Central metabolism is a key player in E. coli biofilm stimulation by sub-MIC antibiotics

Abstract

Exposure of Escherichia coli to sub-inhibitory antibiotics stimulates biofilm formation through poorly characterized mechanisms. Using a high-throughput Congo Red binding assay to report on biofilm matrix production, we screened ~4000 E. coli K12 deletion mutants for deficiencies in this biofilm stimulation response. Mutants lacking acnA, nuoE, or lpdA failed to respond to sub-MIC novobiocin, implicating central metabolism and aerobic respiration in biofilm stimulation. These genes are members of the ArcA/B regulon – controlled by a respiration-sensitive two-component system. Mutants of arcA and arcB had a 'pre-activated' phenotype, where biofilm formation was already high relative to wild type in vehicle control conditions and failed to increase further with the addition of sub-MIC antibiotics. Supporting a role for respiratory stress, the biofilm stimulation response was inhibited when nitrate was provided as an alternative electron acceptor. Deletion of genes encoding the nitrate respiratory machinery abolished its effects, and nitrate respiration increased during growth with sub-MIC antibiotics. In probing the generalizability of biofilm stimulation, we found that the stimulation response to translation inhibitors was minimally affected by nitrate supplementation. Finally, using a metabolism-sensitive dye, we showed spatial co-localization of increased respiration with sub-MIC bactericidal antibiotics. By characterizing the biofilm stimulation response to sub-MIC antibiotics at a systems level, we identified multiple avenues for design of therapeutics that impair bacterial stress management.

A genetic screen identifies a role foroprFinPseudomonas aeruginosabiofilm stimulation by subinhibitory antibiotics

“…Our previous work on antibiotic-induced biofilm formation in E. coli showed that the response involved changes in NADH redox state and was abrogated by disrupting central metabolism and respiration pathways. 69 Periplasmic disulfide bond formation liberates electrons from free thiols that are shuttled through DsbA and DsbB to respiratory quinones, which then participate in the electron transport chain. 70 Our E. coli biofilm stimulation work also implicated ArcB and NlpE, which are involved in signal transduction and whose activity is regulated by disulfide bonding.…”

“…71,72 A dsbA mutant of E. coli failed to respond to sub-MIC novobiocin, and narrowly missed our arbitrary hit cutoff for cefixime and tetracycline. 69 Since antibiotics alter respiratory chain flux, 73 and altering respiration activity modulates antibiotic-induced biofilm formation, 69 it is reasonable to speculate that the ability of DsbA/B to cycle electrons to respiratory quinones could also be disrupted by sub-MIC antibiotics.…”

“…Our prior studies in E. coli suggested that bactericidal and bacteriostatic antibiotics drive biofilm formation through opposing effects on energy metabolism. 69 The biofilm stimulation response could also have evolved under pressure from different kinds of stress, such as phage infection or interbacterial contact-dependent antagonism, 66 and antibiotic stress merely taps into this established phenotype. We also considered the idea that if antibiotics are signalling molecules, 85 they could act as cues for the establishment of a cooperative multispecies biofilm.…”