Fiona H. Crocker

Mineralization of the Cyclic Nitramine Explosive Hexahydro-1,3,5-Trinitro-1,3,5-Triazine by Gordonia and Williamsia spp.

ABSTRACT Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a cyclic nitroamine explosive that is a major component in many military high-explosive formulations. In this study, two aerobic bacteria that are capable of using RDX as the sole source of carbon and nitrogen to support their growth were isolated from surface soil. These bacterial strains were identified by their fatty acid profiles and 16S ribosomal gene sequences as Williamsia sp. KTR4 and Gordonia sp. KTR9. The physiology of each strain was characterized with respect to the rates of RDX degradation and [U-14C]RDX mineralization when RDX was supplied as a sole carbon and nitrogen source in the presence and absence of competing carbon and nitrogen sources. Strains KTR4 and KTR9 degraded 180 μM RDX within 72 h when RDX served as the only added carbon and nitrogen source while growing to total protein concentrations of 18.6 and 16.5 μg/ml, respectively. Mineralization of [U-14C]RDX to 14CO2 was 30% by strain KTR4 and 27% by KTR9 when RDX was the only added source of carbon and nitrogen. The addition of (NH4)2SO4 greatly inhibited KTR9s degradation of RDX but had little effect on that of KTR4. These are the first two pure bacterial cultures isolated that are able to use RDX as a sole carbon and nitrogen source. These two genera possess different physiologies with respect to RDX mineralization, and each can serve as a useful microbiological model for the study of RDX biodegradation with regard to physiology, biochemistry, and genetics.

Biodegradation of the cyclic nitramine explosives RDX, HMX, and CL-20

Cyclic nitramine explosives are synthesized globally mainly as military munitions, and their use has resulted in environmental contamination. Several biodegradation pathways have been proposed, and these are based mainly on end-product characterization because many of the metabolic intermediates are hypothetical and unstable in water. Biodegradation mechanisms for cyclic nitramines include (a) formation of a nitramine free radical and loss of nitro functional groups, (b) reduction of nitro functional groups, (c) direct enzymatic cleavage, (d) α-hydroxylation, or (e) hydride ion transfer. Pathway intermediates spontaneously decompose in water producing nitrite, nitrous oxide, formaldehyde, or formic acid as common end-products. In vitro enzyme and functional gene expression studies have implicated a limited number of enzymes/genes involved in cyclic nitramine catabolism. Advances in molecular biology methods such as high-throughput DNA sequencing, microarray analysis, and nucleic acid sample preparation are providing access to biochemical and genetic information on cultivable and uncultivable microorganisms. This information can provide the knowledge base for rational engineering of bioremediation strategies, biosensor development, environmental monitoring, and green biosynthesis of explosives. This paper reviews recent developments on the biodegradation of cyclic nitramines and the potential of genomics to identify novel functional genes of explosive metabolism.

Functional Characterization of pGKT2, a 182-Kilobase Plasmid Containing the xplAB Genes, Which Are Involved in the Degradation of Hexahydro-1,3,5-Trinitro-1,3,5-Triazine by Gordonia sp. Strain KTR9

ABSTRACT Several microorganisms have been isolated that can transform hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), a cyclic nitramine explosive. To better characterize the microbial genes that facilitate this transformation, we sequenced and annotated a 182-kb plasmid, pGKT2, from the RDX-degrading strain Gordonia sp. KTR9. This plasmid carries xplA, encoding a protein sharing up to 99% amino acid sequence identity with characterized RDX-degrading cytochromes P450. Other genes that cluster with xplA are predicted to encode a glutamine synthase-XplB fusion protein, a second cytochrome P450, Cyp151C, and XplR, a GntR-type regulator. Rhodococcus jostii RHA1 expressing xplA from KTR9 degraded RDX but did not utilize RDX as a nitrogen source. Moreover, an Escherichia coli strain producing XplA degraded RDX but a strain producing Cyp151C did not. KTR9 strains cured of pGKT2 did not transform RDX. Physiological studies examining the effects of exogenous nitrogen sources on RDX degradation in strain KTR9 revealed that ammonium, nitrite, and nitrate each inhibited RDX degradation by up to 79%. Quantitative real-time PCR analysis of glnA-xplB, xplA, and xplR showed that transcript levels were 3.7-fold higher during growth on RDX than during growth on ammonium and that this upregulation was repressed in the presence of various inorganic nitrogen sources. Overall, the results indicate that RDX degradation by KTR9 is integrated with central nitrogen metabolism and that the uptake of RDX by bacterial cells does not require a dedicated transporter.

Microscale patterned surfaces reduce bacterial fouling-microscopic and theoretical analysis

Microscale patterned surfaces have been shown to control the arrangement of bacteria attached to surfaces. This study was conducted to examine the effect of patterned topographies on bacterial fouling using Enterobacter cloacae as the test model. E. cloacae is an opportunistic pathogen involved frequently in nosocomial infections. It is an important model organism to be studied in the context of healthcare associated infections (HAI) and polydimethylsiloxane (PDMS) based urinary catheter fouling. Patterned surfaces, such as Sharklet™, have shown the promise of being a benign surface treatment for prevention of catheter associated urinary tract infections (CAUTI). To the best of our knowledge, inhibition of fouling by E. cloacae has not been demonstrated on microscale patterned PDMS surfaces. In this study, the Sharklet™ and smooth PDMS surfaces were used as controls. All pattern surfaces had statistically significantly lower percentage area coverage compared to the smooth PDMS control. A cross type feature (C-1-PDMS), demonstrated the most significant reduction in percent area coverage, 89% (p<0.01, α=0.05), compared to the smooth PDMS control and all other patterned test surfaces. Additionally, theoretical calculations show that C-1-PDMS is the only surface predicted to hold the thermodynamically stable Cassie state, which occurs due to trapping air pockets at the liquid-solid interface. Combined the results provide new insights for designing environmentally benign, novel, microscale patterned surfaces for restricting bacterial fouling.

Horizontal gene transfer (HGT) as a mechanism of disseminating RDX-degrading activity among Actinomycete bacteria

Aims:  Hexahydro‐1,3,5‐trinitro‐1,3,5,‐triazine (RDX) is a cyclic nitramine explosive that is a major component in many high‐explosive formulations and has been found as a contaminant of soil and groundwater. The RDX‐degrading gene locus xplAB, located on pGKT2 in Gordonia sp. KTR9, is highly conserved among isolates from disparate geographical locations suggesting a horizontal gene transfer (HGT) event. It was our goal to determine whether Gordonia sp. KTR9 is capable of transferring pGKT2 and the associated RDX degradation ability to other bacteria.

Genomic and Transcriptomic Studies of an RDX (Hexahydro-1,3,5-Trinitro-1,3,5-Triazine)-Degrading Actinobacterium

ABSTRACT Whole-genome sequencing, transcriptomic analyses, and metabolic reconstruction were used to investigate Gordonia sp. strain KTR9s ability to catabolize a range of compounds, including explosives and steroids. Aspects of this mycolic acid-containing actinobacteriums catabolic potential were experimentally verified and compared with those of rhodococci and mycobacteria.

Role of Nitrogen Limitation in Transformation of RDX (Hexahydro- 1,3,5-Trinitro-1,3,5-Triazine) by Gordonia sp. Strain KTR9

ABSTRACT The transcriptome of RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine)-degrading strain Gordonia sp. strain KTR9 and its glnR mutant were studied as a function of nitrogen availability to further investigate the observed ammonium-mediated inhibition of RDX degradation. The results indicate that nitrogen availability is a major determinant of RDX degradation and xplA gene expression in KTR9.

The essential role of nitrogen limitation in expression of xplA and degradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in Gordonia sp. strain KTR9

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a widely used explosive and a major soil and groundwater contaminant. Organisms such as Gordonia sp. KTR9, capable of degrading RDX and using it as an N source, may prove useful for bioremediation of contaminated sites. XplA is a cytochrome P450 monooxygenase responsible for RDX degradation. Expression of xplA in KTR9 was not induced by RDX but was strongly induced (50-fold) during N-limited growth. When glnR, encoding a regulatory protein affecting N assimilation in diverse Actinobacteria, was deleted from KTR9, the bacterium lost the ability to use nitrate, nitrite, and RDX as N sources. Deletion of glnR also abolished the inhibition of xplA expression by nitrite. Our results confirm the essential role of GlnR in regulating assimilation of nitrite, but there was no evidence for a direct role of GlnR in regulating XplA expression. Rather, the general availability of nitrogen repressed XplA expression. We conclude that the inability of the glnR mutant to use RDX as an N source was due to its inability to assimilate nitrite, an intermediate in the assimilation of nitrogen from RDX. Regulation of XplA does not seem adaptive for KTR9, but it is important for RDX bioremediation with KTR9 or similar bacteria.

In Situ Degradation and Remediation of Energetics TNT, RDX, HMX, and CL-20 and a Byproduct NDMA in the Sub-Surface Environment

Energetics such as RDX, HMX, and CL-20 exhibit low sorption and natural degradation, resulting in widespread groundwater contamination. Alternatively, TNT exhibits strong sorption and degrades to toxic recalcitrant intermediates. Field scale abiotic, biotic, and coupled abiotic/bioremediation can be more cost effective than pump and treat or sediment removal, but rates of processes in relevant insitu conditions need to be understood.

Biodegradation of insensitive munition formulations IMX101 and IMX104 in surface soils

The biodegradation potential of insensitive munition melt cast formulations IMX101 and IMX104 was investigated in two unamended training range soils under aerobic and anaerobic growth conditions. Changes in community profiles in soil microcosms were monitored via high-throughput 16S rRNA sequencing over the course of the experiments to infer key microbial phylotypes that may be linked to IMX degradation. Complete anaerobic biotransformation occurred for IMX101 and IMX104 constituents 2,4-dinitroanisole (DNAN) and 3-nitro-1,2,4-triazol-5-one during the 30-day incubation period with Camp Shelby (CS) soil. By comparison, soil from Umatilla chemical depot demonstrated incomplete DNAN degradation with reduced transformation rates for both IMX101 and IMX104. Aerobic soil microcosms for both soils demonstrated reduced transformation rates compared to anaerobic degradation for all IMX constituents with DNAN the most susceptible to biotransformation by CS soil. Overall, IMX constituents hexahydro-1,3,5-trinitro-1,3,5-triazine and 1-nitroguanidine did not undergo significant transformation. In CS soil, organisms that have been associated with explosives degradation, namely members of the Burkholderiaceae, Bacillaceae, and Paenibacillaceae phylotypes increased significantly in anaerobic treatments whereas Sphingomonadaceae increased significantly in aerobic treatments. Collectively, these data may be used to populate fate and transport models to provide more accurate estimates for assessing environmental costs associated with release of IMX101 and IMX104.