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(Received for publication, September 26, 1995; and in revised form, November 7, 1995) From the
The modABCD operon, located at 17 min on the Escherichia coli chromosome, encodes the protein components of
a high affinity molybdate uptake system. Sequence analysis of the modA gene (GenBank L34009) predicts that it encodes a
periplasmic binding protein based on the presence of a leader-like
sequence at its N terminus. To examine the properties of the ModA
protein, the modA structural gene was overexpressed, and its
product was purified. The ModA protein was localized to the periplasmic
space of the cell, and it was released following a gentle osmotic
shock. The N-terminal sequence of ModA confirmed that a leader region
of 24 amino acids was removed upon export from the cell. The apparent
size of ModA is 31.6 kDa as determined by gel sieve chromatography,
whereas it is 22.5 kDa when examined by SDS-polyacrylamide gel
electrophoresis. A ligand-dependent protein mobility shift assay was
devised using a native polyacrylamide gel electrophoresis protocol to
examine binding of molybdate and other anions to the ModA periplasmic
protein. Whereas molybdate and tungstate were bound with high affinity
(
Molybdenum is an essential trace metal for most bacteria as well
as for all plants and animals. High affinity uptake of molybdate in the
bacterium Escherichia coli proceeds by a specific transport
system encoded by the modABCD operon (Rech et al.,
1995). Following the uptake of molybdate into the cell, it is
subsequently reduced and then incorporated into the molybdenum
cofactor, molybdopterin guanine dinucleotide, which is required for the
assembly and function of several enzymes including nitrate reductase,
formate dehydrogenase, dimethyl-sulfoxide reductase,
trimethylamine-N-oxide reductase, and biotin-sulfoxide
reductase (Rajagopalan and Johnson, 1992). These enzymes, except
biotin-sulfoxide reductase, are synthesized primarily during the
anaerobic growth of E. coli and other enteric bacteria. They
participate in anaerobic respiration or fermentation reactions to aid
in cellular energy generation. The molybdate (modABCD)
transport operon of E. coli was recently sequenced and
characterized (Maupin-Furlow et al., 1995; Rech et
al., 1995; Johann and Hinton, 1987). It encodes a bacterial ABC
type transport system based on a comparison with other solute uptake
components including the maltose, histidine, and leucine-isoleucine
transporter proteins (Gilson et al., 1982; Higgins et
al., 1982; Ames, 1986; Shuman, 1987). The modA gene
product was predicted to encode a 28.6-kDa protein located in the cell
periplasm (Rech et al., 1995). By analogy to other bacterial
periplasmic binding proteins, the ModA protein binds molybdate and
transfers it to the ModB protein at the outer surface of the
cytoplasmic membrane. ModB in conjunction with the ModC protein then
transports molybdate across the cytoplasmic membrane to the cell
cytoplasm. ModC is proposed to contain an ATP hydrolase activity that
provides energy for the transport process (Rech et al., 1995);
the ATP dependence is predicted by the sequence similarity of the modC gene product to other bacterial ABC transporter genes
including hisP and malK (Ames, 1986). Mutations in
the modC gene severely impair molybdate accumulation and lead
to the inability of the cell to grow under conditions of low molybdate
(approximately below 1 µM). This phenotype can be overcome
by supplementing E. coli cells with exogenous molybdate at 100
µM (Scott and Amy, 1989). Molybdate transporters
similar to the E. coli modABCD have been recently reported in
other bacterial species including Azotobacter vinelandii, Rhodobacter capsulatus, and Haemophilus influenzae Rd based on cloning and DNA sequence studies (Luque et al.,
1993; Wang et al., 1993; Fleischmann et al., 1995).
In the nitrogen fixing bacteria, the molybdate transport process also
provides molybdenum for synthesis of the molybdenum containing cofactor
of nitrogenase as well as for the structurally distinct molybdopterin
cofactor that is made by E. coli and most other bacteria. At present, little is known about the operation of the ModABC
transport systems in bacteria. In this study we report the isolation
and characterization of the modA gene product from E.
coli. It is shown to be located in the cell periplasmic space and
to bind molybdate with high specificity and affinity. The pre-ModA
protein is processed upon secretion to give a mature periplasmic
protein. The binding specificity of the ModA protein is shown to extend
to the molybdate analog, tungstate, but not to other inorganic anions.
Finally, the requirement for ModA protein in growth of E. coli cells in the presence of low concentrations of molybdate is
documented.
Anaerobic cell growth experiments
(see Fig. 7) were performed by growing the indicated strain in
10 ml of anaerobic minimal medium that contained 25 mM sodium
nitrate and 4% glycerol as described previously (Rech et al.,
1995). Where indicated, sodium molybdate was added to a final
concentration of 10 µM. The cells used for inoculation
were grown under the same conditions overnight.
Figure 7:
Effect of sodium molybdate addition on
cell growth of a wild-type and a modA mutant strain. Cell
growth was performed in a minimal salts medium containing nitrate (25
mM) and glycerol (4%) as described (see ``Experimental
Procedures''). Sodium molybdate was added to a final concentration
of 10 µM. The wild-type strain is MC4100, while the modA mutant is LK82RG77; the modA
The dialyzed protein suspension was clarified by
centrifugation for 15 min at 13,000 rpm. The supernatant fraction was
then loaded onto a Mono S HiTrap column (Pharmacia Biotech Inc.), which
was preequilibrated with 50 mM potassium acetate buffer, pH 5,
at room temperature. The ModA protein was eluted from the column using
a linear KCl gradient. The fractions containing the ModA protein were
pooled and dialyzed against potassium acetate buffer. The purified ModA
protein was greater than 99% pure as judged by SDS-PAGE. (
Figure 1:
Purification
and localization of the ModA protein. The indicated E. coli cell or protein fractions were separated by SDS-PAGE as indicated
under ``Experimental Procedures.'' Lane 1, whole
cell proteins of wild-type strain; lane 2, cell shock
fraction; lane 3, whole cell protein of a strain containing
the overexpression plasmid; lane 4, cells following osmotic
shock; lane 5, cell shock protein; lane 6, purified
ModA protein. MW, molecular weight protein
standards.
The cellular location of the ModA
protein was determined by fractionating the cells into the cytoplasmic,
membrane, and periplasmic components (Fig. 1). ModA was
accumulated primarily in the periplasmic space, as revealed by its
release from the cell following an osmotic shock (Fig. 1, lane 5). The ModA protein was estimated to routinely comprise
over 75% of the total periplasmic proteins. Very little ModA protein
was found in either the particulate or soluble cell fractions. The
ModA protein was purified to homogeneity (see ``Experimental
Procedures'') and used for subsequent characterization studies.
The mature ModA protein exhibits an apparent subunit size of 22.5 kDa (Fig. 1). This differs from the 27.4-kDa size predicted from the
DNA sequence analysis (Rech et al., 1995; Maupin-Furlow et
al., 1995).
Figure 2:
Amino acid sequence at the N-terminal end
of the periplasmic ModA protein of E. coli. The predicted
sequence of the pre-ModA protein deduced by DNA sequence analysis is
aligned with the experimentally determined amino acid sequence of the
purified periplasmic ModA protein. The numbering is relative to the
predicted N-terminal methionine (GenBank accession number L34009). The arrow indicates the leader peptidase processing site for the
pre-ModA protein. The underline indicates the experimentally
determined N-terminal amino acid sequence of ModA from E.
coli. Single letter abbreviations of the amino acid code are used. Rcmod, R. capsulatus ModA protein; Avmod, A. vinelandii ModA protein.
Figure 3:
Ligand-dependent mobility shift assay for
the ModA binding protein. The ability of the ModA protein to bind
molybdate, tungstate, and other di- and monovalent anions was
determined as described under ``Experimental Procedures.''
The concentration of the sodium salts of the indicated anions was 10
mM.
Figure 4:
Effect of pH and molybdate on the
migration of the ModA protein in native polyacrylamide gels. The ModA
protein (2 µg) with or without sodium molybdate (100
µM) present was loaded onto individual isoelectric
focusing, pH 3-9, gels and run for 30 min according to the
manufacturer's instructions. Following fixing and staining of the
two gels, they were superimposed and photographed to show the relative
migration of the ModA protein at the indicated pH
values.
Figure 5:
UV-visible spectrum of the ModA
periplasmic binding protein in the presence and absence of molybdate
ions. The spectrum was taken of the ModA protein (0.2 mg/ml) using a 50
mM potassium acetate buffer, pH 5. Sodium molybdate was added
at a final concentration of 10
µM.
This ligand-dependent spectral change in ModA upon
binding of molybdate was further examined by taking the difference
spectra at higher resolution (Fig. 6). As evidenced by the
spectral properties of the ModA protein in the absence as well as in
the presence of increasing amounts of sodium molybdate, it was apparent
that molybdate binding alters the environment of the tryptophan and/or
tyrosine residues in the ModA protein. Upon addition of molybdate, two
absorption maxima at 281 and 287 nm were observed (Fig. 6A). Analysis of the bound versus free
ModA protein, based on the absorption maxima at 281 and 287 nm,
revealed an apparent disassociation constant for sodium molybdate of 3
µM. When analogous studies were performed using sodium
tungstate, a molybdate anion analog (Fig. 6B), the
absorption maxima of the ModA protein was similar to that revealed for
sodium molybdate except that the peaks were shifted slightly in the UV
range (i.e. blue-shifted). Titration of ModA protein with
sodium tungstate revealed an apparent disassociation constant of 7
µM at wavelengths of 280 and 286 nm.
Figure 6:
Effect of molybdate and tungstate ions on
the UV-visible difference spectrum of the ModA Periplasmic molybdate
binding protein. Panel A, Molybdate titration of the ModA
protein. The sodium molybdate concentrations indicated in µM at the right of each spectrum. The ModA protein was 6
µM. Absorbance scale is indicated in B. Panel
B, tungstate titration of the ModA protein. Conditions are as
described above except that sodium tungstate was used. An absorbance of
0.05 units is represented by the vertical
bar.
In this study, we report the overexpression, purification,
and characterization of the ModA periplasmic molybdate-binding protein
of E. coli. The purification procedure we used was similar to
the isolations of previous bacterial binding proteins (Ames, 1994) and
allowed us to rapidly purify ModA to homogeneity. The protein was
released from the periplasmic space using osmotic shock, concentrated,
and dialyzed into buffer at pH 5. The low pH treatment led to the
precipitation of many contaminating proteins and lipopolysaccharides,
but, as shown in previous reports (Ames, 1994), it did not harm the
periplasmic binding protein. The resistance of ModA to low pH allowed
us to load the protein preparation directly onto a cationic exchange
column as a final purification step. The overproduction of ModA
occurred from a multicopy plasmid containing the native modA promoter. Interestingly, ModA only accumulated to high levels when
cells were allowed to grow overnight to stationary phase. The
homogenous preparations of ModA were used to determine the biochemical
characteristics of the protein for comparison to other binding
proteins. The presence of a signal sequence was confirmed by
N-terminally sequencing the purified ModA protein. Comparison to the
nucleotide sequence shows that the first 24 amino acids have been
removed upon transport to the periplasmic space (Fig. 2). This
leader sequence has several characteristics that are common to
prokaryotic signal sequences (Oliver, 1987; Izard and Kendall, 1994).
The amino-terminal end has one positively charged amino acid, arginine
at position 3, which is followed by a stretch of predominantly neutral
amino acids that form the hydrophobic core. The peptide ends in the
consensus processing site AXB as described previously (Oliver, 1987) in
which A and B are alanine. We examined the ModA protein-ligand
interactions using isoelectric focusing, pH titration, a
ligand-dependent gel shift assay, as well as UV-visible spectroscopy
methods. It has been reported previously that the equilibrium for
formation of the enzyme ligand complex can shift the apparent pI of a
protein. Rudnick et al.(1990) showed that the pI of N-myristoyltransferase was shifted by 3 pH units when the
ligand was bound. This shift was not based on the calculated change of
the pI due to the additional charges provided by the ligand. Therefore
the observed shift in pI was thought to indicate a change in the
protein conformation caused by formation of a reaction intermediate. In
the case of the ModA protein, we observed a similar shift in the pI in
the presence of molybdate as seen in the pH titration curve in Fig. 4. When incubated with molybdate, the pI of the protein
decreased by about 1.4 pH units, which we interpret as a change in
protein conformation upon ligand binding. This may be due in part to
the two negative charges of molybdate that may balance positive charges
on the protein upon binding of the anion. Further studies are needed to
resolve this possibility. Both molybdate and tungstate ions had the
ability to influence the mobility of ModA on native PAGE gels (Fig. 3). No other inorganic anions tested had the same effect
even when present at 10 The ModA mobility shift seen using the Native Phast gels
(Pharmacia) also allowed us to determine the apparent dissociation
constants for molybdate and tungstate binding to ModA. These values are
in the range reported for K The
molybdate and tungstate ligand-dependent ModA mobility shift data and
the observation that the ModA pI changes upon ligand binding indicate
that the protein undergoes a conformational change when it binds
ligand. Similar conclusions have been made for the leucine, isoleucine,
valine binding protein of E. coli using x-ray scattering and
computer modeling approaches (Olah et al., 1993). We further
investigated the ligand-dependent changes in ModA protein conformation
by using UV spectroscopy and limited proteolysis methods. Addition of
molybdate resulted in an increase in the absorption maximum observed at
281 nm as well as the appearance of the absorbance peak at a higher
wavelength (i.e. red-shift to 287 nm) (Fig. 5). These
observations indicate that one or more tryptophans or tyrosines
experience a change to a more hydrophobic environment upon binding of
ModA to molybdate (Copeland, 1995). It has been shown recently that
a tryptophan residue is also involved in the interaction of sulfate
with the sulfate periplasmic binding protein (Pflugrath and Quiocho,
1988). The absorbance difference spectra of ModA show two absorption
maxima at about 281 and 287 nm, which increase with the addition of up
to 10 µM of ligand. The K Limited proteolysis of the ModA protein using either trypsin or
chymotrypsin did not yield two polypeptides corresponding to the two
domains typical of several other periplasmic binding proteins including
the leucine, isoleucine, valine and sulfate binding proteins (Adams and
Oxender, 1989). Even though no stable polypeptide intermediates were
observed, the binding of molybdate to ModA slowed the proteolytic
attack. This suggests that the protein-ligand complex has reduced
solvent accessible sites for the protease. In support of the
biochemical data for ModA in its role as the periplasmic molybdate
binding protein, we were able to show that this protein is required for
molybdate uptake by E. coli in vivo. A modA mutant
could not respire with nitrate unless complemented with a modA In summary, using
biochemical methods as well as in vivo studies, we have been
able to demonstrate that modA, the first gene of the modABCD operon, encodes the periplasmic molybdate binding
protein (Table 2). This protein has a characteristically low
homology to other binding proteins except for the presence of a leader
peptide (Fig. 2). The specificity of protein-ligand interaction,
and the requirement of ModA for molybdate transport in vivo confirm its role as a periplasmic binding protein. Analysis of the
ModA crystal structure data should reveal what tertiary features are
shared between ModA and the other periplasmic binding proteins, and it
should aid in elucidating the amino acid interactions of ModA with the
molybdate and tungstate ligands.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank(TM)/EMBL Data Bank with accession number(s)
L34009[GenBank].
Volume 271,
Number 5,
Issue of February 2, 1996 pp. 2557-2562
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
5 µM), sulfate, chromate, selenate, phosphate, and
chlorate did not bind even when tested at 2 mM. A UV spectral
assay revealed apparent K
values of
binding for molybdate and tungstate of 3 and 7 µM,
respectively. Strains defective in the modA gene were unable
to transport molybdate unless high levels of the anion were supplied in
the medium. Therefore the modA gene product is essential for
high affinity molybdate uptake by the cell. Tungstate interference of
molybdate acquisition by the cell is apparently due in part to the high
affinity of the ModA protein for this anion.
Bacterial Strains, Bacteriophages, and
Plasmids
The genotypes and origins of the E. coli K-12
strains, bacteriophage, and plasmids used in this study are given in Table 1.
Cell Growth
For plasmid, phage, and strain
manipulations, cells were grown Luria broth or on solid media. When
required, ampicillin was added to the medium at a concentration of 100
mg/liter. Aerobic growth and anaerobic growth were performed as
described previously (Rech et al., 1995). When desired, sodium
molybdate was added at a final concentration of 100 µM (Rech et al., 1995).
plasmid is pUD10.
Overproduction and Purification of the ModA
Protein
For ModA protein production, strain MC4100 containing
plasmid pModAT7 was grown to stationary phase in a LB medium
supplemented with ampicillin (100 µg/ml). Following cell harvest,
the periplasmic fraction of the cells was isolated using an osmotic
shock protocol as follows. Cells were chilled to 4 °C and harvested
by centrifugation at 8,000 rpm for 8 min in a Sorvall T centrifuge
(DuPont, Inc.). The spent cell growth medium was discarded, and the
cell pellet was resuspended in osmotic shock buffer (0.5 M sucrose, 0.1 M Tris-Cl, pH 8.2, and 1 mM EDTA)
at a ratio of 5 ml/1 g of cells (wet weight). The cells were placed on
ice for 5 min and then centrifuged as described above. The supernatant
was discarded, and the cell pellet was resuspended in distilled water;
a 20 mM MgCl
solution was then added to a give a
final concentration of 1 mM MgCl. The cell
suspension was centrifuged at 10,000 rpm for 10 min in a Sorvall T
centrifuge, and the cell shockate was removed and placed on ice for
subsequent ModA protein purification. The periplasmic proteins were
concentrated by the addition of solid ammonium sulfate to a final
concentration of 60% (w/v). The solution was then stirred gently for 1
h, and the precipitated material was removed by centrifugation for 20
min at 4 °C (10,000 rpm). The supernatant fraction was brought to a
final ammonium sulfate concentration of 90% and then stirred for an
additional hour. The precipitated proteins were harvested by
centrifugation as described above, resuspended in 50 mM potassium acetate buffer at pH 5, and dialyzed overnight against
the same buffer.
)It was stored at -70 °C for subsequent use.
Protein concentration was measured according to Bradford(1976), with
bovine serum albumin as the standard.Gel Electrophoresis of the ModA Protein
SDS-PAGE
analysis was performed as described previously (Laemmli, 1970). Native
protein gel electrophoresis was done according to the instructions
provided with a Pharmacia Phast protein system (Pharmacia). The
isoelectric point of the ModA protein was determined experimentally by
use of a Pharmacia Phast gel system and gels according the
manufacturer's instructions. The pI was also calculated from the
amino acid sequence information by using the PeptideSort routine of the
University of Wisconsin Genetics Software package (Devreux et
al., 1984).Amino Acid Analysis and N-terminal Sequence
Determination
The amino acid composition of the purified ModA
was performed using a Waters HPLC amino acid analyzer. The N-terminal
amino acid sequence of ModA was obtained using an Applied Biosearch
amino acid analyzer. The predicted size from the ModA protein as
determined from the DNA sequence analysis differed from the
experimentally determined sequence (see ``Results'').ModA Protein Gel-shift Assay
A ligand-dependent
protein gel-shift assay was developed to detect molybdate binding to
the ModA protein based on the migration of the pure protein in native
polyacrylamide gels. The standard assay was performed as follows.
Aliquots of purified ModA protein (9 µg) were preincubated with the
indicated concentrations of anion (e.g. sodium molybdate) in
molybdate binding buffer composed of 50 mM potassium acetate,
pH 5, for 20 min on ice. Samples were then applied to a native 12.5%
polyacrylamide gel (Pharmacia), and the protein was electrophoresed in
the Phast System (Pharmacia) at 250 V and 4 °C for 30 min. The gel
was then acid fixed and stained with Commassie R-350 dye (Pharmacia) to
visualize the relative mobility of the ModA protein. The binding of
molybdate to ModA resulted in a marked mobility shift (see
``Results''). The ability of ModA to bind other inorganic
anions was also tested using the same protocol; the anion
concentrations ranged from 2 µM to 10 mM.Spectral Analysis
UV-visible spectra were obtained
using a Kontron Uvikon 810 spectrophotometer. Sodium molybdate and
sodium tungstate stock solutions (1 mM) were used for
titration binding studies with the ModA protein. A ModA protein
concentration of 6 µM was used unless indicated otherwise.
The extinction coefficient of the ModA protein was determined at a
wavelength of 280 nm.Materials
Ampicillin was purchased from Sigma.
Sodium molybdate and sodium tungstate were purchased from Fluka
Chemical Co. (Ronkonkoma, New York). All other reagents used were of
reagent grade.
Overexpression and Purification of the ModA
Protein
The modA gene of E. coli was recently
isolated, and its DNA sequence was determined (see Rech et
al.(1995); sequence accession L34009). In order to purify and
characterize the modA gene product, the modA gene was
cloned into the plasmid expression vector, pGem7-, and the resulting
plasmids that contained modA gene inserted in each orientation
were identified and stocked (see ``Experimental
Procedures''). Following the introduction of each modA expression plasmid into E. coli strain MC4100, cells were
grown to mid-exponential phase (A = 0.9),
and ispropyl-1-thio-
-D-galactopyranoside was added to
induce ModA protein synthesis. At 1, 2, and 3 h postinduction, no
accumulation of ModA protein was observed by SDS-PAGE analysis of total E. coli proteins (data not shown). However, when strain MC4100
containing the modA expression plasmid pModAT7 was grown to
stationary phase (i.e. 18 h postinoculation), a significant
amount of the ModA protein was accumulated as evidenced by the
appearance of a 22.5-kDa protein (Fig. 1, lane 3)
compared with cells that lacked the plasmid (lane 1). No other
growth conditions or induction protocols tested were found to improve
the yield of the ModA protein.
Processing Site of the pre-ModA Protein
The
purified ModA protein was amino acid sequenced at its N-terminal end.
The sequence was DEGKITV (Fig. 2): this revealed that the
N-terminal 24 amino acids of the 257-amino acid pre-ModA protein were
removed following its synthesis and secretion to the periplasmic space,
presumably by the E. coli leader peptidase activity. The
apparent processing site for pre-ModA is similar to that exhibited by a
variety of other E. coli periplasmic proteins including
alkaline phosphatase, and -lactamase (Oliver, 1987). The amino
acid composition of the mature ModA protein was also determined and
found to be in excellent agreement with that predicted from the
nucleotide sequence (data not shown; Fig. 2).
Properties of the ModA Protein
The native size of
the mature ModA protein was estimated by superose gel chromatography
(Pharmacia) and gave an apparent molecular mass of 31.6 kDa (data not
shown). These results suggest that the shape of the ModA protein may be
nonspherical since the denatured size as determined by SDS-PAGE was
22.5 kDa (Fig. 1). The isoelectric point of ModA was
experimentally determined to be 7.0 and thus in good agreement with
that calculated from the amino acid sequence of the mature ModA protein (i.e. pH of 6.82, see ``Experimental Procedures'').
Interestingly, when the ModA protein was preincubated with sodium
molybdate (100 µM), it gave a different apparent pI value
of 5.6. It also showed an increased mobility in native polyacrylamide
gels (see below). These results demonstrate that the ModA protein
undergoes a significant change in its solvent exposed charge and/or
shape.Molybdate Binding to ModA
The molybdate-dependent
ModA mobility-shift phenomenon was exploited to examine the binding
affinity of the ModA protein for molybdate and for other inorganic
anions. A native polyacrylamide gel assay was developed for these
studies (see ``Experimental Procedures''). As shown in Fig. 3, both molybdate and tungstate were bound to ModA when
present at relatively low concentrations (10 µM).
Titration of the ModA protein with sodium molybdate and with sodium
tungstate at lower concentrations ranging from below 1 µM to 10 µM followed by native polyacrylamide gel
electrophoresis allowed us to estimate the apparent disassociation
constant for each anion. A K
value of
approximately 5 µM was revealed for binding of either
molybdate or tungstate.
Specificity of the ModA Protein for Binding Divalent
Anions
The ability of ModA to bind a number of other anions was
also tested (Fig. 3). Of the compounds screened at
concentrations up to 10 mM, none were able to cause a mobility
shift like that seen for either molybdate or tungstate. The anions
include arsenate, sulfate, phosphate, nitrate, chlorate, selenate,
metavanadate, perchlorate, and permanganate. Thus, the ModA protein
appears to exhibit a high specificity for only molybdate and its analog
tungstate.Effect of pH on ModA-Molybdate Binding
To examine
the pH dependence of the mobility shift, ModA was run in a pH gradient
native gel following preincubation with and without molybdate present (Fig. 4). A noticeable mobility shift was seen for ModA across
the entire pH range tested from pH 3 to 9. It appears that ModA protein
is able to effectively bind molybdate at any of these pH values; the
mobility of ModA increases toward the anode at increasing pH. The
relative displacement between the molybdenum bound and unbound forms of
ModA increased from pH 3 to 4.5, and then remained relatively constant
until a pH value of 6.5 to 8 was achieved.
UV-Visible Absorption Spectrum of ModA
The
UV-visible absorption spectrum of the ModA protein was examined in the
absence and presence of sodium molybdate (Fig. 5). As
anticipated from the amino acid content of the ModA protein, an
absorption maximum at 280 nanometers was seen for the protein when no
ligand was present. The addition sodium molybdate to a final
concentration of 10 µM caused a slight but noticeable
change in the ModA spectrum in the high UV wavelength range; a shoulder
appeared on the ModA absorption peak at about 290 nm, while the maximum
absorbance peak at 280 nm was enhanced relative to ModA when no ligand
was present.
Proteolytic Susceptibility of ModA
Limited
proteolysis of the ModA protein was performed in the presence and
absence of sodium molybdate to determine if it undergoes a
conformational change upon binding of ligand. When ModA was incubated
for 40 min with chymotrypsin at 1 µg/ml, it was partially digested
as evidenced by a reduction in the intensity of the 22.5-kDa species
(data not shown). No long-lived intermediate cleavage products were
detected. However, when sodium molybdate was also present during the
digestion, significantly less degradation of ModA was observed. These
studies support the proposal that ModA can exist in either of two
protein conformations depending on whether molybdate is bound or not.
In control experiments to test if molybdate affected the activity of
chymotrypsin, ovalbumin was digested as effectively when molybdate was
present versus when absent.Identification of an E. coli modA Phenotype
To
determine if the wild-type modA gene is
required for molybdate-dependent expression of E. coli fumarate reductase (frdABCD) genes, we screened a number
of previously isolated mod mutants (e.g. chlorate
resistance) for defects in the modA gene using the procedure
of Kalman and Gunsalus(1988, 1990). Based on the ability of a modA
plasmid to restore the
molybdate-dependent nitrate repression of frdA-lacZ gene
expression, one modA mutant was identified. It was designated E. coli strain LK82RG77. The mod phenotype could also
be suppressed when the modA mutant was grown in the presence
of 100 µM sodium molybdate. Additionally, a wild-type copy
of the modA
gene was found to be essential
for molybdate-dependent regulation of the anaerobic respiratory pathway
genes, narGHJI and frdABCD (data not shown). These
genes encode the respiratory nitrate reductase and fumarate reductase
enzymes required for anaerobic respiration to nitrate and fumarate,
respectively (Kälman and Gunsalus, 1990).
Requirement of the ModA Binding Protein for Cell Growth
under Molybdate-limiting Conditions
The phenotype of the modA mutant identified above suggested that a defect in the modA gene might prevent anaerobic cell growth of the mutant on
a glycerol-nitrate medium under molybdenum limiting conditions. To test
this, the modA strain was grown in the minimal medium without
sodium molybdate added (Fig. 7). The mutant was unable to grow,
presumably due to an inability to transport low levels of molybdate,
whereas it could grow when transformed with the modA plasmid. These results demonstrate that
the modA encoded molybdate-binding protein is an essential
component of the high affinity molybdate transport system encoded by
the E. coli modABCD operon. Since one of the complementing
plasmids only contained the modA
gene, the
original modA defect was apparently not due to a polar
mutation. When the modA or wild-type cells were grown in
medium supplemented with 10 µM molybdate, they grew as
well as the wild-type strain in medium lacking molybdate, conditions
where molybdate apparently enters the cell via a different route.
-fold higher levels than used for
molybdate binding (10 mM). The specificity of the
molybdate interactions with ModA supports the previously proposed role
of the periplasmic protein in molybdate uptake (Rech et al.,
1995). These results are also consistent with the in vivo observation that molybdate as well as tungstate can be transported
by the molybdate uptake operon (Miller et al., 1987). It is
noteworthy that sulfate did not bind to the ModA protein, since it has
been suggested previously that when present at high concentrations
(>100 µM), molybdate is taken up via the sulfate
transporter (Lee et al., 1990). Our observations suggest that
sulfate is not likely to enter the cell via the ModABC transport
system.
values of other
binding proteins for their anions such as citrate (2 µM)
and phosphate (0.8 µM) (Tam and Saier, 1993). Therefore
the ligand-induced mobility shift assay appears to be a rapid and
reproducible method that should be applicable to examine the
specificity and affinity of other periplasmic binding proteins. values for
molybdate (3 µM) and tungstate (7 µM),
calculated based on the absorbance changes, show that ModA has a 2-fold
higher affinity for molybdate. The pattern of the difference spectra
confirm that tryptophan and/or tyrosine residues are in close vicinity
of the conformational change occurring in ModA upon molybdate binding.
Additionally the absence of a characteristic third peak at 292 nm
indicates that the largest contributor to the ModA spectrum appears to
be tyrosine (Copeland, 1995). Examination of the deduced amino acid
sequence of ModA (Fig. 2) reveals the presence of a pair of
tryptophans and one tyrosine at positions 106, 124, and 133,
respectively, relative to the ModA N terminus. This region is thus
likely to be involved in molybdate binding. However, direct evidence
for the position of the binding site needs to be established from the
examination of the crystal and/or NMR structures of the ModA protein. plasmid (Fig. 7). The modA mutant also had a similar phenotype to a modC mutant for
chlorate resistance (data not shown and Rech et al.(1995)).
The present studies demonstrate that a modA mutant containing
a defect in the molybdate binding protein is unable to take up the
trace amounts of molybdate present in the medium even though the ModBCD
proteins are apparently present (Fig. 7).
)
We thank A. Fowler for amino acid analysis and
N-terminal sequencing of the ModA protein.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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