High-Throughput Genetic Screens Identify a Large and Diverse Collection of New Sporulation Genes in Bacillus subtilis

Abstract

The differentiation of the bacterium Bacillus subtilis into a dormant spore is among the most well-characterized developmental pathways in biology. Classical genetic screens performed over the past half century identified scores of factors involved in every step of this morphological process. More recently, transcriptional profiling uncovered additional sporulation-induced genes required for successful spore development. Here, we used transposon-sequencing (Tn-seq) to assess whether there were any sporulation genes left to be discovered. Our screen identified 133 out of the 148 genes with known sporulation defects. Surprisingly, we discovered 24 additional genes that had not been previously implicated in spore formation. To investigate their functions, we used fluorescence microscopy to survey early, middle, and late stages of differentiation of null mutants from the B. subtilis ordered knockout collection. This analysis identified mutants that are delayed in the initiation of sporulation, defective in membrane remodeling, and impaired in spore maturation. Several mutants had novel sporulation phenotypes. We performed in-depth characterization of two new factors that participate in cell–cell signaling pathways during sporulation. One (SpoIIT) functions in the activation of σE in the mother cell; the other (SpoIIIL) is required for σG activity in the forespore. Our analysis also revealed that as many as 36 sporulation-induced genes with no previously reported mutant phenotypes are required for timely spore maturation. Finally, we discovered a large set of transposon insertions that trigger premature initiation of sporulation. Our results highlight the power of Tn-seq for the discovery of new genes and novel pathways in sporulation and, combined with the recently completed null mutant collection, open the door for similar screens in other, less well-characterized processes.

The Blueprint of a Minimal Cell: MiniBacillus

“…Of these proteins, those necessary for the synthesis of NAD from nicotinate, the two subunits of the pyridoxal-5-phosphate synthase, the lipoic acid synthase, the enzyme for the final step of CoA synthesis, the S-adenosylmethionine synthetase, the dihydrofolate reductase, and the enzymes for menaquinone synthesis are essential (a total of 14 proteins). It is worth noting that MenH, an enzyme of the menachinone biosynthetic pathway, was recently shown to be dispensable (132,133). While none of the cofactor biosynthetic pathways are present in Mycoplasma species, and thus are also lacking in M. mycoides JCVI-syn3.0, this minimal genome possesses all three general components of the so-called ECF (energy-coupling factor) transporters that are capable of transporting several cofactors (10).…”

Implementation and Data Analysis of Tn-seq, Whole-Genome Resequencing, and Single-Molecule Real-Time Sequencing for Bacterial Genetics

“…The next method to identify over-or underrepresented mutants in a transposon insertion mutant library is to calculate a ratio of mutant abundance for each gene (5,6,9,(16)(17)(18)). An advantage of calculating a ratio of mutant abundance is that the population does not have to be growing exponentially during the experiment, which greatly expands the range of experimental designs amenable to analysis.…”

“…1B). There have been multiple methods reported to determine which of the mutants have statistically significant deviations from the wild-type population (5,6,9,(16)(17)(18). The most straightforward method is to apply a Mann-Whitney U test, which makes no assumptions about the underlying distribution of the data (16).…”

“…Despite decades of research on E. coli DNA repair (24), the authors identified approximately 20 genes not previously known to be important for survival following IR exposure that were missed using classical genetic approaches (18). Similarly, a recent report used Tn-seq to identify genes not previously implicated in the well-studied process of sporulation in Bacillus subtilis (16). Again, despite decades of research (see reviews in references 25 and 26), those authors successfully identified 24 genes not previously known to have a role in sporulation (16).…”

“…Similarly, a recent report used Tn-seq to identify genes not previously implicated in the well-studied process of sporulation in Bacillus subtilis (16). Again, despite decades of research (see reviews in references 25 and 26), those authors successfully identified 24 genes not previously known to have a role in sporulation (16). Those studies underscore the utility of Tn-seq in establishing a comprehensive set of nonessential factors involved in a given biological process.…”