Long-Range PCR Reveals the Genetic Cargo of IncP-1 Plasmids in the Complex Microbial Community of an On-Farm Biopurification System Treating Pesticide-Contaminated Wastewater.

Promiscuous plasmids like IncP-1 plasmids play an important role in the bacterial adaptation to pollution by acquiring and distributing xenobiotic catabolic genes. However, most information comes from isolates and the role of plasmids in governing community-wide bacterial adaptation to xenobiotics and other adaptive forces is not fully understood. Current information on the contribution of IncP-1 plasmids in community adaptation is limited because methods are lacking that directly isolate and identify the plasmid borne adaptive functions in whole-community DNA. In this study, we optimized long-range PCR to directly access and identify the cargo carried by IncP-1 plasmids in environmental DNA. The DNA between the IncP-1 backbone genes trbP and traC, a main insertion site of adaptive trait determinants, is amplified and its content analyzed by high-throughput sequencing. The method was applied to DNA of an on-farm biopurification system (BPS), treating pesticide contaminated wastewater, to examine whether horizontal gene exchange of catabolic functions by IncP-1 plasmids is a main driver of community adaptation in BPS. The cargo recovered from BPS community DNA encoded catabolic but also resistance traits and various other (un)known functions. Unexpectedly, genes with 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 remnants of random gene recruitment, or confer other crucial functions for proliferation in the BPS environment. IMPORTANCE This study presents a long-range PCR for direct and cultivation-independent access to the identity of the cargo of a major insertion hot spot of adaptive genes in IncP-1 plasmids and hence a new mobilome tool for understanding the role of IncP-1 plasmids in complex communities. The method was applied to DNA of an on-farm biopurification system (BPS) treating pesticide-contaminated wastewater, aiming at new insights on whether horizontal exchange of catabolic functions by IncP-1 plasmids is a main driver of community adaptation in BPS. Unexpectedly, catabolic functions represented a small fraction of the cargo genes while multiple other gene functions were recovered. These results show that the cargo of the target insertion hot spot in IncP-1 plasmids in a community, not necessarily relates to the main obvious selective trait imposed on that community. Instead, these functions might contribute to adaptation to unknown selective forces or represent remnants of random gene recruitment.

Targeted metagenomics demonstrates the ecological role of IS1071 in bacterial community adaptation to pesticide degradation.

IS1071, an insertion element that primarily flanks organic xenobiotic degradation genes in cultured isolates, is suggested to play a key role in the formation and distribution of bacterial catabolic pathway gene clusters. However, in environmental settings, the identity of the IS1071 genetic cargo and its correspondence to the local selective conditions remain unknown. To respond, we developed a long-range PCR approach 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 biopurification systems (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 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 a high variety of 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 xenobiotica stressed environments and contributes to community level adaptation towards pesticide biodegradation.

Response of the bacterial community in an on-farm biopurification system, to which diverse pesticides are introduced over an agricultural season.

A biopurification system (BPS) is used on-farm to clean pesticide-contaminated wastewater. Due to high pesticide loads, a BPS represents a hot spot for the proliferation and selection as well as the genetic adaptation of discrete pesticide degrading microorganisms. However, while considerable knowledge exists on the biodegradation of specific pesticides in BPSs, the bacterial community composition of these systems has hardly been explored. In this work, the Shannon diversity, the richness and the composition of the bacterial community within an operational BPS receiving wastewater contaminated with various pesticides was, for the first time, elucidated over the course of an agricultural season, using DGGE profiling and pyrosequencing of 16S rRNA gene fragments amplified from total community DNA. During the agricultural season, an increase in the concentration of pesticides in the BPS was observed along with the detection of significant community changes including a decrease in microbial diversity. Additionally, a significant increase in the relative abundance of Proteobacteria, mainly the Gammaproteobacteria, was found, and OTUs (operational taxonomic units) affiliated to Pseudomonas responded positively during the course of the season. Furthermore, a banding-pattern analysis of 16S rRNA gene-based DGGE fingerprinting, targeting the Alpha- and Betaproteobacteria as well as the Actinobacteria, indicated that the Betaproteobacteria might play an important role. Interestingly, a decrease of Firmicutes and Bacteroidetes was observed, indicating their selective disadvantage in a BPS, to which pesticides have been introduced.

Functional Redundancy of Linuron Degradation in Microbial Communities in Agricultural Soil and Biopurification Systems.

UNLABELLED: The abundance of libA, encoding a hydrolase that initiates linuron degradation in the linuron-metabolizing Variovorax sp. strain SRS16, was previously found to correlate well with linuron mineralization, but not in all tested environments. Recently, an alternative linuron hydrolase, HylA, was identified in Variovorax sp. strain WDL1, a strain that initiates linuron degradation in a linuron-mineralizing commensal bacterial consortium. The discovery of alternative linuron hydrolases poses questions about the respective contribution and competitive character of hylA- and libA-carrying bacteria as well as the role of linuron-mineralizing consortia versus single strains in linuron-exposed settings. Therefore, dynamics of hylA as well as dcaQ as a marker for downstream catabolic functions involved in linuron mineralization, in response to linuron treatment in agricultural soil and on-farm biopurification systems (BPS), were compared with previously reported libA dynamics. The results suggest that (i) organisms containing either libA or hylA contribute simultaneously to linuron biodegradation in the same environment, albeit to various extents, (ii) environmental linuron mineralization depends on multispecies bacterial food webs, and (iii) initiation of linuron mineralization can be governed by currently unidentified enzymes. IMPORTANCE: A limited set of different isofunctional catabolic gene functions is known for the bacterial degradation of the phenylurea herbicide linuron, but the role of this redundancy in linuron degradation in environmental settings is not known. In this study, the simultaneous involvement of bacteria carrying one of two isofunctional linuron hydrolysis genes in the degradation of linuron was shown in agricultural soil and on-farm biopurification systems, as was the involvement of other bacterial populations that mineralize the downstream metabolites of linuron hydrolysis. This study illustrates the importance of the synergistic metabolism of pesticides in environmental settings.

Exploring the complex response to linuron of bacterial communities from biopurification systems by means of cultivation-independent methods.

On-farm biopurification systems (BPSs) treat pesticide-contaminated wastewater at farms through biodegradation and sorption processes. However, information on the microbiota involved in pesticide removal in BPSs is scarce. Here we report on the response of BPS bacterial communities to the herbicide linuron (BPS(+)) compared with the control (BPS(-)) in a microcosm experiment. Both denaturing gradient gel electrophoresis (DGGE) and pyrosequencing of 16S rRNA gene fragments amplified from community DNA indicated shifts in the bacterial community after linuron application. Responding populations belonged to taxa that were previously reported from linuron degrading consortia cultivated from soil (Hyphomicrobiaceae, Comamonadaceae, Micrococcaceae). In addition, numerous taxa with increased relative abundance were identified that were previously not associated with linuron degradation. The relative abundance of IncP-1 korB copies increased in response to linuron application. Amplicon pyrosequencing of IncP-1 trfA genes revealed a high IncP-1 plasmid diversity and suggested that populations carrying IncP-1ß plasmids increased in relative abundance. Transferable mercury resistance plasmids were exogenously captured from BPS(+)/BPS(-), and in three transconjugants from BPS(+) the gene hylA was detected. Our data suggest the existence of a multispecies linuron degrading bacterial food web and an involvement of IncP-1 plasmids in the adaptation of bacterial communities to pesticide pollution in BPSs.

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.

Biopurification systems (BPS) are used on farms to control pollution by treating pesticide-contaminated water. It is assumed that mobile genetic elements (MGEs) carrying genes coding for enzymes involved in degradation might contribute to the degradation of pesticides. Therefore, the composition and shifts of MGEs, in particular, of IncP-1 plasmids carried by BPS bacterial communities exposed to various pesticides, were monitored over the course of an agricultural season. PCR amplification of total community DNA using primers targeting genes specific to different plasmid groups combined with Southern blot hybridization indicated a high abundance of plasmids belonging to IncP-1, IncP-7, IncP-9, IncQ, and IncW, while IncU and IncN plasmids were less abundant or not detected. Furthermore, the integrase genes of class 1 and 2 integrons (intI1, intI2) and genes encoding resistance to sulfonamides (sul1, sul2) and streptomycin (aadA) were detected and seasonality was revealed. Amplicon pyrosequencing of the IncP-1 trfA gene coding for the replication initiation protein revealed high IncP-1 plasmid diversity and an increase in the abundance of IncP-1ß and a decrease in the abundance of IncP-1ε over time. The data of the chemical analysis showed increasing concentrations of various pesticides over the course of the agricultural season. As an increase in the relative abundances of bacteria carrying IncP-1ß plasmids also occurred, this might point to a role of these plasmids in the degradation of many different pesticides.

Characterization of new bacterial catabolic genes and mobile genetic elements by high throughput genetic screening of a soil metagenomic library.

A mix of oligonucleotide probes was used to hybridize soil metagenomic DNA from a fosmid clone library spotted on high density membranes. The pooled radio-labeled probes were designed to target genes encoding glycoside hydrolases GH18, dehalogenases, bacterial laccases and mobile genetic elements (integrases from integrons and insertion sequences). Positive hybridizing spots were affiliated to the corresponding clones in the library and the metagenomic inserts were sequenced. After assembly and annotation, new coding DNA sequences related to genes of interest were identified with low protein similarity against the closest hits in databases. This work highlights the sensitivity of DNA/DNA hybridization techniques as an effective and complementary way to recover novel genes from large metagenomic clone libraries. This study also supports that some of the identified catabolic genes might be associated with horizontal transfer events.

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.

High prevalence of IncP-1 plasmids and IS1071 insertion sequences in on-farm biopurification systems and other pesticide-polluted environments.

Mobile genetic elements (MGEs) are considered as key players in the adaptation of bacteria to degrade organic xenobiotic recalcitrant compounds such as pesticides. We examined the prevalence and abundance of IncP-1 plasmids and IS1071, two MGEs that are frequently linked with organic xenobiotic degradation, in laboratory and field ecosystems with and without pesticide pollution history. The ecosystems included on-farm biopurification systems (BPS) processing pesticide-contaminated wastewater and soil. Comparison of IncP-1/IS1071 prevalence between pesticide-treated and nontreated 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, MGE were less abundant in field ecosystems without pesticide history although some of them still showed a high IS1071 abundance. Our data suggest that MGE-containing organisms were enriched in pesticide-contaminated environments like BPS where they might contribute to spreading of catabolic genes and to pathway assembly.

T4-related bacteriophage LIMEstone isolates for the control of soft rot on potato caused by 'Dickeya solani'.

The bacterium 'Dickeya solani', an aggressive biovar 3 variant of Dickeya dianthicola, causes rotting and blackleg in potato. To control this pathogen using bacteriophage therapy, we isolated and characterized two closely related and specific bacteriophages, vB_DsoM_LIMEstone1 and vB_DsoM_LIMEstone2. The LIMEstone phages have a T4-related genome organization and share DNA similarity with Salmonella phage ViI. Microbiological and molecular characterization of the phages deemed them suitable and promising for use in phage therapy. The phages reduced disease incidence and severity on potato tubers in laboratory assays. In addition, in a field trial of potato tubers, when infected with 'Dickeya solani', the experimental phage treatment resulted in a higher yield. These results form the basis for the development of a bacteriophage-based biocontrol of potato plants and tubers as an alternative for the use of antibiotics.

Bacteriophages LIMElight and LIMEzero of Pantoea agglomerans, belonging to the "phiKMV-like viruses".

Pantoea agglomerans is a common soil bacterium used in the biocontrol of fungi and bacteria but is also an opportunistic human pathogen. It has been described extensively in this context, but knowledge of bacteriophages infecting this species is limited. Bacteriophages LIMEzero and LIMElight of P. agglomerans are lytic phages, isolated from soil samples, belonging to the Podoviridae and are the first Pantoea phages of this family to be described. The double-stranded DNA (dsDNA) genomes (43,032 bp and 44,546 bp, respectively) encode 57 and 55 open reading frames (ORFs). Based on the presence of an RNA polymerase in their genomes and their overall genome architecture, these phages should be classified in the subfamily of the Autographivirinae, within the genus of the "phiKMV-like viruses." Phylogenetic analysis of all the sequenced members of the Autographivirinae supports the classification of phages LIMElight and LIMEzero as members of the "phiKMV-like viruses" and corroborates the subdivision into the different genera. These data expand the knowledge of Pantoea phages and illustrate the wide host diversity of phages within the "phiKMV-like viruses."