RESUMO
Tn5401 is a class II transposable element derived from the gram-positive bacterium Bacillus thuringiensis. The 4,837-bp transposon encodes a Tn3-like transposase (TnpA) and an integrase-like recombinase (TnpI) and is notable for its unusually long 53-bp terminal inverted repeats (TIRs). The tnpA and tnpI genes are transcribed from a common promoter, designated P(R), that is subject to negative regulation by TnpI. The TIRs of Tn5401 each contain a 38-bp sequence that can be aligned with the 38- to 40-bp TIR sequences of Tn3-like transposons and an adjacent 12-bp sequence that binds TnpI. This unique juxtaposition of TnpA and TnpI binding sites suggests that TnpI may regulate the binding or catalytic activity of TnpA. The results of the present study indicate that TnpI, in addition to functioning as a site-specific recombinase and as a transcriptional repressor, is required for TnpA binding to the TIRs of Tn5401.
Assuntos
Bacillus thuringiensis/genética , DNA Nucleotidiltransferases/metabolismo , Elementos de DNA Transponíveis , Recombinação Genética , Proteínas Repressoras/metabolismo , Transposases/metabolismo , Bacillus thuringiensis/metabolismo , Sequência de Bases , Conjugação Genética , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Mutação , Ligação Proteica , Recombinases , Transposon ResolvasesRESUMO
The transformation efficiency of Bacillus thuringiensis depends upon the source of plasmid DNA. DNA isolated from B. thuringiensis, Bacillus megaterium, or a Dam- Dcm- Escherichia coli strain efficiently transformed several B. thuringiensis strains, B. thuringiensis strains were grouped according to which B. thuringiensis backgrounds were suitable sources of DNA for transformation of other B. thuringiensis strains, suggesting that B. thuringiensis strains differ in DNA modification and restriction. Efficient transformation allowed the demonstration of developmental regulation of cloned crystal protein genes in B. thuringiensis.
Assuntos
Bacillus thuringiensis/genética , Plasmídeos , Transformação Bacteriana , Bacillus thuringiensis/crescimento & desenvolvimento , Bacillus thuringiensis/fisiologia , Western Blotting , Clonagem Molecular , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Metilação , Esporos BacterianosRESUMO
Bacillus thuringiensis delta-endotoxin (crystal protein) genes are normally expressed only during sporulation. It is possible to produce crystal protein during vegetative growth by placing B. thuringiensis crystal protein genes downstream of a strong vegetative promoter. By removing a possible transcriptional terminator of the tetracycline resistance gene of pBC16 and inserting a multiple cloning site, delta-endotoxin genes can be cloned downstream from the tetracycline resistance gene promoter. This construct allows for readthrough transcription from the strong vegetative promoter. Crystal protein is then produced during vegetative growth as well as during sporulation in both B. thuringiensis and Bacillus megaterium. This construct also allows for production of delta-endotoxin in B. thuringiensis strains that do not normally produce delta-endotoxin because of a defect in sporulation.
