Mechanisms of Bacterial Envelope Stress From Within and Without

Abstract

The bacterial cell envelope is the frontline protective barrier between the cell and the environment. It is a complex multilayered structure essential for maintaining cellular integrity and plays a crucial defensive role against diverse environmental challenges. Bacteria can encounter stress in the envelope either spontaneously or from external insults such as drug treatments. The mechanism by which drug-induced stress leads to cell death is still poorly understood. How often and to what degree bacteria encounter stresses from within is also not described. Here, I investigate both processes and the results reveal new functions for cell wall biogenesis enzymes whose roles have not yet been clearly defined. Specifically, I attempt to define the downstream events following β-lactam inhibition of PG synthases (PBPs) by understanding how cell wall biogenesis overcomes β-lactam stress. β-lactams not only inhibit PBPs essential for growth, but also induce a lethal malfunctioning of their target cell wall biosynthetic machinery. I used a genetic system to uncouple these two processes by isolating mecillinam suppressors that overcome the toxic Rod machinery under conditions where the target PBP2 is not essential. Subsequent functional profiling uncovered a stress response-independent suppressor encoding an inactivating mutation of a protease, that was implicated to degrade a cell wall hydrolase Spr. Spr cleaves crosslinks between strands in the cell wall matrix toallow for cell wall expansion during growth, but how that is achieved is unclear. Further investigation of Spr and similar enzymes suggests these hydrolases can stimulate PBP activity to promote cell wall synthesis during normal growth. Our results provide evidence for a ‘cut-and-insert’ strategy mediated by hydrolases and PBPs during cell wall expansion. Cell wall expansion during growth requires coordinated concurrent expansion of its surrounding envelope layers. It is not clear how often envelope biogenesis run into problems during normal growth and what these problems might be. Because envelope stress responses are intrinsically tuned to cell envelope physiology, studying them can unveil mechanisms that regulate cell envelope homeostasis. I harnessed envelope stress responses to detect stresses from within by tracking their spontaneous activation using transcriptional fluorescent reporters in unperturbed cells maintained in a constant environment. Rare spikes in activity of the Rcs stress response were observed and correlated with the appearance of membrane blebs in the affected cells. Further characterization demonstrates that these spikes are bona fide indicators of envelope biogenesis errors, and this monitoring system is robust in identifying factors involved in quality control or repair, which have been difficult to detect otherwise. Many gaps remain in our understanding of the bacterial cell envelope, owing to its complexity in both structure and function. While I have used β-lactams and envelope stress responses as chemical and biological probes to interrogate poorly understood aspects of bacterial cell wall regulation in E. coli, these approaches can be extended to address other similar questions in cell envelope biogenesis.