From the Dipartimento di Genetica e Microbiologia
"A. Buzzati-Traverso," Università degli Studi, Pavia 27100 Italy and the ¶ Dipartimento di Fisiologia Veterinaria e
Biochimica, Università degli Studi, Milano 20133, Italy
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ABSTRACT |
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FlgM is an anti-sigma factor of the
flagellar-specific sigma ( In bacteria genes are regulated mainly at the transcriptional
level. In addition to the cis-acting promoter sequences, a
large number of activators and repressors modulate initiation of
transcription. Furthermore initiation depends on alternative sigma
factors that direct the RNA polymerase to specific promoter sequences.
The activity of sigma factors may itself be regulated, either by
stability as is the case for the Escherichia coli stationary
phase FlgM was first identified in Salmonella as a negative
regulator of the flagellar-specific sigma factor The B. subtilis FlgM has now been overproduced in
Escherichia coli, purified, and shown to form a complex with
purified Overexpression and Purification of FlgM--
The flgM
gene (264 base pairs) was obtained by polymerase chain reaction
amplification of Bacillus subtilis chromosomal DNA, using
primers (forward, 5'-CGGAATTCATATGAAAATCAATCAATTTGG-3' and backward
5'-CCCGGATCCTTATTGCTTTTTATAAAAATTAATC-3') with a NdeI and
BamHI site, respectively; after purification and
restriction, this NdeI-BamHI fragment was cloned
into the expression vector pET12a (19). The resulting plasmid, pBG9,
allows high level overproduction of FlgM in E. coli BL21
(DE3) (19).
To purify FlgM, BL21 (DE3) (pBG9) cultures were grown at 37 °C with
shaking in 150 ml of Luria broth medium supplemented with 1% (w/v)
glucose and 100 µg/ml ampicillin. At A600 of
3-4, the cells were collected by centrifugation and resuspended in 1 liter of Luria broth medium with 100 µg/ml ampicillin;
isopropyl- Overexpression and Purification of
Physical Characterization of FlgM--
The molecular mass of
FlgM and its fragments were determined by matrix-assisted laser
desorption and ionization time-of-flight mass spectroscopy using a
matrix of sinapinic acid or of FlgM· Chemical Cross-linking--
Purified proteins were cross-linked
in a 50 µl reaction mixture containing 50 mM HEPES, pH
7.5, 100 mM NaCl, 10 mM MgCl2, 10%
glycerol by adding ethylene glycol-bis(succinic acid
N-hydroxysuccinimide ester) (EGS, Sigma) or
dithio-bis(succinimidyl propionate) (DSP, Sigma) to a final
concentration of 1 mg/ml. EGS and DSP were predissolved in dimethyl
sulfoxide at 10 mg/ml. After incubation at 0 °C for 2 h,
L-lysine was added to a final concentration of 20 mM. Cross-linked products were separated and analyzed by
SDS-PAGE.
Isolelectric Focusing--
Isoelectric focusing was performed in
nondenaturating conditions, using horizontal electrophoresis apparatus
with 1 M NaOH and 1 M
H3PO4 as electrode solutions. The samples
(5-20 µl) were applied to a 1-mm-thick gel using isoelectric
focusing applicator (Pharmacia). After focusing, proteins were
visualized by Coomassie Blue staining.
Gel Mobility Shift Analysis--
DNA binding reactions (10 µl)
contained about 0.5 pmol of 5'-end-labeled DNA fragment and purified
proteins (as indicated) incubated in binding buffer (40 mM
Tris-HCl, pH 8.0, 1 mM dithiothreitol, 0.25 mg/ml bovine
serum albumin, 50 mM NaCl, 1 mM EDTA, pH 8.0, 20% glycerol, 5 ng/µl unspecific plasmid DNA) at 37 °C for 20 min
prior to separation by native 5% PAGE at room temperature. To label
the DNA, the PD-6 promoter containing fragment (300 base pairs) was
amplified by polymerase chain reaction using unphosphorilated primers (5'-GATCAAGTGAAGCTTGGAATT-3' and
5'-AATATTGTGGTTAATTCTCAT-3'), labeled with
[
To analyze the protein components associated with the shifted DNA band,
the corresponding portion of the gel was cut out, rerun in 15%
SDS-PAGE, and transferred to a nitrocellulose filter. The filter was
probed with anti-FlgM polyclonal antibodies, followed by a secondary
antibody conjugated with horseradish peroxidase. The peroxidase
activity was visualized with an enhanced chemiluminescence kit (ECL,
Amersham Corp.)
Limited Proteolytic Digestion--
FlgM (2.5 mg/ml) was
incubated at 21 °C for 30 min in reaction containing 20 mM HEPES, pH 7.5, 50 mM KCl, 10 mM
MgSO4, and 0.01 mg/ml subtilisin. Reactions were stopped by
adding 10 mM phenylmethylsulfonyl fluoride and visualized
by 20% SDS-PAGE. Samples were blotted to polyvinylidene difluoride
membrane and analyzed by automated amino acid sequencing. In a second
experiment the products of limited proteolysis were separated by
reverse phase HPLC (Jasco on an Aquapore RP-300 C 8 column (25 × 0.46 cm). The solvent system used was either 0.1% (v/v)
trifluoroacetic acid in H2O (system A) or 0.075%
trifluoroacetic acid in CH3CN (system B). The elution was
with a linear gradient from 0 to 50% system B in system A, in 70 min
at a flow rate of 0.7 ml min Overproduction and Purification of FlgM--
The B. subtilis FlgM is a 9.8-kDa protein (16). The sequence coding for
FlgM was obtained by polymerase chain reaction amplification of
B. subtilis chromosomal DNA, using primers with a
NdeI and BamHI site, respectively; after
purification and restriction, flgM was cloned in the
expression vector pET12a. The coding sequence was followed by the
original stop codon, and the expected product was only FlgM, without
fusion to any resident sequence. The resultant plasmid, pBG9, was
transformed into E. coli BL21 (DE3). Following induction
with isopropyl- Characterization of FlgM--
N-terminal determination by Edman
degradation of an aliquot of the purified protein gave the sequence
MKINQFG corresponding to the first seven amino acids of FlgM as deduced
from the nucleotide sequence (16). The mass, measured by mass
spectrometry was 9,931 Da, in agreement with the estimated mass of
9,862 Da. We used gel exclusion chromatography to determine whether
FlgM is a dimer in solution. The elution volume on a Sephacryl S-100
column corresponds to a molecular mass of about 21 kDa, suggesting that
FlgM is a dimer. This was further confirmed by PAGE under nondenaturing conditions (Fig. 2). The dimer nature of FlgM
is also corroborated by results of chemical cross-linking experiments.
Incubation of FlgM with cross-linker generated a faster migrating
species, probably due to internal cross-linking, and a band of
approximately 20 kDa, again suggestive of a homodimer (Fig.
3A, lane 3). At least one other anti-sigma factor, SpoIIAB of B. subtilis, has
been reported to be present as a dimer (4, 21). We have no knowledge of
the possible oligomerization of FlgM of Salmonella.
FlgM Forms a Complex with
The purified complex was treated with the homobifunctional
cross-linking agents DSP and EGS. Analysis of the DSP-treated product by SDS-PAGE showed a single band of approximately 40 kDa (Fig. 3A). Similar results were obtained upon treatment with EGS
(data not shown). No bands corresponding to the two proteins, FlgM and
The complex appears to be an heterodimer by the mass of the
cross-linked complex estimated by SDS-PAGE and electrophoresis of
purified complex under nondenaturing gel gradient electrophoresis. In
addition the mass of the EGS cross-linked complex was measured by mass
spectrometry, and the observed value of 43,399 Da is in good agreement
with the theoretical value of 39,311 Da (9, 862 Da of FlgM + 29, 449 Da
of
The complex gave a single band in PAGE under nondenaturing conditions
(Fig. 3B, lane 5). Using antibodies
anti-
To evaluate the specificity of the complex formation, we performed
similar experiments with FlgM and a different sigma factor of B. subtilis, The FlgM·
FlgM inhibits initiation of transcription from
Partial Proteolysis of FlgM--
To gain information on the
structural organization of FlgM, we performed experiments of limited
proteolysis. Partial digestion with subtilisin gave consistently one
major product that in PAGE had a mobility of approximately 5 kDa (Fig.
5A). The same electrophoretic pattern was obtained following incubation at 21 °C for at least one
h (data not shown). Edman degradation of the material transferred to
polyvinylidene difluoride membranes, indicated the presence of the N
terminus of FlgM. In a parallel experiment the products of partial
proteolysis were subjected to separation by HPLC. Only one peak was
observed, but two N-terminal sequences were obtained in approximately
equimolar amounts: MKINQFGTQSVNPY and IENGSYKVDANHIA (Fig.
5B). The first sequence corresponds to the N terminus of FlgM, whereas the second one corresponds to residues 65-78 of the
protein. Various attempts to further purify the two components were
unsuccessful. The same fraction obtained by HPLC was analyzed by mass
spectrometry and gave the values of 5754 and 2284 Da, respectively
(Fig. 5B). The Mr of 5754 is in good
agreement with the calculated value (5753 Da) of a peptide consisting
of the first 51 residues of FlgM; the smaller peptide of
Mr 2284, should correspond to amino acids
Ile65 to Phe84 (calculated mass, 2279 Da)
toward the C terminus of the protein. A minor component of
Mr 5810 was also observed as a shoulder of the
main peak of Mr 5754; we infer that the
proteolytic cleavage can occur either upstream or downstream of
Gly52.
The results of the partial proteolysis are summarized in the Fig.
5C. We conclude that FlgM is organized in two regions: the N-terminal part (residues 1-51) and the C-terminal portion (residues 65-84), which appear structured and resistant to proteolytic cleavage. The two structural motifs are presumably connected by a loop, easily
accessible to cleavage.
We have overexpressed and obtained in soluble form the anti-sigma
factor FlgM of B. subtilis, the main regulator of
FlgM· In the case of bacteriophage T4, the anti-sigma AsiA forms
a tight complex with the The anti-sigma factors themselves are regulated. In the case of SpoIIAB
and RsbW, the sigma factor is set free to accomplish transcription by
the action of an anti-anti-sigma factor (SpoIIAA and RsbV,
respectively), which under certain conditions forms a complex with the
anti-sigma protein (25, 31-33).
As for FlgM we do not yet know the mechanism regulating its activity. A
clue derives from work on the Salmonella homologue of FlgM
that has been shown to be secreted into the medium by the flagellar
secretion system (34, 35). When the flagellar hook-basal body complex
is assembled, the devoted class III secretory apparatus export FlgM
from the cell, thereby relieving the inhibition exerted by the
anti-sigma factor. In solution the free Salmonella FlgM is
mostly unfolded, which may facilitate its secretion; it forms a stable
complex with ) subunit of RNA polymerase in
Bacillus subtilis, and it is responsible of the coupling of
late flagellar gene expression to the completion of the hook-basal body
structure. We have overproduced the protein in soluble form and
characterized it. FlgM forms dimers as shown by gel exclusion
chromatography and native polyacrylamide gel electrophoresis and
interacts in vitro with the cognate
D
factor. The FlgM·
D complex is a stable heterodimer as
demonstrated by gel exclusion chromatography, chemical cross-linking,
native polyacrylamide gel electrophoresis, and isoelectric focusing.
D belongs to the group of sigma factors able to bind to
the promoter sequence even in the absence of core RNA polymerase. The
FlgM·
D complex gave a shift in a DNA mobility shift
assay with a probe containing a
D-dependent
promoter sequence. Limited proteolysis studies indicate the presence of
two structural motifs, corresponding to the N- and C-terminal regions, respectively.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S (1) or by interaction with anti-sigma factors
(2). Anti-sigma factors have been characterized for
B
and
F of B. subtilis (3-5) and for the
flagellar dedicated sigma of Salmonella (6, 7) and
70 of E. coli upon infection with the
bacteriophage T4 (8). Anti-sigma factors have also been described in
Pseudomonas aeruginosa and Myxococcus xanthus
(9-11). The activity of the anti-sigma factors is exercised by
specific protein-protein interaction, even though the details of the
molecular mechanisms involved are still largely to be elucidated
(12-15).
28,
also called FliA (6, 7), and later shown to interact specifically with
the cognate sigma factor, forming a stable complex (15). FlgM has also
been described in B. subtilis, where it appears to
accomplish a similar regulatory function, coupling the early and late
flagellar gene expression (16-18). The action of FlgM is thought to
occur by specific interaction with the late flagellar sigma factor
D.
D protein. Limited proteolysis studies suggest
that FlgM contains a relatively stable N-terminal domain.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-thiogalactopyranoside was added to 0.1 mM, and growth was continued for 3 h at 21 °C, until an A600 of 0.7. The cells were recovered
by centrifugation and resuspended in 100 ml of A buffer (20 mM NaH2PO2, 29.6 mM Na2HPO2, 50 mM NaCl, 0.1 mM EDTA, pH 8.0, 10% (v/v) glycerol). Cells were lysed by
sonication, and the lysate was centrifuged to remove cell debris and
inclusion bodies. The supernatant, containing the soluble fraction of
the protein, was loaded on a
FPLC1 (Pharmacia) S-Sepharose
26/10 column, pre-equilibrated with A buffer. The FlgM protein was
purified by elution with a 0-0.5 M gradient of NaCl in A
buffer; 19 mg of FlgM were recovered, eluting with 0.3 M
NaCl. The protein was further purified by gel exclusion chromatography
on an FPLC Sephacryl S-100 column in B buffer (10 mM
Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM
MgCl2, 1 mM EDTA, pH 8.0, 10% (v/v) glycerol).
B--
B was overexpressed by using a
pT7-5 derivative, kindly supplied by W. G. Haldenwang. For
purification we followed the procedure described by Huang et
al. (20) for
W. The last steps (heparin-Sepharose
and Superdex) were omitted and substituted by a single HiTrap Q column
(Amersham Pharmacia Biotech).
-cyano-4-hydroxycinnamic acid. The
N-terminal amino acid sequences were determined by automated Edman
degradation using an Applied Biosystem 477A liquid pulse protein sequencer.
D Binding Assay--
Purified proteins
FlgM and
D were incubated together at equimolar ratio at
0 °C for 1 h, and the mixture was fractionated by
chromatography on an FPLC Superose HR 12 gel exclusion column in buffer
containing 10 mM Tris-HCl, pH 7.5, 100 mM NaCl,
10% glycerol. Fractions collected were analyzed by 15% SDS-PAGE.
-32P]ATP and T4 polynucleotide kinase and
purified by 5% PAGE.
1. The elution of peptides
was monitored at 220 nm. Aliquots were analyzed by matrix-assisted
laser desorption and ionization time-of-flight mass spectroscopy and
Edman degradation.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-thiogalactopyranoside expression of
FlgM was obtained at high level, but in the insoluble protein fraction.
After several attempts we found conditions at which the majority of
FlgM was in the soluble fraction. The volume of the culture was 1 liter
in a 5-liter volume flask, the incubation temperature was 21 °C, and
the concentration of the
isopropyl-
-D-thiogalactopyranoside inducer 0.1 mM. The soluble fraction was purified by S-Sepharose chromatography, followed by a gel exclusion column of Sephacryl S-100.
After this step, the protein was estimated to be at least 95% pure by
SDS-PAGE and Coomassie staining (Fig. 1). The
final yield was 19 mg of FlgM/liter of culture of E. coli
grown to an A600 of 0.7.
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Fig. 1.
Overexpression of B. subtilis
FlgM in E. coli. Proteins were analyzed by
15% SDS-PAGE. Lane 1, total proteins from E. coli BL21/DE3 (pBG9) not induced. Lanes 2 and
3, pellet and supernatant, respectively, of a culture
induced with 0.1 mM
isopropyl- -D-thiogalactopyranoside at 21 °C for
3 h. Lane 4, purified FlgM, after FPLC Sephacryl S-100.
The positions of the molecular mass standards are shown to the
left of the figure.
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Fig. 2.
Mobility of native FlgM in nondenaturing
polyacrylamide gradient (from 2 to 20%, FASTA system). Standard
proteins used were cytochrome c (Mr = 12,400; lanes 1 and 5) and trypsinogen
(Mr = 24,000; lanes 2 and
3) both from Pharmacia. Lane 4, purified
FlgM.
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Fig. 3.
The
FlgM· D complex is a specific
heterodimer. A, 15% SDS-PAGE analysis of the DSP
cross-linked FlgM·
D complex. Lanes 1 and
8, molecular mass standards (SIGMA); lane 2,
purified FlgM; lane 3, FlgM treated with DSP; lane
4, purified
D; lane 5,
D
treated with DSP; lane 6, FlgM·
D complex
not treated with DSP; lane 7, the complex after DSP
cross-linking. B, specificity of FlgM·
D
complex. Native 8% PAGE of samples incubated at 0 °C for 1 h.
Lane 1,
B (250 pmol); lane 2,
B (125 pmol) + FlgM (250 pmol); lane 3,
B (125 pmol) + FlgM (625 pmol); lane 4,
D (118 pmol); lane 5,
D (59 pmol) + FlgM (250 pmol).
D--
FlgM is an
anti-sigma factor specific for
D as shown by in
vitro and in vivo experiments
(17).2 To investigate the
interaction of the two proteins, we purified
D according
to Chen and Helmann (23). Upon mixing and incubation at 0 °C for
1 h, the two proteins formed a complex that could be resolved by
gel exclusion chromatography on a Superose 12HR column. The complex was
stable even in 0.6 M NaCl, suggesting a participation of
hydrophobic interaction between the two polypeptide chains in the complex.
D, were observed, suggesting that the complex is very
stable and long lived, because it is not in equilibrium with the
monomers. The same cross-linked product was obtained by treating the
two proteins, immediately upon mixing. FlgM and
D
treated separately with DSP did not produce a band of the same mobility
as that obtained when the mixture was treated (Fig. 3A, lanes 3 and 5).
D), considering the presence of an unknown number of
EGS molecules. The same type of heterodimeric structure has been
reported for the Salmonella FlgM.
28 complex
(15). The B. subtilis purified complex, not treated with the
cross-linking agent, had a pI of about 6.8 as determined experimentally
by isoeletric focusing; distinct from the observed pI of
D (5.9) and the deduced pI of 9.9 of FlgM (data not shown).
D to detect free
D in Western
blotting, in nondenaturing conditions, we estimated that the apparent
dissociation constant for the FlgM·
D complex is
10
6 M or less (data not shown).
B, which is known to interact with the
cognate anti-sigma, RsbW (3). We failed to observe any interaction
between the two proteins (Fig. 3B, lanes 2 and
3). (In the electrophoretic system used, FlgM is not
visible, because it moves out of the gel.)
D Complex Binds
DNA--
D belongs to the group of sigma factors devoid
of region 1, responsible for masking the rest of the molecule. Thus
these factors can bind to the promoter sequence even in absence of core
RNA polymerase (23).
D-dependent promoters in
vitro.2 Unexpectedly the FlgM·
D
complex gave a supershift in a gel retardation assay with a probe containing the flagellin,
D-dependent
promoter PD-6 (22). Not only was the probe retarded more than observed
with only
D, but the amount of probe bound to the
complex was much higher. The experiment was performed in the presence
of cold unspecific plasmid DNA. To show that the supershift was indeed
due to the complex, a gel slice corresponding to the shifted band was
subjected to SDS-PAGE and analyzed by Western immunoblotting with
anti-FlgM antibodies. These polyclonal antibodies cross-react with
D and allowed the detection of the presence of both
proteins in the shifted band (Fig. 4). The
same shift was observed when the probe was first incubated with
D and an equimolar amount of FlgM added 5 min prior to
the electrophoresis (Fig. 4, lane 5). In our tests,
D alone gave a weak shift of the probe (Fig. 4,
lane 4), and this is probably due to incomplete renaturation
of the protein solubilized by guanidine hydrochloride. The higher
amount of probe retained in the presence of FlgM suggests that the
anti-sigma factor may facilitate the renaturation of
D.
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Fig. 4.
The
FlgM· D complex binds DNA.
A, 32P-Labeled PD-6 promoter fragment (0.5 pmol)
was incubated for 20 min at 37 °C with increasing concentrations of
D protein (lanes 2-4 contained 0.5, 1.0, and
5.0 pmol/µl, respectively). The sample applied to lane 5 was incubated with 5 pmol/µl of
D for 15 min at
37 °C, and then 5 pmol/µl of FlgM were added for the remaining 5 min of incubation. Lane 6, incubation for 20 min with 5 pmol/µl of purified FlgM·
D complex. Free DNA and
complexes were separated by 4% native PAGE. Lane 1 shows a
probe alone control. B, SDS-PAGE 15% and Western blot
analysis of the proteins present in the shifted DNA band (lane
7). Lane 8, standard of FlgM·
D complex
(0.1 µg).
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Fig. 5.
Partial proteolysis of FlgM.
A, SDS-PAGE 20% analysis of subtilisin (lane 1)
digestion of FlgM. The reaction contained 2.5 mg/ml of FlgM and 0.01 mg/ml of subtilisin. Incubation was at 21 °C for 30 min. Lane
2, untreated FlgM. B, characterization of FlgM
proteolytic fragments, after reverse phase HPLC purification.
C, structure of FlgM. The open bar represents the
88-amino acid FlgM peptide. The shaded boxes highlight the
two structural motifs (residues 1-51 and 65-84), as determined by
subtilisin limited proteolysis.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
D activity. The protein is a dimer in solution, as
determined by gel exclusion chromatography and PAGE electrophoresis
under nondenaturing conditions. The anti-sigma factor FlgM forms a
stable and long lived complex with the cognate sigma factor
D. Highly stable complexes have been reported for two
other B. subtilis sigma-anti-sigma couples,
SpoIIAB·
F and RsbW·
B (24, 25).
D complex still binds to the promoter sequence,
but the fact that FlgM does not interfere with the interaction of
D at the promoter site may have a marginal physiological
significance, as already noted for the DNA binding by the same sigma
factor (23). Therefore, if, as it seems plausible, in vivo
the promoter recognition is performed by the holoenzyme, the specific
anti-transcriptional activity of FlgM could be exerted in three ways:
(i) sequestering of free
D molecules in the
FlgM·
D complex, (ii) removing
D from
the holoenzyme, and (iii) hampering a productive interaction between
the holoenzyme and the promoter.
70 subunit (8, 26).
Nevertheless a detailed analysis of its mechanism of action suggests
that AsiA interacts with the holoenzyme, thereby modifying the
interaction between the enzyme and promoter sequence and not simply
dissociating the sigma factor from the core (27-29). Recently it has
been shown that the Salmonella FlgM, in addition to
sequestering the free sigma factor, binds to the holoenzyme and
increases the rate of dissociation of sigma from the core (30).
28, but upon binding, the C-terminal half
of FlgM becomes structured, with the N terminus still unfolded and thus
potentially competent for secretion (15). The present results from
partial proteolysis indicate a different organization of the B. subtilis FlgM polypeptide. The N-terminal region is structured, at
least as deduced from partial proteolysis and not unfolded as suggested
for the Salmonella FlgM from NMR studies. The structural
differences may hint at different mechanisms in the way to regulate the
anti-sigma factor activity in the cell.
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ACKNOWLEDGEMENTS |
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We thank Daniela Barillà and Michael
Chamberlin for sharing unpublished data; J. Helmann and
W. G. Haldenwang for the gift of anti-D
antibodies and pT7-5 sigB plasmid, respectively; and Giovanna Valentini and Menico Rizzi for helpful discussions.
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FOOTNOTES |
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* This work was supported in part by grants from Ministero dell'Università e della Ricerca Scientifica e Tecnologica and the Consielio Nazionale delle Ricerche Target Project on Biotechnology.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.
§ Supported by a fellowship from International Centre for Genetic Engineering and Biotechnology (Trieste, Italy). Permanent address: Genetic Engineering and Biotechnology Center, Havana, Cuba.
To whom correspondence should be addressed: Dipt. di Genetica
e Microbiologia, Via Abbiategrasso n. 207, 27100 Pavia, Italy. Tel.:
39-382-505548; Fax: 39-382-528469; E-mail:
galizzi{at}pillo.unipv.it.
2 D. Barillà and M. Chamberlin, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: FPLC, fast protein liquid chromatography; EGS, ethylene glycol-bis(succinic acid N-hydroxysuccinimide ester); DSP, dithio-bis(succinimidyl propionate); HPLC, high pressure liquid chromatography; PAGE, polyacrylamide gel electrophoresis.
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