Unmasking Novel Sporulation Genes in Bacillus subtilis

The Bacillus subtilis transcription factor σ E directs the expression of a regulon of 262 genes, but null mutations in only a small fraction of these genes severely impair sporulation. We have previously reported that mutations in seven σ E -controlled genes cause a mild (2- to 10-fold) defect in sporulation. In this study, we found that pairwise combinations of some of these seven mutations led to strong synthetic sporulation phenotypes, especially those involving the ytrHI operon and ybaN . Double mutants of ybaN and ytrH and of ybaN and ytrI had >10,000-fold lower sporulation efficiencies than the wild type. Thin-section electron microscopy revealed a block in cortex formation for the ybaN ytrH double mutant and coat defects for the ybaN single and ybaN ytrI double mutants. Sporulating cells of a ybaN ytrI double mutant and of a ybaN ytrHI triple mutant exhibited a pronounced loss of dipicolinic acid (DPA) between hours 8 and 24 of sporulation, in contrast to the constant levels seen for the wild type. An analysis of the spore cortex peptidoglycans of the ybaN ytrI and ybaN ytrHI mutants showed striking decreases in the levels of total muramic acid by hour 24 of sporulation. These data, along with the loss of DPA in the mutants, suggest that the developing spores were unstable and that the cortex underwent degradation late in sporulation. The existence of otherwise hidden sporulation pathways indicates that functional redundancy may mask the role of hitherto unrecognized sporulation genes.