INVESTIGADORES
TORRES TEJERIZO Gonzalo Arturo
congresos y reuniones científicas
Título:
PLASMID ISOLATION AND CHARACTERIZATION FROM A BIOFILTER SYSTEM USED FOR PESTICIDE REMOVAL
Autor/es:
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.
Lugar:
Copenhagen
Reunión:
Congreso; ISME 14; 2012
Institución organizadora:
ISME
Resumen:
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