Plasmid transformation of Bacillus sphaericus 1593

Plasmids pUB110 and pBC16 were introduced by protoplast transformation into the entomocidal bacterium Bacillus sphaericus 1593. Transformants expressed the antibiotic resistance determinants present on the plasmid and exhibited sporulation frequencies and larvicidal toxicities which were equivalent to those characteristic of the parent strain. These transformations constitute the first report of genetic transfer in B. sphaericus.     

Molecular cloning of the 130-kilodalton mosquitocidal delta-endotoxin gene of Bacillus thuringiensis subsp. israelensis in Bacillus sphaericus

A 3.7-kilobase (kb) XbaI fragment harboring the cryIVB gene (L. Thorne, F. Garduno, T. Thompson, D. Decker, M. A. Zounes, M. Wild, A. M. Walfield, and T. J. Pollock, J. Bacteriol. 166:801-811, 1986) which encoded a 130-kilodalton (kDa) mosquitocidal toxin from a 110-kb plasmid of Bacillus thuringiensis subsp. israelensis 4Q2-72 was cloned into pUC12 and transformed into Escherichia coli. The clone with a recombinant plasmid (designated pBT8) was toxic to Aedes aegypti larvae. The fragment (3.7 kb) was ligated into pBC16 (tetracycline resistant [Tcr]) and transformed by the method of protoplast transformation into Bacillus sphaericus 1593 and 2362, which were highly toxic to Anopheles and Culex mosquito larvae but less toxic to Aedes larvae. After cell regeneration on regeneration medium, the Tcr plasmids from transformants (pBTC1) of both strains of B. sphaericus were prepared and analyzed. The 3.7-kb XbaI fragment from the B. thuringiensis subsp. israelensis plasmid was shown to be present by agarose gel electrophoresis and Southern blot hybridization. In addition, B. sphaericus transformants produced a 130-kDa mosquitocidal toxin which was detected by Western (immuno-) blot analysis with antibody prepared against B. thuringiensis subsp. israelensis 130-kDa mosquitocidal toxin. The 50% lethal concentrations of the transformants of strains 1593 and 2362 against A. aegypti larvae were 2.7 X 10(2) and 5.7 X 10(2) cells per ml, respectively. This level of toxicity was comparable to the 50% lethal concentration of B. thuringiensis subsp. israelensis but much higher than that of B. sphaericus 1593 and 2362 (4.7 X 10(4) cells per ml) against A. aegypti larvae.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning of the 130-kilodalton mosquitocidal delta-endotoxin gene of Bacillus thuringiensis subsp. israelensis in Bacillus sphaericus.

A 3.7-kilobase (kb) XbaI fragment harboring the cryIVB gene (L. Thorne, F. Garduno, T. Thompson, D. Decker, M. A. Zounes, M. Wild, A. M. Walfield, and T. J. Pollock, J. Bacteriol. 166:801-811, 1986) which encoded a 130-kilodalton (kDa) mosquitocidal toxin from a 110-kb plasmid of Bacillus thuringiensis subsp. israelensis 4Q2-72 was cloned into pUC12 and transformed into Escherichia coli. The clone with a recombinant plasmid (designated pBT8) was toxic to Aedes aegypti larvae. The fragment (3.7 kb) was ligated into pBC16 (tetracycline resistant [Tcr]) and transformed by the method of protoplast transformation into Bacillus sphaericus 1593 and 2362, which were highly toxic to Anopheles and Culex mosquito larvae but less toxic to Aedes larvae. After cell regeneration on regeneration medium, the Tcr plasmids from transformants (pBTC1) of both strains of B. sphaericus were prepared and analyzed. The 3.7-kb XbaI fragment from the B. thuringiensis subsp. israelensis plasmid was shown to be present by agarose gel electrophoresis and Southern blot hybridization. In addition, B. sphaericus transformants produced a 130-kDa mosquitocidal toxin which was detected by Western (immuno-) blot analysis with antibody prepared against B. thuringiensis subsp. israelensis 130-kDa mosquitocidal toxin. The 50% lethal concentrations of the transformants of strains 1593 and 2362 against A. aegypti larvae were 2.7 X 10(2) and 5.7 X 10(2) cells per ml, respectively. This level of toxicity was comparable to the 50% lethal concentration of B. thuringiensis subsp. israelensis but much higher than that of B. sphaericus 1593 and 2362 (4.7 X 10(4) cells per ml) against A. aegypti larvae.(ABSTRACT TRUNCATED AT 250 WORDS)     

Construction of hybrid plasmid vectors for cloning inEscherichia coli,Bacillus subtilis,and the cyanobacteriumAnacystis nidulans

Two hybrid plasmids capable of acting as shuttle cloning vectors inAnacystis nidulans andBacillus subtilis were constructed by in vitro ligation. One construct, pMG202, consists of theB. subtilis vector pNN101 and the endogenous cyanobacterial plasmid pUH24. This 14.6 kb plasmid confers chloramphenicol resistance in both hosts and tetracycline resistance inB. subtilis. A second vector, pMG101, consists of pNN101 linked to theA. nidulans-Escherichia coli chimeric plasmid pCB4 and is 12.9 kb in size. The pCB4 portion of the vector enables pMG101 to replicate in the third host,E. coli, and confers ampicillin resistance in this bacterium as well as inA. nidulans. Both plasmids possess identical uniqueStu I sites which permit insertional inactivation of the chloramphenicol resistance gene; and, in addition, identical uniqueXho I sites are present on both vectors. Each vector also has a third unique site:Sma I on pMG101 andXba I on pMG202.     

Plasmid transformation of Bacillus cereus protoplasts

The process of polyethyleneglycol-induced plasmid transformation of Bacillus cereus protoplasts was studied. Plasmid transfer into Bacillus cereus strains was demonstrated with the frequencies 1.3.10(1)-1.6.10(2) transformants per 1 mkg of plasmid DNA. The plasmids transferred are stably inherited by Bacillus cereus cells causing tetracycline resistance (pBC16) or kanamycin resistance (pUB110 and pBD64). The proposed method can be used for construction of Bacillus cereus strains having the plasmid determined characteristics.     

Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance.

The nucleotide sequence of pC194, a small plasmid from Staphylococcus aureus which is capable of replication in Bacillus subtilis, has been determined. The genetic determinant of chloramphenicol (CAM) resistance, which includes the chloramphenicol acetyl transferase (CAT) structural gene, the putative promoter and controlling element of this determinant, have been mapped functionally by subcloning a 1,035-nucleotide fragment which specifies the resistance phenotype using plasmid pBR322 as vector. Expression of CAM resistance is autogenously regulated since the 1,035-nucleotide fragment containing the CAT gene sequence and its promoter cloned into pBR322 expresses resistance inducibly in the Escherichia coli host. A presumed controlling element of CAT expression consists of a 37-nucleotide inverted complementary repeat sequence that is located between the -10 and ribosome-loading sequences of the CAT structural gene. Whereas the composite plasmid containing the minimal CAT determinant cloned in pBR322 could not replicate in B. subtilis, ability to replicate in B. subtilis was seen if the fragment cloned included an extension consisting of an additional 300 nucleotides beyond the 5' end of the single pC194 MspI site associated with replication. This 5' extension contained a 120-nucleotide inverted complementary repeat sequence similar to that found in pE194 TaqI fragment B which contains replication sequences of that plasmid. pC194 was found to contain four opening reading frames theoretically capable of coding for proteins with maximum molecular masses, as follows: A, 27,800 daltons; B, 26,200 daltons; C, 15,000 daltons; and D, 9,600 daltons. Interruption or deletion of either frame A or D does not entail loss of ability to replicate or to express CAM resistance, whereas frame B contains the CAT structural gene and frame C contains sequences associated with plasmid replication.     

Construction and characterization of recombinant phage phi 105 d(Cmrmet) for cloning in Bacillus subtilis

A 1.6 kb fragment of DNA of plasmid pBD64, obtained after partial digestion with HpaII, carrying a chloramphenicol-resistance determinant and a single site for the enzyme Bg/II, was inserted into the genome of defective phage phi 105 d/ys. Two types of phage were subsequently isolated and both transduced cells of Bacillus subtilis to chloramphenicol resistance. One type contained 26 kb and the other 32 kb of DNA. Bacillus subtilis chromosomal DNA fragments generated by cleavage with Bg/II were ligated into the unique Bg/II site within the smaller phage genome. A specialized transducing phage was isolated which carried the metC gene on a 6 kb Bg/II fragment. This phage, denoted phi 105 d(Cmrmet), transduced B. subtilis strain MB79 pheA12 metC3 to Met+ and to chloramphenicol resistance, and the metC3 mutation was complemented in transductants.     

Glycine-induced cryotransformation of plasmids into Bacillus anthracis

Different cloning vectors (pC194, pBC16, pUB110, pBD10, pBD8, pAM beta 1) and Bacillus anthracis plasmid pX02 were introduced into B. anthracis by a transformation method. To induce an artificial competence state for uptake of isolated plasmid DNA, the cultures were treated with glycine, to reduce cross-linking of peptidoglycan, followed by freezing and thawing. The procedure is extremely rapid and relatively efficient (maximum transformation efficiency about 10(3) c.f.u. per micrograms DNA) and allows different cloning vectors with molecular masses ranging from 1.8 to 17.7 MDa to be introduced into B. anthracis.     

Cloning vector system for Corynebacterium glutamicum.

A protoplast transformation system has been developed for Corynebacterium glutamicum by using a C. glutamicum-Bacillus subtilis chimeric vector. The chimera was constructed by joining a 3.0-kilobase cryptic C. glutamicum plasmid and the B. subtilis plasmid pBD10. The neomycin resistance gene on the chimera, pHY416, was expressed in C. glutamicum, although the chloramphenicol resistance gene was not. The various parameters in the transformation protocol were analyzed separately and optimized. The resulting transformation system is simple and routinely yields 10(4) transformants per microgram of plasmid DNA.     

Transformation of Bacillus polymyxa with plasmid DNA

A plasmid transformation system was developed for Bacillus polymyxa ATCC 12321 and derivatives of this strain. The method utilizes a penicillin-treated-cell technique to facilitate uptake of the plasmid DNA. Low-frequency transformation (10(-6) per recipient cell) of plasmids pC194, pBD64, and pBC16 was accomplished with this method. Selection for the transformants was accomplished on both hypertonic and nonhypertonic selective media, with the highest rates of recovery occurring on a peptone-glucose-yeast extract medium containing 0.25 M sucrose. Several additional plasmids were shown to be capable of transferring their antibiotic resistance phenotypes to B. polymyxa through the use of a protoplast transformation procedure which allowed for a more efficient transfer of the plasmid DNA. However, cell walls could not be regenerated on the transformed protoplasts, and the transformants could not be subcultured from the original selective media.     

Transformation of Bacillus polymyxa with plasmid DNA.

A plasmid transformation system was developed for Bacillus polymyxa ATCC 12321 and derivatives of this strain. The method utilizes a penicillin-treated-cell technique to facilitate uptake of the plasmid DNA. Low-frequency transformation (10(-6) per recipient cell) of plasmids pC194, pBD64, and pBC16 was accomplished with this method. Selection for the transformants was accomplished on both hypertonic and nonhypertonic selective media, with the highest rates of recovery occurring on a peptone-glucose-yeast extract medium containing 0.25 M sucrose. Several additional plasmids were shown to be capable of transferring their antibiotic resistance phenotypes to B. polymyxa through the use of a protoplast transformation procedure which allowed for a more efficient transfer of the plasmid DNA. However, cell walls could not be regenerated on the transformed protoplasts, and the transformants could not be subcultured from the original selective media.     

Vegetative expression of the delta-endotoxin genes of Bacillus thuringiensis subsp. kurstaki in Bacillus subtilis.

Bacillus thuringiensis subsp. kurstaki total DNA was digested with BglII and cloned into the BamHI site of plasmid pUC9 in Escherichia coli. A recombinant plasmid, pHBHE, expressed a protein of 135,000 daltons that was toxic to caterpillars. A HincII-SmaI double digest of pHBHE was then ligated to BglII-cut plasmid pBD64 and introduced into Bacillus subtilis by transformation. The transformants were identified by colony hybridization and confirmed by Southern blot hybridization. A 135,000-dalton protein which bound to an antibody specific for the crystal protein of B. thuringiensis was detected from the B. subtilis clones containing the toxin gene insert in either orientation. A toxin gene insert cloned into a PvuII site distal from the two drug resistance genes of the pBD64 vector also expressed a 135,000-dalton protein. These results suggest that the toxin gene is transcribed from its own promoter. Western blotting of proteins expressed at various stages of growth revealed that the crystal protein expression in B. subtilis begins early in the vegetative phase, while in B. thuringiensis it is concomitant with the onset of sporulation. The cloned genes when transferred to a nonsporulating strain of B. subtilis also expressed a 135,000-dalton protein. These results suggest that toxin gene expression in B. subtilis is independent of sporulation. Another toxin gene encoding a 130,000- to 135,000-dalton protein was cloned in E. coli from a library of B. thuringiensis genes established in lambda 1059. This gene was then subcloned in B. subtilis. The cell extracts from both clones were toxic to caterpillars. Electron microscope studies revealed the presence of an irregular crystal inclusion in E. coli and a well-formed bipyramidal crystal in B. subtilis clones similar to the crystals found in B. thuringiensis.     

Improvement of Bacillus sphaericus toxicity against dipteran larvae by integration, via homologous recombination, of the Cry11A toxin gene from Bacillus thuringiensis subsp. israelensis.

Integrative plasmids were constructed to enable integration of foreign DNA into the chromosome of Bacillus sphaericus 2297 by in vivo recombination. Integration of the aphA3 kanamycin resistance gene by a two-step procedure demonstrated that this strategy was applicable with antibiotic resistance selection. Hybridization experiments evidenced two copies of the operon encoding the binary toxin from B. sphaericus in the recipient strain. The Bacillus thuringiensis subsp. israelensis cry11Aal gene (referred to as cry11A), encoding a delta-endotoxin with toxicity against Culex, Aedes, and Anopheles larvae, was integrated either by a single crossover event [strain 2297 (::pHT5601), harboring the entire recombinant plasmid] or by two successive crossover events [strain 2297 (::cry11A)]. The level of the Cry11A production in B. sphaericus was high; two crystalline inclusions were produced in strain 2297 (::pHT5601). Synthesis of the Cry11A toxin conferred toxicity to the recombinant strains against Aedes aegypti larvae, for which the parental strain was not toxic. Interestingly, the level of larvicidal activity of strain 2297 (::pHT5601) against Anopheles stephensi was as high as that of B. thuringiensis subsp. israelensis and suggested synergy between the B. thuringiensis and B. sphaericus toxins. The toxicities of parental and recombinant B. sphaericus strains against Culex quinquefasciatus were similar, but the recombinant strains killed the larvae more rapidly. The production of the Cry11A toxin in B. sphaericus also partially restored toxicity for C. quinquefasciatus larvae from a population resistant to B. sphaericus 1593. In vivo recombination therefore appears to be a promising approach to the creation of new B. sphaericus strains for vector control.     

Identification, cloning and sequence analysis of the Bacillus sphaericus 1593 41.9 kD larvicidal toxin gene

A number of strains of the widespread aerobic soil bacterium, Bacillus sphaericus, possess crystalline inclusions of a toxin lethal to a variety of insect (larvae) which are vectors of major tropical diseases. Partial amino acid sequence data from one strain, B. sphaericus 2362 have permitted us to design oligonucleotide probes for identifying the toxin gene in the closely related B. sphaericus 1593. The gene was found to be contained within an EcoRI-HindIII fragment and was cloned in its entirety in the bacterial plasmid pUC12. The DNA sequence was determined together with the upstream and downstream controlling elements, and a sequence of 370 amino acids was deduced for the toxin protein. This is the first reported sequence of a B. sphaericus toxin gene and will facilitate further work in characterizing the genes from other strains of different virulence and host range. The data do not support the suggestion that the toxin is derived by proteolysis of a protoxin precursor.     

Functional mapping of a trans-activating gene required for expression of a baculovirus delayed-early gene

The temporal regulation of an early gene of the baculovirus Autographa californica nuclear polyhedrosis virus was examined. We constructed a plasmid (plasmid 39CAT) in which the bacterial gene for chloramphenicol acetyltransferase was placed under the control of the promoter for the gene for a A. californica nuclear polyhedrosis virus 39,000-dalton protein (39K). A transient expression assay of plasmid 39CAT revealed that the 39K gene was expressed in infected cells but not in uninfected cells, indicating that the 39K gene should be classified as a delayed-early gene. The 39K promoter also efficiently directed the synthesis of chloramphenicol acetyltransferase when the plasmid was cotransfected with viral DNA which had been restricted with several restriction enzymes. To map the location of the gene(s) required for the synthesis of 39K, plasmid 39CAT was cotransfected with purified restriction fragments of A. californica nuclear polyhedrosis virus DNA. Fragments which mapped between 90.7 and 100.8 map units induced plasmid 39CAT. Plasmid pEcoRI-B, containing EcoRI fragment B (90 to 100 map units), activated plasmid 39CAT. Functional mapping of plasmid pEcoRI-B indicated that the essential region was located between 95.0 and 97.5 map units. The 5' end of this gene was mapped, and the chloramphenicol acetyltransferase gene was inserted under the control of its promoter. Transient assay experiments indicated that the trans-acting regulatory gene was expressed in uninfected cells and is therefore an immediate-early gene. This gene was named IE-1.     

New plasmid vector and host system for genetic engineering in Bacillus sphaericus.

A new plasmid, pNQ116, was constructed in Bacillus sphaericus by cloning a promoter fragment from B. sphaericus Ts-1 into pNQ112. The plasmid (CmrKmr, 5.23 kb) contains a restriction endonuclease polylinker used for cloning foreign genes, and its cat-86 gene is expressed at high levels from the Ts-1 promoter. This plasmid vector has been transformed into B. sphaericus AS 1.270, AS 1.465, AS 1.469, and 2362, at frequencies of 10(2)-10(3) transformants per microgram of DNA, and is maintained stably under nonselective conditions in these host strains. The presence of pNQ116 in B. sphaericus 2362 does ot interfere with the mosquito larvicidal activity of the organism.     

Analysis of promoters in Borrelia burgdorferi by use of a transiently expressed reporter gene.

A transient chloramphenicol acetyltransferase (CAT) expression system was developed for Borrelia burgdorferi. An Escherichia coli vector containing a promoterless Streptococcus agalactiae cat gene was constructed. Promoters for ospA, ospC, and flaB were placed upstream of this cat gene, and CAT assays were performed in E. coli from these stably maintained plasmids. The plasmids with putative promoters ospA and flaB were found to be approximately 20-fold more active than were the plasmids with ospC or no promoter. The level of activity correlated well with the resistance to chloramphenicol that each plasmid provided. Next, the nonreplicative plasmid constructs were transformed by electroporation into B. burgdorferi. CAT assays were performed by both thin-layer chromatography and the fluor diffusion method. Measurement of CAT activity demonstrated that the ospA promoter was again about 20-fold more active than the promoterless cat gene. The flaB and ospC promoters increased the activity seven- and threefold, respectively, over that with the promoterless construct. This simple transient-expression assay was shown to be an effective method to study promoter function in B. burgdorferi in the absence of a well-developed genetic system.     

High copy number plasmid vectors for use in lactic streptococci

Abstract A 3.8 kb DNA fragment from plasmid pBD64 which encoded chloramphenicol and kanamycin resistance genes, but had no replication region, was used as a replicator probe to select for the replication region of the cryptic lactic streptococcal plasmid pSH71 using Bacillus subtilis as host. Three of the resultant recombinant plasmids, pCK1, pCK17 and pCK21 are described. They are vectors in Streptococcus lactis and can be used to clone Bgl II-compatible fragments into their kanamycin resistance gene. All the plasmids have single sites for restriction endonucleases Ava I, Bam HI, Eco RI, Pvu II and Xba I, while plasmids pCK17 and pCK21 have single sites for Cla I.     

Plasmid transformation in Bacillus subtilis: effects of insertion of Bacillus subtilis DNA into plasmid pC194

Summary We have constructed a hybrid plasmid, pBC1, which consists of plasmid pC194 with an insert of B. subtilis DNA at its HindIII restriction site. This plasmid is stably maintained in B. subtilis. In contrast with pC194, monomeric ccc forms of pBC1 are active in transformation. Transformations with these monomeric molecules of pBC1 have a stringent requirement for recombination proficieny., as defined by recE in the recipient cell. The extent of dependence of the transforming activity of oligomeric pBC1 DNA on the recombination proficiency of the recipient cell decreases with increasing oligomer size. A model of DNA proccssing during plasmid transformation of B. subtilis is presented.