Envelope biogenesis and spore formation in Bacillus subtilis

Abstract

The bacterial cell wall exoskeleton (or peptidoglycan) protects cells from osmotic lysis, specifies their shape, and its biosynthetic pathway is a widely-exploited antibiotic target. My thesis research focused on defining how peptidoglycan synthesis is carried out in the model bacterium Bacillus subtilis. My first project explored the transport of cell wall precursors across the lipid bilayer. The precursor lipid II is a lipid-anchored molecule synthesized on the inner surface of the membrane. In E. coli, trans-bilayer movement of lipid II requires the essential flippase MurJ. I discovered that B. subtilis can survive in the absence of its 10 MurJ homologs through an alternate and novel lipid II flippase called Amj. I demonstrated that Amj is upregulated in response to envelope stress, suggesting that it serves as a defense mechanism against environmental stressors or antibiotics. My second project focused on the next step of the peptidoglycan biosynthesis pathway: the polymerization of lipid II into glycan strands. Polymerization is carried out by glycosyltransferases called class A penicillin-binding proteins (APBPs). In most bacteria, these enzymes are essential for viability, but they are dispensable in B. subtilis, implying the existence of an unidentified peptidoglycan synthase. I discovered that cells lacking these enzymes survive by upregulating a widely-conserved gene called rodA, whose homologs are essential for peptidoglycan assembly in virtually all bacteria. My biochemical analysis indicates that RodA has polymerase activity in vitro. Thus, B. subtilis, and likely most other bacteria, use two distinct mechanisms to synthesize their exoskeleton. RodA-like proteins represent appealing targets for broad-spectrum antibiotics. Inspired by high-throughput genetic screens being developed in the lab, I undertook a separate project focused on the developmental process of spore formation in B. subtilis. Genetic screens have identified factors with roles in every step of sporulation. Using a transposon sequencing approach, I identified 24 new sporulation genes, in addition to virtually all of the 148 previously characterized loci. Phenotypic characterization of these mutants uncovered factors involved in diverse aspects of sporulation, including a novel intercellular signaling molecule. My results highlight the power of transposon sequencing, and could inspire similar screens for other developmental processes.