From the
Virginiae butanolides (VBs) A-E are butyrolactone
autoregulators that control virginiamycin production in
Streptomyces virginiae. We have previously reported the
purification and molecular cloning of VbrA, a putative VB binding
protein (Okamoto, S., Nihira, T., Kataoka, H., Suzuki, A., and Yamada,
Y.(1992) J. Biol. Chem. 267, 1093-1098). However, VbrA
[Abstract]
protein overexpressed in Escherichia coli did not show any
detectable VB binding activity nor did the immunoprecipitation of
native VbrA from a cell-free extract of S. virginiae cause any
decrease in such activity, indicating that VbrA is not the true VB
binding protein. This finding prompted us to seek the true VB binding
protein by repurification. After successive purification by anion
exchange, gel filtration, heparin, and hydrophobic interaction
chromatography, a 26-kDa protein (p26k) was identified as the true VB
binding protein. Partial amino acid sequences of p26k were determined,
and the gene (barA) that encodes this protein was isolated and
cloned using degenerate oligonucleotide probes. When the barA gene was expressed in Streptomyces lividans and E.
coli, strong VB binding activity appeared, demonstrating
unambiguously that the S. virginiae p26k protein is the true
VB binding protein.
The genus Streptomyces is of considerable interest
because of its complex life cycle and its ability to produce numerous
antibiotics and extracellular enzymes
(1, 2) . At the
onset of morphological differentiation such as aerial mycelium
formation, the bacteria start to produce a wide variety of secondary
metabolites. The morphological differentiation and/or the production of
secondary metabolites are controlled by low molecular weight compounds
called ``butyrolactone
autoregulators''
(3, 4) . So far, 10 butyrolactone
autoregulators have been isolated (see Fig. 1), of which A-factor
from Streptomyces griseus(5) and virginiae butanolide
from Streptomyces virginiae(6) have been most studied.
Virginiae butanolide (VB)
During the past 5 years, our
laboratory has devoted considerable effort to studying the VB binding
protein at the molecular and biochemical levels. As a putative binding
protein, we have purified a 36-kDa protein (VbrA), cloned the
corresponding gene (vbrA), and sequenced
(15) . However,
through the expression of the vbrA gene in Escherichia
coli and immunological analysis, we found that VbrA is not, in
fact, the true VB binding protein. In this report, we describe the
purification to homogeneity of a 26-kDa protein (p26k) as the true VB
binding protein and the cloning of the gene (barA) encoding
this protein. Expression in Streptomyces lividans and E.
coli demonstrated the barA-dependent production of VB
binding activity, confirming that p26k is the true VB binding protein.
pUC18 was
used for the construction of a genomic library, and M13mp18 was used
for DNA sequencing. pET-3d
(16) was employed for constructing
the expression plasmids. Streptomyces plasmid pIJ486
(18) was kindly provided by D. A. Hopwood (John Innes Institute,
Norwich, UK). DNA manipulations in E. coli and in
Streptomyces were performed as described by Sambrook et
al.(19) and Hopwood et al.(17) ,
respectively.
A KpnI-BssHII fragment
carrying the p26k gene was blunt-ended with T4 DNA polymerase and
introduced into the SmaI site of pUC18 to use as a polymerase
chain reaction template. Polymerase chain reaction was done with
primer-1 (5`-AACCATGGCAGTGCGACACGAACG-3`) and M13 universal primer
(Takara Shuzo) to generate an NcoI site at the 5`-end of the
p26k coding sequence. The polymerase chain reaction product was cleaved
with NcoI and BamHI and cloned into pET-3d vector
resulting in pET-p26k.
BL21 (DE3)/pLysS containing
pET-p26k or pET-3d was grown until A
S. lividans TK21
harboring the indicated plasmid was grown for 72 h as
described
(17) . 1 g of mycelia was suspended in 5 ml of buffer A
and disrupted by sonication. Cell-free extract was stored at -80
°C and later used in the assay of VB binding activity.
E. coli BL21(DE3)/pLysS containing pET-vbrA significantly overproduced a
protein of 38 kDa after IPTG induction, which was not observed in the
uninduced cells (data not shown). The first 6 N-terminal amino acids of
the 38-kDa protein were identical to those deduced from the nucleotide
sequence of the vbrA gene except that the first methionine was
missing (data not shown), confirming that the 38-kDa protein is rVbrA.
rVbrA protein existed in a soluble fraction of cell extract and was
easily purified to homogeneity by ammonium sulfate fractionation and
DEAE-Sephacel chromatography (data not shown).
After localizing the p26k gene on a
1.8-kbp KpnI-SalI fragment, the nucleotide sequence
was determined (Fig. 6). Computer-aided nucleotide sequence
analysis revealed that this fragment contained only one open reading
frame coding for the p26k protein. The p26k gene, ranging from position
454 to position 1152 base pairs, encodes a protein of 232 amino acids,
and all the partial amino acid sequences determined from the purified
protein appear in this frame. The molecular weight of the protein
encoded by the p26k gene is 25,001, which agrees well with that of the
protein purified on SDS-PAGE (
To confirm that the open reading frame actually encoded the
VB binding protein, the coding region of the p26k gene was amplified by
polymerase chain reaction and cloned immediately downstream of an
IPTG-inducible T7 promoter in expression vector pET-3d to construct
pET-p26k. Under both induced and uninduced conditions, BL21(DE3)/pLysS
harboring pET-p26k showed extremely high VB binding activity
(). But specific activity in the IPTG-induced cells was
approximately 50-fold higher than that in the uninduced cells,
indicating that the expression of the p26k gene under the
IPTG-inducible T7 promoter was somewhat leaky. The cells harboring
pET-3d without the insert showed no activity. Scatchard analysis of the
crude VB binding protein expressed in E. coli indicated that
the K
The definite role of the VB binding protein in the VB-mediated
signal transduction pathway still remains to be elucidated. Successful
cloning of the gene encoding the VB binding protein will promote
understanding of the regulatory mechanism at the molecular level.
Further study is needed to clarify the molecular mechanism of the
signal transduction/gene regulation system mediated by VB and its
binding protein.
Data starting from 100 g of mycelia,
wet weight was summarized.
The nucleotide sequence(s) reported
in this paper has been submitted to the GenBank
(
)
A, B, C, D, and E can
induce the production of virginiamycin in wild type S.
virginiae(7, 8, 9) , whereas A-factor
triggers both streptomycin production and aerial mycelium formation in
a streptomycin
-aerial mycelium
mutant of S.
griseus(10, 11) . VBs and A-factor possess a
characteristic 2,3-disubstituted
-butyrolactone skeleton in common
and switch on secondary metabolism and/or cell differentiation at
nanomolar concentration. Although they resemble each other, VBs show no
effect on streptomycin production of S. griseus at
physiological concentration and vice versa
(6, 12) .
Figure 1:
Structures of butyrolactone
autoregulators isolated from Streptomyces species. Absolute
configurations of A-factor (11), VBs (9), and IM-2 (28) have been
assigned to (3R), (2R,3R,6S), and
(2R,3R,6R), respectively, as shown. Although
absolute configurations of Factor I and three factors from
Streptomyces bikiniensis and Streptomyces cyaneofuscatus are not yet determined, the most probable
(2R,3R,6R) and
(2R,3R,6S) forms from spectroscopic data
(29, 30) are depicted, respectively.
[H]VB-C
(54.6 Ci/mmol) indicates a
synthetic
H-labeled ligand for VB binding assay
(13).
Assuming the presence of specific receptors as signal transducers
for these autoregulators, we found a binding protein highly specific to
VBs in the cytoplasmic fraction of S. virginiae using a
H-labeled VB-C analog
([
H]VB-C
) as a ligand (see
Fig. 1
)
(13) . The binding protein had a dissociation
constant (K
) of several nanomolars and
existed at 30-40 molecules/genome
(13) . Later, a similar
binding protein for A-factor was detected in S. griseus (14),
suggesting that receptor-mediated signal transduction is common to
butyrolactone autoregulators.
Bacteria, Bacteriophage, and Plasmids
S. virginiae (strain MAFF 10-06014, National Food
Research Institute, Ministry of Agriculture, Forestry, and Fisheries,
Tsukuba, Japan) was the source of the VB binding protein and was grown
at 28 °C as described previously
(7, 13) . For
genetic manipulation in E. coli, JM105, JM109, and DH5
were used. For expression of the cloned genes in E. coli and
Streptomyces, BL21 (DE3)/pLysS
(16) and S. lividans TK21
(17) were used as hosts, respectively.
Repurification of VB Binding Protein
Cells (100 g) were suspended in 500 ml of buffer A (50 mM triethanolamine HCl, pH 7.0, 0.5 M KCl, 5 mM dithiothreitol, 0.1 mM p-APMSF) and disrupted by
sonication. After centrifugation, cell-free extract was fractionated
with ammonium sulfate (30-50% saturation), and the resulting
pellet was resuspended in 150 ml of buffer B (50 mM triethanolamine HCl, pH 7.0, 0.1 M KCl, 5 mM dithiothreitol, 0.1 mM p-APMSF) and dialyzed against the
same buffer (4 liters 2). After dialysis, the clarified sample
was applied onto a DEAE-Sephacel column (3.3
43 cm, Pharmacia
Biotech Inc.) pre-equilibrated with buffer B. After washing with 1
liter of buffer B, bound proteins were eluted with a linear gradient of
KCl from 0.1 to 0.4 M in 2 liters of buffer B. Active
fractions were pooled, concentrated, and then applied (two separate
runs) onto a Sephacryl S-100 HR gel filtration column (2.6
90
cm, Pharmacia) equilibrated with buffer A. Active fractions were
pooled, concentrated to 21 ml, and stored at -80 °C until
use. A portion (1-3 ml) of each active fraction was dialyzed
against buffer C (50 mM triethanolamine HCl, pH 7.0, 10% (w/v)
glycerol, 1 mM dithiothreitol) and subsequently applied onto
an Econo-Pac heparin column (bed volume, 5 ml; Bio-Rad) equilibrated
with the same buffer. After washing with buffer C, proteins were eluted
with buffer C containing 0.5 M KCl, and active fractions were
pooled. Active material (2 ml/run) was injected onto a Phenyl-5PW HPLC
column (7.5
75 mm, Tosoh) equilibrated with buffer PH (50
mM triethanolamine HCl, pH 7.0, 1 M (NH
)
SO
, 0.3 M KCl, 5
mM dithiothreitol, 0.1 mM p-APMSF). Bound proteins
were eluted at 19-21 °C with a 25-ml linear gradient from
buffer PH to buffer PO (50 mM triethanolamine HCl, pH 7.0, 0.3
M KCl, 5% (v/v) CH
CN, 5 mM dithiothreitol, 0.1 mM p-APMSF) at a flow rate of 0.5
ml/min. Active fractions corresponding to 0 M (NH
)
SO
were collected and
analyzed by SDS-PAGE. The fraction containing the main VB binding
activity was reinjected onto the same column equilibrated with buffer
PL (50 mM triethanolamine HCl, pH 7.0, 0.12 M (NH
)
SO
, 0.3 M KCl, 5
mM dithiothreitol, 0.1 mM p-APMSF) and eluted at
19-21 °C with 12.5 ml of the same buffer followed by a 4.5-ml
gradient to buffer PO at a flow rate of 0.5 ml/min. Active fraction
from the final HPLC step was analyzed by SDS-PAGE to examine the
purity.
Amino Acid Sequence Analysis of p26k and Molecular
Cloning of p26k Gene
N-terminal Sequence
The first Phenyl-5PW active
fraction was separated by SDS-PAGE and electroblotted to an
Immobilon-P polyvinylidene difluoride membrane (Millipore
Corp.) using Trans-Blot SD (Bio-Rad) as recommended by the
manufacturer. The membrane was stained with Coomassie Brilliant Blue
R-250, and the protein band of 26 kDa was excised and subjected to a
protein sequencer (Applied Biosystems 476A protein sequencer). The
obtained N-terminal sequence is shown in Fig. 6, and the 7th to
the 12th amino acid sequences were used to design probe 26k-1
(5`-GTNGCNGTNCG(T/C/G)CA(A/G)GA-3`).
Figure 6:
Nucleotide and deduced amino acid
sequences of p26k gene. Nucleotides are numbered starting at the
KpnI site that is closest to the 5`-end in the 1.8-kbp
KpnI-SalI fragment. The deduced amino acid sequence
is shown in one-letter notation; sequences corresponding to the amino
acid sequences obtained from the purified p26k protein are
underlined. The sixth amino acid in fragment 24 and the
thirteenth and seventeenth amino acids in fragment 42 were His from the
DNA sequence but were not correctly assigned in protein
sequencing.
Fragment Peptide Sequences
For further
purification and desalting, the first Phenyl-5PW active fraction was
separated on a Capcell Pak C HPLC column (type SG, 4.6
250 mm, 300 Å, Shiseido Co., Ltd.) with a linear gradient
of CH
CN from 40 to 60% in 0.1% trifluoroacetic acid. The
purified p26k protein (133 pmol) was hydrolyzed with lysyl
endopeptidase (p2bk/enzyme = 50 mol/mol, Wako Pure Chemicals
Ltd.) in 0.2 ml of 20 mM Tris-HCl, pH 9.0 for 16 h at 30
°C. Fragment peptides were separated on a Capcell pak C
column with a linear gradient of CH
CN from 0 to 60%
in 0.1% trifluoroacetic acid. Four fragments, designated fragments 21,
24, 25, and 42, were collected, lyophilized, and analyzed as above. The
first 8 amino acid residues of fragment 24 were used to design probe
26k-24 (5`-GGNGCNATGTA(T/C)TT(T/C)NN(C/G)TT(T/C)GC-3`).
Molecular Cloning
Total DNA of S. virginiae was obtained by the method of Rao et al.(20) . A
partial genomic library was constructed with size-fractionated
BamHI fragments (2.7-3.5 kbp) and pUC18 using DH5
as a host. The partial genomic library was screened by colony
hybridization using an oligonucleotide probe end-labeled with
[
-
P]ATP (>3000 Ci/mmol, ICN Biomedicals
Inc.). DNA sequence was determined by the dideoxy chain termination
method
(21) on both strands using single-stranded templates of
M13mp18 clones (Sequenase, U.S. Biochemical Corp.). The open reading
frame was identified by the program FRAME
(22) .
Construction of pET-vbrA and pET-p26k
From plasmid pVBR PS2.2 carrying the genes vbrA and
rplk(15) , a 1-kbp EheI-MroI fragment
containing the entire vbrA gene was recovered, blunt-ended
with Klenow enzyme, and ligated into SmaI-digested pUC18. The
1-kbp NspI-BamHI fragment containing a part of the
vbrA gene was ligated into pKK233-2
(23) via an
oligonucleotide linker (5`-pCATGGTTTCTCCAGTATTCGCATG-3`,
5`-CGAATACTGGAGAAAC-3`).From the resulting plasmid, a 1-kbp
NcoI-BamHI fragment was recovered and ligated with
NcoI-BamHI-digested pET-3d. The resulting plasmid was
designated as pET-vbrA.
Preparation of rVbrA and Recombinant p26k Protein
BL21 (DE3)/pLysS harboring pET-vbrA was grown as described
(16) with induction by 0.5 mM IPTG for 2 h. The
harvested cells were resuspended in 10 volumes of buffer B and
disrupted by sonication. Cell-free extract was fractionated with
ammonium sulfate, and the 20-40% protein pellet was resuspended
in 20 ml of buffer B and dialyzed against the same buffer. The dialyzed
solution was applied onto a DEAE-Sephacel column (2.9 30 cm)
previously equilibrated with buffer B and eluted with 2 liters of a
linear gradient of KCl from 0.1 to 0.5 M in the same buffer.
Fractions containing the rVbrA were analyzed by SDS-PAGE, pooled, and
stored at -80 °C until use.
was
0.6-0.9. Induction was done with 1 mM IPTG for 1 h.
Cells were suspended in 5 ml of buffer A and disrupted by sonication.
Cell-free extract was used for SDS-PAGE analysis and the assay of VB
binding activity. For purification of recombinant p26k protein, cells
(3.4 g of cell, wet weight) from a 1-liter culture were suspended in 34
ml of buffer C containing 0.2 M KCl and disrupted by
sonication. Supernatant after centrifugation was adsorbed to a
DEAE-Sephacel column (bed volume, 100 ml) pre-equilibrated with the
same buffer. After washing with 4 bed volumes of the buffer, bound
proteins were eluted with a 600-ml linear gradient of 0.2-0.5
M KCl in buffer C. Active fractions eluting at around 0.35
M KCl were analyzed by SDS-PAGE, and fractions showing a
single p26k protein band were mixed, concentrated by ultrafiltration,
and stored at -80 °C until use.
VB Binding and Protein Assay
VB binding activity was routinely assayed by the ammonium
sulfate precipitation method
(24) with
[H]VB-C
(54.6 Ci/mmol) in the
presence and absence of 2000-fold cold VB-C
. When samples
after the heparin step were assayed, rVbrA was added to the reaction
mixture at a concentration of 110 µg/ml to prevent rapid
inactivation of the VB binding protein due to low protein
concentration. For this purpose, other proteins such as bovine serum
albumin or hemoglobin could be used, but, so far as we have tested, the
most reproducible results have been obtained with rVbrA protein. This
situation is the same for recombinant VB binding protein expressed in
E. coli. Scatchard analysis was performed as described
(13) by the equilibrium dialysis method. Protein concentration
was determined either by dye binding assay (Protein Assay Kit, Bio-Rad)
or by comparison of peak areas on HPLC charts using bovine serum
albumin as a standard.
Immunoprecipitation of VbrA
Cells of S. virginiae (1 g) were suspended in 10
volumes of extraction buffer (20 mM Tris-HCl, pH 7.4, 150
mM NaCl, 10% (w/v) glycerol, 1 mM dithiothreitol, 1
mM EDTA, 1 mM p-APMSF, 5 µg/ml leupeptin, 1%
(v/v) aprotinin, 0.1% (w/v) Nonidet P-40) and sonicated. Cell-free
extract (500 µl) was incubated with anti-VbrA serum (10-200
µl) on ice for 1 h. Protein A beads (100-1,000 µl of 10%
slurry in extraction buffer) were then added and incubated on ice for 1
h. After centrifugation, the supernatant was directly used in the VB
binding assay.
Molecular Mass of Native VB Binding Protein
Gel filtration was done on a Sephacryl S-100 HR column using
the DEAE-Sephacel fraction, and the elution position of the VB binding
protein was determined by VB binding assay. A calibration curve was
prepared using catalase (240 kDa), bovine serum albumin (68 kDa),
albumin (45 kDa), chymotrypsinogen A (25 kDa), and cytochrome c (12.5 kDa) (calibration proteins II, Boehringer Mannheim).
Expression of the vbrA Gene in E. coli
Because
VbrA is only a minor component of cellular proteins
(15) , it was
impossible to perform biochemical analyses using highly purified
protein. To overcome this problem with the aid of recombinant VbrA
(rVbrA), the vbrA gene was expressed in E. coli (see
``Materials and Methods'').
VB Binding Activity of rVbrA
To verify that the
vbrA gene encodes the VB binding protein, we assayed the VB
binding activity of rVbrA. Unexpectedly, neither the crude nor the
purified protein showed any detectable activity (data not shown), a
finding that strongly suggested that VbrA is not the VB binding
protein. However, we could not rule out the possibility that this
inability to bind VB may have been due to improper folding of rVbrA in
its heterologous host or to a lack of some post-translational
modification, such as N-terminal modification of native
VbrA
(15) , essential for VB binding. To further investigate
this, we conducted immunoprecipitation of native VbrA from cell-free
extracts of S. virginiae. Treatment with anti-VbrA antibody
caused no decrease in VB binding activity, and the remaining activity
was very similar to that in control experiments using extraction buffer
or nonimmune serum, although Western blot analysis indicated that up to
50% of the VbrA that existed in the original cell-free extract was
removed by immunoprecipitation (data not shown). From these lines of
evidence, we concluded that VbrA is not the true VB binding protein.
Purification of the True VB Binding Protein
To
obtain the true VB binding protein, we recommenced purification with
100 g of cells as starting material. After successive chromatographies
on a DEAE-Sephacel column, a Sephacryl S-100 HR column, and a heparin
column, hydrophobic chromatography was conducted on a Phenyl-5PW HPLC
column (first Phenyl-5PW) (Fig. 2). VB binding activity existed
in three protein peaks eluting at 60-70 min, with the majority of
VB binding activity existing in Fraction 1. SDS-PAGE analysis of these
fractions (Fig. 3A) indicated that proteins of 38, 35,
34, and 26 kDa, designated as p38k, p35k, p34k, and p26k, respectively,
were present as major components. Because p38k and p34k reacted
strongly with anti-VbrA antibody (Fig. 3B, lanes 2 and 3) and because their N-terminal amino acid sequences
(SDPNLNASHD and ALHVE for p38k and p34k, respectively) matched the
deduced amino acid sequence of VbrA (Ser-Asp
and Ala
-Glu
), these two proteins
were concluded to be derived from VbrA. Elimination of p38k and p34k
left p35k and p26k as possible candidates for the VB binding protein.
Figure 2:
First
Phenyl-5PW HPLC of VB binding protein. An aliquot (2 ml) of pooled
active fraction from heparin chromatography was injected. Every 2 min
for the first hour fractions were collected and three fractions,
indicated as 1, 2, and 3, were collected and
assayed for VB binding activity (). Protein concentration was
monitored by fluorescence (excitation, 280 nm; emission, 340 nm)
(solid line).
Figure 3:
Major proteins in the three active
fractions from Phenyl-5PW HPLC. Three fractions collected as shown in
Fig. 2 were subjected to 10-20% SDS-PAGE. A, Coomassie
Blue staining of the gel. Lane 1, molecular mass standards;
lane 2, fraction 1 (3.9 µg); lane 3, fraction 2
(3.5 µg); lane 4, fraction 3 (3.1 µg). 40% of each
fraction collected in Fig. 2 was applied. Molecular mass standards were
phosphorylase b (94 kDa), bovine serum albumin (67 kDa),
ovalbumin (43 kDa), carbonic anhydrase (30 kDa), and soybean trypsin
inhibitor (20 kDa). B, Western blot analysis. The same samples
were analyzed by Western blotting with anti-VbrA antibody. Prestained
SDS-PAGE standards (Bio-Rad) were used as molecular mass standards.
Polyclonal antiserum against VbrA was prepared in rabbits using rVbrA
and purified as described by Smith and Fisher (31). Western blotting
was performed on electroblotted polyvinylidene difluoride membranes
with the anti-VbrA antibody using horseradish peroxidase-conjugated
anti-rabbit IgG (Amersham Corp.) and enhanced chemiluminescence
detection (ECL system, Amersham Corp.) as recommended by the
supplier.
To identify which protein is the VB binding protein, Phenyl-5PW HPLC
was done at different column temperatures (Fig. 4). At 9 and 16
°C, VB binding activity was eluted mainly in Fraction 1
(Fig. 4A, top and middle panels). At
23 °C, however, the VB binding activity shifted into Fraction 2
(Fig. 4A, bottom panel) with a concomitant
shift of p26k from Fraction 1 at 9 and 16 °C
(Fig. 4B, lanes 2 and 5) to Fraction 2
at 23 °C (Fig. 4B, lane 9). The elution
patterns of other proteins such as p34k, p35k, and p38k were not
influenced and were almost identical at these three temperatures. In
our new purification, we have surveyed a number of different
purification procedures employing Western blotting with anti-VbrA
antibody to monitor elution profiles of VbrA together with the VB
binding activity. In almost all the procedures, the elution profile of
VbrA was nearly identical with or overlapping that of VB binding
activity. Only in the case of hydrophobic HPLC on a Phenyl-5PW column
at or lower than 16 °C was VB binding activity separated
sufficiently from VbrA. We noticed that, during the VB binding assay,
addition of rVbrA is effective in preventing VB binding activity from
rapid inactivation, especially for samples from the latter stage of
purification. This protective effect of VbrA could be the main reason
we were misled earlier to isolate VbrA as the VB binding protein,
because only fractions containing a sufficient amount of VbrA should
retain high VB binding activity. The possibility that p26k protein
might be weakly associating with VbrA remains to be solved.
Figure 4:
A,
hydrophobic Phenyl-5PW HPLC at 9 (top panel), 16 (middle
panel), and 23 °C (bottom panel). Active fractions,
indicated as 1, 2, and 3, were collected and analyzed. B,
effects of column temperature on protein profile in the active
fractions from first Phenyl-5PW. Active fractions from first Phenyl-5PW
HPLC at 9 (lanes 2-4) or 16 (lanes 5-7)
or 23 °C (lanes 8-9) were subjected to 10-20%
SDS-PAGE, and the gel was stained with Coomassie Brilliant Blue G-250.
15% of each fraction containing 1.3-1.9 µg for fraction 1,
2.1-2.6 µg for fraction 2, and 1.0-1.2 µg for
fraction 3 was loaded. Molecular mass standards (lanes 1 and
11) are shown. The most active fraction at each temperature is
shown by underlining the fraction number above the
lane.
To
purify p26k completely, active material from the first Phenyl-5PW HPLC
was rechromatographed on the same column (second Phenyl-5PW) using
different gradient conditions (see ``Materials and
Methods''). Proteins were eluted with three major peaks (fragments
1, 2, and 3 eluting at 6.7-7.9, 17.0-23.5, and
36.3-46.6 min, respectively) (Fig. 5A); only the
second peak (fragment 2) contained VB binding activity (e.g. input, 2.51 pmol; recovery, 1.55 pmol). SDS-PAGE analysis of these
fractions followed by silver staining showed that the active peak
(fragment 2) contained only p26k with no visible contaminant observed
(Fig. 5B), confirming p26k to be the true VB binding
protein.
Figure 5:
Second
Phenyl-5PW HPLC of VB binding protein (A) and SDS-PAGE
analysis (B). A, an aliquot (400 µl) of active
fraction from the first Phenyl-5PW HPLC was injected, and three protein
peaks (fractions 1, 2, and 3) were collected and assayed for VB binding
activity. B, all of the remaining sample after the VB binding
assay was dialyzed against water, lyophilized, and separated on a
4-20% gel, and the gel was
silver-stained.
The purification from 100 g of mycelia is summarized in
. The protein was purified 851-fold with an overall
activity recovery of about 3.4%. The final specific activity of the VB
binding protein was 0.404 nmol/mg, which was only 1% of the theoretical
value (40 nmol/mg) calculated on the assumption of 1:1 binding
stoichiometry between [H]VB-C
and the
p26k protein, indicating that the majority of the purified protein
became inactivated during purification. In our previous purification
(15), we reported that the value of 4.95 nmol/mg for VbrA contaminated
preparation. This discrepancy was due to the fact that during this
purification we focused not on the high recovery of the VB binding
activity itself but on removing the contaminating VbrA, and hence the
inactivation of the VB binding protein was much accelerated. Although
the inactivation during the VB binding assay was reduced by the
addition of rVbrA and VB binding activity became detectable, no VB
binding activity was observed even for the first Phenyl-5PW fraction
without the addition of rVbrA. The instability of the VB binding
protein is evident from the activity recovery after Sephacryl S-100
chromatography (), during which about 50% of activity was
lost on each step of purification.
Molecular Size of VB Binding Protein
The molecular
size of the VB binding protein under native conditions was determined
by gel filtration. The VB binding activity was detected at a molecular
mass of 52 kDa. Purified recombinant VB binding protein expressed in
E. coli also showed a molecular mass of 52 kDa on molecular
sieve HPLC (column: TSK G2000SW, Tosoh Manufacturing Co.,
Ltd.). With molecular masses of 26 and 52 kDa under denatured and
native conditions, respectively, the VB binding protein was concluded
to exist as a homodimer at native state.
Cloning and Sequencing of the Gene Encoding
p26k
In order to clone the gene that encodes p26k, its partial
amino acid sequences were determined, and two probes (probe 26k-1 and
probe 26k-24) were synthesized as described under ``Materials and
Methods.'' Southern hybridization analysis of genomic DNA of
S. virginiae MAFF 10-06014 using probe 26k-24 showed
clear signals, and this probe hybridized to a 2.8-kbp BamHI
fragment (data not shown). A genomic library of S. virginiae constructed from size-fractionated BamHI fragment
(2.7-3.5 kbp) with pUC18 was screened by colony hybridization
using probe 26k-24. From 700 colonies, seven positive clones were
identified. Inserts from all seven clones were identical (a 2.8-kbp
BamHI fragment) and hybridized both to probe 26k-24 and to
probe 26k-1 (data not shown).
26,000). It is noteworthy that p26k
is rich in Ala (38 Ala/232 amino acids = 16.4%), particularly in
the C-terminal region (11 Ala/20 amino acids). Although SWISSPROT
(release number 27) and Northern Biomedical Research Foundation
(release number 38) data bases were searched, no protein with
significant homology was detected.
Heterologous Expression of the p26k Gene
To verify
that the p26k gene encodes the VB binding protein, we expressed this
gene in heterologous hosts (S. lividans and E. coli).
For expression in S. lividans, the 2.8-kbp BamHI
fragment was subcloned into pIJ486, a multicopy vector for
Streptomyces. The resulting plasmid (pAR S701) was introduced
into S. lividans TK21. As expected, the cell extract from
S. lividans TK21 harboring pAR S701 showed high VB binding
activity (). Such high activity was not detected in the
cell extract from S. lividans TK21 harboring pIJ486 or S.
lividans TK21 without any plasmid. Compared with S.
virginiae, S. lividans TK21 (pAR S701) had 4-fold higher
specific activity, probably due to the presence of multiple copies of
the p26k gene. Scatchard analysis indicated that the VB binding protein
expressed in S. lividans possessed a dissociation constant
(K) of 7.1 nM for
[
H]VB-C
, which is similar to that of
the native VB binding protein from S. virginiae (8.6
nM). These observations indicate that the 2.8-kbp
BamHI fragment contains a gene coding for the real VB binding
protein.
value (12.5 nM) was also
comparable with that of the native protein. SDS-PAGE analysis indicated
that IPTG-induced cells harboring pET-p26k significantly overproduced a
protein of 26 kDa (data not shown), the identity of which was confirmed
by its N-terminal amino acid sequence (AVRHERVAVR), which is identical
to that of p26k. Scatchard analysis of the purified recombinant p26k
protein (r-p26k) (Fig. 7A) revealed that it has a
K
value of 30-130 nM and a
B
value of 0.64-1.01 mol of
[
H]VB-C
/mol of r-p26k. The
significant fluctuation in these values could be attributable to the
highly unstable nature of r-p26k. Although the purified r-p26k is
stable at -80 °C and at high protein concentration, simple
dilution of the r-p26k in the absence of rVbrA (Fig. 7B,
0 h of incubation) resulted in a 60% loss of VB binding activity. The
presence of rVbrA was somehow effective in preventing rapid
inactivation, but VB binding activity was lost gradually during
incubation at 25 °C, the temperature at which Scatchard analysis
was performed. From these results, we concluded that the p26k gene
actually encodes the VB binding protein and denoted it as barA (butyrolactone autoregulator receptor).
Figure 7:
Scatchard analysis of purified recombinant
p26k and inactivation by dilution and incubation at 25 °C.
A, Scatchard analysis was done by equilibrium dialysis as
described previously (13). Each 29.2 pmol of purified r-p26k (0.73
µg) in 100 µl of buffer C containing 0.5 M KCl and
purified rVbrA (0.1 mg/ml) was incubated with varying amounts of
[H]VB-C
for 30 min at 25 °C in
the presence and the absence of 2000-fold excess cold VB-C
,
followed by dialysis against 1 ml of the same buffer for 4 h at 25
°C. Calculated K
values varied from
30 to 130 nM, and B
values varied from
0.64 to 1.01 for several experiments. B, VB binding activity
was measured by the ammonium sulfate precipitation method (24).
Purified r-p26k (0.365 mg/ml) was diluted to 1.6 µg/ml with
ice-cold buffer A (
) or buffer A containing rVbrA (0.1 mg/ml)
(
) and incubated at 25 °C. At the indicated time, sample (100
µl) was withdrawn and kept on ice until the VB binding assay. rVbrA
was added immediately after each sampling to the sample incubated in
the absence of rVbrA at a final concentration of 0.1 mg/ml to prevent
further inactivation.
Southern Blot Analysis of DNA from Several Streptomyces
Strains
The distribution of the sequence homologous to barA was investigated by Southern blot analysis for DNA samples of
Streptomyces coelicolor A3(2), S. griseus IFO 13350,
S. lividans TK21, and Streptomyces sp. FRI-5 with
P-labeled barA probe. To our surprise, no DNA
samples except that from S. virginiae showed a hybridization
signal, even under low stringent conditions (2
SSC at room
temperature). The strains used here are all known as producers of
certain butyrolactone autoregulators (i.e. S. coelicolor A3(2)
(25) , S. lividans TK21 (26), and S.
griseus IFO 13350
(10) produce A-factor or A-factor active
compounds, and Streptomyces sp. FRI-5
(4) produces
IM-2). Furthermore, the A-factor binding protein has been detected in
S. griseus IFO 13350
(14) , and IM-2 binding protein has
been detected in Streptomyces sp. FRI-5
(32) . When the
rplK probe coding for ribosomal protein L11
(15, 27) was used as a control, a clear signal was obtained with
every DNA sample, indicating that quality of the DNA samples was
acceptable. Taken together, in these strains, there is no DNA sequence
homologous to barA gene, suggesting that the corresponding
autoregulator receptors of different ligand specificity have no overall
resemblance in protein sequence with that of the VB binding protein.
Table:
Purification of the VB binding protein from S.
virginiae MAFF 10-06014
Table:
Functional expression of the p26k gene
in S. lividans TK21 and E. coli
/EMBL Data Bank
with accession number(s) D32251.
H]VB-C
,
2-(1`-hydroxy-[6`,7`-
H]heptyl-3-(hydroxymethyl)butanolide;
p-APMSF, (p-amidinophenyl)methanesulfonyl fluoride
hydrochloride; IPTG, isopropyl-
-D-thiogalactoside.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.