BBRC

Biochemical and Biophysical Research Communications 325 (2004) 803806

www.elsevier.com/locate/ybbrc

Redox sensing by Escherichia coli: eects of dithiothreitol,

a redox reagent reducing disulphides, on bacterial growth
Gayane Kirakosyan, Karine Bagramyan*, Armen Trchounian*
Department of Biophysics, Yerevan State University, 1 Alex Manoukian Str., Yerevan 375025, Armenia
Received 18 October 2004
Available online 5 November 2004

Abstract
Escherichia coli is able to grow with a high rate under anaerobic conditions upon decrease in redox potential (Eh) both either in
slightly alkaline (pH 7.5) or acidic (pH 5.5) medium. Upon transition of E. coli MC4100 culture to stationary growth phase a
decrease in Eh from the positive values of +120 to +160 mV to the negative ones of 380 to 550 mV, and the H2 production
are observed at various pH. A redox reagent DL -dithiothreitol (DTT) in a concentration of 3 mM reduces Eh to the negative values,
and increases a latent (lag) growth phase duration, as well as delays a logarithmic growth phase independently of pH. At alkaline
and acidic pH the changes in membrane potential (DW) are observed in the presence of 3 mM DTT. K+ uptake is recovered. At pH
5.5 the H2 production is suppressed by DTT only in a higher concentration of 10 mM. The results suggest DTT eects that are in
addition to the eects of Eh. The mechanism of DTT action on bacterial growth might be intermediated through thiol group modulation of the membrane proteins, which is reected as the generation of DW as well as K+ accumulation and the activity of the
membrane-associated enzymes.
2004 Elsevier Inc. All rights reserved.
Keywords: Redox potential;

DL -Dithiothreitol;

Thiol groups; Bacterial growth

Escherichia coli is able to grow well under anaerobic

conditions upon decrease in redox potential (Eh) [1,2]
and within a wide range of external pHeither in alkaline or acidic medium [3,4]. Under anaerobic conditions,
this bacterium performs a mixed-acid fermentation of
sugars (glucose) with production of lactic, formic, acetic,
and other acids. Formic acid is oxidized to carbon dioxide and molecular hydrogen [5]. Meantime, CO2 is likely
not to be evolved and to be used for the anaerobic metabolic processes [4], so, the fermentative gas, H2 is extruded outside.
At bacterial growth under the indicated conditions, a
shift in Eh from positive to negative values is observed
[1,6]. It is worthy of note that the positive values, resulting
from dissolved oxygen, inhibit the growth, whereas the
*

Corresponding authors. Fax: +374 1 554641.

E-mail addresses: KBaghramyan@ysu.am (K.
Trchounian@ysu.am (A. Trchounian).

Bagramyan),

0006-291X/$ - see front matter 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2004.10.119

positive values, created by the presence of other chemicals, are not able to aect the growth [2,7]. However, E.
coli is likely to sense Eh independently of oxygen concentration and of the oxidizers [7]; dierent mechanisms and
models are proposed to explain such a redox taxis [8].
Eh might determine an electron transfer within bacterial membrane [7] and proton-motive force [9]. It is suggested that the eect of Eh on proton-motive force under
anaerobic conditions is induced by the change in pH
gradient across the membrane. The latter resulted from
an alteration in the cytoplasmic pH by fermentation
acids [10,11]; the membrane potential (DW) is changed
slightly [9]. Such a dependence is due to change in the
membrane proton permeability without modication
of the proton-translocating F0F1-ATPase activity
[9,12]. The change in the membrane proton permeability
might depend on thiol groups state and distribution
[11,13,14]. At the same time, a correlation between Eh
and the state and distribution of thiol groups on the bac-

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G. Kirakosyan et al. / Biochemical and Biophysical Research Communications 325 (2004) 803806

terial membrane is found [6,11]. A change in the state

and distribution of thiol groups can be involved in the
mechanism of redox taxis in E. coli so this may lead to
the regulation of the growth by redox reagent oxidizing
thiol groups.
Moreover, a relationship of Eh with both the external
and the cytoplasmic pH is evident [2,15] but not studied
well.
The bacterial growth under anaerobic conditions
upon fermentation of sugars (glucose) at alkaline pH
can be inhibited by oxidizers, which are maintaining
Eh on the positive level, and stimulated by reducers,
decreasing Eh and enhancing thiol groups, reduced state
[6,9]. However, a redox reagent reducing disulphides,
such as DL -dithiothreitol (DTT), is shown to delay the
logarithmic growth phase [9]. This eect possibly depends on the concentration of the reagent, hence
3 mM is usually eective. It should be noted that DTT
became toxic at a higher concentration (10 mM) [9].
In the present work DTT in a concentration of 3 mM
is shown to suppress E. coli growth under anaerobic
conditions independently of external pH. It also aects
the generation of DW and potassium ion accumulation
within bacterial cell. In an acidic medium DTT in a
higher concentration (10 mM) inhibits only H2
production.

Materials and methods

Bacteria, bacterial growth, and preparation for the measurements.
The E. coli MC4100, wild-type strain, supplied by Dr. S.C. Andrews
(School of Zoology and Microbiology, The University of Reading,
Reading, UK) was used throughout.
Bacteria were grown under anaerobic conditions at 37 C in peptone or salt medium with glucose as described earlier [6,16,17]. Growth
pH was adjusted by using NaOH and HCl. Bacterial growth was
monitored and latent (lag) growth phase duration was determined as
described [18]. Specic growth rate was calculated over the interval,
where the logarithm of absorbance of the culture at 600 nm increased
linearly with time and expressed as 0.693/doubling time. Fermentation
under the used growth conditions was detected previously [6,16].
During the growth to the late exponential or stationary phase in media
with an initial pH of 7.5, 6.5 or 5.5, the medium pH decreased to 7.0,
6.1 or 5.2, respectively. Preparation of bacteria for the measurements was described [6,16]; the assay mixture contained: 200 mM Trisphosphate containing 0.4 mM MgSO4, 1 mM NaCl, and 1 mM KCl;
pH was the same like an initial one for the growth of bacteria.
Determination of redox and membrane potentials. The value of Eh in
bacterial suspension was measured using both platinum and titanium
silicate redox electrodes as described [6,16,19]. DW was calculated by
distribution of tetraphenylphosphonium (TPP+) bromide between the
cytoplasm and the medium. It was determined with a selective electrode as described [1]. One micromolar TPP+ was introduced into the
assay mixture. Binding of TPP+ on bacterial surface was determined
after boiling for 35 min; intracellular volumes of 1.16 ll/cell for pH
7.5 and of 1.48 ll/cell for pH 5.5 were employed [17].
H2 production and K+ transport assay. H2 production rate has been
determined as a dierence between the rates of decrease in Eh for
platinum and titaniumsilicate electrodes [16], and is expressed in mV
Eh/min/mg of dry weight. This dierence has become obvious since the

H2 production was conrmed also by a chemical assay [19] and the

Durham tube method [16]. It is interesting to note, CO2 and H2 could
be detected by the latter method, however CO2 is likely to be not
evolved and to be used for the anaerobic metabolic processes by E. coli
[4], while H2 is extruded outside; so the Durham tube method was used
in conrmation with the others.
K+ uxes through the bacterial membrane in whole cells were
measured using selective electrodes as described elsewhere [1,6]. Ion
uxes are expressed as the change in external activity of this ion in
mM/min/1012 cells/ml [16]. Small changes in K+ activity were recorded
using a potentiometer, and they were calibrated by titration with
0.02 mM KCl.
Others and reagents. Bacterial titre and dry weight of bacteria were
determined as described [16]. The average data are presented from two
or three independent measurements, the standard error does not exceed 5%. Agar, peptone, DTT, and TPP+ bromide were from Sigma
(USA), and other reagents were of analytical grade.

Results and discussion

Bacterial growth and redox potential at various pH
The growth of E. coli under anaerobic or aerobic conditions for a wide range of alkaline and acidic media has
been shown in a number of papers [3,4,16,20]. It is a matter of interest that the anaerobic growth of this bacterium at alkaline pH is coupled with a shift of Eh from
the positive to the negative values, which probably determines the growth [1,6,15]. Eh is related with pH [2] and
the growth of E. coli at acidic pH might also be determined by a decrease in Eh. Moreover, an anaerobic
growth of this bacterium has been shown to be aected
by oxidizers [6] so it might be changed with reducers.
Indeed, E. coli MC4100 grows with a high rate under
anaerobic conditions at pH of 7.55.5 (Fig. 1), resulting
in a shift in Eh from the positive values (+120 to
+160 mV) at the beginning of a latent (lag) growth phase
to the negative ones ( 380 to 550 mV) upon transition
to a stationary phase (Fig. 2). The drop of Eh gives evidence of the amplication of reduction processes, which
are apparently characteristic of metabolism under anaerobic conditions. Under these conditions upon the fermentation of glucose the production of H2 is detected
(Fig. 3A).
Thus, the lower values of Eh as well as a deeper decrease in Eh are observed with transition from alkaline
to acidic environment (Fig. 2). These results probably
reect a link between Eh and external pH proposed [15].
Eect of DTT on bacterial growth
The introduction of DTT in the concentration of
3 mM into the growth medium results in a decrease of
Eh value to the negative values of 60 to 220 mV,
which depends on external pH (Fig. 2). Such a decrease
is less than that in the absence of DTT, and a deeper
decrease is observed again with transition from alkaline
to acidic environment. An acceleration of the growth

G. Kirakosyan et al. / Biochemical and Biophysical Research Communications 325 (2004) 803806

805

H2 production rate
(mV Eh/min.mg d.wt)

5
-DTT
+DTT

4
3
2
1
0

Membrane potential (-mV)

pH 7.5

pH 6.5

160

pH 5.5
-DTT
+DTT

150
140
130
120
110
100

pH 7.5

pH 6.5

pH 5.5

K flux (mM/min per 1012 cells)

C
Fig. 1. Eects of DTT on the growth characteristics of E. coli MC
4100 under anaerobic conditions on glucose at various pH. (A) Lag
growth phase duration. (B) Specic growth rate. Bacteria were growing
in salt medium; glucose was in concentration of 0.2%, DTT (3 mM)
was introduced into the growth medium immediately before inoculation of bacteria.

0.2
0.15

-DTT
+DTT

0.1
0.05
0

pH 7.5

pH 6.5

pH 5.5

Fig. 3. Eects of DTT on the molecular hydrogen production (A),

membrane potential (B), and K+ uptake (C) by E. coli MC 4100 grown
under anaerobic conditions at various pH. (A) DTT was added into
the assay mixture in a concentration of 10 mM at pH 5.5. Bacteria
were grown in peptone medium. The other conditions and designations
are the same as in the legends to Fig. 1.

Fig. 2. Change in Eh during E. coli MC4100 growth in the media with

various pH in the absence and in the presence of DTT. The decrease
was the same for bacteria grown either in peptone or salt medium. The
conditions and designations are the same as in the legends to Fig. 1.

processes in E. coli under anaerobic conditions is apparently observed.

Duration of a lag growth phase for E. coli MC4100 is
considerably increased (in 1.5-fold) in the presence of
DTT (Fig. 1A), and the restoration of a specic growth

rate is determined. It appeared to be essentially less in

the acidic medium (Fig. 1B). This is in accordance with
data indicating that a decrease in Eh and pH has
brought about a longer lag phase and a slower growth
rate and even led to growth failure [15].
At acidic pH the Eh drops during the growth of these
bacteria (Fig. 2), and the values of Eh in a late stationary
growth phase (in 2224 h) remain below those observed
in the absence of DTT (Fig. 2). The suppression of bacterial growth in the presence of DTT could have resulted
not only by changes of Eh at various pH but most likely
by direct eects of the reagent on the thiol group of proteins in bacterial membrane.

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G. Kirakosyan et al. / Biochemical and Biophysical Research Communications 325 (2004) 803806

Eect of DTT on transport systems and enzymes

The state of thiol groups might have an eect on the
anity and activity of transport systems and membraneassociated enzymes [13,14,19]. DTT in concentrations of
3 mM (Fig. 3B), 5 or 10 mM (data not shown) results in
decrease of the value of DW irrespective of external pH.
The accumulation of K+ by bacteria in the presence of
3 mM DTT is inhibited either in alkaline or acidic medium (Fig. 3C). The H2 production is suppressed in the
presence of DTT in a higher concentration of 10 mM
only in acidic medium with pH 5.5 (Fig. 3A). These effects most likely testify an inuence of DTT on potassium transport systems, namely TrkA or Kup, and on
formate hydrogenlyase enzyme complex, which are
responsible for K+ accumulation and H2 production,
respectively [5,14,16,19]. Apparently, thiol groups play
an essential role in an activity of these membrane-associated systems.

Concluding remarks
It is assumed that DTT as a reducer itself aects the
growth of E. coli, as well its eects can be intermediated
through Eh and depend on external pH. It is clear that
the oxidationreduction processes play an exclusive role
in the habitability of bacteria. The majority of these processes implemented on bacterial membrane depend on
Eh and play an essential role in metabolism
[12,14,16,17]. For instance, the reduced environment
changes metabolic uxes resulting in decreased or enhanced fermentation products [2123]. But not always,
as it follows from our results (see Figs. 13) this link is
unambiguous and determines the growth of bacteria.
So, the mechanisms of Eh aecting metabolism remain
little known. In this respect it is required to further study
the eects of dierent oxidizers and reducers, which will
allow understanding regulatory paths for the membrane
functions and the mechanisms of bacterial redox taxis.

Acknowledgments
This work is fullled by the support of the Ministry
of Education and Science of the Republic of Armenia

and partially by the US Civilian Research and Development Foundation Grant No. AB1-2307-YE02.
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