Date of Award
Doctor of Philosophy
Robert E. Andrews, Jr.
To investigate the mechanisms of gene expression in Bacillus thuringiensis subsp. kurstaki HD251, HindIII-digested total DNA was ligated into plasmid pKO1, which contains a promoterless galK gene. E. coli HB101 (GalK[superscript]-) transformant colonies resistant to ampicillin and red in color on MacConkey-galactose agar (indicating transformation to the GalK[superscript]+ phenotype) presumably contain B. thuringiensis DNA inserts in pKO1 that function as promoters in E. coli. To determine if such sequences control gene expression in B. thuringiensis, total RNA probes were prepared from cultures at specified times throughout growth and sporulation and used to screen plasmid DNA "dot-blots." Several plasmids were identified that possess veg (transcribed during exponential growth) and spo (transcribed only during sporulation) sequences that function as promoters in E. coli. The plasmids were analyzed by Southern hybridization to verify the observed temporally-dependent pattern of transcription. Two temporal classes of cloned DNA were observed: the first consists of fragments that hybridize to RNAs taken from cells at all times throughout growth and sporulation (veg + spo), and the second consists of fragments that hybridize exclusively to RNAs from sporulating cells (spo only). Plasmids from both classes were tandemly screened with galK DNA and B. thuringiensis RNA probes to reveal temporally expressed DNA subfragments physically juxtaposed to galK. DNA fragments recovered by electroelution from agarose gels were subcloned into the phagemid sequencing vector pUC118 for restriction mapping or into the plasmid pAK15 for in vivo analysis of promoter activity in a Gram-positive background (pAK15 is a shuttle plasmid that replicates in E. coli and B. subtilis). pAK15 derivatives isolated from E. coli were transformed into competent B. subtilis cells; in turn, these were employed as plasmid donors in cell-to-cell filter-matings with recipient B. thuringiensis. Analysis of plasmid and total DNAs from selected transconjugants revealed that pAK15 derivatives had suffered deletions as a consequence of uptake into B. thuringiensis. Thus, pAK15 derivatives containing B. thuringiensis DNAs are not stable in B. thuringiensis. Because such plasmids are stable in B. subtilis, the deletions are likely due to homologous recombination. Galactokinase activity in donor B. subtilis cells was negative throughout growth and sporulation, but galK transcripts were synthesized. Thus, galK transcripts cannot be translated into protein in these hosts. B. subtilis donor cells containing B. thuringiensis DNA inserts in pAK15 synthesize sporulation-specific RNAs homologous to insert DNA.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
John Michael Hurley
Hurley, John Michael, "Isolation of upstream control regions of genes expressed at different times during growth and sporulation in Bacillus thuringiensis " (1989). Retrospective Theses and Dissertations. 9133.