(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
(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.
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 plasmid is pUD10.
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. ()It was stored at -70 °C for subsequent use.
Protein concentration was measured according to Bradford(1976), with
bovine serum albumin as the standard.
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-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.
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 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.
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 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.
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 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).
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].