Family 19 chitinase from rice (Oryza sativa L.): substrate-binding subsites demonstrated by kinetic and molecular modeling studies

A family 19 chitinase (OsChia1c, class I) from rice, Oryza sativa L., and its chitin-binding domain-truncated mutant (OsChia1cCBD, class II) were produced by the Pichia expression system, and the hydrolytic mechanism toward N-acetylglucosamine hexasaccharide [(GlcNAc)₆] was investigated by HPLC analysis of the reaction products. The profile of the time-course of (GlcNAc)₆ degradation obtained by OsChia1c was identical to that obtained by OsChia1cCBD, indicating that the chitin-binding domain does not significantly participate in oligosaccharide hydrolysis. From the theoretical analysis of the reaction time-course of OsChia1cCBD, the free energy changes of sugar residue binding were estimated to be –0.4, –4.7, +3.4, –0.5, –2.3, and –1.0 kcal/mol for the individual subsites of (–3), (–2), (–1), (+1), (+2), and (+3), respectively. The hexasaccharide substrate appears to bind to the enzyme through interactions at the high-affinity sites, (–2) and (+2), and the sugar residues at both ends more loosely bind to the corresponding subsites, (–3) and (+3). The docking study of (GlcNAc)₆ with the modeled structure of OsChia1cCBD supported the subsite structure estimated from the experimental time-course of hexasaccharide degradation. Since the class II chitinase from barley seeds was reported to possess a similar subsite structure from (–3) to (+3) and a similar free energy distribution, substrate-binding mode of plant chitinases of this class would be similar to each other.     

Transport of phenylalanine into vacuoles isolated from barley mesophyll protoplasts

The energy-dependent transport of phenylalanine into isolated vacuoles of barley (Hordeum vulgare L.) mesophyll protoplasts has been studied by silicone-layer floatation filtering. The uptake of this aromatic amino acid into the vacuolar compartment is markedly increased by MgATP, showing saturation kinetics; the K _m values were 0.5 mM for MgATP and 1.2 mM for phenylalanine. V _max for phenylalanine transport was estimated to 140 nmol phenylalanine·(mg·Chl)^-1·h^-1. The transport shows a distinct pH optimum at 7.3 and is markedly inhibited by 40 mM nitrate. Azide (1 mM) and vanadate (400 M) had no or little effect on rates of transport while p-fluorophenylalanine seemed to be an effective inhibitor, indicating a possible competition at an amino-acid carrier. Ionophores such as valinomycin, nigericin or gramicidin were strong inhibitors of phenylalanine transport, indicating that this process is coupled to both the transmembrane pH gradient (pH) and the transmembrane potential ().Abbreviations and symbols BSA bovine serum albumin - Chl chlorophyll - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - pH transmembrane pH gradient - transmembrane potential     

Xanthophyll biosynthesis in chromoplasts: isolation and molecular cloning of an enzyme catalyzing the conversion of 5,6-epoxycarotenoid into ketocarotenoid

The C-terminal propeptide of tobacco (Nicotiana tabacum) chitinase A has been shown to be necessary and sufficient for targeting of chitinases to the plant vacuole. The sequence specificity of this vacuolar targeting peptide (VTP) has now been analysed using transient expression of chitinases in Nicotiana plumbaginifolia protoplasts. An extracellular cucumber chitinase, previously used as a secreted reporter protein in transgenic tobacco, was also secreted into the incubation medium by the transiently transformed protoplasts. Addition of six to seven amino acids at the C-terminus to generate the VTP of tobacco chitinase A were sufficient to cause retention of most of the cucumber chitinase within the protoplasts. The chitinase A itself, as well as a mutant lacking the N-terminal chitin-binding domain, were retained to 80% in the protoplasts when low concentrations of the plasmid were used in the transient expression system. At high concentrations of plasmid, causing high levels of transiently expressed chitinase, retention was reduced, indicating saturation of the sorting system. Deletion of the C-terminal methionine did not affect the intracellular location, but deletion of even a single internal amino acid of the VTP caused predominantly secretion of tobacco chitinase A. In contrast, exchanges of amino acids in the VTP as well as substitution of the VTP with random sequences had intermediary effects that covered the whole range from retention to secretion. The results suggest that the sorting system responsible for the diversion of secretory proteins to the vacuole has a low specificity for the sequence of C-terminal targeting peptides, and that sequence changes in the VTP allow a gradual transition from vacuolar retention to secretion.     

The green fluorescent protein targets secretory proteins to the yeast vacuole

The green fluorescent protein (GFP) was used as a marker to study the intracellular transport of vacuolar and secretory proteins in yeast. Therefore, the following gene constructs were expressed in Saccharomyces cerevisiae under control of the GAL1 promoter: GFP N-terminally fused to the yeast secretory invertase (INV-GFP), the plant vacuolar chitinase (CHN-GFP) and its secretory derivative (CHNDeltaVTP-GFP), which did not contain the vacuolar targeting peptide (VTP), both chitinase forms (CHN and CHNDeltaVTP), GFP without any targeting information and two secretory GFP variants with and without the VTP of chitinase (N-GFP-V and N-GFP). Whereas chitinase without VTP is accumulated in the culture medium the other gene products are retained inside the cell up to 48 h of induction. Independently of a known VTP they are transported to the vacuole, so far as they contain a signal peptide for entering the endoplasmic reticulum. This was demonstrated by confocal laser scanning microscopy, immunocytochemical analysis and subcellular fractionation experiments as well. The transport of the GFP fusion proteins is temporary delayed by a transient accumulation in electron-dense structures very likely derived from the ER, because they also contain the ER chaperone Kar2p/Bip. Our results demonstrate that GFP directs secretory proteins without VTP to the yeast vacuole, possibly by the recognition of an unknown vacuolar signal and demonstrates, therefore, a first limitation for the application of GFP as a marker for the secretory pathway in yeast.     

Substrate specificity and antifungal activity of recombinant tobacco class I chitinases

Endochitinases contribute to the defence response of plants against chitin-containing pathogens. The vacuolar class I chitinases consist of an N-terminal cysteine-rich domain (CRD) linked by a glycine-threonine-rich spacer with 4-hydroxylated prolyl residues to the catalytic domain. We examined the functional role of the CRD and spacer region in class I chitinases by comparing wild-type chitinase A (CHN A) of Nicotiana tabacum with informative recombinant forms. The chitinases were expressed in transgenic N. sylvestris plants, purified to near homogeneity, and their structures confirmed by mass spectrometry and partial sequencing. The enzymes were tested for their substrate preference towards chitin, lipo-chitooligosaccharide Nod factors of Rhizobium, and bacterial peptidoglycans (lysozyme activity) as well as for their capacity to inhibit hyphal growth of Trichoderma viride. Deletion of the CRD and spacer alone or in combination resulted in a modest <50% reduction of hydrolytic activity relative to CHN A using colloidal chitin or M. lysodeikticus walls as substrates; whereas, antifungal activity was reduced by up to 80%. Relative to CHN A, a variant with two spacers in tandem, which binds chitin, showed very low hydrolytic activity towards chitin and Nod factors, but comparable lysozyme activity and enhanced antifungal activity. Neither hydrolytic activity, substrate specificity nor antifungal activity were strictly correlated with the CRD-mediated capacity to bind chitin. This suggests that the presence of the chitin-binding domain does not have a major influence on the functions of CHN A examined. Moreover, the results with the tandem-spacer variant raise the possibility that substantial chitinolytic activity is not essential for inhibition of T. viride growth by CHN A.     

Cloning, expression, and characterization of a highly thermostable family 18 chitinase from Rhodothermus marinus

A family 18 chitinase gene chiA from the thermophile Rhodothermus marinus was cloned and expressed in Escherichia coli. The gene consisted of an open reading frame of 1,131 nucleotides encoding a protein of 377 amino acids with a calculated molecular weight of 42,341 Da. The deduced ChiA was a non-modular enzyme with one unique glycoside hydrolase family 18 catalytic domain. The catalytic domain exhibited 43% amino acid identity with Bacillus circulans chitinase C. Due to poor expression of ChiA, a signal peptide-lacking mutant, chiAsp, was designed and used subsequently. The optimal temperature and pH for chitinase activity of both ChiA and ChiAsp were 70°C and 4.5–5, respectively. The enzyme maintained 100% activity after 16 h incubation at 70°C, with half-lives of 3 h at 90°C and 45 min at 95°C. Results of activity measurements with chromogenic substrates, thin-layer chromatography, and viscosity measurements demonstrated that the chitinase is an endoacting enzyme releasing chitobiose as a major end product, although it acted as an exochitobiohydrolase with chitin oligomers shorter than five residues. The enzyme was fully inhibited by 5 mM HgCl₂, but excess ethylenediamine tetraacetic acid relieved completely the inhibition. The enzyme hydrolyzed 73% deacetylated chitosan, offering an attractive alternative for enzymatic production of chitooligosaccharides at high temperature and low pH. Our results show that the R. marinus chitinase is the most thermostable family 18 chitinase isolated from Bacteria so far.     

Lactate conversion to acetate,CO2 and H2 in cell suspensions of Desulfovibrio vulgaris (Marburg): indications for the involvement of an energy driven reaction

Cell suspensions of Desulfovibrio vulgaris were found to catalyze, in the absence of sulfate, the complete conversion of 1 lactate to 1 acetate, 1 CO₂, and 2 H₂ (G₀=-8.8 kJ/mol) and of 1 pyruvate to 1 acetate, 1 CO₂, and 1 H₂ (G₀=-52 kJ/mol). Protonophores, the proton translocating ATPase inhibitor N,N-dicyclohexylcarbodiimide, and arsenate specifically inhibited H₂ formation from lactate but not from pyruvate. The results suggest that lactate oxidation to pyruvate and H₂ (G ₀=+43.2 kJ/mol) is energy driven.     

Non-PTS uptake and subsequent metabolism of glucose in Pediococcus halophilus as demonstrated with a double mutant defective in phosphoenolpyruvate:mannose phosphotransferase system and in phosphofructokinase

Pediococcus halophilus possesses phosphoenolpyruvate:mannose phosphotransferase system (man:PTS) as a main glucose transporter. A man:PTS defective (man:PTS^d) strain X-160 could, however, utilize glucose. A possible glucose-transport mechanism other than PTS was studied with the strain X-160 and its derivative, man:PTS^d phosphofructokinase defective (PFK^–) strain M-13. Glucose uptake by X-160 at pH 5.5 was inhibited by any of carbonylcyanide m-chlorophenylhydrazone, nigericin, N,N-dicyclohexylcarbodiimide, or iodoacetic acid. The double mutant M-13 could still transport glucose and accumulated intracellularly a large amount of hexose-phosphates (ca. 8 mM glucose 6-phosphate and ca. 2 mM fructose 6-phosphate). Protonophores also inhibited the glucose transport at pH 5.5, as determined by the amounts of accumulated hexose-phosphates (< 4 mM). These showed involvement of proton motive force (P) in the non-PTS glucose transport. It was concluded that the non-PTS glucose transporter operated in concert with hexokinase or glucokinase for the metabolism of glucose in the man:PTS^d strain.Abbreviations BM basal medium - BM-G basal medium containing glucose - CM complex medium - man:PTS phosphoenolpyruvate:mannose phosphotransferase system - CCCP carbonylcyanide m-chlorophenylhydrazone - DCCD N,N-dicyclohexyl carbodiimide - P proton motive force - pH transmembrane pH gradient - transmembrane electrical potential difference - MNNG N-methyl-N-nitro-N-nitrosoguanidine - PIPES piperazine-N,N-bis(-ethanesulfonic acid) - MES 4-morpholineethanesulfonic acid - G-6-P glucose 6-phosphate - F-6-P fructose 6-phosphate - FDP fructose 1,6-bisphosphate - EMP Embden-Meyerhof-Parnas pathway - PFK phosphofructokinase - GK glucokinase - HK hexokinase - IAA iodoacetic acid - II^man enzyme II component of man:PTS     

Functional expression in yeast of an N-deleted form of<Emphasis Type="Italic"> At</Emphasis>-ACA8, a plasma membrane Ca<Superscript>2+</Superscript>-ATPase of<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>, and characterization of a hyperactive mutant

A constitutively active form of At-ACA8, a plasma membrane Ca²⁺-ATPase from Arabidopsis thaliana (L.) Heynh., from which the first 74 amino acids containing the calmodulin-binding domain (74-At-ACA8) had been deleted, was expressed in Saccharomyces cerevisiae strain K616, which lacks the main endogenous active Ca²⁺ transport systems. 74-At-ACA8 complemented the K616 phenotype, making it able to grow in a calcium-depleted medium. 74-At-ACA8 protein, which co-migrated with the endoplasmic reticulum marker BiP in a sucrose-density gradient, catalyzed MgATP-dependent Ca²⁺ uptake and Ca²⁺-dependent MgATP hydrolysis, and retained the biochemical characteristics of the native plant plasma membrane Ca²⁺-ATPase (low specificity for nucleoside triphosphate, high sensitivity to inhibition by the fluorescein derivatives erythrosin B and eosin Y), thus confirming that it is correctly folded and functional. Substitution of the ⁷⁹⁴HE residues (numbers refer to full-length At-ACA8) following the highly conserved TGDG(TV)NDP(AS)L motif in the cytoplasmic headpiece with two lysine residues generated an hyperactive protein, with a catalytic activity 2-fold higher than that of 74-At-ACA8. The ⁷⁹⁴HEKK mutant was also about 6-fold more sensitive than 74-At-ACA8 to inhibition by vanadate, indicating that the mutation determines an increase in the proportion of enzyme in the E₂ state during the catalytic cycle.Abbreviations aa Amino acids - CaM Calmodulin - EB Erythrosin B - ER Endoplasmic reticulum - EY Eosin Y - FITC Fluorescein isothiocyanate - PM Plasma membrane     

SCM2, a tryptophan permease in Saccharomyces cerevisiae, is important for cell growth

SCM2, a novel gene encoding a yeast tryptophan permease, was cloned as a high-copy-number suppressor of cse2-1. The cse2-1 mutation causes cold sensitivity, temperature sensitivity and chromosome missegregation. However, only the cold-sensitive phenotype of cse2-1 cells is suppressed by SCM2 at high copy. SCM2 is located on the left arm of yeast chromosome XV, adjacent to SUP3 and encodes a 65 kDa protein that is highly homologous to known amino acid permeases. Four out of five disrupted scm2 alleles (scm21-4) cause slow growth, whereas one disrupted allele (scm25) is lethal. Cells with both the scm21 and trp1-101 mutations exhibit a synthetic cold-sensitive phenotype and grow much more slowly at the permissive temperature than cells with a single scm21 or trp1-101 mutation. A region of the predicted SCM2 protein is identical to the partial sequence recently reported for the yeast tryptophan permease TAP2, indicating that SCM2 and TAP2 probably encode the same protein.     

Protonmotive force driven 6-deoxyglucose uptake by the oral pathogen,Streptococcus mutans Ingbritt

Streptococcus mutans Ingbritt was grown in glucose-excess continuous culture to repress the glucose phosphoenolpyruvate phosphotransferase system (PTS) and allow investigation of the alternative glucose process using the non-PTS substrate, (³H) 6-deoxyglucose. After correcting for non-specific adsorption to inactivated cells, the radiolabelled glucose analogue was found to be concentrated approximately 4.3-fold intracellularly by bacteria incubated in 100 mM Tris-citrate buffer, pH 7.0. Mercaptoethanol or KCl enhanced 6-deoxyglucose uptake, enabling it to be concentrated internally by at least 8-fold, but NaCl was inhibitory to its transport. Initial uptake was antagonised by glucose but not 2-deoxyglucose. Evidence that 6-deoxyglucose transport was driven by protonmotive force (p) was obtained by inhibiting its uptake with the protonophores, 2,4-dinitrophenol, carbonylcyanide m-chlorophenylhydrazine, gramicidin and nigericin, and the electrical potential difference () dissipator, KSCN. The membrane ATPase inhibitor, N,N¹-dicyclohexyl carbodiimide, also reduced 6-deoxyglucose uptake as did 100 mM lactate. In combination, these two inhibitors completely abolished 6-deoxyglucose transport. This suggests that the driving force for 6-deoxyglucose uptake is electrogenic, involving both the transmembrane pH gradient (pH) and . ATP hydrolysis, catalysed by the ATPase, and lactate excretion might be important contributors to pH.Abbreviations DNP 2,4-dinitrophenol - CCCP carbonylcyanide m-chlorophenylhydrazone - DCCD N,N¹-dicyclohyxyl carbodiimide - p protonmotive force - pH transmembrane pH gradient - transmembrane electrical potential difference     

A nontoxicPseudomonas exotoxin a induces active immunity and passive protective antibody againstPseudomonas exotoxin a intoxication

Pseudomonas exotoxin A (PE) is one of the most potent cytotoxic agents produced byPseudomonas aeruginosa. In this study, we examined the possibility of using PE with a deletion of 38 carboxyl-terminal amino acid residues, designated PE(576–613), for active immunization against PE-mediated disease. We first examined the toxic effects of PE and PE(576–613) on 5- and 9-week-old ICR mice. The results show that the subcutaneous administration of PE(576–613) at a dose of 250 µg was still nontoxic to 5- and 9-week-old ICR mice, while native PE was lethal at a dose of 0.5 and 1 µg, respectively. PE(576–613) was then used to immunize ICR mice. The minimum dose of PE(576–613) that could effectively induce anti-PE antibodies in 5- and 9-week-old ICR mice was found to be 250 ng. However, immunization with 250 ng PE(576–613) failed to protect the immunized mice from a lethal dose of PE. The effective immunization dose of PE(576–613) that could protect mice against a 2 µg PE challenge was found to be 15 µg. In addition, sera obtained from PE(576–613)-immunized ICR mice were able to neutralize PE intoxication and effectively protect mice from PE. Thus, PE(576–613) may be used as an alternative route to new PE vaccine development.     

Expression of Bacillus thuringiensis full-length and N-terminally truncated vip184 gene in an acrystalliferous strain of subspecies kurstaki

Vip3A is an 89-kDa protein secreted by Bacillus thuringiensis during vegetative growth. The 3.5 kb full-length vip184 gene was cloned from a wild-type isolate of B. thuringiensis, and the vip184S gene was constructed by deletion of the putative signal peptide encoding sequence. Both genes were expressed in the acrystalliferous strain Cry^–B of B. thuringiensis. Vip184 protein was observed mainly in the centrifuged pellets of B. thuringiensis Cry^–B(pHPT3), which contains the vip184 gene, and was less abundant in the concentrated supernatant. However, Vip184S proteins were not detected in the concentrated supernatant, but only in the pellets of Cry^–B(pHPT3S), which contains vip184S gene. This indicated that Vip184S proteins were not secreted into the culture medium and that the putative signal peptides were essential for the secretion of Vip184. The toxicity of Cry^–B(pHPT3) and Cry^–B(pHPT3S) were demonstrated against the neonate larvae of Spodoptera exigua and S. litura. Pellets and concentrated supernatant of Cry^–B(pHPT3) showed high activity against S. exigua and S. litura, but the Cry^–B(pHPT3S) strain was not toxic to either because of the deletion of N-terminal putative signal peptides. Therefore, this may suggest that the putative signal peptides are required for lethality.     

Reverse genetic analysis of the glutathione metabolic pathway suggests a novel role of<Emphasis Type="Italic"> PHGPX</Emphasis> and<Emphasis Type="Italic"> URE2</Emphasis> genes in aluminum resistance in<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

We have taken a systematic genetic approach to study the potential role of glutathione metabolism in aluminum (Al) toxicity and resistance, using disruption mutants available in Saccharomyces cerevisiae. Yeast disruption mutants defective in phospholipid hydroperoxide glutathione peroxidases (PHGPX; phgpx1 , phgpx2 , and phgpx3), were tested for their sensitivity to Al. The triple mutant, phgpx1 /2/3, was more sensitive to Al (55% reduction in growth at 300 M Al) than any single phgpx mutant, indicating that the PHGPX genes may collectively contribute to Al resistance. The hypersensitivity of phgpx3 to Al was overcome by complementation with PHGPX3, and all PHGPX genes showed increased expression in response to Al in the wild-type strain (YPH250), with maximum induction of approximately 2.5-fold for PHGPX3. Both phgpx3 and phgpx1/2/3 mutants were sensitive to oxidative stress (exposure to H₂O₂ or diamide). Lipid peroxidation was also increased in the phgpx1/2/3 mutant compared to the parental strain. Disruption mutants defective in genes for glutathione S-transferases (GSTs) (gtt1 and gtt2), glutathione biosynthesis (gsh1 and gsh2), glutathione reductase (glr1) and a glutathione transporter (opt1) did not show hypersensitivity to Al relative to the parental strain BY4741. Interestingly, a strain deleted for URE2, a gene which encodes a prion precursor with homology to GSTs, also showed hypersensitivity to Al. The hypersensitivity of the ure2 mutant could be overcome by complementation with URE2. Expression of URE2 in the parental strain increased approximately 2-fold in response to exposure to 100 M Al. Intracellular oxidation levels in the ure2 mutant showed a 2-fold (non-stressed) and 3-fold (when exposed-to 2 mM H₂O₂) increase compared to BY4741; however, the ure2 mutant showed no change in lipid peroxidation compared to the control. The phgpx1/2/3 and ure2 mutants both showed increased accumulation of Al. These findings suggest the involvement of PHGPX genes and a novel role of URE2 in Al toxicity/resistance in S. cerevisiae.Communicated by D.Y. Thomas     

Sodium ions are necessary for growth and energy transduction in the marine cyanobacterium Oscillatoria brevis

The maximal growth rate of the marine cyanobacterium Oscillatoria brevis was reached at 200–400 mM NaCl and pH 9.0–9.6. NaCl was found (i) to stimulate the rate of the light-supported generation across the cytoplasmic membrane of the cells and (ii) to decrease the sensitivity of level and motility of the O. brevis trichomes to protonophorous uncouplers. The Na⁺/H⁺ antiporter, monensin, increased both and the uncoupler sensitivity of the cells. The data obtained agree with the assumption that O. brevis possesses a primary Na⁺ pump in its cytoplasmic membrane.Abbreviations ATP adenosine-5-triphosphate - TTFB tetrachlortrifluoromethylimidazol - CCCP carbonyl cyanide m-chlorophenylhydrazone - Na⁺ transmembrane electrochemical potential differences of Na⁺ - transmembrane electric potential difference - pNa transmembrane pNa difference     

The ability of acidic pH,growth inhibitors,and glucose to increase the proton motive force and energy spilling of amino acid-fermenting <Emphasis Type="Italic">Clostridium sporogenes</Emphasis> MD1 cultures

Clostridium sporogenes MD1 grew rapidly with peptides and amino acids as an energy source at pH 6.7. However, the proton motive force (p) was only –25 mV, and protonophores did not inhibit growth. When extracellular pH was decreased with HCl, the chemical gradient of protons (ZpH) and the electrical membrane potential () increased. The p was –125 mV at pH 4.7, even though growth was not observed. At pH 6.7, glucose addition did not cause an increase in growth rate, but increased to –70 mV. Protein synthesis inhibitors also significantly increased . Non-growing, arginine-energized cells had a of –80 mV at pH 6.7 or pH 4.7, but was not detected if the F₁F₀ ATPase was inhibited. Arginine-energized cells initiated growth if other amino acids were added at pH 6.7, and and ATP declined. At pH 4.7, ATP production remained high. However, growth could not be initiated, and neither nor the intracellular ATP concentration declined. Based on these results, it appears that C. sporogenes MD1 does not need a large p to grow, and p appears to serve as a mechanism of ATP dissipation or energy spilling.Mandatory disclaimer: Proprietary or brand names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product, and exclusion of others that may be suitable.     

ATP is required for K+ active transport in the archaebacterium Haloferax volcanii

The Archaebacterium Haloferax volcanii concentrates K⁺ up to 3.6 M. This creates a very large K⁺ ion gradient of between 500- to 1,000-fold across the cell membrane. H. volcanii cells can be partially depleted of their internal K⁺ but the residual K⁺ concentration cannot be lowered below 1.5 M. In these conditions, the cells retain the ability to take up potassium from the medium and to restore a high internal K⁺ concentration (3 to 3.2 M) via an energy dependent, active transport mechanism with a K _m of between 1 to 2 mM. The driving force for K⁺ transport has been explored. Internal K⁺ concentration is not in equilibrium with m suggesting that K⁺ transport cannot be accounted for by a passive uniport process. A requirement for ATP has been found. Indeed, the depletion of the ATP pool by arsenate or the inhibition of ATP synthesis by N,N-dicyclohexylcarbodiimide inhibits by 100% K⁺ transport even though membrane potential m is maintained under these conditions. By contrast, the necessity of a m for K⁺ accumulation has not yet been clearly demonstrated. K⁺ transport in H. volcanii can be compared with K⁺ transport via the Trk system in Escherichia coli.Abbreviations CCCP Carbonylcyanide m-chlorophenyl-hydrazone - DCCD N,N-dicyclohexylcarbodiimide - MES 2-[N-morpholino] ethane sulfonic acid - MOPS 3-[N-morpholino] propane sulfonic acid - TRIS Tris (hydroxymethyl) aminomethane - TPP tetraphenyl phosphonium     

Desaturation of oxygenated fatty acids in Lesquerella and other oil seeds

Species of the genus Lesquerella, within the Brassicaceae family, have seed oils containing hydroxy fatty acids. In most Lesquerella species, either lesquerolic (14-hydroxy-eicosa-11-enoic), auricolic (14-hydroxy-eicosa-11,17-dienoic) or densipolic (12-hydroxy-octadeca-9,15-dienoic) acid dominates in the seed oils. Incubations of developing seed from Lesquerella species with 1-¹⁴C-fatty acids were conducted in order to study the biosynthetic pathways of these hydroxylated fatty acids. [¹⁴C]Oleic (octadeca-9-enoic) acid, but not [¹⁴C]linoleic (octadeca-9,12-dienoic) acid, was converted into the hydroxy fatty acid, ricinoleic (12-hydroxy-octadeca-9-enoic) acid, which was rapidly desaturated to densipolic (12-hydroxy-octadeca-9,15-dienoic) acid. In addition, [¹⁴C] ricinoleic acid added to Lesquerella seeds was efficiently desaturated at the 15 carbon. A pathway for the biosynthesis of the various hydroxylated fatty acids in Lesquerella seeds is proposed. The demonstration of desaturation at position 15 of a fatty acid with a hydroxy group at position 12 in Lesquerella prompted a comparison of the substrate recognition of the desaturases from Lesquerella and linseed. It was demonstrated that developing linseed also was able to desaturate ricinoleate at position 15 into densipolic acid. In addition, the linseed 15 desaturase was able to desaturate vernolic (12,13-epoxy-octadeca-9-enoic) acid and safflower microsomal 12 desaturase was able to desaturate 9-hydroxy-stearate. Thus, hydroxy and epoxy groups may substitute for double bonds in substrate recognition for oil-seed 12 and 15 desaturases.Abbreviations GLC gas-liquid chromatography - lysoPC palmitoyl-lysophosphatidylcholine - PC phosphatidylcholine This work was supported by grants from Stifteisen Svensk Oljeväxtforskning, Skanska Lantmännen Foundation, Swedish Farmers Foundation for Agricultural research, The Swedish Natural Science Research Council and The Swedish Council for Forestry and Agricultural Research. Nicki Engeseth was supported by the National Science Foundation under a grant award in 1992.     

Genetic analysis of carbon isotope discrimination and agronomic characters in a bread wheat cross

Carbon isotope discrimination () has been suggested as a selection criterion to improve transpiration efficiency (W) in bread wheat (Triticum aestivum L.). Cultivars Chinese Spring with low A (high W) and Yecora Rojo with high (low W) were crossed to develop F₁, F₂, BC₁, and BC₂ populations for genetic analysis of and other agronomic characters under well-watered (wet) and water-stressed (dry) field conditions. Significant variation was observed among the generations for only under the wet environment. Generation x irrigation interactions were not significant for . Generation means analysis indicated that additive gene action is of primary importance in the expression of under nonstress conditions. Dominance gene action was also detected for , and the direction of dominance was toward higher values of . The broad-sense and the narrow-sense heritabilities for were 61 % and 57% under the wet conditions, but were 48% and 12% under the draughted conditions, respectively. The narrow-sense heritabilities for grain yield, above-ground dry matter, and harvest index were 36%, 39%, and 60% under the wet conditions and 21%, 44%, and 20% under dry conditions, respectively. The significant additive genetic variation and moderate estimate of the narrow-sense heritability observed for indicated that selection under wet environments should be effective in changing in spring bread wheat.     

Anaerobic biosynthesis of unsaturated fatty acids in the cyanobacterium,Oscillatoria limnetica

The mechanism for synthesis of monounsaturated fatty acids under aerobic and anaerobic conditions was studied in the facultative anaerobic cyanobacterium, Oscillatoria limnetica. The hexadecenoic acid (C161) of aerobically grown O. limnetica was shown to contain both the 7 (79%) and 9 (21%) isomers, while the octadecenoic (C181) acid was entirely the 9 acid. Incorporation of [2-¹⁴C] acetate into the fatty acids under aerobic conditions resulted in synthesis of the 7 and 9 C161 and the 9 C181. Synthesis of unsaturated fatty acids in the presence of DCMU required sulfide. Anaerobic incubations in the presence of DCMU and sulfide (less than 0.003% atmospheric oxygen) resulted in a two-fold increase in monounsaturated fatty acids of both 7 and 9 C161 and 9 and 11 C181. The synthesis of these isomers is characteristic of a bacterialtype, anaerobic pathway.Abbreviations DCMU 3(3,4-dichlorophenyl)-1,1-dimethylurea - MFA monounsaturated fatty acid