Biosci. Biotechnol. Biochem.

, 72 (11), 3011–3014, 2008

Note
Anaerobic Regulation of Citrate Fermentation by CitAB in Escherichia coli
Kaneyoshi Y AMAMOTO,1;4; y Fumika M ATSUMOTO,1 Taku O SHIMA,2 Nobuyuki FUJITA,3
Naotake O GASAWARA,2 and Akira I SHIHAMA4
1
Department of Advanced Bioscience, Kinki University, Nakamachi, Nara 631-8505, Japan
2
Graduate School of Information Sciences, Nara Institute of Science and Technology,
Ikoma, Nara 630-0101, Japan
3
Genome Analysis Center, National Institute of Technology and Evaluation, Shibuya-ku, Tokyo 151-0066, Japan
4
Department of Frontier Bioscience and Research Center for Micro-Nano Technology, Hosei University,
Koganei, Tokyo 184-8584, Japan

Received May 1, 2008; Accepted July 16, 2008; Online Publication, November 7, 2008
[doi:10.1271/bbb.80301]

In Escherichia coli, CitB, a cognate response regu-
lator of CitA, specifically bound to the promoter regions
for mdh, citA, citC, and exuT. Transcription of these
genes was induced by citrate under anaerobic conditions
in a CitAB-dependent manner. Taking this together, we
conclude that CitAB is the master regulatory system
that activates the set of genes involved in citrate
fermentation in E. coli.
Key words: citrate fermentation; anaerobic regulation;
two-component signal transduction; CitA/
CitB; Escherichia coli
Escherichia coli is a unique species in that it is unable
to utilize citrate as sole carbon source. Some E. coli
strains that possess a plasmid encoding citrate uptake
systems grow on citrate.1,2) Under anaerobic conditions,
such E. coli strains are able to utilize citrate in the
simultaneous presence of an oxidizable co-substrate
such as glucose or glycerol.3) The citrate fermentation
pathway in E. coli is, however, different from those in
S. typhimurium and K. pneumoniae.4) Citrate is taken
into the cells by CitT transpoter, which is usually
involved in the transport of di- and tricarboxylate,5) and
is split by citrate lyase into acetate and oxaloacetate.
The oxaloacetate formed is subsequently converted to
malate, fumarate, and succinate in that order by malate
dehydrogenase, fumarase, and fumarate reductase re-
spectively. The reduction reactions on this pathway
require the oxidation of co-substrates.3)
In K. pneumoniae, the CitA(HK)/CitB(RR) two-
component system induces the expression of citC and
cisS operons under anaerobic conditions in the presence
of citrate and sodium ions.6) These operons are involved
in citrate fermentation.6–8) In the E. coli genome, CitA/
CitB are homolog of K. pneumoniae. To identify the
complete set of genes under the control of CitA/CitB of
E. coli, we employed the newly developed genomic
SELEX system.9) Acetylphosphate is known to be a
donor of phosphate for HK-independent phosphorylation
of RR in vitro.10) To search for the genes under the
control of phosphorylated CitB (pCitB), purified CitB
was mixed in the presence of acetylphosphate with an
E. coli DNA fragment mixture, which was generated by
PCR using the E. coli DNA library as template. The
CitB-DNA complex was purified by Ni-NTA affinity
chromatography in the presence of acetylphosphate.
When the binding of CitB or purification of the CitB-
DNA complex was carried out in the absence of
acetylphosphate, specific SELEX DNA fragments were
not recovered (data not shown). After two cycles of
genomic SELEX under the conditions employed, spe-
cific DNA fragments were recovered that formed
discrete bands on PAGE. The DNA fragments thus
isolated were cloned into pT7Blue (Novagen, Boston,
MA) for sequencing, and the resulting sequences were
mapped in the E. coli W3110 genome.11) A total of 63
independent SELEX fragments were identified. For
detailed analysis of the pCitB-binding sites, we choose
the SELEX sequences, which were detected in at least
two independent clones. These sequences were mapped
on nine different regions (S1 to S9) on the E. coli
genome (Table 1). Six SELEX fragments (S1, S2, S3,
S5, S7, and S9) included spacer regions between two
different genes, while 3 SELEX fragments (S4, S6, and
S8) were located in the coding regions of the genes
(Table 1). To confirm specific binding of pCitB (not
non-phosphorylated CitB) to these sequences, a gel
mobility shift assay was performed using purified CitB
in the presence and the absence of acetylphosphate.
CitB binding was observed for all nine SELEX-
fragments even in the absence of acetylphosphate, but
the affinity of CitB-binding was enhanced two-fold in
the S1, S2 and S3 fragments in the presence of

y
To whom correspondence should be addressed. Fax: +81-42-387-6225; E-mail: kanyamam@hosei.ac.jp
3012 K. YAMAMOTO et al.
Table 1. Detection of CitB-Binding Sites by Genomic SELEX

Binding affinity of CitB (mM)d

No. Binding positiona Detected fragmentsb Positionc

AcP +AcP
S1 mdh(<-)-S1-argR(->) 12 3384236–3384307 0.09 0.04
S2 uxaC(<-)-S2-exuT(->) 8 3243452–3243598 0.10 0.04
S3 citC(<-)-S3-citA(->) 4 651209–651437 0.10 0.04
S4 yjiR(<-)-S4 (yjiS)-yjiT(->) 4 4576451–4576653 0.08 0.11
S5 ycjK(<-)-S5-ycjL(->) 3 1362765–1362840 0.11 0.12
S6 nadB(->)-S6 (yfiC)-srmB(->) 2 2710941–2711173 0.42 0.42
S7 dapB(->)-S7-carA(->) 2 29239–29406 0.23 0.18
S8 b2653(<-)-S8 (ygaQ)-b2655(->) 2 2785103–2785235 0.13 0.18
S9 yeiC(<-)-S9-fruA(<-) 2 2262814–2262979 ND ND
a
S-number indicates the location of CitB-binding site isolated by the genomic SELEX. Directions of genes are represented by arrows.
b
The number of isolates by genomic SELEX is indicated.
c
The number of the minimum region of the CitB-binding site on SELEX fragments is represented by reference to E. coli W3110 genome (GenBank no. AC 000091).
d
The binding affinity of CitB to each SELEX fragment was measured by gel motility assay, as previously described.10) In brief, the T7 promoter primer (50 -
TAATACGACTCACTATAGGG-30 ) was phosphorylated with [
-32 P]ATP. All 32 P-labeled DNA probes were generated by PCR amplification of CitB target genes
using a set of primers, 32 P-labeled T7 promoter primer and the U-19 primer (50 -GTTTTCCCAGTCACGACGT-30 ), with pT7Blue cloned SELEX fragment as the
template. Mixtures of 32 P-labeled probe and increasing amounts CitB were incubated at 37  C in the absence and the presence of 10 mM acetylphosphate, and were
then subjected to electrophoresis on native polyacrylamide gel. After PAGE, the amount of unbound free DNA probe was determined by measuring the intensity of
radioactivity with BAS1000 (Fuji Film, Tokyo) and associated with free DNA plotted against the concentration of CitB. The apparent CpxB-binding affinity was
determined as the concentration (Con50% ), which gave 50% binding of the labeled probe.

acetylphosphate (Table 1). The increase in CitB-binding
affinity due to acetylphosphate correlated with the
number of independent isolates (Table 1), as observed
in the case of Cra.9) The putative CitB-binding se-
quences among three SELEX fragments (S1 to S3) were
found to be high in A/T content, as in the case of
K. pneumoniae CitB.12) Taking this together we pre-
dicted that at least three regions exist in the E. coli
genome that specifically bind pCitB, between mdh and
argR (S1), between uxaC and exuT (S2), and between
citC and citA (S3).
To examine experimentally the regulation targets
of CitB in vivo, CitB was overproduced in wild-
type E. coli harboring CitB-expression plasmid 6–
12
GFP,13) and the level of mRNA from the predicted
six genes were analysed by S1 nuclease mapping.
Overproduction of CitB is known to induce expression
from the citC promoter in vivo.14) A total of 10
promoters were detected by S1 nuclease mapping, two
each for mdh, argR, exuT and citC, and one each for
uxaC and citA (Fig. 1). Two promoters of the argR gene
have been reported,15) but the argRp1 and argRp2
detected in this study were 1-nt upstream and 1-nt
downstream respectively from the sites described in the
literature. All the other eight promoters, citAp, citCp1p2,
exuTp1p2, mdhp1p2, uxaCp, were newly identified in
this study (Fig. 1, panels a, c, d, e, and f). As expected,
the level of transcripts from citC promoters was
increased by CitB-overproduction, as in previous report
(Fig. 1, panel e). In addition of the increase in citC
transcripts, the transcripts from citAp, exuTp1p2, and
mdhp1p2 increased when CitB was overproduced
(Fig. 1, panels a, d, and f).
Under the aerobic condition (dissolved oxygen
5.5 mg/l at exponential growth phase), no transcription
from five promoters, exuTp1p2, citCp1p2, and citAp was
detected with and without 50 mM citrate, but under the
anaerobic conditions, citrate stimulated transcription
(dissolved oxygen was not detected at exponential
phase) (Fig. 2, panels b, c, and d). No activation of
these promoters was observed in the citAB null mutant
(Fig. 2, panels b, c, and d), supporting the thesis that
CitAB TCS is involved in this anaerobic, citrate-
dependent induction. mdhp2 was active under the
aerobic condition, and was stimulated 2-fold in the
presence of citrate, but also in the citAB null mutant
(Fig. 2a, lanes 2 to 5). Under anaerobic conditions,
citrate also induced the mdhp2 promoter, but not in the
citAB null mutant (Fig. 2a, lanes 8 and 9). Transcription
from three promoters, argRp1, argRp2 and uxaCp, did
not change in the citAB null mutant under any of the
conditions employed (data not shown). These results
indicate that CitAB activates the promoters for four
genes, citA, citC, exuT, and mdh, in the presence of
citrate and under anaerobic conditions.
Both the cit and mdh operons are involved in citrate
fermentation. The cit operon encodes citrate lyase, the
synthase of the citrate lyase prosthetic group, and citrate
carrier8) while the mdh operon encodes one of two
enzymes, Mdh and Mqo, both being involved in the
conversion of oxaloacetate to malate.16) In good agree-
ment with the citrate fermentation pathway,4) all of these
CitAB regulon genes were anaerobically induced by
citrate (see Fig. 2). CitAB induced mdh expression in
the presence of citrate and under anaerobic conditions,
suggesting that Mdh is the principle enzyme in citrate
fermentation. In addition to the genes on the citrate
fermentation pathway, CitAB induced expression of the
exuTR operon under anaerobic conditions (see Fig. 2).
The exuT gene encodes the transporter of glucuronate
and galacturonate, while exuR encodes the regulator of
the exuTR, uxaAC, and uxaB operons.17) Glucuronate
 Characterization of E. coli CitAB Regulon 3013

a b c d e f
citB citB citB citB citB citB
-+ -+ -+ -+ -+ -+

mdhp2 citC p2
(3384325) argR p2
exuTp2 (651262)
(3384458) (3243658)

citAp
(651209)
uxaCp
(3243499)

exuTp1
argR p1 (3243699)
mdhp1 citC p1
(3384170) (3384531)
(651175)
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

Fig. 1. Characterization of the Promoters Regulated by CitB.

E. coli BW25113 strains carrying vector plasmid pCA24N
GFP (lane 2) and 6–12H
GFP (pCA24N
GFP with the cloned citB) (lane 3)
were grown at 37  C in LB medium. At exponential growth phase, total RNAs were prepared and subjected to S1 mapping, as previously
described.18) The 32 P-end-labeled promoter fragments were amplified by PCR using E. coli W3110 genomic DNA. The pair of primers,
32
P-labeled primer and unlabeled reverse primer, were as follows: 32 P-labeled PSL1R (50 -TAACAGTAGTGCAAGCGCCTGGCCAATACC-30 )
and PSL1F (50 -TTAAGTAATGCTTTAAATGCTTTAACTAGT-30 ) for the mdh promoter (a), 32 P-labeled PSL1F and PSL1R for the argR
promoter (b), 32 P-labeled PSL2R (50 -CTCAAGAATGTGTAGTCACGCAAGTTTAGC-30 ) and PSL2-2F (50 -AAACCGGCACGCCCCGAA-
CGTTGCCATGAA-30 ) for the uxaC promoter (c), 32 P-labeled PSL2-2F and PSL2-2R for the exuT promoter (d), 32 P-labeled PSL4R (50 -
ATGTTGTTCAATGTTGCAAACTGATAACCT-30 ) and PSL4-2F (50 - AACCTGCGAGTCATTTCGTAATTACATTAA-30 ) for the citC
promoter (e), and 32 P-labeled PSL4-2F and PSL4R for the citA promoter (f). The AG ladders of the Maxam-Gilbert sequencing reaction are
shown in lane 1. Arrows indicate specific transcripts, while numbers indicate the transcription start sites along the E. coli W3110 genome
(GenBank no. AC 000091).

a mdh b exuT c citC d citA

+ - + - + - + - Aeration
- + - + - + - + - + - + - + - + Citrate
Wild type
Wild type
Wild type

Wild type
Wild type

Wild type

Wild type

Wild type
Wild type

Wild type

Wild type

Wild type

Wild type
Wild type

Wild type
Wild type

∆cit
∆cit
∆cit

∆cit

∆cit
∆cit

∆cit

∆cit
∆cit
∆cit

∆cit

∆cit
∆cit
∆cit

∆cit
∆cit

p2 p2
p2

p1

p1
p1

123 4 5 67 8 9 123 4 5 67 8 9 123 4 5 67 8 9 123 4 5 67 8 9

Fig. 2. The Induction of CitAB Regulon by Citrate under Anaerobic Conditions.

Wild-type E. coli BW25113 (lanes 2, 4, 6, and 8) and BW27876 (citAB) (lanes 3, 5, 7, and 9) were grown at 37  C in LB medium with (lanes
4, 5, 8, and 9) and without (lanes 2, 3, 6, and 7) citrate (50 mM) and under aerobic (lanes 2 to 5) and anaerobic (lanes 6 to 9) conditions. Total
RNAs were prepared from exponential phase cultures and subjected to S1 mapping, as described in Fig. 1. Arrows indicate transcription start
sites.

and galacturonate are then dissimilated by UxaABC Acknowledgment

galacturonate catabolic enzymes to 2-keto-3-deoxyglu-
conate, which is then utilized, after phosphorylation, in
TCA cycle and glycolysis. In E. coli, citrate fermenta-
tion requires the presence of an oxidizable cosubstrate
such as sugar.3) Hence, it is possible that CitAB also
controls the hexuronate dissimilation pathway, provid-
ing oxidizable cosubstrates to be enrolled in citrate
fermentation.
We thank K. Hirao, T. Hirano, and A. Toriyama
(Kinki University) for technical assistance, and J. L.
Hobman (Nottingham University) for critical reading of
the manuscript and useful suggestions. This work was
supported by Grants-in-Aid (18770157, to KY) from the
Ministry of Education, Culture, Sports, Science, and
Technology of Japan.
3014 K. YAMAMOTO et al.
References
1) Ishiguro, N., and Sato, G., Nucleotide sequence of the
gene determining plasmid-mediated citrate utilization.
J. Bacteriol., 164, 977–982 (1985).
2) Sasatsu, M., Misra, T. K., Chu, L., Laddaga, R., and
Silver, S., Cloning and DNA sequence of a plasmid-
determined citrate utilization system in Escherichia coli.
J. Bacteriol., 164, 983–993 (1985).
3) Lutgens, M., and Gottschalk, G., Why a co-substrate is
required for anaerobic growth of Escherichia coli on
citrate. J. Gen. Microbiol., 119, 63–70 (1980).
4) Bott, M., Anaerobic citrate metabolism and its regulation
in enterobacteria. Arch. Microbiol., 167, 78–88 (1997).
5) Pos, K. M., Dimroth, P., and Bott, M., The Escherichia
coli citrate carrier CitT: a member of a novel eubacterial
transporter family related to the 2-oxoglutarate/malate
translocator from spinach chloroplasts. J. Bacteriol.,
180, 4160–4165 (1998).
6) Bott, M., Meyer, M., and Dimroth, P., Regulation of
anaerobic citrate metabolism in Klebsiella pneumoniae.
Mol. Microbiol., 18, 533–546 (1995).
7) Bott, M., and Dimroth, P., Klebsiella pneumoniae genes
for citrate lyase and citrate lyase ligase: localization,
sequencing, and expression. Mol. Microbiol., 14, 347–
356 (1994).
8) Schneider, K., Kastner, C. N., Meyer, M., Wessel, M.,
Dimroth, P., and Bott, M., Identification of a gene cluster
in Klebsiella pneumoniae which includes citX, a gene
required for biosynthesis of the citrate lyase prosthetic
group. J. Bacteriol., 184, 2439–2446 (2002).
9) Shimada, T., Fujita, N., Maeda, M., and Ishihama, A.,
Systematic search for the Cra-binding promoters using
genomic SELEX system. Genes Cells, 10, 907–918
(2005).
10) Yamamoto, K., and Ishihama, A., Characterization of
copper-inducible promoters regulated by CpxA/CpxR in
Escherichia coli. Biosci. Biotechnol. Biochem., 70,
1688–1695 (2006).
11) Hayashi, K., Morooka, N., Yamamoto, Y., Choi, S.,
Ohtsubo, E., Baba, T., Wanner, B. L., Mori, H., and
Horiuchi, T., Highly accurate genome sequences of the
Escherichia coli K-12 strains MG1655 and W3110. Mol.
Syst. Biol., doi:10.1038/msb4100049 (2006).
12) Meyer, M., Dimroth, P., and Bott, M., In vitro binding of
the response regulator CitB and of its carboxy-terminal
domain to A + T-rich DNA target sequences in the
control region of the divergent citC and citS operons of
Klebsiella pneumoniae. J. Mol. Biol., 269, 719–731
(1997).
13) Mori, H., Isono, K., Horiuchi, T., and Miki, T., Func-
tional genomics of Escherichia coli in Japan. Res.
Microbiol., 151, 121–128 (2000).
14) Ingmer, H., Miller, C. A., and Cohen, S. N., Destabilized
inheritance of pSC101 and other Escherichia coli
plasmids by DpiA, a novel two-component system
regulator. Mol. Microbiol., 29, 49–59 (1998).
15) Lim, D. B., Oppenheim, J. D., Eckhardt, T., and Maas,
W. K., Nucleotide sequence of the argR gene of
Escherichia coli K-12 and isolation of its product, the
arginine repressor. Proc. Natl. Acad. Sci. USA, 84, 6697–
6701 (1987).
16) van der Rest, M. E., Frank, C., and Molenaar, D.,
Functions of the membrane-associated and cytoplasmic
malate dehydrogenases in the citric acid cycle of
Escherichia coli. J. Bacteriol., 182, 6892–6899 (2000).
17) Rodionov, D. A., Mironov, A. A., Rakhmaninova, A. B.,
and Gelfand, M. S., Transcriptional regulation of trans-
port and utilization systems for hexuronides, hexuronates
and hexonates in gamma purple bacteria. Mol. Micro-
biol., 38, 673–683 (2000).
18) Yamamoto, K., and Ishihama, A., Transcriptional re-
sponse of Escherichia coli to external copper. Mol.
Microbiol., 56, 215–227 (2005).