Unmasking Novel Sporulation Genes in Bacillus subtilis

A central challenge in the field of microbial development is to identify and characterize the functions of all of the genes that govern morphogenesis during cellular differentiation. An attractive system with which to address this challenge is the process of sporulation in the bacterium Bacillus subtilis, in which a growing cell is transformed via a series of well-defined morphological stages into a dormant cell known as a spore (or, more properly, an endospore) (23, 30). Soon after the commitment to sporulate, a two-chamber cell is formed, consisting of a large compartment called the mother cell and a small compartment called the forespore. Initially, the mother cell and the forespore lay side by side, but at an intermediate stage of development the forespore is wholly engulfed by and therefore resides within the mother cell (23). The engulfed forespore is surrounded by two membranes, one derived from the forespore and the other derived from the mother cell membrane that engulfs the forespore. Later in development, a thick layer of peptidoglycan known as the cortex is deposited in the space between the two membranes (24). Distinctive features of the cortex include the modified sugar muramic δ-lactam and a reduced level of muramic acid residues with peptide cross-links to other glycan chains (10). At approximately the same time as cortex formation, a thick protein shell is deposited around the outside of the forespore to create the spore coat (5). Ultimately, the mature spore is released by lysis of the mother cell. Classical genetic as well as more recent genomic approaches have made it possible to identify genes that are activated during sporulation on a genome-wide scale. Strikingly, only a small subset of these genes appear to be essential for sporulation, based on analyses of null alleles (7, 8, 13, 26, 35). Most likely, in many cases the apparent lack of a sporulation phenotype is due to the mutations having effects that are undetectable under laboratory conditions but significant in the wild. However, it is also the case that the roles of some genes are largely masked by other genes. In other words, to observe the phenotype of a mutation in a given gene, a second mutation is required. An example of this type of redundancy is provided by the genes spoIIB and spoVG, in which mutations only mildly impair sporulation on their own but have a severe impact in combination (i.e., they have a synthetic phenotype) (18). Another synthetic effect involves a double mutant of cwlC and cwlH in which mother cell lysis is strongly blocked, while the corresponding single mutants exhibit little defect (21). A third example involves the activation of the forespore-specific transcription factor σF, which is governed by two independent regulatory pathways that are largely redundant (6). In an effort to determine whether functional redundancy masks the role of other developmentally regulated genes in B. subtilis, we focused on genes under the control of the mother-cell-specific regulatory protein σE. The σE factor directs the transcription of an unusually large regulon, consisting of 262 genes, which are organized into 163 transcription units (excluding genes and operons that are activated under the dual control of σE and the DNA-binding protein SpoIIID) (8). We recently created null mutations in 98 newly identified genes and operons in the regulon. Strikingly, in only three cases did the mutation cause a severe defect in sporulation. In seven cases, however, the mutation caused a slight (2- to 10-fold) but reproducible defect in the efficiency of sporulation, as judged by the production of heat-resistant spores. We wondered whether the impact on sporulation of some of these seven genes is partially masked by redundancy with one or more other genes under the control of σE. As a test of this idea, we created strains bearing mutations in pairs of the seven genes. Here we report that certain pairwise combinations of mutations resulted in striking synthetic phenotypes in which cortex and coat formation were severely impaired.