Department of Microbiology and Molecular
Genetics, Harvard Medical School, Boston, Massachusetts 02115
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TEXT |
During anaerobic sugar utilization,
Escherichia coli carries out mixed-acid fermentation
(33). The products include ethanol (7, 33), which
is formed from acetyl coenzyme A by two consecutive NADH-dependent
reductions. These are catalyzed by the adhE-encoded multifunctional protein which is also a deactivase of pyruvate formate-lyase (9, 13, 16, 20, 29, 30). The adhE
gene is under complex transcriptional, as well as translational,
control. The anaerobic expressions of adhE-lacZ fusions are
about 10-fold higher than the aerobic expressions (5, 19).
It has been suggested that the gene expression responds to a signal
such as the NAD/NADH ratio (5, 19, 23). Recently, the
cra (catabolite repressor-activator at min 2) gene product,
a global transcription regulator of carbon flow (29), was
found to repress adhE expression in the absence of the
effector (24). In addition, translation of the
adhE mRNA depends on cleavage by RNase III, for an unknown physiological reason (1). Here we report another global
regulator that controls adhE transcription.
Fis is essential for anaerobic expression of adhE.
In a
search for trans-acting regulators responsible for the redox
control of adhE expression, we found by chance that strain ECL4001 (Table 1), bearing a
fis::kan mutation (min 73.4), lost the
ability to grow anaerobically on glucose but not on glucuronate, a
phenotype expected of adhE-defective mutants (6).
Fis (factor for inversion stimulation) is known to play a global role
in gene expression (11, 15, 17). Mutations in
adhR (min 72) were also reported to prevent adhE
expression (6). However, the DNA sequence of fis
in an adhR mutant allele was found to be normal (data not
shown).
A comparison of the activity levels of ethanol oxidoreductase and
-galactosidase in strain ECL4000 [adhE+
(adhE-lacZ) fis+] and its
isogenic derivative ECL4001 [adhE+
(adhE-lacZ) fis::kan]
showed that anaerobic growth failed to raise the activity levels of
both enzymes in the fis::kan mutant (Table 2). It therefore appears that Fis
is required for the anaerobic response of adhE expression.
Fis dependence of adhE expression in the presence or
absence of Cra.
In view of the repression of adhE
expression by Cra (24), it is possible that in the absence
of this global regulator, Fis is no longer required for adhE
transcription. We therefore introduced a
cra::Tn10 allele into strains ECL4000
and ECL4001. The resulting strains, ECL4003
[
att::(adhE-lacZ)
fis+ cra::Tn10] and
ECL4004 [att::(adhE-lacZ)
cra::Tn10
fis::kan], were grown aerobically or
anaerobically, and their levels of -galactosidase activity were
assayed. In agreement with a previous study (24), expression
of (adhE-lacZ) in strain ECL4003 lacking Cra was elevated 2 to 3-fold aerobically and 1.5-fold anaerobically (Fig.
1). The double mutant ECL4004, lacking
both Cra and Fis, expressed (adhE-lacZ) at very low
levels either aerobically or anaerobically. Thus, Fis is required for
adhE expression independently of Cra.
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FIG. 1.
-Galactosidase activity levels of aerobically or
anaerobically grown cells of different merodiploid strains
[adhE+ (adhE-lacZ)]. Strains
ECL4000 (fis+ cra+), ECL4003
(fis+ cra::Tn10), ECL4001
(fis::kan cra+), and
ECL4004 (fis::kan
cra::Tn10) were grown at 37°C in
Luria-Bertani medium containing glucose. -Galactosidase activity was
assayed in exponentially growing cultures as described before
(25). Bars indicate the standard deviation of four
independent experiments.
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Expression of (fis-lacZ) transcriptional and
translational fusions under aerobic and anaerobic conditions.
The
cellular levels of Fis are known to vary greatly with growth phase and
environmental conditions (3). However, it is not known
whether the level of Fis depends on the cellular redox state and
whether the low aerobic expression of adhE is due to Fis
limitation. To address these questions, we analyzed strains RJ1835 and
RJ1839 bearing, respectively, a transcriptional and a translational
(fis-lacZ) fusion (3). The expressions of both
fusions were similar in aerobically and anaerobically grown cells (Fig.
2). These results suggest that an
element(s) other than Fis is responsible for the redox control of
adhE expression.
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FIG. 2.
Aerobic and anaerobic expression of a
(fis-lacZ) fusion. Strains RJ1835
[(fis-lacZ) operon fusion] and RJ1839
[(fis-lacZ) protein fusion] were grown aerobically or
anaerobically at 37°C in Luria-Bertani medium containing glucose.
Cell samples were taken at exponential growth phase, and
-galactosidase activity was determined. Bars indicate the standard
deviation of four different experiments.
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Expression of adhE under the control of an
IPTG-inducible promoter.
To test whether Fis specifically acts on
the adhE promoter, we cloned the adhE gene into
plasmid pSE280 (Invitrogen) under the control of the trc
promoter (isopropyl--D-thiogalactopyranoside [IPTG]
inducible). When this plasmid (pADH11) was transformed into strain
ECL4001 (fis::kan), normal anaerobic
growth on glucose occurred only in the presence of IPTG (200 µM).
When assayed for ethanol oxidoreductase activity, anaerobically grown
cells showed fourfold induction of AdhE (Table 2). Thus, the growth
failure of strain ECL4001 (fis::kan)
solely reflects inadequate AdhE activity.
Physical evidence of interaction of Fis with the adhE
promoter.
The adhE promoter region (Fig.
3) contains three perfect matches to the
Fis consensus sequence (35), resembling other promoters known to be under Fis control (27, 28). When a 388-bp DNA fragment (extending from position 
198 to position +172) was used in
an electrophoretic mobility shift assay, the presence of Fis retarded
the migration of the DNA (Fig. 4). The
first detectable complex was formed at a Fis concentration that is
expected for an apparent Kd of 3 × 10
9, which is typical of other established Fis regulatory
binding sites (35). As the Fis concentration was increased,
two additional complexes with lower migration rates were formed. Such a
phenomenon was also previously observed, but its physiological
significance is unknown (34, 35).
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FIG. 3.
The promoter region of adhE. The locations of
the transcriptional start sites are shown in parentheses
(24). Putative Fis consensus sequences I, II, and III are
indicated by thick lines above the sequences. Primers JMH1 and JMH2
(used for Fig. 4) are denoted by arrows. The proposed Cra binding box
is underlined, and the putative 10 box is shown by a square. The
translational start site of adhE is indicated by an open
arrow.
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FIG. 4.
Electrophoretic mobility shift of adhE
promoter DNA by Fis. The 388-bp fragment containing the adhE
promoter region (from position 198 to position +172; Fig. 3) was
32P end labeled. The DNA fragment was gel purified and then
incubated with various amounts of Fis protein (a generous gift from
Reid Johnson) in a 20-µl reaction mixture consisting of 20 mM
Tris-HCl (pH 7.5), 80 mM NaCl, 1 mM EDTA, and 2 µg of sonicated
herring sperm DNA per ml. The binding reaction was performed at 37°C
for 30 min. At the end of the incubation, 5 µl of gel loading buffer
(20 mM Tris-HCl [pH 7.5], 10 mM EDTA, 80 mM NaCl, 100 µg of
sonicated herring sperm DNA per ml, 7.5% Ficoll, 0.1% bromophenol
blue) was added to each mixture. Samples of 20 µl were then subjected
to electrophoresis in a 1× Tris-borate-EDTA-5% polyacrylamide gel
(21, 35). Free DNA and Fis-bound DNA complexes after
autoradiography are indicated. Lanes 1 through 4 contained reaction
mixtures containing, respectively, 0, 0.25, 0.5, and 1 ng of Fis.
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Role of Fis in adhE gene expression.
Fis is known
to be implicated in many different cellular processes (2, 3,
10, 12, 26, 31; for a review, see reference
11), among which is the regulation of gene
expression. It is possible that Fis facilitates adhE
expression by directly interacting with the RNA polymerase, as
previously shown for other Fis-dependent promoters (4, 14).
Alternatively, Fis may act directly on the promoter DNA of
adhE, thereby stabilizing an open complex. Such a mechanism
is consistent with our observation that novobiocin, a specific gyrase
inhibitor, lowered the anaerobic expression of adhE by about
75% at a concentration of 20 µg/ml without appreciable growth
inhibition (data not shown). In any event, it is a mystery why Fis was
recruited evolutionarily for adhE expression, since the
level of the pleiotropic regulatory protein does not seem to vary with
respiratory growth conditions. The activity of Fis as a regulatory
element has generally been associated with additional regulatory
proteins (11, 22), but in the case of adhE
expression, Cra is not the likely partner of Fis.
This is the first report of a gene with an anaerobic function under Fis
control. We have recently isolated suppressor mutants of strains
bearing a fis::kan mutation that
regained the ability to grow anaerobically on glucose as the sole
carbon and energy source. Characterization of these mutants may help to
discover the specific element that actually is responsible for the
redox regulation of adhE.
This work was supported by Public Health Service grants GM40993 and
GM39693. P.D.W. is a postdoctoral D. Collen Fellow of the Belgian
American Educational Foundation.
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