Biodegradation of RDX in Rhodococcus spp.

The manufacture, use and storage of explosives over decades have seriously contaminated the environment. Hexa-hydro-1,3,5-trinitro-1,3,5-triazine (Royal Demolition Explosive - RDX) is one of the most widely used explosives. RDX is a man-made compound, recalcitrant to degradation and proven to be toxic to organisms. Bacteria capable of utilising RDX as a sole nitrogen source for growth have been isolated from RDX polluted sites including Rhodococcus rhodochrous 11Y. The RDX degrading genes, xplA and xplB encoding a novel flavodoxin fused cytochrome P450 and its reductase partner respectively, were first identified in R. rhodochrous 11Y. This unusual P450 system has now been found in almost all of the RDX degrading bacteria isolated so far. To date, XplA/B remains the only characterised RDX degrading P450 system and is encoded on an operon in strain 11Y, which also contains a putative permease (transporter) and transcriptional regulator. This gene cluster is highly conserved amongst other RDX degrading bacteria from geographically distinct regions including the United Kingdom, Belgium, Australia and North America, suggesting the xplA/B gene cluster may have been rapidly distributed across the globe by horizontal gene transfer. The first aim of this study was to characterise Rhodococcus erythropolis HS4, a bacterium that degrades RDX slowly but did not contain xplA and xplB. A number of approaches have been employed to characterise this strain, which includes whole cell assays, western analysis, cell free extract activity assays, PCR amplification and affinity purification of protein in R. erythropolis HS4. While whole cells of HS4 showed low RDX degrading activity, no RDX activity was observed in HS4 cell free extracts. Attempts to purify an RDX degrading enzyme from R. erythropolis HS4 were unsuccessful. The second aim of this project was to characterise the RDX degrading gene cluster in R. rhodochrous 11Y. Four genes encoding xplB, xplA, a putative permease and a MarR type regulator, were individually deleted using an unmarked gene deletion system (pK18mobsacB). The xplB knockout strain metabolised RDX more slowly than the wild-type suggesting XplA was able to obtain reducing equivalents from another source in the xplB knockout strain. The xplA knockout strain did not show RDX activity in whole cell and growth experiments. Neither xplA nor its gene product XplA was detected in the xplA knockout strain. The findings suggested no alternative RDX degrading system is present in strain 11Y. The permease knockout did not show significant difference in the RDX removal rate compared to wild-type. Another attempt to characterise the permease in 11Y was to express it in E. coli. No significant difference of RDX uptake between the permease-expressing clone and the control (non-expressing clone) in the uptake assays. The regulator knockout strain had a lower RDX degradation rate than wild-type in the presence of nitrate/nitrite. Also, when the regulator knockout and wild-type strains were previously exposed to nitrate/nitrite, the knockout showed lower RDX degrading activity. Further investigation of xplA regulation in strain 11Y showed that XplA activity was induced by nitrogen-limiting conditions and further induced by RDX. This was the first observation on the XplA activity could be induced by low nitrogen availability in medium. These results suggested that the regulator is indirectly involved in controlling the expression of XplA in 11Y, which is linked to central nitrogen metabolism.