Bioinformatic and functional analysis of a plasmid pool form bacterial isolates from an on-farm biopurification system used for pesticide removal

MARTINI, C; ALBICORO, F.; LÓPEZ, J. L.; SALAS, M, E.; PISTORIO, M.; SCHLÜTER, A.; LAGARES, A.; DEL PAPA, M.F.

Mar del Plata

Congreso; X Congreso de Microbiologia General; 2014

Sociedad Argentina de Microbiología general

Background. Biopurification systems (BPS) are used on farms as pollution control technique to treat pesticide contaminated water. BPS are efficient systems containing a biological active matrix that retains pesticides and enhances their degradation. Considering that BPSs are used for the pesticide removal, they are target sites for finding genes associated with degradation of these compounds. It is assumed that mobile genetic elements (MGEs) carrying genes coding for enzymes involved in degradation might contribute to the degradation of pesticide and other xenobiotic compounds. In particular, plasmids play an essential role in the evolution and adaptation of bacteria as they encode accessory genes that increase bacterial fitness in a given environment, making them a target for studying the presence of degradative functions. Objectives. The aim of this work was to analyze the information acquired from the sequencing of a plasmid pool obtained from a collection of bacteria recovered with and without selection pressure from a BPS contaminated with several pesticides. Results. In order to investigate the type and diversity of the information encoded in the plasmid pool present in a BPS enriched with several pesticides, a plasmid search was performed using in situ-lysis gel electrophoresis (Eckhardt protocol1). Out of 1,400 isolates screened, a collection of 75 plasmid-containing bacteria was generated. Isolates were classified in 35 groups according to their plasmid profiles. Plasmid sizes ranged from 30 to 300Kpb, being almost 50% of them more than 100 Kbp in length. Also, a phenotypic characterization of the BPS collection comprising i) antibiotic and metal tolerance of isolates, ii) evaluation of the presence of broad host range (BHR) plasmid incompatibility groups and iii) ability of these plasmids to be mobilized to an E. coli host stain, was carried out. In order to go deeper in the characterization of these plasmids, 35 representative isolates comprising at least 50 plasmids were pooled for high throughput sequencing using Illumina MySeq technology. Sequence data was assembled and annotated at the GenDB platform yielding a total of 7Mb (CeBiTec, Uni-Bielefeld, Germany). Preliminary in silico analysis of the obtained data showed a high abundance of plasmid-related functions such as replication, stabilization, partition and mobilization. The variety of these genes gives an idea of the great diversity of the plasmids present in the BPS collection. As expected, the presence of different antibiotic and metal resistance types were detected, in agreement with the multi-resistance phenotypes observed for most of the isolates. In addition, the assembled data allowed us to obtain six complete replicons ranging between 4 and 40 Kbp in length. The detection of InP-1, IncP-7 and IncP-9 broad host range plasmids in our collection that are usually related to degradation of natural and xenobiotic pollutants, gives an indication of the presence of pesticide degrading functions and therefore are of particular interest when studying catabolic genes in the environment. Consequently, a detailed searching of catabolic genes is actually being carried out using the plasmid sequencing data set. Finally, the established database that contains the collective genes and operons with novel activities will be used for further bioinformatics and functional analysis. The presence of putative proteins with potential biotechnological applications justifies further work on gene cassette reservoirs as sources of functionally diverse proteins. References. 1. Eckhardt, T. A rapid method for the identification of plasmid deoxyribonucleic acid in bacteria. Plasmid, 1978. 1: 584-588.