Escherichia coli and Rhizobium leguminosarum response mechanisms to sub-lethal 2,4-dichlorophenoxyacetic acid

Abstract

The chlorophenoxy herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) is used extensively worldwide despite its known toxicity and our limited understanding of how it affects non-target organisms. To determine the global effects of 2,4-D at sub-lethal levels on Rhizobium leguminosarum bv. viciae 3841 (Rlv) and Escherichia coli BL21, I used a novel combination of methods involving advanced microscopy and metabolomics. Rlv showed an oxidative stress response, but showed adaptive capabilities with changes to specific metabolic pathways and consequent changes to its phenotype, surface ultrastructure, and physical properties during 2,4-D exposure. Interestingly, auxin and 2,4-D, its structural analogue, induced common morphological changes in vitro, which were similar in shape to bacteroids isolated from plant nodules, implying that these changes may be related to bacteroid differentiation required for nitrogen fixation. In E. coli, 2,4-D altered biofilm formation and induced a filamentous phenotype in the lab strain and a selection of genotypically diverse strains isolated from the environment. The phenotype was observed at concentrations 1000 times below field application levels, and was reversible upon supplementation with polyamines, implicating DNA damage. Cells treated with 2,4-D had more compliant envelopes, significantly remodelled surfaces that were rougher and more hydrophobic and altered vital metabolic pathways. Most of the observed effects could be attributed to oxidative stress, consistent with increased reactive oxygen species as a function of 2,4-D exposure. The characteristic filamentous phenotype and metabolic changes with 2,4-D exposure implicated impact on cell division. I developed correlative atomic force microscopy-quantitative imaging and laser scanning confocal microscopy to simultaneously probe cell surface alterations at the pico-nanoscale with details of molecular changes inside live cells in real-time. At the mechanistic level, 2,4-D at >1 mM altered FtsZ, FtsA and SulA localization within seconds accompanied by DNA damage resulting in immediate inhibition of Z-ring formation and arrest of cell division. There were simultaneous changes to cell surface roughness, elasticity and adhesion in a time-dependent manner. I propose that 2,4-D rapidly blocks cell division in E. coli likely by affecting the higher order assembly of the divisome complex with inhibition of Z-ring formation resulting from oxidative stress and DNA damage.