GENE-EXPRESSION IN SINGLE CELLS OF BACILLUS-SUBTILIS - EVIDENCE THAT A THRESHOLD MECHANISM CONTROLS THE INITIATION OF SPORULATION

Early during endospore formation in the bacterium Bacillus subtilis, t wo distinct cell types are formed. The initiation of this developmenta l pathway requires several physiological conditions (e.g., nutrient de privation) and is controlled by the Spo0A transcription factor. We hav e found that in a culture of sporulating cells, there are two subpopul ations, one that has initiated the developmental program and activated the expression of early developmental genes and one in which early de velopmental gene expression remains uninduced. We measured the express ion of developmental (spo) genes in single cells of B. subtilis by usi ng spo-lacZ fusions. Cells containing a spo-lacZ fusion were stained w ith a dye that fluoresces upon hydrolysis by beta-galactosidase, and t he fluorescence in individual cells was measured with a flow cytometer . For Spo+ cells, we found that the proportion of the population expre ssing early developmental genes correlates well with the fraction of t he population that eventually produces spores. In addition, mutations that cause a decrease in the amount of activated (phosphorylated) Spo0 A transcription factor cause a decrease in the size of the subpopulati on expressing early developmental genes that are directly activated by Spo0A is similar to P. Again, the size of the subpopulation correlate s well with the fraction of cells that produce spores. These results i ndicate that a threshold level of activated Spo0A (Spo0A is similar to P) or of a component of the phosphorylation pathway must accumulate t o induce sporulation gene expression and that most of the cells that a re able to induce the expression of early genes that are directly acti vated by Spo0A is similar to P go on to produce mature spores. Both po sitive and negative regulatory loops are probably involved in maintain ing the two subpopulations.