Jong Moon Yoon

Biodegradation of Nitro-Substituted Explosives 2,4,6-Trinitrotoluene, Hexahydro-1,3,5-Trinitro-1,3,5-Triazine, and Octahydro-1,3,5,7-Tetranitro-1,3,5-Tetrazocine by a Phytosymbiotic Methylobacterium sp. Associated with Poplar Tissues (Populus deltoides × nigra DN34)

ABSTRACT A pink-pigmented symbiotic bacterium was isolated from hybrid poplar tissues (Populus deltoides × nigra DN34). The bacterium was identified by 16S and 16S-23S intergenic spacer ribosomal DNA analysis as a Methylobacterium sp. (strain BJ001). The isolated bacterium was able to use methanol as the sole source of carbon and energy, which is a specific attribute of the genus Methylobacterium. The bacterium in pure culture was shown to degrade the toxic explosives 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazene (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5-tetrazocine (HMX). [U-ring-14C]TNT (25 mg liter−1) was fully transformed in less than 10 days. Metabolites included the reduction derivatives amino-dinitrotoluenes and diamino-nitrotoluenes. No significant release of 14CO2 was recorded from [14C]TNT. In addition, the isolated methylotroph was shown to transform [U-14C]RDX (20 mg liter−1) and [U-14C]HMX (2.5 mg liter−1) in less than 40 days. After 55 days of incubation, 58.0% of initial [14C]RDX and 61.4% of initial [14C]HMX were mineralized into 14CO2. The radioactivity remaining in solution accounted for 12.8 and 12.7% of initial [14C]RDX and [14C]HMX, respectively. Metabolites detected from RDX transformation included a mononitroso RDX derivative and a polar compound tentatively identified as methylenedinitramine. Since members of the genus Methylobacterium are distributed in a wide diversity of natural environments and are very often associated with plants, Methylobacterium sp. strain BJ001 may be involved in natural attenuation or in situ biodegradation (including phytoremediation) of explosive-contaminated sites.

Expression of glutathione S-transferases in poplar trees (Populus trichocarpa) exposed to 2,4,6-trinitrotoluene (TNT)

Twelve Populus genes were identified from Arabidopsis thaliana sequences previously shown to be induced by exposure to 2,4,6-trinitrotoluene (TNT). Using the resources of the Poplar Genome Project and National Center for Biotechnology Information databases, Populus conserved domains were identified and used to design gene specific primers. RNA extracted from root tissues of TNT-exposed hydroponic poplar plants was used to quantify the expression of genes by reverse-transcriptase real-time polymerase chain reaction. Cyclophilin and 18S ribosomal DNA genes were used as internal standards. Exposure to TNT resulted in a significant increase of gene expression of two glutathione S-transferases (GST), peaking at levels of 25.0 +/- 13.1 and 10 +/- 0.7 fold the expression level of non-exposed plants after 24 h for each of the GST genes, respectively. This paper demonstrates the use of functional genomics information from the model plant species, Arabidopsis, to identify genes which may be important in detoxification of TNT in the model phytoremediation species, Populus trichocarpa.

Leaching of Contaminated Leaves Following Uptake and Phytoremediation of RDX, HMX, and TNT by Poplar

The uptake and fate of 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) by hybrid poplars in hydroponic systems were compared and exposed leaves were leached with water to simulate potential exposure pathways from groundwater in the field. TNT was removed from solution more quickly than nitramine explosives. Most of radioactivity remained in root tissues for 14C-TNT, but in leaves for 14C-RDX and 14C-HMX. Radiolabel recovery for TNT and HMX was over 94%, but that of RDX decreased over time, suggesting a loss of volatile products. A considerable fraction (45.5%) of radioactivity taken up by whole plants exposed to 14C-HMX was released into deionized water, mostly as parent compound after 5 d of leaching. About a quarter (24.0%) and 1.2% were leached for RDX and TNT, respectively, mostly as transformed products. Leached radioactivity from roots was insignificant in all cases (< 2%). This is the first report in which small amounts of transformation products of RDX leach from dried leaves following uptake by poplars. Such behavior for HMX was reported earlier and is reconfirmed here. All three compounds differ substantially in their fate and transport during the leaching process.

Analysis of gene expression in poplar trees (Populus deltoides x nigra DN34) exposed to the toxic explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)

Poplar plants (Populus deltoides × nigra, DN34) growing under hydroponic conditions were exposed to 50 mg L−1 of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) for 24 h. The expression of genes potentially involved in the metabolism of toxic explosives was analyzed by reverse-transcriptase (RT) real-time PCR. Genes under study were selected by reference to corresponding genes that were previously shown to be upregulated in the model plant Arabidopsis thaliana by exposure to 2,4,6-trinitrotoluene (TNT) (Ekman et al., 2003. Plant Physiol., 133, 1397–1406). The target genes investigated include several genes encoding for enzymes known to be involved in the detoxification of xenobiotic pollutants, such as glutathione S-transferases (GSTs), cytochrome P-450s (CYPs), NADPH-dependent reductases, and peroxidases. Starting from A. thaliana TNT-inducible genes, corresponding Populus sequences were retrieved from the JGI Poplar Genome Project database and were used to design gene-specific primers. 18S ribosomal DNA (rDNA) was used as an internal standard and recorded gene expression levels were normalized by reference to nonexposed plants. In three separate experiments, five genes were found to be significantly amplified in leaf tissues by exposure to RDX, including GST (9.7 fold), CYP (1.6 fold), reductases (1.6–1.7 fold), and peroxidase (1.7 fold). In root tissues, only a single GST gene was found to be significantly amplified by exposure to RDX (2.0 fold). These results show, for the first time, that the exposure of poplar plants to RDX results in the induction of several genes that are potentially involved in explosive detoxification.

Influence of carbon to nitrogen ratios on soybean somatic embryo (cv. Jack) growth and composition

Soybean [Glycine max (L.) Merr.] seed are valued for their protein and oil content. Soybean somatic embryos cultured in Soybean Histodifferentiation and Maturation (SHaM) medium were examined for their suitability as a model system for developing an understanding of assimilate partitioning and metabolic control points for protein and oil biosynthesis in soybean seed. This report describes the growth dynamics and compositional changes of SHaM embryos in response to change in the carbon to nitrogen ratio of the medium. It was postulated that at media compositions that were sufficient to support maximal growth rates, changes in the C:N ratio are likely to influence the partitioning of resources between the various storage products, especially protein and oil. As postulated, at steady-state growth rates, embryo protein content was strongly correlated with decreasing C:N ratios and increasing glutamine consumption rates. However, oil content remained relatively unchanged across the C:N ratio range tested, and resources were instead directed towards the starch and residual biomass (estimated by mass balance) pools in response to increasing C:N ratios. Protein and oil were inversely related only at concentrations of sucrose in the medium <88mM, where carbon limited growth and no starch was found to accumulate in the tissues. These observations and the high reproducibility in the data indicate that SHaM embryos are an ideal model system for the application of metabolic flux analysis studies designed to test hypotheses regarding assimilate partitioning in developing soybean seeds.

Metabolic engineering with plants for a sustainable biobased economy.

Plants are bona fide sustainable organisms because they accumulate carbon and synthesize beneficial metabolites from photosynthesis. To meet the challenges to food security and health threatened by increasing population growth and depletion of nonrenewable natural resources, recent metabolic engineering efforts have shifted from single pathways to holistic approaches with multiple genes owing to integration of omics technologies. Successful engineering of plants results in the high yield of biomass components for primary food sources and biofuel feedstocks, pharmaceuticals, and platform chemicals through synthetic biology and systems biology strategies. Further discovery of undefined biosynthesis pathways in plants, integrative analysis of discrete omics data, and diversified process developments for production of platform chemicals are essential to overcome the hurdles for sustainable production of value-added biomolecules from plants.

Engineering Escherichia coli membrane phospholipid head distribution improves tolerance and production of biorenewables

Economically competitive microbial production of biorenewable fuels and chemicals is often impeded by toxicity of the product to the microbe. Membrane damage is often identified as a major mechanism of this toxicity. Prior efforts to strengthen the microbial membrane by changing the phospholipid distribution have largely focused on the fatty acid tails. Herein, a novel strategy of phospholipid head engineering is demonstrated in Escherichia coli. Specifically, increasing the expression of phosphatidylserine synthase (+pssA) was found to significantly increase both the tolerance and production of octanoic acid, a representative membrane-damaging solvent. Tolerance of other industrially-relevant inhibitors, such as furfural, acetate, toluene, ethanol and low pH was also increased. In addition to the increase in the relative abundance of the phosphoethanolamine (PE) head group in the +pssA strain, there were also changes in the fatty acid tail composition, resulting in an increase in average length, percent unsaturation and decreased abundance of cyclic rings. This +pssA strain had significant changes in: membrane integrity, surface potential, electrochemical potential and hydrophobicity; sensitivity to intracellular acidification; and distribution of the phospholipid tails, including an increase in average length and percent unsaturation and decreased abundance of cyclic rings. Molecular dynamics simulations demonstrated that the +PE membrane had increased resistance to penetration of ethanol into the hydrophobic core and also the membrane thickness. Further hybrid models in which only the head group distribution or fatty acid tail distribution was altered showed that the increase in PE content is responsible for the increase in bilayer thickness, but the increased hydrophobic core thickness is due to altered distribution of both the head groups and fatty acid tails. This work demonstrates the importance of consideration of the membrane head groups, as well as a modeling approach, in membrane engineering efforts.