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 ...
Populations with high mutation rates (mutator clones) are being found in increasing numbers of species, and a clear link is being established between the presence of mutator clones and drug resistance. Mutator clones exist despite the fact that in a constant environment most mutations are deleterious, with the spontaneous mutation rate generally held at a low value. This implies that mutator clones have an important role in the adaptation of organisms to changing environments. Our study examines how mutator dynamics vary according to the frequency of environmental fluctuations. Although recent studies have considered a single environmental switch, here we investigate mutator dynamics in a regularly varying environment, seeking to mimic conditions present, for example, under certain drug or pesticide regimes. Our model provides four significant new insights. First, the results demonstrate that mutators are most prevalent under intermediate rates of environmental change. When the environment oscillates more rapidly, mutators are unable to provide sufficient adaptability to keep pace with the frequent changes in selection pressure and, instead, a population of 'environmental generalists' dominates. Second, our findings reveal that mutator dynamics may be complex, exhibiting limit cycles and chaos. Third, we demonstrate that when each beneficial mutation provides a greater gain in fitness, mutators achieve higher densities in more rapidly fluctuating environments. Fourth, we find that mutators of intermediate strength reach higher densities than very weak or strong mutators.
Background Mycobacterium bovis is the aetiological agent of bovine tuberculosis (bTB), an important recrudescent zoonosis, significantly increasing in British herds in recent years. Wildlife reservoirs have been identified for this disease but the mode of transmission to cattle remains unclear. There is evidence that viable M. bovis cells can survive in soil and faeces for over a year.Methodology/Principal FindingsWe report a multi-operator blinded trial for a rigorous comparison of five DNA extraction methods from a variety of soil and faecal samples to assess recovery of M. bovis via real-time PCR detection. The methods included four commercial kits: the QIAamp Stool Mini kit with a pre-treatment step, the FastDNA® Spin kit, the UltraClean™ and PowerSoil™ soil kits and a published manual method based on phenol:chloroform purification, termed Griffiths. M. bovis BCG Pasteur spiked samples were extracted by four operators and evaluated using a specific real-time PCR assay. A novel inhibition control assay was used alongside spectrophotometric ratios to monitor the level of inhibitory compounds affecting PCR, DNA yield, and purity. There were statistically significant differences in M. bovis detection between methods of extraction and types of environmental samples; no significant differences were observed between operators. Processing times and costs were also evaluated. To improve M. bovis detection further, the two best performing methods, FastDNA® Spin kit and Griffiths, were optimised and the ABI TaqMan environmental PCR Master mix was adopted, leading to improved sensitivities.Conclusions M. bovis was successfully detected in all environmental samples; DNA extraction using FastDNA® Spin kit was the most sensitive method with highest recoveries from all soil types tested. For troublesome faecal samples, we have used and recommend an improved assay based on a reduced volume, resulting in detection limits of 4.25×105 cells g−1 using Griffiths and 4.25×106 cells g−1 using FastDNA® Spin kit.
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