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

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.

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

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.