©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Virginiae Butanolide Binding Protein from Streptomyces virginiae
EVIDENCE THAT VbrA IS NOT THE VIRGINIAE BUTANOLIDE BINDING PROTEIN AND REIDENTIFICATION OF THE TRUE BINDING PROTEIN (*)

Susumu Okamoto (§) , Kenji Nakamura , Takuya Nihira , Yasuhiro Yamada (¶)

From the (1) Department of Biotechnology, Faculty of Engineering, Osaka University, 2-1 Yamada-oka, Suita-shi, Osaka 565, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

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)() 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.

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.


MATERIALS AND METHODS

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.

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.

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) CHCN, 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 CHCN 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 CHCN 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.

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.

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.

BL21 (DE3)/pLysS containing pET-p26k or pET-3d was grown until A 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.

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.

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).


RESULTS AND DISCUSSION

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'').

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).

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).

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 (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.

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 Kvalue (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 Kvalue 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 Kvalues 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.

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.

  
Table: Purification of the VB binding protein from S. virginiae MAFF 10-06014

Data starting from 100 g of mycelia, wet weight was summarized.


  
Table: Functional expression of the p26k gene in S. lividans TK21 and E. coli



FOOTNOTES

*
This work was supported in part by Grant BMP-94-V-4-1 from the Ministry of Agriculture, Forestry, and Fisheries, Japan.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) D32251.

§
Present address: National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries, Tsukuba 305, Japan.

To whom correspondence and reprint requests should be addressed: Dept. of Biotechnology, Osaka University, 2-1 Yamada-oka, Suita-shi, Osaka 565, Japan. Tel.: 81-06-879-7431; Fax: 81-06-879-7448.

The abbreviations used are: VB, virginiae butanolide; rVbrA, recombinant VbrA; r-p26k, recombinant p26k; HPLC, high performance liquid chromatography; kbp, kilobase pair(s); PAGE, polyacrylamide gel electrophoresis; [H]VB-C, 2-(1`-hydroxy-[6`,7`-H]heptyl-3-(hydroxymethyl)butanolide; p-APMSF, (p-amidinophenyl)methanesulfonyl fluoride hydrochloride; IPTG, isopropyl--D-thiogalactoside.


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