Engineering of the chloroaniline-catabolic plasmid pDCA-1 and its potential for genetic bioaugmentation

Abstract

Plasmids disseminate pollutant-catabolic genes through horizontal transfer and contribute to the catabolic potential of the host microbial community. Genetic bioaugmentation of conjugative catabolic plasmids has recently been considered an effective and novel approach for long-term bioremediation of contaminated sites. In this study, we engineered an IncP-1β-2 subgroup plasmid pDCA-1 from Achromobacter sp. ANB-1 for its potential application in genetic bioaugmentation. A dcaA1A2B cluster responsible for the deamination of chloroaniline to chlorocatechol and a 1,2-dioxygenase gene (ccdC) for the ring cleavage of chlorocatechol were found to locate on the broad host range plasmid pDCA-1. Insertion of an amidase gene (phh or tccA2) at the accessory region of plasmid pDCA-1 greatly expanded its catabolic substrate spectrum from chloroaniline (3-chloroaniline and 3,4-dichloroaniline) to herbicides (linuron, propanil, propham, and chlorpropham), bacteriostatic agent (triclocarban), plant regulator (forchlorfenuron), and insecticide (diflubenzuron). Taking advantages of high-throughput cell sorting and 16S rRNA gene-based amplicon sequencing, we depicted the diversity of bacterial recipients for the engineered plasmid pDCA-1-gfp-phh in a soil bacterial consortium, showing an excellent conjugative transfer capacity of the pDCA-1 derivative to various Gram negative and even positive strains in natural environment. All in all, the engineered plasmid pDCA-1 had a great potential in genetic bioaugmentation of the sites contaminated with chloroanilines and their derivatives.