Molecular Microbiology (1995) 15(3), 473-482
Regulation of succinate dehydrogenase (sdhCDAB)
operon expression in Escherichia coii in response to
and anaerobiosis: role of ArcA and Fnr
Soon-Jung Park,"*^ Ching-Ping Tseng* and
Robert P. Gunsaius*
Department of Microbiology and Molecular Genetics, and
Mo!ecu!ar Bio!ogy institute. 1602 Mo!ecu!ar Sciences
Building. University of Caiifornia. Los Angeies. Caiifortva
90024. USA.
Summary
Succinate deinydrogenase (SDH) of Escherichia coli,
the soie membrane-bound enzyme of the Iricarboxyiic
acid cycle, participates in the aerobic etectron-
transport pathway to generate energy via oxidative
pbosphorylation reactions. Previous studies have
estabiished that succinate dehydrogenase (SDiH)
syntbesis is elevated by aerobiosis and supressed
during growth with giucose. To examine how the
sdhCDAB genes that encode SDH are regulated by
changes in the environment, sdh~lacZ fusions were
constructed and anaiysed in vivo foiiowing cell
growth under a variety of alternative cuiture con-
ditions. Expression of sdh-lacZ was highest under
aerobic conditions and was decreased 10-foid in the
absence of oxygen. The fnr and arcA gene products
are required for this oxygen controi and each acts to
repress sdhC-lacZ expression. Expression of sdh-
tacZ a\so varied 10- to 14-foid depending on the type
of carbon substrate used or the medium richness.
This control was shown to be independent of the crp
and fruR gene products, and indicates that some
other reguiatory element exists in the ceil to adjust
SDH enzyme ieveis accordingiy. iron and haem avaii-
abiitty affected sdhC-lacZexpression by two- to three-
fold. Lastly, sd/?C-/acZ expression was shown to vary
with the ceii growth rate during aerobic and anaerobic
conditions.
introduction
Succinate dehydrogenase (SDH: E,C, 1,3.99,1) is a
Received 10 December, 1993; revised 3 October, 1994; acceptGd 10
OctobSf, 1994, Present addresses: tDepartment of Biociiemislry,
Stanford University Medical Sciiool, Stanford, California 94305,
USA; :i:institule of Biological Science and Technology, National
Chiao Tung University, Hslnchu, Taiwan. Republic of China, *For
correspondence, Tel, (310) 2068201; Fax (310) 2065231,
membrane-bound enzyme which functions as a member
of the tricarboxylic acid (TCA) cycle in nearly all aerobic
organisms (reviewed by Ackrell et ai, 1992; Hederstecit
and Rutberg, 1981). It catalyses the oxidation ot succlnale
to fumarate and donates electrons to ubiquinone of the
aerobic respiratory chain. The SDH in both prokaryotes
and eukaryotes is similar in its subunit composition and
biochemical properties. The enzyme complex is composed
of four subunits arranged in two domains: the catalytic
domain contains a flavoprotein and a non-haem iron sub-
unit while the hydrophobic domain consists of two small
lipophiiic subunits that anchor the complex to the cell mem-
brane. The catalytic domain contains several cofactors
including one FAD molecule, and nine non-haem-irons
arranged in three iron-sulphur clusters. The hydrophobic
domain has one b-type haem. The SDH enzyme is thought
to be an 'aerobic' enzyme that participates in the TCA cycle.
The genes for SDH, sdbCDAB, map at 16.5 min on the
Esoherichia coli chromosome {Creaghan and Guest,
1972} and have been cloned (Spencer and Guest. 1982)
and sequenced (Darlison and Guest, 1984; Wood et ai,
1984). The sdhCD genes encode the 15 and 13 kDa hydro-
phobic polypeptids subunits whereas the 64 kDa flavo-
protein subunit and the 27 kDa iron-sulphur protein
subunit are encoded by the sdhAB genes, respectively.
The sdtiCDAB genes comprise an operon and are adja-
cent to the gItA gene that encodes citrate synthetase and
the sucABGD genes that encode 7-ketoglutarate dehydro-
genase {sucAB) and succinyl CoA synthetase [sucCD] of
the tricarboxylic acid cycle (Darlison and Guest, 1984;
Wood etai, 1984),
Based on measurement of SDH enzyme activHies in
cells grown under differing conditions, it was demon-
strated a number of years ago that SDH leveis are ele-
vated by aerobic conditions and reduced by anaerobiosis
and/or the presence of giucose (Gray et ai, 1966; Ruiz-
Herrera and Garcia, 1972). More recently, mutations in a
gene called arcA were shown to result in elevated SDH
levefs under anaerobic and, to a limited extent, under
aerobic conditions (luchi and Lin, 1988), This regulatory
gene encodes an anaerobic responsive repressor of
SDH synthesis and of other enzymes of the TCA cycle
(iuchi and Lin, 1988). It also regulates production of addi-
tional enzymes involved in aerobic metabolism including
those for fatty acid degradation, the cytochrome a and d
474 S.-J. Park. C.-P. Tseng and R. P. Gunsalus
oxidases, and superoxide dismutase (luchi and Lin, 1988;
Cotter and Gunsalus, 1992; Fu etai., 1991; Compan and
Touati. 1993).
Transcript analyses of sdhCDAB mRNA using SI
nuclease techniques have suggested that there are two
promoters for controlling expression of the genes (Wilde
and Guest, 1986). One message was reported to iniliate
219bp upstream of the sdhC Iranslational start site (+1)
while lhe second occurred within sdhC a\ position +226,
which is 156 nucleotides before the start of lhe sdhD
gene. Both transcripts were proposed to terminate al the
same location just following sdhB (Wilde and Guest,
1986). The observation that the abundance ot each tran-
script was suppressed by addition of glucose to the cell
culture medium led Wilde and Guest (1986) to propose
four putative CRP-binding sites within the sdhC promoter
region.
To examine how the sd/7CD/\S genes of . co//are regu-
lated in response to anaerobiosis and other conditions of
cell culture including medium richness, several sdhC-
iacZ operon fusions that contained ditferent regions of
upstream DNA were constructed and analysed in vivo.
Transcription of sc//?C-/acZ varied by 10-fold in response
to anaerobiosis. This control was shown to be mediated
by repression by the arcA and fnrgene products. Expres-
sion also varied depending on the type of carbon substrate
used for growth: this control of scy/iC-/acZexpression was
independenl of either lhe crp- or fruR-regulatOiv-gene
products.
Results
Expression of sdh-lacZ fusions containing various
upstream regions of DNA
To determine the region(s) of DNA necessary tor
sdhCDAB expression, four sdh-iacZ fusions were con-
structed that contained different fragments of DNA located
upstream and/or within sdhC (Fig. 1). The fusion located
BomHI Sau3Ai [EcoRI] 3Qu3AI/Br:II
:0) (2541 (6501(6601
I Ji
500 1000 1500 be
PI
r fi Galaotosidase Ac'liuily
t-o.
3 230
3 530
680
14
on SJP58 contains the 555 bp region upstream of sdhC,
the intact sdhCD genes and the 5' 89 bp of sdhA. This frag-
ment was proposed to contain both sdh promoters based
on transcript analysis (Wilde and Guest, 1986). The
sdhC-lacZ fusion in SJP33 contains only the upstream
region and first 226 bp of sdhC (i.e. the proposed minor
promoter, Pi) while the fusion on SJP59 has only the pro-
posed 'P2' or 'major' sdh promoter region (Wilde and
Guest, 1986). The remaining fusion, SJP16, is similar to
SJP33 but lacks the 255 bp sdhC upstream region. Wiid-
type strains containing each phage were grown in a
glucose minimal medium and |H-gatactosidase activities
were determined. Beta-galactosidase expression in the
SJP58 lysogen was 104oid higher during cell growth
under aerobic conditions compared with anaerobic con-
ditions (Fig, 1). Nearly identical values were observed for
the SJP33 lysogen under each condition tested but
virtually no expression was seen for the SJP59 lysogen,
These results suggest tha! there is not an internal pro-
moter within sdhC. Finally, sd/iC-ZacZ expression in the
^SJP16 lysogen, while being oxygen responsive, was
reduced 5-foid under aerobic growth conditions compared
to that of SJP33. Expression of H-galactosidase was a'so
somewhat reduced anaerobically. This indicates that lhe
additional 255 bp region upstream of sdhC present on
SJP33 is somehow necessary for maximal sdhC-lacZ
expression. The scy/7C-/acZ fusion contained on A.SJP33
was used for all further sdhCDAB-geno-expression
studies.
Effect of anaerobic electron acceptors on sdhC-lacZ
expression
In cells grown in glucose minimal medium, sdhC-lacZ
expression was elevated lO-fold by oxygen compared
with anaerobic conditions in which the cell must ferment
to obtain energy (Table 1). However, when the anaerobic
electron acceptors trimethylanine-W-oxide (TMAO) or
fumarate were present during anaerobic conditions,
Fig. 1. Physical map of Ihe sdhCDA gene
region. Locations ol restriction sites are
indicated at the top of the figure anci distances
are given in bp relative to ttie BamH\ site. The
DNA fragments usetl tor construclion ol the
sdhC-tacZ and sdhCDA-lacZ fusions
employed in this study are indicated by
horizontal lines beneath the physical map.
The locations of the two promoters as
0. phage" proposed by Wilde and Guesf (1986). are
indicated by the small arrows and the symbols
330
P, and 'Po'. The level ol sc//i-fac2 expression
seen for each fusion during aerobic and anae-
320 .SJP33
robic cell growth is indicated al Ihe right lor
the wild-type strain. Units of |l-galactosidase
260 ;.sjpi6 activity are in nmoi ol ONPG hydrolyseci per
min per mg proiein. The phages used are
Ii3 ;,S.IP59 described in Table 6.
 Regulafion 0/sdhCDAB by ArcA and Pnr 475

Table 1. Effect ot alternative electron acceptors on . During anaerobic conditions, sdhC-lacZ expression
expression.
was consistently lower by two- to 10-fold on each type of
Beta-galactosidase activity^ medium when compared to aerobic conditions. Interest-
ingly, anaerobic sdhC-lacZ expression in cells grown on
Electron acceptor added'' minimal glucose minimal giyceroi
L-broth, xylose or galactose media was nearly as high as
None 320 NG^ that seen for aerobic growth with glucose.
Oxygen 3530 13900
Nitrate 250 400O
TMAO 710 6540
Fume rate 500 6180 Effect of the arcA and fnr gene products on
sdhC-lacZ expression
a. Strain MC4IO0>,SJP33 was grown in a minimal glucose- or minimal
glycerol-containing medium either aerobicaliy or anaerohically as As a 10-fold difference in scy/7C-/acZ expression occurred
described m ihe texl. Sodium nitrate, TMAO or fumarate was added
at an initial concentration ot 40 mM,
in response to O^ availahiiity, we tested the effect of the
b. Units are given in nmol ot ONPG hydrolysecl per min per mg pro- two known aerohic/anaerobic regulatory proteins, Fnr
tein. and ArcA, in this process. Whereas a deletion of the fnr
c. NG, no growih.
gene did not affect sc/rtC-/acZ expression during aerobic
growth, it resulted in a 5-fold derepression of sdhC-lacZ
expression under anaerobic conditions indicating that
sdhC~lacZ expression was elevated by about twofold
Fnr serves as an anaerobic repressor of sdhC gene
compared to when only glucose or glucose-nitrate
expression (Table 3). A deletion of the arcA gene resuited
medium was used. When cells were grown in a giyceroi
in derepressed sd/7C-/acZ expression during hoth aerobic
medium with any ot these alternative electron acceptors,
(2,5-fold) and anaerobic (70-fold) conditions. Thus, while
conditions in which the cell must derive its energy solely
tho arcA gene product appears to function as a repressor
by anaerobic respiration, sc//?C-/acZ expression was ele-
during aerobic and anaerobic conditions, the greater
vated by nine- to 16-fold compared to when the gtucose-
effect was seen anaerobically. The fnrarcA double mutant
based medium was used. Expression was slightly lower
exhibited a somewhat higher level of ji-galactosidase as
when nitrate was used compared to when either TMAO
seen in the arcA mutant during anaerobic growth: the
or fumarate was the anaerobic electron acceptor. Finally,
effect of the arcA mutation appears to be independent of
it was noted that sc/rtC-facZexpression under anaerobic
the effecf caused by the fnr mutation.
conditions with nitrate, TMAO or fumarate present was
always higher than when cells were grown aerobicaliy on
glucose. Expression of the succinate dehydrogenase
genes is cieariy occurring at significant levels during Effect of himA and fis tnutations on sdhC-lacZ
expression
anaerobic growth conditions. We aiso tested whether a
deletion of the tiarX and narL regulatory genes that are To evaluate whether either of two general gioba! regulatory
involved in nitrate control of many genes in E. coli had
any effect on sdhC-lacZ expression during anaerobic
cell growth with or without nitrate: no significant changes Table 2. Effect of carbon compounds and medium richness on sdhC-
in IVgalactosidase activity were seen (data not shown). /acZ expression.

Beta-galactoatdase activity''

Effect of carbon substrates on sdhC-lacZ expression Compound added"

To determine how other carhon compounds besides Giucose 3530 320

glucose and giyceroi affect sc//7C-/acZ expression, cells Galactose 9740 3480
Xylose 9510 2460
were grown in a minima) medium containing various 6-, Giyceroi 13900 NG*^
5-. 4-. 3-, or 2-carbon compounds as the substrate Acetale 25700 NG
(Table 2), During aerobic growth, sd/iC-tecZ expression Succinate 14 500 NG
Fumarate 20400 NG
varied over a 10-fold range: expression was lowest when Buffered L-broth 6980 4160
glucose was used and it increased in the order xylose, Buffered L-broth-i-glucose 2560 320
galactose, giyceroi, succinate, fumarate, and acetate.
a. Cells containing SJP33 were grown in a minimal basal medium
When a rich medium vvas used (buffered L-broth), sdhC- [pH7,0) wilh the indicated addilions except when buffered L-btoth
lacZ expression was about twice that observed for was used. Aerobic and anaerobic cultures were grown as described
glucose-grown cells. If glucose was added to the L-broth in Ihe lext.
b. Units are given in nmol of ONPG hydrolysed per mm per mg
medium, gene expression was the lowest for any aerobic protein.
condition tested. c. NG, no growih.
476 S.'d. Park. C.-P. Tseng and R. P. Gunsalus
Table 3, Etfect ol arcA. Inr. tiimA and Us mulations on sdtiC-lacl
expression.
Beta-galactosidase
activity
Strain" Relevant genotype +O2
MC4100 wild lype 3530
PC2 fnr 3420
arcA
PC35 9050
SJP6 arcA. fnr 10 300
SJP3 NmA 2340
SJP4 fis 2030
a. Cells containing >.SJP33 were grown in a minimal giueose medium
under aerobic or anaerobic conditions as described in the text.
b. Unils are given in nmnl of ONPG hydrolysed per min per mg
protein.
proteins. Integration host factor (iiHF) atid Fis, affects
sdhC-lacZ expression, himA atid fo deletions were intro-
duced into M C 4 1 0 0 A S J P 3 3 , Expression of sdhC-lacZ
was reduced slightly during aerobic conditions but was ele-
vated by about 30% during anaerobic conditiotis relative to
ttie isogenic parent straiti (Table 3).
Effect of crp and fruR mutations on sdhC-lacZ
expression
As a relatively wide range in sd/7C-/acZ expression was
obseived when cells were grown in different types of
media (Table 2), we tested whether the ap-gene product
contributes to ttiis control as previously proposed (Wilde
and Guest, 1986), A Acrp mutation was introduced into
the MC4100?.SJP33 lysogen and the mutant was grown
as described above. Surprisingly, sdhC-tacZ was not
significantly different in the mutant strain versus the wild-
type strain (Fig. 2). In contto! experiments, this same crp
deletion resulted in a 100-fold decrease in lacZ-gene
expression when driven from the native lac promoter
(data not shown). To determine whether the effect of
carbon type and richness on sd/7C-/acZ expression may
be due to the fruR-gene product, a chromosomal deletion
in Ihis gene was generated and evaluated as described
for the crp mutation. The /mR deletion also did not signifi-
cantly affect sdhC-lacZ expression (Fig, 2). Thus, the
controi of sdhC-lacZ expression in response to carbon
!ype is apparently because of some other type of cellular
regulatory process.
As the 10-folci range in sdhC-lacZ expression seen
when cells were grown otT differing carbon supplies was
independent of crp and fruR (Fig. 3), we carried out
experiments to determine whether or not the arcA gene
product somehow contributes to this control. An arcA-
deletion strain was grown aerobically and anaerobically
on each substrate and (.i-galactosidase activities were
determined (Fig, 2), During aerobic cell culture in most
types of media, sdhC-tacZ expression varied by about
twofold between the wild type and the arcA mutant
{Fig. 3A), The only exceptions were when cells were
grown in a buffered L-broth medium that contained glu-
cose (fourfold increase in sdhC-tacZ expression) or an
-0,
acetate medium (less than 10% difference).
320 During anaerobic cell culture, the arc/\-dependent con-
1490
trol of sd/iC-/acZ expression was tnore pronounced Ihan
22500
27 700 that observed during aerobic conditions (Fig. 3B), The
410 rnagnitude of the AtcA-dependent repression ranged from
430
sixfold when a galactose medium was used, to 70-lold
when a glucose minimal medium was used. The highest
level of gene expression observed was when the arcA
strain was grown in L-broth medium (c. 30 000 units)
which is about 60-fold greater than that seen for the wild-
type strain grown on buffered L-broth glucose rnedium, it
appears that ArcA may somehow help to provide tiie
observed control in response to medium richness or
carbon type; however, it is not evident if this is a direct or
indirect effect.
Effeel of iron availability on sdhC-lacZ expression
The succinate dehydrogenase enzyme complex encoded
by the sdhCDAB operon like other bacterial and eucaty-
otic SDH enzymes contains nine molecules of non-haem
iron (Ackrell etai., 1992), To determine if iron-limiting or
iron-excess cell growth conditions affect sdhC-lacZ
expression, wild type as well as fur mutant cells that are
defective for the iron uptake regulation (DeLorenzo etai.,
1988) were grown in glucose minimal mediutn in the
ioooo
20000-
lOOtJO-
rnedium type
Fig. 2. Eftecl ot a .Scrp mutation and a .MivF! mutation on
sdhC-iacZ expression,
A, Aerobic ceil growth,
B, Anaerot-)ic ceil growth.
The solid bars represent the wild-type strain, Ihe hatched bars
represent the Acrp strain and the open bars indicate the /MruR
strain. Ceils were grown in minimal medium suppiemented with the
indicated carbon compound (40 mM) or in buftered L-broth; glc,
glucose; gal, galaclose; ace, acetate; lum, fumarate; L, L-brotti,
Units are given in nmoi ot ONPG hydrolysed per min per mg
protein.
 Regulation of sdUCDAB by ArcA atid Fnr All

cellular haem-limitation affects sdhC-tacZ expression

medium type
Fig. 3. Eltec! of an !S.arcA mutation on sd/7C-/acZ expression in
cells grown on media ot various carbon types and medium
richness.
A. Aerobic cell growih,
B. Anaerobic ceW growfh.
The open bars represent the wild-type strain and the solid bars
represent Ihe ^aicA strain. Ceils were grown in minimal medium
supplemented with the indicated carbon compound (40 mM) or in
buHered L-bioth as defined in Fig, 2, Units are given innmoi of
ONPG hydrolysed per min per mg protein.
presence of the iron chelator, 2',2-dipyridy(, In a wild-
type strain, anaerobic and aerobic sd/iC-tecZ expression
was decreased from two- to threefold by iron limitation
(Table 4). Addition of excess iron to the dipyridyl-
containing medium partially restored sdtiC-tacZ expres-
(Table 5), A hemA strain that is defective for haem bio-
synthesis was grown in buffered L-broth pyruvate medium.
Haem limitation was evidenced by the inability of the hemA
cells to grow aerobically on sucoinate or glycerol. Wiihout
exogenously added S-aminolevulinic aoid {6-ALA) which
restores haem biosynthesis and thus oell growth on
succinate or glycerol, sd/iC-ZacZ expression in the hemA
strain was decreased by 50% compared to the wild-type
parent strain under either aerobic or anaerobic conditions.
However, <i-ALA addition to the medium restored expres-
sion to almost wild-type levels. Whether this regulatory
effect is direct or indirect is unclear.
Table 5. Effect of haem limitation on sdhC-lacZ expression.
Conditions'* Beta-galactosidase activity'^
6-ALA MC4iao (wild type) PC40 {hemA)
4670 2320
3910 3350
510 2W
430 540
a. Cells were grown in buffered L-broth pyruvate medium aerobically
or anaerobically as described in the text. iS-aminoleuvulinic acid
(6-ALA) was added at an initial conceniration of 40|igml ^
b. Units are given in nmol of ONPG hydroiysed per min per mg
protein.

sion to the levels seen in the wild-type strain. In the fur

mutant strain, the aerobic level of p-galactosidase was Effect of ceti growth rate on sdhC-lacZ expression
similar to that of the wild-type cell indicating that Fur is
not involved in the obsen/ed iron regulation. Continuous-oulture methods were used to examine how
celi growth rate affects sdtiC-tacZexpress'ion {Fig. 4). A
two- to threefold variation was seen during aerobic and
Effect of haem availabiiity on sdhC-lacZ fusion anaerobic growth, respectively. Gene expression was low-
Since the succinate dehydrogenase enzyme contains one est during rapid growth (k = 0,96 per hour) and gradually
b-type haem per complex, we also determined whether increased as the oell growth rate was reduced to 0.3 per
hour (anaerobically} and to 0.12 per hour (aerob'ically).
This pattern was similar to the effect of varying cell growth
rate in batch culture by varying medium richness (data not
Table 4. Effect of iron availabiiity on -ZacZexpression.
shown). A similar growth-rate-dependent pattern was
Addition'' Beta-galactosidase activity'' observed for gItA-iacZ expression during aerobic cell
growth (Park etai, 1994).
O.- Dip MC4100 (wild type) SJP2 {tur)

3530 2930
1070 930 Discussion
3840 3730
2200 1990 From the above studies, it is evident that sdhCDAB operon
320 330
expression is complex. The cell appears to control SDH
180 200
200 1900 synthesis in response not only to anaerobiosis, but also
410 240 to cell carbon and/or medium richness during both anaero-
a. Celis were grown In a minimal glucose medium aerobically or
bic and aerobic conditions. Gene expression varied up to
anaerobically as described in the text. Dipyridyl (Dip) and ferrous sul- 80-fold (Tables 1-3), The sdtiC-lacZ gene expression
phaJe (Fe'""'") were added a! an initial conceniration of 150f)M and data correlate well with the SDH enzyme activity data pre-
80 (iM, respectively, as indicated,
b. Units are given in nmoi of ONPG hydrolysed per min per mg
viously reported {Gray ^f a/,. 1966), Enzyme levels are ele-
protein. vated when oxygen is present and the levels are reduced
478 S.'J. Park. C.-P. Tseng and R. P. Gunsalus
(2 000 -
0 2 0.^ 0,e 08 i.O
Cell Growth Rate (k)
Fig. 4. Effect of celt growth rate on sdhC-iacZ expiess\on during
aerobic and anaerobic continuous culture, Celts were grown at
the indicated growth rates (k) as described in the Experimental
procedures. For aerobic growlii, air was maintained al 100%
saturation in Die culture medium throLighou! the experimenl. For
anaerobic growih, lhe vessel was sparged with O^-lree nilrogen
(200rnlmin '), Beta-galactosidase activity is expressed as nmol
ONPG hydrolysed pec min per mg protein. Cells were grown
aerobicaliy (C) or anaerobicaily (A) cis described in the
Experimenlat procedures.
when cells are grown on glucose. Thus, depending on the
conditions it encounters, the cell continualiy adjusts the
amount of succinate dehydrogenase synthesized and
this control appears to occur primarily by the level of
SdhCDAB gene transcription.
The aerobic/anaerobic control of sdhCDAB gene
expression is provided by the two cellular regulatory pro-
teins, ArcA and Fnr {Table 3, Fig, 3), ArcA functions as
a repressor of sc//7CD/\S-operon expression under both
aerobic and anaerobic conditions. The degree of repres-
sion in an arcA strain under aerobic conditions is about
twofold compared with 70-fold under anaerobic conditions
(Table 3). Assuming tbat ArcA is active only when it is
phosphorylated, some fraction of ArcA must be in the
active state even wben cells are grown in air-saturated
media. If under aerobic conditions ArcA is oniy in an
unphosphorylated form, tben tbis protein may have some
affinity for binding to ArcA regulatory DNA sites to provide
tbe observed aerobic repression. Fnr represses sdhCDAB
expression but oniy during anaerobic conditions,
Wby does tbe aerobic/anaerobic control of sdhCDAB
gene expression require two regulators, ArcA and Fnr?
Perbaps they exert their control at different tbresbolds of
the same celluar signal, or alternatively, eacb regulator
responds to a different ceiluiar/environmenta! signai. The
identity of the signal{s) is unknown although the oxida-
tion/reduction potential of the cell environment bas been
suggested for one regulator (Unden, et al., 1990). Tbe
anaerobic electron acceptors used by E coii have
oxidation/reduction mid-volt potentials well below the
oxygen/water couple wbich is 4-800 mV (i.e.. +430 mV for
nitrate/niti'ite; -i-120i-nV for TMAOATMA: and +33mV for
fumarate/succinate). If ArcB/ArcA were able to detect tbe
mid-volt potential between the oxygen and nitrale mid-
volt potentials, wbicb correlates with the range in wbich
sdhC-tacZ expression responds to the aerobic state
(Table 1), some value above +430 mV would need to be
detected. Alternatively, and more likely, some signal
otber than oxidation/reduction potential of the electron
acceptor used is involved in controlling sc//?C-/acZexpres-
sion. Tbe mid-volt potential for anaerobic respiration witb
nitrate, +430 mV, is clearly not sufficient to allow ArcA to
switch from an inactive state (no repression of sdhC-
lacZ expression) to an active state (repression of sdhC-
/acZexpression), if it was sufficient, a partial derepression
of sdhC-lacZ expression would be observed when the
anaerobic electron acceptors of different mid-voit poten-
tials are used. From the narXL mutant studies (see the
Results), it is noted tbat the nitrate-responsive NarX,
NarQ, NarL regulon of E. coH does not control sdhCDAB
gene expression.
The dual control of the sdhCDAB genes in E. co//by tbe
two aerobic/anaerobic regulators, Fnr and ArcA, is not
uncommon. Tbe cyoABCDE. cydAB and hernA genes
that encode the two cytocbrome oxidase enzymes and
an eariy enzyme in tiie haem biosyntiietic patiiway each
require tbe action of lhe iwo regulatory proteins (Daire
and Gunsalus, 1994), ArcA can function as an activator
(cydAB and hemA) and as a repressor {oyoABCDE and
sdhCDAB) ol gene expression (Fu et a/,,1991; Colter
and Gunsalus. 1992; Daire and Gunsalus, 1994). Fnr func-
tions as a repressor of each of the above operons allhough
it also acts as a transcriptionai activator for the anaerobic
respiratory operons, narGHJI, dmsABC. and frdABCD
(Gunsalus, 1992), Tbese two regulatory proteins are
employed in different combinations to regulate cell meta-
bolic and energy-yieiding pathways. The degree to wbich
tbe ArcA- and Fnr-regulatory proteins control tbe other
TCA cycle genes besides sdhCDAB and gllA wiil be inter-
esting to determine,
Tbere appears to be a significant role for SDH during
anaerobic respiration conditions based on the pattern of
scy/7C-/acZ expression observed {Table 1), When ceils
are grown in a giucose medium, sdhC-lacZ expression
is elevated 10-fold aerobicaliy compared witb anaerobic
growth {i,e, fermentation) conditions. When the cellular
electron acceptors, nitrate, TMAO, or fumarate, were
added to this glucose medium, a modest twofold effect
on sdhC-lacZ gene expression was seen. However,
when giyceroi is used instead of glucose for anaerobic
cell growth, sdhC-lacZ expression is 10- to 2Q-fold
above tbat seen with the giucose media, SDH does not
appear to function solely as an 'aerobic' enzyme. Rather,
it appears to function anaerobicaily to supply biosynthetic
intermediates via fbe TCA cycle.
It is commonly beiieved that decreased syntbesis of
SDH in response to glucose availability during cell growth

is mediated by the Crp protein (Wilde and Guest, 1986). No
sigtiificant change in sdhC-lacZexpression was observed
in the crp or fruR mutants relative to the wild-type strain
(Fig, 2), Thus, some other means of cellular control must
exist to provide appropriate levels ol scJhCDAB expres-
sion in response to the presence of glucose. It is note-
worthy that Crp-independent catabolite repression of
gene expression occurs in many other microorganisms
(Chambliss, 1993; Weickert and Chambliss. 1990), The
effect of the carbon source on sc//TC-/acZ expression is
considerable: an increase of up to 104old was seen during
aerobic conditions while up to a 13-fold increase was seen
anaerobically (Table 2). Expression levels are highest with
acetate, and decrease if fumarate. gfycerol, xylose, galac-
tose or glucose is the energy source. A similar effect was
also seen for expression of the gttA gene that encodes
the citrate synthase of . coti (Park et al.. 1994). These var-
iations in the TCA cycle enzyme leveis appear to occur at
the level of transcription, and provide control of the cellular
capacity to produce biosynthetic intermediates and/or
energy.
Continuous-cell-cuiture methods were used to examine
vwhether or not the 10-fold and the 13-fold range in
sdhC-tacZ expression seen during cell growth on differ-
ent carbon compounds in batoh culture was due to
changes in cell growth rate (Fig. 4). Expresssion varied
by two- to threefold over fhe growth rates tested
(k = 0.12-0.96). Interestingly, when glucose (2.25mM)
was used to limit growth in the chemostat, sdhC-lacZ
expression was eightfold higher than when glucose
(40 mM) was in excess during growth in batoh culture
(Table 2). It is notable that the pattern of expression
seen for sdhC-tacZ in response to growth rate is the
inverse of the pattern observed for control of ribosomal
RNA and protein synthesis (Bremer and Dennis, 1987;
Jinks-Robertson and Nomura, 1987).
By construction and analysis of four sd/i-/acZ fusions
that contained different regions of DNA in the sdhCDAB
regulatory and structural region, we identified a region
upstream of the sdliCgene required for expression of the
operon (Fig, 1). Based on the results of S1 nuclease
experiments of Witde and Guest (1986), transcription of
the operon initiates at a position 219bp before the start
of sdfiC translation. The promoter was proposed to be a
'minor' promoter while a second promoter located within
sdhCvias thought to be the 'major' promoter ('P^'), Accord-
ing to the results of the >.SJP16 and /..SJP33 sdhC-lacZ
fusions (Fig, 1), this second promoter does not appear
to exist. In support of this conclusion, expression in the
SJP58 lysogen (which contains the region proposed to
encompass both promoters) was nearly identical to that
seen for the /.SJP33 lysogen which lacked the 'major' or
V2 promoter. If the sdhCDAB mRNA is somehow rapidly
processed, this would account for the two distinct 5' ends
Regulation of sdhCDAB by ArcA and Fnr 479
observed for the sdhCDAB poiycistronic messages
observed in Ihe earlier transcript study of Wilde and
Guest (1986). The significance ot the two sdh promoters
and two distinct RNAs during steady-state growth is diffi-
cult to reconcile since ihe sucanaie dehydrogenase
complex contains four different subunits in a 1:1:1:1 ratio,
SdhC:SdhD:SdhA:SdhB, that are each required for
enzyme activity (Ackrell et ai. 1992), Comparison of
sdhC-tacZ expression data tor the ASJP33 and the
ASJP16 lysogens demonstrates that lhe 255 bp upstream
region, which is present in A,SJP33 but absent in
XSJP16, is required for normal sdhCDAB expression
(Fig. 1). The pattern of sd/iC-ZacZexpression in ?^SJP16
is consistently lower than that seen for /.SJP33 although
it is still ArcA- and Fnr-dependent (data not shown). The
upstream 255 bp region contains additional regulatory
sequences that appear to enhance the level ot sdhC-
lacZ expression. Experimenls are in progress to identify
the enhancer-like element as well as to locate the sites
for ArcA and Fnr regulation of the sdhCDAB genes.
Experirnental procedures
Bacterial strains, bacteriophages. and plasmids
The genotypes of the E. coli K-12 strains, plasmids, and the
bacter iophages are listed in Table 6. To generate the iso-
genic arcA, himA, fis. fur, fruR and crp mutants, the indicated
alleles were introduced into parental strain MG4100ASJP33
{sdhC-lacZ) by PI transduction (Miller. 1972), A high-titre
lysate of SJP33 was used to introduce the sd/7C-/acZfusion
into the PC2 {fnr) strain (Simons etai., 1987).
Construction of sdh-lacZ operon fusion plasmids
The sdhC-lacZ fusion located on ?,SJP33 which contains
555 bp of DhJA upstream of .sdhCwas constructed by isolating
the 1,2kb eamHI-H/ndlll fragment from plasmid pJTSDi
and inserting it into M13mp19 to give M13SJP1911, Using
oligo-directed mutagenesis (Kunkel, 1985), an EcoRI site
was introduced inio the sc/hC gene ai position +93 relative
to the translationai start site of sdhC to give phage
M13SJP1912 (Fig. 1), The 648bp EcoRI-BamHI fragment
of M13SJP1912 was cloned into pRS1274, a promoterless
/acZ operon fusion vector (Simons ef at.. 1987) to give the
sdhC-lacZ operon fusion piasmid, pSJP33. This fusion con-
tains the upstream 555 bp region from the translational start
site of sdhC. To construct the sc//iC-/acZ fusion SJP16, the
404 bp Sau3AI fragment of plasmid pJTSDI was inserted
into SamHI site of pRS415, a promoterless lacZ operon
fusion vector, to give pSJP16. The resulting fusion contains
the 300 bp region upstream of the translational start site of
the sdhC gene and the first 104 bp ot sdhC DNA- Thfi fusion
junctions between the sdhC an6 /acZ genes were confirmed
by double-strand DNA sequencing (Sanger ei al., 1977),
The sdhC-lacZ fusions on pSJP33 and pSJPIfi were then
transferred to RZ5 to generate SJP33 and SJPI6 (Fig. 1),
480 S.-J. Park, C.-P. Tseng atid R. P. Gunsalus

Table 6. Bacterial sirains, phages and

plasmids. Strain./Phage/
Plasmid Derived from Genotype/Phenotype Source/Reterence

Strain
MC4100 P" araO139(argF-lac) \J^69 Silhavy et at.
rpsLi50relA1 flbS301 deoCI (1984)
ptsF25 rt)sR
PC2 MC4100 Afnr Colter and
Gunsalus (1992}
PC35 MC4100 AarcA Kan''' Park el at. (1994)
PC40 IV1C4100 hetnA'fnmo.-Kan^ Cotter and
Gtjnsakis (1992)
IS4 MC4100 AnarXL Kan'^ 1, Schroder
W3110 fur: Jn.5 fur::'[n5 DeLorenzo tV al.
(1988)
SA2777 Acrp Chl'^ S. Garges
SJP2 MC4100 lur::Tn 5 This work
SJP3 MC4100 hit}iAAB2 Te[^ This work
SJP4 MC4100 fe-767 Kan" This work
SJP5 MC4100 Acrp CU\^ This work
SJP6 MC4100 AarcA A Inr Thi.s work
SJP7 MC4100 AfruPKan'^ This work

Phage
?.RZ5 Simons e{ ai.
(1987)
/.SJP16 pSJPie 'PisdhC-tacZ) tacY^ lacA' This work
/.SJP33 pSJP33 <l>{sdtiC'lacZ) lacY' tacA' This work
X3JP58 pSJP58 ^sdhCDA-lacZ) lacY^ lacA' This work
^SJP59 pSSJ59 <H.sdhDA~lacZ} lacY' lacA' This woik
X.6C1 fruR' Kohara et al.
(1987)
M13mp19 Messing and
ViGira (1982)
M13SJP1911 M13inp19 1.2 kb fiamHI-H/>jdlll This work
fragment
M13SJP1912 (same as M13SJP19n bu! with an This work
additional EcoH\ site at nt +93)

Plasmid
pGem7f(-^) Promega
pSJP48 pGem7{(-(-) fruR* This woik
pUC4K Kan'' P, Cotter
pSJP52 pGem7f{+) MruRKm^ This work
PMAK705 PACYC184 Hamiiton et al.
0989)
pSJP53 pMAK705 AfruR Kan'^ This work
pJTSDI pTZ19 sdhC::Kn903 J. Turna
plS58 pBR322 SdhCDAB sucA 1. Schrrjder
pRS1274 lacZ lacY* tacA' Simons ef al.
(1987)
pRS4l5 lacZ lacY' lacA ' Simons et a!.
(1987)
pSJPie pRS415 'l'(sdhC-lacZ) lacY' lacA " This work
pSJP33 pRS1274 'i>{sdhC-lacZ) tacY* lacA^ This work
PSJP58 PRS1274 'HsdhCOA-lacZ) lacY' lacA' This work
pSJP59 PRS1274 'i^sdtiDA-lacZ) lacY* lacA' This work

^, kanamyoin resistant; Chi", chioramhenicol resistant; T e l " , tetracycline resistant.

respectively, and tben introduced into IV1C4100 as previously
described (Simons etai.. 1987).
To test for a putative second sdh promoter located within tbe
sdhC gene, whicb had been proposed by Wilde and Guest
(1986), two additional sd/i-/acZ fusions were constructed.
Tbe 1373l:)p eamHI-Haelll fragment of plS58 which con-
tains the gItA-sdhCDAB intergenic region, the intact sdhCD
genes and the first 89 bp of sdhA were cloned Into tbo
BamH\-Stnal site of pRS1247 to give pSJP58: tbis fusion
contained the region predicted to tiave botb of the promoters
proposed by transcript analysis (Wilde and Guest, 1986).
The 713bp Bci\-Hae\\\ DNA fragment of plS58 containing
the 344 bp 3' end of sdhC. an intact sdhD gene, and the
89 bp 5' end of sdhA were used to construct the sdhCDA-
/acZ tusion in pSJP59 (proposed to contain only the second
sdh promoter). The intended fusion junction of pSJP58 and

pSJP59 was confirmed by DNA sequence analysis. Each
fusion was transferred onto RZ5 to create /.SJP58 and
A S J P 5 9 (Fig. 1) and then inserted into tbe E co//chromo-
some as a single copy as described above.
Construction of a fruR deletion strain
The 5 kb Kpn\-Bam\-\\ DNA fragment containing the fruR
gene was isolated from A6C1 of the Kohara libraiy of tbe
co//genome and cloned into pGem7f(-i-) to give pSJP48. The
1,9kb /-//ndlll fragment that contains the fruR gene region
was deleted and the flanking DNA regions adjacent to fruR
were ligated with a 1.5 kb HincW Kan*^ fragment of pUC4K to
give pSJP52, The 6,5 kb EcoB\-BamH\ fragment of pSJP52
that contains the kanamycin-resistance gene in place of the
truR gene was then transferred to pMAK705, a plasmid with
a heat-sensitive origin of replication (Hamilton et al.. 1989),
to give pSJP53. The chromosomal wild-type fruR gene was
deleted using the allele-replacement procedure described by
Hamilton et al (1989). The resulting //ufl-deletion strain was
named SJP7,
Cell growth
For strain manipulations and maintenance, cells were grown
in Luria broth or on solid media. When reguired, ampicillin
and chloramphenicol were added to the medium at a con-
centration of lOOmgl""' and 30mgl "', respectively. For fi-
galactosidase assay, cells were grown in glucose (40 mM)
minimal medium (pH7,0) (Cotter and Gunsalus, 1989).
unless otherwise indicated. For assay of cells grown on
other carbon sources, each compound was substituted at
40 mM, Buffered L-broth (50 mM KPO4, pH7.0) was made
with glucose (40 mM) supplements as indicated (Cotter and
Gunsalus, 1992), Aerobic and anaerobic growth was per-
formed as previously described (Cotter and Gunsalus, 1989;
Jones and Gunsalus, 1987), High aeration of cultures during
aerobic growth was accomplished by shaking 10 ml cullure
volumes in 150 mi flasks. Attempts to increase the aeration
o( the culture by reducing the volume of the medium or
by increasing the flask agitation rate did not affect the p-
galactosidase activities. Flasks or tubes containing the indi-
cated medium were inoculated from overnight cultures
grown under the same conditions, and the cells were allowed
to double four or five times under mid-log exponentia) phase
prior to haivesting for analysis (ODsoo = 0-4-0.5, Kontron Uvi-
kon 810 Spectrophotometer). Anaerobic cultures were har-
vested at ODciTO = 0,25. When desired, 2,2'-dipyridyl or
ferrous sulphate was added at a final concentration of
0.15 mM or 0,08 mM, respectively.
For continuous-culture experiments, a Queue Mouse bio-
reactor (Queue Corporation) was fitted with a 2 litre vessel
and operated at a liquid working volume of 1 litre (Tseng ef
al., 1994), Vogel-Bonner medium (pH6,5), modified by
adding Casamino Acids (0,25mgr"') and by reducing the
giucose concentration to 2,25 mM to limit cell growth (i.e. a
carbon limited medium), was used for all studies unless other-
wise noted. Aerobic continuous-cultLire conditions were main-
tained by saturating the culture medium with sterile air at a
rale of 2,01 min' '. Anaerobic conditions were maintained by
continuously sparging the vessel with oxygen-free nitrogen
at a rate of 200 ml m i n " ' ,
Regulation 0/sdhCDAB by ArcA and Fnr 481
Between experiments, the chemostat was maintained at a
ftow rate (F) of 2mlmin"""' (k = 0,12 per hour). To vai7 cell
growth rate (k), the medium-addition rate was adjusted
accordingly. The medium-addition rates ranged from 2 -
16mlmin" ' (k = 0.12 to 0,96 per hour) which corresponded
to cell doubling times (g) of 350 and 58 min, respectJve/y.
The cell doublings per hour (fi) is egual to 1 divided by celt
generation time (g). The cell generation time (g) is equal to
In2 divided hy cell growth rate (k) (see NeidhardI ef al..
1990), When cells were shifted to a new rate of growth, the
steady state was generally achieved in five reactor residence
times.
Beta-galactosidase assay
Beta-galactosidase levels were determined by liydrolysis of
ortho-nitrophenyl-|l-D-galactopyranoside (QNPG) as pre-
viously described (Cotter and Gunsalus, 1989; Miller er al..
1972). Beta-galactosidase values represent the average of
at (east four experiments with a variation of no more (han
10% from the mean.
Note added in proof
After this article was submitted, a paper reporting a similar
eftect of the fnr anc\ arcA gene products on E. coli sdhCDAB
gene expression appearecf (see (uchi eiai. (1994) J Bacteriol
176: 1695-1701).
Acknowtedgements
This work was supported in part by Grant GM49694 from the
Public Health Service and Grant HL16251 from the National
Institutes of Heafth.
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