Mechanisms of Tolerance and High Degradation Capacity of the Herbicide Mesotrione by Escherichia coli Strain DH5-a Luiz R. Olchanheski 1 , Manuella N. Dourado 2 , Fla ´ vio L. Beltrame 3 , Aca ´ cio A. F. Zielinski 4 , Ivo M. Demiate 5 , So ˆ nia A. V. Pileggi 1 , Ricardo A. Azevedo 2 , Michael J. Sadowsky 6 , Marcos Pileggi 1 * 1 Universidade Estadual de Ponta Grossa, UEPG, Departamento de Biologia Estrutural, Molecular e Gene ´tica, Ponta Grossa, PR, Brazil, 2 Escola Superior de Agricultura Luiz de Queiroz, ESALQ, Universidade de Sa ˜ o Paulo, USP, Piracicaba, SP, Brazil, 3 Universidade Estadual de Ponta Grossa, UEPG, Departamento de Cie ˆ ncias Farmace ˆ uticas, Ponta Grossa, PR, Brazil, 4 Programa de Po ´ s-Graduac ¸a ˜o em Engenharia de Alimentos, Universidade Federal do Parana ´, Curitiba, PR, Brazil, 5 Universidade Estadual de Ponta Grossa, UEPG, Departamento de Engenharia de Alimentos, Ponta Grossa, PR, Brazil, 6 Department of Soil, Water, and Climate, and BioTechnology Institute, University of Minnesota, St. Paul, Minnesota, United States of America Abstract The intensive use of agrochemicals has played an important role in increasing agricultural production. One of the impacts of agrochemical use has been changes in population structure of soil microbiota. The aim of this work was to analyze the adaptive strategies that bacteria use to overcome oxidative stress caused by mesotrione, which inhibits 4- hydroxyphenylpyruvate dioxygenase. We also examined antioxidative stress systems, saturation changes of lipid membranes, and the capacity of bacteria to degrade mesotrione. Escherichia coli DH5-a ´ was chosen as a non-environmental strain, which is already a model bacterium for studying metabolism and adaptation. The results showed that this bacterium was able to tolerate high doses of the herbicide (10 6field rate), and completely degraded mesotrione after 3 h of exposure, as determined by a High Performance Liquid Chromatography. Growth rates in the presence of mesotrione were lower than in the control, prior to the period of degradation, showing toxic effects of this herbicide on bacterial cells. Changes in the saturation of the membrane lipids reduced the damage caused by reactive oxygen species and possibly hindered the entry of xenobiotics in the cell, while activating glutathione-S-transferase enzyme in the antioxidant system and in the metabolizing process of the herbicide. Considering that E. coli DH5-a is a non-environmental strain and it had no previous contact with mesotrione, the defense system found in this strain could be considered non-specific. This bacterium system response may be a general adaptation mechanism by which bacterial strains resist to damage from the presence of herbicides in agricultural soils. Citation: Olchanheski LR, Dourado MN, Beltrame FL, Zielinski AAF, Demiate IM, et al. (2014) Mechanisms of Tolerance and High Degradation Capacity of the Herbicide Mesotrione by Escherichia coli Strain DH5-a. PLoS ONE 9(6): e99960. doi:10.1371/journal.pone.0099960 Editor: Marie-Joelle Virolle, University Paris South, France Received February 20, 2014; Accepted May 20, 2014; Published June 12, 2014 Copyright: ß 2014 Olchanheski et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors would like to thank the Brazilian funding agencies Coordination for the Improvement of Higher Level Personnel (www.capes.gov.br), Grant nu. 221/2007, National Council of Technological and Scientific Development (www.cnpq.br), Grant nu. 473438/2010-0, Foundation for Research Support of the State of Sa ˜o Paulo (www.fapesp.br), Grant nu. 2009/54676-6, and Foundation for Research Support of the State of Parana ´ (www.fundacaoaraucaria.org.br), Grant nu. 21724, for the financial support. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: marcos.pileggi@pq.cnpq.br Introduction In recent years, there has been a high demand for increasing agricultural productivity and the arable land area, accompanied by the large scale use and discovery of new pesticides and fertilizers [1–3]. It was estimated that approximately 2.27 million tons of agrochemicals were released into the environment in 2001, 35% of which were herbicides [4]. Despite the fact that the use of pesticides in agriculture has had a positive impact on crop productivity, concerns have been expressed about the adverse effects of these chemicals [5], since only 0.1% of them reach their specific targets. For this reason, there is a large quantity of herbicide residues remaining in the environment, which can be metabolized by microbiota [6–8]. Herbicide application has brought damage to the soil micro- biota, and may have affected the dynamics of biogeochemical cycles and soil fertility. The herbicide napropamide, for example, has been identified as harmful to soil functionality, based on the structural and functional diversity of the soil bacterial community [9]. Other studies have demonstrated the inhibition of nitrification and changes in ammonia oxidation in soils by the herbicide simazine [10]. As regard to the triketone herbicides mesotrione and sulcotrione, their toxicity level was considered equal to or higher than atrazine in studies with model organisms, such as Tetrahymena pyriformis and Vibrio fischeri [11]. Bioremediation has been the main strategy used to eliminate xenobiotics, mainly herbicides, from the environment, and this subject has been the focus of many biotechnological studies [12– 13]. Degradation processes mediated by microorganisms in large part influence the persistence of herbicides in the soil [14]. Reactive oxygen species (ROS), apart from being part of normal aerobic metabolism [15], may increase in concentration as a result of exposure to toxic substances, as for example, when bacteria come into contact with herbicides [16]. An increase in the rate of PLOS ONE | www.plosone.org 1 June 2014 | Volume 9 | Issue 6 | e99960