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Journal of Bacteriology, December 2000, p. 7035-7043, Vol. 182, No. 24
Department of Biochemistry, University of
Dundee, Dundee DD1 5EH, Scotland,1 and
Nitrogen Fixation Laboratory, John Innes Centre, Norwich
NR4 7UH,2 United Kingdom
Received 28 June 2000/Accepted 3 October 2000
The expression of the moa locus, which encodes enzymes
required for molybdopterin biosynthesis, is enhanced under
anaerobiosis but repressed when the bacterium is able to synthesize
active molybdenum cofactor. In addition, moa expression
exhibits a strong requirement for molybdate. The molybdate enhancement
of moa transcription is fully dependent upon the
molybdate-binding protein, ModE, which also mediates molybdate
repression of the mod operon encoding the high-affinity
molybdate uptake system. Due to the repression of moa in
molybdenum cofactor-sufficient strains, the positive molybdate
regulation of moa is revealed only in strains unable to
make the active cofactor. Transcription of moa is
controlled at two sigma-70-type promoters immediately
upstream of the moaA gene. Deletion mutations covering the
region upstream of moaA have allowed each of the
promoters to be studied in isolation. The distal promoter is the site
of the anaerobic enhancement which is Fnr-dependent. The
molybdate induction of moa is exerted at the proximal
promoter. Molybdate-ModE binds adjacent to the Molybdenum is an essential trace
element required for the activity of several enzymes in the form of a
molybdenum cofactor which is found in bacteria, plants, and animals.
All oxo-molybdoenzymes described to date contain a
molybdenum cofactor which consists in its most simple form of a
pterin, molybdopterin, complexed to molybdenum.
Molybdopterin has a terminally phosphorylated, four-carbon
alkyl side chain with a dithiolene group, the two sulfur atoms of which
are ligands to the molybdenum (28). Crystallographic structures of several molybdoenzymes are now available (4, 18, 19,
33).
In Escherichia coli the mo- loci are responsible
for molybdenum transport and the synthesis of the molybdenum cofactor,
and all of these loci have been characterized (12, 13, 15, 26, 29,
30). The moa and moe loci are required for
molybdopterin biosynthesis. The first three genes (moaABC)
of the five genes at the moa locus (30) are
required for the first committed step of molybdopterin biosynthesis,
namely the formation of precursor Z from a guanosine nucleotide or
closely related compound (39).
The modABCD operon of E. coli encodes the
high-affinity molybdate uptake system of the bacterium. Strains
defective at this locus are pleiotropically unable to synthesize active
molybdoenzymes, such as nitrate reductase. The introduction of high
levels of molybdate into the growth medium, however, restores
molybdoenzyme activities to mod strains, indicating that
molybdate can enter the bacterium by another route under such
conditions (6, 27, 31). Transcription of modABCD
is repressed under conditions of high molybdate availability
(23, 29, 31). The repression is mediated by the ModE
protein, which is encoded by a second operon expressed divergently from
modABCD (7, 20, 25, 38).
In vitro studies have demonstrated that dimeric ModE directly binds two
molybdate ions with a Kd of about 0.8 µM and
that the molybdate-ModE complex binds to mod DNA at a site
which spans the modA transcription start site. This is
consistent with its role as a transcriptional repressor of
modA (2, 8, 22). The ModE protein has a wider
role in mediating molybdate regulation of other operons which include
dmsA, the structural operon for the molybdoenzyme
dimethyl sulfoxide reductase (21), hyc, the hydrogenase 3 structural operon (34), and narG,
which encodes the molybdoenzyme nitrate reductase A (34).
Hall and colleagues recently reported the crystallographic structure of
ModE, which appears to possess distinct DNA- and molybdate-binding
domains (9).
Genetic analysis of the regulation of moa has revealed that
its expression is enhanced under anaerobic growth conditions but is
repressed in strains able to synthesize active molybdenum cofactor (3). McNicholas et al. (22) reported that
molybdate-ModE is able to bind at the moa promoter region
and that moa expression displayed a twofold ModE dependency.
However, a dependency of moa expression on molybdate has not
been demonstrated. We report here that internal molybdate availability
exerts a major influence on moa expression via a mechanism
in which ModE acts directly as a positive regulator at one of the two
moa promoters. This effect is distinct from and in addition
to the anaerobic molybdenum-cofactor regulation of moa. This
work exploits our previous finding that ModE is able to bind tungstate,
a molybdate analogue, in a manner that is indistinguishable from that
of molybdate (2).
Bacterial strains, bacteriophage, and plasmids.
All strains
(derivatives of E. coli strain K-12), bacteriophage,
and plasmids used in this study are listed in Table
1. Bacteria were grown in Luria-Bertani
(LB) medium at 37°C, but strains carrying a Mucts prophage
were grown at 30°C. Ampicillin was added, where required, to final
concentrations of 125 µg/ml in liquid media and 50 µg/ml in solid
media. Tetracycline and gentamicin were added, where required, to final
concentrations of 25 and 10 µg/ml, respectively.
5-Bromo-4-chloro-3-indolyl-
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
ModE-Dependent Molybdate Regulation of the
Molybdenum Cofactor Operon moa in Escherichia
coli
and
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
35 region of this
promoter, acting as a direct positive regulator of moa. The
molybdenum cofactor repression also appears to act at the proximal
transcriptional start site, but the mechanism remains to be
established. Tungstate in the growth medium affects moa
expression in two ways. Firstly, it can act as a functional molybdate
analogue for the ModE-mediated regulation. Secondly, tungstate brings
about the loss of the molybdenum cofactor repression of
moa. It is proposed that the tungsten derivative of the
molybdenum cofactor, which is known to be formed under such conditions,
is ineffective in bringing about repression of moa. The
complex control of moa is discussed in relation to the
synthesis of molybdoenzymes in the bacterium.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
-D-galactopyranoside (X-Gal)
was added to media at 125 µg/ml when necessary.
Isopropyl--D-thiogalactopyranoside (IPTG) was used as an
inducer for the lac operon at a concentration of 0.3 mM.
Aerobic growth conditions were achieved by placing 5 to 10 ml of
culture in 50-ml conical flasks and incubating with vigorous shaking on
an orbital shaker. Anaerobic growth was performed in an anaerobic
chamber under an atmosphere containing carbon dioxide and hydrogen
(GasPak; BBL Microbiology).
TABLE 1.
Bacterial strains, bacteriophage, and plasmids
Strain construction.
Generalized transduction between
E. coli strains was carried out using a derivative of
bacteriophage P1 essentially as described previously (35). A
P1vir lysate was prepared with strain RK4921 (zbh-623::Tn10 [near mod])
and used as the donor in a cross with strain SE1597
(modC118). Recombinants selected as Tetr
colonies were screened for loss of molybdenum-dependent formate hydrogenlyase activity by their inability to produce gas during glucose
fermentation and for the nitrate reductase-negative phenotype by the
nitrite overlay procedure (6). A P1vir lysate
prepared with the resultant strain (as SE1597;
zbh-623::Tn10) was used as the donor
with acceptor strains DB1060 [as RK4353;
p
(moa::lacZ)4] and DB1004
[(moa::lacZ)4] to give strains
LA27 (as DB1004; modC118) and TL28 (as DB1060;
modC118), respectively. Strain HORF55 was used as the donor
in P1-mediated crosses with strains LA27 and TL28 to construct strains
LA29 (as LA27; modE::Gm) and LA30 (as TL28;
modE::Gm), respectively. Drug resistance was used
for initial selection in all crosses.
Enzymes, reagents, and recombinant DNA techniques. Restriction endonucleases, T4 DNA ligase, Klenow fragment of DNA polymerase I, and nuclease Bal 31 were purchased from Northumbria Biologicals. Sequenase was obtained from U.S. Biochemicals Corp. through Cambridge Bioscience (Cambridge, Mass.). Avian myeloblastosis virus reverse transcriptase was purchased from International Biotechnologies Inc. Human placental ribonuclease inhibitor and all radionucleotides were obtained from Amersham PLC. The isolation of plasmid DNA, restriction digestion, ligation, E. coli transformation, and agarose gel electrophoresis were essentially as described by Maniatis et al. (17).
The assay for-galactosidase activity was performed essentially as
described by Miller (24). Cells were assayed during early to
mid-log phase following permeabilization by the addition of sodium
dodecyl sulfate and chloroform. All assays were performed in triplicate
and the mean values were reported in Miller units (24).
Individual assays rarely differed from the mean by more than 10%.
Isolation of p(moa-lacZ) phage and construction
of lysogens.
Strain DB1004
[(moaB::lacZ)4] was grown to
mid-log phase, and the cells were harvested and resuspended in a
half-volume of 10 mM magnesium sulfate for lysate preparation. The
suspension was exposed (2 min) to short-wave (260-nm) irradiation,
diluted 10 times in LB medium, and incubated with shaking at 37°C in
foil-covered tubes until lysis occurred (approximately 4 h). Cell
debris was removed by centrifugation and the supernatants were used as
low-titer lysates for production of Lac+ plaques when grown
in the presence of X-Gal and IPTG. A high-titer lysate was used to
construct moa::lacZ reporter lysogens
by transduction into appropriate acceptor strains.
phage DNA, solid polyethylene glycol (PEG
8000) was dissolved at room temperature in a large-scale lysate to a
final concentration of 10% (wt/vol). The phage was pelleted and then
dispersed in a minimum volume (about 5 ml per liter of lysate) of
buffer (50 mM Tris HCl [pH 7.5], 10 mM MgSO4). Following
a phenol-chloroform extraction, the aqueous solution was layered over a
glycerol step-gradient (5 to 40% [wt/vol]) and centrifuged at
70,000 × g for 90 min to pellet the phage. Residual
host-derived DNA and RNA were removed using DNase and RNase. The phage
coat was liberated by suspension in 0.5% (wt/vol) sodium dodecyl
sulfate, 50 mM Tris HCl (pH 7.5), 400 mM EDTA, and 1 mg of proteinase
K/ml. DNA was ethanol precipitated and resuspended in a suitable volume
of sterile water or 10 mM Tris HCl (pH 8.0), and 1 mM EDTA.
Subcloning the moa promoter region and generation of
deletion mutations.
A plasmid carrying the moa promoter
region was isolated by shotgun cloning of EcoRI-digested
phage p(moaB::lacZ) DNA into the
EcoRI-digested promoter-cloning vector pMLB524 and by
selecting for Lac+ transformants of MC4100 (35).
Analysis of transcription initiation sites. Cultures of strain RK4353 carrying plasmid pEM101 were grown to an optical density at 600 nm of 0.6, and total RNA was recovered according to the method of Figueroa et al. (5). The dried RNA was resuspended in 30 µl of sterile H2O and hybridized with 0.1 pmol of 32P-labeled oligonucleotide primer. Primer extension was performed using the method of Inoue and Cech (11). The oligonucleotide primer L9 (see Fig. 2) was used in all the experiments shown. The same oligonucleotide primer was used in DNA sequencing reactions with plasmid pEM101 DNA as the template to produce a reference sequencing ladder, which was electrophoresed adjacent to the transcript-analysis samples in a 6% (wt/vol) denaturing polyacrylamide gel containing 6 M urea.
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Molybdate activates moa expression under anaerobic growth conditions in a molybdenum cofactor-deficient background. To assess the possible molybdate regulation of moa, moa-lacZ reporter strains defective in modC, which encodes part of the high-affinity molybdate transporter, were constructed in order to obtain molybdate starvation conditions in normal growth media.
In strains unable to synthesize molybdenum cofactor, reduction of the internal availability of molybdenum (modC) resulted in a much-reduced expression of moa compared to that found for the parental strain, DB1004 (Fig. 1A). Because strain LA27 (modC) is molybdenum cofactor-deficient, the reduced expression cannot arise from the previously described molybdenum cofactor-mediated repression. This suggests that intracellular molybdenum availability acts positively on moa via a mechanism that is independent of the molybdenum cofactor.
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Molybdate cannot activate moa in a molybdenum
cofactor-sufficient background.
Baker and Boxer (3)
reported that moa is repressed in molybdenum
cofactor-sufficient strains. As anticipated, in the presence of
functional molybdenum cofactor, moa was repressed {strain
DB1060 [moa+/(moaB-lacZ)]},
and the addition of molybdate to the growth medium was without
effect (Fig. 1B). Strain TL28
[moa+/(moaB-lacZ),
modC], in the presence or absence of added molybdate, displayed levels of moa expression similar to the repressed
levels in strain DB1060. Furthermore, loss of ModE function was
without effect, since expression in strain LA30
[moa+/(moaB-lacZ) modC
modE] remained low and uninfluenced by the addition
of molybdate. The clear ModE-dependent molybdate activation of
moa observed in molybdenum cofactor-deficient
backgrounds is not found in strains possessing functional
molybdenum cofactor. It is evident that molybdenum cofactor-dependent
repression is dominant over the ModE-requiring molybdate
activation. However, high expression of moa
clearly requires ModE.
Aerobic expression of moa displays similar control characteristics to those present during anaerobic growth. The aerobic expression of moa was monitored in a manner similar to that described above. The relative patterns of expression were closely similar to those found for anaerobically grown cultures (Fig. 1, compare C and D). In the molybdenum cofactor-deficient strain DB1004, the anaerobic enhancement was four- to fivefold higher, whereas in the molybdenum cofactor-sufficient strain (DB1060), the anaerobic enhancement was much lower. Molybdate availability, therefore, activates moa during both anaerobic and aerobic growth in a ModE-dependent manner and is a major positive control on moa.
Tungstate activates moa expression in a ModE-dependent manner. Tungstate is a close analogue of molybdate in biological systems. Tungstate interacts with isolated ModE indistinguishably from molybdate, and the tungstate-ModE complex also interacts with DNA in a manner similar to that of the molybdate-ModE complex (2). The effects of tungstate on moa regulation were investigated.
The addition of tungstate to the growth media of molybdenum cofactor-deficient strains LA27 (modC) and LA29 (modC modE) influenced moa expression in a way similar to that found for molybdate (Fig. 1A and C). The expression of moa in strain LA27 was enhanced to a comparable level to that found for the parental strain, DB1004, when grown in tungstate. The addition of tungstate to the growth medium for strain LA29 (modE) could not restore the tungstate activation seen in strain LA27. A similar result was also obtained following tungstate addition to the growth medium for strain LA30 (modC modE). We conclude that for a molybdenum cofactor-deficient strain, tungstate, like molybdate, is able to activate moa expression in a ModE-dependent manner.In contrast to molybdate, tungstate relieves molybdenum
cofactor-dependent repression of moa.
The low moa
expression in the (moa+) merodiploid strain
DB1060 can be explained by repression brought about by the molybdenum cofactor sufficiency of the strain. This is revealed by the enhanced expression of moa observed when mutations in other
(mo) genes required for molybdenum cofactor biosynthesis
are introduced into such merodiploids. This repression could be
mediated by the cofactor itself, a derivative of the cofactor, or a
process which is dependent on the active cofactor (3).
Identification of two transcriptional start sites at
moa.
Plasmid pEM101 consists of approximately 0.6 kb of DNA
upstream of the moaA translational start inserted into the
multiple cloning site of the transcriptional (LacZ) reporter plasmid
vector, pAA182. The moa DNA in pEM101 was subcloned from a
Lac+ bacteriophage [(moaB-lacZ)], a
transducing phage induced from strain DB1004. That the moa
promoter region was present in pEM101 was confirmed by the retention of
the moa expression characteristics by the plasmid (data not
shown). This retention also confirmed that moa regulation is
exerted at the level of transcription.
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Analysis of expression from deletion clones.
We determined, by
using deletion analysis, the expression characteristics of the
independent moa promoters in order to assess their
contribution to the overall control of the locus. Three 5'-deleted
plasmids were obtained following Bal 31 digestion from the
EcoRI site in pEM101. These constructs are pEM114 (~0.74
kb insert; carrying only S1), pEM117 (~0.80 kb insert; carrying S1 and S2 with 25 bp upstream of S2), and pEM118 (~0.54 kb; whole promoter region absent). A further deletion, in plasmid pEM220 (insert
0.37 kb), was obtained by Bal 31 digestion originating from
the BamHI site at the 3' limit of the insert in pEM101. The structures of the deletion clones are summarized in Fig.
3.
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indicates that the strain (NS3;
moeB) is unable to synthesize functional molybdenum
cofactor. Each plasmid was examined in both a MoCo+ strain
(RK4353) and a MoCo35 and
10 regions of the proximal promoter. Under all conditions examined,
expression from this clone was lower than that for the parental
plasmid, pEM101. For this clone in the absence of tungstate, expression
was not increased during anaerobic growth. During anaerobic growth the
addition of tungstate to the molybdenum cofactor-sufficient parental
strain carrying pEM114 raised expression. A similar derepression was
also seen when the plasmid was present in the molybdenum
cofactor-deficient (moeB) strain. Overall, these results
suggest that the loss of S2 has two effects. Firstly, the level of
expression is lower. Secondly, the anaerobic enhancement is lost. The
results for pEM117, which has only 25 bp of DNA upstream of S2, were
similar to those found for pEM114.
Transcription-start analysis on pEM114 and pEM117 confirmed that
transcription was from S1 in both plasmids. As would be expected from
the loss of the 35 region of S2, transcription from S2 in pEM117
could not be detected even following anaerobic growth (data not shown).
Expression from these two 5'-deletion clones is much lower than that
from the parental plasmid, which may reflect a requirement for
sequences 5' to S2 being required for full expression from S1. As
anticipated, expression from plasmid pEM118 (both S1 and S2 absent) was
negligible under all conditions tested.
In the absence of S2 (pEM117 and pEM114), there is both a tungstate
enhancement in the molybdenum cofactor-sufficient parental strain
background and a derepression to a similar level in the molybdenum
cofactor-deficient strain. We conclude that the promoter associated with S1 is subject to repression in molybdenum
cofactor-sufficient strains.
If the two moa promoters worked independently in situ,
the expression observed when both promoters are functional should equal the sum of their individual expressions. This is not the case. The proximal promoter clearly overlaps part of the distal promoter.
Expression from the distal promoter is insensitive to
molybdate.
The plasmid pEM101 possesses both promoter regions and
retains the overall characteristics of moa regulation.
Plasmid pEM220 contains only the distal promoter. In order
to establish whether the distal promoter is regulated by
ModE, the plasmids were each transformed into a modE
strain (HORF55) and expression was monitored following anaerobic
growth (Fig. 4). In the modE
strain, expression from pEM101 was reduced approximately twofold from
the level for the parental strain background. Expression from pEM101
was enhanced some 75% by molybdate addition to the growth medium
in the modE+ strain, but in the
modE-deficient strain the addition of molybdate was
without effect. This is consistent with the results
obtained for the chromosomal moa-lacZ strains. When only S2
is present (pEM220), neither the presence of molybdate in the growth
medium nor the lack of modE influences expression. This
strongly indicates that the ModE-dependent molybdate control of
moa is effected through the proximal promoter.
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Fnr is required for the anaerobic regulation of moa at
the distal promoter.
The fnr gene encodes a positive
transcriptional regulator required for the expression of a number of
anaerobically expressed genes (36). A putative Fnr
recognition sequence (GATGAT-N4-ATCAAA), centered at 39.5
nucleotides upstream of S2, was found (Fig. 2A). This site differs from
the consensus sequence in only one base. Baker and Boxer previously
reported that the fnr gene is not required for the anaerobic
expression of moa (3). In our earlier studies,
single-copy moaB-lacZ chromosomal translational gene fusions
which contained the intact promoter region upstream of the
moa operon were used. Since the previous results reflected the combined effects of both promoters and the anaerobic enhancement of
moa can be studied directly using pEM220, we decided to
reinvestigate the possible involvement of fnr in
moa regulation.
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Molybdate addition to the growth medium brings about an activation
of moa expression during both anaerobic and aerobic growth of some four- to fivefold over that found in its absence. Molybdate acts as a major positive regulator of moa and its action
requires the ModE protein. The molybdate activation of moa,
however, is revealed only in a molybdenum cofactor-deficient
background, since in molybdenum cofactor-sufficient strains,
moa is effectively repressed. ModE is a major regulator of
moa, since in modE strains moa
expression levels remain low regardless of growth conditions or
molybdate or molybdenum-cofactor availability. A summary of our
findings regarding the regulation of moa is shown in Fig. 6.
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70 ModE has been best characterized as a transcriptional repressor for the
modABCD operon (8, 38). Although molybdate-ModE has been shown to mediate enhanced expression of dmsA under
certain conditions, its mode of action is thought to be indirect,
namely that of a transcriptional repressor of a direct repressor of
dmsA (21). Recently, molybdate-ModE has been
proposed as a secondary transcriptional activator of both
hyc and narG (34). In the case of
hyc, the ModE-molybdate complex is thought to bind about 95 bp upstream of the main formate-dependent positive transcriptional regulator, FhlA. In the regulation of narG, ModE-molybdate
does not bind directly to the narG promoter region but binds
to the narK-narXL intragenic region. This suggests that its
effect on narG is mediated via the narXL operon
(34). McNicholas et al. (22), by employing
DNaseI-protection experiments, defined the ModE binding site at
moa and we have confirmed this by gel shift analysis (data
not shown). The ModE-molybdate binding site at moa is
centered at 57.5 with respect to the proximal moa
transcription start site and, therefore, is adjacent to the 35 region
of this promoter. The magnitude of the ModE-dependent molybdate
activation of moa, the direct binding of ModE to the
proximal moa promoter region, and the persistent low levels
of moa expression in modE strains point to
molybdate-ModE being a major direct, positive transcriptional regulator
of this operon. Previously, McNicholas et al. (22) reported,
from analysis of a molybdenum cofactor-sufficient strain, a twofold,
ModE-dependent but molybdate-independent enhancement of moa expression.
The molybdate regulation of both the hyc and narG operons also displays a ModE-independent, MoeA-dependent component (10). There is no evidence for such regulation of moa, since all the molybdate effects appear to be totally dependent upon ModE.
Enhanced expression of moa under anaerobic growth conditions has been reported previously (3). This effect, like the molybdate effect, is only fully apparent in strains that lack functional molybdenum cofactor. Only a modest twofold enhancement of moa to a very low level can be seen in molybdenum cofactor-sufficient strains. In molybdenum cofactor-deficient strains also lacking ModE activity, anaerobiosis elevates moa expression about eightfold over the aerobic levels but only to a relatively modest level (~800 Miller units). However, anaerobic growth of such strains with a functional ModE results in a further fourfold increase in moa expression (Fig. 1). Under aerobic growth conditions, the ModE-dependent molybdate activation effects a 10-fold or so enhancement to an intermediate (1,100 units) level of expression. ModE-molybdate, therefore, is required for full moa expression.
The molybdate- and anaerobic-dependent enhancements of moa appear independent and distinct. This interpretation is consistent with our finding of two promoters at moa. Analysis of expression from the individual moa promoters clearly showed that the global anaerobic transcriptional regulator, Fnr, acts positively at the highly active, distal moa promoter. Full expression of moa, during anaerobic growth with high molybdate availability, requires the action of the two positive regulators, ModE and Fnr. ModE and Fnr mediate their effects specifically and distinctly at the proximal and distal promoters, respectively. The promoter subclones containing only the proximal promoter generally displayed poor expression. Given the close proximity of the transcriptional start sites, it is likely that the proximal promoter region overlaps the distal transcription start. Further work is required to illuminate how expression from each of the two promoters is related to that of the other.
The molybdate and anaerobic control of moa described above is apparent only in strains that are molybdenum cofactor-deficient. That molybdenum cofactor sufficiency leads to tight moa repression was identified by Baker and Boxer (3). It is abundantly clear from the present work that the molybdenum cofactor-dependent repression of moa is dominant over both the molybdate and anaerobic effects. The mechanism of this repression has not been established but it appears from the analysis of the promoter subclones that the repression is mediated at the proximal promoter region. Experiments designed to isolate a molybdenum cofactor-dependent repressor are currently under way.
A large enhancement of moa transcription is observed when tungstate is present in the growth medium. ModE binds tungstate in a virtually identical manner to molybdate, and ModE-tungstate can bind to the modA promoter (2). In strains that are deficient in molybdenum cofactor it is clear that tungstate can also bring about a ModE-dependent activation of moa. Under these conditions, tungstate and molybdate are equivalent with respect to their positive effect on moa expression.
In strains genetically able to synthesize molybdenum cofactor, the effect of tungstate in the growth medium is more complicated. Under these conditions tungstate leads to a full derepression of moa via a mechanism that is independent of ModE. This demonstrates a second ModE-independent tungstate effect on moa which is dependent upon the capacity of the bacterium to synthesize active molybdenum cofactor in the absence of tungstate. It is known that tungsten can substitute for molybdenum in the molybdenum cofactor to form a biologically inactive derivative (16). We propose that the tungsten cofactor derivative cannot mediate moa repression. This gives a plausible explanation for this tungstate effect on moa. Either the tungsten-cofactor derivative is unable to bind to the putative molybdenum cofactor-repressor, or the tungsten cofactor-repressor complex cannot productively bind to the proximal transcriptional start region.
The moa operon encodes the enzymes required for the first dedicated step of molybdopterin synthesis. It is appropriate, therefore, that this locus should be subject to transcriptional control, and the expression of moa is indeed highly regulated. The regulation is generally consistent with what is known concerning molybdoenzyme biosynthesis in E. coli. The positive molybdate control should ensure that molybdopterin biosynthesis is down regulated under conditions of limited molybdate availability. When there is sufficient molybdenum cofactor, further molybdopterin synthesis would not be required. Almost all of the E. coli molybdoenzymes are present predominantly during anaerobic growth. The anaerobic molybdoenzymes nitrate reductases and DMSO reductase under fully induced conditions are of high abundance in the bacterium. This points to a relatively high requirement for the molybdenum cofactor during anaerobiosis. The anaerobic regulation of moa, therefore, acts to ensure appropriate molybdopterin availability for the synthesis of these metabolically important enzymes.
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ACKNOWLEDGMENTS |
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This work was supported by a Biotechnology and Biological Sciences Research Council grant to D. H. Boxer and a studentship to L. A. Anderson.
A. Bock, S. Busby, R. Eichenlaub, M. Berman, K. T. Shanmugam, and V. Stewart are thanked for gifts of strains or plasmids.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Biochemistry, MSI/WTB Complex, University of Dundee, Dundee DD1 5EH, Scotland, United Kingdom. Phone: 44 (0) 1382 345561. Fax: 44 (0) 1382 201063. E-mail: d.h.boxer{at}dundee.ac.uk.
Present address: Department of Biochemistry and Molecular Biology,
University of Melbourne, Parkville, Victoria 3010, Australia.
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REFERENCES |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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| 1. |
Amy, N. K., and K. V. Rajagopalan.
1979.
Characterization of molybdenum cofactor from Escherichia coli.
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Journal of Bacteriology, December 2000, p. 7035-7043, Vol. 182, No. 24
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