Properties of the menaquinol oxidase (Qox) and of qox deletion mutants of Bacillus subtilis

Menaquinol oxidase isolated from the membrane of Bacillus subtilis W23 was found to consist of four polypeptides (QoxA, B, C, and D) that were predicted by the sequence of the qox operon of B. subtilis 168 (Santana et al. 1992). The preparation contained 7 mol cytochrome aa ₃ per g protein, which corresponds to 2mol heme A per mol enzyme of 144 kDa molecular mass. Respiration with dimethylnaphthoquinol catalyzed by the enzyme was ten times faster than that with menadiol. Activities with more electropositive quinols were negligible. The activity of the enzyme was inhibited by equimolar amounts of HQNO, while antimycin, myxothiazol, and stigmatellin were more than tenfold less effective. When cells of both strains of B. subtilis (W23 and 168) were grown with glucose, quinol respiration was an order of magnitude more active than respiration with N,N,N,N-tetramethyl-1,4-phenylenediamine plus ascorbate. Surprisingly, the same result was obtained with mutant strains lacking qoxB. As cytochromes a and d were virtually absent, a second quinol oxidase, possibly of the cytochrome o-type, was apparently formed by the mutants.Abbreviations cat Chloramphenicol resistance gene - cta Cytochrome oxidase genes - DMN 2,3-Dimethyl-1,4-naphthoquinone - DMNH ₂ Reduced DMN - HQNO 2-(n-Heptyl)-4-hydroxyquinoline-N-oxide - qox Quinol oxidase genes - TMPD N,N,N,N-tetramethyl-1,4-phenylenediamine     

Phosphorylation of the plasma-membrane H+-ATPase of oat roots by a calcium-stimulated protein kinase

When plasma-membrane vesicles isolated from oat (Avena sativa L.) root cells were incubated with [-³²P]ATP, the H⁺-ATPase was found to be phosphorylated at serine and threonine residues. Phosphotyrosine was not detected. Endogenous ATPase kinase activity was also observed in plasma-membrane vesicles isolated from potato (Solanum tuberosum L.) root cells as well as from yeast (Saccharomyces cerevisiae). Identity of the phosphorylated oat root M_r=100 000 polypeptide as the ATPase was confirmed using conventional glycerol density-gradient centrifugation to purify the native enzyme and by a new procedure for purifying the denatured polypeptide using reversephase high-performance liquid chromatography. Kinase-mediated phosphorylation of the oat root plasma-membrane H⁺-ATPase was stimulated by the addition of low concentrations of Ca²⁺ and by a decrease in pH, from 7.2 to 6.2. These results demonstrate that kinase-mediated phosphorylation of the H⁺-ATPase is a plausible mechanism for regulating activity. They further indicate that changes in the cytoplasmic [Ca²⁺] and pH are potentially important elements in modulating the kinase-mediated phosphorylation.Abbreviations EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid - M_r relative molecular mass - RP-HPLC reverse-phase high-performance liquid chromatography - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Interaction of cytochrome c L with methanol dehydrogenase from Methylophaga marina 42:

The reaction of methanol dehydrogenase with cytochrome c _L from Methylophaga marina and the reactions of the non-physiological substrates, Wurster's blue and ascorbic acid, with both proteins were studied as a function of temperature (4–32 °C), pressure (1–2000 bar) and ionic strength using the optical high pressure stopped-flow method. The thermodynamic parameters H, S and V were determined for all reactions where electron transfers are involved. These data allowed the determination of the Maxwell relationships which proved the internal thermodynamic consistency of the system under study. A conformational change on the cytochrome c _L level was deduced from both breaks in the Arrhenius plots and the variation of the V with temperature.Abbreviations MOPS 4-morpholinepropanesulfonic acid - CHES 2-(cyclohexylamino)ethanesulfonic acid - MDH methanol dehydrogenase - EDTA ethylenedinitrilotetraacetic acid disodium salt - BTB bromothymol blue (3,3-dibromothymolsulfoneph-thalein) - PQQ 2,7,9-tricarboxy-lH-pyrrolo-[2,3f]quinoline-4,5-dione - cytochrome c _HH mammalian horse heart cytochrome c     

Changes of amino acid sequence in PEST-like area and QEEET motif affect degradation rate of D1 polypeptide in photosystem II

The degradation rate of the D1 polypeptide was measured in threeSynechocystis PCC 6803 mutantsin vivo. Mutations were introduced into a putative cleavage area of the D1 polypeptide (QEEET motif) and into the PEST-like area. PEST sequences are often found in proteins with a high turnover rate. The QEEET-motif mutants are CA1 [(E242-E244);Q241H] and E243K, and the third mutation, E229D, was directed to the PEST-like area. During high-light illumination (1500 mol photons m^-2s^-1) that induced photoinhibition of photosystem II (PSII), the half-life time of the D1 polypeptide in mutant E229D (t _1/2=35 min) was about twice as long as in AR (control strain) cells (t _1/2=19 min). In growth light (40 mol photons m^-2s^-1), the degradation rate of the D1 polypeptide in E229D and AR strains was the same (t _1/25 h). In growth light the D1 polypeptide was degraded faster in both QEEET-motif mutants than in the AR strain, but in photoinhibitory light the degradation rates were similar. According to these results, the highly conservative QEEET motif as such is not required for the proteolytic cut of the D1 polypeptide, but it does affect the rate of degradation. No simple correlation existed between the degradation rate of the D1 polypeptide and the susceptibility of PSII to photoinhibition in mutant and AR cells under our experimental conditions.     

Photoinhibition of photosynthesis under natural conditions in ivy (Hedera helix L.) growing in an understory of deciduous trees

We investigated to what extent south-exposed leaves (E-leaves) of the evergreen ivy (Hedera helix L.) growing in the shadow of two deciduous trees suffered from photoinhibition of photosynthesis when leaf-shedding started in autumn. Since air temperatures drop concomitantly with increase in light levels, changes in photosynthetic parameters (apparent quantum yield, _i and maximal photosynthetic capacity of O₂ evolution, P_max; chlorophyll-a fluorescence at room temperature) as well as pigment composition were compared with those in north-exposed leaves of the same clone (N-leaves; photosynthetic photon flux density PPFD< 100 mol · m^–2 · s^–2) and phenotypic sun leaves (S-leaves; PPFD up to 2000 mol · m^–2 · s^–1).In leaves exposed to drastic light changes during winter (E-leaves) strong photoinhibition of photosynthesis could be observed as soon as the incident PPFD increased in autumn. In contrast, in N-leaves the ratio of variable fluorescence to maximum fluorescence (F_V/F_Mm) and _i did not decline appreciably prior to severe frosts (up to -12° C) in January. At this time, _i was reduced to a similar extent in all leaves, from about 0.073 mol O₂ · mol^–1 photons before stress to about 0.020. Changes in _i were linearly correlated with changes in f_v/f_m (r = 0.955). The strong reduction in F_V/F_M on exposure to stress was caused by quenching in F_M. The initial fluorescence (F₀), however, was also quenched in all leaves. The diminished fluorescence yield was accompanied by an increase in zeaxanthin content. These effects indicate that winter stress in ivy primarily induces an increase in non-radiative energy-dissipation followed by photoinhibitory damage of PSII. Although a pronounced photooxidative bleaching of chloroplast pigments occurred in January (especially in E-leaves), photosynthetic parameters recovered completely in spring. Thus, the reduction in potential photosynthetic yield in winter may be up to three times greater in leaves subjected to increasing light levels than in leaves not exposed to a changing light environment.Abbreviations and Symbols F₀, F_M initial and maximal fluorescence yield when all PSII centres are open and closed - F_V variable fluorescence (F_M-F₀) - P_max maximal photosynthetic capacity at 1000 umol · m^–2 · s^–1 PPFD and CO₂ saturation - PPFD photosynthetic photon flux density - _i apparent quantum yield of photosynthetic O₂ evolution - E-leaves, N-leaves shade leaves exposed, not exposed to drastic light changes during winter - S-leaves sun leaves from an open ivy stand Dedicated to Professor Otto Härtel on the occasion of his 80th birthdayThis work was supported by the Austrian Fonds zur Förderung der wissenschaftlichen Forschung.     

Biosynthesis,cDNA and amino acid sequences of a precursor of conglutin δ, a sulphur-rich protein from Lupinus angustifolius

The biosynthesis of conglutin has been studied in developing cotyledons of Lupinus angustifolius L. Precursors of conglutin formed the major sink for [³⁵S]-cysteine incorporated by developing lupin cotyledons, and these precursors were rapidly sequestered into the endoplasmic reticulum. The sequence of a cDNA clone coding for one such precursor of conglutin was determined. The structure of the precursor polypeptide for conglutin predicted from the cDNA sequence contained an N-terminal leader peptide of 22 amino acids directly preceding a subunit polypeptide of M _r 4520, together with a linking region of 13 amino acids and a subunit polypeptide of M _r 9558 at the C-terminus. The amino acid sequence predicted from the cDNA sequence showed minor variations from that established by sequencing of the protein purified from mature dried seeds (Lilley and Inglis, 1986). These were consistent with the existence of a multi-gene family coding for conglutin . Comparison of the sequences of conglutin with those of other 2S storage proteins showed that the cysteines involved in internal disulphide bridges between the mature subunits of conglutin , were maintained throughout this family of proteins but that little else was conserved either at the protein or DNA level.     

Fluorescence microscopy and radiolabeling of C3 and C4 chloroplasts using diisothiocyanatostilbene disulfonic acid as a marker for the phosphate translocator

The usefulness of 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) for in-situ studies of the chloroplast phosphate translocator was evaluated by fluorescence microscopy and radiolabeling of spinach (Spinacia oleracea L.) (C₃ plant) and maize (Zea mays L.) (C₄ plant) chloroplasts. In maize mesophyll and bundle-sheath chloroplasts and in spinach chloroplasts that were either intact, broken or swollen, DIDS fluorescence was only associated with the chloroplast envelope. Intact chloroplasts often had fluorescent patches corresponding to concave regions of the chloroplast which we assume to be regions enriched in DIDS-binding sites.Incubation of intact or broken spinach chloroplasts or maize mesophyll chloroplasts with [³H₂]DIDS resulted in the labeling of a single polypeptide (relative molecular mass, M_r, 30 kDa) in the envelope fraction, in each case. Label in the stromal fraction was not detected when intact chloroplasts were incubated with [³H₂]DIDS. However, when broken chloroplasts were incubated with [³H₂]DIDS, several polypeptides of various molecular masses were labeled, but not the 30×31-kDa polypeptide. In thylakoid fractions from both broken and intact chloroplasts, a single 30×31-kDa polypeptide was labeled inconsistently. When a mixture of intact maize mesophyll and bundle-sheath chloroplasts was labeled with [³H₂]DIDS, extracts of whole chloroplasts displayed radioactivity only in the 30×31-kDa band.We conclude that DIDS is a valuable probe for the in-situ identification and characterization of the 30-kDa protein — the presumptive phosphate translocator — in C₃ and C₄ chloroplasts since DIDS (1) does not penetrate the inner membrane of the envelope of intact chloroplasts and, therefore, (2) does not bind internal sites in intact chloroplasts, and (3) only binds the 30-kDa protein in the inner membrane of the envelope.Abbreviations CBB Coomassie brilliant blue - DIC differential interference contrast optics - DIDS 4,4-diisothiocyanatostilbene-2,2-disulfonic acid - [³H₂]DIDS 1,2-ditritio-1,2-(2,2-disulfo-4,4-diisothiocyano)diphenylethane - kDa kilodalton - M_r relative molecular mass - PGA 3-phosphoglycerate - Pitranslocator phosphate translocator - SDS sodium dodecyl sulfate     

Responses of nitrogen-fixing and nitrate-supplied alfalfa (Medicago sativa L.) to iron chelates in an alkaline hydroponic medium

Ferric ethylenediamine di-(o-hydroxyphenylacetate) (FeEDDHA) and ferric hydroxyethylethylenediaminetriacetic acid (FeHEDTA) were evaluated as Fe sources for hydroponic growth of alfalfa (Medicago sativa L., cv. Mesilla), either dependent on N₂ fixation or supplied with NO₃. The hydroponic medium was maintained at pH 7.5 by addition of CaCO₃. Nitrogen-fixing cultures were inoculated with Rhizobium meliloti 102 F51 and grown in medium without added nitrogen. After five to seven weeks of growth under greenhouse conditions, plants were harvested. Nitrogen fixation was measured by the acetylene reduction method.When FeEDDHA was supplied, growth of alfalfa, whether dependent on N₂ fixation or supplied with NO₃, was severely limited at concentrations typically used in hydroponic medium (10 or 20 M). Maximum yield of NO₃-supplied alfalfa was obtained at 100 M while maximum yield of N₂-fixing alfalfa was obtained in the range of 33 to 200 M FeEDDHA. Nodule fresh weights and N₂ fixation rates increased with FeEDDHA concentration up to 33 M and remained essentially constant up to 200 M. With FeHEDTA, maximum yields of both NO₃-grown and N₂-fixing alfalfa were obtained at 10 M. Growth of NO₃-supplied plants was inhibited at 200 M FeHEDTA while growth of N₂-fixing plants was inhibited at 100 M FeHEDTA. The numbers of nodules per plant increased between 3.3 and 10 M FeHEDTA; however, inhibition of nodule formation occurred at a concentration of 33 M or higher. Nodule weights per plant and N₂ fixation rates were depressed at 3.3 M as well as at 100 M FeHEDTA. The results suggest that alfalfa dependent on N₂ fixation is more sensitive to limited Fe availability than alfalfa supplied with NO₃.     

The membraneous nitrite reductase involved in the electron transport of Wolinella succinogenes

Wolinella succinogenes grown with nitrate as terminal electron acceptor contains two nitrite reductases as measured with the donor viologen radical, one in the cytoplasm and the other integrated in the cytoplasmic membrane. The fumarate-grown bacteria contain only the membraneous species.The isolated membraneous enzyme consists of a single polypeptide chain (M _r 63,000) carrying 4 hemeC groups and probably an iron-sulphur cluster as prosthetic groups. The enzyme amounts to about 1% of the total membrane protein.The isolated enzyme catalyses the reduction of nitrite to ammonium without accumulation of significant amounts of intermediates or alternative products. The Michaelis constant for nitrite was 0.1 mM and the turnover number of the hemeC 1.5 · 10⁵ electrons per min at 37°C.The viologen-reactive site of the enzyme in the membrane is oriented towards the cytoplasm. When the isolated enzyme is incorporated into liposomes, the viologen-as well as the nitrite-reactive site is exposed to thooutside.The cytoplasmic membrane contains a second hemeC protein (M _r 22,000) which may represent a cytochrome c.Abbreviations NQNO 2-(n-nonyl)-4-hydroxyquinoline-N-oxide - MES 2-(N-morpholino)ethanesulfonate - MOPS 3-(N-morpholino)propanesulfonate - HEPES N-2-Hydroxyethylpiperazine-N-2-ethanesulfonate - TES N-tris(hydroxymethyl)methyl-2-aminoethanesulfonate - MK menaquinone     

Silicon amelioration of aluminium toxicity in teosinte (Zea mays L. ssp. mexicana)

The influence of different Al concentrations, (0, 60 and 120 M Al) on growth and internal concentrations of Al, Si and selected organic acids was analysed in plants of teosinte (Zea mays L. ssp. mexicana), a wild form of maize from acid soils from Mexico. The plants were grown in nutrient solutions (pH 4.0) with or without 4 M silicon. Analysis with the GEOCHEM speciation program did not reveal differences between free activities of Al³⁺ in solutions with and without 4 M Si, but solutions with Si yielded lower concentrations of monomeric Al species, [Al]_mono, when analysed by a modified aluminon method. Plants grown on solutions with similar [Al]_mono, but differing in silicon, showed highly significant differences in growth and tissue concentrations of Al and organic acids. Silicon prevented growth inhibition at [Al]_mono concentrations as high as 35 M, while plants grown without Si suffered severe growth reductions with 33 M [Al]_mono. In solutions with similar [Al]_mono concentrations plants with Si had lower tissue Al concentrations and higher concentrations of malic acid than plants without Si. In view of both the significant influence of Si on the response of plants to Al toxicity and the fact that some soluble Si is always present in soil solutions, the addition of low Si concentrations to nutrient solutions used for Al-tolerance screening is recommended.     

Extracellular cross-linking of xylan and xyloglucan in maize cell-suspension cultures: the role of oxidative phenolic coupling

Cell-suspension cultures of maize (Zea mays L.) released soluble extracellular polysaccharides (SEPs) into their medium. Some or all of the SEPs had feruloyl ester groups. Pulse-labelling with [³H]arabinose was used to monitor changes in the SEPs M_r (estimated by gel-permeation chromatography) with time after synthesis. Newly released ³H-SEPs were 1.3–1.6 MDa, but between 2 days and 3 days after radiolabelling (in one experiment) or between 5 days and 6 days (in another), the ³H-SEPs abruptly increased to 17 MDa, indicating extensive cross-linking. The cross-linking involved both [³H]xylan and [³H]xyloglucan components of the SEPs. The cross-links could be cleaved by alkali, returning the SEPs to their original M_r. In 0.1 M NaOH at 37°C, 58% cleavage was effected within 24 h. The requirement for such prolonged alkali treatment indicates that ester-bonded (e.g. diferuloyl) groups were not solely responsible for the cross-linking. Bonds cleaved only by relatively severe alkali could include benzyl ether linkages formed between sugar residues and oxidised phenolics that had quinone methide structures. The ability of alkali to cleave the cross-links was independent of the age of the ³H-SEP molecules. Cross-linking of ³H-SEPs in vivo was delayed (up to approx. 7 days after radiolabelling) by exogenous sinapic acid, chlorogenic acid or rutin—agents predicted to compete with the oxidative coupling of feruloyl-polysaccharides. The cross-linking was promoted by exogenous ferulic acid or l-tyrosine, possibly because these compounds acted as precursors for polysaccharide feruloylation, thus providing additional partner substrates for the oxidative coupling of previously formed ³H-SEPs. The ability of certain phenolics to prevent the cross-linking of ³H-SEPs supports the idea that the cross-linking involved phenolic oxidation.Abbreviations DTT Dithiothreitol - K_av Elution volume relative to those of high-M_r dextran (K_av=0) and sucrose (K_av=1) - MLG Mixed-linkage -(13),(14)-d-glucan - M_r Relative molecular mass - PCW Primary cell wall - SEP Soluble extracellular polysaccharide - TFA Trifluoroacetic acid - V₀ Void volume (centre of elution peak of high-M_r dextran) - V_i Totally included volume (centre of elution peak of sucrose)     

Study ofO-sialylation of glycoproteins in C6 glioma cells treated with retinoic acid

When treated with retinoic acidin vivo, C6 glioma cells show an enhancement of CMP-Neu5Ac:Gal 1–3 GalNAc-R -2,3 sialyltransferase activity. A 300kDa glycoprotein was detected by lectin affinoblotting in retinoic acid-treated C6 cells which stained weakly or not at all in control cells. Comparative studies with different lectins demonstrated that this glycoprotein contains 2,3 Neu5Ac Gal-GalNAc O-glycan moieties. Cultures in the presence of an inhibitor of O-glycan synthesis (N-acetylgalactosaminide -O-benzyl) demonstrated that enhancement of staining of the 300 kDa glycoprotein was not due to the increase of the 2,3 sialyltransferase but to thede novo synthesis of the polypeptide chain of this glycoprotein.Abbreviations RA retinoic acid - Neu5Ac N-acetylneuraminic acid - CMP-Neu5Ac cytidine 5 monophosphosialate - 2,3 ST CMP-Neu5Ac:Gal 1–3 GalNAc-R -2,3 sialyltransferase - GalNAc-O-benzyl N-acetylgalactosaminide -O-benzyl - Gal1-3GalNAc-O-benzyl Galactosyl 1-3N-acetylgalactosaminide -O-benzyl - TBS Tris-HCl buffer 50mm pH 7.5 containing NaCl 0.15m and Tween 20 0.05% - B1 buffer TBS containing MgCl₂ 1mm, MnCl₂ 1mm and CaCl₂ 1mm     

Isolation of the sulphur reductase and reconstitution of the sulphur respiration of Wolinella succinogenes

Wolinella succinogenes can grow at the expense of sulphur reduction by formate. The enzymes involved in the catalysis of this catabolic reaction have been investigated. From the results the following conclusions are drawn: 1. The enzyme isolated as a sulphide dehydrogenase from the cytoplasmic membrane of W. succinogenes is the functional sulphur reductase that operates in the electron transport from formate to sulphur. 2. The enzyme (M_r 200,000) consists essentially of one type of subunit with the M_r 85,000 and contains equal amounts of free iron and sulphide (120 mol/g protein), but no heme. It represents the first functional sulphur reductase ever isolated. 3. The electron transport chain catalyzing sulphur reduction by formate consists merely of formate dehydrogenase and sulphur reductase. A lipophilic quinone which mediates the transfer of electrons between enzymes in other chains, is apparently not involved. This is the first known example of a phosphorylative electron transport chain that operates without a quinone. 4. The same formate dehydrogenase appears to operate in the electron transport both with sulphur and with fumarate as the terminal electron acceptor in W. succinogenes.Abbreviations DMN 2,3-Dimethyl-1,4-naphthoquinone - DTT dithiothreitol - MK menaquinone (vitamin K₂) - PMSF phenylmethane sulfonylfluoride - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]-glycine - Tea triethanolamine - Hepes 4-(2-hydroxyethyl)-1-piperazineethane sulfonate Dedicated to Professor F. Schneider (Philipps-Universität Marburg) on the occasion of his 60th birthday     

Electron transport reactions in respiratory particles of hydrogenase-induced Anacystis nidulans

Respiratory particles from hydrogen-grown Anacystis nidulans were found to oxidize H₂, NADPH, NADH, succinate and ascorbate plus N,N,N,N-tetramethyl-p-phenylenediamine at rates corresponding to 28, 15, 6, 2.5, and 70 nmol O₂ taken up x mg protein^–1xmin^–1, respectively. The particles were isolated by brief sonication of lysozyme-pretreated cells. Respiratory activities were studied in terms of both substrate oxidation and O₂ uptake. The stoichiometry between oxidation of H₂, NADPH, NADH or succinate, and consumption of O₂ was calculated to be 1.95+-0.1 with each substrate.Inhibitors of flavoproteins did not affect the oxyhydrogen reaction while 2-n-heptyl-8-hydroxyquinoline-N-oxide as well as compounds known to block the terminal oxidase impaired the oxidation of both H₂ and of NAD(P)H or succinate in a parallel fashion. No additivity of O₂ uptake was observed when NADPH, NADH or succinate was present in addition to H₂. Instead, H₂ uptake was depressed under such conditions, and also the oxidation of NAD(P)H or succinate was increasingly lowered by increasing H₂ tensions.The results suggest that in Anacystis molecular hydrogen is oxidized through the same type of respiratory chain as are NAD(P)H and succinate. Moreover, the cyanide-resistant branch of respiratory O₂ uptake will be discussed, and a few results obtained with particles prepared from thylakoid-free Anacystis will also be presented.Abbreviations BAL 2,3-dimercaptopropanol-(1) - DCPIP 2,6-dichlorophenolindophenol - HOQNO 2-n-heptyl-8-hydroxyquinoline-N-oxide - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - tricine N-tris-(hydroxymethyl)-methylglycine - Tris tris-(hydroxymethyl)-aminomethane - TTFA thenoyltrifluoroacetone NAD(P)H indicates NADPH and/or NADH     

A novel device for the assessment of shear effects on suspended microbial cultures

Summary A novel device has been designed for the evaluation of the effects of shear on microorganisms. The device consists of a combination of a coaxial cylinder plus cone and plate viscometers and enables cultures of both eukaryotic and prokaryotic microorganisms to be grown under fully defined and controlled fluid dynamic characteristics for serveral generations. In the preliminary tests performed a change in cell length was observed in Escherichia coli, and with Penicillium chrysogenum the septal length, hyphal diameter, and branching frequency all changes as shear was increased.Symbols L Height of liquid (m) - r Radius of cone/bob (m) - r _m Arithmetic mean of R and r (m) - R Radius of cup (m) - T Torque (Nm) - Shear rate (s^-1) - ₀ Angle of cone (rad) - Shear stress (Nm^-2) - Annular velocity (rad s^-1)     

Copper-containing nitrite reductase from Pseudomonas aureofaciens is functional in a mutationally cytochrome cd 1-free background (NirS−) of Pseudomonas stutzeri

The structural gene, nirK, for the respiratory Cu-containing nitrite reductase from denitrifying Pseudomonas aureofaciens was isolated and sequenced. It encodes a polypeptide of 363 amino acids including a signal peptide of 24 amino acids for protein export. The sequence showed 63.8% positional identity with the amino acid sequence of Achromobacter cycloclastes nitrite reductase. Ligands for the blue, type I Cu-binding site and for a putative type-II site were identified. The nirK gene was transferred to the mutant MK202 of P. stutzeri which lacks cytochrome cd ₁ nitrite reductase due to a transposon Tn5 insertion in its structural gene, nirS. The heterologous enzyme was active in vitro and in vivo in this background and restored the mutationally interrupted denitrification pathway. Transfer of nirK to Escherichia coli resulted in an active nitrite reductase in vitro. Expression of the nirS gene from P. stutzeri in P. aureofaciens and E. coli led to nonfunctional gene products. Nitrite reductase activity of cell extract from either bacterium could be reconstituted by addition of heme d ₁, indicating that both heterologous hosts synthesized a cytochrome cd ₁ without the d ₁-group.Abbreviations Cu-NIR Cu-containing nitrite reductase - DDC diethyldithiocarbamate - EPR electron paramagnetic resonance - IPTG isopropyl--D-galactoside - SDS sodium dodecyl sulfate - LB medium Luria-Bertani medium     

Reduction of cytochromes with menaquinol and sulfide in membranes from green sulfur bacteria

Reduction of cytochromes in chlorosome-free membranes of Chlorobia was studied anaerobically, with an LED array spectrophotometer. For Chlorobium tepidum these membranes contained 0.2 moles cytochrome per mole of bacteriochlorophyll a. The observed change upon complete reduction of oxidized membranes with dithionite could be satisfactorily fitted with three cytochrome components having absorption peaks at 553 (cyt c), 558 and 563 nm (cyt b), in relative amounts of 5:1:2. About 20% of total cytochrome 553 were reducible by ascorbate. Menaquinol reduced all of the 553-component, and this reduction was sensitive to stigmatellin, NQNO and antimycin A. The reduction was insensitive to KCN. However, it was transient at low concentrations of menaquinol in the absence of KCN, but permanent in its presence, demonstrating that electron transport into an oxidation pool was blocked. The 563-component was only slightly reduced by menaquinol unless NQNO or antimycin were present. The stimulation of cytochrome 563-reduction by these inhibitors was more pronounced in the presence of ferricyanide. This phenomenon reflects oxidant-induced reduction of cytochrome b and demonstrates that a Q-cycle is operative in Chlorobia. Also, sulfide fully reduced cytochrome 553, but more slowly than menaquinol. KCN inhibited in this case, as did stigmatellin, NQNO and antimycin A. NQNO was a better inhibitor than antimycin A. Cytochrome 563 again was hardly reduced unless antimycin A was added. The effect was more difficult to observe with NQNO. This supports the conclusion that sulfide oxidation proceeds via the quinone pool and the cytochrome bc-complex in green sulfur bacteria.Abbreviations BChl bacteriochlorophyll - cyt cytochrome - NQNO 2-n-nonyl-4-hydroxyquinoline-N-oxide - SQR sulfide-quinone reductase Dedicated to Prof. Dr. Aloys Wild on occasion of his 65th birthday.     

Photoaffinity labeling and partial purification of the putative plant receptor for the fungal wilt-inducing toxin,fusicoccin

The high-affinity fusicoccin-binding protein (FCBP) was solubilized from plasma-membrane vesicles prepared from leaves of Vicia faba L. by aqueous two-phase partitioning. Conditions for the solubilization of intact FCBP-radioligand complexes were worked out. About 60–70% of the complexes can be solubilized with 50–60 mM nonanoyl-N-methylglucamide in the presence of 1 mg· ml^-1 soybean phosphatidylcholine, type IV S, and 20% (v/v) glycerol at pH 5.5. The slow dissociation of the radioligand, 9-nor-fusicoccin-8-alcohol-[³H] from the FCBP at low temperatures permits the purification of FCBP-radioligand complexes at 4–10° C by fast protein liquid chromatography on anion-exchange and gel permeation columns. The FCBP, extracted from plasma membranes with cholate and chromatographed in the presence of this detergent, gave an apparent molecular mass (M_r) of 80±20 kDa on gel permeation columns under the conditions used. By comparison of the elution profiles of the fraction most enriched in FCBP-radioligand complexes with polypeptide patterns obtained on sodium dodecyl sulfate-polyacrylamide gels, a polypeptide with an M_r of approx. 34kDa co-separated with the radioactivity profile. A second, faint band of approx. 31 kDa was sometimes also observed co-electrophoresing. Photoaffinity labeling of plasma-membrane vesicles with the new compound 9-nor-8[(3,5-[³H]-4-azidobenzoy)ethylenediamine]-fusicoccin ([³H]ABE-FC) and subsequent separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis labeled a single band with an M_r of 35±1 kDa. Labeling in this band was strongly reduced when the membranes were incubated with [³H]ABE-FC in the presence of 0.1–1 M fusicoccin. From our data, we conclude (i) that the 34-35-kDa polypeptide represents the FCBP and (ii) that in detergent extracts of plasma membranes this polypeptide is probably present as a di- or trimeric structure.Abbreviations ABE-FC [(4-azidobenzoyl)-ethylenediamine]-fusicoccin - ABE-NHS (4-azidobenzoyl)-N-hydroxysuccinimide ester - FC fusicoccin - FCBP fusicoccin-binding protein - FCol 9-norfusicoccin-8-alcohol - MAB monoclonal antibody - Mega-9(10) nonanoyl(decanoyl)-N-methylglucamide - M_r apparent molecular mass - PMSF phenylmethyl-sulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Biosynthesis of pectic galactan by membrane-bound galactosyltransferase from soybean (<Emphasis Type="Italic">Glycine max</Emphasis> Merr.) seedlings

We investigated the properties of a galactosyltransferase (GalT) that is involved in the synthesis of -(14)-galactan side chains of pectins. A membrane preparation of etiolated 6-day-old soybean (Glycine max Merr.) hypocotyls transferred [¹⁴C]Gal from UDP-[¹⁴C]Gal into intact and partially hydrolyzed lupin -(14)-galactans of various chain lengths as exogenous acceptors, while activity to endogenous acceptors was negligible. Maximal activity occurred at pH 6.5 and 20–25°C in the presence of 25 mM Mn²⁺ and 0.75% Triton X-100. The transfer reaction onto the unmodified commercial pectic galactan (M _r>150,000) from lupin we used was very low but increased when the M _r of the galactan was reduced by partial acid hydrolysis. Among the partially hydrolyzed galactans, high-M _r (average M _r 60,000) -(14)-galactan was a more efficient acceptor [specific activity 2,000–3,000 pmol min^–1 (mg protein)^–1] than low-M _r (average M _r 10,000 and 5,000) polymers. Digestion of the radiolabeled product from high-M _r galactan with endo--(14)-galactanase released mainly radioactive -(14)-galactobiose and Gal, indicating that the transfer of [¹⁴C]Gal occurred through -(14)-linkages. HPLC analysis showed that the enzyme also catalyzes incorporation of Gal into pyridylaminated (PA) -(14)-galactooligomers with degree of polymerization at least 5. Gal₇-PA chains were elongated by attachment of one, two, or three Gal residues leading to the formation of Gal_8–10-PA.Abbreviations AGP Arabinogalactan-protein - Ara Arabinose - DP Degree of polymerization - GalA Galacturonic acid - Gal _n -PA Pyridylaminated -(14)-galactooligosaccharides - GalT Galactosyltransferase - MALDI–TOF–MS Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry - Rha Rhamnose Sugars described in this paper belong to the d-series unless otherwise noted     

Turbulent breakage of protein precipitates in mechanically stirred bioreactors

Experimental data relating to the breakage of isoelectric Soya protein precipitates in a mechanically agitated bioreactor are provided and examined in the light of a proposed mechanistic model which relates the size of the maximum attainable aggregate diameter to the energy dissipation rate in the vessel. The analysis suggests that protein precipitation results in the formation of scale-invariant fractal aggregates with a dimensionality of 2.2. Comparing the fractal dimensionality of the protein precipitates with reported values based on computer simulation studies suggests that the aggregates undergo considerable restructuring during agitation.List of Symbols A Hamaker constant (J) - D impeller diameter (m) - d _p primary particle diameter (m) - d _f maximum aggregate diameter (m) - G shear rate (s^–1) - H ₀ separation distance between two primary particles (m) - k constant in Eq. (5) - K constant in Eq. (6) - N impeller speed (rpm or rps) - r radial position in an aggregate, measured from the centre (m) - t time of exposure to shear (mins) - T _e eddy period (s^–1) - v _f aggregate volume (m³) Greek Symbols aggregate dimensionality constant - energy dissipation rate (W/kg) - dynamic viscosity of particle-free liquid (kg/ms) - kinematic viscosity of particle-free liquid (m²/s) - collision probability (–) - _p aggregate density (kg/m³) - _p continuous phase density (kg/m³) - aggregate mechanical strength (N/m²) - shear stress (N/m²) - particle concentration in an aggregate (m³/m³) - (r) porosity at radial position, r