Cultivation-independent screening revealed hot spots of IncP-1, IncP-7 and IncP-9 plasmid occurrence in different environmental habitats

IncP-1, IncP-7 and IncP-9 plasmids often carry genes encoding enzymes involved in the degradation of man-made and natural contaminants, thus contributing to bacterial survival in polluted environments. However, the lack of suitable molecular tools often limits the detection of these plasmids in the environment. In this study, PCR followed by Southern blot hybridization detected the presence of plasmid-specific sequences in total community (TC-) DNA or fosmid DNA from samples originating from different environments and geographic regions. A novel primer system targeting IncP-9 plasmids was developed and applied along with established primers for IncP-1 and IncP-7. Screening TC-DNA from biopurification systems (BPS) which are used on farms for the purification of pesticide-contaminated water revealed high abundances of IncP-1 plasmids belonging to different subgroups as well as IncP-7 and IncP-9. The novel IncP-9 primer-system targeting the rep gene of nine IncP-9 subgroups allowed the detection of a high diversity of IncP-9 plasmid specific sequences in environments with different sources of pollution. Thus polluted sites are "hot spots" of plasmids potentially carrying catabolic genes.Facultad de Ciencias ExactasInstituto de Biotecnologia y Biologia Molecula

Cultivation-independent screening revealed hot spots of IncP-1, IncP-7 and IncP-9 plasmid occurrence in different environmental habitats

IncP-1, IncP-7 and IncP-9 plasmids often carry genes encoding enzymes involved in the degradation of man-made and natural contaminants, thus contributing to bacterial survival in polluted environments. However, the lack of suitable molecular tools often limits the detection of these plasmids in the environment. In this study, PCR followed by Southern blot hybridization detected the presence of plasmid-specific sequences in total community (TC-) DNA or fosmid DNA from samples originating from different environments and geographic regions. A novel primer system targeting IncP-9 plasmids was developed and applied along with established primers for IncP-1 and IncP-7. Screening TC-DNA from biopurification systems (BPS) which are used on farms for the purification of pesticide-contaminated water revealed high abundances of IncP-1 plasmids belonging to different subgroups as well as IncP-7 and IncP-9. The novel IncP-9 primer-system targeting the rep gene of nine IncP-9 subgroups allowed the detection of a high diversity of IncP-9 plasmid specific sequences in environments with different sources of pollution. Thus polluted sites are "hot spots" of plasmids potentially carrying catabolic genes.Facultad de Ciencias ExactasInstituto de Biotecnologia y Biologia Molecula

Cultivation-independent screening revealed hot spots of IncP-1, IncP-7 and IncP-9 plasmid occurrence in different environmental habitats

IncP-1, IncP-7 and IncP-9 plasmids often carry genes encoding enzymes involved in the degradation of man-made and natural contaminants, thus contributing to bacterial survival in polluted environments. However, the lack of suitable molecular tools often limits the detection of these plasmids in the environment. In this study, PCR followed by Southern blot hybridization detected the presence of plasmid-specific sequences in total community (TC-) DNA or fosmid DNA from samples originating from different environments and geographic regions. A novel primer system targeting IncP-9 plasmids was developed and applied along with established primers for IncP-1 and IncP-7. Screening TC-DNA from biopurification systems (BPS) which are used on farms for the purification of pesticide-contaminated water revealed high abundances of IncP-1 plasmids belonging to different subgroups as well as IncP-7 and IncP-9. The novel IncP-9 primer-system targeting the rep gene of nine IncP-9 subgroups allowed the detection of a high diversity of IncP-9 plasmid specific sequences in environments with different sources of pollution. Thus polluted sites are "hot spots" of plasmids potentially carrying catabolic genes.Facultad de Ciencias ExactasInstituto de Biotecnologia y Biologia Molecula

Generation of probes for Southern blot hybridization.

<p>Generation of probes for Southern blot hybridization.</p

Bacterial densities and PCR-Southern blot hybridization detection of plasmid replicon-specific sequences belonging to the five IncP-1 subgroups, IncP-7 and IncP-9.

<p>Hybridization signal: (+++) very strong, with exposure time up to five minutes; (++) strong, with exposure time up to one hour; (+) weak, with exposure time up to three hours; (−) none, with exposure time of more than three hours; (/) not analyzed.</p

Biopurification systems (BPS).

<p>Hybridization of Southern-blotted PCR products obtained with <i>rep</i> primer system from TC-DNA of BPS with the IncP-7 probe generated from pCAR1. Lanes: 1, 13 and 26, dig ladder; lanes 2 to 4, BPS from Leefdaal, Belgium; lanes 5 to 10, BPS from Belgium (Pcfruit ); lanes 15 to 17, BPS from Lierde, Belgium; lanes 18 to 20, BPS from Kortrijk, Belgium; lanes 21 to 23, BPS from Koksijde, Belgium; lanes 11 and 24, negative control; lanes 12 and 25 IncP-7 positive control pCAR-1. Exposure time of 5 min.</p