Flagellar toxicity: Flagellar synthesis is lytic for Bacillus subtilis in the absence of PBP1.

Abstract

Flagella are large transenvelope nanomachines but how they transit the peptidoglycan in gram-positive bacteria is poorly understood. A recent model suggested that flagellar basal bodies diffuse in the membrane and become captured at locations in the peptidoglycan with a pore diameter that could accommodate the axle-like flagellar rod. To test the role of pore size on flagellar assembly, cells were disrupted for penicillin binding protein 1 (PBP1/PonA), a cell wall synthesis protein thought to decrease peptidoglycan pore frequency and/or diameter. The absence of PBP1 in the ancestral strain of Bacillus subtilis however resulted in a severe growth defect and cell lysis that was dependent on flagellar synthesis. Genetic analysis indicated that toxicity was due to completion of the flagellar hook, which activated the flagellar sigma factor SigD. SigD, in turn, activated a suite of peptidoglycan degrading enzymes that caused cellular lysis when PBP1 was absent. In addition, mutations that resulted in high levels of the stress response transcription factor Spx could lessen the toxicity, while PBPX, a putative teichoic acid D-alanylase, was required for autolysis. In sum, our results indicate that flagellar synthesis, not normally associated with cell viability, causes cell wall stress and under some conditions, cell death. Moreover, the cost of flagellar synthesis on envelope integrity may be underappreciated due to strain domestication, and specialized systems may be needed to compensate for the assembly of transenvelope machines in general.