Disruption of the MreB Elongasome Is Overcome by Mutations in the Tricarboxylic Acid Cycle

Abstract

The bacterial actin homolog, MreB, is highly conserved among rod-shaped bacteria and essential for growth under normal growth conditions. MreB directs the localization of cell wall synthesis and loss of MreB results in round cells and death. Using the MreB depolymerizing drug, A22, we show that changes to central metabolism through deletion of malate dehydrogenase from the tricarboxylic acid (TCA) cycle results in cells with an increased tolerance to A22. We hypothesize that deletion of malate dehydrogenase leads to the upregulation of gluconeogenesis resulting in an increase in cell wall precursors. Consistent with this idea, metabolite analysis revealed that malate dehydrogenase (mdh) deletion cells possess elevated levels of several glycolysis/gluconeogenesis compounds and the cell wall precursor, uridine diphosphate N-acetylglucosamine (UDP-NAG). In agreement with these results, the increased A22 resistance phenotype can be recapitulated through the addition of glucose to the media. Finally, we show that this increase in antibiotic tolerance is not specific to A22 but also applies to the cell wall-targeting antibiotic, mecillinam.

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

“…Mutant #10 has a ∼13kb deletion of many genes involved in chemotaxis and flagella rotation. Due to known connections between metabolism and cell shape we will focus on the role of ptsI on A22 resistance (19, 33-35). It is of note that mutants #3 and #5 have mutations in adjacent residues of MreB which are predicted to be in the A22 binding pocket, yet produce different cell shapes (Table S2) (36).…”

“…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.…”

“…Lack of PtsI will affect the PEP:pyruvate ratio of the cell (58, 59). We have previously proposed that increased pyruvate levels in cells can activate gluconeogenesis and increase the levels of cell wall precursors providing cells with increased tolerance to A22 (19). In Bacillus subtilis , deletion of pyruvate kinase ( pyk ) effects Z-ring formation (60).…”

“…It is possible that deletion of ptsI leads to increased levels of the ftsZ operon providing resistance to A22. We transduced a functional fluorescent copy of FtsZ into the native site of the chromosome as the sole copy of FtsZ in the cell (19). A ptsI deletion strain expressing this construct is still smaller than WT cells (Fig.…”