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.

Multiple stressors in multiple species: Effects of different RDX soil concentrations and differential water-resourcing on RDX fate, plant health, and plant survival.

Response to simultaneous stressors is an important facet of plant ecology and land management. In a greenhouse trial, we studied how eight plant species responded to single and combined effects of three soil concentrations of the phytotoxic munitions constituent RDX and two levels of water-resourcing. In an outdoor trial, we studied the effects of high RDX soil concentration and two levels of water-resourcing in three plant species. Multiple endpoints related to RDX fate, plant health, and plant survival were evaluated in both trials. Starting RDX concentration was the most frequent factor influencing all endpoints. Water-resourcing also had significant impacts, but in fewer cases. For most endpoints, significant interaction effects between RDX concentration and water-resourcing were observed for some species and treatments. Main and interaction effects were typically variable (significant in one treatment, but not in another; associated with increasing endpoint values for one treatment and/or with decreasing endpoint values in another). This complexity has implications for understanding how RDX and water-availability combine to impact plants, as well as for applications like phytoremediation. As an additional product of these greenhouse and outdoor trials, three plants native or naturalized within the southeastern United States were identified as promising species for further study as in situ phytoremediation resources. Plumbago auriculata exhibited relatively strong and markedly consistent among-treatment mean proportional reductions in soil RDX concentrations (112% and 2.5% of the means of corresponding values observed within other species). Likewise, across all treatments, Salvia coccinea exhibited distinctively low variance in mean leaf chlorophyll content index levels (6.5% of the means of corresponding values observed within other species). Both species also exhibited mean wilting and chlorosis levels that were 66% and 35%, and 67% and 84%, of corresponding values observed in all other plants, respectively. Ruellia caroliniensis exhibited at least 43% higher mean survival across all treatments than any other test species in outdoor trials, despite exhibiting similar RDX uptake and bioconcentration levels.

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.

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.

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.

Biotransformation of explosives by Reticulitermes flavipes--associated termite Endosymbionts.

BACKGROUND/AIMS: Termites have an important role in the carbon and nitrogen cycles despite their reputation as destructive pests. With the assistance of microbial endosymbionts, termites are responsible for the conversion of complex biopolymers into simple carbon substrates. Termites also rely on endosymbionts for fixing and recycling nitrogen. As a result, we hypothesize that termite bacterial endosymbionts are a novel source of metabolic pathways for the transformation of nitrogen-rich compounds like explosives. METHODS: Explosives transformation capability of termite (Reticulitermes flavipes)-derived endosymbionts was determined in media containing the chemical constituents nitrotriazolone (NTO) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) that comprise new insensitive explosive formulations. Media dosed with 40 µg/ml of explosive was inoculated with surface-sterilized, macerated termites. Bacterial isolates capable of explosives transformation were characterized by 16S rRNA sequencing. RESULTS: Termite-derived enrichment cultures demonstrated degradation activity towards the explosives NTO, RDX, as well as the legacy explosive 2,4,6-trinitrotoluene (TNT). Three isolates with high similarity to the Enterobacteriaceae(Enterobacter, Klebsiella) were able to transform TNT and NTO within 2 days, while isolates with high similarity to Serratia marcescens and Lactococcus lactis were able to transform RDX. CONCLUSION: Termite endosymbionts harbor a range of metabolic activities and possess unique abilities to transform nitrogen-rich explosives.

Dissemination of bacterial fluoroquinolone resistance in two multidrug-resistant enterobacteriaceae.

Bacterial resistance to antimicrobials has become one of the greatest challenges for clinical microbiologists and healthcare practitioners worldwide. Acquisition of resistance genes has proven to be difficult to characterize and is largely uncontrollable in the environment. Here we sought to characterize conjugal horizontal gene transfer of plasmid-encoded fluoroquinolone resistance genes from two strains of Enterobacteriaceae, one clinical and one from a municipal wastewater treatment plant environment. Conjugation was dissimilar between the two strains. Escherichia coli strain LR09, containing a plasmid with the aac(6')-Ib-cr fluoroquinolone resistance gene, did not conjugate with any of the 15 strains tested, while Enterobacter aerogenes strain YS11 conjugated with two strains of E. coli. The resultant transconjugants were also dissimilar in their stability and potential persistence. The observations presented herein exemplify the difficulties in understanding and controlling the spread of antimicrobial resistance. Thus, it may be prudent to address drug disposal and destruction, incorporating a life-cycle assessment plan 'from cradle to grave', treating antimicrobials as chemical or environmental contaminants.

Draft Genome Sequence of the Multidrug-Resistant Escherichia coli Strain LR09, Isolated from a Wastewater Treatment Plant.

We report the draft genome sequence of Escherichia coli O1:H6 strain LR09, which was isolated from a wastewater treatment plant and displays high resistance to five fluoroquinolone antimicrobials. The assembled data determine that the strain clusters with E. coli phylogroup F and harbors a plasmid conferring resistance to a broad spectrum of antibiotics.

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.

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.

Single nucleotide polymorphism analysis of the major tripartite multidrug efflux pump of Escherichia coli: functional conservation in disparate animal reservoirs despite exposure to antimicrobial chemotherapy.

AcrAB-TolC imparts a strong intrinsic resistance phenotype to many clinically significant molecules in Escherichia coli. This complex is composed of a pump, AcrB, and a periplasmic protein, AcrA, that exports substrates through a common outer membrane porin, TolC. A sequence survey of the pump-specific components, acrA and acrB, was conducted on three discrete animal reservoirs: rodents, bovines, and catfish. Although two of the reservoirs (bovine and catfish) were agrarian, and antibiotic use (ceftiofur and oxytetracycline/Romet 30, respectively) was reported for them, the vast majority of structural polymorphisms were silent except for T104A (AcrA) and Q733R (AcrB), found in certain bovine-derived strains. Overall, the genes were well conserved, with high ratios of synonymous to nonsynonymous substitutions (d(S)/d(N) ratios), consistent with or, in the case of acrB, better than those of standard multilocus sequence typing (MLST) loci. Furthermore, predicted recombination points from single nucleotide polymorphism (SNP) patterns in acrB support a modular evolution of transporter proteins, consistent with an ancient origin. However, functional studies with clones representing the major silent SNPs and the nonsilent mutation in acrB failed to generate significant differences in resistance to a range of common efflux pump substrates. Interestingly, a comparison between log-phase acrA and acrB expression profiles yielded inconsistent trends, with acrB expression increasing modestly (<1.8-fold) in many strains from the antibiotic-enriched pools. Our results suggest that structural polymorphisms in this major efflux pump system may not contribute significantly to adaptive resistance by altering function or substrate specificity but may have a potential use in improving phylogenetic relationships and/or source tracking.