From the Department of Biochemistry, University of Connecticut Health Center, Farmington, Connecticut 06030
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ABSTRACT |
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Small, acid-soluble spore proteins (SASP)
of the /
-type from several Bacillus species were
cross-linked into homodimers, heterodimers and homooligomers with
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in the presence of
linear plasmid DNA. Significant protein cross-linking was not detected
in the absence of DNA. In all four
/
-type SASP examined, the
amino donor in the EDC induced amide cross-links was the
-amino
group of the protein. However, the carboxylate containing amino acid
residues involved in cross-linking varied. In SASP-A and SASP-C of
Bacillus megaterium two conserved glutamate residues, which
form part of the germination protease recognition sequence, were
involved in cross-link formation. In SspC from Bacillus
subtilis and Bce1 from Bacillus cereus the acidic
residues involved in cross-link formation were not in the protease
recognition sequence, but at a site closer to the N terminus of the
proteins. These data indicate that, although there are likely to be
subtle structural differences between different
/
-type SASP, the
N-terminal regions of these proteins are involved in protein-protein
interactions while in the DNA bound state.
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INTRODUCTION |
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Between 5 and 10% of the total protein in spores of the
Bacillus and Clostridium species of bacteria is
/
-type small, acid-soluble spore protein (
/
-type
SASP)1 (1, 2). These proteins
are encoded by four to seven monocistronic genes in each species, and
their amino acid sequences are highly conserved both within and between
Bacillus species (1, 2). The
/
-type SASP are
nonspecific DNA-binding proteins which are synthesized only within the
forespore compartment during sporulation (3, 4). Typically, two major
/
-type SASP accumulate to high levels within the spore, while the
minor
/
-type SASP are found at much lower levels. The level of
total
/
-type SASP in spores is sufficient to saturate the spore
chromosome, and the binding of these proteins to spore DNA is the major
determinant of spore resistance to UV radiation and a significant
determinant of spore heat resistance (1, 2). Bacillus
subtilis spores which lack the two major
/
-type SASP (
and
) are much more sensitive to UV radiation and heat than are wild
type spores (5). During the first few minutes of spore germination,
/
-type SASP are quickly degraded by a sequence-specific protease
termed germination protease (GPR) (1, 2).
Structural studies of purified /
-type SASP and
/
-type
SASP·DNA complexes have shown that significant changes in these proteins' structure occur upon binding to DNA, as
/
-type SASP are predominantly unfolded in solution but acquire significant
-helical content upon binding to
DNA2 (6). The
/
-type
SASP cover 4-6 base pairs of DNA, and binding of these proteins to DNA
is highly cooperative, particularly to DNAs bound with low affinity.
(7). Electron micrographs of
/
-type SASP·DNA complexes indicate
that the protein forms a helical coat along the DNA (8), suggesting
that there are extensive interactions between
/
-type SASP when
bound to DNA, although these proteins are monomers in
solution3 (9). Consequently,
it is possible that interactions between adjacent
/
-type SASP
along the DNA backbone may be important for the
/
-type SASP/DNA
binding interaction.
To determine which regions of the proteins are involved in interactions
between /
-type SASP bound to DNA, we have performed protein
cross-linking studies with the zero-length cross-linking reagent
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). We have
identified EDC-catalyzed protein cross-links in four different
/
-type SASP from Bacillus species, and the
identification of these cross-links has yielded new insights into the
interaction of
/
-type SASP on DNA.
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EXPERIMENTAL PROCEDURES |
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Bacterial Strains and Growth Conditions--
The
Escherichia coli strains used include: JM107 (F'
traD36 proA+ proB+
lacIq lacZ M15/endA1 gyrA96
(Nalr) thi hsdR17 supE44 relA1
(lac-proAB) mcrA) (Life Technologies, Inc.),
JM83 (ara
(lac-proAB) rpsL
80
lacZ
M15) (10), BL21(DE3) (T7 RNA polymerase under control
of the lac promoter) (11), and BMH 71-18 (F' proAB
lacIq lacZ
M15 thi supE
(lac-proAB) mutS::Tn10)
(CLONTECH Laboratories, Inc.). The
Bacillus strains used were Bacillus cereus T
(originally obtained from H. O. Halvorson) and Bacillus
megaterium QMB1551, ATCC no. 12872 (originally obtained from H. Levinson).
Polymerase Chain Reaction Amplification and Site-directed Mutagenesis-- Oligonucleotides were designed to polymerase chain reaction amplify a 512-base pair fragment containing the gene encoding Bce1 (13) from B. cereus genomic DNA; the amplified fragment contained the gene's ribosome binding site and transcription terminator (13). The upstream primer, BCE1-1 (5'-AAAGGATCCTTATTATTTCATAATTTGTAGC; complementary to nucleotides 119-140) (13) and downstream primer, BCE1-2 (5'-AAAGGATCCTTTTAAGTATGCTTTTTCCTGC; complementary to nucleotides 592-613) (13), each contained BamHI restriction sites and 5'-flanking sequences (underlined residues) for cloning purposes. The BamHI-digested polymerase chain reaction product was agarose gel-purified and ligated into BamHIdigested plasmid pET3 (11), generating plasmid pPS2532 in which BclI digestion confirmed that the gene encoding Bce1 was under the control of the T7 promoter. Plasmid pPS2532 was used to transform E. coli strain DE3(BL21) to ampicillin resistance.
The E10K mutant form of Bce1 was generated with the TransformerTM site-directed mutagenesis kit from CLONTECH according to manufacturer's instructions. Phosphorylated primers complementary, except for designed mismatches (underlined bases), to the unique AlwNI restriction site of pET3 (5'-CCTGTTACTAGTGGATGCTGC) and the Gly6-Gly15 coding region of the gene encoding Bce1 (5'-GGAAGTCGTAATAAAGTATTAGTTCGAGGC) (13) were used with plasmid pPS2532 as a template to synthesize a mutagenized plasmid lacking the AlwNI site, and with a lysine codon replacing the codon for glutamate 10 of bce1. The mutagenized plasmid was digested with AlwNI prior to transformation into the mismatch repair deficient E. coli strain (mutS::Tn10, Tetr) supplied with the mutagenesis kit. Mutagenized plasmid was enriched by plasmid isolation, digestion with AlwNI and retransformation into E. coli strain JM83. One clone was isolated and the identity of the mutagenized plasmid, termed pPS2734, was confirmed by DNA sequencing.SASP Expression and Purification--
SspC and SASP-C were
overexpressed in E. coli strain JM107 from pDG148 derived
plasmids containing the /
-type SASP genes under control of an
isopropyl
-D-thiogalactopyranoside inducible promoter as
described previously (14, 15). Bce1 and Bce1E10K were
overexpressed in E. coli strain DE3(BL21) from plasmids pPS2532 and pPS2734, respectively. SspC, Bce1, and Bce1E10K
were extracted from dry ruptured E. coli cells with 3%
acetic acid/30 mM HCl as described previously (16). SASP-C
and SASP-A were extracted with 3% acetic acid (9) from dry ruptured
E. coli cells and dry ruptured spores of B. megaterium strain QMB1551, respectively. All
/
-type SASP
were purified as described previously (9).
Cross-linking of /
-Type SASP with EDC--
/
-Type
SASP (0.5 mg/ml) with or without cesium chloride gradient-purified,
EcoRI-linearized pUC19 plasmid DNA (100 µg/ml) were
incubated in 1 ml of 5 mM sodium phosphate (pH 7.5) at
24 °C for 20 min prior to addition of EDC to 25 mM. The
5:1 (w/w) ratio of protein to DNA is sufficient to saturate the DNA
with
/
-type SASP, although SASP-A binds more weakly than do the
other
/
-type SASP tested (17). Under these conditions,
approximately 50% of the
/
-type SASP are bound to the DNA. The
cross-linking reactions were incubated for 30 min at 24 °C, followed
by dialysis in Spectrapor 3 tubing against 1 liter of 10 mM
sodium phosphate (pH 7.5) at 4 °C for 18 h. Dialyzed
cross-linking reactions were frozen, lyophilized, dissolved in sample
buffer and run on Tris-Tricine SDS-PAGE (18). Gels were stained with
Coomassie Blue, destained, and monomeric and cross-linked proteins were
excised with a clean razor blade. Proteins were electroeluted from
polyacrylamide gel slices into 50 mM
NH4HCO3, 0.1% SDS using ElutrapTM
separation chambers (Schleicher & Schuell). Gel purified proteins were
frozen, lyophilized, dissolved in 100 µl of MilliQ-H2O,
and precipitated with 800 µl of cold acetone. Precipitated proteins were washed with 500 µl of cold acetone and dissolved in freshly prepared 8 M urea prior to trypsin digestion.
Cross-linked Peptide Purification and Analysis-- EDC-treated proteins (~20-40 µg) were digested with trypsin (Worthington, 5 µg) in 100 µl of 0.2 M NH4HCO3, 10 mM CaCl2, 1.2 M urea at 37 °C for 15-18 h. Tryptic digests were run on reverse-phase high performance liquid chromatography (HPLC) using a Waters 680 gradient controller, two Waters 501 pumps, a Waters U6K injector, and a Vydac protein C4 column (3.9 × 150 mm). Tryptic digests were loaded onto the reverse phase column in 100% buffer A (0.06% trifluoroacetic acid) followed by 5 min of washing with 100% buffer A. Peptides were eluted at a flow rate of 1 ml/min with a discontinuous linear gradient as follows: 5-30 min, 0-30% buffer B (0.052% trifluoroacetic acid in 80% acetonitrile); 30-50 min, 30-40% buffer B; 50-70 min, 40-100% buffer B. Peptides were detected by their UV absorption at 214 nm with a Waters 481 spectrophotometer, and fractions containing peptides were collected with an Isco 2150 peak separator and an Isco Foxy fraction collector.
Molecular masses of HPLC-purified peptides were determined by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry using a Perseptive Biosystems Linear MALDI-TOF instrument. External calibration consisting of two standards (angiotensin, 1297.5 Da; and ACTH(18-39), 2466.7 Da) was used for all determinations, resulting in ±0.15% mass accuracy. Peptides from HPLC fractions (1 µl, ~1-10 pmol/µl) were mixed and dried on the instrument stage with an equal volume of ![]() |
RESULTS |
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EDC Cross-linking of /
-Type SASP Is
DNA-dependent--
In an effort to identify the
interacting regions of
/
-type SASP bound to DNA, we decided to
use protein cross-linking to trap interacting amino acid residues for
subsequent biochemical analysis. Most
/
-type SASP do not contain
cysteine residues (1) and therefore several protein cross-linking
reagents which rely upon thiol chemistry could not be used for this
study. The
/
-type SASP are small proteins (6-7.6 kDa) which are
monomeric in solution3 (9) and appear to interact with one
another only when bound to DNA. We were interested in regions of close
contact between
/
-type SASP and therefore decided to use
cross-linking reagents with short- or zero-length linker arms. EDC, a
water-soluble carbodiimide, gave efficient cross-linking of
/
-type SASP only in the presence of DNA (see below).
Consequently, we chose this reagent for further work. Protein
cross-linking with EDC usually involves the formation of an amide bond
between either an N-terminal
-amino or lysine
-amino group and
the carboxyl side chain of aspartate/glutamate residues. Therefore, in
contrast to cross-linkers that contain flexible linker arms several
angstroms in length, EDC induced cross-links should occur only between
residues that are in very close proximity to one another.
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Different /
-Type SASP Interact on DNA--
The DNA
dependence of cross-link formation between
/
-type SASP suggested
that the EDC-generated protein-protein cross-links are formed only
between
/
-type SASP that are adjacent to one another on the DNA
backbone. These in vitro experiments used only a single
/
-type SASP. However, there are multiple
/
-type SASP in
spores, with two proteins present at high levels. Consequently, an
obvious question is whether the different
/
-type SASP interact when bound to DNA. To obtain data pertinent to this question we analyzed protein-protein cross-link formation in reactions with two
different
/
-type SASP bound to DNA. SASP-A and SASP-C from B. megaterium were chosen for the initial
hetero-cross-linking experiments because they are the two major
/
-type SASP found in spores of B. megaterium (9).
These proteins also differ sufficiently in molecular mass (SASP-A = 6,260.1 Da and SASP-C = 7,423.3 Da) to allow resolution of the
three possible dimeric forms by Tris-Tricine SDS-PAGE. Electrophoretic
analysis of cross-linking reactions containing SASP-A, SASP-C, and DNA
revealed the presence of a new predominant band that migrated at the
position expected for a SASP-A/SASP-C heterodimer (Fig.
3, lane A + C), and this band
is indeed a SASP-A/SASP-C heterodimer (see below). Titration experiments demonstrated that the ratio of SASP-A to SASP-C that produces the most heterodimer is ~3:1 (w/w) (data not shown). This
latter ratio approximates the relative levels of these two proteins in
B. megaterium spores (9). Heterodimers were also formed
between SASP-A and SspC from B. subtilis (data not shown). However, in contrast to the SASP-A/SASP-C cross-linking reaction in
which the SASP-A/SASP-C heterodimer was the predominant cross-linked product (Fig. 3), SASP-A/SspC heterodimer formation was much less efficient than formation of the SspC homodimer in these reactions (data
not shown). Since SspC and SASP-C have similar affinities for linear
plasmid DNA in solution, the difference in their formation of
heterodimers with SASP-A is presumably due to differences in the amino
acid sequences of SspC and SASP-C.
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Identification of Cross-links between /
-Type SASP--
There
is presently very little detailed structural information available on
/
-type SASP or the complex they form with DNA. Therefore,
identification of the amino acid residues involved in
EDC-dependent cross-link formation was undertaken to
determine which regions of
/
-type SASP are involved in
protein-protein interactions that occur in the DNA bound state.
Purified monomeric and oligomeric
/
-type SASP from EDC
cross-linking reactions were digested with trypsin and the products
resolved by reverse phase-HPLC. Two types of differences should be
detected between the HPLC tryptic maps of dimeric (or oligomeric) and
monomeric (but EDC treated)
/
-type SASP. First, the digests of
/
-type SASP dimers should show decreases (~50%) in the
relative yield of some peptide(s) as compared with the monomer, because
amino acid residues within this peptide(s) will be in a cross-linked peptide in the dimer. Second, there should be a new peptide peak(s) in
HPLC tryptic maps of
/
-type SASP dimers, which should be the
peptide containing the cross-link. Detailed analyses, including mass
spectrometry, amino acid analysis and amino acid sequencing of the
latter peptides should then allow both the unambiguous identification
of the peptides in the cross-link, as well as the specific amino acid
residues involved. Intramolecular cross-links could also be formed by
EDC, as
/
-type SASP go from an unfolded to a more ordered
structure on binding to DNA. Intramolecular cross-links could be found
within both monomeric and oligomeric proteins, and this modification
could be detected by comparing HPLC tryptic maps of EDC treated
monomers and untreated protein. However, we never saw evidence for
intramolecular cross-link formation in these analyses (data not
shown).
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Elucidation of Amino Acid Residues Involved in Cross-link
Formation--
Various types of information were used to determine the
amino acid residues involved in cross-link formation in the different /
-type SASP. For SASP-A, SASP-C, and SspC, mass spectrometry and
amino acid analysis identified the large peptides whose level was
decreased in tryptic digests of the dimeric species as
Tyr20-Arg37,
Phe29-Arg46, and
Ser8-Lys27, respectively. This identified one
probable partner in the major cross-link formed in these three
proteins. Determination of the mass of each peptide tentatively
identified as a cross-linked species from tryptic digests of both homo-
and heterodimers (Table I), as well as
amino acid analyses (data not shown) allowed assignment of the two
tryptic peptides in the various cross-links. In all cases, the site of
cross-linking was tentatively identified as between the
-amino group
of the protein and an acidic group on a separate tryptic peptide. The
peptides in the two new peaks from tryptic digests of cross-linked
dimers of either SASP-A or SASP-C had virtually identical observed
molecular masses (Table I), suggesting that each peak contained the
same two tryptic peptides linked together, but cross-linked at a
different site. Analyses of the two unique peptides isolated from the
tryptic digest of the SASP-A/SASP-C heterodimer predicted that the
cross-links were between the
-amino group of one protein and a
tryptic peptide in the other protein (Table I).
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Generation and Analysis of Bce1E10K--
The
acidic residues identified as sites of cross-link formation in Bce1 and
SspC are not highly conserved among /
-type SASP (1), and
therefore their involvement in EDC cross-linking was somewhat
unexpected. Consequently, we decided to investigate the role these
specific acidic residues play in DNA binding and to determine whether
in Bce1 the observed cross-link between the
-amino group and
glutamate 10 was the only one generated by EDC. A site-directed mutant
form of Bce1 was generated in which glutamate 10 was changed to a
lysine residue. This change should not destroy Bce1/DNA binding,
because most other
/
-type SASP contain either a lysine
(e.g. SASP-A and SASP-C) or a glutamine residue at this position (1). This type of mutagenesis was not conducted with SASP-A or
SASP-C, because previous work has shown that changes in the glutamate
residues involved in cross-link formation in these proteins
significantly diminish DNA binding (19).
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DISCUSSION |
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Previous studies have demonstrated that /
-type SASP bind to
DNA in a cooperative fashion and that protein-protein interactions probably occur during binding (7). The data reported in this communication confirm that direct protein-protein interactions occur
between
/
-type SASP bound to DNA. The protein-protein interaction
detected by EDC cross-linking is not seen in the absence of DNA, which
is consistent with previous data that suggest that
/
-type SASP
exist as monomers in solution3 (9). It appears that
/
-type SASP/DNA binding involves the polymerization of the
monomeric proteins along the backbone of the DNA double helix. This is
suggested both by direct visualization of
/
-type SASP·DNA
complexes by electron microscopy (8) and by the ability of
/
-type
SASP to protect the DNA backbone from attack by hydroxyl radicals and
orthophenanthroline-Cu2+ (17). It further appears that
/
-type SASP assemble in a polarized fashion along the DNA
backbone, because EDC cross-linked trimers and tetramers of SspC and
Bce1 appear to contain only one type of cross-link, which is consistent
with a head-to-tail arrangement. However, it is not clear whether DNA
polarity itself has any role in organizing
/
-type SASP
binding.
The DNA binding region of /
-type SASP has been postulated to be
within the C-terminal half of these proteins. This region of
/
-type SASP is more highly conserved than the N-terminal region,
and a 29-amino acid residue synthetic peptide corresponding to residues
Thr43-Phe70 of SspC has been shown to bind to
DNA and change the DNA's UV photochemistry (6). However, the binding
of this synthetic peptide to DNA was much weaker than that of
full-length SspC (6). The EDC cross-link sites found in
/
-type
SASP suggest that the N-terminal region of these proteins is involved
in protein-protein interactions. These protein-protein interactions
presumably increase the binding affinity of
/
-type SASP for DNA,
and therefore account, at least in part, for the difference in DNA
binding affinities between the synthetic peptide and full-length SspC.
The N-terminal region of
/
-type SASP is the most variable region
of these proteins, with variations in both sequence and length (Fig. 1)
(1). In fact, many
/
-type SASP are virtually identical proteins
with the exception of their N-terminal regions. This is best
exemplified by SASP-A and SASP-C of B. megaterium, which are
essentially identical with the exception of the longer N terminus of
SASP-C (Fig. 1). However, despite their similarity in primary sequence,
SASP-C has a higher affinity for DNA in solution (9). In light of the
results reported here, it appears that the N-terminal regions of
/
-type SASP are involved in protein-protein interactions, and
therefore could be a major factor in determining the binding affinity
of these proteins for DNA in solution. It is important to note that
other regions of
/
-type SASP may also be involved in
protein-protein interactions, which may not be detected because the
reagent used in this study cross-links only amino and carboxyl groups.
The identification of cross-linked acidic residues within the GPR
recognition sequences of SASP-A and SASP-C is particularly interesting
because previous in vitro studies have demonstrated that
/
-type SASP are resistant to GPR cleavage when bound to DNA (20).
The data presented in this communication suggest that the N termini of
SASP-A and SASP-C are close to the GPR cleavage site, while these
proteins are in the DNA bound state. Thus, the GPR cleavage site may be
inaccessible to the GPR protease due to steric interference by the N
terminus of an adjacent protein. However, other structural changes are
probably also important because purified cross-linked dimeric and
trimeric SspC (which both contain unmodified GPR cleavage sequences)
are partially resistant to GPR cleavage (data not shown). Thus, EDC
cross-linking may stabilize a protein conformation of
/
-type SASP
in which GPR cleavage is inhibited.
The identification of two different sites of DNA dependent cross-link
formation in the /
-type SASP examined was unexpected based upon
the large degree of primary sequence conservation between members of
this protein family. Although the position corresponding to Asp-13 and
Glu-10 in SspC and Bce1, respectively, is far removed from the GPR
recognition site in primary sequence, it is possible that these two
regions are near one another in the three dimensional structure of
/
-type SASP bound to DNA. Because SASP-A and SASP-C contain a
lysine residue at the position corresponding to
Asp13-Glu10, these proteins are unable to form
cross-links with the
-amino group at this position and instead form
cross-links with the glutamate residues of the GPR recognition
sequence. The observed cross-links between the
-amino group and the
two glutamate residues of the GPR recognition sequence in SASP-A and
SASP-C, indicate that the N terminus of each protein is fairly mobile
and may interact electrostatically with an acidic patch that is formed
by the glutamate residues of the GPR recognition sequence. This acidic
patch may also contain Asp13 and Glu10 in SspC
and Bce1, respectively, and could explain the apparent shift in the
cross-link formation site from Glu10 in Bce1 to the GPR
recognition site in Bce1E10K. Although, we believe that we
have identified the major sites of EDC cross-linking in each
/
-type SASP studied, we cannot of course exclude the possibility
that other minor cross-linking sites exist which were not detected by
our HPLC analysis.
Another property of /
-type SASP that has been established in this
study is that different
/
-type SASP make functional protein-protein contacts with one another in the DNA bound state. Indeed, proteins from different species were found to interact as
demonstrated by cross-linking of SspC from B. subtilis to
SASP-A from B. megaterium. However, it appears that the
interaction between SspC and SASP-A is not as favorable as the
interaction between SASP-C and SASP-A. In fact, heterodimers of SASP-A
and SASP-C were the predominant cross-linking products when the ratio
of the two proteins approximated the in vivo ratio.
Preferential heterodimer formation may be due to preferential
association of the two proteins while bound to DNA, or merely to a
greater probability of successful cross-linking occurring between these
two particular proteins. Because SASP-A and SASP-C had identical EDC
cross-linking sites, which were different from those of SspC and Bce1,
it is reasonable to assume that the precise nature of the
protein-protein interactions in these two groups of
/
-type SASP
is similar, yet distinct from one another. Thus, subtle variations in
protein structure may allow some
/
-type SASP to interact with one
another more easily than others. These apparently minor differences in primary sequence between
/
-type SASP may be important for
efficient binding to different regions of the spore chromosome, and
account for the need to maintain multiple
/
-type SASP in each
species.
We are currently using another cross-linking strategy to identify amino
acid residues in /
-type SASP which make close contacts with DNA.
However, the structural information obtained during these types of
studies is limited and efforts to obtain a high resolution structure of
an
/
-type SASP·DNA complex are ongoing. A high resolution
structure of an
/
-type SASP·DNA complex should confirm the
results obtained in cross-linking experiments and also illustrate the
nature of the change in DNA conformation which underlies the change in
UV photochemistry of spore DNA and ultimately spore UV resistance.
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ACKNOWLEDGEMENTS |
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We thank Barbara Setlow and Dave Hutcheson for purification of SASP-A and SspC, respectively. All mass spectrometry and amino acid sequencing were performed by Dr. John Leszyk at the Worcester Foundation for Biomedical Research.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM19698.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.
To whom correspondence should be addressed. Tel. 860-679-2607;
Fax: 860-679-3408; E-mail: setlow{at}sun.uchc.edu
1 The abbreviations used are: SASP, small, acid-soluble spore proteins; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; HPLC, high performance liquid chromatography; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PAGE, polyacrylamide gel electrophoresis; GPR, germination protease.
2 S. C. Mohr and P. Setlow, unpublished results.
3 B. Setlow and P. Setlow, unpublished results.
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REFERENCES |
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