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.

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.

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.

Draft Genome Sequence of Rhodococcus rhodochrous Strain ATCC 21198.

Rhodococcus rhodochrous is a Gram-positive red-pigmented bacterium commonly found in the soil. The draft genome sequence for R. rhodochrous strain ATCC 21198 is presented here to provide genetic data for a better understanding of its lipid-accumulating capabilities.

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.

Effects of C60 on the Salmonella typhimurium TA100 transcriptome expression: Insights into C60 -mediated growth inhibition and mutagenicity.

Rapid advances are being made in the creation and use of nanomaterials, but little is known about the impact these materials might have on key microbial functions if introduced into the environment. Previous studies have generated conflicting results with respect to the impact of fullerenes on microbial activity. In the present study, Salmonella typhimurium TA100 was selected as a model microbial system with which to investigate further the impact of C(60) aggregates on microbial growth, mutagenicity, and global transcript expression. Aggregates of C(60) predominantly less than 100 nm significantly impacted Salmonella growth at concentrations of ≥ 0.5 mg/L. In addition, C(60) aggregates also displayed mutagenic potential at concentrations ≥ 0.1 mg/L. Transcript expression analysis of S. typhimurium TA100 exposed to C(60) for 24 h indicated that 271 transcripts had significant differential expression relative to controls with twofold or more change. Of particular interest was the increased expression of transcripts coding for proteins involved in energy metabolism, amino acid biosynthesis, transcription, and DNA metabolism, and the decreased expression of transcripts coding for proteins involved in protein fate, transport, and binding and bacterial secretion systems. Collectively, these data indicate that C(60) interacts with the outer membrane of S. typhimurium TA100, resulting in delayed growth and mutagenicity, most likely by interfering with key transport functions and inducing a stress response, respectively.

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.

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.