As a means of determining the function of σ29, a sporulation-essential sigma factor, we have isolated and begun to characterize genes that require σ29 for their expression. RNA transcribed in vitro from total Bacillus subtilis DNA by using σ29-containing RNA polymerase (E-σ29) was hybridized to a bank of B. subtilis DNA fragments that had been cloned into bacteriophage λ. Approximately 0.25% of the cloned B. subtilis DNA fragments displayed detectable hybridization with our RNA probe. Five DNA fragments that had strong in vitro template activity for E-σ29 were selected for further study. The DNA fragments which contained in vitro σ29 promoter activity encoded RNAs that were synthesized by B. subtilis during sporulation. Mutant B. subtilis that failed to synthesize σ29 (spoIIA, spoIIE) made less RNA that could hybridize to these cloned DNAs than did a mutant (spoIIC) which did synthesize σ29 but was blocked at a similar stage in development. A detailed analysis of several of the cloned DNAs demonstrated that they encoded RNAs that were transcribed from approximately the same start site in vivo that E-σ29 initiated transcription in vitro. These particular transcripts were present only during the period of σ29 abundance (2 to 4 h after the onset of sporulation) in sporulating cells which carried a wild-type allele of the σ29 structural gene (spoIIG). We conclude that the isolation procedure used in this study identified genes that are transcribed by E-σ29, not only in vitro but also in vivo. Preliminary characterization of the cloned genes indicate that they encoded multiple overlapping RNAs which were each synthesized at unique times during growth or sporulation. This result implies that σ29 does not activate a unique population of genes with a novel function in sporulation but rather that it has a temporal role in spore gene control, transcribing those genes required to be active during its period of abundance regardless of their specific function.
ASJC Scopus subject areas
- Molecular Biology