Hydrogenase activity of mineral-associated and suspended populations of Desulfovibrio desulfuricans Essex 6.

The interactions between sulfate-reducing microorganisms and iron oxides influence a number of important redox-sensitive biogeochemical processes including the formation of iron sulfides. Enzymes, such as hydrogenase which catalyze the reversible oxidation of molecular hydrogen, are known to mediate electron transfer to metals and may contribute to the formation and speciation of ferrous sulfides formed at the cell-mineral interface. In the present study, we compared the whole cell hydrogenase activity of Desulfovibrio desulfuricans strain Essex 6 growing as biofilms on hematite (hematite-associated) or as suspended populations using different metabolic pathways. Hematite-associated cells exhibited significantly greater hydrogenase activity than suspended populations during sulfate respiration but not during pyruvate fermentation. The enhanced activity of the hematite-associated, sulfate-grown cells appears to be dependent on iron availability rather than a general response to surface attachment since the activity of glass-associated cells did not differ from that of suspended populations. Hydrogenase activity of pyruvate-fermenting cells was stimulated by addition of iron as soluble Fe(II)Cl2 and, in the absence of added iron, both sulfate-reducing and pyruvate-fermenting cells displayed similar rates of hydrogenase activity. These data suggest that iron exerts a stronger influence on whole cell hydrogenase activity than either metabolic pathway or mode of growth. The location of hydrogenase to the cell envelope and the enhanced activity at the hematite surface in sulfate-reducing cells may influence the redox conditions that control the species of iron sulfides on the mineral surface.

High-level semi-synthetic production of the potent antimalarial artemisinin.

In 2010 there were more than 200 million cases of malaria, and at least 655,000 deaths. The World Health Organization has recommended artemisinin-based combination therapies (ACTs) for the treatment of uncomplicated malaria caused by the parasite Plasmodium falciparum. Artemisinin is a sesquiterpene endoperoxide with potent antimalarial properties, produced by the plant Artemisia annua. However, the supply of plant-derived artemisinin is unstable, resulting in shortages and price fluctuations, complicating production planning by ACT manufacturers. A stable source of affordable artemisinin is required. Here we use synthetic biology to develop strains of Saccharomyces cerevisiae (baker's yeast) for high-yielding biological production of artemisinic acid, a precursor of artemisinin. Previous attempts to produce commercially relevant concentrations of artemisinic acid were unsuccessful, allowing production of only 1.6 grams per litre of artemisinic acid. Here we demonstrate the complete biosynthetic pathway, including the discovery of a plant dehydrogenase and a second cytochrome that provide an efficient biosynthetic route to artemisinic acid, with fermentation titres of 25 grams per litre of artemisinic acid. Furthermore, we have developed a practical, efficient and scalable chemical process for the conversion of artemisinic acid to artemisinin using a chemical source of singlet oxygen, thus avoiding the need for specialized photochemical equipment. The strains and processes described here form the basis of a viable industrial process for the production of semi-synthetic artemisinin to stabilize the supply of artemisinin for derivatization into active pharmaceutical ingredients (for example, artesunate) for incorporation into ACTs. Because all intellectual property rights have been provided free of charge, this technology has the potential to increase provision of first-line antimalarial treatments to the developing world at a reduced average annual price.

The management of solitary tumours of Hoffa's fat pad.

Hoffa's fat pad (HFP) of the knee is affected by a variety of tumours and tumour-like conditions. HFP can be affected by diffuse or solitary, focal disease. This paper reports a consecutive series of 19 cases of solitary symptomatic HFP tumours. The commonest presenting symptom was anterior knee pain. All patients underwent open excision after diagnostic magnetic resonance imaging (MRI). Histology revealed varied diagnoses with the commonest being pigmented villonodular synovitis (PVNS) and ganglia. American Knee Society scores improved from 76 pre-operatively to 96 post-operatively with an improvement in functional scores from 92 to 100. In conclusion the majority of solitary HFP tumours are benign and may be either cystic or solid. MRI and plain radiographs are the imaging of choice. The definitive treatments of both cystic and solid tumours should be selective arthrotomy and excision biopsy. All patients in this series reported substantial improvement in symptoms following surgery.

Estimates of biogenic methane production rates in deep marine sediments at Hydrate Ridge, Cascadia margin.

Methane hydrate found in marine sediments is thought to contain gigaton quantities of methane and is considered an important potential fuel source and climate-forcing agent. Much of the methane in hydrates is biogenic, so models that predict the presence and distribution of hydrates require accurate rates of in situ methanogenesis. We estimated the in situ methanogenesis rates in Hydrate Ridge (HR) sediments by coupling experimentally derived minimal rates of methanogenesis to methanogen biomass determinations for discrete locations in the sediment column. When starved in a biomass recycle reactor, Methanoculleus submarinus produced ca. 0.017 fmol methane/cell/day. Quantitative PCR (QPCR) directed at the methyl coenzyme M reductase subunit A gene (mcrA) indicated that 75% of the HR sediments analyzed contained <1,000 methanogens/g. The highest numbers of methanogens were found mostly from sediments <10 m below seafloor. By considering methanogenesis rates for starved methanogens (adjusted to account for in situ temperatures) and the numbers of methanogens at selected depths, we derived an upper estimate of <4.25 fmol methane produced/g sediment/day for the samples with fewer methanogens than the QPCR method could detect. The actual rates could vary depending on the real number of methanogens and various seafloor parameters that influence microbial activity. However, our calculated rate is lower than rates previously reported for such sediments and close to the rate derived using geochemical modeling of the sediments. These data will help to improve models that predict microbial gas generation in marine sediments and determine the potential influence of this source of methane on the global carbon cycle.

Diversity of methanotroph communities in a basalt aquifer.

Methanotrophic bacteria play an important role in global cycling of carbon and co-metabolism of contaminants. Methanotrophs from pristine regions of the Snake River Plain Aquifer (SRPA; Idaho, USA) were studied in order to gain insight into the native groundwater communities' genetic potential to carry out TCE co-metabolism. Wells were selected that were proximal to a TCE plume believed to be undergoing natural attenuation. Methane concentrations ranged from 1 to >1000 nM. Carbon isotope ratios and diversity data together suggest that the SRPA contains active communities of methanotrophs that oxidize microbially produced methane. Microorganisms removed from groundwater by filtration were used as inocula for enrichments or frozen immediately and DNA was subsequently extracted for molecular characterization. Primers that specifically target methanotroph 16S rRNA genes or genes that code for subunits of soluble or particulate methane monooxygenase, mmoX and pmoA, respectively, were used to characterize the indigenous methanotrophs via PCR, cloning, RFLP analysis, and sequencing. Type I methanotroph clones aligned with Methylomonas, Methylocaldum, and Methylobacter sequences and a distinct 16S rRNA phylogenetic lineage grouped near Methylobacter. The majority of clone sequences in type II methanotroph 16S rRNA, pmoA, and mmoX gene libraries grouped closely with sequences in the Methylocystis genus. A subset of the type II methanotroph clones from the aquifer had sequences that aligned most closely to Methylosinus trichosporium OB3b and Methylocystis spp., known TCE-co-metabolizing methanotrophs.

H(2)O(2)-forming NADH oxidase with diaphorase (cytochrome) activity from Archaeoglobus fulgidus.

An enzyme exhibiting NADH oxidase (diaphorase) activity was isolated from the hyperthermophilic sulfate-reducing anaerobe Archaeoglobus fulgidus. N-terminal sequence of the protein indicates that it is coded for by open reading frame AF0395 in the A. fulgidus genome. The gene AF0395 was cloned and its product was purified from Escherichia coli. Like the native NADH oxidase (NoxA2), the recombinant NoxA2 (rNoxA2) has an apparent molecular mass of 47 kDa, requires flavin adenine dinucleotide for activity, has NADH-specific activity, and is thermostable. Hydrogen peroxide is the product of bivalent oxygen reduction by rNoxA2 with NADH. The rNoxA2 is an oxidase with diaphorase activity in the presence of electron acceptors such as tetrazolium and cytochrome c. During purification NoxA2 remains associated with the enzyme responsible for D-lactate oxidation, the D-lactate dehydrogenase (Dld), and the genes encoding NoxA2 and Dld are in the same transcription unit. Together these results suggest that NADH oxidase may be involved in electron transfer reactions resulting in sulfate respiration.

Probing the mechanism of a cyanobacterial Delta9 fatty acid desaturase from Spirulina platensis C1 (Arthrospira sp. PCC 9438).

The initial and rate determining step in the mechanism of fatty acid desaturases has been proposed to be breakage of one of the C&z.sbnd;H bonds at the site of the incipient double bond. This has been investigated and supported for a number of eukaryotic fatty acid desaturases through the use of kinetic isotope effect experiments with deuterated substrates. In order to probe the reaction catalyzed by the cyanobacterial Delta9 desaturase and compare it to the eukaryotic desaturases, the desC gene of Spirulina platensis, strain C1 (Arthrospira sp. PCC 9438) was expressed in a desaturase mutant of baker's yeast. Kinetic isotope effects were performed by culturing yeast transformants with deuterated thia-substituted stearic acids. A large kinetic isotope effect was found for the 9 position, in qualitative agreement with results from eukaryotic desaturases.

Rapid micro-assay of camptothecin in Camptotheca acuminata.

A micro-assay has been developed to extract and rapidly quantify the anticancer alkaloid, camptothecin (CPT), from two leaf disks of Camptotheca acuminata Decaisne (Nyssaceae). This assay utilizes thin-layer chromatography in conjunction with fluorescence imaging to obtain reproducible measurements in the nanogram range. A large number of trees can be screened using this procedure to identify high producers of CPT in a relatively short period of time.

Identification and analysis of a gene from Calendula officinalis encoding a fatty acid conjugase.

Two homologous cDNAs, CoFad2 and CoFac2, were isolated from a Calendula officinalis developing seed by a polymerase chain reaction-based cloning strategy. Both sequences share similarity to FAD2 desaturases and FAD2-related enzymes. In C. officinalis plants CoFad2 was expressed in all tissues tested, whereas CoFac2 expression was specific to developing seeds. Expression of CoFad2 cDNA in yeast (Saccharomyces cerevisiae) indicated it encodes a Delta12 desaturase that introduces a double bond at the 12 position of 16:1(9Z) and 18:1(9Z). Expression of CoFac2 in yeast revealed that the encoded enzyme acts as a fatty acid conjugase converting 18:2(9Z, 12Z) to calendic acid 18:3(8E, 10E, 12Z). The enzyme also has weak activity on the mono-unsaturates 16:1(9Z) and 18:1(9Z) producing compounds with the properties of 8,10 conjugated dienes.

Characterization of the regiochemistry and cryptoregiochemistry of a Caenorhabditis elegans fatty acid desaturase (FAT-1) expressed in Saccharomyces cerevisiae.

To characterize the fatty acid desaturase produced by the fat-1 gene from the nematode Caenorhabditis elegans, the functional expression of this enzyme was effected in the yeast Saccharomyces cerevisiae. The GC-MS analysis of desaturated products derived from various fatty acids, including deuterium-labeled thia fatty acids supplied to growing cultures of transformed yeast, has defined the substrate requirements, regiochemistry, and cryptoregiochemistry of the enzyme. The desaturase acts on substrates of 16-20 carbons with a preference for omega-6 fatty acids, and its regioselectivity was confirmed to be that of an omega-3 desaturase. (omega-x refers to a double bond or desaturation between carbons x and x+1, counting from the methyl end of a fatty acid.) The primary deuterium kinetic isotope effects (KIEs) at C-15 and C-16 of a C18 fatty acid analogue were measured via competitive incubation experiments: While k(H)/k(D) at the omega-3 position was shown to be large (7.8 +/- 0.4), essentially no KIE at the omega-2 position was observed (k(H)/k(D) = 0.99 +/- 0.04). This result indicates that omega-3 desaturation is initiated by an energetically difficult C-H bond cleavage at the carbon closer to the carboxyl terminus. The results are discussed in the context of a general model relating the structure and function of membrane-bound fatty acid desaturases featuring differing regioselectivities.

Characterization of the Brassica napus extraplastidial linoleate desaturase by expression in Saccharomyces cerevisiae.

The substrate specificity and regioselectivity of the Brassica napus extraplastidial linoleate desaturase (FAD3) was investigated in vivo in a heterologous expression system. A strain of the yeast Saccharomyces cerevisiae producing the plant enzyme was constructed and cultured in media containing a variety of fatty acids. The products of desaturation of these potential substrates were determined by gas chromatographic and mass spectrometric analysis of the yeast cultures. The results indicate that the enzyme has: (a) omega-3, as opposed to Delta-15 or double-bond-related regioselectivity, (b) the ability to desaturate substrates in the 16 to 22 carbon range, (c) a preference for substrates with omega-6 double bonds, but the ability to desaturate substrates with omega-6 hydroxyl groups or omega-9 or omega-5 double bonds, and (d) a relative insensitivity to double bonds proximal to the carboxyl end of the substrate.

Substrate specificity, regioselectivity and cryptoregiochemistry of plant and animal omega-3 fatty acid desaturases.

In order to define the substrate requirements, regiochemistry and cryptoregiochemistry of the omega-3 fatty acid desaturases involved in polyunsaturated fatty acid formation, the genes Fad3 and fat-1 from Brassica napus and the nematode Caenorhabditis elegans respectively were expressed in baker's yeast (Saccharomyces cerevisiae). Various fatty acids, including deuterium-labelled thia-fatty acids, were supplied to growing cultures of transformed yeast. The results from GC-MS analysis of the desaturated products indicate that both the plant and animal desaturases act on unsaturated substrates of 16-20 carbons with a preference for omega-6-unsaturated fatty acids. The regioselectivities of both enzymes were confirmed to be that of omega-3 desaturases. The primary deuterium kinetic isotope effects at C-15 and C-16 of a C(18) fatty acid analogue were measured via competitive incubation experiments. Whereas k(H)/k(D) at the omega-3 position was shown to be large, essentially no kinetic isotope effect at the omega-2 position was observed for the plant or the nematode enzymes. These results indicate that omega-3 desaturation is initiated by an energetically difficult C-H bond cleavage at the carbon closer to the carboxyl terminus. These results will be discussed in the context of a general model relating the structure and function of membrane-bound fatty acid desaturases featuring different regioselectivities.

The Archaeoglobus fulgidus D-lactate dehydrogenase is a Zn(2+) flavoprotein.

Archaeoglobus fulgidus, a hyperthermophilic, archaeal sulfate reducer, is one of the few organisms that can utilize D-lactate as a sole source for both carbon and electrons. The A. fulgidus open reading frame, AF0394, which is predicted to encode a D-(-)-lactate dehydrogenase (Dld), was cloned, and its product was expressed in Escherichia coli as a fusion with the maltose binding protein (MBP). The 90-kDa MBP-Dld fusion protein was more efficiently expressed in E. coli when coexpressed with the E. coli dnaY gene, encoding the arginyl tRNA for the codons AGA and AGG. When cleaved from the fusion protein by treatment with factor Xa, the recombinant Dld (rDld) has an apparent molecular mass of 50 kDa, similar to that of the native A. fulgidus Dld enzyme. Both the purified MBP-Dld fusion protein and its rDld cleavage fragment have lactate dehydrogenase activities specific for D-lactate, are stable at 80 degrees C, and retain activity after exposure to oxygen. The flavin cofactor FAD, which binds rDld apoprotein with a 1:1 stoichiometry, is essential for activity.

An example of intron junctional sliding in the gene families encoding squalene monooxygenase homologues in Arabidopsis thaliana and Brassica napus.

Sequences of three Arabidopsis thaliana and two Brassica napus cDNAs encoding squalene monooxygenase homologues (Sqp1 and Sqp2) are reported. Southern analysis confirmed that these cDNAs are derived from small gene families in both species. Expression analysis indicates that Sqp1 genes in B. napus are strongly expressed in leaves but not roots or developing seeds. Comparison of cDNA and genomic sequences indicate that the 3' splice site of an intron in these genes has undergone junctional sliding. The evolutionary significance of this phenomenon is discussed.

Comparison of soleus muscles from rats exposed to microgravity for 10 versus 14 days.

The effects of two different duration space-flights on the extent of atrophy, fiber type composition, and myosin heavy chain (MHC) content of rat soleus muscles were compared. Adult male Fisher rats (n=12) were aboard flight STS-57 and exposed to 10 days of microgravity and adult ovariectomized female Spraque-Dawley rats (n=12) were aboard flight STS-62 for 14 days. Soleus muscles were bilaterally removed from the flight and control animals and frozen for subsequent analyses. Muscle wet weights, fiber types (I, IC, IIC, and IIA), cross-sectional area, and MHC content were determined. Although a significant difference was found between the soleus wet weights of the two ground-based control groups, they were similar with regard to MHC content (ca 90% MHCI and ca 10% MHCIIa) and fiber type composition. Unloading of the muscles caused slow-to-fast transformations which included a decrease in the percentage of type I fibers and MHCI, an increase in fibers classified as type IC, and the expression of two fast myosin heavy chains not found in the control rat soleus muscles (MHCIId and MHCIIb). Although the amount of atrophy (ca 26%) and the extent of slow-to-fast transformation (decrease in the percentage of MHCI from 90% to 82.5%) in the soleus muscles were similar between the two spaceflights, the percentages of the fast MHCs differed. After 14 days of spaceflight, the percentage of MHCIIa was significantly lower and the percentages of MHCIId and MHCIIb were significantly higher than the corresponding MHC content of the soleus muscles from the 10-day animals. Indeed, MHCIId became the predominant fast MHC after 14 days in space. These data suggest fast-to-faster transformations continued during the longer spaceflight.

Acetyl-CoA decarbonylase/synthase complex from Archaeoglobus fulgidus.

The acetyl-CoA decarbonylase/synthase (ACDS) multienzyme complex catalyzes the reversible cleavage and synthesis of acetyl-CoA in methanogens. This report of the enzyme complex in Archaeoglobus fulgidus demonstrates the existence of a functional ACDS complex in an organism that is not a methanogen. The A. fulgidus enzyme complex contained five subunits of 89, 72, 50, 49.5, and 18.5 kDa, and it catalyzed the overall synthesis of acetyl-CoA according to the following reaction: CO2 + 2 Fdred(Fe2+) + 2 H+ + CH3 - H4SPt + CoA <==> acetyl-CoA + H4SPt + 2 Fdox(Fe3+) + H2O where Fd is ferredoxin, and CH3-H4SPt and H4SPt denote N5-methyl-tetrahydrosarcinapterin and tetrahydrosarcinapterin, respectively.

Metabolism of Hydroxy Fatty Acids in Developing Seeds in the Genera Lesquerella (Brassicaceae) and Linum (Linaceae).

Members of the genus Lesquerella produce seed oil that contains a high proportion of hydroxy fatty acids (HFAs). There are three groups of Lesquerella species that are distinguished by their most abundant seed oil fatty acid: lesquerolic acid (20:1OH; e.g. Lesquerella fendleri), densipolic acid (18:2OH; e.g. Lesquerella kathryn), and auricolic acid (20:2OH; e.g. Lesquerella auriculata). To investigate the biochemistry of HFA production in Lesquerella species, the conversion of putative radiolabeled intermediates of HFA biosynthesis, including 18:1, 20:1,18:1OH, 18:2OH, and 20:1OH, was examined in developing embryos of L. fendleri, L.kathryn, and L. auriculata. The results are consistent with (a) 18:1OH formation by hydroxylation of 18:1, (b) elongation and desaturation of 18:1OH to produce 20:1OH and 18:2OH, respectively, and (c) desaturation of 20:1OH to produce 20:2OH. The desaturation of 20:1OH was also found to occur in developing embryos of high, but not low, linolenic acid flax. This suggests that the desaturation is catalyzed by the extraplastidial linoleate desaturase. Confirming this suggestion, it was notable that 18:1OH and 18:2OH were found in low and high linolenic flax (Linum usitatissimum) seeds, respectively, at levels of 0.2 to 1%.

In vivo and in vitro expression of a non-mammalian cyclooxygenase-1.

Unlike cyclooxygenase 2 (COX-2), COX-1 has never been identified, purified or cloned in a non-mammalian species. Here we report the RT-PCR cloning of a chicken cDNA that encodes the amphipathic membrane binding region and parts of the dimerization and catalytic domains of COX-1-like enzyme. Sequence comparison showed this putative COX-1 to be evolutionarily less conserved than COX-2. Furthermore, whereas COX-1 in mammals is broadly expressed in tissues as a constitutive enzyme, the mRNA detected by our clone in chicken was almost absent in tissues and embryo fibroblasts (CEF). Highest expression was in brain and seminal vesicle. This transcript was not detectable during chick embryogenesis and, as is the case for mammalian COX-1, was not induced above background by mitogen stimulation. The identification of an avian COX-1 shows that COX-1 and COX-2 existed as separate catalysts for prostaglandin synthesis before the divergence of birds and mammals.

Functional expression of the extraplastidial Arabidopsis thaliana oleate desaturase gene (FAD2) in Saccharomyces cerevisiae.

The functional expression in yeast of the Arabidopsis thaliana FAD2 gene, encoding the extraplastidial oleate desaturase (1-acyl-2-oleoyl-sn-glycero-3-phosphocholine delta 12-desaturase) is reported. Dienoic fatty acids constituted up to 11% (w/w) of the total fatty acids in transformed Saccharomyces cerevisiae cells and were confirmed to be linoleic acid and delta 9, delta 12-hexadecadienoic acid by gas chromatography-mass spectrometry.