On the function of the various quinone species in Escherichia coli

On the function of the various quinone species inEscherichia coliPoonam Sharma, Maarten J. Teixeira de Mattos, Klaas J. Hellingwerf and Martijn Bekker

Molecular Microbial Physiology group, Swammerdam Institute for Life Sciences, University of Amsterdam, The Netherlands

Keywords

cytochrome oxidase;

demethylmenaquinone; fumarate reductase;

physiology; succinate dehydrogenase

Correspondence

M. Bekker, Molecular Microbial Physiology

group, Swammerdam Institute of Life

Sciences, University of Amsterdam, Science

Park 904, 1098XH Amsterdam,

The Netherlands

Fax: +31 20 525 7934

Tel: +31 20 525 6425

E-mail: [email protected]

(Received 30 November 2011, revised 4

April 2012, accepted 18 April 2012)

doi:10.1111/j.1742-4658.2012.08608.x

The respiratory chain of Escherichia coli contains three quinones. Menaqui-

none and demethylmenaquinone have low midpoint potentials and are

involved in anaerobic respiration, while ubiquinone, which has a high mid-

point potential, is involved in aerobic and nitrate respiration. Here, we

report that demethylmenaquinone plays a role not only in trimethylamino-

oxide-, dimethylsulfoxide- and fumarate-dependent respiration, but also in

aerobic respiration. Furthermore, we demonstrate that demethylmenaqui-

none serves as an electron acceptor for oxidation of succinate to fumarate,

and that all three quinol oxidases of E. coli accept electrons from this

naphtoquinone derivative.

Introduction

The respiratory chain of Escherichia coli is character-

ized by a highly variable composition of constituents

compared to mitochondria. In addition to the surpris-

ingly high number of membrane-associated NADH

dehydrogenases (six) [1–5], a formate dehydrogenase, a

lactate dehydrogenase and a succinate dehydrogenase,

it contains many terminal reductases [5–10]. On the

one hand, this allows the organism to donate electrons

to its preferred major terminal acceptor, O2, with a net

H+ ⁄2e) stoichiometry ranging from 2 to 8, and on the

other to use a variety of alternative terminal electron

acceptors, such as dimethylsulfoxide, trimethylamino-

oxide (TMAO) and fumarate [11–14]. These external

electron acceptors relieve the restriction of overall

redox neutrality to which the catabolic reactions are

subjected.

Electrons that are generated during heterotrophic

breakdown of an energy source are funneled to the

quinone pool, and the reduced quinones (quinols) sub-

sequently serve as substrates for reduction of the ter-

minal acceptor(s). In E. coli, three types of quinones

may be involved: menaquinone (MK), demethyl-

menaquinone (DMK) and ubiquinone (UQ) [5,13,15].

When E. coli is grown on substrates that are catabo-

lized via glycolysis and the tricarboxylic acid (TCA)

cycle, NADH is the major electron carrier. Enzyme-

bound FADH2 also performs this function due to suc-

cinate oxidation by succinate dehydrogenase, a key

reaction in the TCA cycle. In this catalytic process,

electrons are assumed to be transferred directly to UQ

[16]. The genome of E. coli encodes three terminal ox-

idases: cytochrome bo oxidase, cytochrome bd I

Abbreviations

DMK, demethylmenaquinone; DW, dry weight; MK, menaquinone; Q, quinone; qO2, O2 flux in mmolÆgÆDW)1Æh)1; Qtot, total of all quinone

pools; TCA, tricarboxylic acid; TMAO, trimethylaminooxide; UQ, ubiquinone.

FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS 1

oxidase and cytochrome bd II oxidase; all three trans-

fer electrons from ubiquinol to oxygen (Fig. 1).

It was recently postulated that cellular redox reac-

tions function optimally if the difference in midpoint

potential between the reductant and the oxidant is

� 150–200 mV [17,18]. This implies that DMK and

MK are mainly involved in respiratory processes

involving terminal acceptors with a less positive redox

potential than O2 ⁄H2O, and hence in anaerobic respi-

ration, whereas UQ is the preferred electron carrier

during aerobic respiration. Indeed, it has been

observed that, under conditions that favor fumarate or

dimethylsulfoxide respiration, MK is virtually the only

naphtoquinone present [19], whereas DMK is the most

abundant quinone during nitrate respiration [19],

although both UQ and MK were shown to be func-

tional in this pathway too [20]. DMK may serve as the

redox carrier in anaerobic respiration with fumarate,

dimethylsulfoxide or TMAO as the terminal electron

acceptor [21]. Aerobic respiration independent of UQ

has been reported, but it was not determined which

naphtoquinone mediated this electron transfer [22].

Given the complexity and variability of the respira-

tory chain [5,13], and the fact that E. coli can employ

a large range of electron donors and acceptors for oxi-

dative energy conservation, quantification of the ener-

getic efficiency of its respiratory module is still a great

challenge [11,23] that is best tackled by a systems-biol-

ogy approach. This type of analysis of growth, and of

the underlying metabolic network in E. coli, has a long

history. These analyses emerged in parallel with use of

the chemostat as a quantitative tool in microbial physi-

ology. Initially the amount of detail incorporated was

limited, and mainly focused on energy (i.e. ATP)

Fig. 1. Simplified stoichiometric model of the catabolic fluxes [39] relevant for Escherichia coli growing aerobically on glucose. Enzyme bio-

energetic efficiency is indicated as the number of protons transported across the membrane per electron (H+ ⁄ e) ratio). NDH I is the coupled

NADH:quinone oxidoreductase, and NDHII represents WrbA (tryptophan (W) repressor binding protein), QOR and QOR2 (quinone oxidore-

ductases), YhdH) and the non-proton translocating NADH:quinone oxidoreductases (NP NDHs). NDH I and NP NDHs transfer electrons to

the quinones (Q) to yield quinol (QH2). Three quinol::oxygen oxidoreductases, cytochromes bo3 (Cybo), bd I (Cybd I) and bd II (Cybd II), oxi-

dize QH2 and reduce O2 to H2O. Acetate formation via acetate kinase (Ack) leads to ATP formation, in contrast to acetate formation by pyru-

vate oxidase (PoxB). Similarly, lactate formation by one of the three lactate dehydrogenases (LDH) results in ATP formation, but lactate

formation via methylglyoxal synthase (MGOS) leads to ATP consumption. The gray area indicates the cytoplasmic membrane. When carbon

atoms from glucose, in the presence of sufficient amounts of oxygen, do not end up in CO2, but are diverted into fermentation products

such as acetic acid and ⁄ or lactic acid, this is often referred to as ‘overflow metabolism’ [42].

The role of DMK in the electron transfer chains of E. coli P. Sharma et al.

2 FEBS Journal (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS

metabolism [24], but extension of the models for inter-

mediary metabolism has taken place gradually [25,26],

culminating in the use of ‘complete’ flux balance analy-

ses [27]. However, in all these approaches, the reac-

tions catalyzed by the free energy-converting

respiratory metabolism were grouped, because of a

lack of knowledge on the underlying biochemistry.

Recently, the SUMO Consortium has taken up the

challenge to clarify these details and provide a kinetic

description of the metabolism of E. coli [28]. More

detailed information regarding the specificity and affin-

ity of the interactions between all components of the

(an)aerobic respiratory system is crucial for the success

of this approach.

Here we have studied the growth and respiration of

two sets of mutants of E. coli. In the first set, the wild-

type organism was compared with mutants containing

UQ or DMK as their sole quinone (note that, as

DMK is the precursor of MK, construction of a strain

that synthesizes MK but not DMK is not possible). In

the second set, mutants containing UQ only, DMK

only, or a combination of DMK and MK were created

in the genetic background of single quinol oxidase

strains (i.e. mutants containing exclusively cytochrome

bo oxidase, cytochrome bd I oxidase or cytochrome

bd II oxidase, Table 1). These latter mutants were ana-

lyzed physiologically in glucose-limited chemostat cul-

tures. Our results demonstrate that DMK functions as

a substrate for all three quinol oxidases in the reduced

form, and as an acceptor of electrons from succinate

in the oxidized form.

Results

In order to identify the relationships between the alter-

native quinone types and their putative electron donors

and acceptors, a series of mutant E. coli strains with

relevant modifications with respect to quinone synthe-

sis and ⁄or composition of their quinol oxidases were

created (see Experimental procedures), and tested for

growth and the rate of oxygen consumption. Using the

growth rate and specific rate of respiration of the wild-

type strain (BW25113) as a reference, it can be con-

cluded from Table 3 that, in the absence of DMK and

MK (i.e. mutants containing UQ exclusively; strain

MB23), respiration is maintained at 67% of the wild-

type rate, whereas in a strain that contains DMK only

(strain MB27), the respiration rate is reduced to 8% of

the wild-type rate. This suggests a small but significant

capacity of demethylmenaquinol (DMKH2) to function

under aerobic conditions, in accordance with previous

observations of UQ-independent respiration [22].

When only UQ is present (strain MB23), a decreased

maximal growth rate is observed (see Table 3). Un-

expectedly, the reduced turnover capacity of the respi-

ratory chain in this strain does not result in an

enhanced rate of production of ‘overflow’ products

such as acetate. Interestingly, this strain has a higher

biomass yield, suggesting more efficient functioning of

Table 1. List of the strains used in this study.

Strain Genotype Cytochrome(s) remaining Quinone(s) remaining

BW2511 K-12 (wild-type) All cytochromes All three

DJ01 BW25113, DcyoB, DappB, DnuoB, DubiCA::kan Cytochrome bd I MK, DMK

DJ02 BW25113, DcydB, DappB, DnuoB, DubiCA::kan Cytochrome bo MK, DMK

DJ03 BW25113, DcyoB, DcydB, DnuoB, DubiCA::kan Cytochrome bd II MK, DMK

DJ11 BW25113, DcyoB, DappB, DnuoB, DubiE::kan Cytochrome bd I DMK

DJ12 BW25113, DcydB, DappB, DnuoB, DubiE::kan Cytochrome bo DMK

DJ13 BW25113, DcyoB, DcydB, DnuoB, DubiE::kan Cytochrome bd II DMK

DJ31 BW25113, DcyoB, DappB, DnuoB, DmenA::kan Cytochrome bd I UQ

DJ32 BW25113, DcydB, DappB, DnuoB, DmenA::kan Cytochrome bo UQ

DJ33 BW25113, DcyoB, DcydB, DnuoB, DmenA::kan Cytochrome bd II UQ

MB23 BW25113, DmenA::kan All cytochromes UQ

MB27 BW25113, DubiE::kan All cytochromes DMK

Table 2. Midpoint potentials of redox intermediates and terminal

electron acceptors relevant to this study. MKH2, menaquinol.

Chemical moiety Midpoint potential (mV)

O2 ⁄ H2O +0.82 [13]

UQ ⁄ UQH2 +0.11 [13]

MK ⁄ MKH2 )0.08 [13]

DMK ⁄ DMKH2 +0.04 [13]

Fumarate ⁄ succinate +0.03 [13]

Dimethylsulfoxide ⁄ DMS +0.16 [13]

TMAO ⁄ TMA +0.13 [41]

NAD ⁄ NADH )0.32 [13]

NADP ⁄ NADPH )0.32 [13]

Nitrate ⁄ Nitrite +0.42 [13]

P. Sharma et al. The role of DMK in the electron transfer chains of E. coli


its catabolism with respect to energy conservation. The

lower biomass yield and the lower rate of production

of acetate are in agreement with this. When DMK

replaced UQ, a yield value close to that of the wild-type

was observed, but the catabolism was found to switch

from acetic acid to lactic acid as the main product.

We have shown ([23], PS, KJH, MJTDM and MB

unpublished results) that, in E. coli strains with a mod-

ified composition of the respiratory chain, acetic acid

and lactic acid may be formed either by the glycolytic

pathway, in combination with acetate kinase and lac-

tate dehydrogenase, respectively, or by the ‘uncoupled

pathway’ of the pyruvate oxidase system for acetic acid

(Fig. 1, dotted arrow), and the methylglyoxal bypass

for lactic acid (Fig. 1, dashed arrow). The actual

contribution of these pathways to fermentation end-

product formation has not been quantified here, but

these ‘uncoupled pathways’ may have significant effects

on the efficiency of cellular energy conservation, and

hence affect the yield values (biomass per glucose) to a

large extent. A carbon flux balance analysis showed

that in none of the three strains could all glucose con-

sumed be accounted for by formation of biomass,

acetic acid and lactic acid (Table 3). As no other prod-

ucts (such as ethanol, formate or succinate) were

detected by HPLC, the missing carbon was most likely

contained in carbon dioxide. Indeed, in subsequent

chemostat studies (see below), it was shown that the

amount of CO2 produced exceeded acetate formation.

Significantly, production of CO2 in excess of acetate

(on a mol ⁄mol basis) and in the absence of ethanol

formation can only be due to activity of the TCA cycle.

As UQ is lacking in MB27, we conclude that TCA

cycle activity can also be maintained by succinate

dehydrogenase-mediated transfer of electrons to DMK.

The DMKH2 must subsequently be oxidized again

by either one or more of the quinol oxidases of E. coli.

To verify which of the three quinol oxidases is

involved in DMKH2 re-oxidation, we physiologically

characterized a set of nine strains that contained only

a single quinol::oxygen oxidoreductase and either UQ

only, DMK only, or DMK plus MK (Table 1). Oxy-

gen consumption rates of all nine strains were assessed

to quantify the ability of each oxidase to accept elec-

trons from the three quinones. All strains showed oxy-

gen consumption rates lower than wild-type (Table 4),

but significantly higher than background oxidation

rates (< 0.2 mmolÆgÆDW)1Æh)1, [23]). Strain DJ03

(lacking UQ and both cytochrome bd I and bo

oxidase) showed the highest respiration rate (4 mmolÆgÆDW)1Æh)1) for all strains that contain DMK with or

without MK. Further analysis of strains DJ11, DJ12

and DJ13 confirmed the absence of MK and UQ in

these strains (Table 5). Hence, we conclude that

Table 3. Physiological analysis of selected quinone mutants. Yields, product formation, growth rates and oxygen consumption rates for qui-

none mutants during exponential growth in Evan’s medium supplied with 50 mM glucose at 37 �C.

Strain

Quinones

present

Yield

(biomass ⁄ glucose)

(% g ⁄ g)

Yield

(acetate ⁄ glucose)

(mol ⁄ mol)

Yield

(lactate ⁄ glucose)

(mol ⁄ mol)

Growth

rate (h)1)

qO2

(mmolÆgÆDW)1Æh)1

BW25113 All 12.7 ± 1.2 1.0 ± 0.1 0.1 ± 0 0.94 ± 0.02 24.9 ± 5.1

MB23 UQ 23.6 ± 3.3 0.3 ± 0.1 0 0.53 ± 0.01 16.8 ± 1.5

MB27 DMK 10.3 ± 0.5 0 1.3 ± 0.1 0.11 ± 0.1 1.9 ± 0.1

Table 4. Yield of biomass and of product formation, rate of oxygen consumption and growth rate for selected mutants in batch culture.

Selected quinone mutants, each expressing only a single quinol oxidase, were grown on Evan’s medium and supplied with 50 mM glucose.

Measurements were taken during exponential growth at 37 �C.

Strain

Yield

(biomass per glucose)

(% g ⁄ g)

Yield

(acetate ⁄ glucose)

(mol ⁄ mol)

Yield

(lactate ⁄ glucose)

(mol ⁄ mol)

Growth

rate (h)1)

DJ01 3.2 ± 0.5 0.0 ± 0.0 1.2 ± 0.6 0.25 ± 0.03

DJ02 4.8 ± 1.2 0.0 ± 0.0 1.8 ± 0.3 0.19 ± 0.01

DJ03 3.7 ± 1.5 0.2 ± 0.2 1.2 ± 0.6 0.13 ± 0.05

DJ11 6.4 ± 2.5 0.1 ± 0.1 1.4 ± 0.6 0.29 ± 0.00

DJ12 6.5 ± 2.1 0.0 ± 0.0 1.8 ± 0.9 0.20 ± 0.00

DJ13 6.1 ± 5.7 0.0 ± 0.0 1.5 ± 0.6 0.12 ± 0.00

DJ31 22.1 ± 0.9 0.9 ± 0.1 0.1 ± 0.1 0.52 ± 0.14

DJ32 24.3 ± 0.3 0.9 ± 0.0 0.1 ± 0.1 0.28 ± 0.03

DJ33 11.2 ± 5.8 1.6 ± 0.9 0.1 ± 0.1 0.18 ± 0.02



DMKH2 serves as an electron donor for all three qui-

nol oxidases.

The data presented in Table 4 show that the

absence of UQ invariably results in decreased acetate

formation in batch cultures, and a shift of metabo-

lism towards lactic acid formation, despite the fact

that these cells respire at a significant rate. Compari-

son of these strains with respect to their molar turn-

over capacity for the various quinones (oxygen

consumption rate per quinone content, Table 4) sug-

gested that the three quinol oxidases are not very

selective with respect to the type of quinol that they

use as their substrate(s). Growth of MB27 under glu-

cose-limited chemostat conditions at a dilution rate of

0.1 h)1 resulted in a high CO2 flux and a relatively

low acetate flux (see Table 5). This again indicates

high DMKH2 oxidase activity. The high CO2 flux

further indicates that significant TCA cycle activity

takes place in MB27 (at a succinate oxidation rate of

� 1.2 mmolÆgÆDW)1Æh)1), indicating electron transfer

from succinate to DMK.

To further compare the relative efficiency of the qui-

nones in this set of mutants, we also calculated their

molar redox turnover, i.e. mol oxygen consumed per

mol total quinone present per unit of time (qO2 ⁄Qtot).

For strain DJ01–0,3 the contents of MK and DMK

were added. These results (sixth column of Table 5)

show even more convincingly that DMKH2 is a suit-

able electron donor for all three quinol oxidases, par-

ticularly the two cytochrome bd oxidases. It is also

important to determine whether or not MK could play

a significant role in reduction of the quinol oxidases.

Although the data do not allow a firm conclusion on

this point, they appear to suggest that MK does not

play such a role, because the molar efficiency of the

quinones in DJ01, DJ02, DJ11 and DJ12 is approxi-

mately constant if we ignore MK content.

Based on the observation that strain MB27 did not

show growth to high attenuance with oxygen as the

terminal electron acceptor (Fig. 2B), and that relatively

high oxygen consumption rates were measured in all

strains containing only DMK and a single quinol oxi-

dase, it was concluded that all the three oxidases are

able to use DMKH2 as a substrate. This conclusion

was confirmed by the observation that strains DJ01,

DJ03, DJ11 and DJ13 all showed good growth when

grown on the non-fermentable carbon source glycerol

(see Fig. 2A). To further quantify the respiratory

capacity of strains DJ11, DJ12 and DJ13, these strains

were grown in a glucose-limited chemostat culture (see

Experimental procedures). From the data presented in

Table 6, it can be seen that catabolism for all these

strains was mostly homolactic, although significant

amounts of CO2 and acetate were produced. This indi-

cates that electrons from succinate are transferred to

DMK, and electrons from DMKH2 are transferred to

all three quinol oxidases. It also indicates that the rate

of respiratory electron flow is so low (or the Km for

DMKH2 so high), that with DMK only strains show

‘overflow catabolism’, resulting in a high rate of

formation of lactic acid.

The above findings suggest low donor ⁄ acceptor spec-ificity at the level of catabolic electron flow. To see

whether similar low specificity is also the case for

anaerobic respiration, strains were grown in batch cul-

tures on the non-fermentable carbon source glycerol,

and supplied with the alternative electron acceptors:

fumarate, dimethylsulfoxide or TMAO. Strain MB23,

containing UQ as the sole quinone species, had virtu-

ally completely lost the capacity to grow under anaero-

bic conditions with all three electron acceptors. This

suggests strongly that fumarate reductase, dimethyl-

sulfoxide reductase and TMAO reductase are unable

to use ubiquinol (UQH2) as their electron donor. In

Table 5. Quinone content, qO2 and qO2 ⁄ Qtot for quinone mutants that contain only a single oxidase. Triplicate measurements were taken

during exponential growth at 37 �C and are presented as means ± standard deviation. ND, not detected. For calculation of the redox turn-

over (qO2 ⁄ Qtot) of mutants containing both DMK and MK, the content of these two quinones was added.

Strain

DMK content

(nmolÆgÆDW)1)

MK content

(nmolÆgÆDW)1)

UQ content

(nmolÆgÆDW)1)

qO2

(mmolÆgÆDW)1Æh)1)

qO2 ⁄ Qtot

(mol O2 ⁄ mmolÆQ ⁄ h)

DJ01 352 ± 171 403 ± 230 ND 1.5 ± 0.1 2.0

DJ02 260 ± 132 282 ± 178 ND 1.9 ± 0.2 3.5

DJ03 633 ± 149 ND ND 3.9 ± 0.2 6.2

DJ11 356 ± 105 ND ND 3.2 ± 0.2 9.0

DJ12 315 ± 173 ND ND 3.4 ± 0.2 10.8

DJ13 450 ± 106 ND ND 2.1 ± 0.1 4.7

DJ31 ND ND 1532 ± 822 16.4 ± 1.3 10.7

DJ32 ND ND 1653 ± 1100 11.6 ± 1.5 7.0

DJ33 ND ND 1863 ± 137 8.4 ± 0.3 4.5



contrast, strain MB27, with DMK as its only quinone

species displayed efficient growth when supplemented

with these alternative electron acceptors, confirming

that the three reductases involved in anaerobic respira-

tion can use DMKH2 as an electron donor [21]. This

conclusion is reinforced by the observations that this

strain converted fumarate into succinate, and that glyc-

erol was oxidized almost completely to carbon dioxide

in the presence of TMAO or dimethylsulfoxide. The

latter observation is a manifestation of significant suc-

cinate-dependent reduction of DMK to DMKH2.

Discussion

The energy conservation system of E. coli comprises a

complex network of alternative parallel pathways

endowing the cell with a choice of biochemical param-

eters, including kinetic and efficiency, to properly fulfil

its most important activity under a broad range of

conditions: conservation of energy to be used for cell

maintenance and proliferation. Indeed, E. coli is able

to respire under fully aerobic, micro-aerobic and

(provided alternative terminal electron acceptors are

present) anaerobic conditions. Here, we assessed the

role that the quinone species demethylmenaquinone

(DMK) plays in anaerobic and aerobic respiration.

We have shown that DMK can act as a redox inter-

mediate for electron transfer towards a number of

terminal electron acceptors. Thermodynamically, this

ability is in agreement with the midpoint potential of

DMK (+36 mV), which is sufficiently close to, or

below, the midpoint potential of the fumarate ⁄ succi-nate couple (+30 mV), the TMAO ⁄ trimethylamine

Table 6. Specific product formation rates and oxygen consumption

rates of selected mutants in the chemostat. Strains MB23, MB27,

DJ11, DJ12 and DJ13 were grown in aerobic glucose-limited che-

mostat cultures at a dilution rate of 0.1 (see Experimental proce-

dures for further details). qAce, acetic acid flux; qLac, lactic acid

flux; qCO2, CO2 flux. All values are in mmolÆgÆDW)1Æh)1. ND, not

detected.

qAce qLac qCO2

MB23 0.3 ± 0.4 1.5 ± 2.5 2.4 ± 0.3

MB27 0.4 ± 0.5 ND 3.9 ± 0.6

DJ11 1.2 ± 0.1 22.2 ± 0.1 1.6 ± 0.8

DJ12 1.4 ± 0.2 27.7 ± 5.4 2.4 ± 0.1

DJ13 1.0 ± 0.1 22.0 ± 1.1 1.8 ± 1.5

A

B

Fig. 2. (A) Growth of selected quinone dele-

tion mutants that contain only a single

quinol::oxygen oxidoreductase on Evan’s

medium supplied with 50 mM glycerol as

the only carbon source under aerobic condi-

tions. Measurements were taken after 72 h

incubation at 37 �C. (B) Growth of selected

quinone deletion mutants on Evan’s med-

ium supplied with 50 mM glycerol with a

variety of terminal electron acceptors after

48 h incubation at 37 �C. Vertical lines, oxy-

gen; hatching, no terminal electron acceptor

added; horizontal lines, 50 mM fumarate;

diagonal lines, 50 mM TMAO; dots, 50 mM

dimethylsulfoxide. When oxygen was not

used as the terminal electron acceptor,

anaerobic conditions were applied.



(TMA) couple (+130 mV), the dimethylsulfoxide ⁄DMS couple (+160 mV) and the O2 ⁄H2O couple

(+820 mV).

Previously, it was shown that DMK can serve as an

electron carrier in anaerobic respiration [21]. However,

involvement of DMK in aerobic respiration had not

been convincingly demonstrated. The only evidence

available was a quinone extraction experiment per-

formed by Hollander [29]. We have furthermore shown

here that electron transfer from DMKH2 is not limited

to a specific oxidase: all three oxidases expressed by

E. coli can use DMKH2 as a substrate, as shown here

by using a set of mutants containing a single oxidase. It

should be mentioned in this respect that growth on

glycerol was only supported by the cytochrome bd I

and cytochrome bd II oxidases. Interestingly, a quanti-

tative metabolic product flux analysis during glucose-

limited continuous growth showed that mutants that

contain only DMK convert glucose almost completely

to carbon dioxide via glycolysis plus the TCA cycle.

Therefore, we conclude that DMK can be used as the

oxidant in conversion of succinate to fumarate, i.e. the

succinate dehydrogenase complex is functional with

DMK as its electron acceptor. Interestingly, it was

shown previously [21], as well as in this study, that

conversion of fumarate to succinate can also use elec-

trons from the DMK pool. Clearly, the small differences

in midpoint potential between the DMK ⁄DMKH2

couple and the succinate ⁄ fumarate couple under physio-

logical conditions allow the redox reaction to operate in

either direction, even though the membrane-bound

fumarate reductase and the succinate dehydrogenase

contain specific MK and UQ binding sites, respectively

[16]. The possibility that the fumarate reductase actually

functions as a succinate:DMK oxidoreductase under

the conditions reported here cannot be excluded. It is

important to note that DMK and MK are structurally

very similar, making the DMK-dependent succinate

oxidase activity of fumarate reductase even more likely.

Our results do not necessarily disagree with the pro-

posal [17,18] that functioning of the respiratory chain

is optimized by an acceptor ⁄donor cascade with a step

size of � 150 mV. As the midpoint potentials of the

first redox-active group of the cytochrome bo and

cytochrome bd oxidase are � 55 mV [30] and 150 mV

[31], respectively, this hypothesis does not exclude

transfer of electrons from DMKH2 to either of these

two oxidases, and in our studies, lower respiration

rates were indeed observed. However, it may be that,

in the mutants used, the contents of some components

of the respiratory chain differ from those in wild-type

strains. The data on the quinone contents (Table 5)

suggest that extensive feedback regulation is operative.

Many studies have shown that the composition, and

thereby the efficiency (Fig. 1), of the respiratory chain

of E. coli is highly flexible and well-adapted to func-

tion properly under a vast range of conditions

[5,13,21]. Flexibility is also observed with respect to

the energetic efficiency of substrate-level phosphoryla-

tion: the inducible activity of non-energy conserving

pathways such the pyruvate oxidase system and the

methylglyoxal bypass (dashed and dotted arrows in

Fig. 1) may (partially) account for the large differences

observed in this investigation in the yield values for

biomass formation during growth on glucose (unpub-

lished results). Further quantitative analysis is required

to confirm this, but this type of analysis is not consid-

ered appropriate here. Nevertheless, the data presented

further emphasize the flexibility of the catabolic

machinery of E. coli, based on the unexpectedly strong

effect of deletion of menaquinone biosynthesis on bio-

mass yield and the concomitant decrease in acetate

production during aerobic batch growth. At the same

time, they demonstrate the complexity of the catabolic

machinery. In applied fields (including synthetic bio-

logy), optimization strategies with respect to biomass

yield are often paramount. Our results imply that these

strategies require further improvement of our under-

standing of catabolism.

The results reported here are part of a larger effort to

obtain a complete systems biology description of the

energetics of respiratory catabolism in E. coli (i.e. the

SysMo-SUMO2 project [28]. Although large amounts of

data are already available on the constituent enzymes of

this respiratory system, which can be used to initiate

quantitative modeling, the data presented here make it

clear that even more detailed information is necessary to

make such models realistic, particularly with regard to

the specificity and affinity of these enzymes for the

various types of quinones that they encounter in the

cytoplasmic membrane of this bacterium, particularly

menaquinone. An even bigger challenge will be to test

experimentally the predicted oxidation ⁄ reduction ratios

of these quinones under physiological conditions.

Experimental procedures

Construction of deletion mutants

The single-deletion strains were obtained from the Keio col-

lection [32,33]. In order to construct strains with multiple

deletions, the kanamycin marker was first removed as

described by Datsenko and Wanner [33]. Mutants with

double and triple deletions were constructed by P1 phage

transduction of the desired mutations. Mutants were

checked by PCR.



Batch cultures

We used a defined simple salts medium described by Evans

et al. [34], with nitrilo-acetic acid (2 mm) rather than citrate

as a chelator, and sodium phosphate buffer (pH 7) at a

concentration of 100 mm instead of 10 mm to increase buf-

fering capacity. In batch culture, glucose was used as the

carbon source at a concentration of 50 mm in most cases

except when glycerol at a concentration of 50 mm was used

(as indicated). Aeration of cultures during aerobic growth

was accomplished by shaking 10 mL cultures in 100 mL

flasks designed for aerobic cultivation. For anaerobic

growth, 50 mL cultures in sealed bottles (50 mL) were used.

In anaerobic cultures, the electron acceptors fumarate,

dimethylsulfoxide and TMAO were used at a concentration

of 50 mm. Cultures were inoculated from LB plates. The

strains were maintained in vials in LB medium with 30%

w ⁄ v glycerol at )70 �C.

Respiratory measurements

Respiration rates were determined amperometrically using a

Clark electrode at 37 �C, with glucose as the substrate, and

the system was saturated with oxygen. Then the rate of

oxygen consumption was monitored.

Continuous cultures

E. coli K-12 strain BW25113 and various deletion derivatives

were grown in an Applikon (Schiedam, The Netherlands)

2 L fermentor at a constant dilution rate of 0.10 ± 0.01 h)1.

Selenite (30 lgÆL)1) and thiamine (15 mg L)1) were added to

the medium. Glucose was used as the sole carbon and energy

source at a final concentration of 50 mm. The dilution rate

was set by adjusting the medium supply rate. The pH was

maintained at 7.0 ± 0.1 by titration with sterile 4 m NaOH,

the temperature was controlled at 37 �C, and the stirring rate

was 600 rpm. The air supply rate was set at 0.5 LÆmin)1.

In all cultures, the steady-state specific rates of fermenta-

tion product formation and glucose and O2 consumption

were measured as described by Alexeeva et al. [35], and are

expressed as q values (Tables 3–6).

Analysis of carbon fluxes

Bacterial dry weights were determined in steady-state che-

mostat cultures as described previously [36]. Physiological

characterization of strains was performed in terms of their

growth rate and the rate of formation of fermentation

products. The growth rate in batch cultures was calculated

based on spectrophotometric measurements of the attenu-

ance of the culture at 600 nm. Metabolic fluxes were

analyzed and calculated using a simple stoichiometric flux

analysis [37–39] (see also Fig. 1).

Glucose, pyruvate, lactate, formate, acetate, succinate

and ethanol contents were determined by HPLC (LKB and

Pharmacia, Oregon City, OR, USA) using a REZEX

organic acid analysis column (Phenomenex, Torrance, CA,

USA) at 45 �C, with 7.2 mm H2SO4 as the eluent, using an

RI 1530 refractive index detector (Jasco, Easton, MD,

USA) and AZUR chromatography software (St. Martin

D’Heres, France) for data integration. All data have a car-

bon balance of 90 ± 10% as calculated from the glucose

consumption and product formation rates.

Quinone analysis

Culture samples (2 mL) were quenched with 6 mL ice-cold

methanol. Then 6 mL petroleum ether (boiling point 40–

60 �C) was added rapidly to each mixture, and the mix-

ture was vortexed for 1 min. After centrifugation of the

mixture at 900 g for 2 min, the upper petroleum ether

phase was removed and transferred to a test tube under a

flow of nitrogen. Then 3 mL of petroleum ether was

added to the lower phase, and the vortexing and centrifu-

gation steps were repeated. The upper phases were com-

bined. After evaporation under a flow of nitrogen to

dryness, the extracts were stored. The extracts could be

kept for at least 7 days under nitrogen at )20 �C without

changes in the quinone ⁄quinol content. Immediately

before use, the extracted quinone ⁄ quinol mixture was re-

suspended using a glass rod in 80 lL ethanol, and frac-

tionated by HPLC (Pharmacia LKB 2249 gradient pump

system with an LKB 2151 variable-wavelength monitor)

using a reversed-phase Lichrosorb RP10 C18 column (par-

ticle size 4.6 lm; internal diameter 250 mm) (Chrompack,

Bergen op Zoom, The Netherlands). The column was

equilibrated with ethanol ⁄methanol (1 : 1 v ⁄ v) as the

mobile phase. The flow rate was set at 1 mLÆmin)1 at

20 �C. Detection of the eluate was performed at 290 nm

for ubiquinones and at 248 nm for menaquinones. The

amount of each quinone species was calculated from the

relevant peak area using ubiquinone-10 and menaquinone-

4 as standards and the method described by Shestopalov

et al. [40]. Methanol, ethanol and petroleum ether were of

analytical grade.

Peaks were identified by UV ⁄ visible spectroscopy and

tandem mass spectral analysis. A UV ⁄ visible absorption

spectrum of demethylmenaquinone was kindly provided by

A.V. Bogachev (Department of Molecular Energetics of

Microorganisms, Moscow State University, Russia). For

mass spectral analysis, fractions collected from the HPLC

were evaporated under nitrogen and re-dissolved in 89%

acetonitrile, 10% water, 1% formic acid (LC grade; Merck,

Frankfurt, Germany). The fractions were analyzed by off-

line electrospray mass spectrometry using coated PicoTips

(Econo12; New Objective, Woburn, MA, USA) and an

electrospray ionization quantitative time-of-flight mass



spectrometer (Micromass, Waters, Manchester, UK). Ions

selected for tandem mass spectrometry were collided with

argon in a hexapole collision cell.

Acknowledgement

This work was supported by the SysMo-SUMO2 pro-

ject and the Erasmus Mundus External Cooperation

Window (EMECW).

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On the function of the various quinone species in Escherichia coli