Cultivation independent analysis of the mobilome of polluted ecosystems: a new approach for mining of novel biocatalysts.

The intensive and widespread use of pesticides over the last decades, has resulted in the contamination of water and soil ecosystems by compounds that are xenobiotic and often persistent. Despite the relative recent introduction of pesticides into our environment, microorganisms have been shown to create novel catabolic pathways allowing to degrade specific xenobiotics. The ability of microorganisms to adapt swiftly to changing environmental conditions and exploit ecological novel niches arises from their genome plasticity and adaptability. A key mechanism in this bacterial genome plasticity is the intra- and extracellular exchange of genetic material facilitated by vehicles called mobile genetic elements (MGEs). Genomic studies from xenobiotic degraders showed that two particular MGEs, I.e., IncP-1 plasmids and IS element IS1071, are often retrieved as carriers of xenobiotic catabolic genes. Therefor it can be hypothesized that IncP-1 and IS1071 play a crucial role in de mobilisation and recruitment of xenobiotic catabolic gene functions in the environment. However, not much is known about their ecology at a community wide base nor about the adaptative genes they carry in complex environmental communities.

The objective of this thesis is to contribute in elucidating the role of IS1071 and IncP‑1 in the dissemination of catabolic gene clusters in the microbiome of pesticide contaminated environments. Specifically, the prevalence of these MGEs in microbiomes of different ecosystems were studied and molecular toolboxes developed to assess their accessory gene loads.

The first objective was to examine whether a relationship existed between the prevalence of IncP-1/IS1071 and pesticide pollution. Therefore, the prevalence and abundance of IncP-1 plasmids and IS1071 in lab and field ecosystems with and without pesticide pollution history was examined. The ecosystems included on-farm biopurification systems (BPS) processing pesticide contaminated wastewater and pesticide treated soil. Comparison of IncP-1/IS1071 prevalence between pesticide treated and non-treated soil and BPS microcosms suggested that both IncP-1 and IS1071 proliferated as a response to pesticide treatment. The increased prevalence of IncP-1 plasmids and IS1071 specific sequences in treated systems was accompanied by an increase in the capacity to mineralize the applied pesticides. Both elements were also encountered in high abundance in field BPS ecosystems that were in operation at farmyards and that showed the capacity to degrade/mineralize a wide range of chlorinated aromatics and pesticides. In contrast, IS1071 and especially IncP‑1 plasmids were less abundant in field ecosystems without pesticide history although some of them still showed a high IS1071 abundance. These data suggest that IS1071 and IncP‑1 containing organisms were enriched in pesticide contaminated environments like BPS where they might contribute to the spreading of catabolic genes and to pathway assembly.

To further unravel and acknowledge the ecological role of these two MGEs, novel cultural independent toolboxes, based on long-range PCR, were developed that allow the recover and identify in a cultivation-independent way the accessory genes carried by IS1071 composite transposons and IncP‑1 plasmids. First a long-range PCR approach was developed that allowed to amplifying accessory genes between two IS1071 copies from community DNA followed by amplicon sequencing. We applied this method to pesticide exposed environments, i.e., linuron-treated agricultural soil and on-farm BPS treating complex agricultural wastewater, as to non-treated controls. Amplicons were mainly recovered from the pesticide exposed environments and the BPS matrix showed a higher amplicon size diversity compared to the agricultural soil. Retrieved gene functions mirrored the main selection pressure as (i) a large fraction of the BPS amplicons contained genes/gene clusters related to the degradation of organics including herbicides present in the wastewater and (ii) in the agricultural soil, recovered genes were associated with linuron degradation. Our metagenomic analysis extends observations from cultured isolates and provides evidence that IS1071 is a carrier of catabolic genes in xenobiotic stressed environments and contributes to community level adaptation towards pesticide biodegradation.

Similarly, a long-range PCR to directly access and identify the cargo carried by IncP-1 plasmids in environmental DNA was developed. The method amplifies the DNA between the IncP-1 backbone genes trbP and traC, a main insertion site of adaptive trait determinants, and analyses its content by high-throughput sequencing, and was applied to DNA of an on-farm BPS, treating pesticide contaminated wastewater. The cargo recovered from BPS community DNA, encoded catabolic but also resistance traits and various other (un)known functions. Unexpectedly, catabolic traits composed only a minor fraction of the cargo, indicating that the IncP-1 region between trbP and traC is not a major contributor to catabolic adaptation of the BPS microbiome. Instead, it contains a functionally diverse set of genes which either may assist biodegradation functions, be co-selected with catabolic genes in other insertion hot spots or confer other crucial functions for proliferation in the BPS environment.

Our data supports the hypothesis that IS1071 and IncP-1 are linked to xenobiotic degradation in pesticide impacted environments, and that especially IS1071 is an important MGE for distributing xenobiotic catabolic gene functions. To fully understand the role of IS1071 and IncP-1 in bacterial adaptation and the type of traits it disperses throughout the microbial community it is essential to study different ecosystems with different environmental stressors.