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Bacillus thuringiensis toxin CryIIIB2 exhibits activity against two agriculturally important pests, the Colorado potato beetle, Leptinotarsa decemlineata, and the Southern corn rootworm, Diabrotica undecimpunctata. CryIIIB2 shows significant structural similarity to Colorado potato beetle-active toxin CryILIA, whose crystal structure has been determined elsewhere [J. Li, J. Carrol, and D. J. Ellar, Nature (London) 353:815-821, 1991]. A clone limited to the putative 7-a-helical bundle domain I peptide of CryIIIB2 was constructed by PCR. The truncated protein was expressed at high levels in Escherichia coli. Domain I peptide was isolated and compared with native CryIIIB2 toxin in promoting ion efflux from synthetic phospholipid vesicles and formation of ion channels in black lipid membranes. The results showed that CryIIIB2 domain I peptide is sulficient for ion channel formation and promotes ion efflux. Both native CryIIIB2 toxin and domain

A bacteriocin produced by Bacillus megaterium ATCC 19213 was identified, purified, and compared with megacin A from B. megaterium 216. The ATCC 19213 bacteriocin was inducible with mitomycin C and showed phospholipase A activity. Both megacin A-216 and megacin A-19213 contained two dissimilar polypeptide subunits. Megacin A-216 contains a 30,000-dalton (x subunit and a 15,000-dalton ,B subunit. Megacin A-19213 is composed of an cx subunit 18,000 daltons in mass and a ,B subunit about 7,500 daltons in mass. No sequence similarities between ot and a subunits of either megacin were detected. The two megacins were further distinguished by quantitative differences in activity spectra and by immunodiffusion analyses. the method of Lowry et al. (16).Protoplast lysis by megacins. Indicator strain B. megaterium 2165 was grown to mid-logarithmic phase in yeast extract-tryptone broth at 37°C. Cells were pelleted by centrifugation and then resuspended in 20% sucrose-50 mM NaPO4, pH 7.0 (PS buffer), 866 on August 5, 2020 by guest

1. Summary Quantitative cloning efficiencies for B. megaterium, B. subtilis, and E. coli were compared. Transformation of B. megaterium is less efficient than transformation of B. subtilis or E. coli. The frequency of recombinant clones was equal in E. coli and B. megaterium; both somewhat higher than in B. subtilis. Equivalent average insert sizes were found in B. megaterium and E. coli clones, but significantly smaller inserts were obtained in B. subtilis clones. Clones obtained and propagated in B. megaterium were structurally stable when grown under plasmid selection.

Megacins A-216 and A-19213 in Bacillus megaterium are plasmid encoded, as shown by analysis of cured, non-megacinogenic (Meg-) derivatives of strains 216 and ATCC 19213 and by polyethylene glycol-mediated protoplast transformation of Megbacteria with plasmid DNA. The results of both techniques implicated a 31-megadalton plasmid, pBM309, in megacin A-216 production and a 29-megadalton plasmid, pBM113, in megacin A-19213 production.