Lateral Transfer of Genes for Hexahydro-1,3,5-Trinitro-1,3,5-Triazine (RDX) Degradation

Abstract

Recent studies demonstrated that degradation of the military explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by species of Rhodococcus, Gordonia, and Williamsia is mediated by a novel cytochrome P450 with a fused flavodoxin reductase domain (XplA) in conjunction with a flavodoxin reductase (XplB). Pulse field gel analysis was used to localize xplA to extrachromosomal elements in a Rhodococcus sp. and distantly related Microbacterium sp. strain MA1. Comparison of Rhodococcus rhodochrous 11Y and Microbacterium plasmid sequences in the vicinity of xplB and xplA showed near identity (6,710 of 6,721 bp). Sequencing of the associated 52.2-kb region of the Microbacterium plasmid pMA1 revealed flanking insertion sequence elements and additional genes implicated in RDX uptake and degradation.Past practices of production, application, and disposal of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) have resulted in widespread contamination. Environmental contamination is aggravated by its high mobility, contributing to more-widespread contamination of groundwater than by other commonly used explosives (27). Ingestion or inhalation of RDX is associated with neurological disorders and organ failure (38), and exposed wildlife show behavioral changes and suffer liver and reproductive damage (38). The U.S. Environmental Protection Agency (EPA) has classified RDX as a possible human carcinogen (37). These adverse effects have provided motivation to better understand the microbiology and biochemistry of RDX degradation.As yet there is relatively limited information concerning natural rates of microbial RDX degradation or degradation mechanisms; such information is needed to predict or control rates of degradation in the environment. Of the three general pathways for RDX degradation or transformation based on metabolite analysis outlined in the review by Crocker and associates (6), aerobic degradation initiated by XplA is among the better-characterized systems. This enzyme, a novel cytochrome P450 with a fused flavodoxin reductive domain (18, 33), was first identified by Seth-Smith et al. in Rhodococcus rhodochrous 11Y as being encoded by xplA (33). This gene has been identified in 24 bacterial isolates of the Corynebacterineae capable of utilizing RDX as a sole nitrogen source (4, 25, 32-34). While mammalian nitric oxide synthase family enzymes are known to be P450-like enzymes with fused flavodoxin domains, there are very few identified examples of this type of protein fusion among characterized microbial species (2,15,18,24). Subsequent studies by Jackson and associates (18) demonstrated that XplA, in association with an electrontransferring flavodoxin reductase (XplB), functions to efficiently denitrate RDX aerobically to the aliphatic nitramine 4-nitro-2,4-diazabutanal (NDAB) (18). NDAB has been shown to serve as a viable nitrogen source for Methylobacterium sp. strain JS178 (13) and to be degraded by Phanerochaete chrysosporium (11). Thus, complete mineralization often appears to be mediated by multiple microbial popul...

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

“…The xplA gene was identified in several types of bacteria able to oxidatively exploit RDX as a source of nitrogen (61,62). XplA and its FAD-binding reductase partner XplB appear to be plasmid-encoded, along with several other genes involved in transport and degradation of RDX (63).…”

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

“…A gene cluster in strain 11Y essential for RDX degradation was identified and shown to contain a novel cytochrome P450, termed XplA, and a redox partner, XplB, which were shown together to be capable of catalyzing the biotransformation of RDX in vitro (5). Near identical xplA and xplB genes have now been identified in strains within the Actinomycetales isolated from geographically distinct sites (6). Interestingly, these genes have been found to be plasmid encoded providing compelling evidence for recent lateral gene transfer.…”

High-Sensitivity Stable-Isotope Probing by a Quantitative Terminal Restriction Fragment Length Polymorphism Protocol

“…Louis, MO) supplied as a nitrogen source. Genomic DNA mixtures were prepared from pure cultures as described previously, with the exception that sucrose was not used in the lysis solution (3). DNA for the SIP experiment was prepared using the soil extraction protocol described below, excluding aluminum sulfate incubation.…”

“…Soil slurries containing 13% (wt/vol) soil were incubated for 3.5 days with an enrichment medium for RDX degraders (3). RDX degradation was monitored using the previously described high-pressure liquid chromatography method (3).…”