Adaptation and diversification of bacterial communities to pesticide contaminants in on-farm biopurification systems via mobile genetic elements

Abstract

Bacteria play important ecological roles and through biodegradation processes, using the pollutants as carbon source to survive, they are fundamental in "cleaning" environments contaminated by anthropogenic activities. We assumed that the main pathways for bacterial adaptation to new contaminants are catabolic genes carried often on plasmids such as IncP-1, IncP-7 and IncP-9 plasmids. To identify "hot spot" environments containing bacterial communities carrying these catabolic plasmids the first step was to understand and predict plasmid dissemination and their functions. A screening of different environments revealed that biopurification systems (BPS), used for the treatment of pesticide contaminated waste water at farms through biodegradation and sorption processes, were "hot spots" of plasmids potentially carrying catabolic genes as high abundances of IncP-1, IncP-7 and IncP-9 plasmids were detected. A suite of different molecular biology tools such as PCR-Southern blot hybridization based detection, denaturing gradient gel electrophoresis (DGGE), clone library and pyrosequencing were applied to analyze total community (TC) DNA or plasmid DNA aiming to unravel the response of bacterial communities to pesticides and to reveal how the degree of pollution influences the abundance and diversity of mobile genetic elements (MGEs) such as plasmids. In order to investigate the influence of high pesticide concentrations on the relative abundance of specific bacterial populations and plasmids likely carrying degradative genes, a microcosm with BPS material spiked with linuron or not was established and monitored over the time. Moreover, for the first time the effects of different pesticides on the abundance and diversity of MGEs and shifts of bacterial communities in an on-farm BPS were investigated over an entire agricultural season. Interesting results were obtained. An increase in pesticide contamination in BPS was associated with an increase of abundance and fluctuations in the diversity of MGEs specifically of IncP-1 plasmids, very "promiscuous" broad-host-range (BHR) often carrying catabolic genes. Through exogenous plasmid isolation, a cultivation-independent technique, several of IncP-1 and IncP-9 plasmids were captured from BPS into Pseudomonas putida recipient cells, allowing a deeper plasmid analysis by sequencing (still in progress). The 16S rRNA gene-based analyses revealed that particular bacterial groups in BPS are responding to pesticides contamination, such as Betaproteobacteria which increased in abundance, while other groups such as Firmicutes and Bacteriodetes had a selective disadvantage in such a polluted environment. Through the methodologies applied in the present work, the dynamics and plasticity of bacterial communities of BPS in response to pesticide exposure was revealed and taxa and plasmids potentially involved in the biodegradation process were identified.