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Previous Article | Next Article 
J Bacteriol, May 1998, p. 2694-2700, Vol. 180, No. 10
Department of Biochemistry, University of
Connecticut Health Center, Farmington, Connecticut 06030
Received 29 December 1997/Accepted 16 March 1998
Methionine residues in Methionine oxidation is a
significant form of protein damage caused by endogenous or
environmental oxidizing agents (35). Methionine residues may
be oxidized to the sulfoxide form with t-butyl hydroperoxide
(tBHP) or hydrogen peroxide (H2O2) under relatively mild conditions (11) or to the sulfone form with other oxidizing agents under harsher conditions (12).
Although oxidation of methionine residues has no effect on the function of some polypeptides (8, 11), in other proteins methionine oxidation severely inhibits biological function (5, 36). Since methionine oxidation can alter protein function, it is not surprising that cells have at least two mechanisms for dealing with
proteins containing oxidized methionine residues. First, it appears
likely that oxidized proteins are preferentially degraded in vivo
(35). Second, an enzyme termed peptidyl methionine sulfoxide reductase (MsrA), which can reduce methionine sulfoxide residues in
proteins to methionine and thus restore protein function (16, 17), has been identified in both prokaryotes and eukaryotes.
Spores of various Bacillus and Clostridium
species can survive for long periods in air and are very resistant to
oxidizing agents (e.g., H2O2) compared to their
corresponding growing cells (22, 27, 32). Since spores of
Bacillus and Clostridium species are
metabolically and enzymatically dormant, they are unable to deal with
proteins containing oxidized methionine residues by either protein
turnover or enzymatic reduction until they initiate spore germination
(26, 32). Consequently, if methionine oxidation in a protein
destroys the protein's function, then dormant spores may have some
mechanism(s) for preventing the initial methionine oxidation. In order
to analyze methionine oxidation in a dormant spore protein, we decided
to study this process in the The Bacteria and plasmids used and conditions for growth and peroxide
killing.
The bacterial strains and plasmids used in this work are
listed in Table 1; all B. subtilis strains are derivatives of strain 168. B. subtilis was transformed as previously described (1), and transformants were selected by their Cmr. B. subtilis was routinely grown and sporulated at 37°C in 2× SG
medium, and spores were purified as described previously
(18). All spores used were free (>98%) of sporulating
cells and growing cell debris. Spore resistance to heat,
H2O2, and tBHP was determined as described
previously (20, 25).
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
In Vitro and In Vivo Oxidation of Methionine
Residues in Small, Acid-Soluble Spore Proteins from
Bacillus Species
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
/
-type small, acid-soluble spore
proteins (SASP) of Bacillus species were readily oxidized
to methionine sulfoxide in vitro by t-butyl hydroperoxide
(tBHP) or hydrogen peroxide (H2O2). These
oxidized /-type SASP no longer bound to DNA effectively, but DNA
binding protected /-type SASP against methionine oxidation by
peroxides in vitro. Incubation of an oxidized /-type SASP with
peptidyl methionine sulfoxide reductase (MsrA), which can reduce
methionine sulfoxide residues back to methionine, restored the
/-type SASP's ability to bind to DNA. Both tBHP and
H2O2 caused some oxidation of the two
methionine residues of an /-type SASP (SspC) in spores of
Bacillus subtilis, although one methionine which is highly
conserved in /-type SASP was only oxidized to a small degree.
However, much more methionine sulfoxide was generated by peroxide
treatment of spores carrying a mutant form of SspC which has a lower
affinity for DNA. MsrA activity was present in wild-type B. subtilis spores. However, msrA mutant spores were no
more sensitive to H2O2 than were wild-type spores. The major mechanism operating for dealing with oxidative damage
to /-type SASP in spores is DNA binding, which protects the
protein's methionine residues from oxidation both in vitro and in
vivo. This may be important in vivo since /-type SASP containing
oxidized methionine residues no longer bind DNA well and /-type
SASP-DNA binding is essential for long-term spore survival.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
/-type small, acid-soluble spore
proteins (SASP) from Bacillus species.
/-type SASP of Bacillus and Clostridium
species are a family of highly abundant, nonspecific DNA binding
proteins, which are synthesized only within the developing forespore
(30). Typically, there are two major /-type SASP and a
number of minor /-type SASP (30) which together
saturate the spore DNA and protect it from a variety of environmental
insults (30, 31); the /-type SASP are the major
determinant of spore UV resistance, and a significant determinant of
spore resistance to both heat and oxidizing agents (14, 27,
32). The primary sequence of these proteins has been highly
conserved both within and between species, and nearly all /-type
SASP identified to date (17 of 20) from species of Bacillus,
Sporosarcina, and "Thermoactinomyces" contain
one highly conserved methionine residue in the middle of the protein
(30). In addition, some /-type SASP (11 of 20) contain
an additional methionine residue at one of two positions near the
carboxy terminus of the protein (30). In this communication,
we report the results of studies on the oxidation of methionine
residues within /-type SASP in vitro and in vivo, as well as the
analysis of the phenotype of an msrA mutant of
Bacillus subtilis.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
TABLE 1.
Bacterial strains and plasmids used in these studies
99.9% killing of spores.
tBHP-treated spores were harvested by centrifugation, washed once with
20 ml of 50 mM dithiothreitol (DTT), and incubated in 20 ml of 50 mM DTT for 1 h on ice. H2O2 treatment of
spores was halted by the addition of 7,000 U of catalase (Worthington).
All peroxide-killed spores were washed once with distilled water and
centrifuged, and the spore pellet was frozen and lyophilized.
Escherichia coli strains were grown at 37°C in 2× YT
medium (16 g of tryptone, 10 g of yeast extract, and 5 g of
NaCl per liter of H2O) with ampicillin (100 µg/ml).
/-type SASP purification.
Large-scale purification of
/-type SASP was performed with E. coli strains
overexpressing these proteins essentially as described previously
(9, 29), but with some modifications. E. coli
PS708 and PS2537 were grown to an optical density at 600 nm of 1.5 and
made to 0.5 mM concentrations in
isopropyl--D-thiogalactopyranoside to induce synthesis
of /-type SASP (24). Cells (2 liters) were harvested
by centrifugation after 1 h of further incubation and washed with
500 ml of cold 150 mM NaCl, and the cell pellet was frozen and
lyophilized. Lyophilized cells (2 to 4 g) were dry ruptured (100 mg at a time) for 2 min in a dental amalgamator (Wig-L-Bug) with glass
beads (150 mg). SspC and SASP-C were purified from acid extracts of
dry-ruptured cells as described previously (9, 29). SspC and
SspCG52A were purified from untreated and large-scale
peroxide-killed spores (~150 mg [dry weight]) essentially as
described previously (9). The yields of these proteins from
peroxide-killed spores were identical to those from untreated spores,
and oxidized proteins copurified with unmodified protein (data not
shown). All /-type SASP used or analyzed were >95% pure as
determined by polyacrylamide gel electrophoresis (PAGE) at low pH and
staining with Coomassie blue (21). All protein
concentrations were determined by the Lowry method (13).
DNase protection assays.
The DNA binding activity of
/-type SASP was determined by measuring their ability to protect
linearized plasmid DNA from DNase I digestion (28). Purified
/-type SASP (1.2 mg/ml) were incubated with
EcoRI-linearized plasmid pUC19 (0.12 mg/ml) in 10 mM sodium
phosphate (pH 7.5)-3 mM MgCl2 for 10 min at 22°C. This
ratio of /-type SASP to DNA is sufficient to saturate the plasmid
with protein (28). DNase I was added to a final
concentration of 0.04 mg/ml, and the solution was incubated at 37°C
for 10 min. The reaction was stopped by adjusting the solution to 1%
sodium dodecyl sulfate (SDS)-20 mM EDTA. Plasmid DNA was precipitated with ethanol and analyzed by 1% agarose gel electrophoresis followed by staining with ethidium bromide (28).
Methionine oxidation and analysis.
In initial studies of
methionine residue oxidation in /-type SASP, purified protein (1 mg/ml) was treated with 100 mM tBHP or H2O2 in
250 µl of 10 mM sodium phosphate (pH 7.5) for 3 h at 22°C
followed by dialysis in Spectrapor 3 tubing against two changes (1 liter of each) of 10 mM sodium phosphate (pH 7.5) at 4°C. These proteins were used directly for DNase protection, enzymatic reduction with partially purified MsrA from E. coli, and reverse-phase
high-performance liquid chromatography (HPLC) analysis (see below). In
later oxidation protection experiments, /-type SASP (1 mg/ml)
were incubated with or without poly(dG) · poly(dC) (0.5 mg/ml;
Sigma) in 100 µl of 10 mM sodium phosphate (pH 7.5) for 20 min at
22°C prior to addition of the oxidizing agent. At this protein-to-DNA
ratio, DNA is in excess and therefore all /-type SASP should be
bound to DNA (28). Oxidizing agents (tBHP or
H2O2) were added to 100 mM, and the reaction
mixtures were incubated for 3 h as described above. Oxidation
reactions were terminated by their injection onto a Shodex KW-800
series HPLC size exclusion column and eluted with 10 mM sodium
phosphate (pH 7.5) at a flow rate of 1 ml/min. The HPLC fractions
containing /-type SASP [with or without poly(dG) · poly(dC)] were frozen and lyophilized, and /-type SASP were further purified by Tris-Tricine-SDS-PAGE (23), followed by staining with Coomassie blue, and electroelution of stained proteins into 50 mM NH4HCO3-0.1% SDS by using the
Elutrap (Schleicher and Schuell). Purified proteins were frozen,
lyophilized, dissolved in 100 µl of Milli-Q water, and precipitated
with 800 µl of cold acetone at 
20°C overnight. Precipitated
proteins were washed with 500 µl of cold acetone and dissolved in 60 µl of freshly prepared 8 M urea.
/-type SASP (~40 µg) were digested in 100 µl of 0.8 M
urea-10 mM CaCl2-200 mM NH4HCO3
for 15 h at 37°C with trypsin (5 µg; Worthington) prior to
analysis of tryptic peptides by reverse-phase HPLC. Tryptic digests
were injected onto a Waters Deltapak C4 (3.9 by 150 mm)
column at a flow rate of 1 ml/min. After the column was washed for 5 min with 98% buffer A-2% buffer B, peptides were eluted with a
linear gradient of 98% buffer A-2% buffer B to 2% buffer A-98%
buffer B over 60 min. Buffer A was 0.06% aqueous trifluoroacetic acid,
and buffer B was 0.052% trifluoroacetic acid in 80% acetonitrile.
Peptides were detected by their UV absorbance at 214 nm. Individual
tryptic peptides were identified by amino acid analyses; methionine
sulfoxide-containing peptides were identified by mass spectrometry.
Quantitation of methionine sulfoxide levels was by integration of HPLC
peaks to determine the areas corresponding to reduced and oxidized
peptides.
To reduce methionine sulfoxide residues in oxidized SspC, tBHP-treated
SspC (1 mg/ml) was incubated with partially purified (~25% pure)
E. coli MsrA (0.04 mg/ml) (a generous gift from H. Weissbach) in 25 mM Tris-HCl (pH 7.4)-15 mM DTT-10 mM
MgCl2 for 4 h at 24°C. The reduced SspC was then
analyzed for DNase protection and digested with trypsin and peptides
analyzed as described above.
Construction of a yppP (msrA) mutant. Oligonucleotide primers were designed to PCR amplify a 253-bp fragment from within the yppP (now termed msrA) coding region. YPPP-1, a 23-mer with the sequence 5'-GGGAATTCGGGACATCGTGAAGC, and YPPP-2, a 23-mer with the sequence 5'-CCGGATCCGTCCGTTACAATCGG (nucleotides 431,735 to 431,749 and nucleotides 431,958 to 431,972 in the Subtilist B. subtilis genome project database, respectively) were designed with additional 5' nucleotides and EcoRI and BamHI restriction sites (underlined residues), respectively, for cloning purposes. The resulting EcoRI-BamHI-digested fragment was ligated with EcoRI-BamHI-digested plasmid pJH101 (7), and the ligation mix was used to transform E. coli JM83 to Ampr. A plasmid resulting from this transformation (pPS2519) was used to transform B. subtilis PS832 and PS356 to Cmr, generating an insertional mutation within the msrA gene which truncated the coding region to 30% of the original open reading frame. Southern blot analysis (34) of chromosomal DNA from Cmr transformants confirmed that the transformation had generated the expected insertion within msrA (data not shown).
Methionine sulfoxide reductase (MsrA) enzyme assays. N-Acetyl-L-[35S]methionine sulfoxide, a radiolabeled substrate for MsrA (3), was synthesized as follows. A 67-µl aliquot (1 mCi) of L-[35S]methionine (>1,000 Ci/mmol; New England Nuclear) was added to 933 µl of 10 mM L-methionine-3% acetic acid followed by the addition of 10 µl of 30% H2O2. After incubation at 22°C for 2 h, the solution was lyophilized and dissolved in 500 µl of glacial acetic acid, and L-[35S]methionine sulfoxide was acetylated by the addition of 500 µl of acetic anhydride. After incubation at 22°C for 2 h, the reaction was quenched with 9 ml of distilled water, lyophilized, and dissolved in 200 µl of distilled water. Conversion of L-[35S]methionine to N-acetyl-L-[35S]methionine sulfoxide was >99.9% complete as determined by analytical reverse-phase HPLC (see below) of the final compound. The specific activity of the synthesized N-acetyl-L-[35S]methionine sulfoxide was 180 cpm/pmol.
Bacterial cells were grown to late log phase (optical density at 600 nm, ~1) in 250 ml of either 2× YT (E. coli) or 2× SG (B. subtilis) (18) medium, harvested by centrifugation, washed with 100 ml of 0.15 M NaCl, and suspended in 1 to 2 ml of cell extraction buffer (20 mM Tris-HCl [pH 7.5]-10 mM MgCl2-10 mM KCl-10 mM DTT-1 mM phenylmethylsulfonyl fluoride-10% glycerol). Cells were disrupted by 4 min of sonication at 0°C with glass beads and centrifuged at 32,000 × g for 1 h at 4°C, and the supernatant fluid was used immediately for enzyme assays. Spores (~150 mg [dry weight]) were decoated as previously described (9), and 200 µl of 10 mM Tris-HCl (pH 8.0)-10 mM EDTA-150 mM NaCl-1 mM DTT-0.1 mM phenylmethylsulfonyl fluoride was added to the decoated spore pellet for resuspension. The decoated spore suspension was digested with lysozyme for 20 min at 37°C, sonicated twice for 20 s, centrifuged in a microcentrifuge (Fisher model 235A) for 15 min at 4°C, and adjusted to 20 mM MgCl2-10 mM DTT-10% glycerol, and the supernatant fluid was used immediately for MsrA assays. MsrA activity was routinely determined as described previously (3). Briefly, cell or spore extracts (30 and 45 µl, respectively) were incubated in cell extract or spore extract buffer with 500 µM N-acetyl-L-[35S]methionine sulfoxide in a final volume of 50 µl at 37°C for 30 min. Reactions were quenched with 250 µl of 0.5 M HCl and extracted with 700 µl of ethyl acetate, and the radioactivity of the organic extract was counted in a scintillation counter. In some experiments a more sensitive assay was used in which any N-acetyl-L-[35S]methionine produced was purified by reverse-phase HPLC and subjected to counting. Briefly, ethyl acetate extracts of reaction mixtures were dried and then dissolved in 18 µl of 0.06% trifluoroacetic acid to which 2 µl of a solution that was 5 mM in both N-acetyl-L-methionine and N-acetyl-L-methionine sulfoxide was added, followed by resolution of N-acetyl-L-methionine and N-acetyl-L-methionine sulfoxide on a Waters µBondapak C18 reverse-phase HPLC column (3.9 by 300 mm) by isocratic elution with 0.06% trifluoroacetic acid. Peaks corresponding to N-acetyl-L-methionine and N-acetyl-L-methionine sulfoxide were collected, dried, dissolved in 100 µl of distilled water, applied to glass fiber filters, dried under an infrared lamp, and counted in a scintillation counter.| |
RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
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Oxidation of /-type SASP in vitro.
The /-type SASP
selected for most of this study was SspC, a minor /-type SASP of
B. subtilis. SspC and its interaction with DNA have been
characterized extensively (28), and this protein has
dramatic effects on spore resistance in vivo (37). In
addition to containing the highly conserved methionine residue (M27)
near the middle of the protein, SspC contains a second, less well
conserved methionine residue near the carboxy terminus (M67)
(6); the translational initiating methionine residues are
removed posttranslationally from all /-type SASP (4, 30). Both methionine residues within SspC were substantially oxidized to methionine sulfoxide residues by treatment with tBHP or
H2O2 for 3 h at room temperature, as
demonstrated by large shifts in the HPLC retention times of the
methionine-containing tryptic peptides of SspC (Fig.
1, peptides 2 and 4). The identities of
these two peptides were confirmed by a combination of amino acid
analysis and matrix-assisted laser desorption, ionization-mass spectrometry; the latter analysis revealed an increase of 16 Da in the
oxidized peptides (data not shown), indicating that the methionine
residues were oxidized to the sulfoxide and not the sulfone form. A
small amount (~8%) of additional oxidation also occurred at lysine
28 in SspC, which resulted in the formation of a larger tryptic peptide
(S9 to R46) that contained methionine sulfoxide at M27 (Fig. 1, peptide
5). The single highly conserved methionine of SASP-C (M28), a major
/-type SASP of Bacillus megaterium (33),
was also oxidized to methionine sulfoxide by tBHP or
H2O2 treatment (Table
2 and data not shown).
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/-type SASP retained DNA binding activity in vitro. Strikingly, oxidation of methionine residues in SspC and SASP-C greatly reduced these proteins' DNA binding activity as measured by the ability of the
oxidized proteins to provide DNase protection to plasmid DNA (Fig.
2). Although the peroxide-treated
/-type SASP also contained some oxidation at lysine residues, the
proportions of these products were much too small (~8 and ~17% in
SspC and SASP-C, respectively) to account for the observed decreases in
DNA binding. Furthermore, DNA binding activity was restored to oxidized
SspC when this protein's methionine sulfoxide residues were reduced back to methionine by MsrA treatment as described in Materials and
Methods (data not shown). Therefore, oxidation of methionine residues
within /-type SASP, in particular the central highly conserved
methionine (see below), eliminates DNA binding in vitro.
|
/-Type SASP complexed to DNA are resistant to methionine
oxidation in vitro.
A recent report demonstrated that only
surface-exposed methionine residues in proteins are oxidized with tBHP,
presumably due to steric constraints imposed by the bulky
tert-butyl group (11). While buried methionine
residues were relatively resistant to oxidation by tBHP, they were
oxidized much more readily by H2O2
(11). Although /-type SASP are relatively unstructured in solution, they undergo a large conformational change upon binding to
DNA as measured by circular dichroism spectroscopy (15). To
determine whether methionine residues within /-type SASP become
more resistant to oxidation when bound to DNA, /-type SASP-DNA
complexes were treated with tBHP or H2O2, and
the /-type SASP were analyzed for methionine oxidation. Untreated
SspC contained only a minute amount of the sulfoxide at the conserved
methionine (M27), although there was significantly more sulfoxide at
the less well conserved methionine (M67) (Table 2). However, the electrophoretic purification scheme used to separate /-type SASP
from DNA caused some methionine oxidation at M27 (Table 2). SspC that
was treated with tBHP while bound to DNA showed no significant change
in the levels of methionine oxidation compared to polyacrylamide gel-purified SspC and contained much less methionine sulfoxide than
SspC treated with tBHP in the absence of DNA (Table 2). H2O2 treatment was more effective at oxidizing
both methionine residues of SspC, but again there was significant
protection afforded by DNA binding, in particular at M27 (Table 2). DNA
binding also protected the single conserved methionine of SASP-C
against oxidation (Table 2), indicating that the protection phenomenon
is probably general to all /-type SASP. These experiments were
also conducted with a linearized plasmid as the protective DNA, and
essentially identical results were obtained (data not shown).
-amino-adipic semialdehyde residues
(2). Although we have not studied this point further, this
oxidation results in a decrease of only 1 Da in the oxidized peptide.
We do not fully understand the reason for this lysine oxidation, but it
appears to correlate with the lyophilization of peroxide-treated
/-type SASP after removal of the oxidizing agent by gel
filtration. Furthermore, trypsin cleavage at other lysine residues in
SspC and SASP-C was unaffected by peroxide treatment, suggesting that
the proximity of the oxidizable lysines 28 and 29 to the highly
conserved M27 and M28 residues (respectively) plays a role in this
oxidation. Accordingly, DNA binding also protected /-type SASP
from lysine oxidation (Table 2).
Oxidation of /-type SASP in vivo.
With the knowledge
that methionine residues in /-type SASP could be oxidized in
vitro and that this had drastic effects on these proteins' function,
an obvious question was whether methionine residues in /-type
SASP could be oxidized in spores. Bacterial spores are much more
resistant to oxidizing agents such as tBHP and
H2O2 than are vegetatively growing cells
(22, 32), and the /-type SASP are significant
determinants of this resistance to these agents; spores which lack the
two major /-type SASP (
spores) show decreased resistance to both H2O2
and tBHP compared to wild-type spores (25, 27). To determine
if methionine residues in /-type SASP are susceptible to
oxidation in vivo, B. subtilis spores which overexpress SspC
as their major /-type SASP (PS2437) (Table 1) (9) were
treated with either H2O2 or tBHP, followed by
purification and analysis of SspC. The levels of methionine sulfoxide
in SspC from untreated spores were similar to levels found in
recombinant SspC purified from E. coli (Tables 2 and 3). However, SspC purified from
peroxide-killed spores contained significantly more methionine
sulfoxide at M67 than did SspC from untreated spores (Table 3).
Methionine sulfoxide levels also rose slightly at M27, but this
conserved methionine residue was much more resistant to oxidation than
the less well conserved M67 residue (Table 3). Interestingly, SspC
purified from H2O2- and tBHP-killed spores
(Table 3) bound to DNA in vitro with an affinity essentially identical
to that of SspC purified from untreated spores or recombinant SspC
purified from E. coli (data not shown).
|
/-type SASP from methionine
oxidation in vitro, we decided to assess whether it was DNA binding that was at least in part responsible for the protection of SspC against methionine oxidation in vivo. For this analysis we used spores
which overexpress SspCG52A, a mutant form of SspC which
does not bind to DNA in vitro with high affinity and fails to confer UV
and heat resistance on spores (37). Previous work has also
shown that although a labile asparagine residue in SspC is protected
against deamidation by binding to DNA in vivo, in SspCG52A
this residue is less well protected (9). Strikingly,
sulfoxide levels at both methionine residues of SspCG52A
increased dramatically as a result of peroxide treatment of PS2438 spores (Table 3); these levels were much higher than the levels measured in SspC from peroxide killed spores, particularly at M27 of
SspC, which was well protected from oxidation in vivo (Table 3).
Substantial oxidation also occurred at lysine 28 in
SspCG52A from peroxide-killed spores (Table 3). The
oxidation of lysine 28 in vivo may be due to hypohalite ions formed by
peroxides and endogenous spore halides, or the enhanced reactivity of
lysine 28 as mentioned above. This lysine oxidation product was not
observed to any significant degree in SspC from peroxide-killed spores (Table 3).
Characterization of an msrA mutant of B. subtilis.
Although at least one critical methionine residue was
relatively well protected from oxidation in spores, we did observe
significant methionine residue oxidation, and it is possible that in
other proteins some methionine residues may be even more readily
oxidized. While it appears likely that /-type SASP with oxidized
methionine residues are degraded during spore germination, as are all
/-type SASP (30), it is possible that oxidized
methionine residues in other proteins might be repaired by MsrA.
Consequently, we wished to determine whether the MsrA protein repair
pathway is present and might function in germinating spores of B. subtilis.
msrA
strains (PS2638 and PS2639) were also no more sensitive to wet heat or
H2O2 than were spores of their respective
parental strains (PS832 and PS356) (data not shown).
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Oxidative damage to proteins within bacterial spores poses a unique problem, because spores can remain dormant for long periods, during which substantial oxidative protein damage may accumulate, yet dormant spores are incapable of degrading or repairing such damaged proteins. One mechanism whereby oxidative damage to spore proteins might be dealt with is damage repair during spore germination and outgrowth. The yppP (now msrA) gene of B. subtilis is predicted to encode a homolog of the MsrA repair enzyme, and peptidyl methionine sulfoxide reductase activity is greatly diminished (>90%) within vegetative cells and spores of an msrA mutant. However, spores of the msrA mutant strains showed no increase in peroxide sensitivity compared to spores of the parental strains. Although the MsrA repair pathway may operate during spore germination, it plays no significant role in spore resistance to oxidizing agents. Similarly, growing cells of the msrA mutant were not sensitive to H2O2, presumably due to the overriding effects of other protective mechanisms. Even in E. coli, only a slight sensitivity to H2O2 was reported in an msrA mutant (17).
A second mechanism for dealing with oxidative damage to spore protein
is to prevent such damage. The low permeability of spores to most
chemicals is one mechanism by which spores resist oxidative damage from
peroxides (32), while /-type SASP protect spore DNA
from oxidative damage (25, 27). However, peroxides can enter
the spore core; as in the absence of /-type SASP, spore killing
by peroxides is in large part through DNA damage (25, 27). A
number of spore enzymes are also inactivated within spores by
H2O2 (19). While the inactivation of
some spore enzymes is significantly slower than spore killing,
inactivation of at least one enzyme parallels spore killing
(19). However, the nature of the oxidative damage resulting
in inactivation of these spore enzymes has not been established.
Because they are significant determinants of spore resistance to
environmental insults, the /-type SASP are potentially important
targets for methionine residue oxidation (30, 31). Indeed,
/-type SASP in which the highly conserved methionine is oxidized
do not bind to DNA effectively in vitro and presumably would also not
confer UV and heat resistance to spores as well as their nonoxidized
counterparts would. However, /-type SASP are resistant to
peroxide-induced methionine oxidation when bound to DNA in vitro.
Interestingly, the two methionine residues of SspC differ somewhat in
their resistance to oxidation in the presence of DNA. In general,
surface-exposed methionine residues are more susceptible to oxidation
than are buried residues (11), and therefore the most
obvious interpretation of the different reactivities of the M27 and M67
residues of SspC with oxidizing agents is that M27 becomes relatively
inaccessible to solvent when the protein binds to DNA, while the region
containing M67 is still somewhat exposed. There is evidence that
protein-protein interactions occur between adjacent /-type SASP
while bound to DNA, and some of the residues involved in these
interactions (for example glutamates 30 and 34 in SASP-C) are very near
the conserved methionine residue (M28 in SASP-C) (10).
Therefore, /-type SASP packing along the DNA backbone could
render the highly conserved methionine inaccessible to solvent.
However, because the M27 residue of SspC is more resistant to oxidation
than M67 in the absence of DNA, it is also possible that M67 is simply
more reactive than M27.
As noted above, killing of wild-type B. subtilis spores with
H2O2 is not due to DNA damage, indicating that
/-type SASP continue to protect DNA even though the spore has
been killed by damage to some other unknown target (27). The
in vivo oxidation data presented in this study are consistent with this
previous finding. Although spores (>99.9%) were killed by the
peroxide treatments used, only ~10% of the highly conserved M27
residue in SspC was oxidized to the sulfoxide form. These data indicate that in spores, the rate of oxidation of this critical methionine residue in SspC is >30-fold lower than the rate of spore killing. Comparison of the latter data with the published rates of enzyme inactivation and spore killing by H2O2
(19) indicates that the rate of oxidation of M27 in SspC in
spores is at least fivefold lower than the rate of inactivation of the
most stable spore enzyme analyzed. Thus, /-type SASP are
extremely well protected against oxidative inactivation in vivo. In
addition, SspC purified from H2O2-killed spores
retained nearly all of its DNA binding ability, even though this
protein contained approximately 60% methionine sulfoxide at the less
well conserved M67 position. This result indicates that the highly
conserved and well protected M27 residue of SspC is critical for DNA
binding but that the less well conserved and less well protected M67
residue is less important (and perhaps not at all important).
Two of the /-type SASP from Bacillus species which
lack the conserved central methionine residue contain phenylalanine at the position corresponding to M27 in SspC, whereas the third contains a
tyrosine (30). Therefore, evolution has selected for
hydrophobic residues at this position, and because the change from a
hydrophobic methionine to a more hydrophilic methionine sulfoxide
residue is not conservative, this change could disrupt hydrophobic
interactions which may be critical to /-type SASP-DNA binding.
Conversely, although all /-type SASP from Bacillus
species contain hydrophobic residues (either leucine or phenylalanine)
at positions corresponding to M67 in SspC, the surrounding C-terminal
region of /-type SASP contains many sequence variations, which
may indicate that this portion of the protein is not critical for
function (30). Therefore, even though M67 of SspC can be
substantially oxidized in vivo, the protein retains function because
the methionine residue (M27) which is critical for DNA binding is
largely protected from oxidation.
SspCG52A from peroxide-killed spores contained much more
methionine sulfoxide at the conserved M27 position than did SspC from peroxide-killed spores. Because SspCG52A does not bind to
DNA with high affinity in vitro and also fails to restore UV and heat
resistance to spores in vivo
(37), it appears that it is DNA binding that protects
/-type SASP from methionine oxidation at the highly conserved M27
residue. Previous work has also shown that DNA binding protects SspC
from asparagine deamidation at a very highly conserved NG sequence in
vitro (9). Deamidation of this asparagine residue in SspC
results in the loss of DNA binding function in vitro, and a mutant form
of SspC in which the labile asparagine is replaced with an aspartate
residue (mimicking the product of deamidation) fails to confer UV and
heat resistance on spores of
B. subtilis (9). However, SspC is well protected from deamidation in vivo due to its interaction with DNA, whereas SspCG52A is protected to a lesser degree (9).
Therefore, in addition to the well-characterized effects whereby
/-type SASP protect spore DNA from a variety of types of damage
(32), DNA also protects /-type SASP from a variety of
protein damage.
| |
ACKNOWLEDGMENTS |
|---|
We are grateful to Stacey Carrington for conducting initial experiments, to Nathan Brot for helpful discussion, and to Herbert Weissbach for generously providing MsrA.
This work was supported by a grant from the National Institutes of Health (GM19698).
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Biochemistry, University of Connecticut Health Center, Farmington, CT 06030. Phone: (860) 679-2607. Fax: (860) 679-3408. E-mail: setlow{at}sun.uchc.edu.
Present address: Escuela de Ciencias Quimico Biologicas,
Universidad Autonoma de Guerrero, Chilpancingo, Guerrero 39000, Mexico.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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