UQ biosynthetic intermediates and structures a multiprotein Ubi
complex (16). Since UbiJ functions exclusively in the O
2
-dependent pathway whereas
UbiT is important only for the O
2
-independent pathway, we propose that UbiT fulfills,
in anaerobiosis, the same functions as UbiJ in aerobiosis. Whether UbiT is part of a
complex and is able to bind UQ biosynthetic intermediates will be addressed in future
studies. Interestingly, PA3911, the homolog of UbiT in P. aeruginosa, was recently
shown to bind phosphatidic acid (54), demonstrating an affinity of UbiT for lipid
molecules.
UbiU and UbiV form a tight heterodimer, suggesting that the proteins function
together, as further supported by the fact that the ubiU and ubiV genes cooccur in
genomes in 99% of the cases and that they are mostly found next to each other (Fig. 3).
We demonstrated that UbiU and UbiV are both required for the O
2
-independent
C6-hydroxylation of DMQ, and the accumulation of OPP in ΔubiU or ΔubiV mutants
suggests that the two proteins also function in C5-hydroxylation. We want to empha-
size our recent demonstration that a single hydroxylase catalyzes all three hydroxyla-
tion steps in the O
2
-dependent UQ pathway of Neisseria meningitidis (19). This result
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showed that three different enzymes are not necessarily required and opens the
possibility that UbiU-UbiV in fact catalyze all three hydroxylation reactions of the
O
2
-independent UQ biosynthesis pathway. Establishing the hydroxylase activity and
the regioselectivity of UbiU-UbiV will require the development of an in vitro assay, a
challenging task given that the oxygen donor of the reaction is currently unknown and
that the substrates are not commercial and are highly hydrophobic. Of note, one of the
O
2
-dependent hydroxylases was shown to hydroxylate DMQ
0
, a substrate analog with
no polyprenyl side chain (58), suggesting that an in vitro assay for UbiU-UbiV can be
developed with soluble analogs.
[Fe-S] clusters in UbiU-UbiV and other members of the U32 peptidase family.
Until now, members of the peptidase family U32 had not been shown to bind [Fe-S]
clusters or to contain any of the ⬃30 cysteine motifs found in well-characterized
iron-sulfur proteins (59). The expression, purification, and spectroscopic characteriza-
tion of UbiV and of the UbiU-UbiV heterodimeric complex clearly showed that each
protein contains one [4Fe-4S] cluster (Fig. 5 and 6). Mutation of the candidate cysteine
ligands, arranged in a CX
6
CX
16
CX
38
C motif in UbiU and in a CX
n
CX
12
CX
3
C motif in UbiV,
disrupted Fe-S binding and abolished in vivo complementation, suggesting a crucial
function of the [4Fe-4S] clusters in these proteins. The conservation of a CX
6
CX
15
CX
3/4
C
motif in other U32 proteases supports that these proteins likely bind [Fe-S] clusters. This
hypothesis should guide and stimulate investigations of U32 members, very few of
which currently have an established molecular function (http://www.ebi.ac.uk/merops/)
(60). Interestingly, RlhA, a member of the U32 protease family involved in the
C-hydroxylation of a cytidine on E. coli 23S rRNA, was recently shown to be connected
to iron metabolism (43), corroborating our suggestion that RlhA is also an Fe-S protein.
In biological systems, [Fe-S] clusters are mainly known to be involved in electron
transfer reactions as well as in substrate binding and activation, in transcription
regulation, in iron storage, and as a sulfur donor (41, 61–63). The role of the [Fe-S]
clusters in UbiU and UbiV is unknown at this stage. Our current working hypothesis is
that they have a role as electron transfer chains between the substrate (the UQ
biosynthetic intermediate to be hydroxylated) and an unidentified electron acceptor
required for the activation of the substrate. Clearly, the [Fe-S] clusters of UbiU-UbiV are
distinct from the molybdenum cofactor present in molybdenum-containing hydroxy-
lases, the only family currently known to catalyze O
2
-independent hydroxylation reac-
tions (64). Together, our results identify UbiU and UbiV as prototypes of a novel class
of O
2
-independent hydroxylases and extend the framework of the chemically fascinat-
ing O
2
-independent hydroxylation reactions.
MATERIALS AND METHODS
Strain construction. Strains used in this study are listed in Table S3 in the supplemental material. We
obtained the collection of E. coli strains containing large and medium deletions from the National
BioResource Project, National Institute of Genetics, Japan (http://www.shigen.nig.ac.jp/ecoli/pec/).
The ΔubiA::cat,ΔubiD::cat,ΔubiT::cat, and ΔubiV::cat mutations were constructed in a one-step
inactivation of ubi genes as described previously (65). A DNA fragment containing the cat gene flanked
with 5=and 3=regions bordering the E. coli ubi genes was amplified by PCR using pKD3 as a template
and oligonucleotides 5wanner and 3wanner (Table S3). Strain BW25113, carrying the pKD46 plasmid, was
transformed by electroporation with the amplified fragment and Cat
r
colonies were selected. The
replacement of chromosomal ubi by the cat gene was verified by PCR amplification in the Cat
r
clones.
Mutations (including ubiU::kan from the Keio strain) were introduced into MG1655 strains by P1 vir
transduction (66), selecting for the appropriate antibiotic resistance. The antibiotic resistance cassettes
were eliminated when needed using plasmid pCP20 as described previously (67).
Plasmid construction. All plasmids generated in this study were verified by DNA sequencing. The
yhbU,yhbT, and yhbV inserts (UniProtKB entries P45527,P64599, and P45475) were obtained by PCR
amplification using E. coli MG1655 as the template and the oligonucleotide pairs yhbU5-yhbU3,yhbT5-
yhbT3, and yhbV5-yhbV3, respectively (Table S3). yhb inserts were EcoRI-SalI digested and inserted into
EcoRI-SalI-digested pBAD24 plasmids, yielding the pBAD-yhbU, pK-yhbT, or pBAD-yhbV plasmid, respec-
tively.
To create a plasmid expressing the ubiV (yhbV) ORF as a C-terminally His-tagged protein, the ubiV
gene was amplified using pET-22-UbiV-FW (introducing the NdeI site) and pET-22-UbiV-RV (introducing
the XhoI site) as primers and pBAD-yhbV as the template. The NdeI- and XhoI-digested amplicon was
ligated to NdeI- and XhoI-digested pET-22b(⫹) plasmid to obtain pET-22-UbiV.
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The plasmid pETDUET-UbiUV, containing UbiU in multiple cloning site 1 (MCS1) and UbiV in MCS2,
was obtained as follows. ubiU was amplified from pBAD-yhbU using pETDUET-UbiU-FW (introducing the
NcoI site) and pETDUET-UbiU-RV (introducing the EcoRI site) as primers. The NcoI- and EcoRI-digested
amplicon was ligated to NcoI- and EcoRI-digested pETDUET-1 plasmid to obtain pETDUET-UbiU. The ubiV
gene was then cloned from pET-22-UbiV into MSC2 of pETDUET-UbiU by PCR amplification with
pET-22-UbiV-FW and pETDUET-UbiV-RV (introducing the C-terminal His
6
tag) as primers. The NdeI- and
XhoI-digested amplicon was ligated to NdeI- and XhoI-digested pETDUET-UbiU to obtain pETDUET-
UbiUV.
A hexahistidine tag was fused at the N-terminal extremity of UbiV to create pBAD-UbiV
6His
. The
ubiV
6His
gene was obtained by PCR amplification (Phusion high-fidelity DNA polymerase) using pBAD-
UbiV as a template and 6HisV5 (introducing the NcoI site) and 6HisV3 (introducing the HindIII site and
the DNA sequence of the 6⫻His tag) as primers. The NcoI/HindIII-digested amplicon was cloned into the
NcoI/HindII-digested pBAD plasmid.
Variants of UbiV and UbiU were constructed using the Q5 site-directed mutagenesis kit (New England
Biolabs) according to the manufacturer’s specifications. The plasmids (pET-22b-UbiV, pETDUET-UbiUV,
pBAD-yhbU, and pBAD-yhbV) were used as templates in conjunction with the appropriate primers for
each respective amino acid substitution.
Culture conditions. E. coli strains were grown at 37°C in lysogeny broth (LB) medium or in synthetic
medium (SM) containing either 0.4% (wt/vol) glycerol, 0.4% (wt/vol) lactate, or 0.2% (wt/vol) glucose as
carbon sources. Autoclaved SM medium was supplemented with 0.5% (wt/vol) Casamino Acids and with
a 1/100 volume of a filter-sterilized solution of 1 mM CaCl
2
, 200 mM MgCl
2
, 1% (wt/vol) thiamine (68).
Ampicillin (50 mg/liter), kanamycin (25 mg/liter), and chloramphenicol (25 mg/liter) were added from
stocks (1,000⫻solution sterilized through 0.22-
␮
m filters and stored at ⫺20°C) when needed. When
needed, 0.02% arabinose was added to induce the expression of genes carried on pBAD and pK plasmids.
External electron acceptors like KNO
3
(100 mM) or dimethyl sulfoxide (DMSO; 50 mM) were added to SM
for anaerobic cultures. Anaerobic cultures were performed in Hungate tubes containing 12 ml medium
deoxygenated by argon (O
2
,⬍0.1 ppm) bubbling for 25 min before autoclaving (in the case of LB
medium, 0.05% antifoam [Sigma] was added). Hungate tubes were inoculated through the septum with
100
␮
l of overnight precultures taken with disposable syringes and needles from closed Eppendorf tubes
filled to the top. Aerobic cultures were performed in Erlenmeyer flasks filled to 1/10 the maximal volume
and shaken at 180 rpm.
For the initial screen, we grew ME strains anaerobically in SMGN (SM medium supplemented with
glycerol and nitrate). Strains that presented a severe growth defect or a low UQ
8
content were
subsequently grown anaerobically in LB medium.
Cultures were cooled on ice before transferring 5- to 10-ml volumes into 15-ml Falcon tubes for
centrifugation at 3,200 ⫻gat 4°C for 10 min. Cell pellets were washed in 1 ml ice-cold phosphate-
buffered saline and transferred to preweighed 1.5-ml Eppendorf tubes. After centrifugation at 12,000 ⫻
gat 4°C for 1 min and elimination of supernatant, the cell wet weight was determined (⬃10 to 20mg),
and pellets were stored at ⫺20°C prior to quinone extraction. We note that these steps were conducted
under normal atmosphere and allowed limited O
2
-dependent UQ biosynthesis in cells grown anaerobi-
cally. Thus, modifications (detailed below) were adopted in additional experiments conducted under
strict anaerobic conditions.
Culture under strict anaerobic conditions and cell quenching. LB medium was supplemented
with 100 mg/liter L-cysteine (adjusted to pH 6 with NaOH) and 2.5 mg/liter resazurin. The medium was
distributed in Hungate tubes and was deoxygenated by argon (O
2
⬍0.1 ppm) bubbling for 45 min at
60°C. The resazurin was initially purple and then quickly turned to pink, and it eventually became
colorless. The Hungate tubes were sealed and autoclaved. Two sequential precultures were performed
in order to dilute the UQ present in the initial aerobic inoculum. The first preculture was performed
overnight and used Eppendorf tubes filled to the top and inoculated with cells grown aerobically on LB
agar. The second preculture was performed for 8 h in Hungate tubes and was used to inoculate Hungate
tubes that were subsequently incubated overnight at 37°C. Disposable syringes (1 ml) and needles
were flushed 5 times with argon prior to inoculating 50
␮
l of preculture through the septum of the
Hungate tubes. The resazurin remained colorless at all steps of the culture, indicating that the medium
in the Hungate tubes was strictly anaerobic. At the end of the culture, the Hungate tubes were cooled
on ice for 45 min, and 2 ml medium was sampled through the septum with argon-flushed syringes (2 ml)
fitted with needles. The cells were immediately quenched by transfer to ⫺20°C precooled glass tubes
containing 6 ml methanol, 0.5-ml glass beads (0.5-mm diameter), and 20 mM KCl. The tubes were
homogenized by vortex for 30 s and kept at ⫺20°C prior to quinone extraction. In parallel, we also
centrifuged 2 ml of culture from the Hungate tubes in order to determine the weight of the cells and
normalized the UQ content of the quenched cells that was subsequently measured.
For the experiments conducted under strict anaerobic conditions (Fig. 2H and I), we used LB medium
instead of SMGN, since nitrite, produced during the anaerobic respiration of nitrate in SMGN medium, is
able to oxidize resazurin (69).
Lipid extraction and quinone analysis. Quinone extraction from cell pellets was performed as
previously described (17).
The method for quinone extraction from cells quenched in methanol was slightly adapted from
reference 9. Briefly, 4
␮
lofa10
␮
MUQ
10
solution was added as an internal standard to the cell-methanol
mixture. Four ml of petroleum ether (boiling range, 40 to 60°C) was added, the tubes were vortexed for
30 s, and the phases were separated by centrifugation for 1 min at 600 rpm. The upper petroleum ether
layer was transferred to a fresh glass tube. Petroleum ether (4 ml) was added to the glass beads and
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methanol-containing tube, and the extraction was repeated. The petroleum ether layers were combined
and dried under nitrogen.
The dried lipid extracts were resuspended in 100
␮
l ethanol, and a volume corresponding to 1 mg
of cell wet weight was analyzed by HPLC-electrochemical detection-mass spectrometry (ECD-MS) with a
BetaBasic-18 column at a flow rate of 1 ml/min with mobile phases composed of methanol, ethanol,
acetonitrile, and a mix of 90% isopropanol, 10% ammonium acetate (1 M), 0.1% trifluoroacetic acid,
mobile phase 1 (50% methanol, 40% ethanol, and 10% mix), and mobile phase 2 (40% acetonitrile, 40%
ethanol, and 20% mix). Mobile phase 1 was used in MS detection on an MSQ spectrometer (Thermo
Scientific) with electrospray ionization in positive mode (probe temperature, 400°C; cone voltage, 80 V).
Single-ion monitoring (SIM) detected the following compounds: OPP (M⫹NH
4
⫹
), m/z 656.0 to 656.8, 5 to
10 min, scan time of 0.2 s; DMQ
8
(M⫹Na
⫹
), m/z 719 to 720, 6 to 10 min, scan time of 0.2 s;
13
C
6
-DMQ
8
(M⫹Na
⫹
), m/z 725 to 726, 6 to 10 min, scan time of 0.2 s; UQ
8
(M⫹NH
4
⫹
), m/z 744 to 745, 6 to 10 min,
scan time of 0.2 s; UQ
8
(M⫹Na
⫹
), m/z 749 to 750, 6 to 10 min, scan time of 0.2 s;
13
C
6
-UQ
8
(M⫹Na
⫹
), m/z
755.0 to 756, 6 to 10 min, scan time of 0.2 s; UQ
10
(M⫹NH
4
⫹
), m/z 880.2 to 881.2, 10 to 17 min, scan time
of 0.2 s. MS spectra were recorded between m/z 600 and 900 with a scan time of 0.3 s. UV detection at
247 nm was used to quantify DMK
8
and MK
8
. ECD, MS, and UV peak areas were corrected for sample loss
during extraction on the basis of the recovery of the UQ
10
internal standard and were then normalized
to cell wet weight. The peak of UQ
8
obtained with electrochemical detection was quantified with a
standard curve of UQ
10
(17). The absolute quantification of UQ
8
based on the m/z 744.6 signal at 8 min
(Fig. 2H and I) was performed with a standard curve of UQ
8
ranging from 0.5 to 150 pmol UQ
8
(the
detection limit was around 0.1 pmol).
Anaerobic
13
C
6
-UQ
8
biosynthesis activity assay. ΔubiC ΔubiF cells containing or not containing the
additional ΔubiU or ΔubiV deletion were grown overnight in MS medium supplemented with 0.2%
glucose. This preculture was used to inoculate, at an optical density at 600 nm (OD
600
) of 0.1, 100 ml of
fresh medium supplemented with 10
␮
M
13
C
7
-4-HB. The culture was grown at 37°C, 180 rpm, until an
OD
600
of 1, at which point 100
␮
M 4-HB was added. The cells were pelleted by centrifugation at
3,200 ⫻gat 4°C for 10 min and suspended in 100 ml SMGN medium. A 10-ml aliquot was taken for
quinone extraction (Fig. 4B to D, aerobiosis), and the rest of the culture was placed at 37°C in an
anaerobic bottle with a two-port cap fitted with plastic tubing used to inject argon (O
2
⬍0.1 ppm)
throughout the experiment in order to create and maintain anaerobiosis. After 5 min of bubbling, a 10-ml
sample was taken corresponding to 0-min anaerobiosis, and then samples were taken every 30 min and
analyzed for quinone content.
Overexpression and purification of proteins. (i) Overexpression and purification of E. coli
wild-type UbiV and variants. The pET-22b(⫹) plasmid, encoding wild-type UbiV or variants, was
cotransformed with pGro7 plasmid (TaKaRa Bio, Inc.) into E. coli BL21(DE3) competent cells. Single
colonies obtained from transformation were grown overnight at 37°C in LB medium supplemented with
ampicillin (50
␮
g/ml) and chloramphenicol (12.5
␮
g/ml). Ten ml of preculture was used to inoculate
1 liter of LB medium with the same antibiotics, and the bacteria were cultured further at 37°C with
shaking (200 rpm). At an OD
600
of 1.2, D-arabinose was added to the cultures at a final concentration of
2 mg/ml. At an OD
600
of 1.8, the culture was cooled in an ice-water bath, and isopropyl 1-thio-
␤
-D-
galactopyranoside (IPTG) was added at a final concentration of 0.1 mM. Cells were then allowed to grow
further at 16°C overnight. All subsequent operations were carried out at 4°C. Cells were harvested in an
Avanti J-26XP high-performance centrifuge from Beckman Coulter with a JLA-8.1000 rotor at 5,000 ⫻g
for 10 min. The cell pellets were resuspended in 5 volumes of buffer A (50 mM Tris-HCl, pH 8.5, 150 mM
NaCl, 15% [vol/vol] glycerol, 1 mM dithiothreitol [DTT]) containing Complete protease inhibitor cocktail
(one tablet per 50 ml) (Roche) and disrupted by sonication (amplitude of 40% for 10 min; Branson digital
sonifier). Cell debris was removed by ultracentrifugation in an Optima XPN-80 ultracentrifuge from
Beckman Coulter with a 50.2 Ti rotor at 35,000 ⫻gfor 60 min. The resulting supernatant was loaded onto
a HisTrap FF crude column (GE Healthcare) preequilibrated with buffer A. The column was washed with
10 column volumes of buffer B (50 mM Tris-HCl, pH 8.5, 150 mM NaCl, 15% [vol/vol] glycerol, 1 mM DTT,
10 mM imidazole) to remove nonspecifically bound E. coli proteins and then eluted with a linear gradient
of 10 column volumes of buffer B containing 500 mM imidazole. Fractions containing WT UbiV or variants
were pooled, and phenylmethylsulfonyl fluoride was added at a final concentration of 1 mM. The
proteins were then loaded on a HiLoad 16/600 Superdex 75 pg (GE Healthcare) preequilibrated in buffer
C (50 mM Tris-HCl, pH 8.5, 25 mM NaCl, 15% [vol/vol] glycerol, 1 mM DTT). The purified proteins were
concentrated to 30 to 40 mg/ml using Amicon concentrators (30-kDa cutoff; Millipore), aliquoted, frozen
in liquid nitrogen, and stored at ⫺80°C. Overall, a high yield of 150 mg UbiV per liter of culture was
obtained.
(ii) Overexpression and purification of UbiU-UbiV complex and variants. The overexpression of
wild-type UbiU-UbiV complex or variants in E. coli BL21(DE3) competent cells was performed by following
the same protocol as that for UbiV. The expression of these proteins was induced by addition of IPTG to
a final concentration of 0.05 mM. Wild-type UbiU-UbiV complex or variants were purified with the same
procedure as that for UbiV, with the exception that the proteins were loaded on the HiLoad 16/600
Superdex 75 pg with buffer A.
[Fe-S] cluster reconstitution. The [Fe-S] cluster(s) reconstitution of holo-UbiV and holo-UbiU/V was
conducted under anaerobic conditions in an Mbraun LabStar glove box containing less than 0.5 ppm O
2
.
Classically, a solution containing 100
␮
M as purified UbiV or UbiU/V complex was treated with 5 mM DTT
for 15 min at 20°C and then incubated for 1 h with a 5-fold molar excess of both ferrous ammonium
sulfate and L-cysteine. The reaction was initiated by the addition of a catalytic amount of the E. coli
cysteine desulfurase CsdA (1 to 2% molar equivalent) and monitored by UV-visible absorption spectros-
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July/August 2019 Volume 10 Issue 4 e01319-19 mbio.asm.org 16

copy. The holo-UbiV or holo-UbiU/V complexes then were loaded onto a Superdex 75 Increase 10/300
GL column (GE Healthcare) preequilibrated with buffer C or A, respectively, to remove all excess iron and
L-cysteine. The fractions containing the holoproteins were pooled and concentrated to 20 to 30 mg/ml
on a Vivaspin concentrator (30-kDa cutoff).
Quantification methods. Protein concentrations were determined using the method of Bradford
(Bio-Rad) with bovine serum albumin as the standard. The levels of iron and acid-labile sulfide were
determined according to the method of Fish (70) and Beinert (71), respectively.
UV-Vis spectroscopy. UV-visible spectra were recorded in a 1-cm-optic-path quartz cuvette under
aerobic conditions on a Cary 100 UV-Vis spectrophotometer (Agilent) and under anaerobic conditions in
a glove box on a XL-100 Uvikon spectrophotometer equipped with optical fibers.
EPR spectroscopy. EPR spectra of frozen solutions were recorded on a Bruker Continuous Wave
X-band ELEXSYS E500 spectrometer operating at 9.39 GHz, equipped with an SHQE cavity cooled by a
helium flow cryostat (ESR 900; Oxford Instruments) under nonsaturating conditions and using the
following parameters: microwave power in the range of 2 to 10 mW and modulation of the magnetic
field at 100 kHz, with a modulation amplitude of 0.6 mT. Holo-UbiV or holo-UbiU-UbiV complex was
treated with a 10-fold molar excess of dithionite to reduce the [Fe-S] cluster. Each solution was
introduced into EPR quartz tubes in a glove box and frozen with liquid nitrogen before the EPR
measurements.
Genome data sets. The protein sequences from 5,750 complete genomes (extended data set) were
downloaded from the NCBI RefSeq database (bacteria and archaea; last accessed in November 2016)
(Table S2A). A representative set of complete genomes from a monophyletic group of bacteria that
potentially harbor the ubiquinone biosynthesis pathway was also created: “reference” and “representa-
tive” genomes were downloaded from the NCBI RefSeq database for 204 Alphaproteobacteria, 103
Betaproteobacteria, and 303 Gammaproteobacteria (last accessed in November 2018). In addition to these
610 genomes, the genome of Phaeospirillum fulvum (99.5% estimated completeness according to
CheckM; http://gtdb.ecogenomic.org/genomes?gid⫽GCF_900108475.1) was included (Table S2B).
HMM protein profile creation. An initial set of protein sequences (so-called curated set) was
retrieved from genomes manually and from a publication (72) to cover the diversity of UQ-producing
organisms. The curated set included 48 pairs of YhbU and YhbV from 10 alpha-, 19 beta-, and 19
gammaproteobacteria, 17 sequences for YhbT, and 64, 181, 69, and 189 sequences for UbiA, MenA, UbiG,
and UbiE, respectively (Table S4). For each gene family these sequences then were aligned with Mafft
(v7.313, linsi) (73), and each alignment was trimmed at its N-terminal and C-terminal extremities based
on the filtering results of BMGE (BLOSUM 30) (74). The core of the alignments were kept as is, and hidden
Markov model (HMM) profiles were created directly from the trimmed alignments using the Hmmbuild
program (Hmmer suite, version 3.1b2) (75).
To ensure a more sensitive search and good delineation between homologs, phylogenetic curation
was used, as YhbU and YhbV are known to be part of the larger U32 protease gene family (43). A search
using the YhbU and YhbV HMM profiles was performed with Hmmsearch (Hmmer suite) on the extended
5,750-genome data set, and sequences with an i-evalue (independent e-value) lower than 10E⫺20 and
a coverage of the profiles higher than 90% were selected. The 4,212 sequences obtained were derep-
licated using Uclust from the Usearch program suite (80% identity level) (76). A phylogenetic tree was
built by maximum likelihood with the IQ-Tree program (best evolutionary model) based on the
alignment (Mafft linsi, BMGE with BLOSUM30) of the 480 selected sequences, including all curated YhbU
and YhbV sequences (73, 74, 77). YhbU and YhbV proteobacterial sequences formed two separate
monophyletic groups, giving credit to our curated set (100% and 80% UF-Boot support, respectively). The
other sequences that formed a large monophyletic group of bacterial sequences were categorized as
U32 proteases (98% UF-Boot support; https://doi.org/10.6084/m9.figshare.7800614.v1). The 98 se-
quences from this U32 protease group (Table S4) were used to recreate an HMM profile as described
above and served as an outgroup for the profile search.
For YhbT, the first profile obtained from the curated set of sequences was used together with the
YhbU, YhbV, and U32 protease profiles for a search in the data set of 611 proteobacterial genomes. A
second profile was created from YhbT sequences (YhbT2) (Table S4) that were colocalizing with YhbU
and YhbV hits (10E⫺20 i-evalue and 80% profile coverage). The two YhbT profiles matched comple-
mentary sets of sequences and therefore were both used for annotating YhbT in genomes.
A similar approach was taken in order to identify the six known aerobic hydroxylases. Fifty-one Coq7,
73 UbiF, 80 UbiH, 58 UbiI, 24 UbiL, and 32 UbiM sequences were extracted manually and from
publications (19, 72) (Table S4, version 1) to serve as a reference, annotated set of sequences. Profiles
were created as described above. To ensure their specificity, we ran the HMM profiles against our
5,570-genome data set and selected the sequences that had an i-evalue lower than 10E⫺20 and a
coverage of the profiles higher than 90%. We built two phylogenetic trees as described above, one for
Coq7 and another one for UbiFHILM, which are known to be part of the large FMO protein family (19).
In the latter case, we dereplicated the 1,619 sequences obtained for the FMO protein family before
performing the alignment, alignment filtering, and tree reconstruction steps (using Uclust at the 60%
identity level). The Coq7 tree obtained showed our reference Coq7 sequences covered the whole
diversity of retrieved sequences, suggesting that they all could be bona fide Coq7 (https://doi.org/10
.6084/m9.figshare.7800680). The FMO tree showed a monophyletic group containing all reference FMO
ubiquinone hydroxylases, forming subgroups for the different homologs (UbiFHILM) in proteobacteria
(https://doi.org/10.6084/m9.figshare.7800620). Further, a large set of sequences formed an outgroup
consisting of sequences from various clades of bacteria, many being found outside of proteobacteria,
robustly separated from the ubiquinone hydroxylases. We split this large clade into four subtrees and
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extracted the corresponding sequences to obtain four new HMM profiles (as described above) to be used
for precise discrimination between ubiquinone hydroxylases and other members of the FMO family
(AlloFMO_1 to AlloFMO_4 in Table S4, version 2). FMO ubiquinone hydroxylase subtrees were also used
to redesign improved HMM profiles for UbiFHILM (36, 168, 198, 139, and 65 sequences; see Table S4,
version 2).
Evaluation of genomic distributions with HMMER and MacSyFinder. Two MacSyFinder models
were created to (i) search sequences of interest in genomes using Hmmer and (ii) investigate their
genetic architecture (78). A first model was created to focus only on YhbTUV-related genes. In this model,
the YhbTUV components were defined as mandatory and U32 protease as accessory. A second, more
comprehensive model, Ubi⫹YhbTUV, was designed to list the families corresponding to the 9 profiles
obtained (UbiA, MenA, UbiE, UbiG, 2 YhbT, YhbU, YhbV, and U32 proteases). For both models, the two
YhbT profiles were set as exchangeable. The parameter inter_gene_max_space was set to 5 in the
YhbTUV model and 10 in the Ubi⫹YhbTUV model. MacSyFinder was set to run HMMER with the options
–i-evalue-select 10E⫺20 and – coverage-profile 0.8. Independently of their genetic context, sequences
corresponding to selected HMMER hits were listed for all profiles in all genomes analyzed in order to
establish the genomic distribution for each of the protein families of interest. When several profiles
matched a sequence, only the best hit (best i-evalue) was considered.
U32 protease sequence analysis. We retrieved from the UniProt-KB database 3,460 protein se-
quences of U32 proteases that were categorized in 12 families by Kimura et al. (43) and created a FASTA
file for each of these families. Fifty sequences from different families could not be retrieved, as they had
been deleted from the Uniprot-KB database (46 were obsolete) or were not found based on the
published accession numbers. As the RlhA1 and RlhA2 families mostly corresponded to two domains
from the same protein sequences that had been split, we put whole sequences together into a single
FASTA file for sequence analysis of the overall RlhA family. For each of the 10 families, sequences were
dereplicated at the 80% identity level with Uclust in order to limit any potential taxonomic sampling bias,
and sequences were aligned (Mafft, linsi). The alignments were visualized in Jalview (79) and used to
create the logo sequences. Images of alignments were created using the ESPript webserver (http://
espript.ibcp.fr/ESPript/ESPript/)(80).
SUPPLEMENTAL MATERIAL
Supplemental material for this article may be found at https://doi.org/10.1128/mBio
.01319-19.
FIG S1, JPG file, 0.1 MB.
FIG S2, JPG file, 0.8 MB.
FIG S3, JPG file, 0.6 MB.
FIG S4, JPG file, 0.5 MB.
FIG S5, TIF file, 2.3 MB.
FIG S6, TIF file, 0.2 MB.
TABLE S1, XLSX file, 0.02 MB.
TABLE S2, XLSX file, 0.5 MB.
TABLE S3, XLSX file, 0.02 MB.
TABLE S4, XLSX file, 0.05 MB.
ACKNOWLEDGMENTS
This work was supported by the Agence Nationale de la Recherche (ANR), ANR Blanc
(An)aeroUbi ANR-15-CE11-0001-02, to F.P. We thank Amélie Amblard for technical
assistance, Louis Givelet for preliminary bioinformatic analyses, Barbara Schoepp-
Cothenet for providing accession numbers to sequences of UbiA, -G, -E and for critical
reading of the manuscript, and the GEM team at TIMC for discussions and suggestions.
C.-D.-T.V., M.L., and M.F. acknowledge support from the French National Research
Agency (Labex program DYNAMO, ANR-11-LABX-0011). C.C. was funded by the
Grenoble Alpes Data Institute, supported by the French National Research Agency
under the “Investissements d’Avenir” program (ANR-15-IDEX-02). We thank the National
Bioresource Project, National Institute of Genetics, for providing ME strains from the
medium- and large-deletion E. coli collection. We have no competing interests to
declare.
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