FEMS MicrobiologyLetters 84 (1991) 319-324
© 1991 Federation of European MicrobiologicalSocieties 0378-1097/91/$03.50
Published by Elsevier
319
FEMSLE 04708
Iron content and FNR-dependent gene regulation
in Escherichia coli
F. N i e h a u s 1, K. H a n t k e ~ a n d G. U n d e n 1
i Institutfiir Biochimie, Heinrich-Heine-Uniuersitiit,Diisseldorf and 2 Lehrstuhlfiir MikrobiologyII, UniversitiitTiibingen,
Tiibingen, F.R.G.
Received 1l September 1991
Accepted 23 September 1991
Key words: FNR; Anaerobic respiration; Anaerobic regulation; Iron; Escherichia coli
1. S U M M A R Y
The significance of intracellular iron levels of
Escherichia coli on the expression of the fumarate
reductase operon (frd), which is regulated by the
transcriptional activator FNR, was studied in vivo.
The iron contents of aerobically and anaerobically grown E. coli were determined and related
to the expression of frd and of genes (flu, fepA,
fhuF) which are regulated by the iron uptake
regulatory protein Fur. The iron contents varied
from 1.6 to 6.9/xmol F e / g protein with no significant difference in aerobic and anaerobic bacteria. Expression of frd was not related to the
different iron levels, but to oxygen supply. Only
severe iron limitation in iron-depleted medium,
which caused lower iron contents (0.8 to 1.6
tzmol/g), reduced the expression of frd under
anaerobic conditions. On the other hand, expression of flu, fepA and fhuF clearly responded to
Correspondence to: G. Unden, Institut fiir Biochemie,Geb~iude
26.42, Heinrich-Heine-Universit~it, Urfiversit~itsstr. 1, 4000
Diisseldorf, F.R.G.
iron supply and cellular content, but only slightly
to changed O 2 supply. Generally, expression of
frd responded only to much stricter iron limitation, than expression of Fur regulated genes. It is
concluded that the functional state of FNR during a e r o b i c / a n a e r o b i c switch is not regulated by
iron content and reversible binding of Fe z+ under physiological conditions. Therefore FNR does
not communicate with the iron pool regulating
the Fur protein.
2. I N T R O D U C T I O N
Metal ions are essential cofactors for various
bacterial gene regulators (for a review see [1]).
Recently it has been shown that FNR, the transcriptional activator of anaerobic respiratory
genes of Escherichia coli [2,3], is also a metalloregulatory protein with Fe 2+ as an essential
cofactor [4-6]. It is not known, however, in which
way Fe 2÷ controls the function of FNR. FNR is
active only under anaerobic conditions and the
functional state appears to be controlled by the
320
Fe 2+ ion [5]. It has been suggested that the
functional state of FNR is regulated by a redox
reaction, presumably of the Fe e+ ion [7]. However, in vivo FNR can also be switched between
the active and inactive state by depletion of Fe z+
or supply with Fe 2+ [8]. Therefore it is not clear
whether a redox reaction at FNR or reversible
binding of iron provides the regulatory signal for
FNR. It will be studied here whether the functional state of FNR during the a e r o b i c / a n a e r o b i c
switch is controlled by iron availability. Iron availability to the bacterial cell will be estimated by
measuring total iron and the expression of Furregulated genes. Fur (ferric uptake regulator) acts
as an aporepressor, which is converted to the
active repressor by binding of Fe 2+ [9,10]. Expression of Fur-regulated genes therefore provides a direct means for estimating the regulatory
pool of intracellular iron, which could not be
investigated so far by chemical or physicochemical methods.
(anaerobic growth). Aerobic and anaerobic growth
were performed as described [8].
3.3. Extraction of iron from M9 medium
M9 medium prepared from distilled water and
analytical grade chemicals contains about 1 ~ M
iron, which is sufficient to support growth of E.
coli (2 ~mol i r o n / g cellular protein) to Asv 8 = 1.5.
To achieve lower levels of iron, M9 medium with
all ingredients, but without CaC12 and MgSO 4,
was pumped slowly through a column of Chelex
• 100 chelating resin (maximally 40 ml m e d i u m / m l
Chelex 100) according to the recommendations of
the supplier (Bio-Rad, Miinchen, F.R.G.) and
made sterile by filtration. The rest of the components (CaC1 z and MgSO 4) were added after the
Chelex 100 treatment. All plastic ware was either
new or incubated with 0.1 M ethylenedinitrilotetraacetic acid EDTA, pH 8) for 48 h prior to use
and rinsed with distilled water. Glassware was
kept for 48 h in 2 M HCI and 24 h in 0.1 M
E D T A before use and rinsed with distilled water.
Only silicone or plastic caps were used.
3. M A T E R I A L S AND M E T H O D S
3.4. Iron determination
3.1. Bacterial strains
For the experiments different derivatives of E.
coli MC4100 ( F - araD139A(argF-lac)U169
rpsL150 reIA1 flbB5301 deoC1 ptsF25 rbsR) were
used. The strains differed from MC4100 as indicated: JB1698 (MC4100 with flu :: hplacMu) [11];
H1858 (MC4100 with fhuF :: M u d l X ) [11]; JB1686
(MC4100 with fepA :: AplacMu). Insertion of
AplacMu in fepA, the receptor for enterochelin,
was demonstrated by the loss of the FepA protein
in the outer membrane (SDS gel electrophoresis) and by resistance to colicin B. Strain
MC4100(AJ100) (MC4100 with frd'-'lacZ) [13]
was kindly provided by R.P. Gunsalus (Los Angeles, CA).
3.2. Media and growth
For growth the M9 mineral salts medium [14]
was used, supplemented with glucose (0.5%), 50
mM fumarate, 0.5 g/1 casein hydrolysate (Gibco,
No 140; Gibco, Eggenstein, F.R.G.) and 75 mg/1
L-tryptophan. Ampicillin was added at concentrations of 5 0 / x g / m l (aerobic growth) or 25 /xg/ml
Bacteria (A578= 0 . 6 - 0 . 8 ) were harvested by
centrifugation in iron-free plastic tubes, washed
twice in distilled water and wet-ashed. Iron was
determined with ferrozine (3-(2-pyridyl)-5,6bis(4-phenylsulfonic acid)-l,2,4-triazine) [15] or
bathophenanthroline [16]. During all steps ironfree, cleaned plastic ware (compare 3.3) was used.
3.5. Other methods
/3-Galactosidase activity was determined from
permeabilised bacteria [14]; protein by the biuret
method with KCN [17].
4. R E S U L T S
4.1. Cellular levels of iron in aerobically and anaerobically respiting bacteria
The cellular levels of total iron and expression
of frd-lacZ were determined in E. coli grown
aerobically or anaerobically in mineral medium
(Fig. 1). The iron contents varied to a considerable degree (1.6 to 6.9 /xmol F e / g protein), but
321
B-Gal
(U/g)
•
800[
~ u
0
u I i
•--
•
I
2
4
Fe ( IJmol/g protein)
I
6
Fig. 1. Iron contents and expression of frd-lacZ in E. coil
MC4100(AJ100) after aerobic ( D , I ) and anaerobic (©, e)
growth. T h e bacteria were grown to As7 s = 0.6-0.8 and analysed for total iron content and/3-galactosidase activity (/3-GAD,
Growth occurred in M9 m e d i u m (closed symbols) or in M9
medium depleted of iron by Chelex 100-treatment prior to
growth (open symbols).
were similar in aerobic and anaerobic bacteria.
By this the contents were similar to values determined earlier for aerobically grown E. coli (4
~ m o l / g dry weight) [18]. At levels > 1.6 p, mol
F e / g protein expression of frd was nearly constant and not affected by the iron contents. In
aerobic bacteria which showed, as expected, low
frd expression, the levels of iron were comparable
to the anaerobic ones. In media which were depleted of iron by treatment with ion exchange
resin the iron contents decreased down to 0.8
p~mol F e / g . This was accompanied by a decrease
of frd-lacZ expression down to 140 U / g in
anaerobic bacteria. Addition of Fe 2+ to the depleted medium restored the original levels of iron
content and frd expression. The experiments
demonstrate that the total iron contents in aerobic bacteria are comparable to anaerobic bacteria, whereas frd expression differs largely under
both conditions.
4.2. Iron content and expression of Fur- and FNRdependent genes
The regulatory pool of 'free' iron within the
cell certainly amounts only to a small proportion
of the chemically determined total iron. It is not
possible as yet to measure a regulatory pool of
free iron by chemical or physicochemical means.
In E. coli, however, the expression of Fur-regu-
lated genes directly responds in a negative manner to the level of regulatory competent iron in
the bacteria, since Fe 2+ acts as a reversible co-repressor of Fur [10]. Therefore the expression of
the fiu gene (Fur-repressible [11,12]) and of the
frd-gene were compared as a function of the
oxygen and iron supply (Table 1). As expected,
the expression of fiu-lacZ was low in the mineral
medium and increased 5- to 6-fold in the iron-depleted medium. The increase of flu expression is
indicative for decreased levels of intracellular
available Fe 2+, which was also reflected by decreased contents of total Fe. Under the same
conditions, the expression of frd-lacZ showed
only a minor response to the change in iron
supply, but a drastic reaction to the O 2 supply.
This argues against a common regulatory signal
for FNR- and Fur-regulated expression. Rather,
it appears that, in contrast to Fur-regulated flu
expression, FNR-dependent expression of frd is
related to O 2 supply but not to Fe supply.
4.3. Effect of ferrozine on the expression of FNR
and Fur-regulated genes
To quantify the effect of iron limitation on Fur
and FNR-dependent expression, E. coli strains
carrying a fusion of lacZ with Fur (fhuF, fepA
and flu) and FNR-regulated frd genes were
Table 1
Iron content, oxygen supply and expression of flu and frd. E.
coli strains JB1698 (fiu::2tplacMu) and MC4100(,~J100)
(frd'-'lacZ) were grown in M9 medium with glucose plus
fumarate
Growth
Fe (total
(/xmolg
protein)
/3-Galactosidase ( U / g )
t JB1698
(flu :: )t
MC4100
( I J100)
(placMu)
(frd'-'lacZ)
Anaerobic
M9
3.5
M9, Chelex treated 1.5
50
310
540
370
Aerobic
M9
3.3
M9, Chelex treated 1.6
60
300
40
45
W h e r e indicated the M9 m e d i u m was depleted of iron by
treatment with Chelex 100 before growth. At .457s = 0.6-0.8
samples were taken and total iron and /3-galactosidase were
determined.
322
Table 2
Expression of Fur (fhuF,
ferrozine (0 to 800 ~ M )
fepA, flu)
and F N R
(frd)
Strain
(relevant genotype)
Regulator
H1858 (fhuF :: M u d l X )
JB1686 ( fepA :: AplacMu)
JB1698 (flu :: AplacMu)
MC (A J100) (frd'-'lacZ)
Fur
Fur
Fur
FNR
d e p e n d e n t genes in
Kso
Kso
grown in M9 medium in the presence of
fl-Galactosidase ( U / g )
(jzM Ferro)
10
16
68
160
E. coli strains
M9
M9 + Ferro
(800 p~M)
800
165
82
800
5010
2040
560
170
gives the concentration of ferrozine in the medium required to achieve 50% of the maximal derepression (fhuF,
inhibition of expression (frd). T h e maximal and minimal values of fl-galactosidase are shown. Ferro = ferrozine.
grown anaerobically in M9 medium containing
ferrozine (0 to 800 /~M) and expression of the
genes was determined. Ferrozine is a chelator of
Fe 2+ which efficiently competes with the bacteria
for the Fe z+ present in the medium. Due to the
presence of two sulfonate residues the molecule
should not be able to penetrate the bacterial cell.
With increasing amounts of ferrozine in the
medium the expression of fhuF, fepA and flu
increased, whereas that of frd decreased logarythmically. The inverse mode of action is due to
the fact that Fe z+ acts as a co-repressor of Fur,
but as a co-activator of FNR. The concentrations
of ferrozine required for 50% derepression (fhuF,
fepA, flu) and 50% inactivation (frd) are given in
Table 2. The concentrations required for the inhibition of frd expression were 2.4-10-fold higher
than those required for flu, fepA and fhuF derepression. This means that for affecting frd expression by iron limitation, the iron deficiency
has to be much stricter than for derepression of
the iron uptake systems. Therefore it is unlikely
that iron availability provides the physiological
signal for a e r o b i c / a n a e r o b i c regulation by FNR.
5. DISCUSSION
In previous experiments it was demonstrated
that F N R can be converted reversibly between
the active and inactive state by supplying or with
fepA, flu)
or
depleting bacteria of iron [8]. It was not clear,
however, whether the supposed reversible binding of Fe 2+ by F N R is part of the normal switch
between aerobic (inactive) and anaerobic (active)
FNR. Here it was shown that the supposed regulation by reversible binding of iron is achieved
only by drastic iron depletion, which is not
achieved normally during a e r o b i c / a n a e r o b i c
shift. Also, the shift from aerobic to anaerobic
respiration does not change the total iron content
of the bacteria significantly, nor does the Feactivated Fur respond to a change in O2-supply.
Thus the shift from aerobic to anaerobic conditions and vice versa does not cause a change in
the iron content of the bacteria. Therefore it is
unlikely that iron concentration (total iron or the
iron pool communicating with Fur) responds
characteristically to aerobiosis or anaerobiosis and
provides the regulatory signal for FNR. It can not
be excluded, however, that other regulatory iron
pools are present which are not recognized by
Fur and respond to other parameters. Thus, it is
possible that there is a specific iron-donating/
-abstracting system which could be regulated by
O 2 and could interact specifically with FNR.
In some metalloregulated bacterial gene regulators the metal iron is bound reversibly and the
reversible binding provides the regulatory signal
(for a review see [1]). For example with the Furprotein, binding of the co-repressor (Fe z+) converts Fur from the apo-repressor to the func-
323
tional repressor. The MerR protein (regulator of
mercury resistance) switches from the repressor
state to a transcriptional activator by binding of
Hg 2+. In the case of FNR, the cofactor appears
to act in a different mode, although experimentally reversible activation and inactivation by iron
supply can be achieved. It appears more likely
that iron acts as a redox-sensitive cofactor of
FNR, as suggested earlier [7]. According to this
assumption, oxygen could oxidize Fe 2+, or an
iron component, in FNR directly or indirectly,
thereby converting FNR to the inactive state.
Again, it is not known whether aerobiosis or
anaerobiosis interact with FNR via a changed
Fe2+/Fe 3+ ratio of cellular iron. It has not been
possible so far to determine the ratio of a regulatory Fe2+/Fe 3+ pool within the bacteria. Only
for two major components of iron metabolism in
E. coli, which presumably have no regulatory
function, the ratio Fe2+/Fe 3+ can be determined
[19]. A better knowledge of the iron metabolism
is therefore required for the understanding of
anaerobic regulation.
ACKNOWLEDGEMENTS
This work was supported by grants of Deutsche
Forschungsgemeinschaft and of the Fonds der
Chemischen Industrie to G.U.
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