Gme. 120 (1992) 17-26 0 1992 Elscvicr

GENE

Science Publishers

B.V. All rights reserved.

17

0378-l 119/92/$05.00

06690

and properties of a novel plasmid vector for Bacillus t huringiensis displaying compatibility with host plasmids

Characterization

(Recombinant

DNA;

B.t. ssp.

vector;

kuvstaki;

replicon;

Pamela H. Game1 * and Jean-Christophe Department,Sandoz

M&c&r

Bio1ng.r

Received

by R.E. Yasbin:

5 February

Agro. Inc., Polo Ah,

plasmid

stability)

Piot CA 94304,

1992; Revised/Accepted:

USA

19 May/25

May 1992; Received

at publishers:

22 June 1992

SUMMARY

A novel plasmid vector, composed of a 1.7-kb Bacillus thuringiensis (B.t.) replicon, a multiple cloning site, and an erythromycin-resistance marker gene from Bacillus subtilis, was constructed for use in B.t. Unlike other vectors which have been reported to be acceptable for B.t., this new B.t. vector was stably maintained in the absence of Er and did not displace host plasmids, some of which carry crystal protein-encoding genes (cry genes). The compatibility of this B.t. vector with native plasmids is highly desirable when introducing new cry genes into a wild-type B.t. strain. When a cryMA gene of B. t. tenebrionis was cloned in this vector and introduced into B. t. kurstaki (kur) HD119, cryZZIA was highly expressed without affecting the level of expression of native cry genes. The stability of this vector and its compatibility with native B. t. plasmids were achieved by subcloning only nucleotide sequences required for the vector to replicate in B.t. The origin of replication was first cloned on a 9.6-kb BglII fragment from a 75-kb plasmid of B.t. kur HD73 and then localized to a 2.4-kb region within the 9.6-kb fragment. Sequencing of the 2.4-kb region revealed the presence of an open reading frame (ORF), encoding a putative 312-amino acid (aa) protein. The deduced aa sequence of the ORF showed no homology to any published aa sequences. Deletion analysis indicated that the B.t. vector required at least the ORF and up to 300 bp surrounding the ORF, in order to replicate.

INTRODUCTION

Biological insecticides in general and B.t.-based products in particular have, in recent years, found increased use as alternatives to broad-spectrum chemical pesticides. The growing acceptance of such products has led to a demand

Correspondence to: Dr. J.-C. Piot, Molecular Biology Department, Agro, Inc., 975 California Ave., Palo Alto, CA 94304, USA. Tel. (415) 354-3580; Fax (415) 8580116.

* Present address: Dr., Chaska,

Sanofi Diagnostics

Pasteur,

Sandoz

erythromycin;

Fax (612) 368-1280.

ORF,

aa, amino

acid(s);

base pair(s); cry, gene encoding extract/salts

(sporulation

B., Bacillus; B.t., B. fhuringiensis;

bp,

crystal toxin protein; CYS, Casitone/yeast

medium;

see legend to Fig. 4); d, deletion;

Er,

open

reading

polyacrylamide-gel PolIk,

Abbreviations:

ExoIII,

exonuclease

III; IS, insertion

sequence(s);

kb, ki-

lobase( or 1000 bp; kur, kurstaki; LB, Luria-Bertani (medium); MCS, multiple cloning site(s); nt, nucleotide(s); oligo, oligodeoxyribonucleotide;

Inc., 1000 Lake Hazeltine

MN 55318, USA. Tel. (612) 4484848;

for insecticides with activity against a wide spectrum of target pests. Several methods are currently being employed to obtain desirable B.t. strains. These include: (I) isolation of B.t. strains containing novel cry genes (Chambers et al., 1991; Visser et al., 1990); and (2) conjugation between naturally

Klenow

resistance/resistant; dodecyl junction

frame;

ori, origin

electrophoresis; (large) SD,

fragment

PCR, of E.

Shine-Dalgamo

sulfate; wt, wild type; (fusion or insertion).

of DNA

replication;

polymerase co/i

DNA

(sequence);

[ 1,denotes plasmid-canier

chain

PAGE, reaction;

polymerase SDS,

I; a, sodium

state; ::, novel

18 occurring strains to produce hybrid strains containing plasmids harboring cry genes with different or complementary host specificities (Karamata and Piot, 1989). Each of these methods suffers from drawbacks and limitations: screening procedures for novel genes or combinations of genes are both time-consuming and tedious while conjugation is limited by the capacity of strains to act as donors or recipients, and transfer of plasmids from one strain to another cannot be controlled. An alternative approach is the use of recombinant DNA techniques to introduce and express cry genes in suitable host strains in a highly controlled fashion. Transformation of B.t. strains using electroporation has been described (&hurter et al., 1989; Bone and Ellar, 1989). An essential element in the successful engineering of B.t. strains is the availability of suitable cloning vectors. Maintenance of an introduced cry gene is dependent upon the stability of the cloning vector within the host strain following transformation. Until recently transformation of B.t. strains had employed genetically engineered shuttle vectors that, after transformation into B. t. strains, would become structurally and/or segregationally unstable. These serious consequences may be due to the fact that the vectors utilized in these studies contained DNA from heterologous species such as E. coli (Miteva et al., 1988) Bacillus cereus (Schurter et al., 1989) or StuphyZococcus clureus (Crickmore et al., 1990; Fischer et al., 1984). In addition, we found that B.t. host cells transformed with cloned cry genes borne on shuttle vectors composed of E. coli and B. cereus DNA exhibited decreased expression of endogenous cry genes, perhaps due to the high copy number of the shuttle vectors. Therefore, construction of a specific cloning vector for B. t. that will be segregationally and structurally stable is of wide-spread interest. To develop a means for stably maintaining cloned genes in B.t. strains, we investigated the use of native B.t. plasmids. In this study, B.t. cloning vectors containing the ori from a 75kb plasmid of B.t. kur HD73 were constructed. Initial constructs displaced native plasmids when they were introduced into wt B.t. strains. Deletion analysis was conducted to determine the minimal DNA region required for replication and exhibiting plasmid compatibility. A plasmid vector, containing the minimal replicon and an ErR gene, was constructed and tested in several B.t. strains. The vector was stably maintained in transformed B.t. strains containing plasmids with similar or identical oris and did not completely displace these native plasmids. When a cryZZZA gene was subcloned in the vector and introduced into B. t. kur HD 119, expression of the cryZZZA gene did not interfere with expression from native cry genes. In this paper we have clearly demonstrated that our B.t. cloning vector is useful for stably introducing cloned genes into B.t. strains without displacing native plasmids.

RESULTS

AND DISCUSSION

(a) Isolation of the ovi from a 75-kb thuringiensis HD73 B.t. kur KToP(Lereclus et al., 1983) plasmids of 7.8, 8.4, and 11.7 kb, two kb, one of which is predominant and

plasmid of Bacillus contains three small large plasmids of 75 contains a cryZA(c)

gene, and the Streptococcus faecalis plasmid pAMP1 that confers Er resistance (Clewell et al., 1974). The presence of pAMp1 and the loss of a small plasmid distinguishes this strain from its progenitor, B.t. kur HD73. Portions of the predominant, cryZA(c+containing 75kb plasmid were subcloned in order to localize regions essential for replication. The 75kb plasmid was purified by sucrose gradient centrifugation and digested to completion with BgZII, yielding restriction fragments of the expected sizes (Kronstad and Whiteley, 1984). The restriction fragments were isolated and ligated separately to BamHI-digested pUC18. These plasmids were then used to transform E. co/i DHSc(. The ErR gene, ermC (Monod et al., 1986) from the B. subtilis plasmid pIM13 (Mahler et al., 1980) was subcloned into each plasmid to provide a selectable marker for B.t. transformation. The resulting plasmids were used to transform an acrystalliferous strain B.t. kur HDl cryB (Stahly et al.,1978). ErR clones were obtained from a single recombinant plasmid, pSB904.1, which contains the 9.6-kb BgZII fragment of the 75-kb HD73 plasmid (Fig. la). This construct thus contains the ori from the 75-kb plasmid. Removal of pUC18 sequences from pSB904.1 yielded plasmid pSB909, which was still able to replicate in B.t. kur HDI cryB (Fig. lb), but could no longer replicate in E. coli. Southern blotting (not presented) was performed on agarose gels containing EcoRI and Hind111 digests of total DNA from KTJand HD73-21 (a derivative of HD73 from which the 75-kb plasmid containing the qlZA(c) gene has been cured; Gonzalez et al., 1982). An 829-bp SalI-AccI probe, generated from the cloned 9.6-kb Bg/II fragment, hybridized to 6.5-kb EcoRI and 7-kb Hind111 fragments of KTofl total DNA. The probe did not hybridize with any restriction fragments of HD73-21 total DNA, confirming that the isolated B.t. kur replicon was derived from the cryZA(c)containing 75-kb plasmid. (b) Localization of the ori in pSB909 The ori within the 9.6-kb BglII fragment was further localized by deleting portions of pSB909 and transforming B.t. kur HDl cryB with the resulting constructs. No ErR was conferred to the cryB strain when the 2.4-kb &z/IEcoRI region of pSB909 was deleted, indicating that the vector could not replicate in cryB without the 2.4-kb region. To verify that the 2.4-kb region from pSB909 was suf-

19 ficient for replication,

1 kb

(a) pSB904.1

Af

I

I

9.6 kb-&/II

(b) pSB909

fragment

I

----



pUC18

than 3 kb (Maciag (c) Nucleotide

ORFl

t

9.6 kb-Q/II

(c) pSB909.3

ermc

ORFl

tT&Gd I

2393

(d) pSB909.4

ermC

ORFl

(e) pSB909.5 S”

AfEWSm/BXPH

*

123

1843

Fig. 1. Cloning

the ori from a 75kb

wise deletions

were

pSB904.1

made.

clone stably replicated

of the 75kb

plasmid

plasmid.

including

in B.r. (a) pSB904.1,

9.6-kb Bg/II fragment

subtilis ErR gene (rrmC); (c) pSB909.3,

B.t. kur HD73

All five constructs

(b) pSB909,

Four stepthe original

the original clone

cloned in pUCl8

with the B.

pUC18 was deleted from pSB904.1;

the 7.2-kb EcoRI fragment

along with pUCl8

was deleted

from pSB904.1 leaving the 2.4-kb San-EcoRI region; (d) pSB909.4, 122 bp from the 5’-end and 550 bp from the 3’-end of the 2.4-kb SalI-EcoRI region were deleted from pSB909.3; into pSB909.4 EarnHI; SmaI;

to produce

(e) pSB909.5,

the B.t. cloning vector.

an MCS was inserted A, AccI; Af, AflIII; B,

C, &I; E, EcoRI; H, HindIII; K, !@I; P, PstI; S, SalI; Sm, locus. Sp, SphI; X, XbaI; Xh, XhoII; umpR, ampicillin-resistance

Methods:

Plasmid

DNA was isolated

line lysis method (Birnboim

from B.t. and E. coli by the alka-

and Doly, 1979), followed by purification

over

Qiagen tip-100 or tip-25 columns (Qiagen Inc., Chatsworth, CA) or isopycnic centrifugation in CsCl density gradients. The 75-kb plasmid of B.t. kur KTJ sucrose

was further

purified

density gradient

tion products

centrifugation

through

a

(5-25”“).

were resolved

(0.089 M Tris.borate/O.O89

by differential

Intact plasmids and restriction digeson O.Sg, agarose gels run in 1 x TBE buffer

M boric acid/O.002

M EDTA).

DNA restric-

tion fragments were isolated on agarose gels followed either by electroelution or treatment with Qiaex (Qiagen Inc.). Transformation of B.t. was performed by clectroporation using a Bio-Rad Gene (Bio-Rad Laboratories, Richmond, CA). Transformants LB

plates

was

released

containing from

50

ng

Er/ml.The

the B. subtilis plasmid

ermC pIM13

Pulser apparatus were isolated on gene

fragment

by digestion

with

Hind111 + &I. The resulting l.l-kb fragment containing ermC was ligated to Hind111 + Accl-digested pUC18 and used to transform E. co/i DHSr.

E. coli DH5r

cells containing

the err&

subclone

only This

et al., 1988).

sequence

of the Bacillus

thuringiensis

replicon Sequence determination of the 2.4-kb SalI-EcoRI region from pSB909 showed that the fragment was 2393 bp in size (Fig. 2) and contained two major ORFs. ORFl, spanning nt 449 to 1387, begins with a GUG and encodes a peptide

I

fragment

containing

plasmid was capable of replicating in B.t. kur HDl cr_vB, confirming that the ori was contained within the 2.4-kb region (Fig. lc). These results are in agreement with previous reports wherein regions of Gram’ plasmids required for replication had been localized to DNA fragments of less

.r

ermC

a plasmid (pSB909.3)

the 2.4-kb region and the ermC gene was constructed.

exhibit resistance

of 312 aa. The sequence of nt 437 to 441 (GGAGG) suggests a ribosome-binding site (Shine and Dalgarno, 1974) and an inverted repeat sequence, located within nt 1411 to 1447, could form a stem-loop transcription termination structure. ORF2, spanning nt 2393 to 1875, is incomplete, consisting of only the C-terminal region of the reading frame. No transcription termination signal was observed following the C terminus of 0RF2. For convenience, we have oriented the 2.4-kb B.t. kur replicon in reference to ORFl so that the Sal1 end (originally the BglII site in the native plasmid) was designated as 5’, or upstream from ORFl, and the EcoRI end as 3’, or downstream from ORFl. The 3’-end of the 2.4-kb B.t. kur replicon contains a portion of IR2150, a large inverted repeat sequence shown on the published restriction map of the 75-kb B.t. kur HD73 plasmid (Kronstad and Whiteley, 1984). A search of the GenBank and UEMBL nucleic acid databases revealed that nt 2393 to 1822 are identical to the 3’-end of insertion sequencelS232 from B. t. thur. (Menou et al., 1990) (Table I). No homology was detected when a comparison was made between ORFl and either the nucleic acid or protein databases. However, the 24-bp PUB 110 oriU sequence, identified by Maciag et al. (1988) had 82 and 85 “/; identity with nt 654 to 670 and nt 964 to 945, respectively, both within to Er but must be plated on LB plates containing 100 pg Er,!ml to prevent background growth of non-transformants. Synthetic oligos used as PCR and sequencing terns 391 DNA

primers

synthesizer

and used without

were synthesized (Applied

purification.

oligos were synthesized:

with an Applied

Biosystems

To produce

MCS 1 (5’-CAT

Inc., Foster

a multiple GTG

cloning

Biosys-

City. CA) site, two

AAT TCC GCG

GTA

CCC GGG GAT CCT CTA GAG TCG ACC TGC AGA) and MCS2 (5’-AGC TTC TGC AGG TCG ACT CTA GAG GAT CCC CGG GTA CCG

CGG

AAT TCA).

MCSl

and MCS2

were purified by the

ohgo-purification-cartridge method recommended by the manufacturer (Applied Biosystems Inc.). These oligos were kinased separately with T4 polynucleotide

kinase, mixed and heated to boiling, allowed to cool grad-

ually to room temperature, and ligated in 1000-fold molar excess with the HindIII-AjfIII fragment from pSB904.2AA3 to construct pSB909.5.

20 TABLE

I 60

tl.r. kur rep&con homology

search” 120

Reginnh

‘Ii) Identity’

Locus”

(1)

(2)

(3)

180 TAATTAAAAAAGAGATTTATGATATAGTTATTTCATATAAACAGGAAATTAAGTTCAATT 240

ORFl,

312 aa

None

s AAAAAATAAAACTCCACCCACCGTTTI'CAATTGATTCGAAGTTTGG CATTTAAAAAAGAC 300

(nt 449-1387) 0RF2,

112 aa

aa 79-250

100

of B.r. rhur.

IS232 orf2 (250 aa prot)

(nt 2393-1873) nt 2393-1822

nt 1612-2183

100

of B.t. rhur.

82

24-bp plJB 110 on’Li sequence (nt 43 1S-4292)

85

nt 964-945

360 420

~CT~~T~TTTT~C~TATC

GGGCTGGAAATATTAG-TTTTTTCGTGTTATT AAAAAATTTTATATTAAGCAAAGC 480 MLLKNFILSKA SD 11

IS232 (2184 bp) nt 654-670

T~CTA~T~TT~CC~CGTTAG~~GTTT~TTAC-TT~TA~ATGGT T~TTATAT~T~CT~T~GTTTTGT~T

24-bp PUB 110 c>n’t/ sequence (nt 43 1S-4292)

AAAACGAG9RCTAAGTCCTC?+ACTG~TCGTATATCTACT~CCAGGAAATATTAATAA 540 31 KRELSPQLDRISTKPGNINK AGCCGT~TTCT~GGAGATTGAT~rCATTCTTTTAG~~CCAG~CTTTAC LKEIDIILLEDQGFT AVXKi

600 51 660 71

_LThe 2.4-kb B.t. kztr replicon GenBank,

EMBL,

nt and aa sequences

PIR, and SwissProt

quenccs using the FASTDB as well as the TFASTA

data bases for homologous

search program search

program

wcrc used to search

(Pearson

and Lipman,

and BESTFIT

analysis

se-

120 91

TTATCTTGTTCACUiAGGTTTTTCTTGTGTGAAACAGGATGCGGG YLVHEGFSCVKQDVIAQKAG

1988) (De-

AATTTCAAAACCCCTTATCCTCTTTCTTGGCTA ISKPLINETLSWLEKLGICH

GAAAAACTTGGTATCTGCCA 780 111

vereux cl al.. 1983). h Regions of the replicon

showing

nt or aa homology

to sequences

data bases. ‘ U, identity between ’ Loci retrieved in column

sequences

given in columns

from the databases

showing

(I) and (3).

homology

840 131

in the

to regions given

(I).

900 TAATAATTATCAAAAAATCATAGATTACTTTAAACACAAATGGCATCTAGCAATTGAGAT 151 NNYQKIIDYFKHKWHLAIEI 960 AACAGAAACTATCTCAAACCTAATATCCAAGTGGACTCTATTAU,?+GAAAX&AAG?+AGA 171 TETISNLISKWTLLKEKKEE

3.7. rhur. is B.I. subsp. thwingiensb.

ORFl (Table I). It is not clear whether these sequences serve the same purpose as those in PUB 110. (d) Determination of the minimal replicon boundaries UnidirectionaI deletions were performed by ExoIII digestion (Henikoff, 1984) into the 3’-end of the 2.4-kb S&IEcoRI region carried in the E. coli vector pSport (Gibco BRL, Gaithersburg, MD). Deletion endpoints were determined by nt sequencing. Constructs with deletions occurring approximately every 200 bp were isolated from E. co/i and electroporated into B.t. kur HDl cryB. At least 481 bp could be deleted from the 3’-end of the 2.4-kb region without destroying the ability to replicate normally (Fig. 3b). However, the replication function was completely destroyed when the region was deleted to nt 1626. Thus, the 3’-end of the mininlum sequence required for replication lies between nt 1626 and 1912. Deletions extending to nt 1500, 1383, and 1190 also destroyed replication capacity (not shown). Deletions extending to nt 1912 eliminated only portions of the IR21.50 sequence (Fig. 3b), but deletions to nt 1626 eliminated 195 nt beyond the probable 3’ -end of IR2150, into regions apparently essential for replication. To determine how much DNA could be removed from the 5’-end of the 2.4-kb region without affecting replication, sequential deletions were performed using available or engineered restriction sites. Only the construct lacking 122 bp, designated pSB904.2.4A3, was capable of replication in

1020 191 I.080 211 1140 ACAAAGAGCATGGCACTATATTATGAGTTCTCCATTCT~CATTTACT~TCTGTCT~GATGC 231 QRAWHYIMSSPFTNLSEKDA ATATGCAATTGC~TAGAATGCCCCA~TA~GACAGAGATGCTTGGTATTTCTTCAG YAIANRMPPDIDRDAWYFFR

L200 251

A~~TGCC~TCG~~G~AGT~G~~T~~GT~~TGCTTA~T~T QAADRFEASKADKSNAAYFI

L260 271

TGAAATATTTAGCCAAAACTATAAATCTTATTTAAAACGT AAAAAAGAAGAAGCTGAAAAL320 291 EIFSQNYKSYLKRKKEEAEK L380 311

ATATGTTTCATCTTTATCCCkU.ACAA?,AGTTAAAAGG YVSSLSKPKQKLIIYDFIKG GGAATAATCTTGCTTAAAATCGCCATTTTTAGCCTTGT _--__----_-> E *

L440 of the B.t. strains examined. The strains were also examined for cry gene content by PCR amplification using primers specific to each crJ>gene. A positive reaction with the B.t. kur replicon primers correlated with a positive reaction with either the q,ZA(b)- or crylil(cj-specific primers. This result suggested that the crlllA(b) and cryIA(c) genes from different B.t. strains are carried on a family of closely related plasmids having replication oris homologous to that of the 75-kb plasmid of B.t. kur HD73.

Subspecies

PCR reactionh r,ryIA(b)

B.t. kur replicon

+

HDI

_ _ _ +

HD12 HD21 HD31 HD52 HD74 HD78

_

HD85 HD88 HD89 HD102

+ + + + + + + + + + +

HD112 HD119 HD125 HD127 HD131 HD132 HD133 HD134 HD136

(f) Characterization of plasmid behavior and compatibility The B.t. cloning vectors constructed in this study exhibited a high degree of concatemerization. When analyzed by agarose gel electrophoresis, the vectors appeared as a ladder of multiple bands which was reduced to a single band by digestion with a restriction enzyme that cleaves the replicon just once. The degree of concatemerization was increased upon deletion of nonessential portions of the replicon. The monomeric conformation predominated with pSB909, which contains the 9.6-kb BglII fragment. In contrast, a high degree of concatemerization was observed with pSB909.3 and pSB909.4, indicating that some function(s) involved in plasmid resolution may be located on portions of the 9.6-kb BglII fragment outside of the boundaries of the 2.4-kb region. Plasmid pSB909, upon transformation into B.t. kur KT,, met1 (a pAMpl-cured auxotrophic derivative of KTJ), as anticipated, displaced the original 75kb plasmid from which it was derived, demonstrating the incompatibility of these two plasmids. When B.t. kur HD 119 was transformed with pSB909, a 68-kb plasmid was lost, and the loss of this plasmid was accompanied by the loss of the cryIA(b) gene (Table IIIA). This result suggested, as discussed earlier in section e, that cr),IA(c) in B. t. kur HD73 and qlIA(b) in B. t. kur HD119 are carried on plasmids composed of homologous, and therefore incompatible, oris. When B.t. kur KT, met1 was transformed with pSB909.3 or pSB909.4, the 75kb plasmid was retained in about 859; of the transformants (Table IIIA), indicating that incompatibility determinants carried on pSB909 had been re-

cryIA(c’)

HD137 HD147 HD162

+ + + + + _ + +

HD198 HD199 HD203 HD207 HD228 HD245 HD248 HD263 HD272

+ + + + _

HD274 HD282 HD283 HD301 HD395

_

+ + + + + _ _ t _ _ _

+

t + + + + + + + + + + + + + + + + + + +

_ t _ _ t _ + _ _ + _ + _ _ _ _ _ _

HD501 HD541 B.t.

t

.I All HD strains were obtained

from the USDA.

B.C. .wtto was obtained

from T. Iiruka, Hokkaido University, Japan. h PCRs were performed with a Perkin Elmer Cetus DNA Thermal cler using Amplitaq CA) according

DNA Polymerase

to the manufacturer’s

(Perkin

recommendations.

Primers

for each cry gene and the B.t. kur replicon were used in separate The following

primers

Cy-

Elmer Cetus, Emeryville,

were used: for cryIAlb), primers

specific

reactions.

TY6 (5’-GGT

CGT GGC TAT ATC CTT CGT GTC ACA GC) and TY 13 (5’-ACA GAA GAA TTG CTT TCA TAG GCT C); for cryIA(c), primers TYlO (5’-GTA (5’-GGT

TGG GGA CCG GAT TCT AGA GAT TGG) and TY 1 I AGA TGT CGA TGG GAA GTG A); for the B.C. kur repli-

con, primers

Btl13 (5’-GAA

TCA AGC CTA GGC

ACT AGG

TTG)

and Bt898 (5’-CTC AAT TGC TAG ATG CCA TTT GTG) were used. ( t ) indicates a positive reaction yielding the correct sire product: ( - ) indicates no reaction. No anomalous products wcrc obtained.

23 TABLE

III

(A) Plasmid

displacement

in B.t. kur strains following

Vector/ Construct”

transformation

Strains: KT,

met1

HD119

cryIAic)

crJllA(b)

O” lossb pSB909 pSB909.3

100 17

100 100

pSB909.4 pSB920

13 NA

31

(B) Segregational

stability

Vector/

Insert‘

0

of B.t. kur HD73 75kb

plasmid

derivatives

in alternate

hosts

Strains

Construct” HD 1 cry B

KT,

met1

KT, met1 [ + 75-kb]

[ - 75kb] V

S

V

S

V

HDll9

HD119

[ - 6%kb]

[

V

S

t 6&kb]

V

S

S

9, ErR ’ pSB909

9.6-kb &/II

100

100

100

100

NA

pSB909.3

2.4-kb SalI-EcoRI

85

100

100

ND

pSB909.4

1.7-kb min. on’

90

75 82

92

94

pSB920

pSB909.5::crrItIA

68

23

Table (A) Displacement and verified by agarose tions without

of native plasmids gel electrophoresis

selective pressure.

Dilutions

selective media. The day cultures 30°C

the sporulatcd

cultures

ND

NA 93

NA

79 ND

NA 100

94

88

29

16

70

following transformation was determined by PCR amplification with primer sets specific for the native cr)’ gcnc of isolated plasmid DNA. Table (B) Plasmid stability was determined after growing cultures for ten generaof the day culture were spread

were also used to inoculate

were heated

90

ND 100

at 65’C

onto LB plates and left at 3O’C overnight.

CYS media to undergo

for 45 min, to kill any vegetative

sporulation

in the absence

cells, and dilutions

Colonies

of selection.

were replica plated onto After 40 h of culturing

at

were plated onto LB plates as above. Colonies

were replica plated onto selective media. ’ Vector or construct introduced by electroporation. h 4; transformed colonies of KT, met1 which lost cryIA(c), or “/, transformed ’ Composition of the vector/construct introduced into host strains. d “J,,colonies containing which contain

the vector/construct,

oris homologous

as determined

by Em. [-,’ t 75kb]

to the ori of the vector/construct.

Abbreviations:

moved. Since the 75kb plasmid was 100% stable in native B.t. kur KT, metl, the slight loss of that plasmid in the transformants containing pSB909.3 or pSB909.4 might be attributed to segregation caused by the increased number of plasmids present in the cells. Mock transformants of B.t. kur KT, metl, electroporated in the absence of any DNA, retain the 75kb plasmid, indicating that loss of the 75kb plasmid is not an artifact of electroporation. When B.t. kur HD119 was transformed with pSB909.3, the crylA(bj-containing 6%kb plasmid was lost (Table IIIA). In contrast, transformants of this strain containing pSB909.4 retained the 68-kb plasmid. These results again suggest that the 75-kb plasmid in HD73 and the 68-kb plasmid in HD 119 share similar incompatibility determinants, and that pSB909.3 still contains sufficient genetic

colonies

of HD I 19 which lost cryIA(b).

and [-I + 68-kb] denotes NA, not applicable;

the absence/presence

ND, not determined;

of plasmids

S, spores:

of given size,

V, vegetative

cells.

information to control replication of the 6%kb plasmid from HD119, but not of the 75-kb plasmid from HD73. Segregational stability of plasmids pSB909, pSB909.3 and pSB909.4 was determined in several B.t. kur strains (Table IIIB). Plasmid pSB909 was highly stable during vegetative growth and sporulation, whereas both pSB909.3 and pSB909.4 showed some loss in stability that may be attributable to incomplete segregation due to their high degree of concatemerization. During sporulation, pSB909.4 in B. t. kur KT, met1 was less stable in the presence of the native cr_vIA(c)-containing 75-kb plasmid than in its absence, indicating that some competition between these two plasmids may have occurred (Table IIIB). Plasmid pSB909.5 readily transformed B. subtilis and B.t. tenebrionis (not shown).

24 (g) Cloning and expression of a cvyZZlA gene The cr_vIZZAgene from B.t. tenebrionis, which encodes a protein that forms a flat square crystal, was placed under the control of the cryZC promoter from B.t. aiznwai and subcloned into pSB909.5, yielding pSB920. The cryZZZA gene was isolated by cloning, into pTZ18R (Pharmacia LKB, Piscataway, NJ), 2.5 to 3.5-kb Hind111 fragments from a digest of B.t. tenebriorzis total DNA. Clones carrying the cryZZZA gene were detected by colony lift hybridization using a 42-nt oligo whose sequence was derived from the published cryZZZA sequence (Sekar et al., 1987). A unique NcoI site was engineered into the clone at the cryZZZA start codon. The c~~~ZCgene from B.t. aizawai HD229 was isolated by cloning 6- to 9-kb EcoRI genomic fragments into i,ZapII (Stratagene, La Jolla, CA). Clones carrying the cry/C gene were detected by plaque lift hybridization using a 972-bp probe generated by amplifying HD229 total DNA with primers specific for the B.t. entonzocidus cryZC gene (Honee et al., 1988). A unique NcoI site was engineered into the clone at the cryZC start codon to facilitate subcloning the promoter fragment. A construct made of the cryZC promoter ligated at the introduced NcoI site to the complete cryZZZA coding region including the terminator, thereby replacing the cryZZZA promoter, was subcIoned into pSB909.5 yielding pSB920. Plasmid pSB920 was introduced into B. t. kur HD 119 by electroporation. B.t. kur HD119 is known to produce CryIA(a), CryIA(b), and CryIA(c) proteins which form bipyramidal crystals as well as CryIIA proteins which form cuboidal crystals. The addition of pSB920 to this strain resulted in the loss of the 68-kb plasmid in 3 1 T’, of the transformants (Table IIIA). A transformant clone maintaining all of the native cry genes was grown in CYS until sporulation was complete. Phase contrast microscopy revealed the presence of three types of crystals (bipyramidal, cuboidal, and flat square) within a single bacterial cell, indicating that the introduced cryZZZA gene was coexpressed along with the native cry genes. Segregational stability of pSB920 in HDl19 was lower than that of pSB909.4, particularly during sporulation (Table IIIB). This decrease in stability may be due to the increased size of the plasmid or to transcription of the cryZZZA gene during sporulation. PCR screening of isolated colonies from native B.t. kur HD119 indicated that the cryZA(bj-containing 68-kb plasmid was inherently unstable with a loss rate of approx. 5 % during sporulation. In transformed B. t. kur HD 119 carrying either pSB909.4 or pSB920, the 68-kb plasmid was lost at a higher frequency (approx. lo-20%, respectively) indicating that the introduced plasmid competed with the native 68-kb plasmid during sporulation. Spore-crystal mixtures from native HDl19 and HD119[pSB920] were analyzed by SDS-PAGE (Fig. 4).

1

2

3

kDa

Fig. 4. SDS-PAGE and transformed

analysts derivatives

_

CryIA

/ ’

CryIIIA CryIIA

01. aporc-cl-yatnl

prcparat~ons from native

of B.t. kur HD119.

Lanes

1; SDS-PAGE

protein size standards; 2; HDI 19; 3, HDl19[pSB920]. Methods. All strains wcrc grown in CYS medium (10 g Difco Bacto Casitonc:5 g glucosc!2 g Difco Bacto Yeast Extract;‘1 g KH,PO, (all per litcr)/O.S mM MgCI,/O.OS mM MnC12/0.05 mM ZnSO,/O.OS mM F&1,/0.2 mM C&I,) at 30°C until fully sporulated were examined

from the lysed cultures resuspended

and lyscd (40 h). Spore-crystal

by phase-contrast

microscopy

wcrc centrifuged

preparations

and SDS-PAGE.

Samples

for 5 min and the pcllcts were

in an equal volume of water. Equal volumes of crystal prep-

aration and double-strength

Laemmli (1970) buffer wcrc mixed and heated

in a boiling water bath for 5 nun prior to running on lo>” polyacrylamide gels. Gels wcrc stained with Coomassie Brilliant Blue R-250.

Quantitation of expression levels of cry genes by laser scanning densitometry showed that expression of the newly introduced cr_vZZZAgene did not interfere with the expression of the native cry genes (not shown). This was further evidenced by results obtained from bioassays conducted with spore-crystal mixtures from HD119 and HD119[pSB920] (Table IV). The activity of the pSB920bearing transformant on Trichoplusiu ni (cabbage looper) and Spodoptem exigua (beet armyworm) was indistinguishTABLE

IV

Insecticidal

activity of wt and recombinant

Strait?

B.I. kur HDI 19 strains”

LC,,,h (for target insect larvae):

HDl19

Spodopteru

Trichoplusict

Leptinotcmu

exiguct

ni

texunu

12,503

HDI 19[pSB920]

9,855

[ f 3.1661 [ f 2,452]

1,191 1,031

[ i 2451 [ i 2511

not active’ 9,399

[ f 1,657]

(’ The wt and recombinant strams were grown in shake flasks for 40 h at 3O’C in CYS to allow complete sporulation. Serial dilutions of the sporulated culture were mixed with the insect diet. Bioassays were conducted at five rates on 25 second-instar larvae per sample dose with three separatc

culturings

h 50”,,

of each isolate.

Mortality

was determined

after three

or four days (Spodoptrru exigua and Trichoplusiu ni). lethal concentrations (LC,,) were dctcrmined by Logit analysis

(Leptirtotursu

texma)

(Berkson, 1953). and arc expressed in ppm of sporulatcd to the insect diet. Standard error is indicated in brackets. ’ No mortality

at 20000 ppm.

culture

added

25 able from that of the wt host, HD119, whereas only the recombinant strain was active against Leptinotarsa texana, a coleopteran beetle.

insect

closely

related

to the false potato

ing bioassays

with quanta1 response

Am. Stat. Assoc. Birnboim,

based on the logistic function.

H.C. and Doly, J.: A rapid alkaline

screening recombinant 1513-1523.

plasmid

DNA.

(h) Conclusions (I) The origin of replication

of the 75kb

plasmid

con-

taining the cryZA(c) gene from B.t. kur HD73 was localized to a 2.4-kb BglII/EcoRI fragment. The nt sequence of the 2.4-kb fragment was determined. Deletion analysis showed that the minimal nt sequence required for replication was 1721 bp in length and contained a single ORF, encoding a putative 3 12-aa protein. (2) Homologous replicons were shown to be present in several B.t. subspecies by PCR analysis, and were correlated with the presence of either the cryIA(b) or cryIA(c) gene, indicating that these genes are carried on closely related plasmids. (3) Removal of nonessential sequences increased concatemerization and decreased segregational stability of vectors derived from the B.t. kur replicon. Segregational stability was predominantly affected during sporulation. (4) Vectors containing the minimal replicon were able to coexist with native plasmids having homologous replicons. In this way, it was possible to introduce a cloned cryIZZA gene into B.t. kur HD 119 while preserving the native cr.y gene complement of this strain. Expression of the introduced cryZIIA gene did not interfere with native cry gene expression. While preparing this manuscript, the sequences for the oris of three large plasmids from B.t. kur HD263 were published (Baum et al., 1991). Nucleotides l-1901 from the 2.4-kb B.t. kur HD73 replicon were identical to nt 349-2249 from ori44, with a nt base change (T30’ +C). However, the report failed to identify the minimal replicon or to demonstrate the removal of incompatibility determinants.

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N.. Nicholls,

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G.M.

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Characterization and properties of a novel plasmid vector for Bacillus thuringiensis displaying compatibility with host plasmids.

A novel plasmid vector, composed of a 1.7-kb Bacillus thuringiensis (B.t.) replicon, a multiple cloning site, and an erythromycin-resistance marker ge...
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