Differential expression of the <i>yfj</i> operon in a <i>Bacillus subtilis</i> biofilm

Type VII protein secretion systems play an important role in the survival and virulence of pathogens and in the competition among some microbes. Potential polymorphic toxin substrates of the type VII secretion system (T7SS) in <i>Bacillus subtilis</i> are important for competition in the context of biofilm communities. Within a biofilm, there is significant physiological heterogeneity as cells within the population take on differential cell fates. Which cells express and deploy the various T7SS substrates is still unknown. To identify which cells express at least one of the T7SS substrates, we investigated the <i>yfj</i> operon. The <i>yfjABCDEF</i> operon encodes at least one predicted T7SS substrate. Starting with an <i>in silico</i> analysis of the <i>yfj</i> operon promoter region, we identified potential regulatory sequences. Using a <i>yfj</i> promoter-reporter fusion, we then identified several regulators that impact expression of the operon, including a regulator of biofilm formation, DegU. In a <i>degU</i> deletion mutant, <i>yfj</i> expression is completely abolished. Mutation of predicted DegU binding sites also results in a significant reduction in <i>yfj</i> reporter levels. Further analysis of <i>yfj</i> regulation reveals that deletion of <i>spo0A</i> has the opposite effect of the <i>degU</i> deletion. Following the <i>yfj</i> reporter by microscopy of cells harvested from biofilms, we find that the <i>yfj</i> operon is expressed specifically in the subset of cells undergoing sporulation. Together, our results define cells entering sporulation as the subpopulation most likely to express products of the <i>yfj</i> operon in <i>B. subtilis</i>.IMPORTANCEDifferential expression of genes in a bacterial community allows for the division of labor among cells in the community. The toxin substrates of the type VII secretions system (T7SS) are known to be active in <i>Bacillus subtilis</i> biofilm communities. This work describes the expression of one of the T7SS-associated operons, the <i>yfj</i> operon, which encodes the YFJ toxin, in the sporulating subpopulation within a biofilm. The evidence that the YFJ toxin may be deployed specifically in cells at the early stages of sporulation provides a potential role for deployment of T7SS in community-associated activities, such as cannibalism.