Cultivation-Independent Screening Revealed Hot Spots of IncP-1, IncP-7 and IncP-9 Plasmid Occurrence in Different Environmental Habitats

Abstract

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.

Shifts in Abundance and Diversity of Mobile Genetic Elements after the Introduction of Diverse Pesticides into an On-Farm Biopurification System over the Course of a Year

“…Several other studies have pointed to the role of IncP-1 plasmids in the adaptation of the bacterial community to xenobiotic compounds such as pesticides released into the environment (6,12; reviewed in references 41, 42, and 43). The high abundance of the subgroups IncP-1␤, -␥, -␦, and -ε revealed in the present study confirmed previously reported findings (15,37). Although Southern blot hybridization does not provide quantitative information on specific gene copy numbers per gram of material as does quantitative PCR (qPCR), changes in the abundances of the different subgroups could be indicated which were confirmed by amplicon pyrosequencing of the trfA amplicon obtained with the same primer sets as those used for the PCR screening.…”

“…The very high relative abundances of the promiscuous IncP-1 plasmids detected in the BPS are comparable to those seen with other "hot spots" of horizontal gene transfer such as manure, sewage, or polluted river sediments (39,40). Our data confirm the results of two recent studies using TC-DNA from different BPS which also found an unusually high abundance of IncP-1 plasmids in the BPS (15,37). Interestingly, the abundance of IncP-1 plasmids seemed to be increased in BPS bacteria, likely in response to the exposure to different types and concentrations of pesticides as recently reported by Jechalke et al (14).…”

“…A semiquantitative but highly sensitive and specific approach was used to detect plasmid replicon-, integrase gene-, and antibiotic resistance gene-specific sequences in TC-DNA. The strong PCR-Southern blot hybridization signals for IncP-1-, IncP-7-, and IncP-9-specific sequences amplified from TC-DNA indicated a high abundance of potentially catabolic plasmids in BPS bacterial communities throughout the agricultural season and thus confirmed previous observations made for single time points from different BPS (15). For the first time, the IncW-, IncQ-, and IncU-specific sequences which typically Sequences were first clustered at 89% nucleotide sequence identity (A), and sequences from large clusters were then reclustered at 97% similarity (B), with small clusters excluded.…”

“…high abundance of IncP-1, IncP-7, and IncP-9 plasmids and insertion sequence (IS) elements typically associated with degradative genes (15,37). Here we monitored fluctuations of a range of different plasmids, integrase genes of class 1 and class 2 integrons, and sulfonamide and streptomycin resistance genes at three times during the agricultural season: before the pesticide spraying season (March), at the height of the spraying season (July), and after the spraying season (September).…”

“…In a PCR-Southern blot-based screening of total community DNA (TC-DNA) from various environments and different geographic locations for the occurrence of potentially catabolic plasmids belonging to the IncP-1, IncP-7, and IncP-9 group, BPS material was revealed as a "hot spot" for these plasmids (15). In accordance with previous findings (14,37), we hypothesize that temporal changes in the abundance and diversity of MGEs in an on-farm BPS might be caused by changes in the composition and concentration of the pesticides added to the BPS over the course of the agricultural season.…”