Substrate selectivity of a 3-nitrophenol-induced metabolic system in Pseudomonas putida 2NP8 transforming nitroaromatic compounds into ammonia under aerobic conditions

The 3-nitrophenol-induced enzyme system in cells of Pseudomonas putida 2NP8 manifested a wide substrate range in transforming nitroaromatic compounds through to ammonia production. All of the 30 mono- or dinitroaromatic substrates except 4-nitrophenol, 2,4-dinitrophenol, 2,4,6-trinitrophenol, 3-nitroaniline, 2-nitrobenzoic acid, and 2-nitrofuran were quickly transformed. Ammonia production from most nitroaromatic substrates appeared to be stoichiometric.     

Synthesis of substituted catechols using nitroarene dioxygenases

The nitroarene dioxygenases are in the class of Rieske iron-containing oxygenases that incorporate atmospheric oxygen into substrates via electrophilic attack on the substrate. In their native role, the nitroarene dioxygenases start degradative pathways by hydroxylating nitro-substituted, and adjacent unsubstituted carbons of nitroaromatic compounds. The reaction yields the corresponding nitro-cis-cyclohexadienediol, which is unstable and spontaneously re-aromatizes to form a catechol and nitrite. In bacterial metabolism, the specificity of the hydroxylation determines subsequent steps in degradation pathways. Experiments were done to find whether the specificity could be exploited to direct the hydroxylation of multiply substituted aromatic substrates and thereby produce novel catechols. Recombinant strains carrying genes for nitroarene dioxygenases were used for transformation of various substituted nitroaromatic compounds. The reactions were analyzed using HPLC to track substrate consumption and product formation, then GC–MS and NMR to identify the reaction products. A number of substituted catechols were obtained using the recombinant biocatalysts. The nitro-substituted carbon was the primary site for dioxygenase hydroxylation. When substrates included nitro and halogen substituents, the halogen-substituted positions were also targeted, but less frequently than the nitro-substituted site. The production of catechols was limited in batch fermentations, likely due to toxicity of the quinones that result from air oxidation of catechols. The nitroarene dioxygenases will serve as catalysts for direct synthesis of highly substituted catechols, however, the reaction conditions must be engineered to overcome product toxicity and allow sustained accumulation of catecholic products.     

Drosophila GSTs display outstanding catalytic efficiencies with the environmental pollutants 2,4,6-trinitrotoluene and 2,4-dinitrotoluene

The nitroaromatic explosive 2,4,6-trinitrotoluene (TNT) and the related 2,4-dinitrotoluene (DNT) are toxic environmental pollutants. The biotransformation and detoxication of these persistent compounds in higher organisms are of great significance from a health perspective as well as for the biotechnological challenge of bioremediation of contaminated soil. We demonstrate that different human glutathione transferases (GSTs) and GSTs from the fruit fly Drosophila melanogaster are catalysts of the biotransformation of TNT and DNT. The human GSTs had significant but modest catalytic activities with both DNT and TNT. However, D. melanogaster GSTE6 and GSTE7 displayed outstanding high activities with both substrates.     

Kinetic and structural basis of reactivity of pentaerythritol tetranitrate reductase with NADPH, 2-cyclohexenone, nitroesters, and nitroaromatic explosives

The reaction of pentaerythritol tetranitrate reductase with reducing and oxidizing substrates has been studied by stopped-flow spectrophotometry, redox potentiometry, and X-ray crystallography. We show in the reductive half-reaction of pentaerythritol tetranitrate (PETN) reductase that NADPH binds to form an enzyme-NADPH charge transfer intermediate prior to hydride transfer from the nicotinamide coenzyme to FMN. In the oxidative half-reaction, the two-electron-reduced enzyme reacts with several substrates including nitroester explosives (glycerol trinitrate and PETN), nitroaromatic explosives (trinitrotoluene (TNT) and picric acid), and alpha,beta-unsaturated carbonyl compounds (2-cyclohexenone). Oxidation of the flavin by the nitroaromatic substrate TNT is kinetically indistinguishable from formation of its hydride-Meisenheimer complex, consistent with a mechanism involving direct nucleophilic attack by hydride from the flavin N5 atom at the electron-deficient aromatic nucleus of the substrate. The crystal structures of complexes of the oxidized enzyme bound to picric acid and TNT are consistent with direct hydride transfer from the reduced flavin to nitroaromatic substrates. The mode of binding the inhibitor 2,4-dinitrophenol (2,4-DNP) is similar to that observed with picric acid and TNT. In this position, however, the aromatic nucleus is not activated for hydride transfer from the flavin N5 atom, thus accounting for the lack of reactivity with 2,4-DNP. Our work with PETN reductase establishes further a close relationship to the Old Yellow Enzyme family of proteins but at the same time highlights important differences compared with the reactivity of Old Yellow Enzyme. Our studies provide a structural and mechanistic rationale for the ability of PETN reductase to react with the nitroaromatic explosive compounds TNT and picric acid and for the inhibition of enzyme activity with 2,4-DNP.     

Reactions Involved in the Lower Pathway for Degradation of 4-Nitrotoluene by Mycobacterium Strain HL 4-NT-1

In spite of the variety of initial reactions, the aerobic biodegradation of aromatic compounds generally yields dihydroxy intermediates for ring cleavage. Recent investigation of the degradation of nitroaromatic compounds revealed that some nitroaromatic compounds are initially converted to 2-aminophenol rather than dihydroxy intermediates by a number of microorganisms. The complete pathway for the metabolism of 2-aminophenol during the degradation of nitrobenzene by Pseudomonas pseudoalcaligenes JS45 has been elucidated previously. The pathway is parallel to the catechol extradiol ring cleavage pathway, except that 2-aminophenol is the ring cleavage substrate. Here we report the elucidation of the pathway of 2-amino-4-methylphenol (6-amino-m-cresol) metabolism during the degradation of 4-nitrotoluene by Mycobacterium strain HL 4-NT-1 and the comparison of the substrate specificities of the relevant enzymes in strains JS45 and HL 4-NT-1. The results indicate that the 2-aminophenol ring cleavage pathway in strain JS45 is not unique but is representative of the pathways of metabolism of other o-aminophenolic compounds.     

Interactions of nitroaromatic compounds with the mammalian selenoprotein thioredoxin reductase and the relation to induction of apoptosis in human cancer cells

Here we described novel interactions of the mammalian selenoprotein thioredoxin reductase (TrxR) with nitroaromatic environmental pollutants and drugs. We found that TrxR could catalyze nitroreductase reactions with either one- or two-electron reduction, using its selenocysteine-containing active site and another redox active center, presumably the FAD. Tetryl and p-dinitrobenzene were the most efficient nitroaromatic substrates with a k(cat) of 1.8 and 2.8 s(-1), respectively, at pH 7.0 and 25 degrees C using 50 muM NADPH. As a nitroreductase, TrxR cycled between four- and two-electron-reduced states. The one-electron reactions led to superoxide formation as detected by cytochrome c reduction and, interestingly, reductive N-denitration of tetryl or 2,4-dinitrophenyl-N-methylnitramine, resulting in the release of nitrite. Most nitroaromatics were uncompetitive and noncompetitive inhibitors with regard to NADPH and the disulfide substrate 5,5'-dithiobis(2-nitrobenzoic acid), respectively. Tetryl and 4,6-dinitrobenzofuroxan were, however, competitive inhibitors with respect to 5,5'-dithiobis(2-nitrobenzoic acid) and were clearly substrates for the selenolthiol motif of the enzyme. Furthermore, tetryl and 4,6-dinitrobenzofuroxan efficiently inactivated TrxR, likely by alkylation of the selenolthiol motif as in the inhibition of TrxR by 1-chloro-2,4-dinitrobenzene/dinitrochlorobenzene (DNCB) or juglone. The latter compounds were the most efficient inhibitors of TrxR activity in a cellular context. DNCB, juglone, and tetryl were highly cytotoxic and induced caspase-3/7 activation in HeLa cells. Furthermore, DNCB and juglone were potent inducers of apoptosis also in Bcl2 overexpressing HeLa cells or in A549 cells. Based on these findings, we suggested that targeting of intracellular TrxR by alkylating nitroaromatic or quinone compounds may contribute to the induction of apoptosis in exposed human cancer cells.     

Biotransformation of 2,4-dinitroanisole by a fungal <Emphasis Type="Italic">Penicillium</Emphasis> sp.

Insensitive munitions explosives are new formulations that are less prone to unintended detonation compared to traditional explosives. While these formulations have safety benefits, the individual constituents, such as 2,4-dinitroanisole (DNAN), have an unknown ecosystem fate with potentially toxic impacts to flora and fauna exposed to DNAN and/or its metabolites. Fungi may be useful in remediation and have been shown to degrade traditional nitroaromatic explosives, such as 2,4,6-trinitrotoluene and 2,4-dinitrotoluene, that are structurally similar to DNAN. In this study, a fungal Penicillium sp., isolated from willow trees and designated strain KH1, was shown to degrade DNAN in solution within 14 days. Stable-isotope labeled DNAN and an untargeted metabolomics approach were used to discover 13 novel transformation products. Penicillium sp. KH1 produced DNAN metabolites resulting from ortho- and para-nitroreduction, demethylation, acetylation, hydroxylation, malonylation, and sulfation. Incubations with intermediate metabolites such as 2-amino-4-nitroanisole and 4-amino-2-nitroanisole as the primary substrates confirmed putative metabolite isomerism and pathways. No ring-cleavage products were observed, consistent with other reports that mineralization of DNAN is an uncommon metabolic outcome. The production of metabolites with unknown persistence and toxicity suggests further study will be needed to implement remediation with Penicillium sp. KH1. To our knowledge, this is the first report on the biotransformation of DNAN by a fungus.     

Mitochondrial permeability transition as a source of superoxide anion induced by the nitroaromatic drug nimesulide in vitro

Nimesulide, a widely used nonsteroidal anti-inflammatory drug containing a nitroaromatic moiety, has been associated with rare but serious hepatic adverse effects. The mechanisms underlying this idiosyncratic hepatotoxicity are unknown; however, both mitochondrial injury and oxidative stress have been implicated in contributing to liver injury in susceptible patients. The aim of this study was, first, to explore whether membrane permeability transition (MPT) could contribute to nimesulide's mitochondrial toxicity and, second, whether metabolism-derived reactive oxygen species (ROS) were responsible for MPT. We found that isolated mouse liver mitochondria readily underwent Ca2+-dependent, cyclosporin A-sensitive MPT upon exposure to nimesulide (at >or=3 microM). Net increases in mitochondrial superoxide anion levels, determined with the fluorescent probe dihydroethidium, were induced by nimesulide only in the presence of Ca2+ and were cyclosporin A-sensitive, indicating that superoxide production was a consequence, rather than the cause, of MPT. In addition, nimesulide caused a rapid dissipation of the inner mitochondrial transmembrane potential (at >or=3 microM), followed by a concentration-dependent decrease in ATP biosynthesis. Because nimesulide, unlike the related nitroaromatic drug nilutamide, did not produce any detectable ROS during incubation with mouse hepatic microsomes, we conclude that mitochondrial uncoupling causes MPT and that ROS production is a secondary effect.     

Deletion of acetyl-CoA synthetases I and II increases production of 3-hydroxypropionate by the metabolically-engineered hyperthermophile Pyrococcus furiosus

The heterotrophic, hyperthermophilic archaeon Pyrococcus furiosus is a new addition to the growing list of genetically-tractable microorganisms suitable for metabolic engineering to produce liquid fuels and industrial chemicals. P. furiosus was recently engineered to generate 3-hydroxypropionate (3-HP) from CO₂ and acetyl-CoA by the heterologous-expression of three enzymes from the CO₂ fixation cycle of the thermoacidophilic archaeon Metallosphaera sedula using a thermally-triggered induction system. The acetyl-CoA for this pathway is generated from glucose catabolism that in wild-type P. furiosus is converted to acetate with concurrent ATP production by the heterotetrameric (α₂β₂) acetyl-CoA synthetase (ACS). Hence ACS in the engineered 3-HP production strain (MW56) competes with the heterologous pathway for acetyl-CoA. Herein we show that strains of MW56 lacking the α-subunit of either of the two ACSs previously characterized from P. furiosus (ACSI and ACSII) exhibit a three-fold increase in specific 3-HP production. The ΔACSIα strain displayed only a minor defect in growth on either maltose or peptides, while no growth defect on these substrates was observed with the ΔACSIIα strain. Deletion of individual and multiple ACS subunits was also shown to decrease CoA release activity for several different CoA ester substrates in addition to acetyl-CoA, information that will be extremely useful for future metabolic engineering endeavors in P. furiosus.     

Quantitative structure–activity relationships in enzymatic single-electron reduction of nitroaromatic explosives: implications for their cytotoxicity

The mechanisms of cytotoxicity of polynitroaromatic explosives, an important group of environmental pollutants, remain insufficiently studied so far. We have found that the rate constants of single-electron enzymatic reduction, and the enthalpies of single-electron reduction of nitroaromatic compounds (ΔHf(ArNO₂^−⋅)), obtained by quantum mechanical calculation, may serve as useful tools for the analysis of cytotoxicity of nitroaromatic explosives with respect to the possible involvement of oxidative stress. The single-electron reduction rate constants of a number of explosives including 2,4,6-trinitrotoluene (TNT) and 2,4,6-trinitrophenyl-N-methylnitramine (tetryl), and model nitroaromatic compounds by ferredoxin:NADP⁺ reductase (FNR, EC 1.18.1.2) and NADPH:cytochrome P-450 reductase (P-450R, EC 1.6.2.4) increased with a decrease in ΔHf(ArNO₂^−⋅). This indicates that the reduction rates are determined by the electron transfer energetics, but not by the particular structure of the explosives. The cytotoxicity of explosives to bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) increased with a corresponding increase in their reduction rate constant by P-450R and FNR, or with a decrease in their ΔHf(ArNO₂^−⋅). This points to an importance of oxidative stress in the toxicity of explosives in this cell line, which was further evidenced by the protective effects of desferrioxamine and the antioxidant N,N′-diphenyl-p-phenylene diamine, and an increase in lipid peroxidation. DT-diaphorase (EC 1.6.99.2) exerted a minor and equivocal role in the cytotoxicity of explosives to FLK cells.     

Residue Phe42 is critical for the catalytic activity of Escherichia coli major nitroreductase NfsA

The major O₂-insensitive nitroreductase (NfsA) of Escherichia coli shares low sequence homology but similar biochemical and structural features with NfsB, the E. coli minor O₂-insensitive nitroreductase. A structural comparison revealed Phe42 was present in the active site of NfsA but not NfsB. F42Y, F42N and F42A were generated and had decreased activity toward nitrofurazone by 52, 96, and 99 %, respectively. The kinetic parameters for other nitroaromatic substrates were also determined. Compared to wild type, the mutants did not have significantly altered K _ms, but had dramatically decreased k _cat and k _cat/K _m values. Far-UV CD spectral analysis of the mutants suggested that there were no significant conformational changes however F42A and F42N had changes from 208 to 222 nm, which was attributed to loss of helix content. These findings revealed that Phe42 is important for maintaining NfsA activity and structure.     

Biotransformation of Hydroxylaminobenzene and Aminophenol by Pseudomonas putida 2NP8 Cells Grown in the Presence of 3-Nitrophenol

Biotransformation products of hydroxylaminobenzene and aminophenol produced by 3-nitrophenol-grown cells of Pseudomonas putida 2NP8, a strain grown on 2- and 3-nitrophenol, were characterized. Ammonia, 2-aminophenol, 4-aminophenol, 4-benzoquinone, N-acetyl-4-aminophenol, N-acetyl-2-aminophenol, 2-aminophenoxazine-3-one, 4-hydroquinone, and catechol were produced from hydroxylaminobenzene. Ammonia, N-acetyl-2-aminophenol, and 2-aminophenoxazine-3-one were produced from 2-aminophenol. All of these metabolites were also found in the nitrobenzene transformation medium, and this demonstrated that they were metabolites of nitrobenzene transformation via hydroxylaminobenzene. Production of 2-aminophenoxazine-3-one indicated that oxidation of 2-aminophenol via imine occurred. Rapid release of ammonia from 2-aminophenol transformation indicated that hydrolysis of the imine intermediate was the dominant reaction. The low level of 2-aminophenoxazine-3-one indicated that formation of this compound was probably due to a spontaneous reaction accompanying oxidation of 2-aminophenol via imine. 4-Hydroquinone and catechol were reduction products of 2- and 4-benzoquinones. Based on these transformation products, we propose a new ammonia release pathway via oxidation of aminophenol to benzoquinone monoimine and subsequent hydrolysis for transformation of nitroaromatic compounds by 3-nitrophenol-grown cells of P. putida 2NP8. We propose a parallel mechanism for 3-nitrophenol degradation in P. putida 2NP8, in which all of the possible intermediates are postulated.     

Screening E3 Substrates Using a Live Phage Display Library

Ubiquitin ligases (E3s) determine specificity of ubiquitination by recognizing target substrates. However, most of their substrates are unknown. Most known substrates have been identified using distinct approaches in different laboratories. We developed a high-throughput strategy using a live phage display library as E3 substrates in in vitro screening. His-ubiquitinated phage, enriched with Ni-beads, could effectively infect E. coli for amplification. Sixteen natural potential substrates and many unnatural potential substrates of E3 MDM2 were identified through 4 independent screenings. Some substrates were identified in different independent experiments. Additionally, 10 of 12 selected candidates were ubiquitinated by MDM2 in vitro, and 3 novel substrates, DDX42, TP53RK and RPL36a were confirmed ex vivo. The whole strategy is rather simple and efficient. Non-degradation substrates can be discovered. This strategy can be extended to any E3s as long as the E3 does not ubiquitinate the empty phage.     

Construction of Escherichia coli strains for conversion of nitroacetophenones to ortho-aminophenols

The predominant bacterial pathway for nitrobenzene (NB) degradation uses an NB nitroreductase and hydroxylaminobenzene (HAB) mutase to form the ring-fission substrate ortho-aminophenol. We tested the hypothesis that constructed strains might accumulate the aminophenols from nitroacetophenones and other nitroaromatic compounds. We constructed a recombinant plasmid carrying NB nitroreductase (nbzA) and HAB mutase A (habA) genes, both from Pseudomonas pseudoalcaligenes JS45, and expressed the enzymes in Escherichia coli JS995. IPTG (isopropyl-beta-D-thiogalactopyranoside)-induced cells of strain JS995 rapidly and stoichiometrically converted NB to 2-aminophenol, 2-nitroacetophenone (2NAP) to 2-amino-3-hydroxyacetophenone (2AHAP), and 3-nitroacetophenone (3NAP) to 3-amino-2-hydroxyacetophenone (3AHAP). We constructed another recombinant plasmid containing the nitroreductase gene (nfs1) from Enterobacter cloacae and habA from strain JS45 and expressed the enzymes in E. coli JS996. Strain JS996 converted NB to 2-aminophenol, 2-nitrotoluene to 2-amino-3-methylphenol, 3-nitrotoluene to 2-amino-4-methylphenol, 4-nitrobiphenyl ether to 4-amino-5-phenoxyphenol, and 1-nitronaphthalene to 2-amino-1-naphthol. In larger-scale biotransformations catalyzed by strain JS995, 75% of the 2NAP transformed was converted to 2AHAP, whereas 3AHAP was produced stoichiometrically from 3NAP. The final yields of the aminophenols after extraction and recovery were >64%. The biocatalytic synthesis of ortho-aminophenols from nitroacetophenones suggests that strain JS995 may be useful in the biocatalytic production of a variety of substituted ortho-aminophenols from the corresponding nitroaromatic compounds.     

Metabolic engineering of β-oxidation to leverage thioesterases for production of 2-heptanone, 2-nonanone and 2-undecanone

Medium-chain length methyl ketones are potential blending fuels due to their cetane numbers and low melting temperatures. Biomanufacturing offers the potential to produce these molecules from renewable resources such as lignocellulosic biomass. In this work, we designed and tested metabolic pathways in Escherichia coli to specifically produce 2-heptanone, 2-nonanone and 2-undecanone. We achieved substantial production of each ketone by introducing chain-length specific acyl-ACP thioesterases, blocking the β-oxidation cycle at an advantageous reaction, and introducing active β-ketoacyl-CoA thioesterases. Using a bioprospecting approach, we identified fifteen homologs of E. coli β-ketoacyl-CoA thioesterase (FadM) and evaluated the in vivo activity of each against various chain length substrates. The FadM variant from Providencia sneebia produced the most 2-heptanone, 2-nonanone, and 2-undecanone, suggesting it has the highest activity on the corresponding β-ketoacyl-CoA substrates. We tested enzyme variants, including acyl-CoA oxidases, thiolases, and bi-functional 3-hydroxyacyl-CoA dehydratases to maximize conversion of fatty acids to β-keto acyl-CoAs for 2-heptanone, 2-nonanone, and 2-undecanone production. In order to address the issue of product loss during fermentation, we applied a 20% (v/v) dodecane layer in the bioreactor and built an external water cooling condenser connecting to the bioreactor heat-transferring condenser coupling to the condenser. Using these modifications, we were able to generate up to 4.4 g/L total medium-chain length methyl ketones.     

Comparison of bacteriocins production from Enterococcus faecium strains in cheese whey and optimised commercial MRS medium

The production of bacteriocins from cheap substrates could be useful for many food industrial applications. This study aimed at determining the conditions needed for optimal production of enterocins SD1, SD2, SD3 and SD4 secreted by Enterococcus faecium strains SD1, SD2, SD3 and SD4, respectively. To our knowledge, this is the first use of cheese whey—a low-cost milk by-product—as a substrate for bacteriocin production by E. faecium; skimmed milk and MRS broths were used as reference media. This cheese manufacturing residue proved to be a promising substrate for the production of bacteriocins. However, the levels of secreted antimicrobial compounds were lower than those achieved by E. faecium strains in MRS broth. Bacteriocin production was affected strongly by physical and chemical factors such as growth temperature, time of incubation, pH, and the chemical composition of the culture medium. The optimal temperature and time of incubation supporting the highest bacteriocin production was determined for each strain. Different types, sources and amounts of organic nitrogen, sugar, and inorganic salts played an essential role in bacteriocin secretion. E. faecium strains SD1 and SD2—producing high bacteriocin levels both in cheese whey and skimmed milk—could be of great interest for potential applications in cheese-making.     

Complex I-Associated Hydrogen Peroxide Production Is Decreased and Electron Transport Chain Enzyme Activities Are Altered in n-3 Enriched fat-1 Mice

The polyunsaturated nature of n-3 fatty acids makes them prone to oxidative damage. However, it is not clear if n-3 fatty acids are simply a passive site for oxidative attack or if they also modulate mitochondrial reactive oxygen species (ROS) production. The present study used fat-1 transgenic mice, that are capable of synthesizing n-3 fatty acids, to investigate the influence of increases in n-3 fatty acids and resultant decreases in the n-6∶n-3 ratio on liver mitochondrial H₂O₂ production and electron transport chain (ETC) activity. There was an increase in n-3 fatty acids and a decrease in the n-6∶n-3 ratio in liver mitochondria from the fat-1 compared to control mice. This change was largely due to alterations in the fatty acid composition of phosphatidylcholine and phosphatidylethanolamine, with only a small percentage of fatty acids in cardiolipin being altered in the fat-1 animals. The lipid changes in the fat-1 mice were associated with a decrease (p<0.05) in the activity of ETC complex I and increases (p<0.05) in the activities of complexes III and IV. Mitochondrial H₂O₂ production with either succinate or succinate/glutamate/malate substrates was also decreased (p<0.05) in the fat-1 mice. This change in H₂O₂ production was due to a decrease in ROS production from ETC complex I in the fat-1 animals. These results indicate that the fatty acid changes in fat-1 liver mitochondria may at least partially oppose oxidative stress by limiting ROS production from ETC complex I.     

Acyl chains of phospholipase d transphosphatidylation products in Arabidopsis cells: a study using multiple reaction monitoring mass spectrometry

Background

Phospholipases D (PLD) are major components of signalling pathways in plant responses to some stresses and hormones. The product of PLD activity is phosphatidic acid (PA). PAs with different acyl chains do not have the same protein targets, so to understand the signalling role of PLD it is essential to analyze the composition of its PA products in the presence and absence of an elicitor.

Methodology/Principal findings

Potential PLD substrates and products were studied in Arabidopsis thaliana suspension cells treated with or without the hormone salicylic acid (SA). As PA can be produced by enzymes other than PLD, we analyzed phosphatidylbutanol (PBut), which is specifically produced by PLD in the presence of n-butanol. The acyl chain compositions of PBut and the major glycerophospholipids were determined by multiple reaction monitoring (MRM) mass spectrometry. PBut profiles of untreated cells or cells treated with SA show an over-representation of 160/18∶2- and 16∶0/18∶3-species compared to those of phosphatidylcholine and phosphatidylethanolamine either from bulk lipid extracts or from purified membrane fractions. When microsomal PLDs were used in in vitro assays, the resulting PBut profile matched exactly that of the substrate provided. Therefore there is a mismatch between the acyl chain compositions of putative substrates and the in vivo products of PLDs that is unlikely to reflect any selectivity of PLDs for the acyl chains of substrates.

Conclusions

MRM mass spectrometry is a reliable technique to analyze PLD products. Our results suggest that PLD action in response to SA is not due to the production of a stress-specific molecular species, but that the level of PLD products per se is important. The over-representation of 160/18∶2- and 16∶0/18∶3-species in PLD products when compared to putative substrates might be related to a regulatory role of the heterogeneous distribution of glycerophospholipids in membrane sub-domains.     

Production of single cell protein from agro-waste using <Emphasis Type="Italic">Rhodococcus opacus</Emphasis>

Livestock and fish farming are rapidly growing industries facing the simultaneous pressure of increasing production demands and limited protein required to produce feed. Bacteria that can convert low-value non-food waste streams into singe cell protein (SCP) present an intriguing route for rapid protein production. The oleaginous bacterium Rhodococcus opacus serves as a model organism for understanding microbial lipid production. SCP production has not been explored using an organism from this genus. In the present research, R. opacus strains DSM 1069 and PD630 were fed three agro-waste streams: (1) orange pulp, juice, and peel; (2) lemon pulp, juice, and peel; and (3) corn stover effluent, to determine if these low-cost substrates would be suitable for producing a value-added product, SCP for aquafarming or livestock feed. Both strains used agro-waste carbon sources as a growth substrate to produce protein-rich cell biomass suggesting that that R. opacus can be used to produce SCP using agro-wastes as low-cost substrates.     

Artificial substrates enhance non-native macroalga and N2 production

The introduction of non-native species occurs within a context of other anthropogenic impacts: thus a holistic approach is needed to understand interactive effects. Installation of shoreline protection structures is increasing in response to rising sea levels and increasing frequency of intense storms. Shoreline hardening structures can facilitate establishment of non-native species with multiple potential consequences. We measured abundances of both native and non-native Codium, a green macroalga, on natural hard substrates (oyster reefs) and artificial substrates (bulkheads and revetments) in two estuaries and assessed the effects of each Codium species on nitrogen fixation and net N₂ fluxes. Native C. decorticatum was the most abundant (86 %) Codium on oyster reefs, while exotic C. fragile dominated (99 %) artificial substrates. N₂ production via denitrification was greater than nitrogen fixation for both species and the net N₂ production associated with non-native C. fragile was greater than with native C. decorticatum. Comparing our results with surveys conducted in the 1940s before C. fragile had invaded, indicates that non-native Codium has outcompeted native Codium on artificial substrates, but that natural substrate provided by oyster reefs remains a refuge for native Codium. Although shoreline hardening with artificial structures can reduce ecosystem services provided by coastal marsh and other habitats, an unexpected beneficial consequence was the N₂ production associated with the non-native Codium, which has the potential to mitigate anthropogenic nutrient loading. Our results illustrate that the interaction between multiple anthropogenic impacts can be positive, and indicate that non-ephemeral macroalgae could be an overlooked component in nitrogen removal from marine ecosystems by enhancing denitrification.