Cloning, Sequencing, and Characterization of the Hexahydro-1,3,5-Trinitro-1,3,5-Triazine Degradation Gene Cluster from Rhodococcus rhodochrous

Abstract

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a high explosive which presents an environmental hazard as a major land and groundwater contaminant. Rhodococcus rhodochrous strain 11Y was isolated from explosive contaminated land and is capable of degrading RDX when provided as the sole source of nitrogen for growth. Products of RDX degradation in resting-cell incubations were analyzed and found to include nitrite, formaldehyde, and formate. No ammonium was excreted into the medium, and no dead-end metabolites were observed. The gene responsible for the degradation of RDX in strain 11Y is a constitutively expressed cytochrome P450-like gene, xplA, which is found in a gene cluster with an adrenodoxin reductase homologue, xplB. The cytochrome P450 also has a flavodoxin domain at the N terminus. This study is the first to present a gene which has been identified as being responsible for RDX biodegradation. The mechanism of action of XplA on RDX is thought to involve initial denitration followed by spontaneous ring cleavage and mineralization.Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is one of the most important and widely used military explosives. Due to its manufacture, decommissioning, and disposal it has become a serious pollutant at many sites, particularly across the United States and Germany, and its recalcitrance in the environment has led to its persistence in soil and groundwater. RDX is a potent convulsant due to its effects on the central nervous system and is also a class C, possible, carcinogen (7).For a long time it was thought that RDX biodegradation could occur only under anaerobic conditions (15, 26); however, aerobic degradation of RDX has been more recently demonstrated by bacterial consortia and pure strains. Coryneform bacteria (45), Stenotrophomonas maltophilia strain PB1 (4), and a rhodococcal strain (9) have been shown to utilize RDX as a sole source of nitrogen, generally degrading RDX at higher rates than anaerobic systems do.The first pathway for the aerobic degradation of RDX by a strain of Rhodococcus was proposed very recently (16) and involves denitration as the first step followed by spontaneous ring cleavage (Fig. 1). Positively identified products include nitrite (NO 2 Ϫ ), nitrous oxide (N 2 O), formaldehyde (HCHO), and carbon dioxide. A dead-end intermediate with the molecular formula C 2 H 5 N 3 O 3 was also found to accumulate, indicating that not all the components of RDX were mineralized. This paper reports the isolation and identification of a Rhodococcus rhodochrous strain which can degrade RDX as a nitrogen source and the first cloning and expression of a gene responsible for RDX degradation. This gene is constitutively expressed, and its product has homology to a cytochrome P450. MATERIALS AND METHODSReagents. RDX (Ͼ95% purity as determined by high-performance liquid chromatography HPLC) was kindly provided by the Defence Science and Technology Laboratory (Dstl), Fort Halstead. Other chemicals were of analytical grade and, unless otherwise stated, were obtained from ...

The 1.5-Å Structure of XplA-heme, an Unusual Cytochrome P450 Heme Domain That Catalyzes Reductive Biotransformation of Royal Demolition Explosive

“…The bioremediation of RDX has thus been the focus of increasing research in recent years, with a number of bacterial strains reported to catalyze its degradation (1)(2)(3). Among these, Bruce and co-workers, using a selective enrichment technique, isolated Rhodococcus rhodochrous strain 11Y from an RDX-contaminated site, which was able to grow on RDX as the sole nitrogen source (4). The products of biotransformation of RDX by this bacterium were shown to be nitrite and formaldehyde.…”

Unusual Spectroscopic and Ligand Binding Properties of the Cytochrome P450-Flavodoxin Fusion Enzyme XplA

“…XplA falls into a different class of P450 redox system, with a flavodoxin-like module (N-terminal) fused to the P450 (1,21,22). XplA receives electrons from a NADPH-dependent flavoprotein reductase (XplB) encoded by the xplB gene immediately upstream of xplA on the R. rhodochrous strain 11Y genome (23).…”

Microbial cytochromes P450: biodiversity and biotechnology. Where do cytochromes P450 come from, what do they do and what can they do for us?

“…CYP177A1 is another new class of cytochrome P450 (Class VI) encoding a natural fusion between an N-terminal flavodoxin domain and a C-terminal haem domain identified by Seth-Smith et al [51]. It was identified in Rhodococcus rhodochrous (strain 11Y) and it allows this bacterium to obtain nitrogen from a high-explosive pollutant called RDX (see earlier).…”