Identification of 3-Sulfinopropionyl Coenzyme A (CoA) Desulfinases within the Acyl-CoA Dehydrogenase Superfamily

In a previous study, the essential role of 3-sulfinopropionyl coenzyme A (3SP-CoA) desulfinase acyl-CoA dehydrogenase (Acd) in Advenella mimigardefordensis strain DPN7^T (Acd_DPN7) during degradation of 3,3′-dithiodipropionic acid (DTDP) was elucidated. DTDP is a sulfur-containing precursor substrate for biosynthesis of polythioesters (PTEs). Acd_DPN7 showed high amino acid sequence similarity to acyl-CoA dehydrogenases but was unable to catalyze a dehydrogenation reaction. Hence, it was investigated in the present study whether 3SP-CoA desulfinase activity is an uncommon or a widespread property within the acyl-CoA dehydrogenase superfamily. Therefore, proteins of the acyl-CoA dehydrogenase superfamily from Advenella kashmirensis WT001, Bacillus cereus DSM31, Cupriavidus necator N-1, Escherichia coli BL21, Pseudomonas putida KT2440, Burkholderia xenovorans LB400, Ralstonia eutropha H16, Variovorax paradoxus B4, Variovorax paradoxus S110, and Variovorax paradoxus TBEA6 were expressed in E. coli strains. All purified acyl-CoA dehydrogenases appeared as homotetramers, as revealed by size exclusion chromatography. Acd_S110, Acd_B4, Acd_H16, and Acd_KT2440 were able to dehydrogenate isobutyryl-CoA. Acd_KT2440 additionally dehydrogenated butyryl-CoA and valeryl-CoA, whereas Acd_DSM31 dehydrogenated only butyryl-CoA and valeryl-CoA. No dehydrogenation reactions were observed with propionyl-CoA, isovaleryl-CoA, succinyl-CoA, and glutaryl-CoA for any of the investigated acyl-CoA dehydrogenases. Only Acd_TBEA6, Acd_N-1, and Acd_LB400 desulfinated 3SP-CoA and were thus identified as 3SP-CoA desulfinases within the acyl-CoA dehydrogenase family, although none of these three Acds dehydrogenated any of the tested acyl-CoA thioesters. No appropriate substrates were identified for Acd_BL21 and Acd_WT001. Spectrophotometric assays provided apparent K_m and V_max values for active substrates and indicated the applicability of phylogenetic analyses to predict the substrate range of uncharacterized acyl-CoA dehydrogenases. Furthermore, C. necator N-1 was found to utilize 3SP as the sole source of carbon and energy.     

Succinyl-CoA:3-Sulfinopropionate CoA-Transferase from Variovorax paradoxus Strain TBEA6, a Novel Member of the Class III Coenzyme A (CoA)-Transferase Family

The act gene of Variovorax paradoxus TBEA6 encodes a succinyl-CoA:3-sulfinopropionate coenzyme A (CoA)-transferase, Act_TBEA6 (2.8.3.x), which catalyzes the activation of 3-sulfinopropionate (3SP), an intermediate during 3,3′-thiodipropionate (TDP) degradation. In a previous study, accumulation of 3SP was observed in a Tn5::mob-induced mutant defective in growth on TDP. In contrast to the wild type and all other obtained mutants, this mutant showed no growth when 3SP was applied as the sole source of carbon and energy. The transposon Tn5::mob was inserted in a gene showing high homology to class III CoA-transferases. In the present study, analyses of the translation product clearly allocated Act_TBEA6 to this protein family. The predicted secondary structure indicates the lack of a C-terminal α-helix. Act_TBEA6 was heterologously expressed in Escherichia coli Lemo21(DE3) and was then purified by Ni-nitrilotriacetic acid (NTA) affinity chromatography. Analytical size exclusion chromatography revealed a homodimeric structure with a molecular mass of 96 ± 3 kDa. Enzyme assays identified succinyl-CoA, itaconyl-CoA, and glutaryl-CoA as potential CoA donors and unequivocally verified the conversion of 3SP to 3SP-CoA. Kinetic studies revealed an apparent V_max of 44.6 μmol min⁻¹ mg⁻¹ for succinyl-CoA, which corresponds to a turnover number of 36.0 s⁻¹ per subunit of Act_TBEA6. For 3SP, the apparent V_max was determined as 46.8 μmol min⁻¹ mg⁻¹, which corresponds to a turnover number of 37.7 s⁻¹ per subunit of Act_TBEA6. The apparent K_m values were 0.08 mM for succinyl-CoA and 5.9 mM for 3SP. Nonetheless, the V. paradoxus Δact mutant did not reproduce the phenotype of the Tn5::mob-induced mutant. This defined deletion mutant was able to utilize TDP or 3SP as the sole carbon source, like the wild type. Complementation of the Tn5::mob-induced mutant with pBBR1MCS5::acd_DPN7 partially restored growth on 3SP, which indicated a polar effect of the Tn5::mob transposon on acd_TBEA6, located downstream of act_TBEA6.     

Dihydrolipoamide Dehydrogenases of Advenella mimigardefordensis and Ralstonia eutropha Catalyze Cleavage of 3,3′-Dithiodipropionic Acid into 3-Mercaptopropionic Acid

The catabolism of the disulfide 3,3′-dithiodipropionic acid (DTDP) is initiated by the reduction of its disulfide bond. Three independent Tn5::mob-induced mutants of Advenella mimigardefordensis strain DPN7^T were isolated that had lost the ability to utilize DTDP as the sole source of carbon and energy and that harbored the transposon insertions in three different sites of the same dihydrolipoamide dehydrogenase gene encoding the E3 subunit of the pyruvate dehydrogenase multi-enzyme complex of this bacterium (LpdA_Am). LpdA_Am was analyzed in silico and compared to homologous proteins, thereby revealing high similarities to the orthologue in Ralstonia eutropha H16 (PdhL_Re). Both bacteria are able to cleave DTDP into two molecules of 3-mercaptopropionic acid (3MP). A. mimigardefordensis DPN7^T converted 3MP to 3-sulfinopropionic acid, whereas R. eutropha H16 showed no growth with DTDP as the sole carbon source but was instead capable of synthesizing heteropolythioesters using the resulting cleavage product 3MP. Subsequently, the genes lpdA_Am and pdhL_Re were cloned, heterologously expressed in Escherichia coli applying the pET23a expression system, purified, and assayed by monitoring the oxidation of NADH. The physiological substrate lipoamide was reduced to dihydrolipoamide with specific activities of 1,833 mkat/kg of protein (LpdA_Am) or 1,667 mkat/kg of protein (PdhL_Re). Reduction of DTDP was also unequivocally detected with the purified enzymes, although the specific enzyme activities were much lower: 0.7 and 0.5 mkat/kg protein, respectively.In Advenella mimigardefordensis strain DPN7^T (15, 42), three independent mutants with an insertion of Tn5::mob in the lpdA gene coding for the E3 component of the pyruvate dehydrogenase multi-enzyme complex revealed an interesting phenotype: these mutants were fully impaired in utilizing 3,3′-dithiodipropionic acid (DTDP) as the sole carbon and energy source, whereas the growth on no other tested carbon sources was affected (41). Our main interest in the catabolism of DTDP is to unravel the pathway and to identify the involved enzymes. Furthermore, the application of this disulfide as precursor substrate for biotechnological production of polythioesters (PTE) (22) is of interest. Since poly(3-mercaptopropionate) (PMP) biosynthesis depends hitherto on supplying the harmful thiol 3-mercaptopropionic acid (3MP) (35), an improvement of the recombinant Escherichia coli system by heterologous expression of enzymes capable of cleaving the less toxic DTDP symmetrically into two molecules of 3MP, which are then polymerized, could be an important achievement toward large-scale biotechnological production of PMP.Two different enzyme systems catalyzing the conversion of disulfides into the corresponding thiols are already known and have been described in detail. (i) Enzymes belonging to the well-characterized family of pyridine-nucleotide disulfide oxidoreductases (25) contain a redox center formed by a disulfide bridge coupled to a flavin ring. They catalyze a simultaneous two-electron transfer via the enzymatic active disulfides associated with the pyridine nucleotides and flavin, toward the substrate (39, 40). (ii) An alternative disulfide reduction is catalyzed by enzymes using iron-sulfur clusters to cleave of disulfide substrates in two one-electron reduction steps (37). The disrupted gene in A. mimigardefordensis was designated lpdA_Am (EC 1.8.1.4), and it encodes a homodimeric flavoprotein, the dihydrolipoamide dehydrogenase LpdA_Am (i.e., the E3 component of the pyruvate dehydrogenase multi-enzyme complex of A. mimigardefordensis strain DPN7^T) belonging to the above-mentioned family of pyridine nucleotide-disulfide oxidoreductases. Enzymes of this class share high sequence and structural similarities and catalyze reduction of compounds which are linked by disulfide bonds (38). Alkylhydroperoxide reductases, coenzyme A disulfide reductases, glutathione reductases, mycothione reductases, thioredoxin reductases, and trypanothione reductases also, in addition to dihydrolipoamide dehydrogenases, belong to this family (3, 38). The physiological function of LpdA_Am is most probably the conversion of lipoamide to dihydrolipoamide, but the reduction of DTDP into two molecules of 3MP (Fig. ​(Fig.1)1) is also predicted, enabling the first step in DTDP catabolism in A. mimigardefordensis strain DPN7^T (41).Open in a separate windowFIG. 1.Reactions catalyzed by LpdA_Am and PdhL_Re. Presented are the enzymatic conversions of DTDP into two molecules of 3MP (A), lipoamide into dihydrolipoamide (B), and DTNB into two molecules of NTB (C). Abbreviations: DTDP, 3,3′-dithiodipropionic acid; 3MP, 3-mercaptopropionic acid; DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid); NTB, 2-nitro-5-thiobenzoic acid.Ralstonia eutropha H16 synthesizes copolymers of 3-hydroxybutyrate and 3MP, if 3MP (23) or DTDP (22) is supplied as a precursor in addition to a second utilizable carbon source. Although R. eutropha is not able to grow with DTDP as the sole carbon source, it must be capable of cleaving this organic disulfide symmetrically, because it synthesizes from it heteropolymers containing the resulting 3MP. Thus, R. eutropha must possess at least one gene encoding a DTDP-cleaving enzyme. Five genes coding for homologues of a dihydrolipoamide dehydrogenase (DHLDH), which in A. mimigardefordensis DPN7^T is obviously involved in DTDP degradation, are known to exist in the genome of R. eutropha H16 (27; M. Raberg, J. Bechmann, U. Brandt, J. Schlüter, B. Uischner, and A. Steinbüchel, unpublished data). Therefore, LpdA_Am and the five DHLDH paralogues of R. eutropha H16 were aligned and compared (Fig. ​(Fig.2).2). Subsequently, lpdA_Am and the gene encoding the DHLDH belonging to the pyruvate dehydrogenase complex of R. eutropha H16 (pdhL_Re) were cloned, heterologously expressed in Escherichia coli, purified, and assayed.Open in a separate windowFIG. 2.Phylogenetic relationships of the A. mimigardefordensis strain DPN7^T LpdA (boldface), R. eutropha H16 PdhL (boldface), and homologues. The neighbor-joining plot was derived from a CLUSTAL X alignment of amino acid sequences closely related to LpdA_Am. The amino acid sequence of the outer membrane protein P64K from Neisseria meningitidis was used as the outgroup. GenBank accession numbers are given in parentheses. Scale bar, 10% sequence divergence.     

Water-relation Parameters of Individual Mesophyll Cells of the Crassulacean Acid Metabolism Plant Kalanchoë daigremontiana

Water-relation parameters of leaf mesophyll cells of the CAM plant Kalanchoë daigremontiana have been determined directly in cells of tissue slices using the pressure-probe technique. Turgor pressures measured in cells of the second to fourth layer from the cut surface showed an average of 1.82 ± 0.62 bar (mean ± sd; n = 157 cells). This was lower than expected from measurements of the osmotic pressure of the cell sap. The half-time (T_1/2) for water-flux equilibration of individual cells was 2.5 to 8.8 seconds. This is the fastest T_1/2 found so far for higher-plant cells. The calculated values of the hydraulic conductivity were in the range of 0.20 to 1.6 × 10⁻⁵ centimeters second⁻¹ bar⁻¹, with an average of (0.69 ± 0.46) × 10⁻⁵ centimeters second⁻¹ bar⁻¹ (mean ± sd; n = 8 cells). The T_1/2 values of water exchange of individual cells are consistent with the overall rates of water-flux equilibration measured for tissue slices.The volumetric elastic moduli (∈) of individual cells were in the range 13 to 128 bar for turgor pressures between 0.0 and 3.4 bar; the average ∈ value was 42.4 ± 27.7 bar (mean ± sd; n = 21 cells). This ∈ value is similar to that observed for other higher-plant cells.The water-storage capacity of individual cells, calculated as C_c = V/(∈ + πⁱ) (where V = cell volume and πⁱ = internal osmotic pressure) was 9.1 × 10⁻⁹ cubic centimeters bar⁻¹ per cell, and the capacity for the tissue was 2.2 × 10⁻² cubic centimeters bar⁻¹ gram⁻¹ fresh weight. The significance of the water-relation parameters determined at the cellular level is discussed in terms of the water relations of whole leaves and the high water-use efficiency characteristic of CAM plants.     

Novel pathway for catabolism of the organic sulfur compound 3,3'-dithiodipropionic acid via 3-mercaptopropionic acid and 3-Sulfinopropionic acid to propionyl-coenzyme A by the aerobic bacterium Tetrathiobacter mimigardefordensis strain DPN7

The hitherto unstudied microbial degradation of the organic disulfide 3,3'-dithiodipropionic acid (DTDP) was investigated with the recently described bacterium Tetrathiobacter mimigardefordensis strain DPN7(T) (DSM 17166(T); LMG 22922(T)), which is able to use DTDP as the sole carbon source for growth. 3-Mercaptopropionic acid (3MP) and 3-sulfinopropionic acid (3SP) were detected in the growth medium and occurred as intermediates during DTDP degradation. To identify genes coding for enzymes of DTDP catabolism, Tn5::mob-induced mutants of T. mimigardefordensis were generated. Screening of transposon mutant libraries yielded many mutants fully or partially impaired in utilizing DTDP as a carbon source. Mapping of the insertion loci in some mutants identified four disrupted open reading frames (ORFs) with putative metabolic functions. The ORFs were assigned function on the basis of homologies with lpdA (EC 1.8.1.4), cdo (EC 1.13.11.20), sucCD (EC 6.2.1.5), and acnB (EC 4.2.1.3). Tn5::mob insertions occurred additionally in the vicinity of heat shock protein-encoding genes. The predicted function of the LpdA homologue in T. mimigardefordensis is cleavage of the disulfide bond of DTDP to form two molecules of 3MP. Cdo catalyzes the conversion of the sulfhydryl group of 3MP, yielding the corresponding sulfinic acid, 3SP. SucCD exhibits thiokinase activity, ligating coenzyme A (CoA) with 3SP to form 3SP-CoA. Afterwards, an elimination of sulfite via a putative desulfinase is expected. acnB encodes a putative 2-methylisocitrate dehydratase. Therefore, a new pathway is proposed for the catabolism of DTDP via 3MP, 3SP, and 3SP-CoA toward propionyl-CoA, which is then further catabolized via the 2-methylcitric acid cycle in T. mimigardefordensis.     

Methanocella conradii sp. nov., a thermophilic, obligate hydrogenotrophic methanogen, isolated from Chinese rice field soil

Background

Methanocellales contributes significantly to anthropogenic methane emissions that cause global warming, but few pure cultures for Methanocellales are available to permit subsequent laboratory studies (physiology, biochemistry, etc.).

Methodology/Principal Findings

By combining anaerobic culture and molecular techniques, a novel thermophilic methanogen, strain HZ254^T was isolated from a Chinese rice field soil located in Hangzhou, China. The phylogenetic analyses of both the 16S rRNA gene and mcrA gene (encoding the α subunit of methyl-coenzyme M reductase) confirmed its affiliation with Methanocellales, and Methanocella paludicola SANAE^T was the most closely related species. Cells were non-motile rods, albeit with a flagellum, 1.4–2.8 µm long and by 0.2–0.3 µm in width. They grew at 37–60°C (optimally at 55°C) and salinity of 0–5 g NaCl l⁻¹ (optimally at 0–1 g NaCl l⁻¹). The pH range for growth was 6.4–7.2 (optimum 6.8). Under the optimum growth condition, the doubling time was 6.5–7.8 h, which is the shortest ever observed in Methanocellales. Strain HZ254^T utilized H₂/CO₂ but not formate for growth and methane production. The DNA G+C content of this organism was 52.7 mol%. The sequence identities of 16S rRNA gene and mcrA gene between strain HZ254^T and SANAE^T were 95.0 and 87.5% respectively, and the genome based Average Nucleotide Identity value between them was 74.8%. These two strains differed in phenotypic features with regard to substrate utilization, possession of a flagellum, doubling time (under optimal conditions), NaCl and temperature ranges. Taking account of the phenotypic and phylogenetic characteristics, we propose strain HZ254^T as a representative of a novel species, Methanocella conradii sp. nov. The type strain is HZ254^T ( = CGMCC 1.5162^T = JCM 17849^T = DSM 24694^T).

Conclusions/Significance

Strain HZ254^T could potentially serve as an excellent laboratory model for studying Methanocellales due to its fast growth and consistent cultivability.     

Burkholderia dabaoshanensis sp. nov., a Heavy-Metal-Tolerant Bacteria Isolated from Dabaoshan Mining Area Soil in China

Heavy-metal-tolerant bacteria, GIMN1.004^T, was isolated from mine soils of Dabaoshan in South China, which were acidic (pH 2–4) and polluted with heavy metals. The isolation was Gram-negative, aerobic, non-spore-forming, and rod-shaped bacteria having a cellular width of 0.5−0.6 µm and a length of 1.3−1.8 µm. They showed a normal growth pattern at pH 4.0–9.0 in a temperature ranging from 5°C to 40°C.The organism contained ubiquinone Q-8 as the predominant isoprenoid quinine, and C_16∶0, summed feature 8 (C_18∶1 ω7c and C_18∶1 ω6c), C_18∶0, summed feature 3 (C_16∶1 ω7c or iso-C_15∶0 2-OH), C_17∶0 cyclo, C_18∶1 ω9c, C_19∶0 cyclo ω8c, C_14∶0 as major fatty acid. These profiles were similar to those reported for Burkholderia species. The DNA G+C % of this strain was 61.6%. Based on the similarity to 16S rRNA gene sequence, GIMN1.004^T was considered to be in the genus Burkholderia. The similarities of 16S rRNA gene sequence between strain GIMN1.004^T and members of the genus Burkholderia were 96−99.4%, indicating that this novel strain was phylogenetically related to members of that genus. The novel strain showed the highest sequence similarities to Burkholderia soli DSM 18235^T (99.4%); Levels of DNA-DNA hybridization with DSM 18235^T was 25%. Physiological and biochemical tests including cell wall composition analysis, differentiated phenotype of this strain from that closely related Burkholderia species. The isolation had great tolerance to cadmium with MIC of 22 mmol/L, and adsorbability of 144.94 mg/g cadmium,and it was found to exhibit antibiotic resistance characteristics. The adsorptive mechanism of GIMN1.004^T for cadmium depended on the action of the amide,carboxy and phosphate of cell surface and producing low-molecular-weight (LMW ) organic acids to complex or chelated Cd²⁺.Therefore, the strain GIMN1.004^T represented a new cadmium resistance species, which was tentatively named as Burkholderia dabaoshanensis sp. nov. The strain type is GIMN1.004^T ( = CCTCC M 209109^T =  NRRL B-59553^T ).     

Relative Bioavailability of Chlorothiazide from Mucoadhesive Compacts in Pigs

The relative bioavailability of chlorothiazide from mucoadhesive polymeric compacts is compared to commercial oral suspension in pigs. A single-dose randomized study was conducted in 12 healthy pigs that are 9–10 weeks old. After overnight fasting, pigs were divided into two groups of six animals. To the first group, a reference product containing 50 mg of chlorothiazide suspension, and in the second group, test product (mucoadhesive compacts) chlorothiazide (50 mg) was administered with 75 mL of water via gastric tubes. Blood samples were collected between 0 to 24 h using catheters inserted into the jugular vein. Plasma was separated by protein precipitation, and chlorothiazide concentrations were determined using a high-performance liquid chromatography method. The mean T_max and the C_max of chlorothiazide following the administration of oral suspension and mucoadhesive compacts were 0.58 ± 0.20 h and 682.97 ± 415.69 ng/mL and 2.17 ± 0.98 h and 99.42 ± 124.08 ng/mL, respectively. The K_el and T_1/2 of chlorothiazide were found to be 1.06 ± 0.28 h⁻¹ and 0.70 ± 0.21 h from suspension and 0.95 ± 1.11 h⁻¹ and 2.05 ± 1.90 h from the compacts, respectively. The T_max of mucoadhesive compacts were significantly longer (p < 0.05; 2.17 h) than the reference products (0.58 h), whereas the C_max of compacts were significantly lower (99 ng/mL) than the reference product (683 ng/mL; p < 0.05). The area under the curve (AUC) of compacts accounts only 50.15% (404.32 ± 449.93 ng h/mL) of the reference product’s AUC (806.27 ± 395.97 ng h/mL). The relative bioavailability of the compacts was lower than that of the suspension, and this may be due to the narrow window of absorption for chlorothiazide.Key words: bioavailability, chlorothiazide, mucoadhesive compacts, pigs     

Fat Oxidation,Fitness and Skeletal Muscle Expression of Oxidative/Lipid Metabolism Genes in South Asians: Implications for Insulin Resistance?

Background

South Asians are more insulin resistant than Europeans, which cannot be fully explained by differences in adiposity. We investigated whether differences in oxidative capacity and capacity for fatty acid utilisation in South Asians might contribute, using a range of whole-body and skeletal muscle measures.

Methodology/Principal Findings

Twenty men of South Asian ethnic origin and 20 age and BMI-matched men of white European descent underwent exercise and metabolic testing and provided a muscle biopsy to determine expression of oxidative and lipid metabolism genes and of insulin signalling proteins. In analyses adjusted for age, BMI, fat mass and physical activity, South Asians, compared to Europeans, exhibited; reduced insulin sensitivity by 26% (p = 0.010); lower VO_2max (40.6±6.6 vs 52.4±5.7 ml.kg⁻¹.min⁻¹, p = 0.001); and reduced fat oxidation during submaximal exercise at the same relative (3.77±2.02 vs 6.55±2.60 mg.kg⁻¹.min⁻¹ at 55% VO_2max, p = 0.013), and absolute (3.46±2.20 vs 6.00±1.93 mg.kg⁻¹.min⁻¹ at 25 ml O₂.kg⁻¹.min⁻¹, p = 0.021), exercise intensities. South Asians exhibited significantly higher skeletal muscle gene expression of CPT1A and FASN and significantly lower skeletal muscle protein expression of PI3K and PKB Ser473 phosphorylation. Fat oxidation during submaximal exercise and VO_2max both correlated significantly with insulin sensitivity index and PKB Ser473 phosphorylation, with VO_2max or fat oxidation during exercise explaining 10–13% of the variance in insulin sensitivity index, independent of age, body composition and physical activity.

Conclusions/Significance

These data indicate that reduced oxidative capacity and capacity for fatty acid utilisation at the whole body level are key features of the insulin resistant phenotype observed in South Asians, but that this is not the consequence of reduced skeletal muscle expression of oxidative and lipid metabolism genes.     

Specificity of RSG-1.2 peptide binding to RRE-IIB RNA element of HIV-1 over Rev peptide is mainly enthalpic in origin

Rev is an essential HIV-1 regulatory protein which binds to the Rev responsive element (RRE) present within the env gene of HIV-1 RNA genome. This binding facilitates the transport of the RNA to the cytoplasm, which in turn triggers the switch between viral latency and active viral replication. Essential components of this complex have been localized to a minimal arginine rich Rev peptide and stem IIB region of RRE. A synthetic peptide known as RSG-1.2 binds with high binding affinity and specificity to the RRE-IIB than the Rev peptide, however the thermodynamic basis of this specificity has not yet been addressed. The present study aims to probe the thermodynamic origin of this specificity of RSG-1.2 over Rev Peptide for RRE-IIB. The temperature dependent melting studies show that RSG-1.2 binding stabilizes the RRE structure significantly (ΔT _m = 4.3°C), in contrast to Rev binding. Interestingly the thermodynamic signatures of the binding have also been found to be different for both the peptides. At pH 7.5, RSG-1.2 binds RRE-IIB with a K_a = 16.2±0.6×10⁷ M⁻¹ where enthalpic change ΔH = −13.9±0.1 kcal/mol is the main driving force with limited unfavorable contribution from entropic change TΔS = −2.8±0.1 kcal/mol. A large part of ΔH may be due to specific stacking between U72 and Arg15. In contrast binding of Rev (K_a = 3.1±0.4×10⁷ M⁻¹) is driven mainly by entropy (ΔH = 0 kcal/mol and TΔS = 10.2±0.2 kcal/mol) which arises from major conformational changes in the RNA upon binding.     

Carbohydrate uptake in Advenella mimigardefordensis strain DPN7T is mediated by periplasmic sugar oxidation and a TRAP‐transport system

In this study, we investigated an SBP (DctP_Am) of a tripartite ATP‐independent periplasmic transport system (TRAP) in Advenella mimigardefordensis strain DPN7^T. Deletion of dctP_Am as well as of the two transmembrane compounds of the tripartite transporter, dctQ and dctM, impaired growth of A. mimigardefordensis strain DPN7^T, if cultivated on mineral salt medium supplemented with d ‐glucose, d ‐galactose, l ‐arabinose, d ‐fucose, d ‐xylose or d ‐gluconic acid, respectively. The wild type phenotype was restored during complementation studies of A. mimigardefordensis ΔdctP_Am using the broad host vector pBBR1MCS‐5::dctP_Am. Furthermore, an uptake assay with radiolabeled [¹⁴C(U)]‐d ‐glucose clearly showed that the deletion of dctP_Am, dctQ and dctM, respectively, disabled the uptake of this aldoses in cells of either mutant strain. Determination of K_D performing thermal shift assays showed a shift in the melting temperature of DctP_Am in the presence of d ‐gluconic acid (K_D 11.76 ± 1.3 µM) and the corresponding aldonic acids to the above‐mentioned carbohydrates d ‐galactonate (K_D 10.72 ± 1.4 µM), d ‐fuconic acid (K_D 13.50 ± 1.6 µM) and d ‐xylonic acid (K_D 8.44 ± 1.0 µM). The sugar (glucose) dehydrogenase activity (E.C.1.1.5.2) in the membrane fraction was shown for all relevant sugars, proving oxidation of the molecules in the periplasm, prior to transport.     

Floral thermogenesis of three species of Hydnora (Hydnoraceae) in Africa

Background and Aims

Floral thermogenesis occurs in at least 12 families of ancient seed plants. Some species show very high rates of respiration through the alternative pathway, and some are thermoregulatory, with increasing respiration at decreasing ambient temperature. This study assesses the intensity and regulation of respiration in three species of African Hydnora that represent the Hydnoraceae, an unusual family of holoparasitic plants from arid environments.

Methods

Long-term respirometry (CO₂ production) and thermometry were carried out on intact flowers of H. africana, H. abyssinica and H. esculenta in the field, and short-term measurements were made on floral parts during the protogynous flowering sequence.

Key Results

For H. africana, there was no temperature elevation in either the osmophores or the gynoecial chamber in any phase, and mass-specific respiration rates of the flower parts were low (maximum 8·3 nmol CO₂ g⁻¹ s⁻¹ in osmophore tissue). Respiration tracked ambient and floral temperatures, eliminating the possibility of the inverse relationship expected in thermoregulatory flowers. Hydnora abyssinica flowers had higher respiration (maximum 27·5 nmol g⁻¹ s⁻¹ in the osmophores) and a slight elevation of osmophore temperature (maximum 2·8 °C) in the female stage. Respiration by gynoecial tissue was similar to that of osmophores in both species, but there was no measurable elevation of gynoecial chamber temperature. Gynoecial chamber temperature of H. esculenta could reach 3·8 °C above ambient, but there are no respiration data available. Antheral tissue respiration was maximal in the male phase (4·8 nmol g⁻¹ s⁻¹ in H. africana and 10·3 nmol g⁻¹ s⁻¹ in H. abyssinica), but it did not raise the antheral ring temperature, which showed that thermogenesis is not a by-product of pollen maturation or release.

Conclusions

The exceptionally low thermogenesis in Hydnora appears to be associated with scent production and possibly gynoecial development, but has little direct benefit to beetle pollinators.Key words: Pollination biology, Hydnora, thermogenesis, respiration rate, temperature, flowers, insects     

Temporal changes of soil physic‐chemical properties at different soil depths during larch afforestation by multivariate analysis of covariance

Soil physic-chemical properties differ at different depths; however, differences in afforestation-induced temporal changes at different soil depths are seldom reported. By examining 19 parameters, the temporal changes and their interactions with soil depth in a large chronosequence dataset (159 plots; 636 profiles; 2544 samples) of larch plantations were checked by multivariate analysis of covariance (MANCOVA). No linear temporal changes were found in 9 parameters (N, K, N:P, available forms of N, P, K and ratios of N: available N, P: available P and K: available K), while marked linear changes were found in the rest 10 parameters. Four of them showed divergent temporal changes between surface and deep soils. At surface soils, changing rates were 262.1 g·kg⁻¹·year⁻¹ for SOM, 438.9 mg·g⁻¹·year⁻¹ for C:P, 5.3 mg·g⁻¹·year⁻¹ for C:K, and −3.23 mg·cm⁻³·year⁻¹ for bulk density, while contrary tendencies were found in deeper soils. These divergences resulted in much moderated or no changes in the overall 80-cm soil profile. The other six parameters showed significant temporal changes for overall 0–80-cm soil profile (P: −4.10 mg·kg⁻¹·year⁻¹; pH: −0.0061 unit·year⁻¹; C:N: 167.1 mg·g⁻¹·year⁻¹; K:P: 371.5 mg·g⁻¹ year⁻¹; N:K: −0.242 mg·g⁻¹·year⁻¹; EC: 0.169 μS·cm⁻¹·year⁻¹), but without significant differences at different soil depths (P > 0.05). Our findings highlight the importance of deep soils in studying physic-chemical changes of soil properties, and the temporal changes occurred in both surface and deep soils should be fully considered for forest management and soil nutrient balance.     

Establishing zebrafish as a novel exercise model: swimming economy, swimming-enhanced growth and muscle growth marker gene expression

Background

Zebrafish has been largely accepted as a vertebrate multidisciplinary model but its usefulness as a model for exercise physiology has been hampered by the scarce knowledge on its swimming economy, optimal swimming speeds and cost of transport. Therefore, we have performed individual and group-wise swimming experiments to quantify swimming economy and to demonstrate the exercise effects on growth in adult zebrafish.

Methodology/Principal Findings

Individual zebrafish (n = 10) were able to swim at a critical swimming speed (U_crit) of 0.548±0.007 m s⁻¹ or 18.0 standard body lengths (BL) s⁻¹. The optimal swimming speed (U_opt) at which energetic efficiency is highest was 0.396±0.019 m s⁻¹ (13.0 BL s⁻¹) corresponding to 72.26±0.29% of U_crit. The cost of transport at optimal swimming speed (COT_opt) was 25.23±4.03 µmol g⁻¹ m⁻¹. A group-wise experiment was conducted with zebrafish (n = 83) swimming at U_opt for 6 h day⁻¹ for 5 days week⁻¹ for 4 weeks vs. zebrafish (n = 84) that rested during this period. Swimming zebrafish increased their total body length by 5.6% and body weight by 41.1% as compared to resting fish. For the first time, a highly significant exercise-induced growth is demonstrated in adult zebrafish. Expression analysis of a set of muscle growth marker genes revealed clear regulatory roles in relation to swimming-enhanced growth for genes such as growth hormone receptor b (ghrb), insulin-like growth factor 1 receptor a (igf1ra), troponin C (stnnc), slow myosin heavy chain 1 (smyhc1), troponin I2 (tnni2), myosin heavy polypeptide 2 (myhz2) and myostatin (mstnb).

Conclusions/Significance

From the results of our study we can conclude that zebrafish can be used as an exercise model for enhanced growth, with implications in basic, biomedical and applied sciences, such as aquaculture.     

In vitro propagation of female Ephedra foliata Boiss. & Kotschy ex Boiss.: an endemic and threatened Gymnosperm of the Thar Desert

Ephedra foliata Boiss. & Kotschy ex Boiss., (family – Ephedraceae), is an ecologically and economically important threatened Gymnosperm of the Indian Thar Desert. A method for micropropagation of E. foliata using nodal explant of mature female plant has been developed. Maximum bud-break (90 %) of the explant was obtained on MS medium supplemented with 1.5 mg l⁻¹ of benzyl adenine (BA) + additives. Explant produces 5.3 ± 0.40 shoots from single node with 3.25 ± 0.29 cm length. The multiplication of shoots in culture was affected by salt composition of media, types and concentrations of plant growth regulators (PGR’s) and their interactions, time of transfer of the cultures. Maximum number of shoots (26.3 ± 0.82 per culture vessel) were regenerated on MS medium modified by reducing the concentration of nitrates to half supplemented with 200 mg l⁻¹ ammonium sulphate {(NH₄) ₂SO₄} (MMS3) + BA (0.25 mg l⁻¹), Kinetin (Kin; 0.25 mg l⁻¹), Indole-3-acetic acid (IAA; 0.1 mg l⁻¹) and additives. The in vitro produced shoots rooted under ex vitro on soilrite moistened with one-fourth strength of MS macro salts in screw cap bottles by treating the shoot base (s) with 500 mg l⁻¹ of Indole-3-butyric acid (IBA) for 5 min. The micropropagated plants were hardened in the green house. The described protocol can be applicable for (i) large scale plant production (ii) establishment of plants in natural habitat and (iii) germplasm conservation of this endemic Gymnosperm of arid regions.     

Formation of the Stable Structural Analog of ADP-sensitive Phosphoenzyme of Ca2+-ATPase with Occluded Ca2+ by Beryllium Fluoride: STRUCTURAL CHANGES DURING PHOSPHORYLATION AND ISOMERIZATION*

As a stable analog for ADP-sensitive phosphorylated intermediate of sarcoplasmic reticulum Ca²⁺-ATPase E1PCa₂·Mg, a complex of E1Ca₂·BeF_x, was successfully developed by addition of beryllium fluoride and Mg²⁺ to the Ca²⁺-bound state, E1Ca₂. In E1Ca₂·BeF_x, most probably E1Ca₂·BeF₃⁻, two Ca²⁺ are occluded at high affinity transport sites, its formation required Mg²⁺ binding at the catalytic site, and ADP decomposed it to E1Ca₂, as in E1PCa₂·Mg. Organization of cytoplasmic domains in E1Ca₂·BeF_x was revealed to be intermediate between those in E1Ca₂·AlF₄⁻ ADP (transition state of E1PCa₂ formation) and E2·BeF₃⁻·(ADP-insensitive phosphorylated intermediate E2P·Mg). Trinitrophenyl-AMP (TNP-AMP) formed a very fluorescent (superfluorescent) complex with E1Ca₂·BeF_x in contrast to no superfluorescence of TNP-AMP bound to E1Ca₂·AlF_x. E1Ca₂·BeF_x with bound TNP-AMP slowly decayed to E1Ca₂, being distinct from the superfluorescent complex of TNP-AMP with E2·BeF₃⁻, which was stable. Tryptophan fluorescence revealed that the transmembrane structure of E1Ca₂·BeF_x mimics E1PCa₂·Mg, and between those of E1Ca₂·AlF₄⁻·ADP and E2·BeF₃⁻. E1Ca₂·BeF_x at low 50–100 μm Ca²⁺ was converted slowly to E2·BeF₃⁻ releasing Ca²⁺, mimicking E1PCa₂·Mg → E2P·Mg + 2Ca²⁺. Ca²⁺ replacement of Mg²⁺ at the catalytic site at approximately millimolar high Ca²⁺ decomposed E1Ca₂·BeF_x to E1Ca₂. Notably, E1Ca₂·BeF_x was perfectly stabilized for at least 12 days by 0.7 mm lumenal Ca²⁺ with 15 mm Mg²⁺. Also, stable E1Ca₂·BeF_x was produced from E2·BeF₃⁻ at 0.7 mm lumenal Ca²⁺ by binding two Ca²⁺ to lumenally oriented low affinity transport sites, as mimicking the reverse conversion E2P· Mg + 2Ca²⁺ → E1PCa₂·Mg.Sarcoplasmic reticulum Ca²⁺-ATPase (SERCA1a),² a representative member of the P-type ion transporting ATPases, catalyze Ca²⁺ transport coupled with ATP hydrolysis (Fig. 1) (1–9). The enzyme forms phosphorylated intermediates from ATP or P_i in the presence of Mg²⁺ (10–13). In the transport cycle, the enzyme is first activated by cooperative binding of two Ca²⁺ ions at high affinity transport sites (E2 to E1Ca₂, steps 1–2) (14) and autophosphorylated at Asp³⁵¹ with MgATP to form the ADP-sensitive phosphoenzyme (E1P, step 3), which reacts with ADP to regenerate ATP in the reverse reaction. Upon this E1P formation, the two bound Ca²⁺ are occluded in the transport sites (E1PCa₂). Subsequent isomeric transition to the ADP-insensitive form (E2PCa₂), i.e. loss of ADP sensitivity at the catalytic site, results in rearrangement of the Ca²⁺ binding sites to deocclude Ca²⁺, reduce the affinity, and open the lumenal gate, thus releasing Ca²⁺ into the lumen (E2P, steps 4–5). Finally Asp³⁵¹-acylphosphate in E2P is hydrolyzed to form the Ca²⁺-unbound inactive E2 state (steps 6 and 7). Mg²⁺ bound at the catalytic site is required as a physiological catalytic cofactor in phosphorylation and dephosphorylation and thus for the transport cycle. The cycle is totally reversible, e.g. E2P can be formed from P_i in the presence of Mg²⁺ and absence of Ca²⁺, and subsequent Ca²⁺ binding at lumenally oriented low affinity transport sites of E2P reverses the Ca²⁺-releasing step and produces E1PCa₂, which is then decomposed to E1Ca₂ by ADP.Open in a separate windowFIGURE 1.Ca²⁺ transport cycle of Ca²⁺-ATPase.Various intermediate structural states in the transport cycle were fixed as their structural analogs produced by appropriate ligands such as AMP-PCP (non-hydrolyzable ATP analog) or metal fluoride compounds (phosphate analogs), and their crystal structures were solved at the atomic level (15–22). The three cytoplasmic domains, N, P, and A, largely move and change their organization state during the transport cycle, and the changes are coupled with changes in the transport sites. Most remarkably, in the change from E1Ca₂·AlF₄⁻·ADP (the transition state for E1PCa₂ formation, E1PCa₂·ADP·Mg^‡) to E2·BeF₃⁻ (the ground state E2P·Mg) (23–25), the A domain largely rotates by more than 90° approximately parallel to the membrane plane and associates with the P domain, thereby destroying the Ca²⁺ binding sites, and opening the lumenal gate, thus releasing Ca²⁺ into the lumen (see Fig. 2). E1PCa₂·Ca·AMP-PN formed by CaAMP-PNP without Mg²⁺ is nearly the same as E1Ca₂·AlF₄⁻·ADP and E1Ca₂·CaAMP-PCP in their crystal structures (17, 18, 22).Open in a separate windowFIGURE 2.Structure of SERCA1a and its change during processing of phosphorylated intermediate. E1Ca₂·AlF₄⁻·ADP (the transition state analog for phosphorylation E1PCa₂·ADP·Mg^‡) and E2·BeF₃⁻ (the ground state E2P analog (25)) were obtained from the Protein Data Bank (PDB accession code 1T5T (17) and 2ZBE (21), respectively). Cytoplasmic domains N (nucleotide binding), P (phosphorylation), and A (actuator), and 10 transmembrane helices (M1–M10) are indicated. The arrows on the domains, M1′ and M2 (Tyr¹²²) in E1Ca₂·AlF₄⁻·ADP, indicate their approximate motions predicted for E1PCa₂·ADP·Mg^‡ → E2P·Mg. The phosphorylation site Asp³⁵¹, TGES¹⁸⁴ of the A domain, Arg¹⁹⁸ (tryptic T2 site) on the Val²⁰⁰ loop (DPR¹⁹⁸AV²⁰⁰NQD) of the A domain, and Thr²⁴² (proteinase K site) on the A/M3-linker are shown. Seven hydrophobic residues gather in the E2P state to form the Tyr¹²²-hydrophobic cluster (Y122-HC); Tyr¹²²/Leu¹¹⁹ on the top part of M2, Ile¹⁷⁹/Leu¹⁸⁰/Ile²³² of the A domain, and Val⁷⁰⁵/Val⁷²⁶ of the P domain. The overall structure of E1Ca₂·AlF₄⁻·ADP is virtually the same as those of E1Ca₂·CaAMP-PCP and E1PCa₂·Ca·AMP-PN (17, 18, 22).Despite these atomic structures, yet unsolved is the structure of E1PCa₂·Mg, the genuine physiological intermediate E1PCa₂ with bound Mg²⁺ at the catalytic site without the nucleotide. Its stable structural analog has yet to be developed. E1PCa₂·Mg is the major intermediate accumulating almost exclusively at steady state under physiological conditions. Its rate-limiting isomerization results in Ca²⁺ deocclusion/release producing E2P·Mg as a key event for Ca²⁺ transport. In E1Ca₂·CaAMP-PCP, E1Ca₂·AlF₄⁻·ADP, and E1PCa₂·Ca·AMP-PN, the N and P domains are cross-linked and strongly stabilized by the bound nucleotide and/or Ca²⁺ at the catalytic site, thus they are crystallized (17, 18, 22). Kinetically, E1PCa₂·Ca formed with CaATP is markedly stabilized due to Ca²⁺ binding at the catalytic Mg²⁺ site, and its isomerization to E2P is strongly retarded in contrast to E1PCa₂·Mg (26, 27). Thus, the bound Ca²⁺ at the catalytic Mg²⁺ site likely produces a significantly different structural state from that with bound Mg²⁺.Therefore, it is now essential to develop a genuine E1PCa₂·Mg analog without bound nucleotide and thereby gain further insight into the structural mechanism in the Ca²⁺ transport process. It is also crucial to further clarify the structural importance of Mg²⁺ as the physiological catalytic cation. In this study, we successfully developed the complex E1Ca₂·BeF_x, most probably E1Ca₂·BeF₃⁻, as the E1PCa₂·Mg analog by adding beryllium fluoride (BeF_x) to the E1Ca₂ state without any nucleotides. For its formation, Mg²⁺ binding at the catalytic site was required and Ca²⁺ substitution for Mg²⁺ was absolutely unfavorable, revealing a likely structural reason for its preference as the physiological cofactor. In E1Ca₂·BeF₃⁻, two Ca²⁺ ions bound at the high affinity transport sites are occluded. It was also produced from E2·BeF₃⁻ by lumenal Ca²⁺ binding at the lumenally oriented low affinity transport sites, mimicking E2P·Mg + 2Ca²⁺ → E1PCa₂·Mg. All properties of the newly developed E1Ca₂·BeF₃⁻ fulfilled the requirements as the E1PCa₂·Mg analog, and hence we were able to uncover the hitherto unknown nature of E1PCa₂·Mg as well as structural events occurring in the phosphorylation and isomerization processes. Also, we successfully found the conditions that perfectly stabilize the E1Ca₂·BeF₃⁻ complex.     

Crystal structure of Archaeoglobus fulgidus CTP:inositol-1-phosphate cytidylyltransferase, a key enzyme for di-myo-inositol-phosphate synthesis in (hyper)thermophiles

Many Archaea and Bacteria isolated from hot, marine environments accumulate di-myo-inositol-phosphate (DIP), primarily in response to heat stress. The biosynthesis of this compatible solute involves the activation of inositol to CDP-inositol via the action of a recently discovered CTP:inositol-1-phosphate cytidylyltransferase (IPCT) activity. In most cases, IPCT is part of a bifunctional enzyme comprising two domains: a cytoplasmic domain with IPCT activity and a membrane domain catalyzing the synthesis of di-myo-inositol-1,3′-phosphate-1′-phosphate from CDP-inositol and l-myo-inositol phosphate. Herein, we describe the first X-ray structure of the IPCT domain of the bifunctional enzyme from the hyperthermophilic archaeon Archaeoglobus fulgidus DSMZ 7324. The structure of the enzyme in the apo form was solved to a 1.9-Å resolution. The enzyme exhibited apparent K_m values of 0.9 and 0.6 mM for inositol-1-phosphate and CTP, respectively. The optimal temperature for catalysis was in the range 90 to 95°C, and the V_max determined at 90°C was 62.9 μmol · min⁻¹ · mg of protein⁻¹. The structure of IPCT is composed of a central seven-stranded mixed β-sheet, of which six β-strands are parallel, surrounded by six α-helices, a fold reminiscent of the dinucleotide-binding Rossmann fold. The enzyme shares structural homology with other pyrophosphorylases showing the canonical motif G-X-G-T-(R/S)-X₄-P-K. CTP, l-myo-inositol-1-phosphate, and CDP-inositol were docked into the catalytic site, which provided insights into the binding mode and high specificity of the enzyme for CTP. This work is an important step toward the final goal of understanding the full catalytic route for DIP synthesis in the native, bifunctional enzyme.     

HCO3− Fixation by Naturally Occurring Tufts and Pure Cultures of Thiothrix nivea

Naturally occurring tufts of the mixotroph Thiothrix nivea blanketed the East Everglades (Dade County, Fla.) Chekika artesian well and runoff areas. The rate of HCO₃⁻ fixation by these Thiothrix tufts was determined to be 14.0 ± 5.4 nmol of HCO₃⁻ per min per mg of dry weight, which reflected a growth rate of 5.0%/h. The addition of 10 mM glucose, ribose, acetate, or pyruvate or 0.05% Casamino Acids (Difco Laboratories, Detroit, Mich.) did not appear to alter the HCO₃⁻ fixation rate. Whereas 1 mM acetate or 10 mM lactate, ethanol, glycerol, α-ketoglutarate, succinate, fumarate, or citrate slightly stimulated HCO₃⁻ fixation, 5 to 10 mM malate inhibited HCO₃⁻ fixation by 90%. Pure Thiothrix cultures isolated from Chekika fixed HCO₃⁻ at rates as high as 29.9 ± 2.8 nmol of HCO₃⁻ per min per mg of dry weight in the presence of growth medium. Malate did not have a suppressive effect but rather slightly stimulated in vivo HCO₃⁻ fixation.     

Kinetic and Thermodynamic Characterization of Glucoamylase from Colletotrichum sp. KCP1

Extracellular glucoamylase of Colletotrichum sp. KCP1 produced through solid state fermentation was purified by two steps purification process comprising ammonium sulphate precipitation followed by gel permeation chromatography (GPC). The Recovery of glucoamylase after GPC was 50.40 % with 19.3-fold increase in specific activity. The molecular weight of enzyme was found to be 162.18 kDa by native-PAGE and was dimeric protein of two sub-units with molecular weight of 94.62 and 67.60 kDa as determined by SDS-PAGE. Activation energy for starch hydrolysis was 26.45 kJ mol⁻¹ while temperature quotient (Q₁₀) was found to be 1.9. The enzyme was found to be stable over wide pH range and thermally stable at 40–50 °C up to 120 min while exhibited maximum activity at 50 °C with pH 5.0. The pKa₁ and pKa₂ of ionisable groups of active site controlling V_max were 3.5 and 6.8, respectively. V_max, K_m and K_cat for starch hydrolysis were found to be 58.82 U ml⁻¹, 1.17 mg (starch) ml⁻¹ and 449 s⁻¹, respectively. Activation energy for irreversible inactivation (E_a(d)) of glucoamylase was 74.85 kJ mol⁻¹. Thermodynamic parameters of irreversible inactivation of glucoamylase and starch hydrolysis were also determined.