A Novel Catabolic Plasmid for 1,2-dichloroethane Biodegradation  — ASN Events
  1. Schedule
  2. Division 3 – General, Applied and Environmental Microbiology
  3. Symposium 3 - Bioremediation
  4. A Novel Catabolic Plasmid for 1,2-dichloroethane Biodegradation 

A Novel Catabolic Plasmid for 1,2-dichloroethane Biodegradation  (#58)

Jacob E Munro 1 , Nicholas V Coleman 1
  1. School of Molecular Bioscience, The University of Sydney, Sydney, NSW, Australia

Organochlorines are problematic environmental contaminants. The groundwater at the Botany Industrial Park in South Sydney is heavily contaminated with the organochlorine compound 1,2-dichloroethane (DCA), which is used for plastics manufacture. We previously isolated five different DCA-degrading bacteria from this site, and detected haloalkane and haloacid dehalogenase genes dhlA and dhlB in 4/5 strains. 

Fosmid libraries were constructed from genomic DNA of three Botany isolates: Starkeya EL1, Xanthobacter EL4, and Xanthobacter EL8. Clones containing dhlA and dhlB were detected by hybridisation and PCR, and sequenced using Illumina and Ion-Torrent. The sequences were difficult to assemble, and needed to be manually completed with using Sanger sequencing (gap-filling by PCR and primer walking). The assembled sequences were analysed.

The dhlB genes are relatively diverse in sequence across the strains, and are located on the chromosomally. The dhlA genes are identical in all strains, and are associated with plasmid and transposase genes. The complete sequence of a novel 35 kb plasmid (pDCA) was reconstructed from the fosmid sequences. Unique variants of this plasmid exist in each of the three strains.

The pDCA plasmid has replication, partitioning, and conjugation genes similar to those of described α-proteobacterial plasmids, eg. from Agrobacterium. The plasmid contains many Tn3 and IS4 transposases, and a type III restriction/modification system. Surprisingly, a dhlB homolog is also present on the dhlA-containing plasmid, which is divergent (36% amino acid identity) from the chromosomal dhlB genes detected in fosmids.

Previous studies suggested that dhlA genes in Xanthobacter were carried on a 200 kb linear plasmid. Contrary to this, we have shown that circular ~35 kb plasmids carry both dhlA genes and dhlB-homologs in isolates from the Botany site. The location of DCA biodegradation genes on a plasmid predicted to be conjugative has significance for understanding the evolution of the DCA pathway, and for guiding future bioremediation work with DCA.