A Bacillus Spore-Based Display System for Bioremediation of Atrazine

Abstract

Owing to human activities, a large number of organic chemicals, including petroleum products, industrial solvents, pesticides, herbicides (including atrazine [ATR]), and pharmaceuticals, contaminate soil and aquatic environments. Remediation of these pollutants by conventional approaches is both technically and economically challenging. Bacillus endospores are highly resistant to most physical assaults and are capable of long-term persistence in soil. Spores can be engineered to express, on their surface, important enzymes for bioremediation purposes. We have developed a Bacillus thuringiensis spore platform system that can display a high density of proteins on the spore surface. The spore surface-tethered enzymes exhibit enhanced activity and stability relative to free enzymes in soil and water environments. In this study, we evaluated a B. thuringiensis spore display platform as a bioremediation tool against ATR. The Pseudomonas sp. strain ADP atzA determinant, an ATR chlorohydrolase important to the detoxification of ATR, was expressed as a fusion protein linked to the attachment domain of the BclA spore surface nap layer protein and expressed in B. thuringiensis Spores from this strain are decorated with AtzA N-terminally linked on the surface of the spores. The recombinant spores were assayed for ATR detoxification in liquid and soil environments, and enzyme kinetics and stability were assessed. We successfully demonstrated the utility of this spore-based enzyme display system to detoxify ATR in water and laboratory soil samples.IMPORTANCE Atrazine is one of the most widely applied herbicides in the U.S. midwestern states. The long environmental half-life of atrazine has contributed to the contamination of surface water and groundwater by atrazine and its chlorinated metabolites. The toxic properties of ATR have raised public health and ecological concerns. However, remediation of ATR by conventional approaches has proven to be costly and inefficient. We developed a novel B. thuringiensis spore platform system that is capable of long-term persistence in soil and can be engineered to surface express a high density of enzymes useful for bioremediation purposes. The enzymes are stably attached to the surface of the spore exosporium layer. The spore-based system will likely prove useful for remediation of other environmental pollutants as well.

Keywords: Bacillus thuringiensis; atrazine; bioremediation; exosporium; spore display.