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 analysed 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, 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 selective trait imposed on that community. Instead these functions might contribute to adaptation to unknown selective forces or represent remnants of random gene recruitment.

A Pesticide Biopurification System: A Source of Biosurfactant-Producing Bacteria with Environmental Biotechnology Applications

Biosurfactants, a wide group of compounds produced by different microorganisms, generally have less toxicity and are more biodegradable than synthetic surfactants. Biosurfactant-producing bacteria can be found in contaminated environments, such as soils receiving pesticide applications constantly, or in pesticides treatment systems where microorganisms are adapted to biodegrading pesticides. Five pesticide-tolerant bacteria previously isolated from a pesticide biopurification system were evaluated as biosurfactant-producers. Pseudomonas rhodesiae C4, Rhodococcus jialingiae C8 and Pseudomonas marginalis C9 strains were positive in qualitative tests. Biosurfactant production by these strains using Bushnell-Haas medium with olive oil at 2% (w/v) was evaluated as emulsification index, oil displacement, droplet collapse test and surface tension. After 144 h, these strains showed a similar emulsification index of >55%. The two Pseudomonas (C4 and C9) strains showed lower superficial tension compared with Rhodococcus strain (C8)—34.47, 37.44 and 47.55 mN/m for strains C4, C9 and C8, respectively. The chemical characterization of the biosurfactants revealed the presence of glycolipids in P. rhodesiae (C4) and glycopeptides in P. marginalis (C9). The degradation of chlorpyrifos increased from 39.2% to 51.6% when biosurfactants produced by P.rhodesiae (C4) were added (10%) with respect to the control. Therefore, biopurification systems are a relevant source of biosurfactant-producing bacteria with environmental biotechnology applications.

Biopurification system as a source of pesticide-tolerant bacteria able to degrade the commonly used pesticides chlorpyrifos and iprodione

Intensive use of pesticides applied simultaneously in field to improve the effectiveness of pest control increase the environmental contamination, affecting the soil and water quality. Some of the commonly used pesticides are the insecticide chlorpyrifos and the fungicide iprodione; being thus critically essential to develop bioremediation methods to remove these contaminants by tolerant-bacteria. In this study we selected and characterized different pesticides-tolerant bacteria isolated from a biomixture of a biopurification system that had received continuous applications of a mixture of the pesticides chlorpyrifos and iprodione. Out of the 10 isolated bacterial colonies, only six strains presented adequate growth in presence of the both pesticides at 100 mg L-1. Biochemical and enzymatic characterization using API ZYM showed that all isolates (100%) were positive for esterase, leucine aminopeptidase, acid phosphatase, and naphthol-AS-BI-phosphohydrolase. According to the molecular level study of the 16S ribosomal gene and MALDI TOF/TOF MS, it was possible to determine that the isolated bacteria belong to the genera Pseudomonas, Rhodococcus and Achromobacter. Bacterial growth decreased proportionally (R2 > 0.96) as been as both pesticide concentrations increased from 10 to 100 mg L-1. Achromobacter sp. strain C1 showed the best chlorpyrifos removal (between 56–29%) after 120 h of incubation. On the other hand, the highest iprodione removal (between 91.2–98.9%) was observed for the Pseudomonas sp. strain C9, which was not detected after 48 h of incubation. According with their identification and ability to remove the contaminants, Achromobacter sp. strain C1 and Pseudomonas sp. strain C9 appear as promising microorganisms for their use in the treatment of matrices contaminated with chlorpyrifos, iprodione or their mixture. The results of this study will help to improve current technologies for the biodegradation of this commonly used insecticide and fungicide, in order to give a response to the problem of contamination by pesticides.