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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