Mode of Growth and Temperature Dependence on Expression of Atrazine-degrading Genes in Pseudomonas sp. strain ADP Biofilms

Bacterial strain Pseudomonas sp. strain ADP is capable of metabolizing atrazine, a synthetic herbicide, and uses atrazine as a sole nitrogen source for growth. The microbe completely mineralizes the substrate in a catabolic pathway comprised of six enzymatic steps. All enzymes, AtzA-AtzF, encoded by corresponding genes, AtzA-AtzF, are located on a self-transmissible plasmid, pADP-1. (Souza, M. L., Wackett, L.P., and Sadowsky, M.J Appl. and Environ. Microbiol. 64(6): 2323-2326, 1998) RT-qPCR was used to differentiate gene expression in atrazine-degrading genes in Pseudomonas sp. strain ADP cells grown as suspended cells and as biofilms. Relative gene expression was also evaluated for biofilms grown at 25{degrees}C, 30{degrees}C, and 37{degrees}C. Complementary atrazine kinetic data was collected using GC-MS for both modes of growth and temperature variance. No significant difference in expression was observed for all atrazine-degrading genes in biofilm-mediated cells relative to planktonic cells, suggesting neither decreased or increased catabolic activity at the mRNA level. In contrasting experiments concerning biofilm growth, expression was downregulated at 37{degrees}C for genes AtzA, AtzB, and AtzC and upregulated for genes AtzD, AtzE, AtzF, signifying Pseudomonas sp. strain ADP biofilms catabolic activity may change in response to substantial temperature changes. Gradual decreases in atrazine concentration were apparent in cells grown in shake flasks, while biofilm-mediated cells showed transient increases and decreases in reactor effluent. The complex extracellular matrix components, quorum sensing, and genetic transfer may account for accumulation and rapid degradation of atrazine. The data collected suggest biofilm-mediated bioremediation may give insight into catabolic activity and atrazine degradation potential.\n\nImportanceAtrazine is the second most applied herbicide in the United States. It is applied to crops including sorghum, corn, and sugarcane to prevent the growth of broad-leaved weeds. Once used, it can permeate the soil and contaminate proximal groundwater sources, which provide drinking water for over 90-million people. The Environmental Protection Agency sets the maximum contaminant level at 3 parts per billion for atrazine in drinking water, however this is frequently exceeded in rural regions which presents a public safety concern. Atrazine is an endocrine disruptor compound and a suspected teratogen in humans and freshwater species, respectively. This research is significant in evaluating the use an atrazine-degrading strain, Pseudomonas sp. strain ADP, grown in a biofilm mode of growth to increase the degradation potential compared to suspended cells. Our results concerning expression and kinetics will aid the development of biofilm reactors for ex situ bioremediation and understanding environmental biofilms.