Exposure to Sub-lethal 2,4-Dichlorophenoxyacetic Acid Arrests Cell Division and Alters Cell Surface Properties in Escherichia coli

Abstract

Escherichia coli is a robust, easily adaptable and culturable bacterium in vitro, and a model bacterium for studying the impact of xenobiotics in the environment. We have used correlative atomic force – laser scanning confocal microscopy (AFM-LSCM) to characterize the mechanisms of cellular response to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). One of the most extensively used herbicides world-wide, 2,4-D is known to cause hazardous effects in diverse non-target organisms. Sub-lethal concentrations of 2,4-D caused DNA damage in E. coli WM1074 during short exposure periods which increased significantly over time. In response to 2,4-D, FtsZ and FtsA relocalized within seconds, coinciding with the complete inhibition of cell septation and cell elongation. Exposure to 2,4-D also resulted in increased activation of the SOS response. Changes to cell division were accompanied by concomitant changes to surface roughness, elasticity and adhesion in a time-dependent manner. This is the first study describing the mechanistic details of 2,4-D at sub-lethal levels in bacteria. Our study suggests that 2,4-D arrests E. coli cell division within seconds after exposure by disrupting the divisome complex, facilitated by dissipation of membrane potential. Over longer exposures, 2,4-D causes filamentation as a result of an SOS response to oxidative stress induced DNA damage.

Correlative atomic force microscopy quantitative imaging-laser scanning confocal microscopy quantifies the impact of stressors on live cells in real-time

“…S3 ), disruption of the Z-ring, changes to surface ultrastructure, elasticity and adhesion (Fig. 3 ), consistent with our previous study 34 .…”

“…To demonstrate proof-of-principle and dramatic responses during the correlative imaging assay, we chose E . coli exposed to the chemical stressor 2,4-D, which we previously showed to induce filamentation, ROS, the SOS response, cell envelope remodelling, along with the relocalization of components of the cell division machinery (FtsA, FtsZ) 34 . As expected, E .…”

“…AFM-QI-LSCM was used to simultaneously track the ROS and FtsZ-GFP signals, along with changes to surface adhesion and elasticity following the addition of 2,4-D. Further detail on the impact of 2,4-D in E . coli is described in our previous work 34 .

Figure 3 Simultaneous AFM-QI-LSCM of E .

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“…coli surface 37 is enhanced by bridging, which is more prevalent at higher ionic strengths 38 . Adhesion, even when non-specific, is an important comparative characteristic that reflects the macromolecular arrangement of the cell surface which can impact important physiological processes such as biofilm formation and host cell invasion 9 , 33 , 34 , 39 – 41 .…”

“…The entire system is housed on a vibration isolation platform and in a home-built acoustic enclosure. Our AFM-LSCM setup consists of the Nano Wizard 3 AFM (JPK Germany) mounted on an inverted LSCM 780 (Carl-Zeiss) as described previously 34 . The custom designed AFM stage (JPK) was positioned in place of the LSCM stage and the sample mounted as described above.…”