APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1990, p. 3420-3428 Vol. 56, No. 11 0099-2240/90/113420-09$02.00/0 Copyright © 1990, American Society for Microbiology Novel Cloning Vectors for Bacillus thuringiensis JAMES A. BAUM,* DOLORES M. COYLE, M. PEARCE GILBERT, CHRISTINE S. JANY, AND CYNTHIA GAWRON-BURKE Ecogen Inc., 2005 Cabot Boulevard West, Langhorne, Pennsylvania 19047-1810 Received 28 June 1990/Accepted 22 August 1990 Seven replication origins from resident plasmids of Bacillus thuringienis subsp. kurstaki HD263 and HD73 were cloned in Escherichia coli. Three of these replication origins, originating from plasmids of 43, 44, and 60 MDa, were used to construct a set of compatible shuttle vectors that exhibit structural and segregational stability in the Cry- strain B. thuringiensis HD73-26. These shuttle vectors, pEG597, pEG853, and pEG854, were designed with rare restriction sites that permit various adaptations, including the construction of small recombinant plasmids lacking antibiotic resistance genes. The crylA(c) and cryllA insecticidal crystal protein genes were inserted into these vectors to demonstrate crystal protein production in B. thuringiensis. Introduction of a cloned crylA(c) gene from strain HD263 into a B. thuringiensis subsp. aizawai strain exhibiting good insecticidal activity against Spodoptera exigua resulted in a recombinant strain with an improved spectrum of insecticidal activity. Shuttle vectors of this sort should be valuable in future genetic studies of B. thuringiensis as well as in the development of B. thuringiensis strains for use as microbial pesticides. The gram-positive soil bacterium Bacillus thuringiensis approach will depend on the availability of suitable cloning produces proteinaceous parasporal crystals that are toxic to vectors. a select variety of insect species. Over two dozen varieties of In this report, we describe the cloning of seven replication B. thuringiensis representing different flagellar antigens (5) origins derived from resident plasmids of B. thuringiensis and insecticidal activities against lepidopteran, dipteran, or subsp. kurstaki HD263 and HD73 and the construction of coleopteran larvae have been identified (11). Since its intro- cloning vectors based on three of these replication origins. duction as a product in the early 1960s, B. thuringiensis has These vectors have features that should prove useful in the become the major biological pesticide in use worldwide, with development of commercial strains of B. thuringiensis and in several subspecies currently being used as active ingredients future genetic studies of this important organism. (3). The components of the parasporal crystals, often referred MATERIALS AND METHODS to as delta-endotoxins or insecticidal crystal proteins (ICPs), represent a diverse group of proteins that differ extensively Bacterial strains and plasmids. B. thuringiensis subsp. in structure and insecticidal activity (11). The composition of kuirstaki HD263 and HD73 were obtained from the collection ICPs found in B. thuringiensis strains varies considerably; of Dulmage (8). Strain HD73-26 is a cured derivative of even strains of the same serotype can exhibit substantial HD73 that contains a cryptic 4.9-MDa plasmid (7). Strain differences in insecticidal activity. ICPs are encoded by HD73-26-10 is an HD73-26 transconjugant strain containing genes typically found on large plasmids (>30 MDa) (10, 13), a crylA(c) ICP-encoding 44-MDa plasmid from HD263 as some of which can be transferred conjugatively. Conjugal well as the 4.9-MDa plasmid. Strain HD263-6 is a cured transfer of ICP-encoding plasmids has been successfully derivative of HD263 lacking the 44-MDa plasmid (2). B. employed at the commercial level to construct B. thurin- thuringiensis subsp. aizawai EG6346 is a cured derivative of giensis strains with improved insecticidal activities (3). Al- strain EG6345 that contains several non-crylA ICP genes. though it provides a "natural" means of altering the ICP Strain EG6345 contains, in addition to the ICP genes found gene composition of B. thuringiensis, the use of conjugation in EG6346, a cryIA(b) gene located on a 45-MDa plasmid. is limited to mobilizable genes and strains that are amenable Both EG6345 and EG6346 were obtained from the Ecogen to conjugation and by plasmid incompatibility. A recombi- strain collection. Escherichia coli TG1 (Amersham Corp.), nant DNA approach to B. thuringiensis strain construction XL-1 Blue (Stratagene Corp.), and GM2163 (kindly provided offers a greater degree of flexibility than that afforded by by New England BioLabs Inc.) were used as host strains for conjugation. subcloning. Plasmids pTZ18u and pTZ19u (U.S. Biochemi- Numerous ICP genes have been cloned, and their prod- cal Corp.) were used as cloning vectors. Plasmid pMI1101, ucts have been assessed for insecticidal activity (11). In which harbors the chloramphenicol acetyltransferase gene addition, an efficient transformation system for B. thurin- (cat) from pC194 (12), was a gift from Michelle Igo. giensis has been developed by employing electroporation DNA manipulations. Standard recombinant DNA proce- (15, 17, 23). Thus, it should be possible to manipulate the dures were performed as described by Maniatis et al. (19). production, regulation, and activity of ICPs by molecular Plasmids from B. thuringiensis HD73-26-10 and HD263-6 genetic techniques and to construct improved B. thuringien- were isolated as described by Kronstad et al. (13). Plasmids sis strains for use as microbial pesticides. The success of this from E. coli were prepared by a small-scale alkaline lysis procedure (19). For Southern blot analysis, DNAs were resolved on 1% agarose gels (Tris-phosphate buffer [19]) and transferred to Zeta-probe membranes (Bio-Rad Corp.) by * Corresponding author. using the alkaline blotting procedure recommended by the 3420 VOL. 56, 1990 NOVEL CLONING VECTORS FOR B. THURINGIENSIS 3421 manufacturer. Hybridization probes were prepared by using 500 bp the random primer method of Feinberg and Vogelstein (9). Transformants of B. thuringiensis HD73-26 harboring amp f recombinant plasmids were analyzed on 0.8% agarose gels cat- plac ori - by using a modified Eckhardt lysis procedure (10). For ........4.- restriction enzyme analysis, B. thuringiensis transformants 8u E were grown for 6 h at 30°C in brain heart infusion (Difco) EI- _ ~~~~~pTZ1 The cells were pelleted in a ~~~~~~~~~~~~.................. containing 0.5% glycerol. microfuge, frozen on dry ice, and thawed at room tempera- S BSmEStKSmBXbSPSpH ture. DNA was extracted from the cell pellets by using the E. FIG. 1. Linear restriction map of replicon cloning vector coli alkaline lysis procedure. DNAs were sequenced accord- pEG588. An EcoRI fragment from pMI1101 harboring the chloram- ing to the dideoxy chain termination method (22) with phenicol acetytransferase (cat) gene of pC194 (dark-shaded box) [cx-355]dATP and the Sequenase DNA sequencing kit (U.S. was inserted into the EcoRI site of the E. coli vector pTZ18u (light-shaded box) as shown. Restriction sites: B, BamHI; E, EcoRI; templates were prepared Biochemical Corp.). Sequencing H, Hindlll; K, KpnI; P, PstI; S, Sall; S, SmaI; Sp, SphI; St, SstI; from double-stranded DNA by procedures outlined in the Xb, XbaI. Other abbreviations: f, fl phage replication origin; ori, Sequenase manual. Synthetic oligonucleotides were gener- replication origin of pTZ18u; amp, beta-lactamase gene; cat, chlor- ated on an Applied Biosystems 380B DNA Synthesizer and amphenicol acetyltransferase gene. purified by the oligonucleotide purification cartridge method recommended by the manufacturer. Transformation of B. thuringiensis. Transformation was ics model 300A computing densitometer and purified performed by the electroporation procedure of Mettus and CryIA(c) and CryllA proteins as standards. Macaluso (20) with the Bio-Rad Gene Pulser apparatus. Western blot analysis. Crystal proteins resolved on 7.5% Electroporated cells were grown in Luria broth containing SDS-polyacrylamide gels were transferred to nitrocellulose 0.2 ,ug of chloramphenicol per ml for 1 to 2 h at 37°C before filters (Millipore HATF, 0.45-,um pore size) by electropho- plating on NSM plates (23 g of Bacto nutrient agar per liter, resis in 12 mM Tris-96 mM glycine-20% (vol/vol) methanol. 1 mM MgCl2, 0.7 mM CaCl2, 0.05 mM MnCl2) containing 5 The filters were blocked by incubation in 5% (wt/vol) nonfat ,ug of chloramphenicol per ml. dry milk-10 mM Tris hydrochloride (pH 7.5)-0.9% (wt/vol) temper- Cloning of an ICP gene from HD263. A cryIA(c) gene NaCl-0.09% (wt/vol) sodium azide for 1 h at room were located on the 44-MDa plasmid of B. thuringiensis HD263 ature. After a 10-min rinse in 0.3% Tween 80, the filters antibodies at a was cloned in E. coli by using the bacteriophage cloning incubated with CryIA(c) protein-specific vector Lambda-Dash (Stratagene). Plasmid DNA from the 1:200 dilution in TBSN (10 mM Tris hydrochloride, 0.9% which harbors the 44- NaCl, 0.1% [wt/vol] globulin-free bovine serum albumin, transconjugant strain HD73-26-10, 0.09% sodium azide, 0.05% [vol/vol] Triton X-405) for 1 h. was partially digested with MboI to yield MDa plasmid, After subsequent washes with TBSN, TBSN-0.05% SDS, DNA fragments in the 15- to 30-kb range. This DNA was intestinal alkaline phos- and TBSN, the filters were incubated for several hours with then dephosphorylated with calf a second antibody consisting of anti-mouse alkaline phos- phatase (Boehringer Mannheim Corp.), ligated to BamHI- phatase-conjugated immunoglobulin G (Sigma Chemical digested bacteriophage vector DNA, and packaged into Co.) at a 1:1,000 dilution in TBSN. After thorough washing phage particles by using packaging extracts prepared from E. with TBSN and double-distilled water, the alkaline phos- coli BH2688 and BH2690 (19). Strain NM539 (Stratagene) phatase-specific color reaction was developed with 5-bromo- was used as the host strain for phage propagation. Clones 4-chloro-3-indoyl phosphate and Nitro Blue Tetrazolium harboring the crylA(c) gene were identified by plaque hy- (Sigma). bridization with a 720-bp EcoRI fragment from the cryIA(a) Bioassay.