Determination of Key Metabolites during Biodegradation of Hexahydro-1,3,5-Trinitro-1,3,5-Triazine with Rhodococcus sp. Strain DN22

Rhodococcus sp. strain DN22 can convert hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) to nitrite, but information on degradation products or the fate of carbon is not known. The present study describes aerobic biodegradation of RDX (175 μM) when used as an N source for strain DN22. RDX was converted to nitrite (NO₂⁻) (30%), nitrous oxide (N₂O) (3.2%), ammonia (10%), and formaldehyde (HCHO) (27%), which later converted to carbon dioxide. In experiments with ring-labeled [¹⁵N]-RDX, gas chromatographic/mass spectrophotometric (GC/MS) analysis revealed N₂O with two molecular mass ions: one at 44 Da, corresponding to ¹⁴N¹⁴NO, and the second at 45 Da, corresponding to ¹⁵N¹⁴NO. The nonlabeled N₂O could be formed only from -NO₂, whereas the ¹⁵N-labeled one was presumed to originate from a nitramine group (¹⁵N-¹⁴NO₂) in RDX. Liquid chromatographic (LC)-MS electrospray analyses indicated the formation of a dead end product with a deprotonated molecular mass ion [M-H] at 118 Da. High-resolution MS indicated a molecular formula of C₂H₅N₃O₃. When the experiment was repeated with ring-labeled [¹⁵N]-RDX, the [M-H] appeared at 120 Da, indicating that two of the three N atoms in the metabolite originated from the ring in RDX. When [U-¹⁴C]-RDX was used in the experiment, 64% of the original radioactivity in RDX incorporated into the metabolite with a molecular weight (MW) of 119 (high-pressure LC/radioactivity) and 30% in ¹⁴CO₂ (mineralization) after 4 days of incubation, suggesting that one of the carbon atoms in RDX was converted to CO₂ and the other two were incorporated in the ring cleavage product with an MW of 119. Based on the above stoichiometry, we propose a degradation pathway for RDX based on initial denitration followed by ring cleavage to formaldehyde and the dead end product with an MW of 119.     

Chemotaxis-mediated biodegradation of cyclic nitramine explosives RDX, HMX, and CL-20 by Clostridium sp. EDB2

Cyclic nitramine explosives, RDX, HMX, and CL-20 are hydrophobic pollutants with very little aqueous solubility. In sediment and soil environments, they are often attached to solid surfaces and/or trapped in pores and distribute heterogeneously in aqueous environments. For efficient bioremediation of these explosives, the microorganism(s) must access them by chemotaxis ability. In the present study, we isolated an obligate anaerobic bacterium Clostridium sp. strain EDB2 from a marine sediment. Strain EDB2, motile with numerous peritrichous flagella, demonstrated chemotactic response towards RDX, HMX, CL-20, and NO(2)(-). The three explosives were biotransformed by strain EDB2 via N-denitration with concomitant release of NO(2)(-). Biotransformation rates of RDX, HMX, and CL-20 by the resting cells of strain EDB2 were 1.8+/-0.2, 1.1+/-0.1, and 2.6+/-0.2nmol h(-1)mgwet biomass(-1) (mean+/-SD; n=3), respectively. We found that commonly seen RDX metabolites such as TNX, methylenedinitramine, and 4-nitro-2,4-diazabutanal neither produced NO(2)(-) during reaction with strain EDB2 nor they elicited chemotaxis response in strain EDB2. The above data suggested that NO(2)(-) released from explosives during their biotransformation might have elicited chemotaxis response in the bacterium. Biodegradation and chemotactic ability of strain EDB2 renders it useful in accelerating the bioremediation of explosives under in situ conditions.     

Position effects due to chromosome breakpoints that map approximately 900 Kb upstream and approximately 1.3 Mb downstream of SOX9 in two patients with campomelic dysplasia

Campomelic dysplasia (CD) is a semilethal skeletal malformation syndrome with or without XY sex reversal. In addition to the multiple mutations found within the sex-determining region Y-related high-mobility group box gene (SOX9) on 17q24.3, several chromosome anomalies (translocations, inversions, and deletions) with breakpoints scattered over 1 Mb upstream of SOX9 have been described. Here, we present a balanced translocation, t(4;17)(q28.3;q24.3), segregating in a family with a mild acampomelic CD with Robin sequence. Both chromosome breakpoints have been identified by fluorescence in situ hybridization and have been sequenced using a somatic cell hybrid. The 17q24.3 breakpoint maps approximately 900 kb upstream of SOX9, which is within the same bacterial artificial chromosome clone as the breakpoints of two other reported patients with mild CD. We also report a prenatal identification of acampomelic CD with male-to-female sex reversal in a fetus with a de novo balanced complex karyotype, 46,XY,t(4;7;8;17)(4qter-->4p15.1::17q25.1-->17qter;7qter-->7p15.3::4p15.1-->4pter;8pter-->8q12.1::7p15.3-->7pter;17pter-->17q25.1::8q12.1-->8qter). Surprisingly, the 17q breakpoint maps approximately 1.3 Mb downstream of SOX9, making this the longest-range position effect found in the field of human genetics and the first report of a patient with CD with the chromosome breakpoint mapping 3' of SOX9. By using the Regulatory Potential score in conjunction with analysis of the rearrangement breakpoints, we identified a candidate upstream cis-regulatory element, SOX9cre1. We provide evidence that this 1.1-kb evolutionarily conserved element and the downstream breakpoint region colocalize with SOX9 in the interphase nucleus, despite being located 1.1 Mb upstream and 1.3 Mb downstream of it, respectively. The potential molecular mechanism responsible for the position effect is discussed.     

Systemic Induction of Photosynthesis via Illumination of the Shoot Apex Is Mediated Sequentially by Phytochrome B,Auxin and Hydrogen Peroxide in Tomato

Systemic signaling of upper leaves promotes the induction of photosynthesis in lower leaves, allowing more efficient use of light flecks. However, the nature of the systemic signals has remained elusive. Here, we show that preillumination of the tomato (Solanum lycopersicum) shoot apex alone can accelerate photosynthetic induction in distal leaves and that this process is light quality dependent, where red light promotes and far-red light delays photosynthetic induction. Grafting the wild-type rootstock with a phytochome B (phyB) mutant scion compromised light-induced photosynthetic induction as well as auxin biosynthesis in the shoot apex, auxin signaling, and RESPIRATORY BURST OXIDASE HOMOLOG1 (RBOH1)-dependent hydrogen peroxide (H₂O₂) production in the systemic leaves. Light-induced systemic H₂O₂ production in the leaves of the rootstock also was absent in plants grafted with an auxin-resistant diageotropica (dgt) mutant scion. Cyclic electron flow around photosystem I and associated ATP production were increased in the systemic leaves by exposure of the apex to red light. This enhancement was compromised in the systemic leaves of the wild-type rootstock with phyB and dgt mutant scions and also in RBOH1-RNA interference leaves with the wild type as scion. Silencing of ORANGE RIPENING, which encodes NAD(P)H dehydrogenase, compromised the systemic induction of photosynthesis. Taken together, these results demonstrate that exposure to red light triggers phyB-mediated auxin synthesis in the apex, leading to H₂O₂ generation in systemic leaves. Enhanced H₂O₂ levels in turn activate cyclic electron flow and ATP production, leading to a faster induction of photosynthetic CO₂ assimilation in the systemic leaves, allowing plants better adaptation to the changing light environment.As a consequence of their sessile lifestyle, plants have evolved a high capacity for the regulation of physiology, growth, and development that facilitates survival in a constantly changing environment. Environmental stimuli perceived within an organ not only influence morphogenetic and physiological changes within that organ but also generate systemic effects in other organs that are remote from the site of signal perception. This crucial phenomenon is called systemic signaling or systemic regulation. Systemic signaling prepares other tissues of a plant for future challenges that may initially only be sensed by a few local tissues or cells. Several types of systemic responses are known. These include systemic acquired resistance, which is typically activated by pathogens such as viruses, bacteria, and fungi (Fu and Dong, 2013), induced systemic resistance, which is triggered by beneficial soil microorganisms or others (Pieterse and Dicke, 2007), and systemic acquired acclimation, which is initiated by abiotic stresses such as high light, UV radiation, heat, cold, and salinity (Mittler and Blumwald, 2015).The light utilization efficiency of photosynthesis is important for the survival of understory plants and plants growing in canopies. In particular, the efficient use of the energy contained in light (sun) flecks is important because light flecks contribute up to 60% to 80% of photosynthetically active radiation received by understory plants (Pearcy and Seemann, 1990; Leakey et al., 2003, 2005). Earlier studies have shown the existence of systemic regulation of stomatal development and of photosynthesis in developing leaves in response to environmental signals perceived by mature leaves, such as changing irradiance and atmospheric CO₂ conditions (Lake et al., 2002; Coupe et al., 2006; Araya et al., 2008). Phytochome B (phyB) is important in the transmission of the systemic signals that modulate stomatal development in young leaves of Arabidopsis (Arabidopsis thaliana; Casson and Hetherington, 2014). In tomato (Solanum lycopersicum), there are two forms of phyB, phyB1 and phyB2, that work together to mediate red (R) light-induced responses, such as hypocotyl elongation and greening in seedlings (Hauser et al., 1995; Weller et al., 2000).Photosynthesis is completely switched off in the dark, specifically to prevent futile cycling of metabolites through the reductive and oxidative pentose phosphate pathways. Hence, leaves need time to reactivate the enzymes of carbon assimilation after a period of darkness. The time taken to reach maximum net rates of photosynthesis upon illumination is called photosynthetic induction (Walker, 1973). Systemic signaling also has been observed for the regulation of photosynthesis in relation to leaf ontology in understory plants (Montgomery and Givnish, 2008). The uppermost leaves, which are generally the first to receive sunlight, display faster photosynthetic induction times than understory leaves (Bai et al., 2008). Photosynthetic induction in understory leaves is enhanced by the preillumination of upper leaves but not lower leaves, suggesting a directional signal transfer (Hou et al., 2015). While this process allows plants to use the light energy in sun flecks more efficiently, the nature of the systemic signals and their transmission pathways remain largely unresolved. Although systemic signaling between different leaf ranks has been suggested to occur through the xylem (Thorpe et al., 2007) and also via electrical signals (Zimmermann et al., 2009), it is likely that systemic signals also pass through the phloem (Turgeon and Wolf, 2009; Hou et al., 2015). In addition, the phytohormone auxin is produced in the shoot apex and redistributed throughout the shoot by rapid nonpolar phloem transport (Ljung et al., 2001). Changes in the light environment can dramatically alter auxin homeostasis, which is regulated in a light quality- and photoreceptor-dependent manner (Halliday et al., 2009).The photosynthetic electron transport chain exhibits enormous flexibility in the relative rates of NADPH and ATP production in order to accommodate the varying requirements of metabolism (Foyer et al., 2012). Noncyclic, pseudocyclic, and cyclic electron flow (CEF) pathways operate in the photosynthetic electron transport chain to drive the proton gradient across the thylakoid membrane (Allen, 2003). Photosynthetic induction is not only associated with the activation of the light- and thiol-dependent activation of carbon assimilation enzymes but also dependent on a high rate of CEF to drive ATP synthesis (Foyer et al., 1992). Considerable overreduction of the electron transport acceptors occurs during the photosynthetic induction period, and this continues until carbon assimilation can be activated. CEF around PSI, an essential component of photosynthesis, drives the proton gradient in a situation when NADP reduction has reached its highest capacity and this essential electron acceptor is no longer available (Yamori et al., 2015; Yamori and Shikanai, 2016). CEF is particularly sensitive to the reduction-oxidation (redox) status of the chloroplast, which in turn is responsive to cellular redox homeostasis. Oxidants such as hydrogen peroxide (H₂O₂), which are produced by pseudocyclic electron flow in the chloroplasts, play a crucial role in the activation of CEF through modulation of the activity of the NADPH-plastoquinone reductase complex (Strand et al., 2015). Hormone-mediated generation of H₂O₂ also can stimulate CO₂ assimilation (Jiang et al., 2012).Auxins such as indole-3-acetic acid (IAA) generate H₂O₂ (Ivanchenko et al., 2013; Peer et al., 2013) and can regulate CO₂ assimilation (Bidwell and Turner, 1966; Hayat et al., 2009; Peng et al., 2013). Therefore, we used tomato plants to test the hypothesis that the systemic signaling that regulates photosynthetic induction in understory leaves arises from light-induced changes in auxin and H₂O₂ homeostasis involving the modulation of CEF in systemic leaves. We present evidence showing that R light perceived in the shoot apex by a phyB-dependent pathway alters IAA signaling in a systemic manner. IAA signals from the apex, perceived in distal leaves, trigger systemic H₂O₂ production that accelerates photosynthetic induction by increasing CEF-dependent ATP production in the systemic leaves. These findings provide new insights into the elaborate plant regulatory network that allows light adaptation in different organs.     

Metabolite production during transformation of 2,4,6-trinitrotoluene (TNT) by a mixed culture acclimated and maintained on crude oil-containing media

Metabolites formed during 2,4,6-trinitrotoluene (TNT) removal by a mixed bacterial culture (acclimated and maintained on crude oil-containing medium and capable of high rates of TNT removal) were characterized. In resting cell experiments in the absence of glucose, 46.2 mg/l TNT were removed in 171 h (87.5% removal), with a combined total formation of 7.7 mg/l amino-4,6-dinitrotoluene (ADNT) and 0.3 mg/l 4,4-azoxytetranitrotoluene and 2,4-azoxytetranitrotoluene, leaving 70% of the initial TNT unaccounted for. In the presence of glucose, resting cells removed 45.4 mg/l TNT in 49 h (95.5% removal), with 9.1 mg/l ADNT and 2.4 mg/l azoxy compounds being produced, leaving 70.3% of the TNT unaccounted for. Growing cells (glucose present) were capable of removing 44.2 mg/l TNT within 21 h (97.9% removal), with the concomitant formation of 1.8 mg/l ADNTs and 2.2 mg/l azoxy compounds. Denitrated TNT in the form of 2,6-dinitrotoluene was also produced in growing cells with a maximum amount of 1.31 mg/l after 28 h, followed by a slight decrease with time, leaving 88.5% of the initial TNT unaccounted for after 171 h. Radiolabeled ¹⁴C-TNT studies revealed 4.14% mineralization after an incubation period of 163 days with growing cells.     

Fluorinated aminoglycosides and their mechanistic implication for aminoglycoside 3'-phosphotransferases from Gram-negative bacteria

Aminoglycoside 3'-phosphotransferases [APH(3')s] are important bacterial resistance enzymes for aminoglycoside antibiotics. These enzymes phosphorylate the 3'-hydroxyl of these antibiotics, a reaction that inactivates the drug. A series of experiments were carried out to shed light on the details of the turnover chemistry by these enzymes. Quench-flow pre-steady-state kinetic analyses of the reactions of Gram-negative APH(3') types Ia and IIa with kanamycin A, neamine, and their respective difluorinated analogues 4'-deoxy-4',4'-difluorokanamycin A and 4'-deoxy-4',4'-difluoroneamine were carried out, in conjunction with measurements of thio effect and viscosity studies. The fluorinated analogues were shown to be severely impaired as substrates for these enzymes. The magnitude of the effect of the impairment of the fluorinated substrates was in the same range as when the D198A mutant APH(3')-Ia was studied with nonfluorinated substrates. Residue 198 is the proposed active site base that promotes the aminoglycoside hydroxyl for phosphorylation. These findings collectively argue that the Gram-negative APH(3')s show significant nucleophilic participation in the transition state for the phosphate transfer reaction.     

NPR1-dependent salicylic acid signaling is not involved in elevated CO2-induced heat stress tolerance in Arabidopsis thaliana

Elevated CO₂ can protect plants from heat stress (HS); however, the underlying mechanisms are largely unknown. Here, we used a set of Arabidopsis mutants such as salicylic acid (SA) signaling mutants nonexpressor of pathogenesis-related gene 1 (npr1-1 and npr1-5) and heat-shock proteins (HSPs) mutants (hsp21 and hsp70-1) to understand the requirement of SA signaling and HSPs in elevated CO₂-induced HS tolerance. Under ambient CO₂ (380 µmol mol⁻¹) conditions, HS (42°C, 24 h) drastically decreased maximum photochemical efficiency of PSII (Fv/Fm) in all studied plant groups. Enrichment of CO₂ (800 µmol mol⁻¹) with HS remarkably increased the Fv/Fm value in all plant groups except hsp70-1, indicating that NPR1-dependent SA signaling is not involved in the elevated CO₂-induced HS tolerance. These results also suggest an essentiality of HSP70-1, but not HSP21 in elevated CO₂-induced HS mitigation.     

Enhanced antinociception by intracerebroventricularly and intrathecally-administered orexin A and B (hypocretin-1 and -2) in mice

Orexins are neuropeptides located exclusively in neurons of the lateral hypothalamic area, which send projections to most monoaminergic nuclei, such as noradrenergic locus coeruleus, dopaminergic ventral tegmental areas, and histaminergic tuberomammillary nuclei. The present work was carried out to examine the role of orexins in nociception in mice. C57BL/6 mice were administered with orexin A and B intracerebroventricularly (i.c.v.), intrathecally (i.t.) and subcutaneously (s.c.) to reveal the sites of action of these peptides and to examine the pain thresholds using four kinds of nociceptive tasks. Orexins showed antinociceptive effects in all four types of assays for thermal (hot-plate, tail-flick, paw-withdrawal), mechanical (tail-pressure), chemical (formalin, capsaicin and abdominal stretch) nociceptions and nociceptin-induced behavioral responses, when administered i.c.v. or i.t., whereas the s.c. administration was ineffective. The antinociceptive effects of orexin A were more remarkable than those of orexin B. The i.c.v. administration of orexin A was as effective as, or more potent than the i.t. administration. The effects of orexin A were completely blocked by adenosine type 1 receptor antagonists, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) and theophylline, but not by naloxone, suggesting a possible involvement of the adenosine-containing neurons and/or the adenosine pathway in these orexin actions. The i.c.v. administration of nociceptin had no significant effects on orexin expression in the brain and spinal cord. The present findings suggest that orexins have an antinociceptive role in at least four different types of pains, probably acting on both the brain and spinal cord.