Draft Genome Sequences of Bacteria with Cyanocidal Potential Isolated from Eutrophic Waters Associated with Cyanobacterial Harmful Algal Blooms.

Here, we report the draft genome sequences of nine bacterial species isolated from eutrophic waters associated with cyanobacterial harmful algal blooms with cyanocidal potential.

Genomic investigations of acute munitions exposures on the health and skin microbiome composition of leopard frog (Rana pipiens) tadpoles.

Natural communities of microbes inhabiting amphibian skin, the skin microbiome, are critical to supporting amphibian health and disease resistance. To enable the pro-active health assessment and management of amphibians on Army installations and beyond, we investigated the effects of acute (96h) munitions exposures to Rana pipiens (leopard frog) tadpoles and the associated skin microbiome, integrated with RNAseq-based transcriptomic responses in the tadpole host. Tadpoles were exposed to the legacy munition 2,4,6-trinitrotoluene (TNT), the new insensitive munition (IM) formulation, IMX-101, and the IM constituents nitroguinidine (NQ) and 1-methyl-3-nitroguanidine (MeNQ). The 96h LC50 values and 95% confidence intervals were 2.6 (2.4, 2.8) for ΣTNT and 68.2 (62.9, 73.9) for IMX-101, respectively. The NQ and MeNQ exposures caused no significant impacts on survival in 96h exposures even at maximum exposure levels of 3560 and 5285 mg/L, respectively. However, NQ and MeNQ, as well as TNT and IMX-101 exposures, all elicited changes in the tadpole skin microbiome profile, as evidenced by significantly increased relative proportions of the Proteobacteria with increasing exposure concentrations, and significantly decreased alpha-diversity in the NQ exposure. The potential for direct effects of munitions exposure on the skin microbiome were observed including increased abundance of munitions-tolerant phylogenetic groups, in addition to possible indirect effects on microbial flora where transcriptional responses suggestive of changes in skin mucus-layer properties, antimicrobial peptide production, and innate immune factors were observed in the tadpole host. Additional insights into the tadpole host's transcriptional response to munitions exposures indicated that TNT and IMX-101 exposures significantly enriched transcriptional expression within type-I and type-II xenobiotic metabolism pathways, where dose-responsive increases in expression were observed. Significant enrichment and increased transcriptional expression of heme and iron binding functions in the TNT exposures served as likely indicators of known mechanisms of TNT toxicity including hemolytic anemia and methemoglobinemia. The significant enrichment and dose-responsive decrease in transcriptional expression of cell cycle pathways in the IMX-101 exposures was consistent with previous observations in fish, while significant enrichment of immune-related function in response to NQ exposure were consistent with potential immune suppression at the highest NQ exposure concentration. Finally, the MeNQ exposures elicited significantly decreased transcriptional expression of keratin 16, type I, a gene likely involved in keratinization processes in amphibian skin. Overall, munitions showed the potential to alter tadpole skin microbiome composition and affect transcriptional profiles in the amphibian host, some suggestive of potential impacts on host health and immune status relevant to disease susceptibility.

Modeling a synthetic aptamer-based riboswitch biosensor sensitive to low hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) concentrations.

RNA aptamers are relatively short nucleic acid sequences that bind targets with high affinity, and when combined with a riboswitch that initiates translation of a fluorescent reporter protein, can be used as a biosensor for chemical detection in various types of media. These processes span target binding at the molecular scale to fluorescence detection at the macroscale, which involves a number of intermediate rate-limiting physical (e.g., molecular conformation change) and biochemical changes (e.g., reaction velocity), which together complicate assay design. Here we describe a mathematical model developed to aid environmental detection of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) using the DsRed fluorescent reporter protein, but is general enough to potentially predict fluorescence from a broad range of water-soluble chemicals given the values of just a few kinetic rate constants as input. If we expose a riboswitch test population of Escherichia coli bacteria to a chemical dissolved in media, then the model predicts an empirically distinct, power-law relationship between the exposure concentration and the elapsed time of exposure. This relationship can be used to deduce an exposure time that meets or exceeds the optical threshold of a fluorescence detection device and inform new biosensor designs.

Metal accumulation capacity in indigenous Alaska vegetation growing on military training lands.

Permafrost thawing could increase soil contaminant mobilization in the environment. Our objective was to quantify metal accumulation capacities for plant species and functional groups common to Alaskan military training ranges where elevated soil metal concentrations were likely to occur. Plant species across multiple military training range sites were collected. Metal content in shoots and roots was compared to soil metal concentrations to calculate bioconcentration and translocation factors. On average, grasses accumulated greater concentrations of Cr, Cu, Ni, Pb, Sb, and Zn relative to forbs or shrubs, and bioconcentrated greater concentrations of Ni and Pb. Shrubs bioconcentrated greater concentrations of Sb. Translocation to shoots was greatest among the forbs. Three native plants were identified as candidate species for use in metal phytostabilization applications. Elymus macrourus, a grass, bioconcentrated substantial concentrations of Cu, Pb, and Zn in roots with low translocation to shoots. Elaeagnus commutata, a shrub, bioconcentrated the greatest amounts of Sb, Ni, and Cr, with a low translocation factor. Solidago decumbens bioconcentrated the greatest amount of Sb among the forbs and translocated the least amount of metals. A combination of forb, shrub, and grass will likely enhance phytostabilization of heavy metals in interior Alaska soils through increased functional group diversity.

Effects of chitin and temperature on sub-Arctic soil microbial and fungal communities and biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and 2,4-dinitrotoluene (DNT).

Climate warming in the Arctic and the thawing of frozen carbon stocks are leading to uncertainty as to how bacterial communities will respond, including pollutant degrading bacteria. This study investigated the effects of carbon stimulation and temperature on soil microbial community diversity and explosive biodegradation in two sub-Arctic soils. Chitin as a labile carbon source stimulated overall microbial activities as reflected by increases in basal respiration (three to tenfold) and potential nitrification activity (two to fourfold) compared to unamended soil. This stimulation extended to 2,4-dinitroluene- (DNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)-degrading microorganisms either directly or via co-metabolic reaction mechanisms. A stimulatory effect of the incubation temperature (2, 12, or 22 °C) on these microbial activities was also observed, but the chitin stimulation caused greater shifts in the structure of the bacterial and fungal communities. The first reported occurrence of an associated role of chitinolytic bacteria belonging to Cellulomonadaceae and chitinolytic fungi belonging to Mortierellaceae in explosive biodegradation is described. This study found that sub-Arctic soil microbial communities were adapted to respond quickly to an increase in labile carbon sources over the range of temperatures used in this study. The warming climate in the Arctic could benefit explosive contaminated soil clean-up by providing non-recalcitrant carbon sources that stimulate overall microbial activity and correspondingly explosive biodegradation.

Enhanced plasmid-mediated bioaugmentation of RDX-contaminated matrices in column studies using donor strain Gordonia sp. KTR9.

Horizontal gene transfer (HGT) is the lateral movement of genetic material between organisms. The RDX explosive-degrading bacterium Gordonia sp. KTR9 has been shown previously to transfer the pGKT2 plasmid containing the RDX degradative genes (xplAB) by HGT. Overall, fitness costs to the transconjugants to maintain pGKT2 was determined through growth and survivability assessments. Rhodococcus jostii RHA1 transconjugants demonstrated a fitness cost while other strains showed minimal cost. Biogeochemical parameters that stimulate HGT of pGKT2 were evaluated in soil slurry mating experiments and the absence of nitrogen was found to increase HGT events three orders of magnitude. Experiments evaluating RDX degradation in flow-through soil columns containing mating pairs showed 20% greater degradation than columns with only the donor KTR9 strain. Understanding the factors governing HGT will benefit bioaugmentation efforts where beneficial bacteria with transferrable traits could be used to more efficiently degrade contaminants through gene transfer to native populations.

Detection of hexahydro-1,3-5-trinitro-1,3,5-triazine (RDX) with a microbial sensor.

Explosives such as hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) are common contaminants found in soil and groundwater at military facilities worldwide, but large-scale monitoring of these contaminants at low concentrations is difficult. Biosensors that incorporate aptamers with high affinity and specificity for a target are a novel way of detecting these compounds. This work describes novel riboswitch-based biosensors for detecting RDX. The performance of the RDX riboswitch was characterized in Escherichia coli using a range of RDX concentrations from 0-44 µmol l-1. Fluorescence was induced at RDX concentrations as low as 0.44 µmol l-1. The presence of 4.4 µmol l-1 RDX induced an 8-fold increase in fluorescence and higher concentrations did not induce a statistically significant increase in response.

Effects of acute exposures of 2,4,6-trinitrotoluene and inorganic lead on the fecal microbiome of the green anole (Anolis carolinensis).

Microbiome studies focused on ecologically relevant vertebrate models like reptiles have been limited. Because of their relatively small home range, fast maturation, and high fecundity, lizards are an excellent reptilian terrestrial indicator species. For this study we used the green anole, Anolis carolinensis, to assess the impact of military relevant contaminants on fecal microbiome composition. Fourteen day sub-acute exposures were conducted via oral gavage with 2,4,6-Trinitrotoluene (TNT) and inorganic lead at doses of 60 mg/kg and 20 mg/kg of body weight, respectively. Body weights and food consumption were monitored and fecal samples were collected for high-throughput 16S rRNA gene amplicon sequencing and analytical chemistry at days 0 and 15. At the end of the study, liver and gut were harvested for body burden data. Chemical analysis confirmed accumulation of TNT, TNT transformation products, and lead in liver tissue and fecal samples. Bacterial community analysis of fecal material revealed significant differences between day 0 and day 15 of TNT exposed anoles with an operational taxonomic unit (OTU) within the genus Erwinia representing 32% of the microbial community in TNT exposed anoles. Predictable changes in gut microbiome composition could offer an easily assayed, noninvasive biomarker for specific chemical exposure providing enhanced scientific support to risk assessments on military installations.

Rhizosphere Microbial Communities of Spartina alterniflora and Juncus roemerianus From Restored and Natural Tidal Marshes on Deer Island, Mississippi.

The U. S. Gulf of Mexico is experiencing a dramatic increase in tidal marsh restoration actions, which involves planting coastal areas with smooth cordgrass (Spartina alterniflora) and black needlerush (Juncus roemerianus) for erosion control and to provide habitat for fish and wildlife. It can take decades for sedimentary cycles in restored marshes to approach reference conditions, and the contribution of the sediment microbial communities to these processes is poorly elucidated. In this study, we addressed this gap by comparing rhizosphere microbiomes of S. alterniflora and J. roemerianus from two restored marshes and a natural reference marsh located at Deer Island, MS. Our results revealed that plants from the restored and reference areas supported similar microbial diversity indicating the rapid colonization of planted grasses with indigenous soil microbiota. Although close in composition, the microbial communities from the three studied sites differed significantly in the relative abundance of specific taxa. The observed differences are likely driven by the host plant identity and properties of sediment material used for the creation of restored marshes. Some of the differentially distributed groups of bacteria include taxa involved in the cycling of carbon, nitrogen, and sulfur, and may influence the succession of vegetation at the restored sites to climax condition. We also demonstrated that plants from the restored and reference sites vary in the frequency of culturable rhizobacteria that exhibit traits commonly associated with the promotion of plant growth and suppression of phytopathogenic fungi. Our findings will contribute to the establishment of benchmarks for the assessment of the outcome of coastal restoration projects in the Gulf of Mexico and better define factors that affect the long-term resiliency of tidal marshes and their vulnerability to climate change.

RDX degradation in bioaugmented model aquifer columns under aerobic and low oxygen conditions.

Degradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in laboratory columns following biostimulation and bioaugmentation was investigated using sediment and groundwater from a contaminated aquifer at a US Navy facility. No RDX degradation was observed following aerobic biostimulation with either fructose or lactate (both 0.1 mM) prior to bioaugmentation. Replicate columns were then bioaugmented with either Gordonia sp. KTR9, Pseudomonas fluorescens I-C (Ps I-C), or both strains. Under aerobic conditions (influent dissolved oxygen (DO) >6 mg/L), RDX was degraded following the addition of fructose, and to a lesser extent with lactate, in columns bioaugmented with KTR9. No degradation was observed in columns bioaugmented with only Ps I-C under aerobic conditions, consistent with the known anaerobic RDX degradation pathway for this strain. When influent DO was reduced to <2 mg/L, good RDX degradation was observed in the KTR9-bioaugmented column, and some degradation was also observed in the Ps I-C-bioaugmented column. After DO levels were kept below 1 mg/L for more than a month, columns bioaugmented with KTR9 became unresponsive to fructose addition, while RDX degradation was still observed in the Ps I-C-bioaugmented columns. These results indicate that bioaugmentation with the aerobic RDX degrader KTR9 could be effective at sites where site geology or geochemistry allow higher DO levels to be maintained. Further, inclusion of strains capable of anoxic RDX degradation such as Ps I-C may facilitate bimodal RDX removal when DO levels decrease.

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.

Comparative Genomics and Metabolic Analysis Reveals Peculiar Characteristics of Rhodococcus opacus Strain M213 Particularly for Naphthalene Degradation.

The genome of Rhodococcus opacus strain M213, isolated from a fuel-oil contaminated soil, was sequenced and annotated which revealed a genome size of 9,194,165 bp encoding 8680 putative genes and a G+C content of 66.72%. Among the protein coding genes, 71.77% were annotated as clusters of orthologous groups of proteins (COGs); 55% of the COGs were present as paralog clusters. Pulsed field gel electrophoresis (PFGE) analysis of M213 revealed the presence of three different sized replicons- a circular chromosome and two megaplasmids (pNUO1 and pNUO2) estimated to be of 750Kb 350Kb in size, respectively. Conversely, using an alternative approach of optical mapping, the plasmid replicons appeared as a circular ~1.2 Mb megaplasmid and a linear, ~0.7 Mb megaplasmid. Genome-wide comparative analysis of M213 with a cohort of sequenced Rhodococcus species revealed low syntenic affiliation with other R. opacus species including strains B4 and PD630. Conversely, a closer affiliation of M213, at the functional (COG) level, was observed with the catabolically versatile R. jostii strain RHA1 and other Rhodococcii such as R. wratislaviensis strain IFP 2016, R. imtechensis strain RKJ300, Rhodococcus sp. strain JVH1, and Rhodococcus sp. strain DK17, respectively. An in-depth, genome-wide comparison between these functional relatives revealed 971 unique genes in M213 representing 11% of its total genome; many associating with catabolic functions. Of major interest was the identification of as many as 154 genomic islands (GEIs), many with duplicated catabolic genes, in particular for PAHs; a trait that was confirmed by PCR-based identification of naphthalene dioxygenase (NDO) as a representative gene, across PFGE-resolved replicons of strain M213. Interestingly, several plasmid/GEI-encoded genes, that likely participate in degrading naphthalene (NAP) via a peculiar pathway, were also identified in strain M213 using a combination of bioinformatics, metabolic analysis and gene expression measurements of selected catabolic genes by RT-PCR. Taken together, this study provides a comprehensive understanding of the genome plasticity and ecological competitiveness of strain M213 likely facilitated by horizontal gene transfer (HGT), bacteriophage attacks and genomic reshuffling- aspects that continue to be understudied and thus poorly understood, in particular for the soil-borne Rhodococcii.

Evaluation of Biostimulation and Bioaugmentation To Stimulate Hexahydro-1,3,5-trinitro-1,3,5,-triazine Degradation in an Aerobic Groundwater Aquifer.

Hexahydro-1,3,5-trinitro-1,3,5,-triazine (RDX) is a toxic and mobile groundwater contaminant common to military sites. This study compared in situ RDX degradation rates following bioaugmentation with Gordonia sp. strain KTR9 (henceforth KTR9) to rates under biostimulation conditions in an RDX-contaminated aquifer in Umatilla, OR. Bioaugmentation was achieved by injecting site groundwater (6000 L) amended with KTR9 cells (10(8) cells mL(-1)) and low carbon substrate concentrations (<1 mM fructose) into site wells. Biostimulation (no added cells) was performed by injecting groundwater amended with low (<1 mM fructose) or high (>15 mM fructose) carbon substrate concentrations in an effort to stimulate aerobic or anaerobic microbial activity, respectively. Single-well push-pull tests were conducted to measure RDX degradation rates for each treatment. Average rate coefficients were 1.2 day(-1) for bioaugmentation and 0.7 day(-1) for high carbon biostimulation; rate coefficients for low carbon biostimulation were not significantly different from zero (p values ≥0.060). Our results suggest that bioaugmentation with KTR9 is a feasible strategy for in situ biodegradation of RDX and, at this site, is capable of achieving RDX concentration reductions comparable to those obtained by high carbon biostimulation while requiring ~97% less fructose. Bioaugmentation has potential to minimize substrate quantities and associated costs, as well as secondary groundwater quality impacts associated with anaerobic biostimulation processes (e.g., hydrogen sulfide, methane production) during full-scale RDX remediation.

Metagenomic analysis of denitrifying wastewater enrichment cultures able to transform the explosive, 3-nitro-1,2,4-triazol-5-one (NTO).

Removal of 3-nitro-1,2,4-triazol-5-one (NTO) was investigated in conjunction with heterotrophic and autotrophic denitrifying growth conditions by a microbial consortium from a wastewater treatment plant. Microcosms were supplemented with molasses, methanol, or thiosulfate. Cultures were passaged twice by transferring 10 % of the culture volume to fresh media on days 11 and 21. Rates of NTO removal were 18.71 ± 0.65, 9.04 ± 2.61, and 4.34 ± 2.72 mg/L/day while rates of nitrate removal were 20.08 ± 1.13, 21.58 ± 1.20, and 24.84 ± 1.26 mg/L/day, respectively, for molasses, methanol, or thiosulfate. Metagenomic analysis showed that Proteobacteria and Firmicutes were the major phyla in the microbial communities. In molasses supplemented cultures, the community profile at the family level changed over time with Pseudomonadaceae the most abundant (67.4 %) at day 11, Clostridiaceae (65.7 %) at day 21, and Sporolactobacillaceae (35.4 %) and Clostridiaceae (41.0 %) at day 29. Pseudomonadaceae was the dominant family in methanol and thiosulfate supplemented cultures from day 21 to 29 with 76.6 and 81.6 % relative abundance, respectively.

Evaluation of microbial transport during aerobic bioaugmentation of an RDX-contaminated aquifer.

In situ bioaugmentation with aerobic hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)-degrading bacteria is being considered for treatment of explosives-contaminated groundwater at Umatilla Chemical Depot, Oregon (UMCD). Two forced-gradient bacterial transport tests of site groundwater containing chloride or bromide tracer and either a mixed culture of Gordonia sp. KTR9 (xplA (+)Km(R)), Rhodococcus jostii RHA1 (pGKT2 transconjugant; xplA (+)Km(R)) and Pseudomonas fluorescens I-C (xenB (+)), or a single culture of Gordonia sp. KTR9 (xplA (+); i.e. wild-type) were conducted at UMCD. Groundwater monitoring evaluated cell viability and migration in the injection well and downgradient monitoring wells. Enhanced degradation of RDX was not evaluated in these demonstrations. Quantitative PCR analysis of xplA, the kanamycin resistance gene (aph), and xenB indicated that the mixed culture was transported at least 3 m within 2 h of injection. During a subsequent field injection of bioaugmented groundwater, strain KTR9 (wild-type) migrated up to 23-m downgradient of the injection well within 3 days. Thus, the three RDX-degrading strains were effectively introduced and transported within the UMCD aquifer. This demonstration represents an innovative application of bioaugmentation to potentially enhance RDX biodegradation in aerobic aquifers.

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.

Expression and characterization of an N-oxygenase from Rhodococcus jostii RHAI.

Nitro group-containing natural products are rare in nature. There are few examples of N-oxygenases, enzymes that incorporate atmospheric oxygen into primary and secondary amines, characterized in the literature. N-oxygenases have yet to be characterized from the Corynebacterineae, a metabolically diverse group of organisms that includes the genera Rhodococcus, Gordonia, and Mycobacterium. A preliminary in silico search for N-oxygenase AurF gene orthologs revealed multiple protein candidates present in the genome of the Actinomycete Rhodococcus jostii RHAI (RHAI_ro06104). Towards the goal of identifying novel biocatalysts with potential utility for the biosynthesis of nitroaromatics, AurF ortholog RHAI_ro6104 was cloned, expressed and purified in E. coli and amine and nitro containing phenol substrates tested for activity. RHAI-ro06104 showed the highest activity with 4-aminophenol, producing a Vmax of 18.76 µM s(-1) and a Km of 15.29 mM and demonstrated significant activities with 2-aminophenol and 2-amino-5-methylphenol, producing a Vmax of 12.86 and 12.72 µM s(-1) with a Km of 8.34 and 2.81 mM, respectively. These findings are consistent with a substrate range observed in other N-oxygenases, which seem to accommodate substrates that lack halogenated substitutions and side groups directly flanking the amine group. Attempts to identify modulators of RHAI-ro06104 gene activity demonstrated that aromatic amino acids inhibit expression by almost 50%.

Laboratory evaluation of bioaugmentation for aerobic treatment of RDX in groundwater.

The potential for bioaugmentation with aerobic explosive degrading bacteria to remediate hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) contaminated aquifers was demonstrated. Repacked aquifer sediment columns were used to examine the transport and RDX degradation capacity of the known RDX degrading bacterial strains Gordonia sp. KTR9 (modified with a kanamycin resistance gene) Pseudomonas fluorescens I-C, and a kanamycin resistant transconjugate Rhodococcus jostii RHA1 pGKT2:Km+. All three strains were transported through the columns and eluted ahead of the conservative bromide tracer, although the total breakthrough varied by strain. The introduced cells responded to biostimulation with fructose (18 mg L(-1), 0.1 mM) by degrading dissolved RDX (0.5 mg L(-1), 2.3 µM). The strains retained RDX-degrading activity for at least 6 months following periods of starvation when no fructose was supplied to the column. Post-experiment analysis of the soil indicated that the residual cells were distributed along the length of the column. When the strains were grown to densities relevant for field-scale application, the cells remained viable and able to degrade RDX for at least 3 months when stored at 4 °C. These results indicate that bioaugmentation may be a viable option for treating RDX in large dilute aerobic plumes.

The effects of putative lipase and wax ester synthase/acyl-CoA:diacylglycerol acyltransferase gene knockouts on triacylglycerol accumulation in Gordonia sp. KTR9.

Previously, we demonstrated triacylglycerol (TAG) accumulation and the in vivo ability to catalyze esters from exogenous short chain alcohol sources in Gordonia sp. strain KTR9. In this study, we investigated the effects that putative lipase (KTR9_0186) and wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT; KTR9_3844) gene knockouts had on TAG accumulation. Gene disruption of KTR9_0186 resulted in a twofold increase in TAG content in nitrogen starved cells. Lipase mutants subjected to carbon starvation, following nitrogen starvation, retained 75 % more TAGs and retained pigmentation. Transcriptome expression data confirmed the deletion of KTR9_0186 and identified the up-regulation of key genes involved in fatty acid degradation, a likely compensatory mechanism for reduced TAG mobilization. In vitro assays with purified KTR9_3844 demonstrated WS/DGAT activity with short chain alcohols and C16 and C18 fatty acid Co-As. Collectively, these results indicate that Gordonia sp. KTR9 has a suitable tractable genetic background for TAG production as well as the enzymatic capacity to catalyze fatty acid esters from short chain alcohols.