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.

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.

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.

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.

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.

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%.

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.

Role of nitrogen limitation in transformation of RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) by Gordonia sp. strain KTR9.

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.

Physiological characterization of lipid accumulation and in vivo ester formation in Gordonia sp. KTR9.

Previous work has demonstrated the feasibility of in vivo biodiesel synthesis in Escherichia coli, however, ethyl ester formation was dependent on an external fatty acid feedstock. In contrast to E. coli, actinomycetes may be ideal organisms for direct biodiesel synthesis because of their capacity to synthesize high levels of triacylglcerides (TAGs). In this study, we investigated the physiology and associated TAG accumulation along with the in vivo ability to catalyze ester formation from exogenous short chain alcohol sources in Gordonia sp. KTR9, a strain that possesses a large number of genes dedicated to fatty acid and lipid biosynthesis. Total lipid fatty acids content increased by 75 % and TAG content increased by 50 % under nitrogen starvation conditions in strain KTR9. Strain KTR9 tolerated the exogenous addition of up to 4 % methanol, 4 % ethanol and 2 % propanol in the media. Increasing alcohol concentrations resulted in a decrease in the degree of saturation of recovered fatty acid alcohol esters and a slight increase in the fatty acid chain length. A linear dose dependency in fatty alcohol ester synthesis was observed in the presence of 0.5-2 % methanol and ethanol compared to control KTR9 strains grown in the absence of alcohols. An inspection of the KTR9 genome revealed the presence of several putative wax ester synthase/acyl-coenzyme A : diacylglycerol acyltransferase (WS/DGAT) enzymes, encoded by atf gene homologs, that may catalyze the in vivo synthesis of fatty acid esters from short chain alcohols. Collectively, these results indicate that Gordonia sp. KTR9 may be a suitable actinomycete host strain for in vivo biodiesel synthesis.

Photosynthetic accumulation of carbon storage compounds under CO2 enrichment by the thermophilic cyanobacterium Thermosynechococcus elongatus.

The growth characteristics of Thermosynechococcus elongatus on elevated CO2 were studied in a photobioreactor. Cultures were able to grow on up to 20% CO2. The maximum productivity and CO2 fixation rates were 0.09 ± 0.01 and 0.17 ± 0.01 mg ml⁻¹ day⁻¹, respectively, for cultures grown on 20% CO2. Three major carbon pools--lipids, polyhydroxybutyrates (PHBs), and glycogen--were measured. These carbon stores accounted for 50% of the total biomass carbon in cultures grown on atmospheric CO2 (no supplemental CO2), but only accounted for 30% of the total biomass carbon in cultures grown on 5-20% CO2. Lipid content was approximately 20% (w/w) under all experimental conditions, while PHB content reached 14.5% (w/w) in cultures grown on atmospheric CO2 and decreased to approximately 2.0% (w/w) at 5-20% CO2. Glycogen levels did not vary significantly and remained about 1.4% (w/w) under all test conditions. The maximum amount of CO2 sequestered over the course of the nine-day chemostat experiment was 1.15 g l⁻¹ in cultures grown on 20% CO2.

Anaerobic oxidation of methane: mechanisms, bioenergetics, and the ecology of associated microorganisms.

Microbially mediated anaerobic oxidation of methane (AOM) moderates the input of methane, an important greenhouse gas, to the atmosphere by consuming methane produced in various marine, terrestrial, and subsurface environments. AOM coupled to sulfate reduction has been most extensively studied because of the abundance of sulfate in marine systems, but electron acceptors otherthan sulfate are more energetically favorable. Phylogenetic trees based on 16S rRNA gene clone libraries derived from microbial communities where AOM occurs show evidence of diverse, methanotrophic archaea (ANME) closely associated with sulfate-reducing bacteria, but these organisms have not yet been isolated as pure cultures. Several biochemical pathways for AOM have been proposed, including reverse methanogenesis, acetogenesis, and methylogenesis, and both culture-dependent and independent techniques have provided some clues to howthese communities function. Still, questions remain regarding the diversity, physiology, and metabolic restrictions of AOM-related organisms.

Thermotolerant hydrogenases: biological diversity, properties, and biotechnological applications.

Hydrogenases are metalloproteins that catalyze the oxidation and reduction of molecular hydrogen and play a crucial role in many microbial metabolic processes. A subset of hydrogenases capable of functioning at temperatures from 50 to 125 degrees C is found in thermophilic microorganisms. Most known thermotolerant hydrogenases contain a [NiFe] active site and are either bidirectional or uptake type. Although no exhaustive survey has been done of the ecological diversity of thermophilic hydrogen-reducing or oxidizing bacteria, they appear to exist in virtually every thermophilic environment examined to date. Thermotolerant hydrogenases share many similarities with their mesophilic counterparts, but they have several features in addition to thermotolerance that make them especially well suited for biotechnological applications. Ongoing research is focused on potential applications of thermotolerant H2 ases in biosynthesis, H2 production, bioremediation, and biosensors.