Bruce C. Carlton

CORRELATION BETWEEN SPECIFIC PLASMIDS AND DELTA-ENDOTOXIN PRODUCTION IN BACILLUS THURINGIENSIS

Abstract Five strains of Bacillus thuringiensis that produce crystalline δ-endotoxin were used as parental strains in an effort to isolate acrystalliferous (Cry−) mutants: HD-2 (B. thuringiensis var. thuringiensis, flagellar serotype 1); HD-1 and HD-73 (both var. kurstaki, serotype 3ab); HD-4 (var. alesti, serotype 3a); and HD-8 (var. galleriae, serotype 5ab). The parental strains contain complex plasmid arrays that have been previously characterized ( Gonzalez and Carlton, 1980 ). The plasmid patterns of both Cry− and Cry+ variants were analyzed and compared to the parental strains using a modified Eckhardt (1978) lysate-electrophoresis method. Most Cry− mutants derived from strain HD-2 were found to exhibit a distinctive colony morphology which facilitated their isolation. Loss of crystal production was associated with loss of a 75-Md plasmid. A 50-Md plasmid of strain HD-73 was lost in the Cry− mutants. Crystal production in strain HD-4 appears to be associated with a plasmid about 105 Md in size; in strain HD-1, a smaller plasmid (29 Md in size) seems to be involved. In strain HD-8, a large plasmid (~130 Md in size) is implicated in crystal production. Direct bioassay of several of the mutant strains has confirmed the loss of δ-endotoxin activity in the acrystalliferous isolates. The evidence obtained supports the notion of a relationship between specific extrachromosomal DNA elements and δ-endotoxin production in B. thuringiensis, and suggests that in each strain only a single plasmid is involved, although the size of the implicated plasmid varies from one strain to another.

A large transmissible plasmid is required for crystal toxin production in Bacillus thuringiensis variety israelensis

Bacillus thuringiensis var. israelensis (BTI), serotype 14, which produces parasporal crystals toxic to certain dipteran larvae, was analyzed by agarose gel electrophoresis and found to contain a complex plasmid array. Eight plasmids were detected, with approximate sizes of 3.3, 4.2, 4.9, 10.6, 68, 75, 105, and 135 MDa, as well as a plasmidlike linear DNA element of approximately 10 MDa. Partially cured mutants of BTI implicated the 75-MDa plasmid in crystal production. Fifteen independently isolated acrystalliferous (Cry-) mutants were found to lack this plasmid. In plasmid transfer experiments, several of the BTI plasmids transferred into a plasmid-free, Cry- BTI recipient, but only transfer of the 75-MDa plasmid converted the recipient to crystal toxin production. The presence or absence of mosquito-toxic activity in all Cry+ and Cry- variants of BTI was confirmed by bioassay of sporulated cultures against larvae of Aedes aegypti. Southern blot analyses revealed that in one unusual Cry+ variant in which no 75-MDa plasmid band was detectable, plasmid sequences were still present, possibly integrated into the chromosome. The 75-MDa plasmid could also apparently recombine with the 68-MDa plasmid, to which it was partially homologous.

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

Abstract Five strains of Bacillus thuringiensis which produce crystalline δ-endotoxin, and two acrystalliferous strains, have been examined for the presence of circular duplex DNA. Extrachromosomal DNA from each strain, obtained by a cleared lysate technique, was analyzed by agarose gel electrophoresis and, in one case, by electron microscopy. Each strain, whether crystal producing or acrystalliferous, contained a different complex array of plasmids. Plasmid sizes ranged from approximately 2 to 80 megadaltons, and the complexity of the size distribution ranged from as few as 3 in one strain to as many as 11 distinct plasmids in another. These results show that lack of crystalline toxin production is not necessarily accompanied by absence of all plasmids.

Selection for resistance to Bacillus thuringiensis subspecies israelensis in field and laboratory populations of the mosquito Aedes aegypti.

Abstract One laboratory and two wild populations of the yellow fever mosquito, Aedes aegypti , were used in an attempt to artificially select for resistance to Bacillus thuringiensis subsp. israelensis . After 14 generations of LC 50 selection pressure, a small but statistically significant shift (twofold increase) in response was observed in the F 15 generation of one wild strain only. Regression lines, LC 50 values, and slopes of parental populations between strains did not differ significantly.

Molecular cloning of the delta-endotoxin gene of Bacillus thuringiensis var. israelensis

A transformant of Bacillus megaterium, VB131, was isolated which carries a 6.3-kb XbaI segment of the crystal toxin gene of Bacillus thuringiensis var. israelensis (BTI) cloned in a vector plasmid pBC16 to yield pVB131. The chimeric plasmid DNA from VB131 was introduced into a transformable Bacillus subtilis strain by competence transformation. Both the B. megaterium VB131 strain and the B. subtilis strain harboring the chimeric plasmid produced irregular, parasporal, phase-refractile, crystalline inclusions (Cry+) during sporulation. The sporulated cells as well as the isolated crystal inclusions of the pVB131-containing B. megaterium and B. subtilis strains were highly toxic to the larvae of Aedes aegypti. Also, the solubilized crystal protein preparation from VB131[pVB131] showed clear immuno cross-reaction with antiserum to the BTI crystal toxin. 32P-labeled pVB131 plasmid DNA showed specific hybridization with a 112-kb plasmid DNA of Cry+ strains of BTI, and no hybridization with other plasmid or chromosomal DNA of either Cry+ or Cry- variants. These results are in agreement with our previous findings (González and Carlton, 1984) that the 112-kb plasmid of BTI is associated with the production of the crystal toxin.

Construction and application of a modified "gene machine": a circular concentrating preparative gel electrophoresis device employing discontinuous elution.

Abstract A modified version of a preparative circular gel electrophoresis apparatus, first described by Edwin Southern (Medical Research Council, University of Edinburgh, Edinburgh, Scotland), has been constructed. The apparatus fractionates a large volume of sample into concentric bands which migrate toward a small circular collection chamber. Samples exiting the gel into the collection chamber are concentrated against a dialysis membrane which encloses the inner electrode and are pumped from this center chamber into a fraction collector at fixed time intervals. The apparatus has been employed to fractionate samples of DNA (10 mg) by electrophoresis through either agarose or acrylamide gels. Two examples of nucleic acids which have been successfully fractionated are given: restriction endonuclease cleavage fragments of total soybean DNA, and a heterogeneous mixture of covalently closed circular plasmid DNA from Bacillus megaterium. Franctionated DNA is suitable for molecular cloning directly from acrylamide and, after one additional treatment, from agarose. The run time for DNA treated with restriction endonuclease is from 24 to 48 h. Purification of 60- to 200-fold is common for a DNA restriction fragment from a total genome.

Conjugal Plasmid Transfer in Bacillus Thuringiensis

Bacillus thuringiensis (BT) is a gram-positive, sporeforming bacterium distinguished by its ability to produce parasporal protein crystals. These proteins, collectively known as δ-endotoxins, are toxic to a number of lepidopteran insects of economic importance as well as to certain dipterans which are important vectors of human disease (7). Over 20 different varieties of B. thuringiensis are currently recognized on the basis of flagellar antigens (6) and immunological (19) and toxicity (8) properties of the parasporal crystals.

Physical mapping of a plasmid from Bacillus megaterium by restriction endonuclease cleavage.

Abstract A 4-Mdalton plasmid from Bacillus megaterium strain 216 has been physically mapped by restriction endonuclease digestion. A combination of single and double digests with seven restriction enzymes, together with a terminal labeling procedure, has produced a physical map containing 21 apparently unique cleavage sites. The data are most consistent with the view that this plasmid does not contain extensive variability in sequence.

Characterization of the plasmids of Bacillus megaterium: Restriction endonuclease digestions, Southern blotting analysis, and partial denaturation mapping

Abstract Three plasmids of the complex plasmid system of Bacillus megaterium strain 216 have been physically characterized by restriction endonuclease digestions, DNA-DNA hybridizations by the Southern blotting technique, and partial denaturation mapping by electron microscopy. The results show that each plasmid size class produces a unique pattern of restriction endonuclease digestion fragments and anneals only to molecules of the same plasmid size. Partial denaturation reveals unique and consistent differential melting patterns for each plasmid. It is concluded that each of these three plasmid species, and probably the larger ones as well, represents a molecular entity of unique and distinct nucleotide sequence, unrelated to each other or to the chromosome of the bacterium.

Selective inhibition of plasmid DNA production in Bacillusmegaterium by 6-(p-hydroxy-phenylazo)-uracil: Evidence for multiple maintenance systems

Summary The substituted pyrimidine analog 6-(p-hydroxy-phenylazo)-uracil, a specific inhibitor of DNA replication in Gram-positive bacteria, inhibits incorporation of radioactive nucleotides into DNA, but not RNA, of Bacillus megaterium at sub-bacteriocidal levels. Incorporation into chromosomal DNA is much more severely inhibited than into the polydisperse circular DNA of unknown function produced by the organism. The effect on the circular fraction appears to be primarily on the larger molecules, since incorporation into the two smallest plasmids appears insensitive to the drug. These results suggest that at least two maintenance systems are involved in the production of this complex array of plasmid molecules.