Abstract

Three distinct fumarases, FumA, FumB and FumC, have been reported in Escherichia coli. While the fumA and fumC gene products are expressed under aerobic cell growth conditions, the FumB fumarase appears to be more abundant during anaerobic growth. To study the transcriptional regulation of the fumB gene, a fumB-lacZ operon fusion was constructed and analyzed in a single copy under a variety of cell culture conditions. Expression of fumB-lacZ was fourfold higher under anaerobic than aerobic growth conditions. This anaerobic response is modulated by the ArcA and Fnr proteins, which function independently as anaerobic activators of fumB gene expression. Cellular iron limitation in a fur mutant caused fumB-lacZ expression to decrease sevenfold while cellular heme limitation decreased fumB gene expression twofold. In addition, fumB-lacZ expression was shown to vary depending on the DNA superhelicity. This study further delineates the regulation of the fumB gene in cell growth.

Introduction

Escherichia coli contains three fumarase genes, fumA, fumB, and fumC, that catalyze the interconversion of fumarate and l-malate [1, 2]. The fumA and fumC genes are located at 35.5 min of the E. coli linkage map [3] while the fumB gene is located at 93.5 min [1]. The physiological function of each E. coli fumarase has been studied by using a triple mutant transformed with a plasmid containing one of the three fumarase genes. The FumA enzyme appeared to be a component of the tricarboxylic acid (TCA) cycle since it was synthesized predominantly under aerobic conditions [4]. Expression of the fumA gene was lowest during anaerobic cell growth suggesting a role for FumA as an aerobic fumarase. Anaerobic expression of fumA-lacZ from the fumA promoter was derepressed in both an arcA and an fnr mutant indicating that both ArcA and Fnr function as anaerobic repressors [5]. The FumB enzyme was shown to be more abundant under anaerobic conditions, especially during anaerobic respiratory growth with glycerol plus fumarate [4]. Furthermore, the anaerobic expression of the fumB gene is reduced in an fnr mutant suggesting that Fnr is a transcriptional activator [4]. Transcription of the fumC gene was shown to be complex: it was dependent on both the fumA and fumC promoters [5]. It has been reported that synthesis of FumC was increased by the addition of oxidizing agents, and this increase was assumed to be dependent on the soxRS gene products [6]. Recently, it has been proved that both the superoxide control and the iron control of fumC expression require the SoxR regulatory protein. FumC was thus proposed to substitute for FumA when environmental iron is limiting or when superoxide radicals accumulate [5]. In this study, we further examined the regulation of fumB gene using a fumB-lacZ operon fusion.

Materials and methods

Bacterial strains, bacteriophages, and plasmids

The genotypes of the E. coli K-12 strains, plasmids and the bacteriophage are listed in Table 1. The arcA, fur, topA and fis strains were constructed by introducing the indicated mutation into strain MC4100 λCPT7 (fumB-lacZ) by P1 transduction followed by selection for resistance to the appropriate drug [7]. The PC2 (fnr) λCPT7 lysogen and PC10 (hemA) λCPT7 lysogen were constructed by infecting PC2 and PC10 with a high titer λCPT7 lysate as previously described [8].

1

Escherichia coli strains, phages and plasmids

Strain, phage, plasmid Derivation Genotype or phenotype Reference or source 
Strains    
MC4100  FaraD139 Δ(argF−lac)U169rpsL 150 relA1 flb-5301 deoC1 ptsF25 rbsR [11
PC2 MC4100 fnr [13
C35 MC4100 arcA Kanr [13
PC40 MC4100 hemA41 Kanr [5
SJP 2 MC4100 fur::Tn5 [5
SJP 4 MC4100 fis-767Kanr [5
SJP 6 PC2 fnr arcA Kanr [5
RS 2 MC4100 pyrF287topAgal125rpsλ [16
Phages    
λRZ5   [11
λCPT7 pCPT7 Φ(fumB-lacZ)lacY+lacA+ This study 
M13mp19   [9
M13CPT5 M13mp but with 1.48-kb BglII-PstI fragment This study 
M13CPT6 M13 CPT5 but with an EcoRI site This study 
Plasmids    
pRS1247  lacZ lacY+lacA+ [11
pCPT4 pTZ19 fumB+ This study 
pCPT7 pRS1247 Φ(fumB-lacZ)lacY+lacA+ This study 
Strain, phage, plasmid Derivation Genotype or phenotype Reference or source 
Strains    
MC4100  FaraD139 Δ(argF−lac)U169rpsL 150 relA1 flb-5301 deoC1 ptsF25 rbsR [11
PC2 MC4100 fnr [13
C35 MC4100 arcA Kanr [13
PC40 MC4100 hemA41 Kanr [5
SJP 2 MC4100 fur::Tn5 [5
SJP 4 MC4100 fis-767Kanr [5
SJP 6 PC2 fnr arcA Kanr [5
RS 2 MC4100 pyrF287topAgal125rpsλ [16
Phages    
λRZ5   [11
λCPT7 pCPT7 Φ(fumB-lacZ)lacY+lacA+ This study 
M13mp19   [9
M13CPT5 M13mp but with 1.48-kb BglII-PstI fragment This study 
M13CPT6 M13 CPT5 but with an EcoRI site This study 
Plasmids    
pRS1247  lacZ lacY+lacA+ [11
pCPT4 pTZ19 fumB+ This study 
pCPT7 pRS1247 Φ(fumB-lacZ)lacY+lacA+ This study 

Construction of fumB-lacZ operon fusion

To construct the fumB-lacZ fusion, the 3.16-kb HindIII-HpaI fragment of fumB gene was cloned from the Kohara library into pTZ19 to give plasmid pCPT4. The 1.49-kb BegII-PstI fragment was then inserted into M13 mp19 to give M13CTP5 [9]. By using oligonucleotide-directed mutagenesis, a new EcoRI site was introduced into the fumB gene at position +86 relative to the start of fumB translation to give M13CPT6. A 896-bp BglII-EcoRI fragment containing the 5′ end of the fumB gene and the upstream 810 bp was isolated from M13CPT6 and inserted into the BamHI-EcoRI sites of pRS1247 to generate the fumB-lacZ operon fusion designated pCPT7. The junction between the fumB promoter regions and the lacZ gene was confirmed by double-strand DNA sequencing analysis [10]. The fusion was transferred to RZ5 to generate the corresponding λCPT7, which was then introduced into the MC4100 chromosome as previously described [11]. Single lysogen was identified and purified for subsequent study. The wild-type locus of fumB was stably integrated at the lambda attachment site on the chromosome.

Cell growth

For strain manipulation and maintenance, cells were grown in Luria broth or on solid medium. When required, ampicillin and kanamycin were added to the medium at concentrations of 100 and 50 μg ml−1, respectively. For β-galactosidase assay, cells were grown in glucose (40 mM) minimal medium (pH 7.0) [12], unless otherwise indicated [13]. Aerobic and anaerobic growth were performed as previously described [13]. Flasks or tubes containing medium were inoculated from the overnight cultures grown under the same conditions, and the cells were allowed to double four of five times while in mid- exponential phase prior to harvest for analysis (optical density at 600 nm of 0.4–0.5; Kontron Uvikon 810 spectrophotometer). Trimethylamine-N-oxide (TMAO), sodium nitrate, or fumarate was added at an initial concentration of 40 mM [12]. Where indicated, δ-aminolevulinic acid (δ-ALA), 2,2-dipyridyl or ferrous sulfate was added at final concentrations of 24 μg ml−1 or 150 or 80 μM, respectively.

β-Galactosidase assay

β-Galactosidase assays were performed as previously described [12]. One unit of β-galactosidase is defined as the hydrolysis of 1 nmol o-nitrophenyl-β-d-galactopyranoside (ONPG) per min per mg protein. All values shown represent the average of at least three determinations and did not vary more than 10% from the mean.

Results

Effect of oxygen and other electron acceptors on fumB-lacZ expression

To examine the effect of anaerobic respiratory conditions on fumB expression, an E. coli wild-type strain containing a fumB-lacZ fusion was grown in the presence and absence of the alternative electron acceptors oxygen, nitrate, TMAO, or fumarate (Table 2). Consistent with the earlier observations of Woods and Guest [4], fumB expression was lowest during aerobic cell growth and elevated fourfold when cells were grown anaerobically under fermentative conditions (minimal glucose medium). When cells were grown anaerobically with nitrate present, fumB-lacZ expression was twofold lower than when no electron acceptors were added. Interestingly, fumB expression was highest when fumarate and TMAO were present as electron acceptors during anaerobic growth in a glucose minimal medium. When glycerol was substituted for glucose as the carbon source, conditions in which the cells derive energy from electron transport-linked phosphorylation reactions, fumB-lacZ expression was lowest in the presence of oxygen. It was elevated four- to seven-fold when nitrate, TMAO or fumarate was added in glycerol minimal medium (Table 2). The results of these fumB-lacZ studies suggest that FumB is operative during conditions of anaerobic respiration since fumB expression is elevated during these conditions.

2

Effect of alternative electron acceptors on fumB-lacZ expression

Electron acceptor addeda β-Galactosidase activity (U) 
 Minimal glucose Minimal glycerol 
None 120 NGb 
Oxygen 30 32 
Nitrate 74 140 
TMAO 150 190 
Fumarate 140 230 
Electron acceptor addeda β-Galactosidase activity (U) 
 Minimal glucose Minimal glycerol 
None 120 NGb 
Oxygen 30 32 
Nitrate 74 140 
TMAO 150 190 
Fumarate 140 230 

aCells were grown in 40 mM glucose or glycerol minimal medium either aerobically or anaerobically as described in the text. Sodium nitrate, TMAO, or fumarate was added at an initial concentration of 40 mM.

bNG, no growth.

Effect of the arcA, fnr, topA and fis gene products on fumB-lacZ expression

The aerobic/anaerobic control of the fumA, fumB and fumC genes is mediated by the fnr gene product [4, 5]. Expression of the fumA and fumC genes is also negatively regulated by the ArcA protein [5]. To examine the individual and combined effects of mutations in these two genes, the λCPT7 fusion was introduced into isogenic fnr, arcA, and fnr arcA strains. Single lysogens were identified and purified for subsequent study. A fourfold difference in fumB-lacZ expression occurred in response to oxygen availability in the wild-type parent strain (Table 3). Fnr acts as a positive regulator of fumB-lacZ expression during anaerobic conditions as previously proposed [4]. Expression of fumB-lacZ was decreased by 50% in an fnr mutant. The results showed that ArcA was also a positive regulator of fumB gene expression. The level of fumB-lacZ expression in the arcA deletion strain decreased 30% under anaerobic growth conditions relative to the wild-type strain, whereas aerobic expression was unaffected. In addition, fumB-lacZ expression decreased fourfold in the fnr and arcA double mutant strain anaerobically. Thus, the Fnr and ArcA proteins appear to function independently of each other to regulate fumB gene expression.

3

Effect of arcA, fnr, topA, himA, and fis mutations on fumB-lacZ gene expressiona

Genotype β-Galactosidase activity (U) 
 Aerobic Anaerobic 
Wild-type 30 120 
fnr 30 56 
arcA 28 85 
fnr arcA 18 35 
topA 12 94 
fnr topA 12 46 
arcA topA 12 31 
fnr arcA topA 14 26 
fis 45 160 
Genotype β-Galactosidase activity (U) 
 Aerobic Anaerobic 
Wild-type 30 120 
fnr 30 56 
arcA 28 85 
fnr arcA 18 35 
topA 12 94 
fnr topA 12 46 
arcA topA 12 31 
fnr arcA topA 14 26 
fis 45 160 

aCells were grown in glucose minimal medium under aerobic or anaerobic conditions as described in the text.

There is increasing evidence that DNA supercoiling varies in response to environmental signals such as osmolarity or anaerobic growth [14, 15]. In a topA mutant strain, expression of fumB-lacZ decreased threefold under aerobic conditions and lowered 30% under anaerobic conditions (Table 3). When the topA allele was introduced into arcA and fnr arcA double mutant strains [16], fumB-lacZ expression was lowered about fourfold and fivefold, respectively. These results indicate that a change of DNA superhelicity negatively regulates fumB gene expression. The Fis protein is known to be involved in regulation of a variety of genes in E. coli[17]. Under the conditions tested, a fis mutation activated fumB gene expression about 30% during anaerobic conditions relative to the parent strain (Table 3).

Effect of iron and heme availability on fumB-lacZ expression

Iron is an essential component of the FumA and FumB fumarase activities [18]. Because cellular iron limitation stimulates fumC gene expression [5], the effect of if iron limitation on fumB gene expression was tested. When cells were grown in the presence of the iron chelator 2,2-dipyridyl to limit iron, fumB-lacZ expression was decreased sevenfold during anaerobic growth (Table 4). When iron was added in excess, fumB-lacZ returned to the level seen when no 2,2-dipyridyl was present. Under aerobic conditions, expression of fumB-lacZ was elevated twofold when iron was added in excess to the medium. To test whether a fur mutant which is defective for iron regulation and uptake affects fumB-lacZ expression, a fur allele was introduced into the fumB-lacZ fusion strain [19]. Anaerobic levels of β-galactosidase were reduced to the level seen under aerobic conditions (Table 4). Addition of excess iron to the medium did not restore fumB-lacZ expression. These results indicate that Fur is involved in the observed iron regulation.

4

Effect of iron availability on fumB-lacZ expression

Additiona β-Galactosidase activity (U) 
O2 Dip Fe2+ Wild-type fur 
− − 30 20 
− 15 14 
− 57 22 
50 15 
− − − 120 28 
− − 16 15 
− − 134 30 
− 125 32 
Additiona β-Galactosidase activity (U) 
O2 Dip Fe2+ Wild-type fur 
− − 30 20 
− 15 14 
− 57 22 
50 15 
− − − 120 28 
− − 16 15 
− − 134 30 
− 125 32 

aCells were grown in a minimal glucose medium aerobically or anaerobically as described in the text. Dipyridyl (Dip) and ferrous sulfate (Fe2+) were added at initial concentrations of 150 μM and 80 μM, respectively, as indicated.

Heme is a cofactor of several enzymes needed for energy generation during anaerobic growth [20]. We examined the effects of heme availability on fumB-lacZ expression since it affects fumA and fumC gene expression [5]. In the hemA mutant strain which is defective for heme biosynthesis, fumB-lacZ expression was reduced twofold during anaerobic growth (Table 5). Expression was partially restored to wild-type levels by providing the cells exogenously with δ-ALA, the end product of the reaction catalyzed by the hemA gene product. Whether this regulatory effect is direct or indirect is unclear.

5

Effect of heme availability on fumB-lacZ gene expression

Additiona β-Galactosidase activity (U) 
Oxygen δ-ALA Wild-type hemA 
− 30 22 
28 25 
− − 120 63 
− 110 92 
Additiona β-Galactosidase activity (U) 
Oxygen δ-ALA Wild-type hemA 
− 30 22 
28 25 
− − 120 63 
− 110 92 

aCells were grown in a minimal glucose medium aerobically or anaerobically as described in the text. δ-ALA was added at an initial concentration of 24 μg ml−1.

Discussion

Fumarase participates in the TCA cycle during aerobic growth, and in the reductive pathway from oxaloacetate to succinate during anaerobic growth. It was previously shown that the fumA and fumC genes are most highly expressed during aerobic conditions [5], whereas the fumB gene was expressed at a higher level under anaerobic cell growth conditions [4]. As the other TCA cycle genes are regulated by ArcA or both ArcA and Fnr repressors [5, 19, 21], ArcA and Fnr also function as negative regulators of the fumA and fumC genes while Fnr is an anaerobic activator of the fumB gene [4]. In this study, we further demonstrate that this anaerobic regulation of fumB gene expression is mediated by both ArcA and Fnr proteins that function independently as anaerobic activators of fumB-lacZ expression. It is similar to their effects on the respiratory genes cydAB, frdABCD, dmsABC and narGHJK[22]. The fact that anaerobic cell growth in a glycerol medium with the electron acceptor fumarate present results in a relatively high level of fumB gene expression suggested that FumB contributes to the fermentative and non-cyclic TCA pathway [4].

Iron is an essential component of both the FumA and FumB fumarases, while FumC does not require iron for its activity [23]. However, iron limitation only affects the fumC gene but has no effects on fumA gene expression [5]. In contrast to the observation that fumC gene expression was activated by 2,2-dipyridyl, fumB-lacZ expression decreased when 2,2-dipyridyl was added. This repression can partially be restored by adding excess iron (Table 4). Since iron is required for Fnr activity [24], the decrease of fumB-lacZ expression by the chelator may be due to lowered Fnr activity. FumA and FumB are members of the iron-dependent hydrolases [3, 23]. Earlier studies suggested that the fumA gene is activated by Fur whereas the fumB and fumC genes were not significantly affected by Fur under aerobic conditions [25]. In this study, we found that the expression of the fumB gene was abolished in the fur deletion strain under anaerobic conditions where fumB is more highly expressed. Thus, iron control of fumB expression was fur-dependent. Although the fumA and fumB genes are homologous and their encoded products are structurally similar, FumA has a higher affinity for fumarate than for l-malate whereas FumB exhibits the reverse pattern of affinities for these two compounds [23]. Therefore, a hierarchical control of fumA and fumB genes by ArcA and Fnr during anaerobic growth suggested that the synthesis of the FumB fumarase under anaerobic conditions gives E. coli the flexibility to meet its requirement for diverse environmental conditions.

Acknowledgements

We thank Dr. Robert P. Gunsalus for providing strains for this study. This work was supported by Grant NSC84-2321-B-009-001 from the National Science Council of the Republic of China.

References

1
Guest
J.R.
Miles
J.S.
Robert
R.E.
Woods
S.A.
(
1985
)
The fumarase genes of Escherichia coli: location of the fumB gene and discovery of a new gene (fumC)
.
J. Gen. Microbiol.
 
131
,
2971
2984
.
2
Guest
J.R.
Roberts
R.E.
(
1983
)
Cloning and mapping and expression of the fumarase gene of Escherichia coli K-12
.
J. Bacteriol.
 
153
,
588
596
.
3
Miles
J.S.
Guest
J.R.
(
1984
)
Complete nucleotide sequence of the fumarase gene fumA of Escherichia coli
.
Nucleic Acids Res.
 
12
,
3631
3642
.
4
Woods
S.A.
Guest
J.R.
(
1988
)
Differential roles of the Escherichia coli fumarases and fnr-dependent expression of fumarase B and aspartase
.
FEMS Microbiol. Lett.
 
48
,
219
224
.
5
Park
S.-J.
Gunsalus
R.P.
(
1995
)
Oxygen, iron, carbon, and superoxide control of the fumarase fumA and fumC genes of Escherichia coli: role of the arcA, fnr, and soxR gene products
.
J. Bacteriol.
 
177
,
6255
6262
.
6
Liochevand
S.I.
Fridovich
I.
(
1992
)
Fumarase C, the stable fumarase of Escherichia coli, is controlled by the soxRS regulon
.
Proc. Natl. Acad. Sci. USA
 
89
,
5892
5896
.
7
Miller
J.H.
(
1972
)
Experiments in Molecular Genetics
 .
Cold Spring Harbor Laboratory
,
Cold Spring Harbor, NY
.
8
Silhavy
T.
Besman
M.
Enquist
L.
(
1984
)
Experiments with Gene Fusions
 .
Cold Spring Harbor Laboratory
,
Cold Spring Harbor, NY
.
9
Messing
J.
Vieira
J.
(
1982
)
A new pair of M13 vectors for selecting either DNA strand of double digest restriction fragments
.
Gene
 
19
,
269
276
.
10
Sanger
F.
Nicklen
B.J.
Coulson
A.R.
(
1977
)
DNA sequencing with chain-terminating inhibitors
.
Proc. Natl. Acad. Sci. USA
 
74
,
5463
5467
.
11
Simons
R.W.
Houman
R.
Kleckner
N.
(
1987
)
Improved single and multicopy lac-based cloning vectors for protein and operon fusions
.
Gene
 
53
,
85
96
.
12
Cotter
P.A.
Gunsalus
R.P.
(
1989
)
Oxygen, nitrate, and molybdate regulation of dmsABC gene expression in Escherichia coli
.
J. Bacteriol.
 
171
,
3817
3823
.
13
Cotter
P.A.
Gunsalus
R.P.
(
1992
)
Contribution of the fnr and arcA gene products in coordinate regulation of the cytochrome o and d oxidase (cyoABCDE and cydAB) genes in Escherichia coli
.
FEMS Microbiol. Lett.
 
91
,
31
36
.
14
Dorman
C.J.
Barr
G.C.
Bhriain
N.N.
Higgins
C.F.
(
1988
)
DNA supercoiling and the anaerobic and growth phase regulation of tonB gene expression
.
J. Bacteriol.
 
170
,
2816
2826
.
15
Higgins
C.F.
Dorman
C.J.
Waddell
D.A.
Booth
I.R.
May
G.
Bremer
E.
(
1988
)
A physiological role for DNA supercoiling in the osmotic regulation of gene expression in S. typhimurium and E. coli
.
Cell
 
52
,
569
584
.
16
Miller
W.G.
Simons
R.W.
(
1993
)
Chromosomal supercoiling in Escherichia coli.
Mol. Microbiol.
 
10
,
675
684
.
17
Tsui
P.
Haung
C.
Freudlich
M.
(
1991
)
Integration host factor binds specifically to multiple sites in the ompB promoters of Escherichia coli and inhibits transcription
.
J. Bacteriol.
 
173
,
5800
5807
.
18
Flint
D.H.
Emptage
M.H.
Guest
J.R.
(
1992
)
Fumarase A from Escherichia coli: purification and characterization as an iron-sulfur cluster containing enzyme
.
Biochemistry
 
31
,
10331
10337
.
19
Park
S.-J.
Tseng
C.P.
Gunsalus
R.P.
(
1995
)
Regulation of the Escherichia coli succinate dehydrogenase (sdhCDAB) operon in response to anaerobiosis and medium richness: role of the ArcA and Fnr proteins
.
Mol. Microbiol.
 
15
,
473
482
.
20
Darie
S.
Gunsalus
R.P.
(
1994
)
Effect of heme and oxygen availability on hemA gene expression in Escherichia coli: role of the fnr, arcA, and himA gene products
.
J. Bacteriol.
 
176
,
5270
5276
.
21
Park
S.-J.
Cotter
P.A.
Gunsalus
R.P.
(
1995
)
Regulation of malate dehydrogenase (mdh) gene expression in Escherichia coli in response to oxygen, carbon, and heme availability
.
J. Bacteriol.
 
177
,
6652
6656
.
22
Gunsalus
R.P.
(
1992
)
Control of electron flow in Escherichia coli: coordinated transcription of respiratory pathway genes
.
J. Bacteriol.
 
174
,
7069
7074
.
23
Woods
S.A.
Schwartzbach
S.D.
Guest
J.R.
(
1988
)
Two biochemically distinct classes of fumarases in Escherichia coli
.
Biochim. Biophys. Acta
 
954
,
14
26
.
24
Engel
P.
Trageser
M.
Unden
G.
(
1991
)
Reversible interconversion of the functional state of the gene regulator FNR from Escherichia coli in vivo by O2 and iron availability
.
Arch. Microbiol.
 
156
,
463
470
.
25
Gruer
M.J.
Guest
J.R.
(
1994
)
Two genetically distinct and differentially regulated aconitases (AcnA and AcnB) in Escherichia coli
.
Microbiology
 
140
,
2531
2541
.