a20-kilodalton protein preserves cell viability promotes
TRANSCRIPT
Vol. 175, No. 16JOURNAL OF BACTERIOLOGY, Aug. 1993, p. 5276-5280
0021-9193/93/165276-05$02.00/0 Copyright © 1993, American Society
for Microbiology
A 20-Kilodalton Protein Preserves Cell Viability and Promotes CytA Crystal Formation during Sporulation in
Bacillus thuringiensis DONG WU1 AND BRIAN A. FEDERICI' 2*
Department ofEntomology and Interdepartmental Graduate Program in Genetics,2 University of California, Riverside, California 92521
Received 1 February 1993/Accepted 10 June 1993
The effect of a 20-kDa protein on cell viability and CytA crystal production in its natural host, Bacils thuringiensis, was studied by expressing the cyt4 gene in the absence or presence of this protein. In the absence of the 20-kDa protein, B. thuringiensis cells either were killed during sporulation (strain cryB) or produced very small CytA crystals (strain 4Q7). Expression of cyt4 in the presence of the 20-kDa protein, however, preserved cell viability, especially in strain cryB, and in both strains yielded bipyramidal crystals of the CytA protein that were larger than those of wild-type B. thuringiensis. These results suggest that the 20-kDa protein promotes crystal formation, perhaps by chaperoning CytA molecules during synthesis and crystallization, concomitantly preventing the CytA protein from interacting lethally with the bacterial host cell.
The CytA protein of Bacillus thuringiensis is a hydropho- bic, cytolytic 27.3-kDa protein toxic for certain dipterous insects in vivo and for many vertebrate and invertebrate cells in vitro (10, 18, 19, 22). The cytolytic properties of this protein are attributed to its affinity for unsaturated fatty acids in cell membranes, in which it apparently aggregates, leading to the formation of pores that cause cell lysis (19). CytA was first identified in the mosquitocidal subspecies B. thuringiensis subsp. israelensis (20) but also occurs in the PG-14 isolate of B. thuringiensis subsp. morrisoni (11). In both, CytA occurs along with at least three other mosquito- cidal proteins, CryIVA (128 kDa), CryIVB (134 kDa), and CryIVD (72 kDa). The genes that code for these proteins are on the same plasmid, and all are highly expressed during sporulation, resulting in the formation of distinct paracrys- talline inclusions that are assembled into a large spherical parasporal body about 1 pm in diameter (12). The toxicity of each of these proteins is from 10- to 100-fold less than that of the parasporal body, and to explain this it has been proposed that the proteins potentiate each other, with the CytA protein playing a particularly important role in this potenti- ation owing to its hydrophobic properties and because it is the dominant protein in the parasporal body (27). The specific role that the CytA protein plays in toxicity remains controversial, however, because deletion of this protein from the parasporal body results in little change in its toxicity (5). One way to test the role of (ytA in toxicity would be to
overexpress this protein and then determine its toxicity alone and in various combinations with CryIV proteins. However, several recent studies have shown that efficient production of CytA in Escherichia coli requires the presence of a 20-kDa protein that is coded for by a gene located immediately downstream from the cryIVD gene (1, 7, 15, 21). Expression of this gene during sporulation is apparently driven by the cryIVD promoter, with both cryIVD and the 20-kDa gene being expressed as a single transcriptional unit (1). In fact, expression of the cytA gene in E. coli in the
* Corresponding author.
absence of the 20-kDa protein is typically lethal (7). More- over, although incorporation of the 20-kDa open reading frame (ORF) in cytA and cryIVD constructs expressed in E. cofl resulted in detectable levels of CytA and CryIVD proteins, the levels of synthesis were very low, especially in comparison with those obtained in wild-type B. thuringien- sis, and no detectable inclusions were formed (1). In general, similar results were obtained with bacilli when the cytA gene was expressed in the absence of the 20-kDa protein. In B. megaterium (6), no inclusions were observed, and in B. subtilis, only very small inclusions were obtained (23). Larger inclusions were reported to be obtained when a construct containing the cyt4 gene and the 20-kDa ORF, the latter without a promoter, was expressed in B. thuringiensis, but no data on the level of CytA production or inclusion size or shape were provided (3). Enhancement of CytA production by the 20-kDa protein in
E. coil (1, 15, 21) suggested that this protein functions similarly in the subspecies of B. thuringiensis in which it occurs naturally and that this could be tested by placing the 20-kDa protein gene under the control of a strong promoter. Our objective was to manipulate the expression of the 20-kDa protein and thereby contribute to our knowledge of this protein's function and its effect on CytA crystal forma- tion. Thus, in the present study, we expressed the cytA gene with and without the 20-kDa protein gene in the constructs and to test its effect on cell viability and crystal production, ensured expression of this gene by placing it under control of the cryL4(c) promoters.
Plasmid construction and transformation of B. thuringien- sis. To ensure that the 20-kDa protein gene was expressed and to determine the effect of the 20-kDa protein on produc- tion of the CytA protein in B. thuingiensis, the following plasmids were constructed: pWF27, containing the cytA gene alone; pWF38, containing the cytA gene and most of the cryIVD gene; pWF32, containing the cytA gene and the 20-kDa protein gene along with the region upstream, up to and including the 350 codons that code for the C terminus of CryIVD (Fig. 1A). In addition, because the 20-kDa protein gene in WF32 would lack its putative promoter because of the deletion of the cryIVD promoter, we constructed WF45,
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20KD
20KD P FIG. 1. Schematic illustration of the (
the CytA protein in the presence or absl The constructs were made in pBluesci giensis-E. coli shuttle vector pHT3101, expressed individually in acrystalliferou: as described in the text. (A) Physical derivative constructs of cytA, cryWVD, from this 9.4-kb HindIII fragment origii ringiensis subsp. momsoni (9). (B) Fi construct made by placing the 20-kDa cry4(c) promoters, which was used promoter region of the cryL(c) gene c promoters (24).
in which the cryA(c) promoters (e used to drive the expression of tl ensuring synthesis of the 20-kDa pr in B. thuringiensis (Fig. 1B). To construct WF27 and WF38, ti
fragment (containing the cytA gene fragment (containing the cytA gene gene) were isolated from a 9.4-kb I
of plasmid pMl (9) and inserted into San Diego, Calif.). In pWF38-Blues oriented toward the SmaI and SacI cryIVD gene was oriented toward ti
1 kb construct WF32, the 2.1-kb ClaI-PvuII fragment of pM1 was inserted into pWF27-Bluescript at the ClaI-EcoRV sites. To
EcoRI Hindil add the cryLA(c) promoters to the 20-kDa ORF, the 1.1-kb NsiI-KpnI fragment (NsiI in codon 54 of the C terminus of
UIIIIIIHII | CryIVD and the KpnI site in the polylinker of pBluescript) cytA from pWF32-Bluescript was inserted into HBmpl8, an M13-
crylA(c) clone (25) containing the promoter region and 647 Sad codons of the crylA(c) gene to yield WF40 (Fig. 1B). smal Because the NsiI site in HBmpl8 is located at codon 8 ofE|RI cryL4(c) and the KpnI site is in the polylinker, this insertion
replaced most of the coding region of the crylA(c) gene, cytA concomitantly placing the 20-kDa gene under control of the
Sad crylA(c) promoters. To construct WF45, the HincII-Sall Smal fragment from WF40 (Fig. 1B) was inserted into pWF27-EcoI Bluescript at the EcoRV-SalI sites. All of the above-de-
I,,,,,,,uI |scribed constructs made in pBluescript were isolated from E. cytA coli JM101 and recloned into the Sacl and SailI sites of
Sad pHT3101, an E. coli-B. thuingiensis shuttle vector (14), to ufl/EcoRV sian yield the corresponding derivative plasmids pWF27, pWF32,
EcoRI pWF38, and pWF45 (Fig. 1). Effect of 20-kDa protein on cell viability. The above-
described plasmids were introduced by electroporation (26)CytA into the acrystalliferous strains of B. thuingiensis, cryB (17) and 4Q7 (a derivative of 4Q2, from D. H. Dean, Department
Sad of Microbiology, Ohio State University, Columbus). The AVEcoRV Ee~l . transformed cells were then plated on G-Tris medium (26) or
nutrient agar (Difco) containing 25 ~tg of erythromycin per ml and grown at 300C (16).
cytA Cells of both cryB and 4Q7 transformed with the above- described plasmids grew normally during the vegetative stage. When grown on G-Tris medium, however, cryB cells transformed with constructs that contained the cytA gene but lacked the 20-kDa protein gene (pWF27 and 38), or the construct that lacked the ability to express the 20-kDa protein gene (pWF32), died when they began to sporulate. No spores or CytA inclusions were observed in these cells by phase microscopy 2 days after sporulation began, but the CytA protein was detected in cells by dissolving the cells in
(HBmp18) USDP buffer (8 M urea, 1% sodium dodecyl sulfate [SDS], constructs made to produce 50 mM dithiothreitol, 2 mM phenylmethylsulfonyl fluoride, ence of the 20-kDa protein. 50mM Tris-HCl [pH 6.8] [25]) and separating the proteins by ript, cloned into B. thurin- SDS-polyacrylamide gel electrophoresis (PAGE) (13). More- and then transformed and over, no living cells were detected when these cultures were
s strains of B. thuringiensis plated on nutrient agar (containing 25 pug of erythromycin per maps of pMl and various ml) 4 days after sporulation was initiated, even though very and the 20-kDa ORF made few of the cells were able to complete sporulation. To nally obtained from B. thu- quantify the mortality rate of these cells, the cultures were ORE underpcontrol of the suspended in buffer (50 mM NaCl, 5 mM Tris-HCl [pH 7.5]) to construct pWF45. pt and plated on G-Tris medium (26) or nutrient agar (Difco) ontaining the BtI and BtII containing 25 pg of erythromycin per ml in 6-cm-diameter
petri dishes at 105 cells per dish and grown at 30'C. On the basis of counts of viable colonies made 24 h after plating, no CryB cells that expressed the CytA protein in the absence of the 20-kDa protein survived when grown on G-Tris, and
arly and late [24]) were survival on nutrient agar was less than 0.1% (Table 1). ie 20-kDa protein gene, Similar results were obtained with pWF32 and pWF38 in rotein during sporulation cryB cells.
The cell mortality caused by cytA gene expression differed he 2.8-kb EcoRV-EcoRI in 4Q7 cells. When these were transformed with plasmids .) and the 5.3-kb EcoRI incapable of expressing the 20-kDa protein gene (pWF27, and most of the cryIVD pWF38, and pWF32), they grew and sporulated normally on findIII fragment (Fig. 1) plates of either G-Tris medium or nutrient agar. When plated pBluescript (Stratagene, at 102 cells per dish, a density that facilitated counting of ,cript, the cytA gene was viable colonies, a much lower percentage of these cells in sites whereas the partial comparison with cryB cells succumbed to CytA production he SalI site (Fig. 1A). To (Table 1).
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TABLE 1. Survival of B. thuringiensis cryB and 4Q7 in the presence or absence of the 20-kDa protein f ng expression of the cyt4 gene
Plasmid Gene(s) Host cell Medium No. of cells plated' Mean no. of surviving % Survivalcells ±t SEMb
pWF27 cytA cryB G-Tris 105 0 <0.001 Nutrient agar 105 545 ± 45 0.05-0.1
pWF27 cytA 4Q7 G-Tris 8 x 102 679 ± 40 85 Nutrient agar 8 x 102 758 ± 43 94
pWF45 cyt4, cryL(c), 20-kDa ORF' cryB G-Tris 8 x 102 809 ± 37 100
a The approximate number of cells plated was estimated by counting cells in a Fisher Scientific hemocytometer. Much higher numbers of cryB cells containing pWF27 were plated because preliminary tests indicated a high mortality rate.
b There were four replicates per experiment. c Expression was under control of the cqyL4(c) promoters (24).
In contrast to the above-described results, cells of cryB and 4Q7 transformed with pWF45, which contained the cytA gene and the 20-kDa protein gene under control of the cryL4(c) promoters, grew and sporulated very well. Cell viability, colony size, and sporulation were comparable to those of typical wild-type strains of B. thuringiensis subsp. israelensis (Table 1), and the CytA protein was synthesized in very large amounts (Fig. 2 and 3).
Effect of 20-kDa protein on CytA crystal formation. Pro- duction of CytA protein in the absence of the 20-kDa protein _ (cells transformed with pWF27, WF32, and WF38) was detected by SDS-PAGE (13), but no inclusions (strain cryB) or very small, irregular inclusions (strain 4Q7), less than 200 nm in diameter, were observed by phase microscopy. In marked contrast to this, the cells of either cryB or 4Q7 transformed with pWF45, expressing both the cytA gene and 20-kDa ORF, produced very large crystalline inclusions of the CytA protein when the cells sporulated (Fig. 2). Inclu- sions were apparent by 36 h when the cells were grown at 30'C. Initially the inclusions were amorphous in shape, but as they increased in size they became ovoid (Fig. 2a). The mature inclusions released upon cell lysis were typically polyhedral and usually had a bipyramidal shape (Fig. 2b), averaging 1.3 pnm (long axis) by 0.7 pum (short axis).
Purification and characterization of CytA protein. The bipyramidal shape of the inclusion produced by CytA in transformed cells was not typical of that observed in wild- type parasporal bodies of B. thuringiensis (11, 12). To dem- onstrate that these inclusions were composed of CytA pro- tein, the inclusions were purified on sodium bromide gradients and analyzed by SDS-PAGE and Western blotting (immunoblotting) (2, 13). These experiments showed that the inclusions consisted of a single protein which migrated slightly faster than the CytA protoxin from the inclusions of wild-type B. thuringiensis subsp. israelensis (Fig. 3). When digested with trypsin, the CytA protein yielded a protein of 25 kDa, the same size as the trypsin-cleaved protein pro- duced by the wild-type cells (Fig. 3). When the solubilized and trypsin-treated CytA protein
was added to cultures of Spodoptera frugiperda cells, the cytoplasm of the cells became granular within 30 mm at a concentration of 1 pLg/ml. Most cells had lysed within 1 h FIG. 2. Morphology and size of C7ytA inclusions produced in after the CytA protein had been added to the culture medium B. thurngiensis subsp. israelensis 407 cells transformed with (Fig. 4). Similar results were obtained with the toxins of the pWF45. (a) Light micrograph of sporulating cells illustrating largeovoid inclusions of CytA protein (arrows) adjacent to the cell spore.wild-type mixture, but the protein concentration for lysis of Magnification, x3,800. (b and c) Scanning (b) and transmission (c) 90% of the cells in 1 h was three- to fivefold higher. electron micrographs of purified CytA inclusions illustrating their
Implications for the mechanism of action of the 20-kDa polyhedral shape. Magnification, x12,300 and x32,700, respec- protein. The high level of CytA production and formation of tively.
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FIG. 3. SDS-PAGE analysis of CytA inclusions produced in B. thuringiensis subsp. israelensis 4Q7 transformed with pWF45. (a) Coomassie blue-stained gel. Lanes: 1, molecular mass standards; 2, intact wild-type parasporal bodies from B. thuringiensis subsp. israelensis; 3, purified CytA inclusions; 4, wild-type parasporal bodies from B. thuringiensis subsp. israelensis treated with trypsin for 1 h; 5, CytA inclusions treated with trypsin for 4 h. (b) Western blot of untransformed 4Q7 cells (lane 1) and 4Q7 cells transformed with pWF45 (lane 2). In panel a, all lanes contained 5 pg of protein, except lane 2, which contained 10 ,ug. In panel b, both lanes contained 10 pg of protein.
large CytA inclusions obtained in B. thuingiensis cells transformed with pWF45 are apparently due to high-level expression of the 20-kDa protein gene when driven by the cryl(c) promoters. Visick and Whiteley (21) provided evi- dence that in E. coli, the 20-kDa protein protected newly synthesized proteins from proteolysis and furthermore showed that this protein binds to the CytA protein. On the
FIG. 4. Cytolytic activity of the CytA protein derived from inclusions produced in B. thuringiensis subsp. israelensis 4Q7 cells transformed with pWF45. Cells from an established S. frugiperda cell line (Sf21) were treated with solubilized, trypsin-treated CytA protein at a concentration of 1 pg/ml and photographed after 1 h. Control cells were treated with buffer. Panels: a, control cells; b, cells treated with CytA.
basis of their results, they postulated that the 20-kDa protein might be involved in crystal assembly. In this report, we have presented direct evidence that the 20-kDa protein significantly promotes CytA crystal formation, leading to very large crystals in B. thuringiensis. In general, proteins can assemble by themselves, but in most cases, especially in vivo, assembly is aided by specific molecular chaperones (8). Thus, our results and those of Visick and Whiteley (21) suggest that the 20-kDa protein acts as a chaperone protein. A precedent exists for this in B. thuringiensis in that Crick- more and Ellar (4) have suggested that ORF2 in the CryIIA operon might be a chaperonin. The diminished crystal production and poor survival of E.
coli (7) and B. thunrngiensis cryB cells obtained when the CytA protein is synthesized demonstrate that this protein inhibits cell growth and viability and can be lethal in the absence of the 20-kDa protein or other proteins with a similar function. The lethal mechanism is not known but may be attributable to the hydrophobic activity of CytA and thus its capacity to interact with the inner membrane of the bacterial cell (7). In any case, if the 20-kDa protein does act as a chaperone-like protein, by binding to the CytA protein during or upon synthesis, it could protect CytA from pro- tease attack, thereby promoting crystal formation, concom- itantly protecting the cell plasma membrane from the lytic activity. The reason for the lower sensitivity of the 4Q7 strain to CytA in comparison with the cryB strain is not known. It may be that 4Q7, which is derived from B. thu- ringiensis subsp. israelensis, a wild-type host for the CytA protein, synthesizes other proteins with a function similar to that of the 20-kDa protein. CytA inclusions have been observed in transformed cells
of B. subtilis (24), B. megaterium (6), and B. thuningiensis (3), but these were typically very small, e.g., much smaller than spores, and irregular in shape. Thus, the CytA inclu- sions produced in cryB and 4Q7 cells with the cryLA(c) promoters to drive the expression of the 20-kDa protein gene are interesting from the standpoints of both their size and their shape. In wild-type B. thuringiensis cells, such as those of B. thuringiensis subsp. israelensis, the CytA inclusion occurs in a spherical parasporal body, delimited by a fibrous envelope, along with inclusions of the CryIVA, CryIVB, and CryIVD proteins. In this parasporal body, the inclusions containing the CytA protein tend to be round with some flat surfaces; this shape is apparently the result of the presence of the envelope and other inclusions in the parasporal body (11, 12). Expression of the CytA protein in large quantities independently of other parasporal-body proteins and the parasporal envelope, as in the present study, made it possi- ble to determine what is probably the actual morphology of the CytA crystal. The large size of the CytA crystals also indicates that this type of construct may be useful in pro- ducing large quantities of 8-endotoxins and other proteins in B. thunrngiensis.
We thank Jeffrey J. Johnson for excellent technical assistance during the course of this study and S. S. Gill for antisera to CytA.
This research was supported in part by grants from the University of California Mosquito Research Program, the University of Cali- fornia Biotechnology Research and Education Program, and USDA competitive grant 92-37302-7603 to B.A.F.
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20-kilodalton protein is required for efficient production of the Bacillus thuringiensis subsp. israelensis 27-kilodalton crystal protein in Escherichia coli. J. Bacteriol. 171:521-530.
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2. Chang, C., S. M. Dai, R. Frutos, B. A. Federici, and S. S. Gill. 1992. Properties of a 72-kilodalton mosquitocidal protein from Bacillus thuingiensis subsp. mormsoni PG-14 expressed in B. thuringiensis subsp. kurstaki by using the shuttle vector pHT3101. Appl. Environ. Microbiol. 59:507-512.
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12. Ibarra, J. E., and B. A. Federici. 1986. Isolation of a relatively nontoxic 65-kilodalton protein inclusion from the parasporal body of Bacillus thuringiensis subsp. israelensis. J. Bacteriol. 165:527-533.
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A 20-Kilodalton Protein Preserves Cell Viability and Promotes CytA Crystal Formation during Sporulation in
Bacillus thuringiensis DONG WU1 AND BRIAN A. FEDERICI' 2*
Department ofEntomology and Interdepartmental Graduate Program in Genetics,2 University of California, Riverside, California 92521
Received 1 February 1993/Accepted 10 June 1993
The effect of a 20-kDa protein on cell viability and CytA crystal production in its natural host, Bacils thuringiensis, was studied by expressing the cyt4 gene in the absence or presence of this protein. In the absence of the 20-kDa protein, B. thuringiensis cells either were killed during sporulation (strain cryB) or produced very small CytA crystals (strain 4Q7). Expression of cyt4 in the presence of the 20-kDa protein, however, preserved cell viability, especially in strain cryB, and in both strains yielded bipyramidal crystals of the CytA protein that were larger than those of wild-type B. thuringiensis. These results suggest that the 20-kDa protein promotes crystal formation, perhaps by chaperoning CytA molecules during synthesis and crystallization, concomitantly preventing the CytA protein from interacting lethally with the bacterial host cell.
The CytA protein of Bacillus thuringiensis is a hydropho- bic, cytolytic 27.3-kDa protein toxic for certain dipterous insects in vivo and for many vertebrate and invertebrate cells in vitro (10, 18, 19, 22). The cytolytic properties of this protein are attributed to its affinity for unsaturated fatty acids in cell membranes, in which it apparently aggregates, leading to the formation of pores that cause cell lysis (19). CytA was first identified in the mosquitocidal subspecies B. thuringiensis subsp. israelensis (20) but also occurs in the PG-14 isolate of B. thuringiensis subsp. morrisoni (11). In both, CytA occurs along with at least three other mosquito- cidal proteins, CryIVA (128 kDa), CryIVB (134 kDa), and CryIVD (72 kDa). The genes that code for these proteins are on the same plasmid, and all are highly expressed during sporulation, resulting in the formation of distinct paracrys- talline inclusions that are assembled into a large spherical parasporal body about 1 pm in diameter (12). The toxicity of each of these proteins is from 10- to 100-fold less than that of the parasporal body, and to explain this it has been proposed that the proteins potentiate each other, with the CytA protein playing a particularly important role in this potenti- ation owing to its hydrophobic properties and because it is the dominant protein in the parasporal body (27). The specific role that the CytA protein plays in toxicity remains controversial, however, because deletion of this protein from the parasporal body results in little change in its toxicity (5). One way to test the role of (ytA in toxicity would be to
overexpress this protein and then determine its toxicity alone and in various combinations with CryIV proteins. However, several recent studies have shown that efficient production of CytA in Escherichia coli requires the presence of a 20-kDa protein that is coded for by a gene located immediately downstream from the cryIVD gene (1, 7, 15, 21). Expression of this gene during sporulation is apparently driven by the cryIVD promoter, with both cryIVD and the 20-kDa gene being expressed as a single transcriptional unit (1). In fact, expression of the cytA gene in E. coli in the
* Corresponding author.
absence of the 20-kDa protein is typically lethal (7). More- over, although incorporation of the 20-kDa open reading frame (ORF) in cytA and cryIVD constructs expressed in E. cofl resulted in detectable levels of CytA and CryIVD proteins, the levels of synthesis were very low, especially in comparison with those obtained in wild-type B. thuringien- sis, and no detectable inclusions were formed (1). In general, similar results were obtained with bacilli when the cytA gene was expressed in the absence of the 20-kDa protein. In B. megaterium (6), no inclusions were observed, and in B. subtilis, only very small inclusions were obtained (23). Larger inclusions were reported to be obtained when a construct containing the cyt4 gene and the 20-kDa ORF, the latter without a promoter, was expressed in B. thuringiensis, but no data on the level of CytA production or inclusion size or shape were provided (3). Enhancement of CytA production by the 20-kDa protein in
E. coil (1, 15, 21) suggested that this protein functions similarly in the subspecies of B. thuringiensis in which it occurs naturally and that this could be tested by placing the 20-kDa protein gene under the control of a strong promoter. Our objective was to manipulate the expression of the 20-kDa protein and thereby contribute to our knowledge of this protein's function and its effect on CytA crystal forma- tion. Thus, in the present study, we expressed the cytA gene with and without the 20-kDa protein gene in the constructs and to test its effect on cell viability and crystal production, ensured expression of this gene by placing it under control of the cryL4(c) promoters.
Plasmid construction and transformation of B. thuringien- sis. To ensure that the 20-kDa protein gene was expressed and to determine the effect of the 20-kDa protein on produc- tion of the CytA protein in B. thuingiensis, the following plasmids were constructed: pWF27, containing the cytA gene alone; pWF38, containing the cytA gene and most of the cryIVD gene; pWF32, containing the cytA gene and the 20-kDa protein gene along with the region upstream, up to and including the 350 codons that code for the C terminus of CryIVD (Fig. 1A). In addition, because the 20-kDa protein gene in WF32 would lack its putative promoter because of the deletion of the cryIVD promoter, we constructed WF45,
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20KD P FIG. 1. Schematic illustration of the (
the CytA protein in the presence or absl The constructs were made in pBluesci giensis-E. coli shuttle vector pHT3101, expressed individually in acrystalliferou: as described in the text. (A) Physical derivative constructs of cytA, cryWVD, from this 9.4-kb HindIII fragment origii ringiensis subsp. momsoni (9). (B) Fi construct made by placing the 20-kDa cry4(c) promoters, which was used promoter region of the cryL(c) gene c promoters (24).
in which the cryA(c) promoters (e used to drive the expression of tl ensuring synthesis of the 20-kDa pr in B. thuringiensis (Fig. 1B). To construct WF27 and WF38, ti
fragment (containing the cytA gene fragment (containing the cytA gene gene) were isolated from a 9.4-kb I
of plasmid pMl (9) and inserted into San Diego, Calif.). In pWF38-Blues oriented toward the SmaI and SacI cryIVD gene was oriented toward ti
1 kb construct WF32, the 2.1-kb ClaI-PvuII fragment of pM1 was inserted into pWF27-Bluescript at the ClaI-EcoRV sites. To
EcoRI Hindil add the cryLA(c) promoters to the 20-kDa ORF, the 1.1-kb NsiI-KpnI fragment (NsiI in codon 54 of the C terminus of
UIIIIIIHII | CryIVD and the KpnI site in the polylinker of pBluescript) cytA from pWF32-Bluescript was inserted into HBmpl8, an M13-
crylA(c) clone (25) containing the promoter region and 647 Sad codons of the crylA(c) gene to yield WF40 (Fig. 1B). smal Because the NsiI site in HBmpl8 is located at codon 8 ofE|RI cryL4(c) and the KpnI site is in the polylinker, this insertion
replaced most of the coding region of the crylA(c) gene, cytA concomitantly placing the 20-kDa gene under control of the
Sad crylA(c) promoters. To construct WF45, the HincII-Sall Smal fragment from WF40 (Fig. 1B) was inserted into pWF27-EcoI Bluescript at the EcoRV-SalI sites. All of the above-de-
I,,,,,,,uI |scribed constructs made in pBluescript were isolated from E. cytA coli JM101 and recloned into the Sacl and SailI sites of
Sad pHT3101, an E. coli-B. thuingiensis shuttle vector (14), to ufl/EcoRV sian yield the corresponding derivative plasmids pWF27, pWF32,
EcoRI pWF38, and pWF45 (Fig. 1). Effect of 20-kDa protein on cell viability. The above-
described plasmids were introduced by electroporation (26)CytA into the acrystalliferous strains of B. thuingiensis, cryB (17) and 4Q7 (a derivative of 4Q2, from D. H. Dean, Department
Sad of Microbiology, Ohio State University, Columbus). The AVEcoRV Ee~l . transformed cells were then plated on G-Tris medium (26) or
nutrient agar (Difco) containing 25 ~tg of erythromycin per ml and grown at 300C (16).
cytA Cells of both cryB and 4Q7 transformed with the above- described plasmids grew normally during the vegetative stage. When grown on G-Tris medium, however, cryB cells transformed with constructs that contained the cytA gene but lacked the 20-kDa protein gene (pWF27 and 38), or the construct that lacked the ability to express the 20-kDa protein gene (pWF32), died when they began to sporulate. No spores or CytA inclusions were observed in these cells by phase microscopy 2 days after sporulation began, but the CytA protein was detected in cells by dissolving the cells in
(HBmp18) USDP buffer (8 M urea, 1% sodium dodecyl sulfate [SDS], constructs made to produce 50 mM dithiothreitol, 2 mM phenylmethylsulfonyl fluoride, ence of the 20-kDa protein. 50mM Tris-HCl [pH 6.8] [25]) and separating the proteins by ript, cloned into B. thurin- SDS-polyacrylamide gel electrophoresis (PAGE) (13). More- and then transformed and over, no living cells were detected when these cultures were
s strains of B. thuringiensis plated on nutrient agar (containing 25 pug of erythromycin per maps of pMl and various ml) 4 days after sporulation was initiated, even though very and the 20-kDa ORF made few of the cells were able to complete sporulation. To nally obtained from B. thu- quantify the mortality rate of these cells, the cultures were ORE underpcontrol of the suspended in buffer (50 mM NaCl, 5 mM Tris-HCl [pH 7.5]) to construct pWF45. pt and plated on G-Tris medium (26) or nutrient agar (Difco) ontaining the BtI and BtII containing 25 pg of erythromycin per ml in 6-cm-diameter
petri dishes at 105 cells per dish and grown at 30'C. On the basis of counts of viable colonies made 24 h after plating, no CryB cells that expressed the CytA protein in the absence of the 20-kDa protein survived when grown on G-Tris, and
arly and late [24]) were survival on nutrient agar was less than 0.1% (Table 1). ie 20-kDa protein gene, Similar results were obtained with pWF32 and pWF38 in rotein during sporulation cryB cells.
The cell mortality caused by cytA gene expression differed he 2.8-kb EcoRV-EcoRI in 4Q7 cells. When these were transformed with plasmids .) and the 5.3-kb EcoRI incapable of expressing the 20-kDa protein gene (pWF27, and most of the cryIVD pWF38, and pWF32), they grew and sporulated normally on findIII fragment (Fig. 1) plates of either G-Tris medium or nutrient agar. When plated pBluescript (Stratagene, at 102 cells per dish, a density that facilitated counting of ,cript, the cytA gene was viable colonies, a much lower percentage of these cells in sites whereas the partial comparison with cryB cells succumbed to CytA production he SalI site (Fig. 1A). To (Table 1).
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TABLE 1. Survival of B. thuringiensis cryB and 4Q7 in the presence or absence of the 20-kDa protein f ng expression of the cyt4 gene
Plasmid Gene(s) Host cell Medium No. of cells plated' Mean no. of surviving % Survivalcells ±t SEMb
pWF27 cytA cryB G-Tris 105 0 <0.001 Nutrient agar 105 545 ± 45 0.05-0.1
pWF27 cytA 4Q7 G-Tris 8 x 102 679 ± 40 85 Nutrient agar 8 x 102 758 ± 43 94
pWF45 cyt4, cryL(c), 20-kDa ORF' cryB G-Tris 8 x 102 809 ± 37 100
a The approximate number of cells plated was estimated by counting cells in a Fisher Scientific hemocytometer. Much higher numbers of cryB cells containing pWF27 were plated because preliminary tests indicated a high mortality rate.
b There were four replicates per experiment. c Expression was under control of the cqyL4(c) promoters (24).
In contrast to the above-described results, cells of cryB and 4Q7 transformed with pWF45, which contained the cytA gene and the 20-kDa protein gene under control of the cryL4(c) promoters, grew and sporulated very well. Cell viability, colony size, and sporulation were comparable to those of typical wild-type strains of B. thuringiensis subsp. israelensis (Table 1), and the CytA protein was synthesized in very large amounts (Fig. 2 and 3).
Effect of 20-kDa protein on CytA crystal formation. Pro- duction of CytA protein in the absence of the 20-kDa protein _ (cells transformed with pWF27, WF32, and WF38) was detected by SDS-PAGE (13), but no inclusions (strain cryB) or very small, irregular inclusions (strain 4Q7), less than 200 nm in diameter, were observed by phase microscopy. In marked contrast to this, the cells of either cryB or 4Q7 transformed with pWF45, expressing both the cytA gene and 20-kDa ORF, produced very large crystalline inclusions of the CytA protein when the cells sporulated (Fig. 2). Inclu- sions were apparent by 36 h when the cells were grown at 30'C. Initially the inclusions were amorphous in shape, but as they increased in size they became ovoid (Fig. 2a). The mature inclusions released upon cell lysis were typically polyhedral and usually had a bipyramidal shape (Fig. 2b), averaging 1.3 pnm (long axis) by 0.7 pum (short axis).
Purification and characterization of CytA protein. The bipyramidal shape of the inclusion produced by CytA in transformed cells was not typical of that observed in wild- type parasporal bodies of B. thuringiensis (11, 12). To dem- onstrate that these inclusions were composed of CytA pro- tein, the inclusions were purified on sodium bromide gradients and analyzed by SDS-PAGE and Western blotting (immunoblotting) (2, 13). These experiments showed that the inclusions consisted of a single protein which migrated slightly faster than the CytA protoxin from the inclusions of wild-type B. thuringiensis subsp. israelensis (Fig. 3). When digested with trypsin, the CytA protein yielded a protein of 25 kDa, the same size as the trypsin-cleaved protein pro- duced by the wild-type cells (Fig. 3). When the solubilized and trypsin-treated CytA protein
was added to cultures of Spodoptera frugiperda cells, the cytoplasm of the cells became granular within 30 mm at a concentration of 1 pLg/ml. Most cells had lysed within 1 h FIG. 2. Morphology and size of C7ytA inclusions produced in after the CytA protein had been added to the culture medium B. thurngiensis subsp. israelensis 407 cells transformed with (Fig. 4). Similar results were obtained with the toxins of the pWF45. (a) Light micrograph of sporulating cells illustrating largeovoid inclusions of CytA protein (arrows) adjacent to the cell spore.wild-type mixture, but the protein concentration for lysis of Magnification, x3,800. (b and c) Scanning (b) and transmission (c) 90% of the cells in 1 h was three- to fivefold higher. electron micrographs of purified CytA inclusions illustrating their
Implications for the mechanism of action of the 20-kDa polyhedral shape. Magnification, x12,300 and x32,700, respec- protein. The high level of CytA production and formation of tively.
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FIG. 3. SDS-PAGE analysis of CytA inclusions produced in B. thuringiensis subsp. israelensis 4Q7 transformed with pWF45. (a) Coomassie blue-stained gel. Lanes: 1, molecular mass standards; 2, intact wild-type parasporal bodies from B. thuringiensis subsp. israelensis; 3, purified CytA inclusions; 4, wild-type parasporal bodies from B. thuringiensis subsp. israelensis treated with trypsin for 1 h; 5, CytA inclusions treated with trypsin for 4 h. (b) Western blot of untransformed 4Q7 cells (lane 1) and 4Q7 cells transformed with pWF45 (lane 2). In panel a, all lanes contained 5 pg of protein, except lane 2, which contained 10 ,ug. In panel b, both lanes contained 10 pg of protein.
large CytA inclusions obtained in B. thuingiensis cells transformed with pWF45 are apparently due to high-level expression of the 20-kDa protein gene when driven by the cryl(c) promoters. Visick and Whiteley (21) provided evi- dence that in E. coli, the 20-kDa protein protected newly synthesized proteins from proteolysis and furthermore showed that this protein binds to the CytA protein. On the
FIG. 4. Cytolytic activity of the CytA protein derived from inclusions produced in B. thuringiensis subsp. israelensis 4Q7 cells transformed with pWF45. Cells from an established S. frugiperda cell line (Sf21) were treated with solubilized, trypsin-treated CytA protein at a concentration of 1 pg/ml and photographed after 1 h. Control cells were treated with buffer. Panels: a, control cells; b, cells treated with CytA.
basis of their results, they postulated that the 20-kDa protein might be involved in crystal assembly. In this report, we have presented direct evidence that the 20-kDa protein significantly promotes CytA crystal formation, leading to very large crystals in B. thuringiensis. In general, proteins can assemble by themselves, but in most cases, especially in vivo, assembly is aided by specific molecular chaperones (8). Thus, our results and those of Visick and Whiteley (21) suggest that the 20-kDa protein acts as a chaperone protein. A precedent exists for this in B. thuringiensis in that Crick- more and Ellar (4) have suggested that ORF2 in the CryIIA operon might be a chaperonin. The diminished crystal production and poor survival of E.
coli (7) and B. thunrngiensis cryB cells obtained when the CytA protein is synthesized demonstrate that this protein inhibits cell growth and viability and can be lethal in the absence of the 20-kDa protein or other proteins with a similar function. The lethal mechanism is not known but may be attributable to the hydrophobic activity of CytA and thus its capacity to interact with the inner membrane of the bacterial cell (7). In any case, if the 20-kDa protein does act as a chaperone-like protein, by binding to the CytA protein during or upon synthesis, it could protect CytA from pro- tease attack, thereby promoting crystal formation, concom- itantly protecting the cell plasma membrane from the lytic activity. The reason for the lower sensitivity of the 4Q7 strain to CytA in comparison with the cryB strain is not known. It may be that 4Q7, which is derived from B. thu- ringiensis subsp. israelensis, a wild-type host for the CytA protein, synthesizes other proteins with a function similar to that of the 20-kDa protein. CytA inclusions have been observed in transformed cells
of B. subtilis (24), B. megaterium (6), and B. thuningiensis (3), but these were typically very small, e.g., much smaller than spores, and irregular in shape. Thus, the CytA inclu- sions produced in cryB and 4Q7 cells with the cryLA(c) promoters to drive the expression of the 20-kDa protein gene are interesting from the standpoints of both their size and their shape. In wild-type B. thuringiensis cells, such as those of B. thuringiensis subsp. israelensis, the CytA inclusion occurs in a spherical parasporal body, delimited by a fibrous envelope, along with inclusions of the CryIVA, CryIVB, and CryIVD proteins. In this parasporal body, the inclusions containing the CytA protein tend to be round with some flat surfaces; this shape is apparently the result of the presence of the envelope and other inclusions in the parasporal body (11, 12). Expression of the CytA protein in large quantities independently of other parasporal-body proteins and the parasporal envelope, as in the present study, made it possi- ble to determine what is probably the actual morphology of the CytA crystal. The large size of the CytA crystals also indicates that this type of construct may be useful in pro- ducing large quantities of 8-endotoxins and other proteins in B. thunrngiensis.
We thank Jeffrey J. Johnson for excellent technical assistance during the course of this study and S. S. Gill for antisera to CytA.
This research was supported in part by grants from the University of California Mosquito Research Program, the University of Cali- fornia Biotechnology Research and Education Program, and USDA competitive grant 92-37302-7603 to B.A.F.
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