PLASMID ISOLATION AND CHARACTERIZATION FROM A BIOFILTER SYSTEM USED FOR PESTICIDE REMOVAL

DEL PAPA M. F; MARTINI MC; SALAS ME; LOPEZ, JL; SALTO, I.; GIUSTI, M. A.; LOZANO M; TORRES TEJERIZO GA; PISTORIO, M.; SCHLÜTER, A.; PUHLER, A.; LAGARES, A.

Copenhagen

Congreso; ISME 14; 2012

ISME

A systematic search of genetic markers of interest, was performed over different terrestrial and aquatic habitats towards their deeper metagenomic analysis. Based on the result of such genetic pre-screening, a biofilter system implemented for decontamination of pesticides used in agriculture was selected for further studies. The target biofilter consists of a biological active matrix that retains/degrades pesticides into its organic matter. In order to investigate the type and diversity of the information encoded in the plasmid mobilome present in the biofilter, a plasmid search over more than 1,400 randomly selected bacterial clones was performed using in situ-lysis gel electrophoresis (Eckhardt protocol). The experimental approach allowed for the identification of 75 plasmid-containing clones, with molecular weights ranging from 30 to 300Kpb. Interestingly, almost 50% of the identified plasmids presented more than 100 Kbp in length. According to the plasmid profiles observed, the Isolates could be classified into 35 different diversity groups. From them, a sub-collection of clones representing all observed plasmid profiles were analyzed for the presence of replication functions present in the broad host range and conjugative IncP, IncN, IncW plasmids, and in the mobilizable IncQ plasmids. Over the purified collection of plasmids, a PCR-based screening was performed to identify clones carrying genes encoding for specific aliphatic and aromatic halogenases and dehalogenases, and for other enzymes of industrial interest such as laccases. Several plasmids resulted positive for these assays. Based on the observed diversity, the high molecular weight of most plasmids, and the marker genes that we found on them, we approached the deep sequencing of our plasmid collection by using the Ion Torrent technology. The total non-redundant DNA sequence accounted for more than 7 Mb, and has been automatically annotated via the GenDB pipeline available at the CeBiTec (Uni-Bielefeld, Germany). Genes of interest, as identified from the sequencing and bioinformatics analysis, are physically available in the conserved plasmid collection and can be recovered for activity evaluation. The quite diverse replication/mobilization elements that were identified and sequenced also provide a diverse set of functional modules that can be useful for the design of new (cloning/expression) plasmid vectors suitable for their evaluation and use in non-conventional environmental bacterial hosts