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INTRODUCTION |
Disulfide bonds are essential for the correct folding and
stability of many exocytoplasmic proteins (2). The oxidation of
cysteine residues to form disulfide bonds can occur spontaneously in
the presence of molecular oxygen. However, air oxidation is a slow,
mechanistically complex reaction whose time scale is much longer than
what is required for the folding of proteins under biosynthetic
conditions, i.e. in the cell. As a result, both prokaryotic and eukaryotic cells have evolved elaborate enzymatic mechanisms for
the catalysis of disulfide bond formation and for maintaining the
proper thiol-disulfide redox balance in various cellular compartments.
In Gram-negative bacteria, disulfide bond formation normally occurs
following export into the periplasmic space, which is topologically
equivalent to the endoplasmic reticulum, albeit substantially more
oxidizing (3, 4). Genetic and biochemical studies have unequivocally
defined four proteins (DsbA, DsbB, DsbC, and DsbD) that are involved in
the formation of disulfide bonds in secreted proteins (5). All the
known Dsb proteins contain a Cys-X-X-Cys sequence
that is characteristic of the thioredoxin superfamily (5). DsbA is a
soluble periplasmic enzyme that serves as a potent catalyst of protein
and peptide cysteine oxidation (6-8). It also exhibits some disulfide
isomerization activity that may be important under some conditions
in vivo (9-11). Once it has transferred its disulfide bond
to a substrate, DsbA is rapidly reoxidized by the membrane protein
DsbB, which in turn transfers its electrons either to molecular oxygen
or to the quinone system (12, 13). The oxidation of protein cysteines
by DsbA is very rapid but often results in the formation of incorrect disulfide bonds. The rearrangement of nonnative disulfides is catalyzed
primarily by the dimeric periplasmic enzyme DsbC (8, 14, 15). For DsbC
to be able to catalyze disulfide bond isomerization, its active site
Cys-X-X-Cys sequence must be present in the
dithiol form. Although the redox potentials of DsbA and DsbC are
comparable,
89 mV (7) and
96 mV (calculated from Ref. 8),
respectively, under steady state conditions in the periplasm DsbA is
oxidized, whereas DsbC is almost exclusively reduced (10, 16, 17). Reduction of DsbC is mediated by cytoplasmic membrane protein DsbD and
also depends on the cytoplasmic proteins thioredoxin (TrxA) and
thioredoxin reductase (TrxB) (10, 17).
In addition to dsbA, dsbB, dsbC, and
dsbD, Missiakas and Raina (18) have isolated additional
genes that affect sensitivity to dithiothreitol
(DTT).1 One of these genes
was named dsbE and has been proposed to play a role in
oxidative protein folding. However, dsbE is identical to
ccmG, which has been shown to be an inner membrane protein involved in cytochrome biosynthesis in Escherichia coli and
other bacteria (19-21). It is not yet known whether DsbE affects the oxidation state of proteins other than cytochrome c.
Recently, Raina and co-workers (1) isolated a gene that functioned as a
multicopy suppressor of DTT sensitivity in a
dsbB
background. The same gene
(dsbG) was also isolated in a search for mutants conferring
increased sensitivity to DTT and an increase in
E-dependent periplasmic heat shock response.
Interestingly, Andersen et al. (1) reported that
dsbG is required for growth unless the cells are provided
with exogenous oxidants. This is surprising, since none of the other
dsb genes, including dsbA, which encodes the main
catalyst of protein oxidation in the periplasm, is essential. In
addition, Andersen et al. (1) reported that dsbG
can catalyze insulin reduction in vitro and is partially
responsible for the oxidation of alkaline phosphatase in the periplasm.
We independently cloned and expressed DsbG and characterized its
in vivo and in vitro function in detail. Contrary
to the previous report, we show that dsbG is not an
essential gene in E. coli; it is maintained by DsbD
exclusively in reduced form, and it does not appear to affect protein
oxidation in the periplasm. On the other hand, multicopy expression of
dsbG suppressed the effect of a
dsbC
mutation on the folding of heterologous
multidisulfide substrates. DsbG was found to have a very unstable
disulfide much like DsbA and DsbC, but unlike these two proteins it
appears to have narrow substrate specificity. Our results indicate that
DsbG functions predominantly either as a reductant or as a catalyst for
disulfide isomerization.
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EXPERIMENTAL PROCEDURES |
Cloning and Expression--
The strains and plasmids used in
this work are listed in Table I. The 5-kbp
Eco52I/SalI fragment containing the E. coli
dsbG gene from Kohara clone 166 (22) was isolated and cloned into Eco52I/SalI-digested pBR322, generating plasmid
pPBdsbG. The dsbG coding region was amplified from pPBdsbG
by polymerase chain reaction (PCR) using the primers
5'-AGGAATTCAGGAGGTCTCTCATGTTAAAAAAGATACTTTTAC-3' and
5'-CCATCCATGAGGATCCTTTTATTTATTCCCCATAAT-3'. The PCR product was
digested with BsaI and BamHI and ligated into
NcoI/BamHI-digested pTrc99A (Amersham Pharmacia
Biotech) or pET-11d (Novagen, Madison, WI), generating plasmids
pTrcdsbG2 and pETdsbG2, respectively. Likewise, dsbG without
its stop codon was amplified from pPBdsbG with the same forward primer
and reverse primer 5'-CTAGAGGATCCTCGAGTTTATTCCCCATAATGATATT-3', digested with BsaI and XhoI, and ligated into
pET-28a (Novagen, Madison, WI) that had been cut with NcoI
and XhoI. The resulting plasmid, designated pETdsbG2his,
contains the coding sequence for DsbG fused to a 6× histidine tag at
its C terminus and under the control of the T7 promoter. Plasmid
constructions were verified by automated DNA sequencing.
Expression and Purification of DsbG--
Plasmid pETdsbG2his was
transformed into strain BL21(DE3), and histidine-tagged DsbG was
purified from the osmotic shockate using nickel-chelate chromatography,
following standard protocols (Qiagen, Santa Clarita, CA). The purity of
DsbG in the nickel-SepharoseTM eluant, was approximately 95% as judged
by SDS-PAGE and Coomassie Brilliant Blue staining. This material was
used to raise polyclonal antisera in mice using standard protocols
(23). Rabbit polyclonal antisera against DsbA and DsbC were a gift of
John Joly (Genentech, S. San Francisco, CA). Purified DsbA was
purchased from Boehringer Mannheim. Purified DsbC was a kind gift of
John Joly. All other chemicals were purchased from Sigma except as noted.
To purify DsbG expressed without a hexahistidine affinity tag, E. coli BL21(DE3) harboring the plasmid pETdsbG2 were grown in LB
medium with 50 µg/ml ampicillin at 37 °C to midlog phase (A600 ~ 0.5). Cells were then transferred to a
25 °C water bath and induced with 0.5 mM IPTG for 8 h to maximize the concentration of soluble, mature protein in the
periplasm. Immediately after induction, the cells were collected by
centrifugation, and periplasmic fractions were obtained by the cold
osmotic shock procedure as modified by Thorstenson et al.
(24). The periplasmic fraction was dialyzed against 30 mM Tris-HCl, pH 8.5, 0.5 mM EDTA, and 50 mM NaCl and applied to a DEAE anion exchange column
(Bio-Rad), equilibrated with the same buffer. The column was developed
with a linear gradient of NaCl from 50 to 300 mM. Analysis
of the collected fractions by SDS-PAGE showed that DsbG eluted between
100 and 150 mM NaCl. The peak fractions were then pooled,
concentrated, and dialyzed against 10 mM Tris-HCl, pH 8.0, 0.5 mM EDTA buffer and applied to a
SephadexTM-75 sizing column (Amersham Pharmacia Biotech)
previously equilibrated with the same buffer. Densitometric analysis of
a scanned SDS-PAGE gel revealed that following gel filtration the
protein was at least 95% pure. Matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry was performed on
a Perseptive Biosystems Voyager Biospectrometry Workstation with a mass
resolution of greater than 95 and error of less than 0.07%.
Construction of dsbG
Mutant
Strains--
Plasmid pPBdsbG was digested with AflII and
Sse8387I; the large fragment was isolated, and its ends
blunted with T4 polymerase. This fragment was then ligated with
either the 1.4-kbp BsaAI fragment of pACYC184 containing the
chloramphenicol resistance gene to generate pPB
dsbG::Cm or
the 1.3-kbp kanamycin resistance cassette from pUC-4K to generate
pPB
dsbG::Kan. Plasmid pPB
dsbG::Cm was linearized and transformed into the recD
strain D301 (25), and CmR, AmpS colonies were
selected. Additionally, plasmid pPB
dsbG::Kan was digested
with MluI and SphI, and the fragment containing
the KanR cassette with the dsbG chromosomal
flanking regions was recovered and cloned into pBAD39, a vector
containing a conditional IPTG-requiring replicon, an ampicillin
resistance gene, and a wild type rpsL gene (Table
I). MC4100 cells transformed with the
resulting plasmid, pBAD
dsbG::Kan, were grown
first in liquid medium without IPTG and then plated on LB agar
containing kanamycin, and streptomycin for counterselection. Colonies
that were KanR, StrepR, and AmpS
were selected, and correct constructs were confirmed both by immunoblotting to verify the absence of a DsbG band and by PCR using
primers flanking the deletion region. The
dsbG::Cm and
dsbG::Kan
mutations were transduced into different strains using P1vir following
standard protocols (26).
Expression of BPTI and Urokinase--
Bovine pancreatic trypsin
inhibitor (BPTI) expression was monitored by enzyme-linked
immunosorbent assay as described previously (27). Urokinase activity
was detected by indirect chromogenic assay as follows. Cultures were
grown at 37 °C to A600 = 0.7, and protein
synthesis was induced by adding IPTG to 1 mM. The cells
were harvested 3 h later and lysed by French®
pressure cell. Following centrifugation to remove insolubles, the total
soluble protein was quantified by the Bradford assay (Bio-Rad), using
bovine serum albumin as a standard, and then diluted to 0.3 µg/µl
in 50 mM Tris-HCl, pH 7.4, 0.01% Tween 80. In a microtiter
plate, 50 µl of whole cell lysate soluble fraction was mixed with 50 µl of human plasminogen (Calbiochem), 0.1 µg/µl in 50 mM Tris-HCl, pH 7.4, 0.01% Tween 80, 6 mM
6-aminohexanoic acid. The plasminogen substrate, 50 µl of 4 mM Spectrozyme® PL (American Diagnostica,
Greenwich, CT), was added immediately, and the plate was incubated at
room temperature for 60 min. The absorbance at 405 nm was measured and,
after subtracting the background activity of a strain not expressing
urokinase, compared with a standard curve prepared using human
urokinase (Calbiochem).
Redox Properties of DsbG--
The in vivo redox state
of DsbG was assayed by derivatization of free thiols by
4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid (AMS) (Molecular
Probes, Inc., Eugene, OR) under denaturing conditions essentially
as described for DsbC (10). The oxidized standard was purified DsbG
without AMS added. The reduced standard was generated by reducing
purified DsbG in 40 mM DTT for 20 min at room temperature
followed by removal of reducing agent using a gel filtration spin
column (Bio-Rad) and AMS derivatization.
The redox equilibrium of DsbG with glutathione was assayed as described
for DsbA (7). In this assay, the change in fluorescence intensity
(excitation wavelength 280 nm) was measured at the wavelength of
maximum emission (330 nm for DsbG and 324 nm for DsbA). Experiments were carried out in 100 mM sodium phosphate, pH 7.0, and
1.0 mM EDTA. Oxidized DsbG or DsbA (0.45 µM)
was incubated at 25 °C in the presence of 0.1 mM GSSG
and 0-2.0 mM GSH for 12 h before recording the
fluorescence emission in an SLM-Aminco Luminescence Spectrometer (series 2). The equilibrium concentrations of GSH and GSSG were calculated according to Equations 1-3,
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(Eq. 1)
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(Eq. 2)
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(Eq. 3)
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where [GSH]0 and [GSSG]0 are the
initial concentrations of GSH and GSSG, R is the relative
amount of reduced protein at equilibrium, [Dsb]0 is the
initial concentration of DsbG or DsbA in the oxidized form,
F is the measured fluorescence intensity, and
Fox and Fred are the
fluorescence intensities of completely oxidized and reduced protein.
The equilibrium constant Keq was estimated from
nonlinear regression analysis of the data according to Equation 4
(28).
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(Eq. 4)
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Chemical Cross-linking--
Chemical cross-linking was used to
determine the oligomeric state of DsbG. The reactions were performed by
adding 0.1 volumes of Me2SO containing various
concentrations (0-5 mM) of the amine cross-linker ethylene
glycol-bis(succinimidylsuccinate) (EGS) (Pierce) to protein solutions
containing 250 µg/ml of purified DsbG or DsbA. Each reaction was
incubated at 4 °C for 30 min and then quenched by the addition of
glycine to 75 mM. The samples were resolved by SDS-PAGE
(10-20% resolving gels), and the cross-linked products were
visualized by Coomassie Brilliant Blue staining.
In Vitro Oxidoreductase Activity Assays--
The ability of
DsbA, DsbC, and DsbG to catalyze the reduction of human insulin (Sigma
catalog no. I-5523) in the presence of DTT was tested as described
previously by Holmgren (29). A stock solution of 5 mM
insulin was freshly prepared in 0.1 M potassium phosphate
buffer, pH 7.0, and 2 mM EDTA before each assay. The
reaction mixtures were prepared directly in cuvettes using 0.1 M potassium phosphate buffer, pH 7.0, 2 mM
EDTA, 131 µM insulin, and various concentrations of Dsb
proteins (typically between 5 and 15 µM) in a final
volume of 0.8 ml. The reactions were started by adding DTT to a final
concentration of 0.35 mM. After thorough mixing, the
cuvettes were placed in the spectrophotometer, and measurements were
performed at 650 nm every 30 s. In all of the experiments, the
uncatalyzed reduction of insulin by DTT was monitored in a control
reaction without the addition of Dsb proteins.
The preparation of fully reduced, denatured RNase and refolding assays
were performed as described previously (30). The recovery of
ribonuclease activity resulting from oxidative renaturation was
measured in a UV-visible spectrophotometer using cCMP as a substrate
for the RNase. The reaction mixtures consisted of 50 mM
Tris acetate buffer, pH 8.0; 4.5 mM cCMP; GSH and GSSG (at predetermined concentrations to provide a redox buffer); pure DsbG or
DsbC at concentrations ranging from 1 to 20 µM or 1.5 µM bovine PDI as a positive control. After equilibrating
the reaction at 25 °C, the assay was initiated by the addition of
reduced, denatured RNase to a final concentration of 8 µM. Hydrolysis of cCMP by the refolded RNase was
monitored every 30 s for 1 h as an increase in the absorbance
at 296 nm. As a negative control, the uncatalyzed reaction was
recorded in parallel under identical redox conditions. The
concentration of active RNase at any time in each assay was calculated
as described by Lyles and Gilbert (30).
 |
RESULTS |
Cloning and Expression of dsbG--
A BLAST (31) search of the
E. coli genome indicated the presence of an open reading
frame,2 having 49%
similarity and 30% identity to 220 residues of the E. coli
DsbC protein. Because of the high degree of similarity between DsbC and
the identified hypothetical protein, we reasoned that it may have a
role in the formation of disulfide bonds in the periplasmic space.
While this work was in progress, Raina and co-workers (1) identified
the same open reading frame in a genetic screen for resistance to
dithiothreitol and named it dsbG (GenBankTM
accession no. AF000956). We cloned the complete 248-amino acid coding
region of dsbG into a T7 expression vector fused to a 6×
histidine tag and purified the expressed protein by immobilized metal
affinity chromatography. The N-terminal sequence of the mature purified
protein was verified by automated Edman degradation, and cleavage of
the signal peptide was shown to occur after Ala17.
Antibodies were raised against the resulting material and used to probe
a Western blot of whole cell extracts of E. coli not carrying any plasmids. A band of the expected mobility could be detected in lysates of exponential phase cells growing aerobically in
rich media, but the corresponding band was not detected in strains in
which the dsbG coding region had been deleted (see below).
These results demonstrate that dsbG is indeed normally expressed in wild type cells under these conditions. The relative amounts of DsbG, DsbC, and DsbA in exponentially grown cells were determined by quantitative Western blotting, using serial dilutions of
the respective purified proteins as standards. Immunoreacting proteins
were visualized by ECLTM and quantified by densitometry. It
was found that in strain MC4100, DsbG is present at approximately
one-fourth the level of DsbC, which, in turn, is approximately 6-fold
less abundant than DsbA (not shown).
Construction and Characterization of dsbG Null Mutants--
In
earlier studies Andersen et al. (1) reported that a
dsbG::
Tet mutation could be crossed onto the
chromosome only when the cells were grown in the presence of low
molecular weight oxidants such as cystine or oxidized DTT. Despite
finding that a dsbG::
Kan mutation could be
transduced without supplementation of oxidants, they nevertheless
hypothesized that this observation resulted from the accumulation of
second site suppressor mutations. On the basis of these results, they
concluded that mutations in dsbG are conditionally lethal.
To evaluate this hypothesis, we first constructed a large deletion in
dsbG comprising 187 codons and marked it by the insertion of
fragments of either 1.4 or 1.3 kbp containing, respectively, a
CmR or a KanR gene. First, the
CmR-marked dsbG deletion was integrated into the
chromosome by homologous recombination in a
recD
strain. Hundreds of CmR,
AmpS colonies were obtained, as expected for a gene that is
not essential for viability. Colonies were picked at random and proven
to contain
dsbG::Cm by PCR using the
appropriate primers. Subsequently, the allele was transferred to
different strains by P1 transduction.
In a second approach, the
dsbG::Kan mutation
was inserted into the suicide vector pBAD39 that can only replicate in
cells grown in the presence of IPTG. The plasmid
pBAD
dsbG::Kan was transformed into MC4100, and
cells that had lost the plasmid and had recombined the
dsbG::Kan allele on the chromosome were
selected for resistance to kanamycin and streptomycin and sensitivity
to ampicillin on plates that also lacked IPTG. KanR,
StrepR, and AmpS colonies were tested by PCR
and were all found to carry the expected deletion in dsbG.
In order to test whether suppressor mutations had arisen in our
dsbG
strains, we transduced the
dsbG::Kan mutation to either a wild type strain or the
same strain carrying dsbG+ on a multicopy plasmid (pTrcdsbG2). For comparison, the same procedure was repeated in order
to recombine the degP41 mutation, a nonlethal
KanR-marked deletion in the gene encoding the periplasmic
protease DegP (32), into the chromosome of the same two acceptor
strains. The ratio of KanR transductants in RI89 to RI89
(pTrcdsbG2) was the same (3:2) regardless of whether
dsbG::Kan or the unrelated degP41
mutation was transduced. If the introduction of a dsbG gene
disruption in the haploid strain were lethal, and growth could only
occur via the accumulation of second site suppressor mutations, as
suggested by Andersen et al. (1), then we would expect that
transduction of the
dsbG::Kan allele into RI89
to result in a significantly lower number of KanR colonies
relative to the number of colonies obtained by transducing the
unrelated degP41 allele. This, however, was not the case. In
summary, the above results clearly show that dsbG is not
necessary for growth in RI89. Also, the frequency with which
dsbG
transductants could be obtained in
MC4100, RI90, and SR3324 strains suggests that dsbG is not
essential in these genetic backgrounds either. The dsbG
mutation was found to have no effect on alkaline phosphatase activity,
growth at 42 °C, or the growth rate in rich or minimal media (data
not shown).
The dsbG deletion mutants were also tested for their effect
on the folding of heterologous proteins containing multiple disulfide bonds, such as bovine pancreatic trypsin inhibitor (BPTI) and mouse
urokinase. BPTI is a 6.5-kDa protein with three disulfides, whereas
mouse urokinase is a 48-kDa protein containing 12 disulfides. The
folding of both proteins had been shown to be strongly dependent on the
presence of functional dsbA, dsbB,
dsbC, and dsbD genes (14, 17, 33, 34). However,
the levels of folded BPTI and active urokinase in the
dsbG
strain were identical to their wild type
counterparts (data not shown).
We furthermore examined the effect of overexpressed DsbG on disulfide
formation in heterologous proteins. While overexpression of dsbG from
the trc promoter in a wild type strain could not improve the
yield of BPTI or urokinase, it could complement the folding defect in a
dsbC
strain. In the case of BPTI, the yield of
native protein was restored to wild type levels in the
dsbC
strain; dsbG overexpression
could not, however, complement a null mutation in dsbD (Fig.
1A). With urokinase, the
activity was only restored to approximately 15% of the wild type level (Fig. 1B).

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Fig. 1.
DsbG overexpression complements
dsbC for expression of multiple
disulfide-containing heterologous proteins. A, BPTI
expression was measured by enzyme-linked immunosorbent assay using
whole cell soluble protein from strains RI89 (w.t.), RI179
(dsbC ), and RI242
(dsbD ) transformed with BPTI expression
plasmid pPBBPTI and either control plasmid pTrc99A (open
bars) or dsbG expression plasmid pTrcdsbG2
(filled bars). B, mouse urokinase
expression was quantified by indirect chromogenic assay. Activity is
shown in units (compared with a human urokinase standard) per mg of
total soluble protein from strains MC4100 (wt) and SR3324
(dsbC ) transformed with urokinase expression
plasmid puPA184 and either control plasmid pTrc99A (open
bars) or dsbG expression plasmid pTrcdsbG2
(filled bars).
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In Vivo Redox State--
Unlike DsbC, which contains a structural
disulfide in addition to its active site disulfide, mature DsbG has a
single pair of cysteine residues. We examined the in vivo
redox status of the active site disulfide in DsbG under normal growth
conditions in wild type cells as well as in null mutants of
dsb family genes. Briefly, exponentially grown cells were
lysed in the presence of 10% trichloroacetic acid to denature DsbG
while preventing the rearrangement of free thiols. Subsequently, free
thiols were blocked with AMS, and the AMS-conjugated (reduced) and
unconjugated (oxidized) proteins were resolved by SDS-PAGE under
nonreducing conditions (10, 12, 35). We found that at steady state DsbG is present exclusively in reduced form in wild type cells and in
dsbA
, dsbB
, and
dsbC
mutants. In contrast, in
dsbD
cells the protein was completely oxidized
(Fig. 2A). Furthermore, there
was no significant change in the expression level of DsbG in any of the
dsb mutants. Upon introduction of a plasmid overexpressing DsbG from an inducible promoter, a small amount of DsbG is found to be
oxidized. The reduced state, however, is overwhelmingly favored in all
strains except, of course, the dsbD null (Fig. 2B).

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Fig. 2.
In vivo redox state of DsbG active
site. Whole cells were precipitated with 10% trichloroacetic
acid, and free thiols were derivatized with AMS, which adds 490 Da
per thiol reacted, therefore reducing the electrophoretic mobility.
Proteins were separated by 12% SDS-PAGE and visualized by Western
blotting using DsbG antisera with ECLTM (Amersham Pharmacia
Biotech) development. Strains used are as follows: RI89
(wt), RI90 (dsbA ), RI317
(dsbB ), RI179 (dsbC ),
RI242 (dsbD ), PB303
(dsbG ). A contains proteins from
cells not carrying any plasmids; B contains proteins from
cultures carrying the plasmid pTrcdsbG2, with dsbG induction
by 1 mM IPTG for 30 min before trichloroacetic acid
precipitation. Oxidized and reduced standards were generated from
purified DsbG without AMS derivatization (ox.) or subjected
to reduction by 40 mM DTT followed by AMS
derivatization (red.).
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In Vitro Characterization of DsbG--
DsbG was purified from the
periplasmic fraction of a culture harboring a plasmid containing the
dsbG gene under the control of the T7 promoter. The
overexpressed periplasmic protein was extracted by cold osmotic shock
(Fig. 3A, lane
3) and partially purified by DEAE anion exchange
chromatography, yielding a preparation that contained more than 90%
pure DsbG protein (Fig. 3A, lane 4).
To further remove other protein contaminants, peak fractions were
loaded onto a SephadexTM-75 size exclusion column.
Fractions containing DsbG eluted slightly ahead of the ovalbumin (44 kDa) marker and yielded a preparation that was over 95% pure (Fig.
3A, lane 5). The molecular weight of
the purified protein was determined by matrix-assisted laser desorption/ionization time-of-flight spectrometry and shown to agree
with the calculated molecular weight within the error of measurement.

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Fig. 3.
DsbG purification and oligomeric state.
A, SDS-PAGE analysis of recombinant DsbG purified from
E. coli strain BL21(DE3). Cells containing the plasmid
pETdsbG2 were induced with 0.5 mM IPTG for 8 h at
25 °C to overexpress DsbG (lane 2), subjected
to osmotic shock (lane 3), and purified in two
successive chromatographic steps using an ion exchange column
(lane 4) and a size exclusion column
(lane 5) as described under "Experimental
Procedures." Lane 1 shows the migration of
molecular weight markers (M) with mass in kDa indicated to
the left. B, the oligomeric state of DsbG was
examined by incubating purified protein in the presence of increasing
concentrations (0-0.5 mM, lanes
2-6) of the cross-linking reagent EGS and analyzing by
SDS-PAGE. Molecular weight markers (M) are indicated in
lane 1.
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Upon gel filtration, DsbG eluted as a 45-50-kDa protein, raising the
possibility that DsbG (subunit molecular mass 25.7 kDa) may form a
homodimer as has been shown for DsbC (8). As an independent means to
establish the quaternary structure of DsbG, purified protein was
subjected to cross-linking with EGS, and the migration of the complex
was analyzed by SDS-PAGE and Coomassie Brilliant Blue staining (Fig.
3B). These data revealed that, indeed, with increasing
concentration of EGS, the electrophoretic mobility of DsbG shifted to
approximately twice the expected 25.7-kDa mass of the monomer (Fig.
3B, lanes 3-5). As a negative
control, purified DsbA, known to be a monomer, did not shift in
mobility when subjected to the same concentrations of cross-linking
agent (data not shown). These results suggest that DsbG exists
predominantly in the homodimer form in the periplasmic space of
E. coli, which is consistent with its estimated molecular
weight from gel filtration analysis.
It has been previously shown that the fluorescence emission spectra of
thioredoxin and DsbA are strongly dependent on the redox states of the
enzymes (7, 36-38). The fluorescence intensity at 324 nm of reduced
DsbA at pH 7.0 is 3-fold higher than that of the oxidized protein.
Reduced DsbG likewise exhibits increased fluorescence emission at 330 nm at pH 7.0, compared with the oxidized form (Fig.
4A), but the difference is
smaller than that of DsbA. In contrast, there is no difference in the
spectroscopic properties of active site reduced and oxidized DsbC
(8).

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Fig. 4.
Calculation of the redox equilibrium
constants (Keq) for DsbA and DsbG with
glutathione. A, the fluorescence emission spectra of
oxidized and reduced DsbG were recorded at protein concentrations of
0.45 µM in 0.1 M sodium phosphate buffer, pH
7.0, 1 mM EDTA. Excitation was at 280 nm, and the maximum
emission was recorded at 330 nm. B, the relative amounts of
reduced DsbA and DsbG (R) at equilibrium with glutathione
were determined using the specific change in fluorescence of oxidized
and reduced DsbA (324 nm) and DsbG (330 nm) (excitation at 280 nm).
Oxidized DsbA or DsbG (0.45 µM) was incubated for 12 h in 100 mM sodium phosphate buffer, pH 7, 1 mM
EDTA, containing 100 µM GSSG and 4 µM to 2 mM GSH. The equilibrium concentrations of DsbA, DsbG, GSSG,
and GSH were calculated using Equations 1-3, and the equilibrium
constants were determined by fitting the data to Equation 4 (see
"Experimental Procedures"). Nonlinear regression analysis yielded
Keq values of 130 µM for DsbA and
140 µM for DsbG (correlation coefficients were 0.999 and
0.987, respectively).
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The observed differences in fluorescence intensities of reduced
or oxidized DsbA and DsbG were used to measure the equilibrium concentrations of the oxidized and reduced forms in the presence of
different ratios of oxidized (GSSG) and reduced (GSH)
glutathione. The DsbG/glutathione redox equilibrium and its equilibrium
constant are given by Equations 5 and 6.
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(Eq. 5)
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(Eq. 6)
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DsbA or DsbG was incubated in the presence of 100 µM
GSSG and increasing concentrations of GSH (0-2 mM), and
the relative amount of reduced enzyme at equilibrium (R) was
measured over the range from fully oxidized to fully reduced protein
(Fig. 4B). Nonlinear regression was used to fit the data to
Equation 4. The equilibrium constant Keq for the
reduction of DsbA by glutathione was determined to be 130 µM. This result agrees well with the values of 120 and 80 µM reported by Wunderlich and Glockshuber (7) and Zapun
et al. (6), respectively, for DsbA. Under identical
conditions, the equilibrium constant for DsbG was estimated to be 140 µM. Thus, the DsbG disulfide bond is nearly as unstable as that of DsbA and slightly less so than that of DsbC
(Keq = 200 µM) (8). The general
behavior of the DsbG titration by glutathione indicates that its redox
potential is comparable with those of DsbA and DsbC.
The ability of DsbG to catalyze the reduction of insulin in the
presence of DTT was evaluated. Reduction of insulin leads to cleavage
of the two interchain disulfide bonds, causing the
-chain of
insulin, which is insoluble, to aggregate and form a precipitate whose
formation can be recorded by measuring the turbidity in the sample at
650 nm. Assays were performed exactly as described by Holmgren and
co-workers (29). As expected, the addition of 5.0 µM DsbC
or DsbA resulted in the rapid precipitation of insulin, which could be
detected after 6 min for DsbC and 18 min for DsbA (Fig.
5A). At lower concentrations
of either protein (2.5 µM), the time at which the
turbidity could initially be detected was increased, and the
precipitation rate was slower (data not shown). Andersen et
al. (1) had reported that DsbG has insulin reduction activity
comparable with that of DsbA. However, in our hands DsbG was not able
to reduce insulin above the background levels observed in the control
reaction with DTT alone (Fig. 5A). No activity was observed
with different preparations of DsbG expressed with or without a
hexahistidine tail at 25 or 37 °C (Fig. 5A) or with high
concentrations of DsbG (up to 20 µM). We also failed to
see any activity with protein that had been prereduced with 15 mM DTT (data not shown). On the basis of these results, we concluded that DsbG is unable to catalyze the reduction of insulin, at
least under the standard assay conditions.

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Fig. 5.
Oxidoreductase activity of DsbG compared with
other disulfide catalysts. A, the enzymatic reduction
of insulin in the presence of DTT was assayed by turbidimetric assay as
described previously (29). Reactions were performed in a final volume
of 800 µl containing 0.1 M potassium phosphate buffer, pH
7; 2 mM EDTA; 0.35 mM DTT; 0.13 mM
insulin; and purified DsbA, DsbC, or DsbG at 5 µM. The
background reduction by DTT without any enzyme present was used as a
control. B, analysis of the kinetics of oxidative refolding
of RNase. The concentration of refolded RNase was determined by
measuring the absorbance change at 296 nm produced by RNase-catalyzed
hydrolysis of cCMP (initial concentration 4.5 mM) at pH 8.0 in 50 mM Tris acetate buffer (see "Experimental
Procedures") in the presence of redox buffer (0.2 mM
GSSG, 1.0 mM GSH). Concentrations of PDI, DsbC, DsbG, and
DsbG·His are indicated in the key. Reactions were started
with the addition of 8 µM reduced, denatured RNase.
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We also examined whether DsbG could catalyze the oxidative renaturation
of reduced, denatured RNase (30). The addition of reduced, denatured
RNase to a glutathione redox buffer containing a catalytic protein and
the RNase substrate cCMP results in an increase of RNase activity,
which is monitored as an increase in the rate of cCMP hydrolysis
measured at 296 nm. The concentration of active RNase can be calculated
at any time from the instantaneous slope of the absorbance
versus time trace after considering the change in cCMP
concentration and the formation of the RNase inhibitor CMP (for
calculations, see Lyles and Gilbert (30)). In a 60-min assay, there is
no substantial gain in RNase activity with catalytic concentrations of
enzyme in the absence of glutathione redox buffer, suggesting that
oxygen-dependent activation of RNase does not provide a
significant source of oxidizing equivalents under these conditions.
Consistent with previous studies, bovine PDI was shown to catalyze the
oxidative refolding of RNase (Fig. 5B). Similarly, DsbC was
also efficient in catalyzing the reactivation of RNase. The
kcat and Km values for this
reaction were 0.24 ± 0.01 min
1 and 16 ± 3 µM, which are 3-fold slower and 2-fold higher,
respectively, than those reported for PDI (39). The presence of DsbG (5 µM) resulted in a rate of RNase reactivation that was
reproducibly slightly above background. Identical results were obtained
with either native DsbG or DsbG·His-tagged protein (Fig.
5B). These results indicate that DsbG is a poor catalyst of
oxidative protein refolding, at least using RNase as the substrate.
Furthermore, DsbG (5 µM) could not act synergistically
with PDI or DsbC, failing to give a reactivation rate higher than PDI
or DsbC alone.
 |
DISCUSSION |
We first identified DsbG based on its homology with
E. coli DsbC. A data base search also revealed that DsbG is
highly similar to an unidentified open reading
frame3 from Pseudomonas
aeruginosa (47% identity, 70% similarity, allowing for
conservative substitutions), distinct from the putative DsbC (GenBankTM accession no. AF057031) of P. aeruginosa, with the two exhibiting similarity (25% identity,
43% similarity) comparable with that between the E. coli
DsbC and DsbG.
The E. coli dsbG gene was shown to encode a dimeric
periplasmic protein, which in exponentially grown cells is expressed at a level about 25% that of DsbC. The two proteins share a number of
similarities; they are both dimeric, form an unstable disulfide bond,
and are maintained in the reduced state in the periplasm via the action
of DsbD (10, 17). High level expression of DsbG could fully complement
the defect in the folding of BPTI and partially complemented the
formation of active urokinase in a dsbC
mutant
background. In vitro, the folding pathway of BPTI has been
shown to involve the formation and subsequent rearrangement of
intermediates with nonnative disulfide bonds. There is evidence that
DsbC catalyzes kinetically important disulfide bond isomerization steps
in the folding of BPTI in the periplasm and most likely has a similar
effect on urokinase (17, 34). The ability of DsbG to fully restore the
expression of BPTI and partially restore that of urokinase suggests
that DsbG also is able to effect disulfide bond isomerization.
Remaining to be determined is whether DsbG is a bona fide catalyst of
disulfide isomerization or whether its overexpression acts indirectly,
for example by altering the redox state of the periplasm.
DsbG contains a Cys-X-X-Cys sequence that is
found in the active site of proteins belonging to the thioredoxin
superfamily. This is consistent with DsbG's being a redox active
protein that may play a role in disulfide bond isomerization. The
Cys-Pro-Tyr-Cys sequence in DsbG forms an unstable disulfide bond that
is readily reduced by glutathione. Interestingly, DsbA, DsbC, and DsbG
all exhibit equilibrium constants with glutathione in the 80-200
µM range (6-8), considerably lower than that of
thioredoxin (10 M) (40). Despite the highly oxidizing
nature of the periplasmic space, DsbG is found exclusively in the
reduced state in wild type cells. Only under conditions of
overexpression did we observe a small amount of oxidized protein (Fig.
2B). On the other hand, DsbG was completely oxidized in a
dsbD
background, suggesting that, in the
absence of a membrane protein reductant, DsbG becomes completely oxidized.
While this work was in progress, Andersen et al. isolated
dsbG genetically and examined its function in
vivo (1). We have found several discrepancies between our results
and those of Andersen et al. First of all, they reported
that a dsbG null linked to TetR could not be
crossed onto the chromosome unless the cells were supplemented with
high concentrations of low molecular weight oxidants. Although a
dsbG::
Kan null allele could be transduced without supplementation by oxidants, they attributed the appearance of
transductants to suppressor mutations and went on to conclude that
dsbG is essential. In contrast, we found the following: 1) a
KanR- or a CmR-marked null allele could be
transduced from a recD
strain into a variety
of recipient strains at a normal frequency; 2) a null allele could be
readily recombined into the chromosome from a suicide plasmid, again at
a high frequency; and 3) finally, confirming the absence of second site
suppressors, we found that the frequencies with which the
dsbG
allele can be transduced to a wild type background with or without
dsbG+ present in multicopy are comparable. These results
prove unequivocally that dsbG is not required for growth.
In contrast to our results in Fig. 2A, Andersen et
al. (1) reported that DsbG is present as a mixture of oxidized and
reduced protein in vivo. There are two possible reasons for
this discrepancy. First, in their experiments they expressed DsbG from
a T7 promoter. As shown in Fig. 2B, under conditions of
overexpression a small portion of DsbG is indeed oxidized. Second, they
used a methodology for trapping free thiols that has been shown to be
subject to artifacts (16).
Another discrepancy between our results and those of Andersen et
al. (1) is that in our hands DsbG exhibited no insulin reduction
activity, although different high purity protein preparations were
tested. Furthermore, DsbG exhibits substantially lower activity than
DsbC in catalyzing the in vitro refolding of RNase,
indicating that it is a poor protein oxidant, at least for this substrate.
The inability of DsbG to catalyze the reduction of insulin or the
oxidative refolding of RNase in vitro or to restore the defect in alkaline phosphatase formation in a dsbA mutant
background (data not shown) argues that its biological function is to
facilitate disulfide bond isomerization rather than the oxidation of
protein thiols or net disulfide bond reduction. Consistent with this
hypothesis, DsbG is maintained in a reduced state in the periplasm in a
manner analogous to DsbC. Also, the fact that DsbG can partially rescue the defect in the formation of active multidisulfide proteins in a
dsbC
background strongly suggests that it can
play a role in protein folding in vivo. In agreement with
this conclusion, Andersen et al. (1) reported that the
synthesis of an htrA-lacZ fusion, which is dependent on the
E factor, is restored to wild type levels by
overexpression of DsbG in a dsbC
background.
Induction of a
E-dependent heat shock
response has been shown to occur under conditions that lead to the
accumulation of misfolded periplasmic proteins (41). In our hands, the
deletion of dsbG had no negative effect on expression of
disulfide-containing proteins in the periplasm, indicating that it may
be redundant under the conditions tested or have a limited set of
substrates for which it is necessary. Studies to identify the in
vivo substrates of DsbG in the bacterial periplasm are under way.