USE OF THE TRANSPOSON TN917 TO STUDY TEMPORALLY REGULATED GENE EXPRESSION IN BACILLUS SUBTILIS

Spore formation in Bacillus subtilis involves the sequential activation of gene sets in a highly ordered temporal program. The purpose of this dissertation was to use the transposon Tn917 as an insertional mutagen to generate developmental mutations and to examine the regulation of late-expressed sporulation genes defined by Tn917 insertions.;The expression and regulation of two fusions, to spoIVC and cotA (pig) was studied in detail. The fusions were crossed into a lysogenic phage and transferred to a variety of developmental mutants. spoIVC transcription was prevented in early-blocked mutants, partially impaired in mutants blocked at intermediate states (III and IV) and unaffected in late-blocked mutants. Transcription of cotA (pig) was highly correlated with colony color, indicating that pigmentation was a reliable indicator of cotA expression. Using colony color and/or (beta)-galactosidase activity to monitor cotA transcription, I found that almost all mutants blocked before stage V did not transcribe cotA, but mutants blocked after stage V expressed cotA at normal levels, or in one case (gerE) at a several-fold higher level. These findings suggest that some morphological or physiological aspect of the stage IV bacterium must be a prerequisite for the induction of cotA. Nuclease mapping experiments and deletion analysis established that the sequences needed for the expression and regulation of cotA were within 90 bases of the cotA coding sequence.;Twenty-four insertional sporulation mutations were generated with Tn917. Each insertion was mapped to the chromosome and its phenotypic effects on sporulation were characterized. Ten of the mutations define new loci, which suggests that the B. subtilis genetic map is not saturated with regard to sporulation loci. One mutation blocked the appearance of the sporulation-specific brown pigment but not spore formation and was found to map to pig, a previously known pigment-determining gene which is shown here to correspond to a spore coat protein gene, cotA. A recombinational replacement procedure was used to incorporate lacZ into the chromosomal copy of Tn917 in several of the mutants so as to construct transcriptional lacZ fusions. The expression of the interrupted genes was determined by measuring (beta)-galactosidase activity during growth and sporulation. These spo::Tn917lac fusions were found to exhibit diverse temporal patterns of sporulation-induced gene expression.