Unmasking developmental genes and untranslated RNAs in Bacillus subtilis

The developmental process of sporulation in the bacterium Bacillus subtilis has been the subject of genetic investigation for many decades. An important goal of this research has been to identify comprehensively all genes that are involved in the conversion of a growing a cell into a spore. In this study I attempted to discover additional genes involved in sporulation that had been missed by classical genetic and genomic approaches. Here I describe two separate approaches that were employed in search of novel developmental genes.; In one approach I uncovered synthetic defects in sporulation by creating a large number of double mutants for genes under the control of the sporulation transcription factor sigmaE. Several single mutants from this regulon exhibited only a small defect in sporulation, whereas double mutants of ytrH and ybaN and ytrI and ybaN had >10,000--fold lower sporulation efficiencies compared to wild type. Using thin-section electron microscopy and analysis of spore peptidoglycan, my collaborators and I found defects in the spore coat and cortex for these mutants. These data suggested that developing spores of these two double mutants were unstable and that the cortex underwent degradation late in sporulation. From these results I suspect that the functions of other developmentally controlled genes that are redundant or act in synergy with other pathways remain to be revealed by the creation of multiply mutant strains.; In the second approach my collaborators and I utilized microarrays and a computational comparative genomics program, QRNA, to search for small, untranslated RNAs (sRNAs) from the intergenic regions of B. subtilis. A total of 109 intergenic regions were predicted by QRNA to contain structural RNAs, while transcripts were also detected from 13 intergenic regions using Affymetrix microarrays. I conducted Northern blots for 25 of these intergenic regions and detected seventeen sRNAs, three of which were produced under developmental control, including two that were synthesized under the control of cell-specific regulatory proteins. In the course of these experiments we also discovered a chromosomally-encoded toxin-antitoxin locus in which a small RNA prevents cell death caused by a toxic peptide. In sum, genetic, genomic and computational approaches have been used to unmask previously unrecognized genes that play a critical role in sporulation or are expressed under sporulation control.