We have developed a strategy for isolating cry genes from Bacillus thuringiensis. The key steps are the construction of a DNA library in an acrystalliferous B. thuringiensis host strain and screening for the formation of crystal through optical microscopy observation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses. By this method, three cry genes-cry55Aa1, cry6Aa2, and cry5Ba2-were cloned from rice-shaped crystals, producing B. thuringiensis YBT-1518, which consists of 54-and 45-kDa crystal proteins. cry55Aa1 encoded a 45-kDa protein, cry6Aa2 encoded a 54-kDa protein, and cry5Ba2 remained cryptic in strain YBT-1518, as shown by SDS-PAGE or microscopic observation. Proteins encoded by these three genes are all toxic to the root knot nematode Meloidogyne hapla. The two genes cry55Aa1 and cry6Aa2 were found to be located on a plasmid with a rather small size of 17.7 kb, designated pBMB0228.

Aims:  To elaborate an effective electroporation protocol for large plasmids and wild type strains of Bacillus thuringiensis. Methods and Results:  The effect of DNA desalting, wall‐weakening agency, cell growth conditions, electroporation solutions, and electric fields on electroporation efficiency was evaluated to optimize electroporation conditions for B. thuringiensis. By using this improved method, the greatest efficiency was reached 2 × 1010 CFU μg−1 with pHT304, which is 104 times higher than previously reported. Four large plasmids (29·1, 44·9, 58 and 60 kb) were successfully transferred into the acrystalliferous B. thuringiensis strain BMB171; these results have not been achieved with previous protocols. Three wild type B. thuringiensis strains which could not be transformed previously were also transferred successfully. Conclusions:  This improved method is more efficient for small plasmids; it is also appropriate for large plasmids and wild type B. thuringiensis strains which were not transformed by previous procedures. Significance and Impact of the Study:  The present study established an effective electroporation protocol for large plasmids and wild type strains of B. thuringiensis. This method is well suited for the cloning and expression of huge DNA fragments such as gene clusters in B. thuringiensis. It also can be used as a reference method for other Bacillus strains that are refractory to electroporate.

The nucleotide sequence of a 2711bp DNA segment which contains the N-terminal coding sequence and the 5' flanking region of a crystal protein gene (bta) from Bacillus thuringiensis subsp. aizawai 7.29 has been determined. The coding region encodes an 824 amino-acid polypeptide corresponding to a carboxy-terminally truncated delta-endotoxin specifically active against the cotton leaf worm Spodoptera littoralis. Comparison of the deduced amino acid sequence of the bta gene with that of the 4.5, 5.3 and 6.6 kb classes of lepidopteran-active delta-endotoxins revealed that the Bta sequence contains a very high level of amino acid substitutions in the N-terminal part of the protoxin molecule. The substitutions are grouped in several highly variable segments separated by highly conserved regions. These conserved domains are also present in the dipteran- and coleopteran-active delta-endotoxins. The control region of the bta gene shows considerable DNA identity with the control regions of the other lepidopteran-active genes. Deletions of the 3' region of the gene were carried out and the toxic fraction of the bta delta-endotoxin was identified with the N-terminal half of the molecule.

Bacillus thuringiensis has long been recognized to carry numerous extrachromosomal molecules. Of particular interest are the strains belonging to the B. thuringiensis subsp. israelensis lineage, as they can harbor at least seven extrachromosomal molecules. One of these elements seems to be a cryptic molecule that may have been disregarded in strains considered plasmid-less. Therefore, this work focused on this cryptic molecule, named pBtic235. Using different approaches that included transposition-tagging, large plasmid gel electrophoresis and Southern blotting, conjugation and phage-induction experiments, in combination with bioinformatics analyses, it was found that pBtic235 is a hybrid molecule of 235,425 bp whose genome displays potential plasmid- and phage-like modules. The sequence of pBtic235 has been identified in all sequenced genomes of B. thuringiensis subsp. israelensis strains. Here, the pBtic235 sequence was considered identical to that of plasmid pBTHD789-2 from strain HD-789. Despite the fact that the pBtic235 genome possesses 240 putative CDSs, many of them have no homologs in the databases. However, CDSs coding for potential proteins involved in replication, genome packaging and virion structure, cell lysis, regulation of lytic-lysogenic cycles, metabolite transporters, stress and metal resistance, were identified. The candidate plasmidial prophage pBtic235 exemplifies the notable diversity of the extrachromosomal realm found in B. thuringiensis.

Cointegrate plasmids are useful models for the study of plasmid evolution if their evolutionary processes can be replicated under laboratory conditions. pBMB0228, a 17 706 bp native plasmid originally isolated from Bacillus thuringiensis strain YBT-1518, carries two nematicidal crystal protein genes, cry6Aa and cry55Aa. In this study, we show that pBMB0228 is in fact a cointegrate of two plasmids and contains two functional replication regions and two functional mobilization regions. Upon introduction into B. thuringiensis strain BMB171, pBMB0228 spontaneously resolves into two constituent plasmids via recombination at its oriT1 and oriT2 sites. The resolution does not require conjugation but can be promoted by conjugation. We further confirm that the resolution is mediated by oriT site-specific recombination requiring Mob02281 or Mob02282. Additionally, the two constituent plasmids of pBMB0228 are mobilizable, and can fuse back via oriT site-specific integration after entering into the same cell by conjugation. Our study confirms that native plasmid can reversibly interconvert between a cointegrate structure and its constituent plasmids. This study provides insight into the evolution of cointegrate plasmids, linking plasmid evolution with conjugation and the oriT site-specific recombination function of relaxase.