Thursday, March 21, 2013

Ibuprofen-degrading bacterium found using CopyControl™ Fosmid Library

Disposal of pharmaceuticals into the environment through wastewater treatment systems, landfills or recycling systems is an ongoing problem that can cause serious unwanted changes in the fauna of our planet. Consequences include extinction of species, or genetic changes that can disrupt bio/ecosystems. There are microbes that can enzymatically break down many compounds and use much of it as a carbon/food source. In this report, Murdoch and Hay describe a strain of Sphingomonas that was able to at least partially degrade the anti-inflammatory/analgesic ibuprofen. Sphingomonas Ibu-2 has the unusual ability to cleave the acid side chain from the pharmaceutical ibuprofen and related arylacetic acid derivatives to yield corresponding catechols under aerobic conditions via a previously uncharacterized mechanism.

To locate the gene(s) responsible for this activity, the researchers generated a large-insert fosmid library using the CopyControl™ Fosmid Library Production Kit and screened the clones obtained by mutagenesis of the fosmids using transposon mutagenesis in standard gene knock-out experiments. Screening a chromosomal library of Ibu-2 DNA in E. coli EPI300 allowed identification of one fosmid clone that conferred the ability to metabolize ibuprofen to isobutylcatechol. Characterization of fosmid-specific loss-of-function transposon mutants permitted identification of five ORFs, ipfABDEF, whose predicted amino acid sequences bore similarity several known genes that are involved in an aromatic dioxygenase system, respectively, were also identified in a second fosmid isolated from the same library. Complementation of a markerless loss-of-function of a certain deletion mutant restored catechol production as did complementation of the Transposon mutant. Expression of subcloned ibuprofen degradation operon (ipfABDEF) alone in E. coli did not impart full metabolic activity unless it was coexpressed with the ipfHI gene. This work provides preliminary insights into the mechanism behind this novel arylacetic acid-deacylating, catechol-generating activity during ibuprofen metabolism.

ResearchBlogging.orgMurdoch RW, et al. (2013). Genetic and chemical characterization of ibuprofen degradation by Sphingomonas Ibu-2. Microbiology (Reading, England), 159 (Pt 3), 621-32 PMID: 23329679