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

Abstract

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-1β 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-1β plasmids also occurred, this might point to a role of these plasmids in the degradation of many different pesticides.

FIG 1

Rooted neighbor-joining trees of clusters of IncP-1 trfA amplicon sequences from BPS TC-DNA. Each branch represents one cluster of sequences. 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. Known reference sequences were included in the clustering, and their locations and subgroup names are indicated by colored boxes. The tables show the mean relative abundances of sequences from each primer set. There is a small overlap in targets between primer sets αβε and δ. Dotted circles indicate significant changes in abundance between March, July, and September (one-way ANOVA; n = 3; *, P < 0.05; **, P < 0.01; ***, P < 0.001). Numbers of branches represent percentages of bootstrap support of 1,000 iterations.