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Microbial adaptations towards utilisation of the explosive RDX in soil

Sabir, Dana (2015) Microbial adaptations towards utilisation of the explosive RDX in soil. PhD thesis, University of York.

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Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a synthetic toxic explosive compound which was introduced into the environment during the Second World War. Microorganisms have adapted to degrade RDX and the enzymes involved include an unusual cytochrome P450, termed XplA, that is N-terminally-fused to a flavodoxin domain, and a flavodoxin reductase partner, XplB. To discover new RDX-degrading enzymes, selective enrichments were performed on explosive-contaminated soil samples from the United Kingdom, Belgium, Germany, Czech Republic, Ukraine and Moldova. Thirteen RDX-degrading bacteria were isolated and all identified as Rhodococcus spp. The xplA gene was identified in all isolates and TNT found to inhibit RDX-degradation. The evolutionary origin of xplA was analysed in eleven aerobic RDX-degrading bacteria belonging to four different genera: Rhodococcus spp., Microbacterium, Gordonia and Williamsia. Only six single nucleotide polymorphisms were found in the xplA/xplB region, emphasising the recent evolution of these genes. Additionally, genes flanking xplA/xplB were nearly identical between the four genera and together comprise a genomic island approximately 36 kbp in size which has been transmitted horizontally within a transposable element. In Gordonia sp. KTR9 several gene re-arrangements were found on the xplA/xplB-containing plasmid, including the fusion of xplB to glnA. The XplB portion of the fusion was found to be inactive due to a serine to tryptophan substitution, whereas the GS portion, encoded by glnA, has activity, despite missing 15 % of the C-terminal region. Finally, the evolutionary origin of xplA was investigated by characterising three putative cytochromes P450: Gt-XplA from Gordonia terrae strain NBRC 100016; and CYPA and CYPB from Gordonia polyisoprenivorans NBRC 16320. Although purified Gt-XplA, CYPA and CYPB did not have activity towards RDX, activity was detected following the substitution of amino acids into the putative active site of GT-XplA and truncated-CYPA.

Item Type: Thesis (PhD)
Academic Units: The University of York > Biology (York)
Identification Number/EthosID: uk.bl.ethos.647077
Depositing User: Mr Dana Sabir
Date Deposited: 08 May 2015 11:23
Last Modified: 24 Jul 2018 15:20
URI: http://etheses.whiterose.ac.uk/id/eprint/8836

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