Evidence is presented for an alternative to the superoxide
dismutase (SOD)-catalase oxidative stress defense system in
Desulfovibrio vulgaris (strain Hildenborough). This
alternative system consists of the nonheme iron proteins, rubrerythrin
(Rbr) and rubredoxin oxidoreductase (Rbo), the product of the
rbo gene (also called desulfoferrodoxin). A
rbo strain of D. vulgaris was found to be
more sensitive to internal superoxide exposure than was the wild type.
Unlike Rbo, expression of plasmid-borne Rbr failed to restore the
aerobic growth of a SOD-deficient strain of Escherichia coli. Conversely, plasmid-borne expression of two different Rbrs from D. vulgaris increased the viability of a
catalase-deficient strain of E. coli that had been exposed
to hydrogen peroxide whereas Rbo actually decreased the viability. A
previously undescribed D. vulgaris gene was found to encode
a protein having 50% sequence identity to that of E. coli
Fe-SOD. This gene also encoded an extended N-terminal sequence with
high homologies to export signal peptides of periplasmic redox
proteins. The SOD activity of D. vulgaris is not affected
by the absence of Rbo and is concentrated in the periplasmic fraction
of cell extracts. These results are consistent with a superoxide
reductase rather than SOD activity of Rbo and with a peroxidase
activity of Rbr. A joint role for Rbo and Rbr as a novel cytoplasmic
oxidative stress protection system in D. vulgaris and other
anaerobic microorganisms is proposed.
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INTRODUCTION |
Although sulfate-reducing bacteria
are classified as strict anaerobes, many species are aerotolerant and
some species have been shown to accumulate at oxic-anoxic interfaces
(28). Therefore, sulfate-reducing bacteria must deal with
oxidative stress. Some sulfate-reducing bacteria have been shown to
contain superoxide dismutases (SODs) and catalases, but other species
do not demonstrate the activities expected for these enzymes (15,
18, 43). The apparent absence of SOD and catalase can be
rationalized on the basis that their catalytic dismutation reactions
(reactions 1 and 2) generate O2, which may be
disadvantageous for strict anaerobes.
Evidence for an alternative oxidative stress protection mechanism in
sulfate-reducing bacteria has begun to emerge. Rubredoxin oxidoreductase (Rbo), which has also been named desulfoferrodoxin (29), is the product of the rbo gene in several
sulfate-reducing bacteria (7). A Desulfovibrio
vulgaris strain with the rbo gene deleted was shown to
be more air sensitive than the wild type (45). The Rbo
from Desulfoarculus baarsii was shown to complement a
sodA sodB defect in Escherichia coli
(31). Rbo has no detectable sequence homologies to known
SODs, and little or no SOD activity has been found for Rbos in vitro
(26, 35). Nevertheless, the D. baarsii Rbo was
reported to reduce the steady-state level of intracellular superoxide
in the E. coli sodA sodB strain (25). This Rbo
in vitro showed evidence for superoxide reductase (SOR) activity
(26), i.e., reduction of O2
,
presumably to H2O2, without dismutation
(reaction 3).
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(1)
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(2)
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(3)
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Rbo is a homodimeric protein, each subunit of which contains two
mononuclear nonheme iron centers (11). Center I contains a
distorted rubredoxin-type [Fe(SCys)4] coordination sphere
and is presumably an electron transfer center. Center II contains a
unique [Fe(NHis)4(SCys)] site that is rapidly oxidized by
O2 and is, therefore, the likely site of
superoxide reduction (26). The only iron site in a blue
nonheme iron protein, neelaredoxin (Nlr) from Desulfovibrio
gigas (10), closely resembles that of Rbo center II
(11).
A second type of nonheme iron protein, rubrerythrin (Rbr), has also
been implicated in oxidative stress defense in anaerobic microbes
(1, 2, 24). Rbr is a homodimeric protein that contains
both a rubredoxin-like [Fe(SCys)4] center and a
nonsulfur, oxo-bridged diiron site (14). The
best-characterized Rbr is that from the anaerobic sulfate-reducing
bacterium D. vulgaris (strain Hildenborough). In vitro,
D. vulgaris Rbr is able to function as the terminal
component of an NADH peroxidase, catalyzing the reduction of hydrogen
peroxide to water (reaction 4) (12, 13):
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(4)
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D. vulgaris contains a second Rbr-like protein, called
nigerythrin (Ngr) (27, 32), which also exhibits NADH
peroxidase activity (13).
In the present work we present evidence for the roles of Rbo and Rbr in
the protection of D. vulgaris against exposure to air,
superoxide, and hydrogen peroxide. We also describe a D. vulgaris gene with high sequence homology to bacterial Fe-SODs. A
joint role for Rbo and Rbr as a novel oxidative stress protection system in anaerobic microorganisms is proposed.
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MATERIALS AND METHODS |
Molecular biology reagents and procedures.
Molecular biology
procedures not explicitly described below followed those in references
3 or 36. Restriction enzymes were
from either Boehringer Mannheim (Indianapolis, Ind.) or Promega, Inc.
(Madison, Wis.).
Bacterial strains and plasmids.
The bacterial strains and
plasmids used in this work are listed in Table
1.
Media and growth conditions.
D. vulgaris
(Hildenborough) strains were maintained in liquid culture in
Postgate's anaerobic medium C (33), supplemented with
kanamycin (50 µg/ml) and chloramphenicol (10 µg/ml) when appropriate. Agar plates were poured aerobically using Postgate's medium E (33). After 24 h, the plates were
transferred to a Coy anaerobic chamber (Coy Laboratory Products, Inc.,
Grass Lake, Mich.) containing an atmosphere of 5% hydrogen, 10%
carbon dioxide, and 85% nitrogen and were allowed to equilibrate for 4 days prior to use. E. coli strains were grown aerobically at
37°C in Luria-Bertani (LB), M9, or M63 minimal medium containing
ampicillin (100 µg per ml) when needed for plasmid maintenance. Where
noted, M63 minimal medium was supplemented with 0.1% Casamino Acids
and 0.5 mg of vitamin B1 per liter.
Cell extracts.
To prepare periplasmic and cytoplasmic
fractions of D. vulgaris cells, 1-liter anaerobic cultures
were grown overnight at 37°C to early stationary phase. All further
workup was done under an aerobic atmosphere. Periplasmic and
cytoplasmic fractions were prepared essentially as described previously
(32, 42). The cells were harvested by centrifugation for
20 min at 4,000 × g and washed once with 50 mM
Tris-HCl (pH 8.0). The cells were resuspended in 10 ml of 50 mM
Tris-HCl-50 mM EDTA (pH 9.0) and were incubated on ice, with
occasional stirring, for 45 min. After centrifugation, the red-colored
supernatant, designated the periplasmic fraction, was removed.
Cytochrome c3, a periplasmic protein, was
measured by absorbance at 410 nm for oxidized samples and at 419 nm for dithionite reduced samples. The pellet was resuspended in 10 ml of 50 mM Tris-HCl-50 mM EDTA (pH 9.0) and was lysed to release the
cytoplasmic fraction by three passes through a French pressure minicell
at 18,000 psi. Desulfoviridin, a cytoplasmic protein, was detected
in cytochrome fractions spectrophotometrically, as determined by
A628 
(A608 + A648)/2 (21). The periplasmic and cytoplasmic fractions were dialyzed against 50 mM phosphate buffer (pH
7.8). Protein quantification was performed by the Bio-Rad DC protein
assay using bovine serum albumin standards.
Superoxide dismutase activity.
A standard assay that relies
on the inhibition of cytochrome c reduction by superoxide
generated from xanthine/xanthine oxidase was used (6).
Aliquots (~100 µl) of cytoplasmic and periplasmic fractions were
added to achieve ca. 50% inhibition of cytochrome c under
standard assay conditions.
D. vulgaris oxidative stress assays.
For
exposure to O2 and superoxide, 100-ml cultures of D. vulgaris wild type and strain L2 were grown in anaerobic medium C at 37°C until mid-log phase (optical density at 600 nm
[OD600], ~0.6). Forty-five-milliliter aliquots of the
anaerobic cultures were transferred to sterile 250-ml flasks and
continuously stirred with a magnetic stirrer in air at room
temperature. For exposure to superoxide, paraquat (methyl viologen)
(PQ) was added to a concentration of 10 µM. Aliquots (0.1 to 1 ml) of
these air-exposed cultures were removed hourly, transferred to the
anaerobic chamber, diluted (100 to 106) into
anaerobic medium C, and immediately plated onto medium E plates in
triplicate. After incubating the cultures for 5 days at 37°C,
colonies were counted and the number of surviving CFU/ml was
determined. As noted previously (45), the oxidatively
stressed D. vulgaris cells produced variable sized colonies,
making them difficult to enumerate. For the experiments reported in the
present work, all colonies (large and small) were enumerated after 5 days of anaerobic incubation, and each reported CFU/ml number is the average from three plates.
To test for hydrogen peroxide sensitivity, anaerobic mid-log-phase
cultures were spread onto medium E plates. Hydrogen peroxide (2.5 or
5.0 µmol in 10 µl of final volume) was spotted onto circular glass-fiber membranes placed on the cell lawns. The plates were incubated anaerobically for 3 days, and the diameters of growth inhibition surrounding the filters were measured to compare sensitivities.
Cloning of rbo, rbr, ngr, and
nlr.
The D. vulgaris genes encoding Rbr, Ngr, and
Rbo and the D. gigas gene encoding Nlr were amplified by PCR
and cloned into the overexpression vector pCYB1. rbr and
ngr were amplified from template plasmids pDK3-5 and pNS1,
respectively. rbo was amplified from D. vulgaris
chromosomal DNA. D. gigas nlr was cloned from D. gigas chromosomal DNA using a procedure similar to that described by Silva et al. (38), and pNLR119 (Table
1) was used as the template. Both
D. vulgaris and D. gigas chromosomal DNAs had
been isolated as previously described (27). Sequences of
the oligonucleotide primers duplicated the N- and complementary
C-terminal 14 bases of each gene using sequences from GenBank accession
numbers M28848 (rbo), U82323 (rbr), U71215
(ngr), and AF034965 (nlr). An NdeI
restriction site was incorporated into the start codon of each
N-terminal primer. A stop codon (TGA) and an appropriate restriction
site, either a SapI (rbo, ngr),
BamHI (rbr), or HindIII (nlr) site, were added to the 3' end of each C-terminal
primer. All PCRs were carried out in 50-µl volumes in 1× PCR buffer
(Boehringer Mannheim) containing 1.5 mM MgCl2, 200 mM
deoxynucleoside triphosphate, 0.05 mM primers, and 2.5 U of
Taq DNA polymerase (Promega). The amplification procedure
consisted of a hot start of the template DNA and primers at 94°C for
10 min, followed by 30 cycles of 94°C for 1 min, 60°C for 1 min,
and after addition of the remaining reaction components, 72°C for 2 min. The purified PCR products were digested with the appropriate
restriction enzymes and ligated into corresponding restriction sites of
pCYB1. Nucleotide sequences of the genes in the recombinant plasmids
were verified by DNA sequencing, which was carried out in the Molecular
Genetics Instrumentation Facility at the University of Georgia.
E. coli H2O2 survival
assay.
All steps were carried out aerobically. Cultures of
E. coli strain NC202 (katE katG) harboring the
pCYB1-based vectors listed in Table 1 were grown at 37°C to an
OD600 of 1.0 in LB-ampicillin medium.
Isopropyl-
-D-thiogalactopyranoside (IPTG) was added to 2 mM and the culture was allowed to grow to an OD600 of 2.2. Aliquots of the cultures were diluted into 50 ml of LB medium in a
250-ml flask without antibiotics to an initial OD600 of
0.2. Hydrogen peroxide from a 30% aqueous stock was added to achieve a
concentration of 2.5 mM H2O2. Aliquots of the
cultures were removed immediately before and 30 min after the
H2O2 challenge. These aliquots were diluted in
LB medium containing catalase (100 µg/ml) and plated in triplicate on
LB agar plates containing catalase (100 µg/ml). Colonies were
enumerated after 3 or 4 days at 37°C.
SOD complementation assay.
E. coli QC774 (sodA
sodB) cultures harboring one of the pCYB1-derived expression
vectors were grown aerobically to saturation in M63-ampicillin medium
supplemented with amino acids and vitamin B1 and were diluted to an
initial OD600 of ca. 0.06 in aerobic M63-ampicillin medium
without supplements. The cultures were incubated with shaking at
37°C, and the growth rate was monitored by measuring the
OD600.
Cloning of a D. vulgaris SOD gene.
A conserved
amino-acid-sequence region found in several bacterial Fe-SODs,
DVWEHAYYLDYQN, was used to design a degenerate oligonucleotide probe with the sequence
5'-GAYGTNTGGGARCAYGCNTAYTAYCTNGAYTAYCARAAY-3'. The 3' end of
the probe was labeled with digoxigenin-ddUTP and then used in Southern
blotting of D. vulgaris (Hildenborough) chromosomal DNA,
both according to the protocol for the Genius system (Boehringer
Mannheim). The probe hybridized to a 4.5-kbp EcoRI/HindIII restriction fragment of
D. vulgaris chromosomal DNA. Therefore,
EcoRI/HindIII restricted DNA fragments in the 4- to 5-kb range were isolated from an 0.8% low melting agarose gel by
electroelution and were ligated into pUC18. The ligation mixture was
transformed into E. coli DH5
and plated onto
M9-ampicillin agar plates containing X-Gal
(5-bromo-4-chloro-3-indolyl--D-galactopyranoside) at 2 mg/ml. White colonies were selected to inoculate 1.5-ml cultures grown
in LB-ampicillin. Plasmids were purified by alkaline lysis from six
combined cultures and were screened for the presence of the
sod gene by Southern hybridization. Screening of 100 plasmid pools resulted in the isolation of a plasmid containing a 4.5-kbp EcoRI/HindIII fragment containing the
D. vulgaris sod. Sequencing of the cloned gene was performed
at the Molecular Genetics Instrumentation Facility at the University of Georgia.
Nucleotide sequence accession number.
The newly determined
sequence was deposited in the GenBank database under accession no.
AF034841.
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RESULTS |
Rbo protects D. vulgaris from exposure to superoxide
but not to hydrogen peroxide.
Survival of the D. vulgaris rbo strain, L2, was previously shown to be
lower than that of the wild type when anaerobically growing cells were
diluted into aerobic medium and then exposed to air for several hours
(45). PQ can be used to generate a steady-state level of
superoxide within bacterial cells under aerobic conditions. We
therefore compared the survival of D. vulgaris wild type
with that of strain L2 after exposure to air + 10 µM PQ
([air + PQ]). The viability of log-phase D. vulgaris
cells, as surviving CFU/ml exposed to [air + PQ] over a time
course of 4 h, is shown in Fig. 1A,
normalized to the air-only surviving CFU/ml. The unnormalized data are
plotted in Fig. 1B. For cells of both the wild type and strain L2 that
were exposed to air only, we observed a steeper drop-off in cell
survival versus time than observed previously (45). This
phenomenon can be attributed to the larger surface area for aeration
used in our experiments. As can be seen in Fig. 1B, at 4 h
exposure to air only, the surviving CFU/ml for strain L2 (1.03 ± 0.04 × 108) was greater than that of wild type
(9 ± 4 × 106). This artifact could be due to
the greater initial viable cell density we used for strain L2 than for
wild type, thereby affording greater protection against oxidative
stress at the earlier exposure times. By 8 h of air-only exposure,
the surviving CFU/ml of strain L2 (9 ± 1 × 104)
had decreased below that of wild type (2.1 ± 0.1 × 105), consistent with the greater air sensitivity of strain
L2 previously observed (45). Strain L2 continued to
exhibit lower cell survival at 12 h of air-only exposure (data not
shown). By 24 h of air-only exposure neither the wild type nor
strain L2 showed any survival under our conditions.
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FIG. 1.
Viabilities of D. vulgaris wild type and L2
strains following exposure to either air or air plus 10 µM PQ. (A)
Surviving CFU versus the time of the [air+PQ]-exposed cells
normalized to the surviving CFU of the air-only exposed cells. (B) The
actual CFU/ml versus the time for both air only- and [air + PQ]-exposed cells.
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The novel observation in the present work occurred upon the addition of
10 µM PQ to the air-exposed D. vulgaris cells. Both the
wild type and strain L2 lost viability faster when exposed to
[air + PQ] than to air only but the effect of PQ was much
greater on strain L2 (Fig. 1A). After 4 h the survival of
wild-type cells exposed to [air + PQ] had decreased by a factor
of ~2 relative to those exposed to air only (9 ± 4 × 105 versus 4 ± 2 × 106), whereas
the survival of the L2 [air + PQ]-exposed cells decreased by a
factor of ~500 (1.03 ± 0.04 × 108 versus
1.9 ± 0.2 × 105) relative to the cells exposed
to air only. The much greater [air + PQ] versus air-only
sensitivity of strain L2 compared to wild type was verified in two
other experiments (data not shown). The addition of up to 100 µM PQ
did not affect the anaerobic growth (measured as OD600) of
either D. vulgaris wild type or L2 cultures (data not shown).
Anaerobic peroxide sensitivity was tested by growth inhibition on agar
plates containing lawns of either wild type or L2 and filter disks
saturated with hydrogen peroxide solutions. We found no significant
difference in the zones of growth inhibition between wild type and L2
using two different amounts of hydrogen peroxide (Table
2).
SOD activity of D. vulgaris is not affected by the
absence of Rbo and is concentrated in the periplasmic fraction.
A
low level of SOD activity had previously been reported in D. vulgaris cell extracts (18). In order to localize and
quantitate this activity we tested periplasmic and cytoplasmic
fractions of D. vulgaris wild type and strain L2 for SOD
activity using a standard assay (Table 2). The fidelity of periplasmic
and cytoplasmic preparations were checked by comparing cytochrome
c3 (a periplasmic protein) with desulfoviridin
(a cytoplasmic protein) levels using a previously described
spectrophotometric method (32, 42). No desulfoviridin
could be detected in the periplasmic fractions, while more than 70% of
the total cytochrome c3 detected was seen in the
periplasmic fractions (data not shown). The specific SOD activities
(per milligram of total protein) of these subcellular fractions were
unaffected by the absence of the rbo gene. For both strains,
the specific SOD activity was approximately five times greater in the
periplasmic fractions than in the cytoplasmic fractions (Table 2),
consistent with the presence of a periplasmic SOD in D. vulgaris.
D. vulgaris has an Fe or Mn SOD-like gene.
Using a
degenerate oligonucleotide probe that was designed to encode a
conserved region of Fe-SOD sequences, we cloned a D. vulgaris gene encoding a protein with 50% sequence identity (59% similarity) to that of E. coli Fe-SOD, when aligned as shown
in Fig. 2. The N terminus for the
D. vulgaris SOD was taken as the first methionine that codes
for a continuous open reading frame and has a recognizable ribosome
binding site (AGGAG) 6 bp upstream. Analysis of the amino acid sequence
using SignalP (30) suggested the existence of a 34-residue
export leader peptide with the most likely cleavage site between
residues A34 and A35. As shown by the sequence alignments in Fig.
3, the putative leader peptide of the
D. vulgaris SOD resembles those that are commonly used for
export of bacterial redox proteins (5) with the notable exception that one of the two residues of a conserved arginine pair is
replaced by a serine. We have independently confirmed this replacement
by examination of the preliminary genome sequence for D. vulgaris (Hildenborough) (The Institute for Genomic Research, personal communication). The implication is that the D. vulgaris Fe- or Mn-SOD is periplasmic, in contrast to the Fe-SOD
from E. coli, which is known to be cytoplasmic. Our database
searches have not located a complete amino acid or nucleotide sequence of a recognizable SOD from any other sulfate-reducing bacterium. None
of our attempts to express this D. vulgaris SOD gene in
E. coli, including QC774, have resulted in an active SOD.
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FIG. 2.
Amino acid sequence alignment of the putative
sod gene product from D. vulgaris with the
Fe-containing SOD from E. coli (GenBank accession no.
J03511.1). The putative leader sequence cleavage site in the D. vulgaris SOD sequence is shown in italics between residues A34 and
A35. Asterisks (*) indicate iron ligand residues in E. coli SOD (22). +, amino acids are similar to each
other.
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FIG. 3.
Sequence alignment of export signal peptides from
selected bacterial and archaeal redox proteins with the putative signal
peptide of the D. vulgaris (Hildenborough) SOD (DvH_SOD).
Alignment of the sequences follows that in reference
5. Sequence sources (GenBank accession numbers) are
as follows: Db_HysB, Desulfomicrobium baculatum NiFeSe
hydrogenase (P13063); Ws_HydA, Wolinella succinogenes NiFe
hydrogenase (S33852); Sa_SoxF, Sulfolobus acidocaldarius
Rieske iron-sulfur protein (S56156); Ec_HybA, E. coli
hydrogenase (P37179); Ec_NrfC, E. coli putative iron-sulfur
protein (P32708); Hi_NrfC, Haemophilus influenzae putative
iron-sulfur protein (P45015); Sm_NosZ Sinorhizobium meliloti
nitrous oxide reductase (Q59746); and Ps_NosZ, Pseudomonas
stutzeri nitrous oxide reductase (P19573).
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Rbo but not Rbr or Nlr can complement E. coli sodA sodB.
E. coli strain QC774 is deficient in both sodA
and sodB encoding the Mn and Fe SODs, respectively
(9). Under aerobic conditions this strain is unable to
grow in minimal medium lacking amino acid supplements. This phenotype
is attributed to superoxide damage to iron-sulfur enzymes in the
biosynthetic pathway of branched chain amino acids and to Fenton
chemistry resulting from increased free iron levels (41).
Figure 4 shows that plasmid-borne
expression of D. vulgaris Rbo is able to restore aerobic
growth to this strain whereas expression of D. vulgaris Rbr
and Ngr and D. gigas Nlr are not. Strain QC774 will grow
aerobically in rich medium (LB) but unlike the wild type, this growth
is inhibited by 10 µM PQ. Rbo expression completely restored growth
to 10 µM PQ-stressed QC774 in LB, whereas once again expression of
Rbr, Ngr, or Nlr did not (data not shown). Preinduction of protein
overexpression by the addition of IPTG to 0.4 mM did not change these
outcomes. The plasmid-expressed Rbr, Rbo, and Nlr have all been
isolated from QC774 (E. D. Coulter, N. V. Shenvi, and D. M. Kurtz, Jr., unpublished data), demonstrating that these proteins can
be expressed in this strain.
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FIG. 4.
Aerobic growth versus the time at 37°C in M63 medium
of E. coli QC774 (sodA sodB) containing
pCYB1-based plasmids expressing D. vulgaris Rbo and Rbr and
D. gigas Nlr.
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D. vulgaris Rbr and Ngr but not Rbo are able to rescue
E. coli kat from H2O2 stress.
E. coli strain NC202 lacks both katG and
katE genes encoding catalase/peroxidase (HPI) and catalase
(HPII), respectively (39). Plasmid-borne expression of
D. vulgaris Rbr or Ngr increased the survival of this strain
when exposed to H2O2 under aerobic conditions, as shown in Fig. 5.
H2O2-exposed NC202 cells expressing Rbr showed at least 25% higher viability than those containing the parent plasmid, and cells expressing Ngr were fully protected against H2O2 exposure. Expression of Rbo failed to
provide any protection to the H2O2-exposed
NC202 strain and in fact reduced its viability to a few percent upon
peroxide stress. Overexpression of these heterologous proteins in NC202
was verified by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (data not shown).
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FIG. 5.
Viability of aerobically grown E. coli NC202
(katG katE) expressing plasmid-borne D. vulgaris
Rbr, Ngr, or Rbo genes following a 30-min exposure to 2.5 mM
H2O2. IPTG (0.4 mM) was added to induce
overexpression of the genes prior to H2O2
exposure.
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DISCUSSION |
The results reported here indicate that D. vulgaris Rbo
functions as an intracellular superoxide scavenger but not as an SOD, that D. vulgaris Rbr and Ngr protect against hydrogen
peroxide stress in vivo, and that D. vulgaris contains a
periplasmic Fe- (or possibly Mn-)SOD.
Sequence analysis of the D. vulgaris Fe-SOD gene indicated
the presence of an export leader peptide commonly found in periplasmic redox proteins. The replacement of one of the two residues of a highly
conserved arginine pair by a serine is unusual if not unique for redox
protein leader peptides, including those previously reported in
sulfate-reducing bacteria (44). Consistent with the
implied periplasmic localization, we found that the majority of the SOD
activity in D. vulgaris is associated with the periplasmic fraction of cell extracts, even in the rbo-deleted strain.
We have found no data addressing the localizations of SODs in other sulfate-reducing bacteria. A few other periplasmic Fe/Mn-SODs have been
reported, but only from aerobes (4, 16, 37).
A D. vulgaris rbo strain was previously found
to be more air sensitive than wild type (45) but our
results show that this air sensitivity is greatly magnified by PQ, a
known intracellular superoxide generator. Presumably, the reduction in
viability of the rbo strain exposed to air only is also
due to intracellular generation of superoxide, but at a much slower
rate than in the [air + PQ]-exposed cells. The D. vulgaris rbo strain showed no significant difference
in the SOD activity of cellular extracts relative to wild type,
indicating that Rbo is not an SOD. We therefore conclude that Rbo
protects D. vulgaris against internal superoxide stress by
functioning as an SOR (reaction 3). The reduction in viability of the
aerobically grown E. coli catalase-deficient strain upon
overexpression of Rbo can be explained by Rbo-catalyzed production of
excess hydrogen peroxide from endogenously generated superoxide. A
BLAST search of the preliminary D. vulgaris genome sequence
(The Institute for Genomic Research, personal communication) revealed
no other Rbo or Fe-SOD homologs. All available evidence indicates that
Rbos are cytoplasmic proteins whereas our results strongly suggest that
the D. vulgaris SOD is periplasmic. A proposed model for
compartmentalized superoxide generation and scavenging in D. vulgaris is presented in Fig. 6.
We found that neither D. vulgaris Rbr nor Ngr is capable of
restoring growth to the E. coli sodA sodB strain in aerobic
minimal medium. Rbr and Ngr are, however, capable of increasing the
survival of an E. coli catalase-deficient strain that was
exposed to hydrogen peroxide. This protection by Rbr and Ngr apparently
occurs in competition with at least some of the 30 or more other
proteins that are induced in response to peroxide stress in E. coli (34). As is the case for Rbo, the protection
against hydrogen peroxide by Rbr and Ngr presumably requires an
endogenous electron source, perhaps functioning analogously to the in
vitro NADH peroxidase activity (reaction 4) (12, 13). Rbo
and Rbr (and Ngr) may thus be complementary components of an
alternative oxidative stress defense in D. vulgaris, as
shown in Fig. 6.
Heme-containing catalases have been isolated from D. vulgaris (Hildenborough) and D. gigas (15,
18), and a catalase gene has previously been reported from
D. vulgaris (Miyazaki F) (GenBank accession no. AB020341).
Neither the Miyazaki nor the Hildenborough catalase genes (The
Institute for Genomic Research, personal communication) contain a
recognizable signal peptide sequence, which indicates that D. vulgaris catalase is cytoplasmic, as is Rbr (32).
Since heme catalases typically have Kms for
H2O2 in the millimolar to molar range
(46), the D. vulgaris catalase might complement the intracellular Rbr-catalyzed hydrogen peroxide removal by
diminishing high concentrations of extracellular hydrogen peroxide,
which is freely diffusible across cell membranes (Fig. 6).
Endogenous electron donors to Rbo or Rbr have not been identified
either when complementing the E. coli strains or in the native organism. The inability of plasmid-expressed D. gigas
Nlr to restore aerobic growth to E. coli sodA sodB implies
that center I {[Fe(SCys)4]}, which is presumably the
electron transfer center of Rbo (and not present in Nlr), as well as
center II, is required for complementation by Rbo. In D. vulgaris, one potential redox partner is rubredoxin, whose gene,
rub, is cotranscribed with rbo (7).
A rub gene is also adjacent to the rbo gene in
D. baarsii (Fig. 7)
(31). Based on genetic proximity, Rbr could also use
rubredoxin-like proteins as redox partners. In D. vulgaris (Hildenborough), Rbr is cotranscribed with Rdl, yet another class of
rubredoxin-type protein (23, 27).
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FIG. 7.
Diagrams of open reading frames encoding adjacent
rbo, rbr, and rub homologs from
anaerobic bacteria and archaea. rub, rubredoxin gene;
rbo, Rbo homolog gene; nlr, neelaredoxin homolog
gene; rdl, rubredoxin-like protein gene; fur, Fur
homolog gene. Horizontal distances between boxes are proportional to
the relative spacings of the genes. Arrow-shaped orientations of boxes
show putative transcription directions. Open reading frames were
derived from sequences deposited in GenBank with the following
accession numbers: X99543 (D. baarsii), AE001047
(Archaeoglobus fulgidus), AF156097 (P. furiosus),
AJ248285 (Pyrococcus abysii), AE001739 (Thermotoga
maritima), AF202316 (Moorella thermoacetica), U67520
(Methanococcus jannaschii), AE000854 (Methanobacterium
thermoautotrophicum), M28848 [Desulfovibrio vulgaris
(Hildenborough) rbo-rub] and U82323 [D.
vulgaris (Hildenborough) fur-rbr-rdl].
|
|
The rbr gene in D. vulgaris (Hildenborough) is
transcribed with a third gene designated fur
(27), which encodes a protein showing highest sequence
homology to PerR, a peroxide-responsive regulatory protein in
Bacillus subtilis (8, 47). The product of the
fur-like gene may, therefore, regulate the Rbo-Rbr oxidative stress response in D. vulgaris. The genes encoding Rbo and
Rbr (and Ngr) occur in separate operons in D. vulgaris
(7, 27). However, as diagrammed in Fig. 7, in other
anaerobic microorganisms Rbo- and Rbr-like genes are located either in
succession or within what appears to be the same operon.
To date, Rbo and Rbr (or their genes) have been reported only in
anaerobic or microaerophilic bacteria and archaea, whereas we have
found no Rbo or Nlr or Rbr homologs in sequenced genomes of aerobes (as
of June 2000). We therefore propose that Rbo-Rbr constitutes an
alternative oxidative stress protection system in anaerobes and
microaerophiles operating similarly to that diagrammed in Fig. 6. Some
aspects of our proposal resemble those recently suggested for oxidative
stress protection in the anaerobic hyperthermophilic archaeon
Pyrococcus furiosus (19). P. furiosus contains an Nlr-like protein with superoxide reductase
activity as well as an Rbr, the genes for which are tandemly located
(Fig. 7). The microorganismic segregation of SOD-catalase between
aerobes and anaerobes appears to be less distinct than for Rbo-Rbr
(20). The latter segregation suggests that the Rbo-Rbr
oxidative stress protection system is well suited to anaerobic life in
an aerobic world.
This research was supported by NIH grant GM40388 to D.M.K. and a
grant from the Natural Sciences and Engineering Research Council
(NSERC) to G.V.
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Abstract
Introduction
