Metabolism of the aliphatic nitramine 4-nitro-2,4-diazabutanal by Methylobacterium sp. strain JS178

Abstract

The aliphatic nitramine 4-nitro-2,4-diazabutanal (NDAB; C2H5N3O3) is a ring cleavage metabolite that accumulates during the aerobic degradation of the energetic compound hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by various Rhodococcus spp. NDAB is also produced during the alkaline hydrolysis of either RDX or octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) and during the photolysis of RDX. Traces of NDAB were observed in a soil sampled from an ammunition-manufacturing facility contaminated with both HMX and RDX, suggesting natural attenuation. In this study, we report the isolation of a soil bacterium that is able to degrade NDAB under aerobic conditions. The isolate is a pink-pigmented facultative methylotroph affiliated with the genus Methylobacterium. The strain, named Methylobacterium sp. strain JS178, degrades NDAB as a sole nitrogen source, with concomitant growth and formation of 1 molar equivalent of nitrous oxide (N2O). Comparison of the growth yield of strain JS178 grown on NDAB, nitrite (NO2-), or ammonium (NH4+) as a nitrogen source revealed that 1 N equivalent is assimilated from each mole of NDAB, which completes the nitrogen mass balance. In radiotracer experiments, strain JS178 mineralized 1 C of the [14C]NDAB produced in situ from [14C]RDX by Rhodococcus sp. strain DN22. Studies on the regulation of NDAB degradation indicated that allantoin, an intermediate in the purine catabolic pathway and a central molecule in the storage and transport of nitrogen in plants, up-regulated the enzyme(s) involved in the degradation of the nitramine. The results reveal the potential for the sequential participation of rhodococci and methylobacteria to effect the complete degradation of RDX.

FIG. 1.

Structures of RDX, HMX, NDAB, MEDINA, and allantoin.

FIG. 2.

Growth of Methylobacterium sp. strain JS178 in M-succinate and NDAB (1,300 nmol) as a sole N source. Shown are amounts of NDAB (▵), N₂O (□), and NO₂⁻ (×). Growth is expressed as A₆₀₀ (○). Error bars, standard deviations from duplicate experiments.

FIG. 3.

Growth yield of Methylobacterium sp. strain JS178 in M-succinate, with nitrite (▵), ammonium (□), and NDAB (○) as N sources. The linear regressions have the following gradients: 0.00165 (R = 0.995) for nitrite, 0.00158 (R = 0.999) for ammonium, and 0.00173 (R = 0.999) for NDAB. Error bars, standard deviations from triplicate experiments.

FIG. 4.

Incubation of [¹⁴C]RDX with Rhodococcus sp. strain DN22 (medium was supplemented with 10 mM succinate at day 25) (○), with Methylobacterium sp. strain JS178 (×), with both strains DN22 and JS178 (JS178 was reinoculated to the microcosms at day 42) (▵), and with the sequential addition of strain DN22 (used to transform [¹⁴C]RDX to [¹⁴C]NDAB) and NDAB-grown JS178 (added when 30% of [¹⁴C]CO₂ was liberated from [¹⁴C]RDX, after the removal of DN22 cells by filtration) (□). Values are averages and standard deviations of duplicate experiments.