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

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-1 β -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.