S25: The Molecular Basis of RDX Degradation in Gordonia sp. KTR9: the Role of a Catabolic Plasmid, Horizontal Gene Transfer, and Nitrogen Limitation

Hexahydro-1,3,5-trinitro-1,3,5,-triazine (RDX) is a cyclic nitramine explosive that is a major component in many high-explosive formulations. Towards the goal of identifying genes and pathways involved in RDX transformation, the genome of RDX-degrading strain Gordonia sp. KTR9 was sequenced and annotated. Genome sequencing revealed the presence of a 182 kb plasmid, pGKT2, which contains the RDX degrading genes xplAB. Plasmid pGKT2 was shown to be both necessary and sufficient for RDX degradation, for KTR9 strains cured of pGKT2 were unable to transform or grow on RDX as a nitrogen source. In addition, successful conjugal transfer of pGKT2 was demonstrated from Gordonia sp. KTR9 to Gordonia polyisoprenivorans, Rhodococcus jostii RHA1 and Nocardia sp. TW2. The presence of pGKT2 conferred to transconjugants the ability to degrade and utilize RDX as a nitrogen source. Transcriptome analysis of strain KTR9 grown on different nitrogen sources which included 40 ppm RDX; 4 mM ammonium sulfate; or 40 ppm RDX supplemented with 4mM ammonium sulfate, revealed a significant overlap between differentially expressed gene sets common to the transcriptomes of cells grown on ammonium sulfate versus RDX and cells grown on the RDX versus RDX-and-ammonium together.  Specifically, genes involved in transport, nitrogen assimilation, nucleic acid/amino acid/fatty acid catabolism, and most importantly, RDX degradation are common to both comparisons. Collectively, these results strongly suggest that KTR9’s response to RDX is primarily one of nitrogen limitation and that xplAB was likely acquired by horizontal gene transfer and subsequently integrated into the cells global response to nitrogen limitation.