Cell density-dependent antibiotic tolerance to inhibition of the elongation machinery requires fully functional PBP1B

Abstract

The peptidoglycan (PG) cell wall provides shape and structure to most bacteria. There are two systems to build PG in rod shaped organisms: the elongasome and divisome, which are made up of many proteins including the essential MreB and PBP2, or FtsZ and PBP3, respectively. The elongasome is responsible for PG insertion during cell elongation, while the divisome is responsible for septal PG insertion during division. We found that the main elongasome proteins, MreB and PBP2, can be inhibited without affecting growth rate in a quorum sensing-independent density-dependent manner. Before cells reach a particular cell density, inhibition of the elongasome results in different physiological responses, including intracellular vesicle formation and an increase in cell size. This inhibition of MreB or PBP2 can be compensated for by the presence of the class A penicillin binding protein, PBP1B. Furthermore, we found this density-dependent growth resistance to be specific for elongasome inhibition and was consistent across multiple Gram-negative rods, providing new areas of research into antibiotic treatment.

Role of EnvC and the phosphoenolpyruvate:sugar phosphotransferase system in resistance to MreB disruption

“…It is also unclear if MreB plays other roles in the cell, independent from regulating rod shape. We have previously reported that two gene deletions, envC and tusE , result in cells with increased sensitivity to A22, although the mechanism for this is unknown (18). Interestingly, deletion of either of these genes leads to slower growth, a condition normally thought to promote growth without MreB (Fig S1) (18).…”

“…We further characterized the effects of A22 on each of these mutants by measuring the change in rod shape and size of the cells. Using the metric, coefficient of variation of intracellular diameter deviation (IDD), we can measure the “rodness” of cells (4, 12, 18).…”

“…MreB is a highly conserved protein essential for the regulation of rod shape and viability, making it a potentially good target for antibiotic development (1, 15). While we have previously reported on conditions that help cells grow without MreB, there is little known about how cells can become hypersensitive to the disruption of MreB (18, 19). Here, we performed a screen of the Keio collection to identify gene deletions that lead to the inhibition of growth in sublethal levels of A22, an MreB depolymerizing agent.…”

“…FtsN activates PBP1B at the division site (50). PBP1B mutants are sensitive to A22, and PBP1B is necessary to overcome the loss of the elongation machinery (18, 19, 51). One possible reason for the increased sensitivity of an envC mutant is a lowering of the presence of denuded glycans leading to less active FtsN and therefore less active PBP1B.…”

“…We have previously explored conditions in which Escherichia coli cells are better able to withstand the loss of MreB function. Using A22 as a method for disrupting MreB we have shown that MreB and the elongasome in general are dispensable for growth but not cell shape once cells reach a threshold density (18). In addition, we have shown that mutations in metabolic genes that lead to an increase in cell wall precursors allow cells to better tolerate A22 treatment (19).…”