The widespread presence in the environment of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), one of the most widely used military explosives, has raised concern owing to its toxicity and recalcitrance to degradation. To investigate the potential of plants to remove RDX from contaminated soil and water, we engineered Arabidopsis thaliana to express a bacterial gene xplA encoding an RDX-degrading cytochrome P450 (ref. 1). We demonstrate that the P450 domain of XplA is fused to a flavodoxin redox partner and catalyzes the degradation of RDX in the absence of oxygen. Transgenic A. thaliana expressing xplA removed and detoxified RDX from liquid media. As a model system for RDX phytoremediation, A. thaliana expressing xplA was grown in RDX-contaminated soil and found to be resistant to RDX phytotoxicity, producing shoot and root biomasses greater than those of wild-type plants. Our work suggests that expression of xplA in landscape plants may provide a suitable remediation strategy for sites contaminated by this class of explosives.

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a widely used explosive and a serious environmental pollutant. Nineteen strains of Rhodococcus spp. capable of utilizing RDX as the sole nitrogen source have been isolated. The cytochrome P450 system XplA-XplB, which is responsible for RDX breakdown, is present in 18 of these strains.The explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a toxic anthropogenic compound that persists as an environmental pollutant as a result of decades of weapon manufacture, deployment, and decommissioning. Although RDX is a xenobiotic compound, bacteria have been shown to be able to degrade it. An unusual cytochrome P450 system comprising a flavodoxin domain fused to the N terminus of a cytochrome P450 (CYP177A1, XplA) and a partner flavodoxin reductase XplB (6, 7) is responsible for RDX degradation in Rhodococcus rhodochrous strain 11Y. The presence of oxygen influences which of two RDX degradation pathways is catalyzed by XplA (4).Selective enrichments were performed with minimal medium containing RDX as the sole source of nitrogen, using previously reported methods (1). Samples were provided by the Defence Science and Technology Laboratory of the United Kingdom Ministry of Defence, taken from an explosive-contaminated Ministry of Defence site, and stored at 4°C prior to enrichment. Nineteen pure bacterial isolates were able to remove RDX at a concentration of 1 mM from growth medium in 5 days, as assayed by thin-layer chromatography performed using a previously described method (7). None of the isolates were able to utilize RDX supplied as the sole carbon and nitrogen source (data not shown).The isolates were identified using the 16S rRNA gene, amplified from extracted genomic DNA using primers fD1 and rD1 (9) and PfuTurbo DNA polymerase (Stratagene). PCR (95°C for 2 min; 30 cycles of 95°C for 30 s, 55°C to 62°C for 30 s, and 72°C for 2 min; and 72°C for 10 min) yielded products of approximately 1.6 kb, which underwent sequencing. Each strain possesses a unique 16S rRNA sequence, differing from those of other strains by at least one nucleotide. BLAST searches showed that all bacteria belong to the genus Rhodococcus. These data, in conjunction with a phenotypic analysis of colony morphology on different media, determined that all strains were distinct, and strain names were assigned (Table 1).Resting-cell incubations were used to compare the RDXdegrading rates of the isolates. Cells were grown at 30°C in minimal medium containing 0.5 mM RDX as the sole nitrogen source and harvested 24 h after all the RDX had been removed from the growth medium, as assayed by high-performance liquid chromatography using a method previously described (7). Cells were rinsed twice in 40 mM potassium phosphate buffer, pH 7.5, and resuspended in the same buffer to a concentration of 500 mg (wet weight) per ml. Resting-cell incubations containing 250 M RDX and 100 l of cell suspension in 1 ml of potassium phosphate buffer, pH 7.5, were performed at 30°C with shaking at 110 rpm. The amounts of RD...

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Neisseria meningitidis is an important human pathogen that is capable of killing within hours of infection. Its normal habitat is the nasopharynx of adult humans. Here we identify a genomic island (the prp gene cluster) in N. meningitidis that enables this species to utilize propionic acid as a supplementary carbon source during growth, particularly under nutrient poor growth conditions. The prp gene cluster encodes enzymes for a methylcitrate cycle. Novel aspects of the methylcitrate cycle in N. meningitidis include a propionate kinase which was purified and characterized, and a putative propionate transporter. This genomic island is absent from the close relative of N. meningitidis, the commensal Neisseria lactamica, which chiefly colonizes infants not adults. We reason that the possession of the prp genes provides a metabolic advantage to N. meningitidis in the adult oral cavity, which is rich in propionic acid-generating bacteria. Data from classical microbiological and sequence-based microbiome studies provide several lines of supporting evidence that N. meningitidis colonization is correlated with propionic acid generating bacteria, with a strong correlation between prp-containing Neisseria and propionic acid generating bacteria from the genus Porphyromonas, and that this may explain adolescent/adult colonization by N. meningitidis.

Here, we report the overexpression, purification, and characterization of the transcriptional activator fumarate and nitrate reductase regulator from the pathogenic bacterium Neisseria meningitidis (NmFNR). Like its homologue from Escherichia coli (EcFNR), NmFNR binds a 4Fe-4S cluster, which breaks down in the presence of oxygen to a 2Fe-2S cluster and subsequently to apo-FNR. The kinetics of NmFNR cluster disassembly in the presence of oxygen are 2-3؋ slower than those previously reported for wild-type EcFNR, but similar to constitutively active EcFNR* mutants, consistent with earlier work in which we reported that the activity of FNR-dependent promoters in N. meningitidis