ERDC/EL TR-12-33 Strategic Environmental Research and Development Program Identification of Microbial Gene Biomarkers for in situ RDX Biodegradation Project ER-1609 Fiona. H. Crocker, Karl J. Indest, Carina M. Jung, December 2012 Dawn E. Hancock, Megan E. Merritt, Christine Florizone, Hao-Ping Chen, Gordon R. Stewart, Songhua Zhu, Nicole Sukdeo, Marie-Claude Fortin, Steven J. Hallam, William W. Mohn, Lindsay D. Eltis, Nancy N. Perreault, Jian-Shen Zhao, Louise Paquet, Annamaria Halasz, and Jalal Hawari Environmental Laboratory Environmental Approved for public release; distribution is unlimited. Strategic Environmental Research and ERDC/EL TR-12-33 Development Program December 2012 Identification of Microbial Gene Biomarkers for in situ RDX Biodegradation Project ER-1609 Fiona. H. Crocker, Karl J. Indest, Carina M. Jung, Dawn E. Hancock, and Megan E. Merritt Environmental Laboratory U.S. Army Engineer Research and Development Center 3909 Halls Ferry Road Vicksburg, MS 39180-6199 Christine Florizone, Hao-Ping Chen, Gordon R. Stewart, Songhua Zhu, Nicole Sukdeo, Marie-Claude Fortin, Steven J. Hallam, William W. Mohn, and Lindsay D. Eltis Department of Microbiology and Immunology University of British Columbia 2350 Health Sciences Mall Vancouver, British Columbia Canada V6T 1Z3 Nancy N. Perreault, Jian-Shen Zhao, Louise Paquet, Annamaria Halasz and Jalal Hawari Biotechnology Research Institute, NRC 6100 Royalmount Avenue Montreal, Quebec Canada H4P 2R2 Final report Approved for public release; distribution is unlimited. Prepared for U.S. Army Corps of Engineers Washington, DC 20314-1000 ERDC/EL TR-12-33 ii Abstract Objectives of this project were to: (a) elucidate RDX degradation pathways in model RDX-degrading bacteria, (b) design and develop molecular tools to identify genes responsible for RDX biodegradation, and (c) correlate the response of biomarker(s) to concentrations of RDX and/or rates of RDX degradation. Gordonia sp. KTR9 and Shewanella oneidensis MR-1 served as model bacterial systems for the aerobic and anaerobic degradation of RDX, respectively. Genome annotation and functional characterization of the plasmid pGKT2 in KTR9 revealed that xplA gene is both necessary and sufficient for RDX degradation. Shewanella oneidensis MR-1 was shown to efficiently degrade RDX anaerobically via two initial routes: (a) sequential N-NO2 reduction to the corresponding nitroso (N-NO) derivatives; and (b) mono- denitration followed by ring cleavage. The qPCR molecular tools described in this report have the potential to be used by remediation specialists for site characterization, treatment recommendations, and for evaluation and optimization of the treatment process. The fundamental information gained in this study suggests that XplA- mediated aerobic denitration of RDX may be subjected to inhibitory effects in response to nitrogen availability. Additional research is required to determine reliable guidelines to inform site managers of specific field concentrations of ammonium and nitrate that will increase RDX treatment times. Also, techniques to effectively lower the inorganic nitrogen concentrations to non-inhibitory levels for the aerobic RDX biodegradation pathway will need to be determined. DISCLAIMER: The contents of this report are not to be used for advertising, publication, or promotional purposes. Citation of trade names does not constitute an official endorsement or approval of the use of such commercial products. All product names and trademarks cited are the property of their respective owners. The findings of this report are not to be construed as an official Department of the Army position unless so designated by other authorized documents. DESTROY THIS REPORT WHEN NO LONGER NEEDED. DO NOT RETURN IT TO THE ORIGINATOR. ERDC/EL TR-12-33 iii Contents Figures and Tables .................................................................................................................................. v Acronyms ................................................................................................................................................ix Preface ................................................................................................................................................. xiii 1 Project Objectives .......................................................................................................................... 1 2 Project Background ....................................................................................................................... 6 3 Gordonia sp. KTR9 Genome Annotation and Regulation of RDX Biodegradation ............... 13 Introduction ............................................................................................................................ 13 Gordonia sp. KTR9 genome assembly, annotation, and functional characterization of pGKT2 ..................................................................................................... 13 Methods ...................................................................................................................................... 14 Results and discussion .............................................................................................................. 19 Conclusions ................................................................................................................................ 31 Proteomic analysis of KTR9 RDX Biodegradation ................................................................ 31 Introduction ................................................................................................................................ 31 Methods ...................................................................................................................................... 32 Results and discussion .............................................................................................................. 35 Conclusions ................................................................................................................................ 38 Regulation of RDX biotransformation and gene expression in Gordonia sp. KTR9 ........... 39 Introduction ................................................................................................................................ 39 Methods ...................................................................................................................................... 40 Results and discussion .............................................................................................................. 44 Conclusion .................................................................................................................................. 58 Role of nitrogen limitation and GlnR on RDX degradation ................................................... 59 Introduction ................................................................................................................................ 59 Methods ...................................................................................................................................... 59 Results and discussion .............................................................................................................. 62 Conclusions ................................................................................................................................ 75 4 Determination of RDX Biodegradation Pathways in Shewanella oneidensis MR-1 ............. 76 Introduction ............................................................................................................................ 76 Growth physiology of Shewanella oneidensis MR-1 on RDX as a carbon or nitrogen source or electron acceptor .................................................................................... 77 Introduction ................................................................................................................................ 77 Methods ...................................................................................................................................... 77 Results and discussion .............................................................................................................. 81 Conclusions ................................................................................................................................ 84 Gene disruption and characterization of RDX pathways in mutant strains ........................ 86 Introduction ................................................................................................................................ 86 Methods ...................................................................................................................................... 86 ERDC/EL TR-12-33 iv Results and discussion .............................................................................................................. 89 Conclusions ................................................................................................................................ 94 Transcriptomic analysis of MR-1 RDX biodegradation ......................................................... 95 Introduction ................................................................................................................................ 95 Methods ...................................................................................................................................... 95 Results and discussion .............................................................................................................