Correlation between specific plasmids and δ-endotoxin production in Bacillus thuringiensis

PLASMID

$351-365

Correlation

(1981)

between

Jo& M. GONZALEZ, *Department

Specific Plasmids and 6-Endotoxin in Bacillus thuringiensis JR.,* HOWARD

T. DULMAGE,~

Production

AND BRUCE C. CARLTON*

of Molecular and Population Genetics, University of Georgia, Athens, Georgia 30602, and Wotton insects Research AR-SEA-USDA, Brownsville, Texas 78520 Received

December

1, 1980

Five strains of Bacillus thuringiensis that produce crystalline Sendotoxin were used as parental strains in an effort to isolate acrystalliferous (Cry-) mutants: HD-2 (B. thuringiensis var. thuringiensis, flagellar serotype 1); HD-1 and HD-73 (both var. kurstaki, serotype 3ab); HD-4 (var. alesti, serotype 3a); and HD-8 (var. galleriae, serotype Sab). The parental strains contain complex plasmid arrays that have been previously characterized (Gonzalez and Carlton, 1980). The plasmid patterns of both Cry- and Cry+ variants were analyzed and compared to the parental strains using a modified Eckhardt (1978) lysate-electrophoresis method. Most Cry- mutants derived from strain HD-2 were found to exhibit a distinctive colony morphology which facilitated their isolation. Loss of crystal production was associated with loss of a 75-Md plasmid. A 50-Md plasmid of strain HD-73 was lost in the Crymutants. Crystal production in strain HD-4 appears to be associated with a plasmid about 105 Md in size; in strain HD-1, a smaller plasmid (29 Md in size) seems to be involved. In strain HD-8, a large plasmid (- 130 Md in size) is implicated in crystal production. Direct bioassay of several of the mutant strains has confirmed the loss of Sendotoxin activity in the acrystalliferous isolates. The evidence obtained supports the notion of a relationship between specific extrachromosomal DNA elements and g-endotoxin production in B. thuringiensis, and suggests that in each strain only a single plasmid is involved, although the size of the implicated plasmid varies from one strain to another.

The gram-positive bacterium Bacillus thuringiensis is of special interest among members of the genus Bacillus because it produces an insecticidal toxin (the &endotoxin) which is lethal to a wide variety of lepidopteran larvae (Norris, 1970). S-Endotoxin is produced by sporulating cells as a large, crystalline inclusion, the parasporal crystal, which is bipyramidal and phase refractile (Bulla et ul., 1977). The crystal apparently consists of a single 1.3 x 105dalton glycoprotein protoxin which is converted to an active 68,000-dalton polypeptide toxin after solubilization at alkaline pH (Bulla et al., 1979). Attempts to establish the genetic basis for 6-endotoxin production have been hindered by the lack of a genetic exchange system in this organism. However, strains that normally produce &endotoxin can readily lose the ability to form a 351

parasporal crystal; insecticidal activity is lost simultaneously (Vankova, 1957). Reversion to a crystal-producing, insecticidal phenotype occurs rarely, if at all (Norris, 1970). The irreversible loss of &endotoxin production has suggested a possible plasmidmediated inheritance for this property, and several reports on the presence of extrachromosomal DNA in B. thuringiensis have dealt with its possible involvement in crystal production (Debabov et al., 1977; Galushka and Azizbekyan, 1977; Stahly et al., 1978; Miteva, 1978; Gonzalez and Carlton, 1980). The results of these studies have been contradictory. Several groups have reported the isolation of acrystalliferous derivatives that have lost all the plasmids present in the crystal-producing parental strain (Debabov et al., 1977; Galushka and Azizbekyan, 1977; Stahly et al., 1978). On the other hand, 0147-619X/81/030351-15$02.00/0 Copyright AI1 rights

0 1981 by Acxdemic Press, Inc. of reproduction in any form reserved.

352

GONZALEZ,

DULMAGE,

Miteva (1978) has described acrystalliferous mutants which had apparently unchanged plasmid patterns. The analysis of seven standard varieties of B. thuringiensis in our laboratory (Gonzalez and Carlton, 1980) showed that all seven strains, whether crystal-producing or acrystalliferous, possessed three or more species of plasmid DNA. No correlation was apparent between ability to produce the parasporal crystal and the presence of a particular plasmid or plasmids. The two acrystalliferous strains that we studied were not directly derived from any of the five crystal-producing strains, and their plasmid patterns were so different that no definitive conclusions could be derived by comparing them. We have attempted to resolve this uncertainty in 6-endotoxin inheritance by isolating new acrystalliferous mutants directly from several crystal-producing varieties of B. thuringiensis whose plasmid patterns were previously characterized (Gonzalez and Carlton, 1980). Because the genetic origin of each mutant strain would then be known, the plasmid pattern of each mutant could be reliably compared with that of the crystal-producing parent. For these studies, Eckhardt’s lysate-electrophoresis method (Eckhardt, 1978) was modified for use with B. thuringiensis cells. Using this procedure, both small (< 10 Md)’ and very large (> 100 Md) plasmid molecules could be readily detected and resolved on agarose gels. The modified Eckhardt method was used to further characterize the plasmid arrays of the crystal-producing parental strains and to analyze the plasmid patterns of mutant strains. The results presented here establish a strong correlation between loss of parasporal crystal production and the absence or change in mobility of specific plasmid DNA species. In addition, direct bioassay of sev-

AND CARLTON

era1 of the mutant strains has confirmed the loss of 6-endotoxin activity in the new acrystalliferous derivatives. MATERIALS

AND METHODS

Reagents and Special Materials

Nutrient agar, nutrient broth, yeast extract, vitamin-free casamino acids, Bactoagar, Bacto-dextrose, and tryptose-phosphate broth were purchased from Difco Laboratories, Detroit, Michigan. Bovine pancreatic ribonuclease A (RNase A), egg.white lysozyme, novobiocin (sodium salt), sodium dodecyl sulfate (SDS), agarose (Type I, low EEO), and Trizma base (Tris) were obtained from Sigma Chemical Company, St. Louis, Missouri. Strains and Media

Five standard crystal-producing (Cry’) strains ofBacillus thuringiensis were chosen as prototype strains from which to isolate acrystalliferous variants. Their complex plasmid arrays, as well as those of two acrystalliferous strains, have been partially characterized (Gonzalez and Carlton, 1980). These strains and their properties are listed in Table 1. The bacterial cultures were maintained as sporulated colonies on Difco nutrient agar plates. The cultures were streaked on nutrient agar and incubated at 30°C for 3-5 days. Material from each plate was examined by phase-contrast microscopy to monitor the progress of sporulation. After sporulation was complete, plates were stored at 5°C. All derivatives of the crystalliferous strains in Table 1 which showed a total absence of parasporal crystals after sporulation on nutrient agar were classified as acrystalliferous (Cry-). SCG agar contained 0.1% (w/v) vitamin1 Abbreviations used: Md, megadalton; SDS, sodium free casamino acids, 0.5% glucose, and dodecyl sulfate; SCG agar, 0.1% vitamin-free casamino 1.5% Bacto-agar in 1x Spizizen’s minimum acids, 0.5% glucose, 1.5% Bacto-agar in 1 x Spizizen’s minimum phosphate medium; SCGY agar, SCG agar phosphate medium (Spizizen, 1958). Gluplus 0.01% yeast extract; CCC, covalently closed cir- cose was added as a sterile 50% solution. cular; OC, open circular. SCGY agar contained the ingredients of

PLASMIDS AND &ENDOTOXIN

IN

353

B. thuringiensis

TABLE 1 Bacillus

Strain Crystalliferous strains HD-2: B. thuringiensis

thuringiensis

STRAINSAND THEIR PLASMIDS

Serotype”

No. of plasmids

1

10

5.2, 6.2, 7.2,?, 75, -150

11

-1.4, 4.9, 5.2, 5.4, 9.3, -lo’, 44, 52, -110, -120

var. thuringiensis

HD-1:

B. thuringiensis var. kurstaki

3ab, K-l

HD-73:

B. thuringiensis

3ab, K-73

var.

Plasmid masses (megadalton#

6

4.9, 5.2, 5.4,1.5,

29,

50, 50

kurstaki

HD-4:

B. thuringiensis var. alesti

3a

10

HD-8:

B. thuringiensis

Sab

4

5.2,8.J,

1

5

-3.5,

3ab

3

5.2, 9.3, 46

var.

32, 37, 54, 51,

4.6, 4.9, 5.4, 6.2, 6.4, 35, 37, 39, 46, -105 10.3, -130

galleriae

Acrystalliferous strains HD-42: B. fhuringiensis

5.2, 7.1, 9.6, 52

var. thuringiensis

HD-31:

B. thuringiensis var. kurstaki

n Gives flagellar serotype (deBatjac and Bonnefoi, 1973), except for K-l and K-73, which designate crystal serotype (Krywienczyk et al., 1978). * Masses of the HD-2 plasmids were determined by electron microscopy (Gonzalez and Carlton, 1980), with the exception of the - 150-Md plasmid. All HD-2 plasmids thus measured, along with previously measured B. megaterium plasmids (Carlton, 1976), were used as molecular-weight standards on agarose gels to obtain molecular weights for the other B. thuringiensis plasmids. Masses falling below 3.95 or above 75 Md are preceded by (-) to emphasize that they are only approximations, since they are derived by extrapolation from the ends of the standard curve. Underlined masses are those of plasmids isolated predominantly in the relaxed (OC) form. In the case of the 7.6-Md HD-2 plasmid, no CCC DNA has ever been detected. r This extrachromosomal DNA element is isolated in the linear form; due to a lack of linear DNA standards, the figure given is only a rough estimate of its molecular weight.

SCG agar, plus 0.01% yeast extract. It was used for growth of strain HD-8 and its derivatives. Isolation of Partially Cured Derivatives and Spontaneous Acrystalliferous Mutants Strain derivatives cured of one or more of the plasmids originally present were isolated either spontaneously or after growth in the presence of curing agents (SDS or novobiotin). Curing with SDS was done essentially as described by Bernhard et al. (1978). The growth medium was nutrient broth, the con-

centration of SDS was 0.002%, and incubation was at 30°C. The same procedure was used for curing with novobiocin (at a concentration of 2-5 pg/ml of the sodium salt), except that the cultures were incubated for 3-6 days at 30°C because cell growth was very slow. After exposure to the curing agent and plating on nutrient agar, 30 to 40 single colonies were analyzed by the modified Eckhardt method (described below) to determine whether curing had occurred. Derivatives spontaneously cured of one or two plasmids were often detected as colonies having an unusual morphology compared to that of the parental strain (i.e., a

354

GONZALEZ,

DULMAGE,

AND CARLTON

change in shape, color, translucency, tex- ,&ml preboiled RNAseA in TES buffer ture, etc.). Analysis of the plasmid content (30 mM Tris-base, 5 mM disodium EDTA, of such unusual colonies or sectors of col- 50 mM NaCl, pH 8.0). The cell suspensions onies often revealed the loss of a plasmid. were incubated in a 37°C water bath for 30 Some colonies, although unchanged in ap- to 120 min to generate spheroplasts. The pearance, serendipitously proved to be lack- appearance of spheroplasts was monitored by phase-contrast microscopy. ing one or more of the parental-strain In Eckhardt’s original lysis protocol, both plasmids. Spontaneous acrystalliferous derivatives an SDS mixture and an overlay mixture were generated on solid media, usually nu- were used, and the slots were sealed with trient agar plates. A few were picked from molten agarose. Our simplified protocol SCG or SCGY plates. Streaked cultures of a used only the SDS mixture, modified to concrystal-producing strain were incubated at tain 2% sodium dodecyl sulfate, 5% sucrose, and 0.05% bromphenol blue in Tris-borate 30°C and the plates examined periodically for the presence of colonies (or of sectors electrophoresis buffer (89 mM Tris base, within a colony or streak) of atypical mor- 2.5 mM disodium EDTA, 89 mM boric acid, phology. Material from such colonies or pH 8.3-8.5). Twenty microliters of the SDS sectors was examined by phase-contrast mixture was pipetted into each slot of a vermicroscopy. Only isolates producing a tical agarose gel (dimensions: 125 mm long, normal number of sporulated cells on nu- 150 mm wide, 3 mm thick) and allowed to trient agar, with the spore lying in a clear stand for 20 min. Ten microliters of each cytoplasm yet lacking a parasporal inclusion spheroplast suspension was then pipetted of any kind, were considered to be true under the SDS mixture, without mixing the acrystalliferous derivatives. Some Cry- mu- two layers, and electrophoresis was begun tants were first detected by phase-contrast immediately. The slots were not sealed with microscopy of the confluent section of agarose. sporulated streaked cultures of the Cry+ Electrophoresis was usually at a constant parent strain. Whenever Cry- cells were current of 3.0 mA (IO- 14 V) for li/-2 h, detected, material from that part of the plate followed by a change to constant voltage: was restreaked for single colonies, and the first to 40 V (8-10 mA) for 50 min, then to process was repeated until pure colonies of 120 V (25-29 mA) for 2% to 3% h.The gels Cry cells were obtained. were agarose slabs (concentration of 0.4 or 0.5%) in Tris-borate buffer, essentially as described by Meyers et al. (1976), and ModiJied Eckhardt Lysate Electrophoresis Gonzalez and Carlton (1980). The gel slab was prevented from slipping between the The plasmid patterns of B. thuringiensis strains and derivatives were analyzed using glass plates of the apparatus by a sponge and a modification of the method of Eckhardt a 25-ml plug of 2% agarose. (1978). Our simplified protocol involved streaking cells on plates of SCG or SCGY Bioassay of Strains for Endotoxin Activity agar, and incubation overnight at 30°C. Incubation for lo- 16 h, depending on the The insecticidal activities of a representastrain, usually yielded sufficient material for tive sample of wild-type strains and various analysis. A loopful of cells (lo”- 10Rcells) mutant derivatives were evaluated by the from each streak was resuspended in 50 ~1 following procedures. A loopful of spores of lysozyme mixture by vortexing vigor- from nutrient agar slants was initially inocuously. This lysozyme mixture contained lated into a 500-ml Erlenmeyer flask con2 mg/ml lysozyme, 20% sucrose, and 100 taining 100 ml of tryptose-phosphate broth.

PLASMIDS AND SENDOTOXIN

IN B.

thuringiensis

355

After 24 h incubation on a rotary shaking might not be detected due to low recoveries. incubator (340 r-pm) at 30-32°C 3 ml of this Eckhardt (1978) has reported a method of first-passage seed was inoculated into 100ml rapid detection of plasmid DNA which inof the same medium, and growth was con- volves the lysis of cells directly in the slots tinued an additional 18-24 h. Of this second- of vertical agarose gels. In his report two passage seed, 2 ml was used to inoculate large plasmids (with sizes of 96 Md and over 100 ml of the cottonseed flour-glucose140 Md) formed bands on agarose gels as peptone Medium B-4 (Dulmage and de intense as those formed by small (2-4 Md) Barjac, 1973). Incubation at 30-32°C in this plasmids. The Eckhardt method was theremedium was allowed to continue for 96 h. fore simplified and adapted for use with Microscopic examination and cell and spore B. thuringiensis, as detailed above under counts were conducted on each sample at Materials and Methods. Since cells of B. 24, 48, 72, and 96 h. At 72 and 96 h, sam- thuringiensis are often resistant to lysoples of the whole “beer” were bioassayed zyme, even at high concentrations of the against the cabbage looper, Trichoplusia ni, enzyme, several growth media were tested and the tobacco budworm, Heliothis vires- to determine which gave the best results. tens, essentially by the procedures of Dul- Two solid media, SCG agar and SCGY agar, mage ef al. (1976). Strain HD-4 and its consistently yielded cell preparations that derivatives were bioassayed against the silk- were lysozyme sensitive. For the most reworm, Bombyx mori. At 96 h the remainder producible results, the cells had to be grown of each culture was harvested by centrifuga- at 30°C on either SCG or SCGY agar for no tion, and the spore-endotoxin complexes longer than 12- 16 h. Generally a sufficiently were recovered by the acetone coprecipita- large fraction of these cells was sensitive to tion procedure and assayed as described by lysozyme so that a reproducible plasmid Dulmage et al. (1970). All samples were pattern appeared after electrophoresis. bioassayed against strain HD-1-S-1971 (DulFigure 1 shows the results of modified mage and de Barjac, 1973). All bioassay re- Eckhardt lysate electrophoresis using cells sults presented are based on comparative of the seven B. thuringiensis strains listed in Table 1. This procedure revealed the presassays with this standard strain. ence of large plasmids in strains HD-8 (size -130 Md), HD-2 (-150 Md), HD-1 (-110 RESULTS Md and - 120 Md), and HD-4 (- 105 Md). Strains HD-42, HD-73, and HD-31 had no Modified Eckhardt Lysate Electrophoresis detectable plasmids larger than about 50 We have previously reported the exist- Md. The peculiar relaxed-form plasmids ence of complex plasmid arrays in the seven present in strains HD-8 (8.7 Md), HD-2 (7.6 B. thuringiensis strains listed in Table 1 Md), and HD-4 (6.2 and 6.4 Md), which were (Gonzalez and Carlton, 1980); in that study reported earlier (Gonzalez and Carlton, plasmid DNA was isolated by a modification 1980), were also detectable by the Eckhardt of the SDS-NaCl-cleared lysate method of method. The plasmid profile of strain HD-1 Guerry et al. (1973). The largest plasmid reveals another previously undetected exthat was reproducibly detected by that trachromosomal DNA element, indicated method was the plasmid of 75 Md from strain by an (L). This molecule appears to be presHD-2. This method has been reported to ent in the linear form, since its mobility ingive progressively lower yields with increas- creases relative to the mobilities of CCC ing plasmid size (Currier and Nester, 1976; and OC DNA molecules when the agarose Hansen and Olsen, 1978), so that any large concentration of the gel is increased. This plasmids (~100 Md) present in the strains linear element is removed during isolation

356

GONZALEZ,

DULMAGE,

AND CARLTON

HD8 HD2 HO42 HD73 HD I HD31 HD4

;“,“-

FIG. 1. Agarose gel electrophoresis patterns of seven standard B. thuringiensis strains, using a modified Eckhardt method of lysing spheroplasts directly on the gel slots. In this gel, 30 ~1 of the SDS mixture was used in each slot. Electrophoresis was at constant current, 3.0 mA (12 V) for 2!4 h, followed by electrophoresis at constant voltage: 40 V (9.5 mA) for 1 h, then at 120V (28 mA) for 2 h 14min. The gel was 0.5% agarose. The strain number is listed above each slot; the properties of each strain are given in Table 1, except for the strain on the far right slot labeled HD4 Cry-, which was an acrystalliferous derivative of HD-4. The major CCC plasmid bands are indicated with arrows (+). The major open circular plasmid bands are marked with (0); these bands are generated by plasmids which occur predominantly in the relaxed form. Brackets ([) indicate the position of chromosomal DNA fragments in each slot. An (L) marks the plasmid-like linear DNA element present in strain HD-1. The low intensity of the plasmid bands of strain HD-3 1 is due to poor lysis of the cells, which are very lysozyme resistant. The molecular weights of the HD-2 plasmids are listed on the left-hand margin for use as an approximate size scale.

procedures involving extraction with chloro- believed to represent multimeric forms of form or phenol, which possibly explains major plasmids and are not listed here. why this unusual extrachromosomal DNA molecule was not detected earlier, using the Partially Cured Cry+ and Cry- Mutants cleared lysate method (Gonzalez and Carlof Strain HD-2 ton, 1980). Strain HD-2 yielded the largest number of The revised plasmid arrays of the seven B. thuringiensis strains originally examined Cry- mutants: a total of 20 independent Cryare summarized in Table 1. The new data mutants were obtained. Isolation of Cryfrom Eckhardt lysate electrophoresis is in- mutants from this strain was facilitated by a cluded. A few minor changes have been distinctive colony morphology exhibited by made in the molecular weights (both those all of these mutants (except HD2-19, below). Figures 2 and 3 show the plasmid patterns from electron microscopy and those from electrophoretic mobility) previously re- of the HD-2 wild-type strain and of several ported. Plasmids present in minor amounts partially cured derivatives. In Fig. 2, HD2-1 (less than 1% of total plasmid DNA) are ,is the crystal-producing wild-type strain and

PLASMIDS AND &ENDOTOXIN

has 10 distinct plasmid bands. Figure 2 also shows several derivatives which lack one or more plasmids but still produce parasporal crystals. These are HD2-3, which has lost the -150-Md plasmid; HD2-5, which has lost both the -150- and 5J-Md plasmids; HD2-13, which has been cured of the 3J-Md plasmid; and HD2-23, in which the 7.2-Md plasmid band has decreased in intensity. Figure 2 also includes the plasmid patterns of several spontaneous acrystalliferous mutants of HD2-1 and of its derivatives: HD2-2 (derived from HD2-l), HD2-4(from HD2-3), HD2-6 (from HD2-5), and HD2-24 (from HD2-23). These Cry- mutants have all lost the J5-Md plasmid. Figure 3 shows the patterns of HD2-11, a derivative of HD2-5 which has lost the 7.6-Md (relaxed) plasmid, and its derivative HD2-35, which has addi-

IN

357

B. thuringiensis

tionally lost the 6.2-Md plasmid; both are Cry+. Figure 3 also shows the two spontaneous Cry- mutants HD2-12 (derived from HD2-11) and HD2-36 (from HD2-35); both have lost the J5-Md plasmid. Several other interesting derivatives of HD-2 are presented in Fig. 3. Some show only the loss of a single plasmid band; others show the loss of a plasmid band accompanied by the appearance of a new plasmid band of different mobility. For example, HD2-16 was isolated from HD2-5 after growth in the presence of SDS. The J5-Md band of HD2-5 has been replaced by a new band -65 Md in size; however, the crystal is still produced. HD2-17 is a spontaneous Cry- mutant of HD2-16. The -65-Md band present in HD2-16 has been lost, as has the 6.2-Md band; a new band of -7.7 Md has

+

-

+

-

+

-

2-l

2-2

2-3

2-4

2-5

2-6

I-

+

-

2-13 2-232-24

FIG. 2. Modified Eckhardt lysate-electrophoresis of cells of strain HD-2 and derivatives. The patterns of the Cry+ parental strain HD2-1 and of eight partially cured derivatives (including four Cry- mutants) are shown. The gel was 0.5% agarose and was electrophoresed at a constant current of 4.0 mA for 90 min, then at constant voltage: at 40 V for 50 min, then at 120V for 3 h 50 min. The molecular weights (in megadaltons) of the parental HD-2 plasmids are shown on the left margin, adjacent to the corresponding bands in slot HD2-1. The bracket shows the position of chromosomal DNA fragments. The strain number of each derivative is written above its slot. A (+) above a slot indicates crystal production; a (-) indicates loss of crystal production. The properties of the derivatives (crystal production, change in plasmid pattern, method of derivation) are discussed in the text and summarized in Table 2.

358

GONZALEZ,

-+-+-+++-+

2-36 2-35

2-12

DULMAGE,

2-11

AND CARLTON

2-19 2-26

2-30

2-29

2-17 2-16

FIG. 3. Eckhardt lysate-electrophoresis of several additional partially cured derivatives of strain HD-2. Electrophoresis was in a 0.5% agarose gel run at constant current (4.0 mA) for 2 h, then at constant voltage: 40 V for 30 min, then at 120 V for 3 h. The properties of the derivatives are discussed in the text and summarized in Table 2. The strain number of each derivative is written above its slot. Molecular weights (in megadaltons) for some of the plasmids in wild-type HD-2 are shown on the lefthand margin to serve as an approximate molecular weight scale. A (+) above the slot indicates that the derivative produces crystals; a (-) indicates that the derivative is acrystalhferous.

appeared. HD2-29 is a spontaneous Cry+ derivative of HD2-23 (Fig. 2) showing a change in plasmid size (75 Md + 65 Md) similar to that in HD2-16. HD2-30 is a Cry+ derivative of HD2-23, isolated after growth in the presence of novobiocin. The 5.2-Md band has been replaced by a new band -6.6 Md in size; the 10.3-Md minor band present in HD-2 has also disappeared. HD2-28 is Cry+ and was derived from HD2-23 after growth in the presence of SDS. The 7.6-Md relaxed-form plasmid has been replaced by a new relaxed-form plasmid band of -10 Md in size. This open-circular plasmid is almost too large to migrate stably at 120 V, which accounts for the blurred appearance of the band. HD2-19 was a spontaneous Cryderivative of HD2-13; interestingly, the 75-Md plasmid is still present but the 54-Md plasmid has disappeared. HD2-19 is the only Cry- derivative so far isolated that still carries the 75-Md plasmid. The Cry- mutant HD2-41 (pattern not shown) was derived

from HD2-19; it had lost the 75-Md plasmid. The Cry+ mutant HD2-42 (not shown), derived from HD2-1, spontaneously lost both the 37- and 32-Md plasmids. Another Cry+ mutant, HD2-46 (not shown), derived from HD2-3, spontaneously lost the 54-Md plasmid. The origins, properties, and alterations in plasmid array of the HD-2 mutants are summarized in Table 2. The first spontaneous Cry- mutants of HD-2 were discovered by chance while examining some peculiar sectors of colonies for the presence of spores and crystals by phase-contrast microscopy. Colonies of these Cry- mutants showed a distinctive morphology following sporulation (after 2-4 days’ growth on nutrient agar at 30°C). Sporulated Cry- colonies or sectors were paler than sporulated Cry+ colonies and became even paler during storage at 5°C. The Cry- cells were also separated from contiguous Cry+ cells by a clear “line of demarcation”: a thin (-0.2-0.5 mm in width),

PLASMIDS AND &ENDOTOXIN

nearly transparent zone between the two colony types (or between a Cry- sector and the Cry+ part of the colony). This “line of demarcation” was invariably associated with the loss of the 75-Md plasmid. Figure 4 shows the morphology of typical Cry- colonies of HD-2. The mutant HD2-19 (Fig. 3) was picked from a pale sector of an HD2-13 colony growing on nutrient agar. The usual

IN B. thuringiensis

359

“line of demarcation” seen in Fig. 4 and characteristic of the other Cry- mutants was absent, so that the morphology of HD2-19 was unlike that of a typical Cry- sector. Loss of the 75-Md plasmid by HD2-19 gave rise to the colony morphology shown in Fig. 4; strain HD2-41 was detected and isolated by searching plate cultures of HD2-19 for such colonies.

TABLE 2 MUTANTS OF Bacillus

Strain No.

Derivation”

HD2-1 HD2-2 HD2-3 HD2-4 HD2-5 HD2-6 HD2-11 HD2-12 HD2-13 HD2-16 HD2-17 HD2-19 HD2-23 HD2-24 HD2-28 HD2-29 HD2-30 HD2-35 HD2-36 HD2-4 1 HD2-42 HD2-46

Wild type HD2-1, spont. HD2- 1, spont . HD2-3, spont. HD2-3, SDS HD2-5, spont . HD2-5, SDS HD2-11, spont . HD2-5, SDS HD2-5, SDS HD2-16, spont. HD2-13, spont. HD2-13, SDS HD2-23, spont. HD2-23, SDS HD2-23, spont. HD2-23, novo. HD2-11, novo. HD2-35, spont. HD2-19, spont. HD2-1, spont. HD2-3, spont.

HD73- 1 HD73-2 HD73-3 HD73-4 HD73-5 HD73-6 HD73-7

Wild type HD73-1, spont. HD73-2, spont. HD73-2, spont. HD73-4, spont. HD73-2, spont. HD73-6, spont.

crystal production6

thuringiensis

&Endotoxin activityc

+ + + +

ND ND

+ +

+ + + + -

+

ND + ND ND ND

-

+ +

ND ND ND +

+ + -

STRAINS HD-2 AND HD-73

Change in plasmid pattemd (Wild type: plasmid array shown in Table 1) 75-Md plasmid lost - 150-Md plasmid lost 75-Md plasmid lost 57-Md plasmid lost 75-Md plasmid lost 7.6-Md OC plasmid lost 75-Md plasmid lost 37-Md plasmid lost 75-Md plasmid lost; new 65-Md plasmid 65-Mdand6.2-Mdplasmids lost; new 7.7-Mdplasmid 54-Md plasmid lost 7.2-Md plasmid band reduced in intensity 75-Md plasmid lost 7.BMd OC plasmid lost; new IO-Md OC plasmid 75-Md plasmid lost; new 65-Md plasmid 5.2-Md plasmid lost; new 6.6-Md plasmid 6.2-Md plasmid lost 75-Md plasmid lost 75-Md plasmid lost 37-Md and 32-Md plasmids lost 54-Md plasmid lost (Wild type: plasmid array shown in Table 1) 5.4-Md plasmid lost; new 6.7-Md plasmid 50-Md plasmid band greatly reduced in intensity New 47-Md plasmid SO-Mdplasmid lost New 43-Md plasmid 50-Md plasmid lost

n The number of the immediate predecessor strain is given, followed by the means of mutant derivation: spont., the mutant arose spontaneously after growth under normal conditions; SDS, the mutant arose after growth in the presence of sodium dodecyl sulfate; novo., the mutant arose after growth in the presence of novobiocin. * Presence (+) or absence (-) of phase-refractile parasporal crystals after growth on nutrient agar. c Insecticidal activity of sporulated preparations determined by direct insect bioassay; + , active; 2, low activity; -, no activity; ND, activity not determined. d Indicates the change in plasmid pattern as compared to its immediate predecessor.

360

GONZALEZ,

DULMAGE,

AND CARLTON

FIG. 4. Colony morphologies of normal cells and acrystalliferous mutants of strain HD-2. Spores of strain HD2-1 (crystalliferous parental strain) were streaked on a plate of nutrient agar and incubated at 30°C for 72 h. The plate was photographed by reflected light, against a dark background. Wild-type, crystal-producing colonies are yellowish-white and opaque. Here they appear grayish-white. Three typical spontaneous acrystalliferous (Spa+ Cry-) colonies are indicated by arrows. These colonies are yellowish-white but not as opaque as the wild-type colonies. Here the Cry- colonies appear darker because they were photographed against a dark background. The Cry- colonies are separated from the adjacent Cry+ colonies by a thin transparent zone, here appearing as a dark line. These three colonies were examined by phase microscopy and Eckhardt lysate-electrophoresis. They were verified as being Spa+ Cry- and as having lost the 75-Md plasmid.

The Cry+ mutant HD2-11 (Fig. 3) and its derivatives (whether Cry+ or Cry-) were difficult to maintain as pure cultures because of an unusually high rate of plasmid loss and alteration. Spontaneous Cry- mutants of HD2-11 (of the HD2-12 type) arise at a high frequency (up to 50% of the cells in any colony). The plasmid array of these Crymutants has been observed commonly to undergo characteristic, spontaneous alterations. Replacement of the 54-Md plasmid with a new -4O-Md plasmid has been de-

tected several times. Replacement of the 37-Md plasmid with a new -80-Md plasmid has also been seen. The faint, low-mobility band visible in the HD2-12 slot in Fig. 3 is probably due to the presence of this new -80-Md plasmid in a significant fraction of the cells. The mobility of the 75Md plasmid in HD2-11 seems to be slightly less than in the parental strain, suggesting the possible insertion of -2-3 Md of DNA. This barely detectable alteration in the 75Md plasmid might be the reason for the observed in-

PLASMIDS AND &ENDOTOXIN

stability of HD2-11 and of its derivatives (such as HD2-35 and HD2-36). In addition to the loss of plasmids, increases or decreases in the size (i.e., electrophoretic mobility) of plasmids were common, and occurred both spontaneously and after exposure to “curing” conditions: in Fig. 3, HD2-16, HD2-17, HD2-29, HD2-30, and HD2-28 exhibited such alterations of the wild-type plasmid array. Cry+ and Cry- Derivatives

of Strain HD-73

Figure 5 presents the agarose gel plasmid patterns of wild-type HD-73 and of six spontaneous mutants, including three which are Cry-. HD73-1 is the wild type, while HD73-2 is a spontaneous derivative of HD73- 1; HD73-2 differs from HD73- 1 in the substitu-

IN B. thuringiensis

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tion of a 6.7-Md band for the 5.4-Md band present in HD73-1. HD73-3 is a spontaneous Cry- mutant of HD73-2. The only change evident in the plasmid pattern is an approximately fivefold reduction in the intensity of the 50-Md band. HD73-4 is a spontaneous Cry+ derivative of HD73-2; what is apparently a new plasmid band of -47 Md is seen running ahead of (below) the SO-Md band. HD73-5 is a spontaneous Cry- mutant of HD73-4; here the 50-Md band has now been lost. HD73-6 is a Cry+, spontaneous derivative of HD73-2 which contains a new band of -43 Md. HD73-7 is a spontaneous Cry- mutant of HD73-6; as in HD73-5, the 50-Md band has been lost. Biotoxicity assays (Table 2) corroborate the involvement of the 50-Md plasmid in Sendotoxin production. The colonies

-+-+-++ 73-7 73-6 73-6 m4

73-3 737

73-l

FIG. 5. Eckhardt lysate-electrophoresis of cells from strain HD-73. The patterns of the Cry+ parental strain HD73-1 and of six derivatives (including three Cry- mutants) are shown. Electrophoresis was in a 0.4% agarose gel run at a constant current of 2.0 mA (9 V) for 3 h, then at constant voltage: at 40 V for 50 min, then at 120 V for 3 h. The serial number of each derivative is written above its slot. A (+) above the slot indicates crystal production, a (-) indicates loss of crystal production. The molecular weights (in megadaltons) of the plasmids in the parental HD-73 strain are listed on the right-hand margin, to serve as an approximate size scale.

362

GONZALEZ,

DULMAGE,

of the Cry- mutants were morphologically identical to those of the Cry+ parental strains. The mutants of strain HD-73, and their characteristics, are summarized in Table 2.

AND CARLTON

eluding HD2-41) possessed a distinctive colony morphology; this property made possible the routine derivation of Cry- mutants. Although it is possible that loss of the 75-Md plasmid, loss of crystal formation, and the appearance of the Cry- colony morphology Acrystalliferous Derivatives of Strains could all result from chromosomal mutaHD-4, HD-I, and HD-8 tions with pleiotropic effects, the high deThree independent, spontaneous Crymutants of strain HD-4 have been isolated; gree of correlation (100%) between disapa representative plasmid pattern is shown in pearance of the 75-Md plasmid and the other Fig. 1. The - 105Md band of the HD-4 wild- two phenotypic effects suggests that spontype pattern was lost in all three Cry- taneous curing of this plasmid is responsible for the Cry- mutants which arise in strain mutants. Several partially cured derivatives of HD-2 with such high frequency and in the strain HD-1 have been isolated (Fig. 6). Cry+ absence of selective conditions. Although all mutants lacking the 75-Md derivatives were obtained that lacked the 44-, 9.3-, -120-, and -1 lo-Md bands, as plasmid invariably exhibit the Cry- phenowell as a Cry+ derivative that showed a re- type, the occurrence of mutant HD2-19 (Fig. duction in the intensity of the 52-Md band. 3) shows that it is possible to lose crystal A single Cry- derivative of strain HD-1 was formation and retain the 75-Md plasmid. isolated, in which the 29-Md plasmid was re- HD2-19 is Cry-, but still contains the 75-Md placed by a larger plasmid of -34 Md (Fig. 6, plasmid and does not show the “typical” Cry- colony morphology of the other HD-2 slot D). Several derivatives of HD-8 have been Cry- mutants. Although no phase-refractile obtained that have altered plasmid arrays crystals could be seen in the sporangia, (Fig. 6). Absence of the 8.7- (OC) and 10.3- HD2-19 showed low but detectable &endoMd plasmids has no effect on crystal produc- toxin activity (Table 2), suggesting that a tion. A single spontaneous Cry- mutant of mutation in the 75-Md plasmid itself has reHD-8 has been isolated; the only change in sulted in production of 6-endotoxin at a rethe plasmid pattern was the replacement of duced level, or in an altered form which does the -130-Md plasmid by a new plasmid not form a normal crystal. In addition, the isolation of HD2-46, a mutant that lacks the -90 Md in size (Fig. 6, slot G). 5CMd plasmid but is still Cry+ (Table 2), DISCUSSION further indicates that absence of the 54-Md The studies reported in this paper provide plasmid in HD2-19 is not the reason for the strong evidence for the correlation of para- absence of parasporal crystals. sporal crystal (bendotoxin) production with Many Cry+ derivatives of strain HD-2 specific extrachromosomal plasmid DNA have been isolated that lack one or more of molecules in several strains of Bacillus the other plasmids present in the wild type, thuringiensis. The most extensive evidence as seen in Table 2 and Figs. 2 and 3. These for such a correlation was obtained from results suggest that crystal synthesis does analysis of mutants of strain HD-2 (B. not require the presence of the -150-, 57-, thuringiensis var. thuringiensis, serotype 1). 37-, 32-, 7.6- (OC), or 6.2-Md plasmids. No A total of 20 independently derived Cryderivatives of HD-2 clearly lacking the 5.2mutants of HD-2 have been examined and or 7.2-Md plasmids have yet been isolated, all but one (HD2-19) have lost a 75Md plas- so that these two plasmids cannot be exmid present in the Cry+ parent. All of the cluded from involvement in crystal syntheCry- mutants lacking the 75Md band (in- sis at this time.

PLASMIDS AND &ENDOTOXIN

Several spontaneous acrystalliferous derivatives of strain HD-73 were also isolated. Their plasmid patterns, shown in Fig. 5, suggest that a 50-Md plasmid is involved in crystal production in this strain. The 50-Md plasmid band in this strain is apparently a doublet, which complicates the results somewhat. The simplest interpretation is that loss of the more prominent member of this doublet leads to loss of crystal and Sendotoxin production. The second, or minor, 50-Md plasmid, with a lower copy number, has presumably undergone deletion in strains HD73-4 and HD73-6, with an increase in its mobility that resolves its band from that of the major 50-Md plasmid. The data at present do not indicate whether or not the minor 50-Md plasmid, or any of the smaller plasmids present in strain HD-73, are required for parasporal crystal production, since no variants have been recovered which have lost any of these plasmids. Three Cry mutants of strain HD-4 were isolated; the plasmid pattern of one mutant is shown in Fig. 1. The disappearance of the largest plasmid (size - 105 Md) suggests that it may carry information necessary for crystal production. Biotoxicity assay of one of these Cry- mutants showed that it no longer produced 6-endotoxin. In strain HD-1, a single Cry- derivative was isolated, in which the 29-Md plasmid present in the parental Cry+ strain had been replaced by a larger plasmid (-34 Md in size). Possibly a segment of DNA (-5 Md in size) has been inserted into the 29-Md plasmid and is responsible for the Cry- phenotype-implicating this plasmid in crystal production. Digestion of the purified 29-Md (wild type) and -34-Md (mutant) plasmids with the restriction endonuclease EcoRI revealed that a fragment of about 3 Md in the 29-Md plasmid digest was replaced by a fragment of -8 Md in the 34-Md plasmid digest (K. Bernhard, unpublished observations). Biotoxicity assay of the HD-1 parental strain and the Cry- mutant confirmed that the Crymutant no longer produced Sendotoxin.

IN B. rhuringiensis

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100 5a

5

FIG. 6. Eckhardt lysate-electrophoresis of cells from strains HD-1 and HD-8. The gel was 0.5% agarose and electrophoresis was at a constant current of 3.0 mA (12 V) for 2M h, followed by electrophoresis at constant voltage: 30 V (8 mA) for 1 h, then at 120 V (30 mA) for 3 h. The numbers along the left-hand margin provide an approximate molecular weight scale. A (+) above the slot indicates crystal production, a (-) indicates loss of crystal production. Slots A, B, C, and D were loaded with cells of strain HD-1. Slot A shows the plasmid pattern of wild-type HD-1. Slot B shows a derivative of HD-1 that has lost the 9.3-, 44-, and -120-Md plasmids and is Cry+. Slot C shows a derivative of(B) which has in addition lost the - 1IO-Md plasmid; it is still Cry+. Slot D shows a second derivative of(B) which shows an increase in the size of the 29-Md plasmid and a decrease in the size of the - 1lOMd plasmid; this mutant is Cry. Slots E, F, and G were loaded with cells of strain HD-8. Slot E shows the plasmid pattern of wild-type HD-8. Slot F shows a Cry+ derivative of(E) which has lost the 8.7- (OC) and 10.3Md plasmids. Slot G shows a derivative of(F) in which the -130-Md plasmid has been replaced by a new -9O-Md plasmid; this mutant is Cry-.

The isolation of several Cry+ variants of strain HD-1 that lacked the 44-,9.3-, -120-, or -1 IO-Md plasmids, suggests that these plasmids are not required for crystal synthesis. In strain HD-8, a single Cry- mutant was isolated in which the largest (- 130 Md) plasmid of the wild-type strain had been replaced by a smaller (-90 Md) plasmid (Fig. 6). This suggests that the - 130-Md plasmid contains genes required for crystal produc-

364

GONZALEZ,

DULMAGE,

tion, which have undergone deletion in the Cry- mutant. Most of the previous reports on the relationship between endotoxin and plasmids (Debabov et al., 1977; Galushka and Azizbekyan, 1977; Stahly et al., 1978) have suggested that the loss of crystalline toxin production was accompanied by the loss of all extrachromosomal DNA. On the other hand, our preliminary results (Gonzalez and Carlton, 1980) agreed with Miteva’s (1978) in suggesting that Cry- strains may retain a complex array of plasmid molecules. Our present results corroborate our earlier data. As detailed above, in every strain the loss of crystal production is associated with the loss or modification of one specific plasmid molecule; the rest of the complex plasmid array is retained. In screening derivatives of several strains of B. thuringiensis, we have observed that loss of one plasmid was sometimes accompanied by the appearance of a new plasmid of different electrophoretic mobility, suggesting that rearrangements such as deletions, insertions, and perhaps recombinations are relatively common among these plasmids. The combination of these plasmid alterations in a strain could explain how the Cry+ strains HD- 1and HD-73, both of flagellar serotype 3ab, apparently have only three plasmids in common (of sizes 4.9, 5.2, and 5.4 Md). These alterations in the plasmid arrays also suggest how, in theory, the Crystrain HD-42 could be descended from a serotype-1 strain similar to strain HD-2, or how the Cry- strain HD-3 1 could have originated in a serotype-3ab strain such as HD-1 or HD-73. Such changes might be interpreted as examples of the evolution of these complex plasmid arrays under laboratory conditions. Outside the laboratory, of course, genetic exchange of plasmids might be an added factor contributing to evolution of plasmid systems. Whether the genetic determinants we have identified with specific plasmid DNA molecules represent the structural genes

AND CARLTON

which code for the 6-endotoxin polypeptides of the various strains, or whether they are regulatory functions, remains to be determined. In addition, a conclusive proof of the postulated association of 6-endotoxin determinants with the plasmids we have implicated will ultimately require the demonstration that acrystalliferous mutants can regain the ability to produce S-endotoxin by genetic transfer of the presumed toxin-coding plasmid from the Cry+ to the Cry- strains. Experiments along both of these lines are now in progress. ACKNOWLEDGMENTS We acknowledge the assistance of Professor Keio Aizawa for conducting bioassays on silkworms. We also thank Dr. D. Vapnek for his critical reading of the manuscript. One of us (J.M.G., Jr.) thanks Laura Carreira for helpful discussions and encouragement. This work was supported in part by National Science Foundation Research Grant PCM 7923814.

REFERENCES BERNHARD, K., SCHREMPF,H., AND GOEBEL, W. (1978). Bacteriocin and antibiotic resistance plasmids in Bacillus cereu~ and Bacillus subtilis. .I. Bacteriol.

133, 897-903.

BULLA, L. A., JR., KRAMER, K. J., AND DAVIDSON, L. I. (1977). Characterization of the entomocidal parasporal crystal of Bacillus thuringiensis. J. Bacteriol.

130, 375-383.

BULLA, L. A., JR., DAVIDSON, L. I., KRAMER, K. J., AND JONES,B. L. (1979). Purification of the insecticidal toxin from the parasporal crystal of BaciNus thuringiensis subsp. kurstaki. Biochem. Biophys. Res. Commun. 91, 1123-1130. CARLTON, B. C. (1976). Complex plasmid system of Bacillus megaterium. In “Microbiology1976” (D. Schlessinger, ed.), pp. 394-405. Amer. Sot. Microbiol., Washington, D. C. CURRIER,T. C., AND NESTER,E. W. (1976). Isolation of covalently closed circular DNA of high molecular weight from bacteria. Anal. Biochem. 76, 431-441. DEBABOV, V. G., AZIZBEKYAN, R. R., KHLEBALINA, 0. I., D’YACHENKO, V. V., GALUSHKA, F. P., AND BELYKH, R. A. (1977). Isolation and preliminary characterization of extrachromosomal elements of Bacillus thuringiensis DNA. Genetika 13, 496-501. DEBARJAC,H., AND BONNEFOI, A. (1973). Mise au point sur la classification des Bacillus thuringiensis. Entomophaga 18, S- 17. DULMAGE, H. T., CORREA,J. A., AND MARTINEZ,

PLASMIDS AND &ENDOTOXIN A. J. (1970). Coprecipitation with lactose as a means of recovering the spore-crystal complex of Bncillus thuringiensis. .I. Invertebr. Pathol. 15, 15-20. DULMAGE, H. T., AND DEBARJAC, H. (1973). HD-187; A new isolate of Bacillus thuringiensis that produces high yields of &endotoxin. J. Invertebr. Pathol. 22, 273-277. DULMAGE, H. T., MARTINEZ, A. J., AND PENA, T. (1976). “Bioassay ofBacillus thuringiensis (Berliner) 6-Endotoxin Using the Tobacco Budworm,” Tech. Bull. No. 1528. U. S. Dept. Agr., Agr. Res. Serv. ECKHARDT, T. (1978). A rapid method for the identification of plasmid desoxyribonucIeic acid in bacteria. Plasmid 1, 584-588. GALUSHKA, F. P., AND AZIZBEKYAN, R. R. (1977). Investigation of plasmids of different variants of

IN B. thrtringiensis

365

P2 incompatibility group plasmids pMG1 and pMG5. J. Bacterial.

135, 227-238.

Piasmid 3, 92-98. GUERRY, P., LEBLANC, D. J., AND FALKOW, S. (1973).

KRYWIENCZYK,J., DULMAGE, H. T., AND FAST, P. G. (1978). Occurrence of two serologically distinct groups within Bacillus thuringiensis serotype 3ab var. kurstaki. J. Invertebr. Pathol. 31, 372-375. MEYERS, J. A., SANCHEZ, D., ELWELL, L. P., AND FALKOW, S. (1976). Simple agarose gel electrophoretic method for the identification and characterization of plasmid deoxyribonucleic acid. J. Bacteriol. 127, 1529- 1537. MITEVA, V. I. (1978). Isolation of plasmid DNA from various strains of Bacillus thuringiensis and Bacillus cereus. C. R. Acad. Bulg. Sci. 31,913-916. NORRIS, J. R. (1970). Sporeformers as insecticides. J. Appl. Bacterial. 33, 192-206. SPIZIZEN, J. (1958). Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate. Proc. Nat. Acad. Sci. USA 44, 1072-1078. STAHLY, D. P., DINGMAN, D. W., BULLA, L. A., JR., AND ARONSON,A. I. (1978). Possible origin andfunction of the parasporal crystals in Bacillus thuringien-

General method for the isolation of plasmid deoxyribonucleic acid. J. Bacterial. 116, 1064- 1066. HANSEN, J. B., AND OLSEN, R. H. (1978). Isolation of large bacterial plasmids and characterization of the

VANKOVA, J. (1957). Study of the effects of Bacillus thuringiensis on insects. Folia Biol. (Prague) 3, 175182.

Bacillus

thuringiensis.

Dokl. Akad. Nauk SSR 236,

1233- 1235. GONZALEZ, J. M. JR., AND CARLTON, B. C. (1980).

Patterns of plasmid DNA in crystalliferous and acrystalliferous strains of Bacillus thuringiensis.

sis. Biochem. Biophys. Res. Commun. 84,581-588.