Evidence That the CryIA Crystal Protein from Bacillus thuringiensis Is ...

2 downloads 0 Views 4MB Size Report
Apr 15, 2016 - This paper is dedicated to the memory of Jean Louis Roustan, ...... Bulla, L. A., Jr., Kramer, K. J., and Davidson, L. I. (1977) J. Bacteriol. 20.
THEJOURNAL OF BIOLOGICAL CHEMISTRY

Vol. 268, No. 11, Issue of April 15, pp. 82404245,1993 Printed in U.S.A.

0 1993 hy The American Society for Biochemistry and Molecular Biology,, Inc

Evidence Thatthe CryIA Crystal Protein from Bacillus thuringiensis Is Associated with DNA* (Received for publication, December 18,1992)

Henri P. Bietlot, Johann P. Schernthaner, Ross E. MilneS, Franpois R. Clairmont, Resham S. Bhellaj, and Harvey KaplanT From the Department of Chemistry, University of Ottawa, Ottawa, Ontario K I N 6N5, Canada

Toxin generated by activation of the Bacillus thuringiensis CryIA(c) crystal protein (protoxin) with bovine trypsin was separated into two components by anionexchange chromatography. One component (T2) was DNA-associated toxin, and the other was the DNA-free toxin (Tl). Only one major protoxin component was observed, and it wasfound to be associated with DNA. The DNA from the T2 toxin varied in sizefrom 100 to 300 base pairs, whereas the crystal and the solubilized protoxin contained 20-kilobase DNA as the major DNA component. DNase treatment converted the T2 toxin to the DNA-free T1 toxin. In contrast, the DNA in the crystal and the solubilized protoxin was resistant to DNase digestion and was not dissociated from the protein by 1.5 M NaCl. The protoxin and DNA appeared to elute as a complex with a molecular mass of >2 X lo6Da ongel-filtration chromatography.No toxin was generated from the protoxin with trypsin after extensive digestion of the protoxin with DNase or dissociation of the DNA bysuccinylation of the lysine residues. It is proposed that DNA binds to the COOH-terminal half of the crystal protein and is essential for maintaining the conformational integrity required for crystal formation and generation of toxin.

Bacillus thuringiensis is characterized by the production of a proteinaceous crystalline inclusion body during sporulation. Many strains of B. thuringiensis have been identified that differ greatly in their insecticidal activities toward various host larvae (1-3). The genes encoding the crystal proteins from various strains have been classified by Hofte and Whiteley (4) on the basis of their structural similarities and their insect host spectra. Genes coding for lepidopteran-specific proteins have been designated as cry1 genes, and their protein products as CryI proteins. These proteins vary in molecular mass from 130 to 140 kDa (3,4); and, in the crystal, they are linked by a network of symmetrical interchain disulfide bonds

* This work was supported by a strategic grant from the Natural Sciences and Engineering Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. This paper is dedicated to the memory of Jean Louis Roustan, whose scientific and teaching excellence, honesty, and integrity will be missed. 4 Present address: Forest Pest Management Inst., Forestry Canada, Sault St. Marie, Ontario P6A 5M7, Canada. Present address: Plant Biotechnology Inst., National Research Council of Canada, Saskatoon, Saskatchewan S7N OW9,Canada. ll To whom correspondence should be addressed: Dept. of Chemistry, University of Ottawa, 140 Louis Pasteur, Ottawa, OntarioK1N 6N5,Canada. Tel.: 613-564-5872; Fax: 613-564-9562.

(5). On ingestion by susceptible lepidopteran larvae, the crystal protein is released by cleavage of the disulfide linkages under the alkaline conditions of the insect gut. The solubilized crystal protein is then acted on by larval gut proteinases and proteolyzed to a 60-70-kDa toxin (3, 6).This activation process appears to be required for all CryI crystal proteins; and therefore, they are protoxins. The CryI protoxins contain -1175 amino acids (4) and are composed of an NH2-terminal toxic moiety and a COOHterminal region that is removed during the activation process. In uitro, the COOH-terminal cleavage site is dependent on the length of enzymatic digestion and the specific enzyme used (7-9). The trypsin-generated toxin has been shown to be composed of three domains ( 7 ) and appears to be very similar in overall architecture tothe coleopteran-specific CryIIIA toxin for which the x-ray crystallographic structure has been reported (10). Other than proteolytic cleavage, no postsynthetic covalent modifications or cofactors have been found to be required for insecticidal activity. During the course of purification of B. thuringiensis insecticidal proteins by anion-exchange chromatography, B. thuringiensis protein eluting at unusually high salt concentration was observed. Further investigation showed that DNA was associated with the B. thuringiensis protein. In this study, evidence is presented that DNA has an important role in the structure and function of the B. thuringiensis crystal protein. EXPERIMENTALPROCEDURES

Crystal Preparation-B. thuringiensis subsp. kurstaki HD-73 was grown in half-strength Trypticase soy broth. The cells were lysed in double-distilled water, and the crystals were purified by Renografin (Squibb) gradients as previously described (11).Purified crystals were stored in distilled water at 4 “C. Preparation of CAM Protoxin and Toxin-Crystals were suspended in 10 mM Tris, pH 7.0,and treatedwith diisopropyl fluorophosphate (1 pl/mg of protein; Sigma) to inactivate serine proteinases. The crystals were solubilized in 0.1 M CAPS,’ pH 10.5,with 1% (v/v) 2mercaptoethanol. CAM protoxin was prepared as previously described (12).Toxin was generated from solubilized protoxin by a 24-h incubation with 5% (w/w) bovine trypsin as previously described (13). Shorter incubations were carried out for 3 hwith 0.1% (w/w) trypsin. Protein Quantification-Toxin and protoxin concentrations were estimated by absorbance at 280 nm (13).The protoxin and toxin solutions were also quantified using a Bio-Rad protein assay. Zon-exhange Chromatography of B. thuringiensis Toxin and CAM Protoxin-Toxin was purified on a Pharmacia LKB Biotechnology fast protein liquid chromatography system equipped with a Mono Q HR 10/10 anion exchanger. Elution was carried out atroom temperature with a 0-1 M NaCl gradient in 0.1 M CAPS, pH 10.5.The flow rate of the system was 0.2 ml/min, and theproteins were detected by The abbreviations used are: CAPS, 3-(cyclohexylamino)propanesulfonic acid; CAM, carbaminomethyl; SCAM, succinylcarbaminomethyl; HPLC, high pressure liquid chromatography; kb, kilobase(s).

8240

D N A Associated with B. thuringiensis Crystal Protein UV absorbance at 280 nm. CAM protoxin was purified by HPLC on a Bio-Rad MA7Q column (50 X 7.8 mm). Elution was also carried out atroom temperature with a 0-1 M NaCl gradient in 0.1 M CAPS, pH 10.5. The flow rate was 1.5ml/min, and theproteins were detected at 280 nm. All materials collected were thoroughly dialyzed against distilled water. Acetic acid was added to pH 5, and the precipitated protein was removed and stored in distilled water a t 4 "C. Extraction of DNA from Protoxin and T2 Toxin-Renografinpurified crystals were solubilized in CAPS at pH10.5 in the presence of 1%(v/v) 2-mercaptoethanol. The extraction of the DNA from the protoxin was achieved by the addition of an equal volume of phenol/ chloroform a t 65 "C previously equilibrated in 0.1 M CAPS at pH 10.5. The aqueous phase was then re-extracted with an equal volume of chloroform, and the DNA was precipitated by the addition of 0.3 M sodium acetate, pH 5.5, and 2 volumes of ethanol. The DNA was then run on a Tris/acetic acid/EDTA-agarose gel (O.S%, w/v). DNA was extracted from the T2toxin with phenol/chloroform in the same fashion as thecrystal protein,except that thephenol/chloroform was equilibrated inTris/EDTA a t pH 8.0 a t room temperature. The samples were then run on agarose gels as described above. Fluorometric DNA Detection-The nature of the nucleic acid associated with the crystal protein was determined using the DNAspecific dye bisbenzimidazole (Hoechst 33258) according to the procedure of Labarca and Paigen (14). Succinylation of CAM Protoxin-CAM protoxin (25 mg) was succinylated in the absence of denaturants as previously described to give SCAM protoxin (12). The SCAM protoxin was divided into two equal portions to demonstrate that SCAM protoxin renatured after treatment with 8 M urea. As a control, one portion was dialyzed extensively against water adjusted to pH 8.5 by the addition of NH3 (Fig 7A). The other portion (Fig. 7 B ) was made 8 M in urea at pH 4 and allowed to stirfor 30 min. The pH of the solution was then raised to 8.5, and the solution was left stirring for another 30 min before being dialyzed against distilled water/NH3 at pH8.5. A second 25-mg aliquot of CAM protoxin was dissolvedin 8 M urea at pH4. This solution was left stirring for 30 min before the pHwas raised to 9 by making the solution 1% in NaHC03. Thissample was then divided into two equal portions to demonstrate the effect of succinylating buried amino groups on the binding of DNA.One portion (Fig. 7C) was succinylated by the addition of succinic anhydride and then dialyzed against distilled water/NHs at pH 8.5. As a control, the other portion (Fig. 7 0 ) was dialyzed against distilled water/NH3 a t pH 8.5. A final dialysis of all four samples was carried out against distilled water, and the precipitated protein was removed by centrifugation. Equal amounts of the precipitated proteins were then digested with trypsin (O.OOl%,w/w) at pH10.5 as described previously (7). DNase Sensitivity of CAM Protoxin-Three samples of CAM protoxin (2 mg) were dissolved in 2 ml of buffer (40 mM Tris, pH9.5, 10 mM NaCI, 6 mM MgCl,, and 2 mM spermidine) a t 25 "C.The first sample (Fig. 8, A and B ) was used as a control and had no DNase added. To the second sample (Fig. 8, C and D ) was added 2% (w/w) DNase (Sigma), and the incubation was continued for up to 3 days. The third sample (Fig. 8, E and F) contained 10 mM EDTA, which was added prior to theaddition of the DNase and digested under the same conditions as described above. All samples were stirred for the duration of the experiment. At each time point, two aliquots were removed, one to determine the protoxin content and the other tobe digested with 0.1% (w/w) trypsin for 1 h. Polyacryhmide Gel Electrophoresis-SDS-polyacrylamide gels (1015% gradient) were run on a PharmaciaPhast electrophoresis system with preformed gels and other material supplied by Pharmacia. Gels were stained with Coomassie Blue. Cell Bioassays-The relative biological activity of the T1 and T2 toxins was determined using CF-1 insect cells in a lawn assay (15). Microscopy-B. thuringiensis subsp. kurstaki HD-1 was grown for 24 h at 30 "Cin half-strength Trypticasesoy broth (100 ml in 300-ml baffled flasks). Freshly grown culture was incubated for 30 min a t room temperature with ethidium bromide at 1 pg/ml in Tris/acetic acid/EDTA. Cells were observed with phase-contrast and fluorescence microscopy using a 100 X objective and oil immersion.

8241

tion, nucleic acid condenses in the region where the spore forms. After spore formation, the fluorescence from this region vanishes and appears in the region where the crystalline inclusion body is assembled (Fig. 1). The elution profile of trypsin-generated toxin on a Mono Q quaternary ammonium anionexchanger is shown in Fig. 2. Two major peaks elute, one at 0.3 M salt (Tl) and the other 0.9 M salt(T2).Therelativeproportions of T1 and T2 obtained depended on the source of the trypsin, the amount used, and the length of treatment. Overnight treatmentof the crystal protein withhigh concentrations (5%, w/w) of trypsin yielded mostly T1,whereas shorter treatments of a few hours yielded mostly T2. On SDS-polyacrylamide gels, both these peaks gave a protein band with an apparentmolecular mass of 67 kDa characteristic of the toxin and hadequal cytolytic activities toward CF-1cells (15).The difference spectra of T2 and T1 (Fig. 3) showed that T2 had an absorption 260 at nm A

FIG. 1. Phase-contrast and fluorescence microscopy of B. thuringiensis subsp. kurstaki HD-1. The culture shown consists of mature unlysed cells at stage VI1 (magnification X 5000) (20). A, phase contrast. The four light areasare mature spores, and the RESULTS adjacent dark areas are the regions of crystal formation. E , fluorescence observed after incubation of the cells with ethidium bromide. Cells from sporulating B. thuringiensis cultures incubated The cells were photographed under fluorescence conditions first to with ethidium bromide showed a shifting pattern of nucleic record maximum fluorescence and then under phase-contrast condiacid distribution within the bacterium. Just prior to sporula-tions in an effort to match the two views.

B. thuringiensis Crystal Protein

D N A Associated with

8242 1.0

100

/" / /

T1

/

/

W

u Z

a

E

5:m

//

T2

m

I

8

/

a5

/

-

'

/

50

/

&

-1

""

a

I ,

I

I

0.5

0

10

15

u)

TlME(h)

FIG.2. Fast protein liquid chromatography elution profile of CryIA(c) toxin. Toxin generated with bovine trypsin was run on a Pharmacia fast protein liquid chromatographysystemequipped with a Mono Q HR 10/10 anion exchanger. Elution was carried out a t room temperature with a 0-1 M NaCl gradient in 0.1 M CAPS, pH 10.5. The flow rate of the system was 0.2 ml/min, and the proteins were detected by UV absorbance a t 280 nm.

FIG.4. DNA associated with B. thuringiensisproteins. DNA was subjected to electrophoresis on a 0.8% (w/v) agarose gel in 40 mM Tris acetate, 1mM EDTA. The DNA was stained with ethidium bromide, and the gel was photographed. Lane 1, X phage Hind111 digest; lane 2, DNA isolated from B. thuringiensis crystal protein; lane 3, DNA isolated from T2 toxin; lane 4, 100-base pair ( b p ) DNA ladder. .20.

.5(1-

:

/

.

W

.16.

z a

I2.

0 v)

m

a

\ \

L '

.";

/

,

/

.08

/ /

/

. ..

..

m

\ I \

,: j

/

m

a

I

,

0

100

x- 7 1

I

/

.. ..

50

L .

\

.

8

7

.04 I

0

W

YU

m

a

0

v,

m

a

-2.5

10

20 TlME(min)

30

0

FIG.5. HPLC elution profileof CAMprotoxin. CAM protoxin was purified on a Bio-Rad MA7Q column (50 X 7.8 mm). Elution was also carried out a t room temperature with a 0-1 M NaCl gradient in 0.1 M CAPS, pH 10.5. The flow rate was 1.5 ml/min, and the protein was detected a t 260 nm (. . . .) and 280 nm (-).

greater at 260 nm than at280 nm, indicating thepresence of nucleic acid. Incubation of the CAM protoxin with DNase for