From the Department of Cell and Organism Biology,
Lund University, Sölvegatan 35, SE-22362 Lund, Sweden and the
¶ Centre for Metalloprotein Spectroscopy and Biology, School of
Chemical Sciences and Pharmacy, University of East Anglia,
Norwich NR4 7TJ, United Kingdom
Received for publication, January 6, 2003, and in revised form, February 25, 2003
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ABSTRACT |
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Covalent attachment of heme to apocytochromes
c in bacteria occurs on the outside of the cytoplasmic
membrane and requires two reduced cysteinyls at the heme binding site.
A constructed ResA-deficient Bacillus subtilis strain was
found to lack c-type cytochromes. Cytochrome c
synthesis was restored in the mutant by: (i) in trans
expression of resA; (ii) deficiency in BdbD, a
thiol-disulfide oxidoreductase that catalyzes formation of an intramolecular disulfide bond in apocytochrome c after
transfer of the polypeptide across the cytoplasmic membrane; or (iii)
by addition of the reductant dithiothreitol to the growth
medium. In vivo studies of ResA showed that it is
membrane-associated with its thioredoxin-like domain on the outside of
the cytoplasmic membrane. Analysis of a soluble form of the protein
revealed two redox reactive cysteine residues with a midpoint potential
of about Thiol-disulfide oxidoreductases contain the active site motif
Cys-Xxx-Yyy-Cys in which the two cysteine residues reversibly cycle
between oxidized disulfide and reduced dithiol forms, and thus
participate in redox reactions. Cytoplasmically located thiol-disulfide oxidoreductases, such as thioredoxin, have low redox potentials and are
involved in maintaining the reducing environment of the cytoplasm. The
more recently discovered thiol-disulfide oxidoreductases located on the
outside of the cytoplasmic membrane in bacteria are found to be
involved in: oxidation reactions, e.g. Escherichia coli DsbA, which catalyzes the formation of disulfide bonds in proteins transported across the membrane (1); disulfide bond isomerization reactions, e.g. E. coli DsbC, which
functions in the redistribution of disulfide bonds among the cysteine
residues of target proteins (2); and reduction reactions,
e.g. Bradyrhizobium japonicum TlpA, which
functions in the assembly of cytochrome aa3
(3).
Thiol-disulfide oxidoreductases play a central role in the assembly of
c-type cytochromes, in which the heme group is covalently attached via (usually) two thioether bonds between the thiol side chains of cysteine residues, occurring in the conserved motif Cys-Xxx-Yyy-Cys-His, and the vinyl side chains of the heme (4). Bacterial c-type cytochromes are localized to the outside of
the cytoplasmic membrane where they function in electron transport processes. The mechanisms by which cytochromes c assemble
are currently of great interest. Assembly takes place on the outside of
the cytoplasmic membrane, involving separate transport of heme and the
apocytochrome across the membrane (5); the latter occurs via the
general protein secretory (Sec) pathway of the cell (6). Following
translocation, assembly proceeds via a pathway involving a number of
specific proteins. To date, two distinct systems have been identified
in bacteria. System I is found in Bacterial Strains and Growth Media--
Bacterial strains and
plasmids used in this work are listed in Table
I. E. coli cells were grown at
37 °C in LB broth or on LA plates (23). B. subtilis
strains were cultivated at 37 °C in LB or NSMP (24), or on tryptose
blood agar base (Difco) plates. Strains with an insertion mutation in
resA were grown in the presence of 1 mM
IPTG.1 Antibiotics were used
at the following concentrations when appropriate: spectinomycin, 150 mg/liter; erythromycin, 1 mg/liter; chloramphenicol, 5 mg/liter;
phleomycin, 1.5 mg/liter (for B. subtilis); and ampicillin, 50-100 mg/liter; chloramphenicol, 20-25 mg/liter (for E. coli).
DNA Techniques--
Standard molecular genetics techniques were
employed (23). Plasmid DNA was isolated using Quantum miniprep
(Bio-Rad) or Qiagen midiprep (Qiagen) kits, or by CsCl density gradient
centrifugation. Chromosomal DNA from B. subtilis was
isolated according to Marmur (25). E. coli was transformed
by electroporation and B. subtilis was grown to natural
competence as described by Hoch (26). PCR was carried out using Pfu
(Promega) or Taq (Roche Diagnostics GmbH) DNA polymerases,
and B. subtilis 1A1 chromosomal DNA used as template.
Cloning was carried out using E. coli JM109 as host. All
constructs obtained by PCR were verified by DNA sequence analysis.
Construction of Plasmids--
Primers used for DNA amplification
by PCR are listed in Table II. pLLE36 was
constructed by amplifying an internal fragment of resA using
PCR with primers LE032 and LE033. The 0.35-kb PCR product was cut with
HindIII and EcoRI and cloned into pMutin2 cut
with the same enzymes.
pRAN1 was constructed by amplifying a DNA fragment containing the
resA gene and its natural promoter using PCR with primers resA4 and resA5. The 0.73-kb product was cut with PstI and
SalI and ligated into pHPSK cut with the same enzymes.
pRAN1Es was obtained by cloning the resA fragment of pRAN1
into pHP13Es.
pLLE56 and pLLE57 were constructed by amplifying two DNA fragments,
containing the natural resA promoter and a truncated
resA reading frame, using PCR with primer pairs LE040/LE041
and LE040/LE042, respectively. The PCR products were cut with
BamHI and KpnI and cloned into pPHO1 digested
with the same enzymes. The resulting plasmids were cut with
BamHI and HindIII and the appropriate fragments cloned into pHPKS cut with the same enzymes.
pRAN11 and pRAN8 were constructed by amplifying DNA fragments using PCR
with primer pairs resA8/resA3 (soluble ResA) and resA8/resA2 (His-tagged soluble ResA). The products were ligated into
SmaI-cut pUC18, generating pRAN10 and pRAN6, respectively.
These were digested with NdeI and XhoI (pRAN6) or
NdeI and EcoRI (pRAN10) and the resulting
resA fragments ligated into pET21a(+) cut with the same enzymes.
Construction of B. subtilis LUL9--
Strain LUL9 was isolated
by transforming 1A1 with pLLE36, and selection on plates containing
erythromycin and 1 mM IPTG. The pMutin2 insertion in
resA was confirmed by PCR amplification of a DNA fragment
using primers LE040 and MUT01 (which hybridize upstream of
resA and within pMutin2, respectively); and by gene linkage
analysis using strain 1A1C, which carries a chloramphenicol resistance
marker within the spmAB locus located just upstream of
resA in the B. subtilis chromosome. >90% of the
erythromycin-resistant transformants obtained when strain 1A1C was
transformed with limiting amounts of LUL9 chromosomal DNA were found to
be chloramphenicol-sensitive.
Cytochrome c Oxidase Activity Assay--
Isolated membranes from
B. subtilis strains were added at a final protein
concentration of 40 µg/ml to a 40 µM solution of reduced Saccharomyces cerevisiae cytochrome c in
20 mM MOPS, pH 7.4, in a stirred 3-ml cuvette at 30 °C.
Oxidation of the c-type cytochrome was followed at the
wavelength pair 540 and 550 nm (27), using Immunoblot Analysis--
SDS-PAGE was carried out using the
Schägger and von Jagow system (29). Proteins were blotted onto a
polyvinylidene difluoride membrane (Millipore) by wet
electroblotting using 20 mM Tris, 150 mM
glycine, and 20% methanol. Rabbit antiserum raised against His-tagged
soluble ResA or E. coli alkaline phosphatase was used as the
primary antibody. Peroxidase-labeled anti-rabbit antibodies were used
as secondary antibodies and SuperSignal West pico chemiluminescent substrate (Pierce) was used for visualization of bound primary antibodies.
Purification of His-tagged and Soluble ResA--
His-tagged
soluble ResA (htsResA) and soluble ResA (sResA) were purified from
BL21(DE3)/pRAN8 and BL21(DE3)/pRAN11, respectively, after induction of
resA expression with 1 mM IPTG. Harvested cells were washed and resuspended in 20 mM sodium phosphate, 0.5 M NaCl, 10 mM imidazole, pH 7.4, and sonicated
while on ice. For htsResA, the soluble fraction obtained after
centrifugation was applied to, and eluted from, a HiTrap metal
chelation affinity column (Amersham Biosciences) according to the
manufacturer's instructions. Purified htsResA was exchanged into 100 mM sodium phosphate, 100 mM NaCl, 1 mM EDTA, pH 7.0, using an ultrafiltration unit (Amicon). For sResA, the soluble fraction obtained after centrifugation was
subjected to ammonium sulfate fractionation. The pellet from the
50-80% (w/v) (NH4)2SO4 fraction
was resuspended in 100 mM sodium phosphate, 100 mM NaCl, 1 mM EDTA, pH 7.0, desalted using a
HiTrap desalting Sephadex G-25 Superfine column (Amersham Biosciences), and applied to a HiTrap Q-Sepharose HP anion exchange column (Amersham Biosciences) equilibrated in the same buffer. Proteins were eluted using a linear gradient of 0.1-1 M NaCl in 10 column
volumes of the same buffer. Fractions containing sResA were pooled,
exchanged into 100 mM sodium phosphate, 100 mM
NaCl, 1 mM EDTA, pH 7.0, and applied to a XK26/60 Sephacryl
S-100 gel filtration column equilibrated in the same buffer, giving
pure sResA. Concentrations of htsResA and sResA were calculated using
extinction coefficients, Reactivities of Thiols and Determination of Redox
Potentials--
The number of reactive thiols per protein was
determined using iodoacetamide and iodoacetate, as previously described
(31), except that samples were separated using PAGE according to
Laemmli (32) with 8 M urea in place of SDS. The
reactivities of cysteine residues of ResA as isolated and following
reduction were determined using 5,5'-dithiobis(2-nitrobenzoic acid)
(Ellman's reagent) as previously described (33).
For redox potential determinations, 20 µM ResA (sResA or
htsResA) in deoxygenated 100 mM sodium phosphate, 1 mM EDTA, 100 mM NaCl, pH 7.0, was incubated
with 5 mM oxidized or reduced DTT for 3 h at 4 °C
to generate the fully oxidized or reduced species, respectively.
Treatment with diamide was also used to generate the fully oxidized
form (14). DTT or diamide was removed using a HiTrap desalting Sephadex
G-25 Superfine column, and ResA was diluted to a final concentration of
1 µM. Varying ratios of oxidized and reduced DTT, at a
total concentration of 2 or 5 mM, were added to generate
different potentials and the reaction was allowed to equilibrate for
3 h at 25 °C. The redox state of ResA was followed by measuring
fluorescence emission intensity at 350 nm, following excitation at 280 nm, using a PerkinElmer LS55 fluorimeter with excitation and emission
slit widths set to 10 nm. Measurements indicated that equilibrium was
achieved within 1 h. Similar results were obtained at different
total DTT concentrations; when starting with fully oxidized or fully
reduced ResA protein; and, when using de-oxygenated buffers in an
anaerobic glove box in which oxygen was <2 ppm (Faircrest
Engineering). Fluorescence intensities at ratios of 399:1 and 1:399
oxidized to reduced DTT were taken to represent 100% oxidized and
reduced ResA, respectively. The data were fitted using the following
equation, which is derived from the Nernst expressions for the two
redox couples at equilibrium,
Analytical Gel Filtration Chromatography--
Molecular
weights/association states of sResA and htsResA were determined using a
Superdex 75 HR 10/30 column (Amersham Biosciences). sResA or htsResA (3 mg/ml) were applied to the column equilibrated with 100 mM
sodium phosphate, 100 mM NaCl, 1 mM EDTA, pH
7.0, and eluted in the same buffer at a flow rate of 1.0 ml
min Other Methods--
Sporulation efficiency assays were performed
as described previously (28). Radiolabeling of cytochromes using
5-[4-14C]aminolevulinic acid (51 mCi mmol Inactivation of resA--
To investigate the function of ResA in
B. subtilis, a derivative of pMutin2 was constructed
(pLLE36), containing bp 55-409 of resA (537 bp). A
Campbell-type integration of the entire plasmid into the chromosome
resulted in strain LUL9 (Table I), in which resA was
disrupted. pMutin2 contains an IPTG-inducible spac promoter that, following integration, can drive the expression of genes located
downstream of the insertion (37). Growth of the resA knockout mutant LUL9 was similar to the parental strain 1A1 in the
presence of 1 mM IPTG. However, in the absence of IPTG it grew poorly. This indicates that the pMutin2 insertion into
resA has a polar effect on resBC and/or
resDE expression. In subsequent experiments involving strain
LUL9 the cells were grown in the presence of 1 mM IPTG. The
properties of LUL9 show that the resA gene is not
essential for viability of the cell.
A ResA-deficient Strain Is Defective in Cytochrome c
Synthesis--
Colonies of LUL9 on NSMP plates did not oxidize TMPD,
indicating a deficiency in cytochrome caa3
activity. This was confirmed by analysis of membranes isolated from
LUL9 cells grown in NSMP medium, which showed <0.1% activity of
wild-type membranes. Plasmid pRAN1Es, containing the resA
gene with its natural promoter, in LUL9 complemented the defect in
TMPD-oxidation activity.
Under denaturing conditions cytochromes lose their heme unless it is
covalently bound to the polypeptide. In a wild-type B. subtilis strain, grown in the presence of
[14C]aminolevulinic acid (a precursor to heme), the four
membrane-bound cytochromes c and QcrB are observed as
radioactive bands by SDS-PAGE. QcrB is the b subunit of the
cytochrome bc complex and one of its two hemes is probably
covalently bound to a cysteine residue in the polypeptide (38). Fig.
1 shows that strain LUL9 lacks all four
c-type cytochromes but contains QcrB. The cytochrome c content in LUL9 was restored when resA was
expressed in trans from pRAN1Es. These results combined show
that ResA is required for cytochrome c synthesis.
The tested properties of LUL9 were found to be very similar to those of
mutants defective in CcdA, e.g. those of strain LU60A1 (Fig.
1). Strain LUL14, defective in both ccdA and
resA, showed the same TMPD-oxidation phenotype as LUL9.
The ResA-deficient Phenotype Can Be Suppressed by Inactivation of
BdbD or Addition of DTT to the Growth Medium--
B.
subtilisBdbD and BdbC are thiol-disulfide oxidoreductases that
catalyze formation of disulfide bonds in proteins on the outer side of
the cytoplasmic membrane (28, 39). Strain LUL15, deficient in both ResA
and BdbD, was found to be TMPD-oxidation positive. The same result was
obtained when strain LUL9 was grown on plates in the presence of the
reducing thiol reagent DTT (15 mM). Addition of DTT to the
medium did not restore TMPD oxidation in strains deficient in ResB and
ResC (28). Our in vivo findings indicate that ResA is a
thiol-disulfide oxidoreductase involved in disulfide reduction during
biosynthesis of c-type cytochromes.
A ResA-deficient Strain Is Not Defective in Sporulation--
Some
proteins in the B. subtilis spore coat are rich in cysteine
residues and are heavily cross-linked by disulfide bonds in the final
spore (40). B. subtilis strains lacking CcdA are defective,
but not completely blocked, in the synthesis of endospores (20). The
exact role of CcdA in the sporulation process is not understood but
sporulation in a ccdA mutant can be restored by inactivation
of bdbD or bdbC (28). CcdA is therefore thought to function in breaking or isomerization of disulfide bonds during spore maturation and perhaps also in germination. The ResA-deficient strain LUL9, however, showed normal sporulation efficiency when compared with the parental strain (data not shown). Thus, ResA, in
contrast to CcdA, does not play an important role in spore synthesis.
ResA Is a Protein of 179 Amino Acid Residues--
The B. subtilis resA gene has been reported to encode a putative
181-amino acid residue protein (21, 41). Sequencing of the
resA insert of pRAN1, and two other independent
resA constructs, revealed a guanine residue that is
not present in the data base resA sequence (Fig.
2A). Thus, the reading frame
either upstream or downstream of the extra residue is different from
that indicated in the data base. Alignment of ResA with thiol-disulfide
oxidoreductases that are known to be involved in cytochrome
c assembly showed significant sequence similarity,
particularly around the dicysteine motif, but not in the N-terminal
region (not shown). Therefore, we concluded that the reading frame
upstream of the point of insertion is affected by the correction. Fig.
2A shows an ATG start codon 7 bp downstream of the
originally proposed GTG translation start codon (21) and gives the
sequence of ResA shown in Fig. 2B. Alignment of the revised
ResA sequence with other system II thiol-disulfide oxidoreductases
(data not shown) showed that similarity does extend into the N-terminal
region, thus supporting our conclusion.
ResA Is a Membrane Protein--
Analysis of the original erroneous
ResA sequence led to the prediction that it is a soluble protein (10).
However, the revised sequence of the resA gene encodes a
protein with one predicted transmembrane segment (42, 43) and a
putative type I signal peptidase cleavable N-terminal signal peptide
(44) (Fig. 2B). Using immunoblot analysis of B. subtilis subcellular fractions we found that ResA is a
membrane-bound protein (Fig. 3) and is probably anchored to the membrane by the N-terminal hydrophobic segment
(Fig. 2C). Strain 1A1 contained membrane-bound ResA but LUL9
containing the vector pHP13Es did not. LUL9/pRAN1Es overproduced membrane-bound ResA, as expected. Thus, the disruption of
resA with pMutin2 in LUL9 abolishes formation of stable ResA
antigen in the cells.
The transmembrane topology of ResA was analyzed by using N-terminal
segments of ResA fused to E. coli alkaline phosphatase (PhoA) lacking its native signal sequence. Active alkaline phosphatase is only formed if it is transported to the outer side of the
cytoplasmic membrane in E. coli (45). Lysates of E. coli cells harboring plasmid pLLE56 or pLLE57 (which encode
residues 1-35 and 1-140 of ResA, respectively, fused to PhoA)
contained PhoA antigen of the expected sizes (immunoblot not shown) and
contained alkaline phosphatase activity (0.22 and 0.17 µmol × min Production and Isolation of the Thioredoxin-like Domain of
ResA--
A version of resA encoding a shortened protein
missing the first 35 amino acid residues that anchor ResA to the
membrane and with the 36th, Ile, changed to Met, was
cloned, E. coli expression vectors encoding His-tagged and
non-tagged forms were constructed, and the proteins were purified.
N-terminal sequence analysis indicated that the N terminus is
processed, resulting in the removal of the initial Met residue. EI-MS
gave masses of 15921.9 and 16988.8 Da for sResA and htsResA,
respectively, which are 134.4 and 132.6 Da lower than the predicted
masses, consistent with the processing of the N-terminal Met residue
(131.2 Da).
Molecular weights of 15,900 ± 120 Da and 16,500 ± 130 Da
were determined for sResA and htResA, respectively, using analytical gel filtration. These are in good agreement with predicted values and
clearly indicate that both proteins are monomers in solution.
Reactivity of ResA Cysteine Thiols--
The number of
thiol-containing residues in sResA and htResA was investigated using
the cysteine modification reagents iodoacetamide and iodoacetate, as
described under "Experimental Procedures." Urea-polyacrylamide gels
of sResA (not shown) and htsResA (Fig. 4)
contained three bands corresponding to the protein modified with no,
one, and two carboxymethyl groups, consistent with the presence of 2 cysteine thiol residues.
Investigation of cysteine thiol reactivity with Ellman's reagent
showed that, as isolated, both sResA and htsResA contained ~0.5
reactive cysteine residues per protein. Reduction with DTT followed by
removal of the excess reductant immediately prior to the addition of
Ellman's reagent showed that, as expected, sResA contained 2 reactive
cysteine residues per protein, although only 1.4 reactive cysteine
residues could be detected per htsResA. The reason for this difference
is unknown, but may indicate a difference in the cysteine reactivities
of the two proteins.
Thiol-disulfide oxidoreductases such as E. coli thioredoxin,
calf liver protein-disulfide isomerase, and B. japonicum
TlpA (49-51) exhibit an enhanced fluorescence signal at ~350 nm when in their reduced state. This is because of the significant quenching, in the oxidized state, of the fluorescence arising from tryptophan residues close to the dicysteine motif. This can be used to monitor the
redox state of the protein and, when allowed to reach equilibrium with
varying ratios of oxidized and reduced thiol reagents, can be used to
determine the midpoint redox potential (Em) of the
thiol-disulfide oxidoreductase (15, 51). Like thioredoxin, ResA has two
tryptophan residues close to the dicysteine motif (i.e.
WXXWCXXC) and, as isolated, both sResA and
htsResA gave fluorescence intensity at 350 nm. Excess reduced
glutathione did not affect the fluorescence signal, whereas reduced DTT
caused a significant increase in intensity. Comparison of the
fluorescence intensities of the isolated and reduced proteins indicated
that ~29% of the proteins were in the reduced state following
purification, consistent with the Ellman's reagent assay. DTT has a
significantly lower standard redox potential than glutathione ( In this paper we have shown that ResA is a low potential
thiol-disulfide oxidoreductase that is involved in cytochrome
c assembly on the outer side of the B. subtilis
cytoplasmic membrane. The revised sequence of resA presented
here leads to the prediction that ResA has one transmembrane segment
and a thioredoxin-like motif. Our results confirmed that the protein is
membrane-associated and showed that the C-terminal domain, containing
the thioredoxin-like motif, is exposed on the outside of the membrane.
We have previously proposed that ResA accepts reducing equivalents from
CcdA (28). The ResA-deficient strain LUL9 described here showed the
same pleiotropic cytochrome c defect as a CcdA-deficient strain. That the deficiency can be complemented by inactivation of
BdbD, or by supplementing the medium with DTT is consistent with the
proposed roles of ResA and CcdA in breaking disulfide bonds during
cytochrome c synthesis. In Fig.
6 we present a scheme for the proposed
functions of ResA and CcdA. Apocytochrome c is transported
across the membrane in an unfolded state by the Sec-protein transport
machinery. BdbD and BdbC catalyze the formation of an intramolecular
disulfide bond in the heme binding site. This disulfide bond is
specifically broken by the action of reduced ResA; once broken, heme
can be covalently attached to the cysteine residues of the
apocytochrome. ResA is re-reduced by the activity of CcdA. The
ResA/CcdA pathway seems to be dispensable in the absence of BdbD/BdbC;
that is, if the disulfide bond-forming pathway is disrupted, the
disulfide bond-breaking pathway is not required. This is consistent with the findings of Daltrop et al. (56) who showed that
heme can be covalently attached to apocytochrome c in vitro
without biosynthetic enzymes if the two cysteine residues in the heme binding site are reduced.
340 mV at pH 7. We conclude that ResA, probably together with another thiol-disulfide oxidoreductase, CcdA, is required for the
reduction of the cysteinyls in the heme binding site of apocytochrome
c.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
- and
-proteobacteria, including E. coli, Rhodobacter capsulatus,
B. japonicum, and Paraccocus denitrificans
(7-9). System II seems more widespread than system I, occurring in a
range of bacteria including cyanobacteria, Gram-positive bacteria such
as Mycobacterium species,
-,
-, and
-proteobacteria such as Bordetella pertussis, Thiobacillus
ferrooxidans, and Helicobacter pylori, respectively,
and some extremophiles such as Aquifex aeoliticus (10, 11).
Common to both systems is that the apocytochrome cysteine thiols and
the heme iron must be in their reduced states for thioether bond
formation to occur. Thiol-disulfide oxidoreductases are required for
this, and specific proteins have been identified in a number of system
I organisms, including E. coli (CcmG), R. capsulatus (HelX), B. japonicum (CycY), and P. denitrificans (CcmG) (12-16), and one system II organism,
B. pertussis (CcsX) (11). We are studying cytochrome
c assembly in the system II Gram-positive bacterium
Bacillus subtilis. To date three genes required for this
assembly have been identified: ccdA, resB, and
resC (10, 17, 18). CcdA, ResB, and ResC are all
polytopic integral membrane proteins. CcdA shows sequence
similarity to a domain of E. coli DipZ (DsbD) that functions
in the transfer of reducing equivalents from the cytoplasm to the outer
side of the cytoplasmic membrane (19). In the B. subtilis
chromosome the ccdA gene is not located close to other genes
that are important for cytochrome c synthesis (20). The
resBC genes reside in the resABCDE cluster in the chromosome. resD and resE encode a two-component
signal transduction system (21, 22), whereas resA encodes a
putative thiol-disulfide oxidoreductase, which has been proposed to be
involved in cytochrome c assembly (9, 10). Previous attempts
to disrupt resA were unsuccessful, leading to the conclusion
that it (along with resB and resC) is an
essential gene (21). Here, by generating a resA knockout
mutant, we demonstrate that ResA is not essential for cell viability
and that it is required for cytochrome c assembly. We show
that ResA is located on the outside of the cytoplasmic membrane,
attached to it via a single transmembrane segment and is shown to be a
low potential thiol-disulfide oxidoreductase. The specific role of ResA
in cytochrome c assembly-associated redox processes has
been investigated and is discussed.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Bacterial strains and plasmids used in this work
Primers used in this work
550-540 = 19.5 mM
1 cm
1 to calculate
activities. TMPD oxidation by colonies on NSMP plates was
assayed as described by Le Brun et al. (10) or by Erlendsson and Hederstedt (28) for cells grown on NSMP plates containing DTT.
, at 278 nm of 18.25 and 18.15 mM
1 cm
1, respectively,
determined as previously described (30).
where r is the fraction of protein in the reduced
form, Eh is the potential of the DTT couple,
Em is the midpoint redox potential of ResA.
n, the number of electrons involved in the reaction, was set
to 2, the expected value for a thiol-disulfide oxidoreductase. Setting
n = 1 gave very poor fits. All Eh calculations were based on the value of
(Eq. 1)
330 mV for the standard redox
potential (Em) of DTT at pH 7.0 (34).
Em values for DTT at other pH values were calculated
using a correction of
59 mV per pH unit increase.
1. Molecular weights were determined by reference to a
calibration curve prepared using bovine lung aprotinin (6,500),
horse heart cytochrome c (12,400), bovine erythrocyte
carbonic anhydrase (29,000), bovine serum albumin (66,000), and blue
dextran (~2,000,000).
1)
was performed as described by Schiött et al. (18).
Membranes were isolated from bacteria grown in NSMP at 37 °C as
described previously (35). Membrane protein concentrations were
determined using the bicinchoninic acid protein assay (Pierce Chemical
Co.), with bovine serum albumin as standard. N-terminal sequencing was carried out by Edman degradation (Biomolecular Analysis Facility, University of Leeds, UK) on proteins separated by SDS-PAGE, blotted onto polyvinylidene difluoride membranes, and stained with 0.005% (w/v) sulforhodamine B in 30% (w/v) methanol, 0.2% (v/v) acetic acid.
Blots were dried and stored at
20 °C. EI-MS was performed using a
VG platform electrospray mass spectrometer. For this, ~20 pmol of
protein was prepared in 20 µl of 1:1:0.005, acetonitrile:water:formic acid. Horse heart myoglobin (16951.48 Da) was used as a calibrant. Alkaline phosphatase activities of cell lysates of E. coli
were measured using p-nitrophenyl phosphate as substrate
(36). UV-visible absorbance spectra were recorded on a PerkinElmer
800 spetrophotometer.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Cytochrome c contents of
B. subtilis membranes. Autoradiograph of a
SDS-polyacrylamide gel of membranes of B. subtilis strains
labeled with 14C-heme. The strains and plasmids are
presented in Table I. The proteins indicated in the figure are subunit
II (CtaC) of cytochrome caa3, cytochrome
b (QcrB), and cytochrome c (QcrC) subunits of the
cytochrome bc complex, and one band corresponding to
cytochrome c550 (CccA) and cytochrome
c551 (CccB) which co-migrate. 40 µg of protein
was loaded in each lane.
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Fig. 2.
Analysis of B. subtilis resA
and its encoded protein product. A, nucleotide
sequence flanking the translational start point of resA. The
original designated start codon is underlined (21). A G
residue that was missing in the original published sequence and
revealed in this work is indicated by the asterisk and
bold lettering. The ATG translational start codon predicted
from the corrected sequence is indicated in bold.
B, corrected amino acid sequence of ResA. The
asterisks indicate the positions at which truncated ResA was
fused to PhoA for topology studies (see text). The cysteine residue at
a position characteristic of lipoproteins is underlined.
C, schematic representation of the deduced topology of ResA
(corrected sequence) based on the transmembrane segment prediction
program TMHMM (43) and data from the topology and localization studies
described in the text. The model features one transmembrane -helical
segment, and a major domain with the thioredoxin-like motif on the
outside of the cytoplasmic membrane. The numbers indicate
amino acid residues.
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Fig. 3.
Immunoblot analysis of isolated membranes
from B. subtilis strains using ResA antiserum.
The genotypes of the different strains and plasmids are presented in
Table I. The position of ResA is indicated on the right.
Mr indicates relative molecular mass. 5 µg of
protein was loaded in each lane.
1 × (mg protein)
1, respectively),
whereas lysates of E. coli cells containing the vector pHPKS
showed no activity (<0.01 µmol × min
1 × (mg
protein)
1). The alkaline phosphatase activity was found
to be associated with the particulate subfraction of the lysates.
Therefore, the N-terminal part of ResA probably functions as a membrane
anchor and the C-terminal thioredoxin-like domain is exposed on the
outer side of the cytoplasmic membrane, see Fig. 2C. ResA
contains one cysteine residue in the N-terminal region of the
polypeptide, in a position characteristic of lipoproteins (Fig.
2B). However, the protein does not satisfy other prediction
criteria for lipoproteins (46, 47), and analysis by SDS-PAGE and
Western botting of ResA antigens in strains LUH102 and LUH104, which
are deficient in Lgt (diacylglyceride transferase) and Lsp (type II
signal peptidase), respectively (48), showed the same apparent size of
ResA as found in 1A1 (data not shown). Therefore, ResA is most likely not a lipoprotein.
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Fig. 4.
Carboxymethylation of htsResA cysteine
thiols. Urea-polyacrylamide gel of htsResA following the addition
of iodoacetamide, iodoacetate, or mixtures of the two as described
under "Experimental Procedures." htsResA reacted with: lane
1, iodoacetamide only; lanes 2-4, 1:1, 3:1, and 9:1
ratios of iodoacetamide to iodoacetate; lane 5, iodoacetate
only. Lane 6 contains a mixture of equal portions of the
protein samples applied to lanes 1-5.
330 mV
compared with
245 mV at pH 7 (34, 52)) and was subsequently used for the determination of the midpoint potential of ResA (53-55). Fig. 5, A and B, show
plots of the fraction of reduced protein versus the
potential generated by DTT for sResA and htsResA, respectively. Fitting
the data from multiple titrations gave an Em of
340 ± 10 mV at pH 7 for both ResA proteins. The pH dependence of the midpoint potential of sResA was investigated between pH 6.0 and
8.0, see Fig. 5C. The data were fitted to a straight line with a gradient of
59 ± 1 mV per pH unit. This is
characteristic of the involvement of two protons and two electrons in
the redox process, as expected for a thiol-disulfide oxidoreductase in
this pH range.
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Fig. 5.
Redox titrations of ResA. A,
titration of sResA at pH 7.0. The reaction mixture contained oxidized
sResA at a concentration of 1 µM and varying ratios of
reduced and oxidized DTT (total concentration 5 mM). The
mixtures were equilibrated for 3 h prior to measurement of
fluorescence intensities, as described under "Experimental
Procedures." The solid line represents a fit to Equation 1
derived from the Nernst equation, with n = 2. This gave
an Em value of 342 mV. B, titration of
htsResA at pH 7.0. Conditions and analysis as in A. The fit
gave Em =
344 mV. C, pH dependence of
Em of sResA. Reaction conditions were as in
A. The line represents a least squares fit of the
data and has a slope of
59 mV per pH unit.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 6.
Scheme illustrating the function of ResA and
other membrane-bound thiol-disulfide oxidoreductases in cytochrome
c synthesis in B. subtilis.
Part I, reduced apocytochrome domain is translocated across
the cytoplasmic membrane. Part II, an intramolecular
disulfide bond in apocytochrome c, formed by the action of
BdbC/BdbD, is reduced by the action of ResA. ResA is reduced by CcdA,
which mediates transmembrane electron transfer from thioredoxin
(TrxA) in the cytoplasm. Part III, heme is
transported by a carrier (HT) from the cytoplasm to
apocytochrome c on the outer side of the cytoplasmic
membrane and holocytochrome c is formed. + and indicate the positive and the negative sides of the cytoplasmic
membrane.
In contrast to ResA, CcdA is also involved in spore synthesis. This indicates that CcdA, in addition to transferring reducing equivalents for cytochrome c assembly, also transfers reducing equivalents to components required for sporulation (28).
The redox potentials of numerous thiol-disulfide oxidoreductases have
been determined. In general terms, they fall into one of three
approximate types: high potential (about 100 mV and above),
e.g. E. coli DsbA (
89 mV) (57, 58); mid
potential (about
100 to
200 mV), e.g. eukaryotic
protein-disulfide isomerase (
110 to
190 mV) (59, 60); and low
potential (about
200 and below), e.g. thioredoxins (about
230 to
270 mV) (61-63). Each of these types is characteristic of a
different cellular function. The low potential, cytoplasmic
thioredoxins are involved in maintaining protein cysteine residues in
their reduced form. The mid potential protein-disulfide isomerases are
involved in thiol-disulfide interchanges and an
intermediate redox potential apparently facilitates a role in breaking
(reducing) and making (oxidizing) disulfide bonds. High potential DsbA
is located on the outside of the cytoplasmic membrane and functions in
the oxidation of protein thiols to form disulfide bonds in periplasmic
proteins. A number of characterized thiol-disulfide oxidoreductases in
Gram-negative bacteria are involved specifically in cytochrome
c assembly, including E. coli CcmG, R. capsulatus HelX, B. japonicum CycY, and P. denitrificans CcmG (12-16, 55). Where measured, these have
generally been found to be of the low potential, reductant-type, but
they are distinct from thioredoxins because they are located on the
outside of the cytoplasmic membrane, where they are believed to be
involved in the reduction of specific cellular components, for example,
apocytochrome c prior to heme attachment. Assuming that the
redox properties of sResA and full-length B. subtilis ResA
are similar, the Em =
340 mV obtained here for
sResA indicates that it is a reductant-type thiol-disulfide
oxidoreductase. This is consistent with a role for ResA in reducing the
cysteine residues of apocytochrome c immediately prior to
covalent heme attachment.
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FOOTNOTES |
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* This work was supported by a travel grant from The Swedish Royal Academy of Sciences and grants from the Swedish Research Council (contract 621-2001-3125) (to L. H.), the Biotechnology and Biological Sciences Research Council of the United Kingdom, The Wellcome Trust, and The Royal Society (to N. L. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this work.
To whom correspondence should be addressed: School of Chemical
Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, UK.
Tel.: 01603-592003; Fax: 01603-592003; E-mail:
n.le-brun@uea.ac.uk.
Published, JBC Papers in Press, March 7, 2003, DOI 10.1074/jbc.M300103200
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ABBREVIATIONS |
---|
The abbreviations used are:
IPTG, isopropyl--D-thiogalactopyranoside;
DTT, dithiothreitol;
EI-MS, electrospray ionization-mass spectrometry;
MOPS, 3-morpholinopropanesulfonic acid;
NSMP, nutrient sporulation medium
with phosphate;
TMPD, N,N,N',N'-tetramethyl-p-phenylenediamine.
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