Institut für Mikrobiologie und
Weinforschung, Johannes Gutenberg- Universität Mainz, 55099 Mainz, Germany
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INTRODUCTION |
In Escherichia coli
the switch from aerobic to anaerobic metabolism is regulated at
the transcriptional level in response to the presence of the electron
acceptors O2, nitrate, and fumarate (8, 9, 11,
25, 27, 28). This regulation ensures that in the presence of
oxygen only aerobic metabolism and not anaerobic respiration or
fermentation is functional. Under anoxic conditions, nitrate (and
nitrite) represses the synthesis of the enzymes associated with
fumarate respiration. The sensor-regulator systems controlling gene
expression in response to O2 and nitrate are known and have
been studied in detail. Regulation by O2 is effected
by the two-component regulatory system ArcB/A (aerobic respiratory control) and by the cytoplasmic one-component
regulator FNR (fumarate-nitrate reductase regulator) (8, 11,
27). Nitrate and nitrite regulate via two
homologous two-component regulatory systems, NarX/L and
NarP/Q (Nar is an acronym for nitrate reductase) (25).
Fumarate is also an important electron acceptor for respiration, and
fumarate and related C4-dicarboxylates are known to induce a variety of genes required for anaerobic fumarate metabolism, such as
the structural genes for fumarate reductase (frdABCD) (8, 12), the proton-pumping NADH dehydrogenase I
(nuoA to -N) (3, 26, 28), and
dicarboxylate carriers (dcu genes) (7, 24, 29).
In aerobic growth, synthesis of succinate dehydrogenase
(sdhCDAB) is stimulated by the same substrates
(18). Therefore, there is a large group of genes which
should be transcriptionally regulated by fumarate or other
C4-dicarboxylates. For Rhizobium leguminosarum and Rhodobacter capsulatus,
the two-component sensor-regulators, DctSR and DctBD, which
control gene expression in response to C4-dicarboxylates are known (10, 21). In the
present study a two-component regulatory system was identified in
E. coli. It is responsible for regulation of the genes of
fumarate respiration, including fumarate reductase and a fumarate
carrier (DcuB), in response to the presence of
C4-dicarboxylates.
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MATERIALS AND METHODS |
Bacterial strains and growth.
For genetic experiments the
bacteria (Table 1) were grown aerobically
in Luria Bertani broth (22). For expression studies the
bacteria were grown in M9 mineral medium (15) supplemented with acid-hydrolyzed casein (1 g/liter) (26). Anaerobic
growth was performed in gastight stoppered tubes under an atmosphere of
N2 (3). Aerobic growth was performed in flasks
filled to 5% of the maximal volume with vigorous shaking. For
anaerobic growth the carbon sources were added at 20 mM, and for
aerobic growth the carbon sources were added at 10 mM. Cell densities were measured as the absorbance at 578 nm. Cells were harvested at an
A578 of 0.5 to 0.7. 
-Galactosidase assays
were performed according to Miller (15).
Inactivation of dcuR (yjdG),
dcuS (yjdH), and citB.
The genes
were inactivated by replacing their central portions with resistance
cassettes. The flanking regions upstream and downstream of the genes
were amplified by PCR. The downstream region of dcuR was
amplified with primers yjdG-Hin (5'-TGA CAT CAA GAC CGC CCG AAG CTT
GCA AGG-3') and yjdG-Eco (5'-GCG TCC AGT TTA CCG TTA CCG AAT TCA
GGC-3'), generating a 848-bp fragment with flanking
HindIII and EcoRI sites. The upstream region
of dcuR was amplified with primers yjdG-Pst (5'-TGT TCG
TTG GAG CTG CAG CCG TGG ATT AGC-3') and yjdH-Xba (5'-CAG TGA AAG
CCA GCT TCT AGA CAG CGG CAG-3'), producing a 815-bp fragment with
flanking PstI and XbaI sites. The flanking region
upstream of dcuS was amplified with primer YjdH-Eco (5'-CTC
TCT GCG AAT TCT TTG TGC ATC-3'), introducing an EcoRI site,
and primer YjdH-Bam-2 (5'-CTT CAG GAT CCG AGT AGC GAA GAC-3'),
introducing a BamHI site, generating a 1,091-bp
fragment. The downstream flanking region of dcuS was amplified with primer YjdH-Xba (5'-TGA GCG CCT CTA GAA AGC GGG AAG-3'), with a XbaI site, and primer YjdH-Bam-1 (5'-GGC GTT
ATC ATC GGA TCC ATT TC-3'), with another BamHI site,
generating a 1,020-bp fragment. The upstream region of citB
was amplified with primers cri-Sac (5'-AAG ATG CTG GGG CTG AGC TCC-3')
and cri-Bam (5'-ATT CCG CAT GGA TCC CTG CC-3'), generating a 929-bp
fragment with SacI and BamHI cloning sites. The
downstream region of citB was amplified with primers
cri-Hind (5'-ATG TTT AAA GCT TAT GCT CGC G-3') and cri-Cla (5' GAT CAT
CGG TGT ATC GAT TTT TG-3'), producing a 918-bp fragment with
HindIII and ClaI cloning sites. For each
gene, the flanking regions were cloned into pKS
(Stratagene). For the dcuR and citB genes the
Kanr and Spcr resistance cassettes derived from
pGS607 and pGS606, respectively (24), were cloned into the
EcoRI-PstI and the BamHI sites,
respectively, resulting in
dcuR::Kanr (pMW75) and
citB::Spcr (pMW92). For
dcuS, the flanking regions were separated by a single BamHI site (pMW107). A Camr resistance cassette
was amplified from pACYC184 (6) by PCR with primers CAMLIB2
(5'-CAA TAA CTG GAT CCA AAA AAT TAC GC-3') and CAMREB (5'-ATA TCC TGG
ATC CCA TAT TCT GC-3'), both introducing a BamHI site. The
resistance cassette was then cloned into the BamHI site of
pMW107, resulting in pMW108
(dcuS::Camr). Any possible terminating
sequences downstream of the Camr resistance cassette were
removed to enable transcription of dcuR located downstream
of dcuS. The plasmids were transformed into E. coli JC7623 and were used for replacement of the intact genes by
homologous recombination (16). Presence of the
dcuR::Kanr,
dcuS::Camr, and
citB::Spcr alleles was confirmed by
PCR of the genomic DNA with the corresponding primers, yielding
fragments corresponding to the sizes of the inactivated genes. The
inactivated genes were transferred to strains with suitable genetic
backgrounds by P1 transduction (15).
Construction of protein fusions.
For creating protein
fusions (dcuB'-'lacZ, dcuC'-'lacZ, and
dctA'-'lacZ) plasmid pJL28 or its derivative pJL29 was used
(13). The dcuB'-'lacZ fusion was obtained by
cloning the 0.65-kb PCR fragment generated with primer dcuB-Bam (5'-AAG
TTG GAT CCT AAA TAA CAT GTG TGA ACC-3') and primer yjdG-Eco into
the BamHI and EcoRI sites of pJL29, yielding
pMW99. For the dcuC'-'lacZ fusion (pMW98), the
dcuB promoter region was amplified with primers dcuC-Bam (5'-CCC CAA TAA GGA TCC CAA TG-3') and dcuC-Eco (5'-CCA GCG GTG AAT TCC
AGA CC-3'), and the 1.1-kb fragment was cloned into the BamHI and EcoRI sites of pJL29. The
dctA'-'lacZ fusion (pMW103) was obtained by cloning the
0.5-kb PCR fragment generated with primers dctA-Bam (5'-CAG AGA GGG ATC
CAT AGG GTG TCC-3') and dctA-Eco (5'-CGC TGG ATG AAT TCG GCA TGG G-3')
into the respective restriction sites of pJL28. The dcuB'-
and dcuC'-'lacZ fusions were transferred to the chromosome
with phage 
RZ5 (12, 17), and monolysogens were identified
and used for further work (3).
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RESULTS |
Fumarate induction of dcuB and frdA
depends on the dcuSR regulatory genes.
In a search for
potential fumarate-responsive regulators, the E. coli
data base was screened for gene products similar to the
sensor-regulators DctRS and DctBD of R. capsulatus and
Rhizobium leguminosarum, which stimulate the synthesis of
the C4-dicarboxylate carriers in response to
C4-dicarboxylates (10, 21). Both systems showed
only low levels of similarity to two-component regulators of
E. coli (<28% sequence identity). The genes for two
of these systems, yjdHG and citAB, were
located next to genes involved in anaerobic fumarate metabolism
(Fig. 1). The yjdHG genes
are in the dcuB fumB to lysU intergenic
region at 93.7 min on the E. coli map (2).
The dcuB fumB genes encode the anaerobically expressed
fumarate carrier (dcuB) and fumarase (fumB)
(1, 24). The citA citB (formerly criR)
genes on the other hand are positioned at 14.1 min on the
E. coli map between genes encoding an alternative fumarate carrier (dcuC) and the citC to
citT gene cluster for anaerobic citrate metabolism (2,
20, 29). The citAB genes encode proteins homologous to
the citrate sensor-regulators from Klebsiella pneumoniae
(4, 5, 20). Anaerobic citrate metabolism of E. coli is related to C4-dicarboxylate metabolism due to
the production and excretion of succinate (5, 14).
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FIG. 1.
Map positions and arrangement of the dcuSR
(previously yjdHG) (A) and citAB (B) genes on
the E. coli genome. The scale gives the DNA length in
kilobases. The positions of the dcuSR and the
citAB genes on the calculated E. coli map
are shown. Data are from reference 2 and the
E. coli data bank.
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The genes for the putative response regulator yjdG and
the sensor kinase yjdH were genetically inactivated by
replacement with genes carrying resistance cassettes. The mutant
strains were tested for fumarate regulation of the dcuB and
frdA genes (Table 2).
Expression of the genes was determined with lacZ
fusions, and growth was performed under anaerobic
conditions on glucose or glycerol plus dimethyl sulfoxide
(DMSO). DMSO has to be included as an electron acceptor for
growth on glycerol, which cannot be fermented by E. coli. In the wild type, the expression of dcuB was
stimulated 5.6-fold or 10.9-fold after growth on glucose or glycerol,
respectively, when fumarate was present in the medium. When DMSO
was omitted from the glycerol medium, a similar stimulation was found
with the addition of fumarate. The lower expression of dcuB
during growth on glucose could be due to glucose repression. In the
yjdG (dcuR) and yjdH
(dcuS) mutants the expression of dcuB was
decreased to background levels, and the expression was not stimulated
by fumarate (Table 2). Therefore both genes are required for fumarate
stimulation of dcuB expression.
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TABLE 2.
Regulation of dcuB and frdA
expression by fumarate, dcuR (formerly yjdG),
and dcuS (formerly yjdH) under
anaerobic conditionsa
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Expression of frdA is stimulated by the presence of
fumarate in the medium, too, but this stimulation is lower (about
twofold [Table 2]). In the yjdG (dcuR) and
yjdH (dcuS) mutants background expression of
frdA was still high, but the fumarate-dependent stimulation was lost completely.
The citB gene encoding the response regulator of the second
two-component system (CitAB) was inactivated, too. The
inactivation of citB, however, had no effect on
expression and fumarate stimulation of the dcuB and
frdA genes (not shown). Therefore, the
yjdHG genes, but not the citB gene, are
required for fumarate stimulation of dcuB and
frdA expression. For this reason the genes
yjdH and yjdG were termed
dcuS (sensor kinase) and dcuR (response
regulator).
Genes regulated by DcuR: not all
C4-dicarboxylate-regulated genes respond to DcuSR.
Other genes which are transcriptionally regulated by
C4-dicarboxylates were tested in the same way for
dcuR involvement (Table 3).
The genes tested encode the proton-pumping NADH dehydrogenase I
(nuoA to -N genes), an alternative
anaerobic C4-dicarboxylate carrier (dcuC gene),
succinate dehydrogenase (sdhCDAB), and a C4-dicarboxylate carrier for aerobic growth
(dctA). The increase in the expression of the genes
stimulated by fumarate or succinate was between 1.4- (nuoAB'-'lacZ) to 2.8-fold (dctA'-'lacZ) (Table 3). However, the fumarate- or succinate-dependent stimulation of
nuoA, dcuC, and sdhC expression was
not significantly affected in the dcuR mutant. Expression of
the dctA'-'lacZ fusion was decreased in the dcuR
mutant, but the succinate stimulation was retained and the increases
were similar for the wild type (2.8-fold) and the mutant (3.2-fold).
Thus, from the genes tested, only dcuB and
frdA were clearly regulated by DcuR and DcuS. In the
citB mutant neither of the genes was affected in
C4-dicarboxylate-stimulated expression (not shown).
C4-dicarboxylates affecting regulation by DcuR.
The effects of various carboxylates on the expression of
dcuB'-'lacZ were studied by including the respective
substrates in the medium (Table 4).
Growth was performed under anaerobic conditions in the presence of
glycerol plus DMSO, which enables high expression of dcuB
when suitable carboxylates are added (see Tables 2 and 4). Each of the
C4-dicarboxylates fumarate, succinate, malate, tartrate,
aspartate, and maleate caused a strong induction of dcuB
expression compared to growth with glycerol plus DMSO alone. Even with
succinate and maleate, which are not metabolized under the respective
(anaerobic) conditions, the induction amounted to at least 63% of the
maximal induction found with fumarate. Most remarkably, maleate, which
is not even taken up by the anaerobic Dcu carriers (24),
induced the expression of dcuB strongly. For all the
C4-dicarboxylates the stimulation was completely lost in
the dcuR mutant. Therefore neither uptake nor metabolism of the C4-dicarboxylates is required for induction by the
DcuSR system. The results suggest that the
C4-dicarboxylates bind to the sensor at the periplasmic
aspect of the membrane and that the sensor is able to react with each
of the C4-dicarboxylates. Butyrate and acetate, on the
other hand, had no stimulating effect. During anaerobic growth on
glucose, the respective C4-dicarboxylates and aspartate
showed similar stimulating effects, but expression was generally lower,
possibly due to glucose repression (not shown). Expression of
frdA'-'lacZ responded in a similar way to the
C4-dicarboxylates (not shown).
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TABLE 4.
Effectors for dcuR-dependent regulation of
dcuB'-'lacZ expression during anaerobic growth with glycerol
plus DMSO
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| |
DISCUSSION |
Physiology and significance of fumarate regulation in
E. coli.
Transcriptional regulation by fumarate
and other C4-dicarboxylates plays an important
role in E. coli. The DcuSR two-component regulators identified here apparently exert this fumarate regulation for the genes of fumarate respiration, that is, frdABCD
and dcuB. The expression of fumB (encoding
anaerobic fumarase B), which is located downstream of
dcuB and is possibly expressed from the dcuB
promoter (1, 24), could also be subject to DcuR
regulation. Expression of other genes which are transcriptionally
stimulated by C4-dicarboxylates was not (nuoA to
-N, sdhCDAB, and dcuC) or was only
partially (dctA) dependent on DcuR. This indicates that DcuSR is required specifically for the regulation of the anaerobic fumarate respiratory pathway. The C4-dicarboxylate
regulation of other genes apparently is effected by a different system,
and the CitA-CitB two-component regulatory system obviously does not serve this function either, as shown here.
DcuRS as a C4-dicarboxylate-sensing two-component
system.
The dcuSR genes, and the derived DcuS and DcuR
proteins, show the typical properties of two-component regulatory
systems. Both genes overlap by 4 bp, which is a strong indication
for a joint transcription similar to that of the genes of other
two-component regulators, which are mostly organized in one
transcriptional unit. The DcuR protein contains a helix-turn-helix
DNA-binding motif in the C-terminal half and an N-terminal receiver
domain with a conserved aspartate residue (Asp56) as a potential
phosphorylation site.
The DcuS protein contains the elements typical for sensory histidine
kinases, and the arrangement is very similar to that found in the CitA
protein of K. pneumoniae (4, 5) (Fig. 2). The CitA protein consists of an
N-terminal sensory domain with two transmembrane helices which are
separated by a long periplasmic domain of about 130 amino acids
(5). The kinase domain is separated from the sensor domain
by an extra domain of about 80 amino acids. The similarity of DcuS to
CitA extends over the complete range, including the periplasmic and the
extra domain. In the kinase domain the H, N, F, and G boxes, which are
designated according to the characteristic amino acid residues
(19), are present in an arrangement very similar to that of
CitA. His349, which is supposed to be the phosphorylation site, is
conserved in the H box.
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FIG. 2.
Overview of the suggested domain structure of the sensor
kinase DcuS. The positions of characteristic sequence features and of
the domains are indicated by the numbers. The transmembrane helices
(TM) were predicted from the sequence. The Q linkers (Q) and the
signature segments of the kinase domain (H, N, G1, F, and G2)
(19) were identified by sequence alignments. For the
segments, the positions of the naming amino acid residue are given
(drawn according to reference 5).
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DcuS has significantly higher levels of similarity with the CitA
citrate sensors of K. pneumoniae and E. coli
than with the C4-dicarboxylate sensors DctB and DctS
of Rhizobium sp. strains and R. capsulatus
(not shown). Both the citrate (CitA) and the C4-dicarboxylate (DcuS, DctB, and DctS) sensors have
similar N-terminal sensory domains consisting of two transmembrane
helices and a long intervening periplasmic domain. The periplasmic
domains of DctB and DctS, however, are about twice the size of the CitA
or DcuS periplasmic domain, which presumably acts in ligand binding (5). Such a location of the sensory domain in the periplasm suggests sensing of the C4-dicarboxylates from without.
This is also supported from the functioning of maleate as a signal
which apparently is not taken up by the bacteria (24). In
agreement with the postulated fumarate sensing by DcuSR from without,
the fumarate carrier (DcuB) shows a strong induction by fumarate (up to
10.9-fold), whereas fumarate reductase shows only a weak induction by
fumarate (up to twofold). These different responses to external fumarate appear to be sensible since DcuB is required in particular when external fumarate is present. FrdA on the other hand is also required when internal fumarate is produced from intermediary metabolism.
This study was supported by grants from the Deutsche
Forschungsgemeinschaft and the Fonds der Chemischen Industrie.
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Abstract
Introduction
