Cytochromes,rubredoxin, and sulfur metabolism of the non-thiosulfate-utilizing green sulfur bacterium Pelodictyon luteolum

Two soluble c-type cytochromes (c-553 and c-555) and the nonheme iron-containing protein rubredoxin of the non-thiosulfate-utilizing green sulfur bacterium Pelodictyon luteolum were highly purified by ion exchange column chromatography, gel filtration and ammonium sulfate fractionation. Both cytochrome are small and basic hemoproteins, while rubredoxin is an acidic small nonheme iron protein. Cytochrome c-553 has a molecular weight of 13,000 determined by Sephacryl S-200 chromatography and of 10,700 by electrophoresis on SDS acrylamide gel, an isoelectric point at pH 10.2, a redox-potential of +220 mV. It shows maxima at 413 nm in the oxidized form, and the characteristic three maxima in the reduced state (-band at 553 nm, -band at 523 nm, -band at 417 nm). The best purity index (A ₂₈₀/A ₄₁₇) obtained was 0.18. Cytochrome c-555 (best purity index obtained: A ₂₈₀/A ₄₁₈=0.17) has an isoelectric point at pH 10.5, a molecular weight of 9,500 (by electrophoresis on SDS acrylamide gel) and a redox-potential of +160mV. The reduced form of this cytochrome shows the typical bands of c-type cytochromes at 555 (551) nm (-band), 523 nm (-band) and 418 nm (-band), while the oxidized form has the -band at 413 nm.Rubredoxin (best purity index obtained: A ₂₈₀/A ₄₉₀=3.5) is an acidic small protein. Its molecular weight estimated by gel filtration and SDS acrylamide gel electrophoresis is 27,000 and 6,300 respectively. The monomer of this protein contains one iron atom per molecule. Rubredoxin has an isoelectric point at pH 2.8 and shows maxima at 570 nm, 490 nm and 370 nm in the oxidized form.During anaerobic sulfide oxidation of a growing culture of Pelodictyon luteolum elemental sulfur is the first main product, which appears in the medium. Elemental sulfur is further oxidized to sulfate, after the available sulfide is completely consumed by the cells.Non-common abbreviations C Chlorobium - P Pelodictyon - SDS sodium dodecylsulfate - HIPIP high-potential-iron-sulfur-protein Offprint requests to: U. Fischer     

Functional and spectral characterization of the respiratory chain of Chloroflexus aurantiacus grown in the dark under oxygen-saturated conditions

In this paper we attempt a functional and spectral characterization of the membrane-bound cytochromes involved in respiratory electron transport by membranes from cells of Chloroflexus aurantiacus grown in the dark under oxygen saturated conditions. We conclude that the NADH-dependent respiration is carried out by a branched respiratory chain leading to two oxidases which differ in sensitivity to CN^- and CO. The two routes also show a different sensitivity to the ubiquinone analogue, HQNO, the pathway through the cytochrome c oxidase being fully blocked by 5 M HQNO, whereas the alternative one is insensitive to this inhibitor. The cytochrome c oxidase containing branch is composed by at least two c-type haems with E _m 7.0 of +130 and +270 mV ( bands at 550/553 nm and 549 nm, respectively), plus a b-type cytochrome with E _m 7.0 of +50 mV ( band at 561 nm). From this, and previous work, we conclude that respiratory and photosynthetic electron transport components are assembled together and function on a single undifferentiated plasma membrane.Abbreviations HQNO heptylhydroxy-quinoline-N-oxide - UHDBT undecyl-hydroxydioxobenthiazole - Q/b-c ubiquinol/cytochrome c oxidoreductase complex - BChl bacteriochlorophyll     

Cytochromes in Thiobacillus tepidarius and the respiratory chain involved in the oxidation of thiosulphate and tetrathionate

Thiobacillus tepidarius was shown to contain cytochrome(s) c with absorption maxima at 421, 522 and 552 nm in room temperature reduced minus oxidized difference spectra, present at 1.1–1.2 nmol per mg dry wt and present in both membrane and soluble fractions of the cell. The membrane-bound cytochrome c (1.75 nmol per mg membrane protein) had a midpoint potential (E_m, pH 7.0) of 337 mV, while the soluble fractions appeared to contain cytochrome(s) c with E_m (pH 7.0) values of about 270 and 360 mV. The organism also contained three distinct membrane-bound b-type cytochromes (totalling 0.33 nmol per mg membrane protein), each with absorption maxima in reduced minus oxidized difference spectra at about 428, 532 and 561 nm. The E_m (pH 7.0) values for the three cytochromes b were 8 mV (47.8% of total), 182 mV (13.7%) and 322 mV (38.5%). No a- or d-type cytochromes were detectable spectrophotometrically in the intact organism or its membrane and soluble fractions. Evidence is presented for both CO-binding and CO-unreactive cytochromes b or o, and CO-binding cytochrome(s) c. From redox effects observed with CO it is proposed that a cytochrome c donates electrons to a cytochrome b, and that a high potential cytochrome b or o may be acting as the terminal oxidase in substrate oxidation. This may be the 445 nm pigment, a photodissociable CO-binding membrane haemoprotein. Substrate oxidation was relatively insensitive to CO-inhibition, but strongly inhibited by cyanide and azide. Thiosulphate oxidation couples directly to cytochrome c reduction, but tetrathionate oxidation is linked (probably via ubiquinone Q-8) to reduction of a cytochrome b of lower potential than the cytochrome c. The nature of possible electron transport pathways in Thiobacillus tepidarius is discussed. One speculative sequence is: c^– b₈ b₁₈₂ c₂₇₀ c₃₃₇ b₃₂₂/c₃₆₀ O₂ Abbreviations E_m midpoint electrode potential - E _inf0 ^sup pH 7, standard electrode potential at pH 7.0 - Q-8 coenzyme Q-8 (ubiquinone-40)     

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     

Utilization of sulfide and elemental sulfur by Ectothiorhodospira halochloris

Ectothiorhodospira halochloris grows photoheterotrophically with a variety of sulfur sources. During sulfide oxidation to elemental sulfur considerable amounts of polysulfides may be accumulated transiently. When grown on elemental sulfur no sulfate was produced by oxidation, but sulfide and polysulfide were formed by reduction. Only one soluble cytochrome c-551 was isolated and purified. It was a small acidic hemeprotein with a molecular weight of 6,300, an isoelectric point of 3.1 and a redox potential of-11 mV at pH 7.0. It showed three absorption maxima in the reduced state (=551 nm; =523 nm; =417 nm). The addition of various c-type cytochromes to a suspension of spheroplasts stimulated the velocity of sulfide oxidation. This stimulation was best with the small acidic cytochromes from E. halochloris or Ectothiorhodospira abdelmalekii. Sulfide oxidation was stopped by several uncoupling agents, ionophores and electron transport inhibitors. Antimycin A, rotenone and cyanide had no effect on sulfide oxidation.Dedicated to Prof. Dr. H. G. Schlegel on the occasion of his 60th birthday     

Spectroscopic studies of bound cytochrome c and an iron-sulfur center in a purified reaction center complex from the green sulfur bacterium Chlorobium tepidum

Flash-induced optical kinetics at room temperature of cytochrome (Cyt) c ₅₅₁ and an Fe-S center (CF_A/CF_B) bound to a purified reaction center (RC) complex from the green sulfur photosynthetic bacterium Chlorobium tepidum were studied. At 551 nm, the flash-induced absorbance change decayed with a t _1/2 of several hundred ms, and the decay was accelerated by 1-methoxy-5-methylphenazinium methyl sulfate (mPMS). In the blue region, the absorbance change was composed of mPMS-dependent (Cyt) and mPMS-independent component (CF_A/CF_B) which decayed with a t _1/2 of 400–650 ms. Decay of the latter was effectively accelerated by benzyl viologen (Em –360 mV) and methyl viologen (–440 mV), and less effectively by triquat (–540 mV). The difference spectrum of Cyt c had negative peaks at 551, 520 and 420 nm, with a positive rise at 440 to 500 nm. The difference spectrum of CF_A/CF_B resembled P430 of PSI, and had a broad negative peak at 430435 nm.Abbreviations (B)Chl (bacterio)chlorophyll - Cyt cytochrome - F_A, F_B and F_X iron-sulfur center A, B and X of Photosystem I - CF_A, CF_B and CF_X F_A-,F_B- and F_X-like Fe-S center of Chlorobium - mPMS 1-methoxy-5-methylphenazinium methyl sulfate - PSI Photosystem I - RC reaction center     

Conformational Change of the Rieske [2Fe–2S] Protein inCytochrome bc 1 Complex

Structures of mitochondrial bc ₁ complex have been reported based on four different crystalforms by three different groups. In these structures, the extrinsic domain of the Rieske [2Fe–2S]protein, surprisingly, appeared at three different positions: the c ₁ position, where the [2Fe–2S]cluster exists in close proximity to the heme c ₁; the b position, where the [2Fe–2S] clusterexist in close proximity to the cytochrome b; and the intermediate position where the[2Fe–2S] cluster exists in between c ₁ and b positions. The conformational changes betweenthese three positions can be explained by a combination of two rotations; (1) a rotation of theentire extrinsic domain and (2) a relative rotation between the cluster-binding fold and thebase fold within the extrinsic domain. The hydroquinone oxidation and the electron bifurcationmechanism at the Q_P binding pocket of the bc ₁ complex is well explained using theseconformational changes of the Rieske [2Fe–2S] protein.     

Characterisation of cytochrome c6from Chlorella fusca

A c-type monohaem, cytochrome c₆was isolated from a soluble extract of the green alga Chlorella fusca. The isolated protein shows an apparent molecular mass of 10 kDa by SDS-PAGE, but behaves as a dimer of 20.3 kDa in gel-filtration; the isoelectric point is 3.6. The N-terminal sequence shows high identity with other green algae cytochromes c₆. The mid-point redox potential is about +350 mV between pH 5 and 9. The ferric and ferrous forms, and their pH equilibria, have been studied using visible, CD and EPR spectroscopies. The visible spectrum of the reduced cytochrome c₆is typical of a c-type haem protein, with maxima at 274 nm, 318 nm (-peak), 416 nm (-peak), 522 nm (-peak), 552–553 nm (-peak). A 690 nm band, characteristic of a haem Met-His axial coordination of the haem group, is present in the oxidized form. At high pH values ( 8), cytochrome c₆undergoes an alkaline transition, with a pKa of 8.7. Between pH 3 and 9 the EPR spectrum is dominated by two rhombic species, with g-values at 3.32, 2.05, 1.05 and 2.96, 2.30, 1.43, which interconvert with a pK_aof 4. CD spectrum of Chlorella fusca cytochrome c₆shows that the proteins must be mainly built up by -helices. Even though there are similarities between Chlorella fusca cytochrome c₆and that isolated from Monoraphidium braunii, no cross-reactivity with the antibodies raised against the Chlorella fusca cytochrome has been detected for the protein from Monoraphidium braunii.     

The Cytochrome cbo from the Obligate Methylotroph Methylobacillus flagellatus KT Is a Cytochrome c Oxidase

The cbo-type oxidase of Methylobacillus flagellatus KT was purified to homogeneity by preparative native gel electrophoresis, and the kinetic properties and substrate specificity of the enzyme were studied. Ascorbate and ascorbate/N,N,N,N-tetramethyl-p-phenylenediamine (TMPD) were oxidized by cytochrome cbo with a pH optimum of 8.3. With TMPD as an electron donor for the cbo-type oxidase, the optimal pH (7.0 to 7.6) was determined from the difference between respiration rates in the presence of ascorbate/TMPD and only ascorbate. The kinetic constants determined at pH 7.0 were as follows: oxidation by the enzyme of reduced TMPD was characterized by K _M = 0.86 mM and V _max = 1.1 mol O₂/(min mg protein), and oxidation of reduced horse heart cytochrome c was characterized by K _M = 0.09 mM and V _max = 0.9 mol O₂/(min mg protein). Cyanide inhibited ascorbate/TMPD–oxidase activity (K _i = 4.5–5.0 M). The soluble cytochrome c _H (12 kDa), partially purified from M. flagellatus KT, was found to serve as a natural electron donor for the cbo-type oxidase.     

A flash-photolysis study of the reactions of acaa 3-ttype cytochrome oxidase with dioxygen and carbon monoxide

The time course of absorbance changes following flash photolysis of the fully-reduced carboxycytochrome oxidase fromBacillus PS3 in the presence of O₂ has been followed at 445, 550, 605, and 830 nm, and the results have been compared with the corresponding changes in bovine cytochrome oxidase. The PS3 enzyme has a covalently bound cytochromec subunit and the fully-reduced species therefore accommodates five electrons instead of four as in the bovine enzyme. In the bovine enzyme, following CO dissociation, four phases were observed with time constants of about 10 s, 30 s, 100 s, and 1 ms at 445 nm. The initial, 10-s absorbance change at 445 nm is similar in the two enzymes. The subsequent phases involving hemea and Cu_A are not seen in the PS3 enzyme at 445 nm, because these redox centers are re-reduced by the covalently bound cytochromec, as indicated by absorbance changes at 550 nm. A reaction scheme consistent with the experimental observations is presented. In addition, internal electron-transfer reactions in the absence of O₂ were studied following flash-induced CO dissociation from the mixed-valence enzyme. Comparisons of the CO recombination rates in the mixed-valence and fully-reduced oxidases indicate that more electrons were transferred from hemea ₃ toa in PS3 oxidase compared to the bovine enzyme.     

Synthetic substrate analogues for UDP-GlcNAc: Manα1-6R β(1-2)-N-acetylglucosaminyltransferase II. Substrate specificity and inhibitors for the enzyme

UDP-GlcNAc: Man1-6R (1-2)-N-acetylglucosaminyltransferase II (GlcNAc-T II; EC 2.4.1.143) is a key enzyme in the synthesis of complexN-glycans. We have tested a series of synthetic analogues of the substrate Man1-6(GlcNAc1-2Man1-3)Man-O-octyl as substrates and inhibitors for rat liver GlcNAc-T II. The enzyme attachesN-acetylglucosamine in 1-2 linkage to the 2-OH of the Man1-6 residue. The 2-deoxy analogue is a competitive inhibitor (K _i=0.13mm). The 2-O-methyl compound does not bind to the enzyme presumably due to steric hindrance. The 3-, 4- and 6-OH groups are not essential for binding or catalysis since the 3-, 4- and 6-deoxy and -O-methyl derivatives are all good substrates. Increasing the size of the substituent at the 3-position to pentyl and substituted pentyl groups causes competitive inhibition (K _i=1.0–2.5mm). We have taken advantage of this effect to synthesize two potentially irreversible GlcNAc-T II inhibitors containing a photolabile 3-O-(4,4-azo)pentyl group and a 3-O-(5-iodoacetamido)pentyl group respectively. The data indicate that none of the hydroxyls of the Man1-6 residue are essential for binding although the 2- and 3-OH face the catalytic site of the enzyme. The 4-OH group of the Man-O-octyl residue is not essential for binding or catalysis since the 4-deoxy derivative is a good substrate; the 4-O-methyl derivative does not bind. This contrasts with GlcNAc-T I which cannot bind to the 4-deoxy-Man- substrate analogue. The data are compatible with our previous observations that a bisectingN-acetylglucosamine at the 4-OH position prevents both GlcNAc-T I and GlcNAc-T II catalysis. However, in the case of GlcNAc-T II, the bisectingN-acetylglucosamine prevents binding due to steric hindrance rather than to removal of an essential OH group. The 3-OH of the Man1-3 is an essential group for GlcNAc-T II since the 3-deoxy derivative does not bind to the enzyme. The trisaccharide GlcNAc1-2Man1-3Man-O-octyl is a good inhibitor (K _i=0.9mm). The above data together with previous studies indicate that binding of the GlcNAc1-2Man1-3Man- arm of the branched substrate to the enzyme is essential for catalysis. Abbreviations: GlcNAc-T I, UDP-GlcNAc:Man1-3R (1-2)-N-acetylglucosaminyltransferase I (EC 2.4.1.101); GlcNAc-T II, UDP-GlcNAc:Man1-6R (1-2)-N-acetylglucosaminyltransferase II (EC 2.4.1.143); MES, 2-(N-morpholino)ethane sulfonic acid monohydrate.     

Cloning and comparison of Aα mating-type alleles of the basidiomycete Schizophyllum commune

Summary An A mating-type allele (A4) was isolated by walking the chromosome from the closely linked PAB1 gene. A cosmid clone containing the A1 allele isolated from the walk was used as a probe to recover the A1 allele from another cosmid library. Cosmids encoding mating-type activity were identified by transforming Schizophyllum cells and screening for activation of A-regulated development. Putative mating-type transformants were confirmed in mating tests and genetic analyses of progeny. The identity of the specific alleles isolated was demonstrated by showing that their effectiveness in transforming for mating type is limited to recipient strains possessing an A allele different from the one encoded by the cloned sequences. Transforming DNA is active in trans, suggesting that A encodes a diffusible product. Restriction mapping shows that A1 and A4 are coded in the same physical region of the genome, but within a subregion that contains extensive sequence divergence. In addition, Southern analyses show that there is only one copy of A1 or A4 per haploid genome, and that they do not cross-hybridize to one another or to any of the other A alleles. A1 and A4 were subcloned as 2.8 and 1.2 kb fragments, respectively, retaining in transformation all the mating-type activity demonstrated of the original cosmids.     

Biogenesis of mitochondrialc-type cytochromes

Cytochromesc andc ₁ are essential components of the mitochondrial respiratory chain. In both cytochromes the heme group is covalently linked to the polypeptide chain via thioether bridges. The location of the two cytochromes is in the intermembrane space; cytochromec is loosely attached to the surface of the inner mitochondrial membrane, whereas cytochromec ₁ is firmly anchored to the inner membrane. Both cytochromec andc ₁ are encoded by nuclear genes, translated on cytoplasmic ribosomes, and are transported into the mitochondria where they become covalently modified and assembled. Despite the many similarities, the import pathways of cytochromec andc ₁ are drastically different. Cytochromec ₁ is made as a precursor with a complex bipartite presequence. In a first step the precursor is directed across outer and inner membranes to the matrix compartment of the mitochondria where cleavage of the first part of the presequence takes place. In a following step the intermediate-size form is redirected across the inner membrane; heme addition then occurs on the surface of the inner membrane followed by the second processing reaction. The import pathway of cytochromec is exceptional in practically all aspects, in comparison with the general import pathway into mitochondria. Cytochromec is synthesized as apocytochromec without any additional sequence. It is translocated selectively across the outer membrane. Addition of the heme group, catalyzed by cytochromec heme lyase, is a requirement for transport. In summary, cytochromec ₁ import appears to follow a conservative pathway reflecting features of cytochromec ₁ sorting in prokaryotic cells. In contrast, cytochromec has invented a rather unique pathway which is essentially non-conservative.     

Evolutionary relationship between Enterobacteriaceae: comparison of the ATP synthases (F1F0) of Escherichia coli and Klebsiella pneumoniae

The ATP synthase complex of Klebsiella pneumoniae (KF₁F₀) has been purified and characterized. SDS-gel electrophoresis of the purified F₁F₀ complexes revealed an identical subunit pattern for E. coli (EF₁F₀) and K. pneumoniae. Antibodies raised against EF₁ complex and purified EF₀ subunits recognized the corresponding polypeptides of EF₁F₀ and KF₁F₀ in immunoblot analysis. Protease digestion of the individual subunits generated an identical cleavage pattern for subunits , , , , a, and c of both enzymes. Only for subunit different cleavage products were obtained. The isolated subunit c of both organisms showed only a slight deviation in the amino acid composition. These data suggest that extensive homologies exist in primary and secondary structure of both ATP synthase complexes reflecting a close phylogenetic relationship between the two enterobacteric tribes.Abbreviations ACMA 9-amino-6-chloro-2-methoxyacridine - DCCD N,N-dicyclohexylcarbodiimide - FITC fluorescein isothiocyanate - SDS sodium dodecyl sulfate - TTFB 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole     

Spectral characterization of c-type cytochromes purified from Beggiatoa alba

Three c-type cytochromes were purified from the filamentous sulfur-oxidizing bacterium, Beggiatoa alba strain B18LD, by ammonium sulfate fractionation, flat bed isoelectric focusing and gel filtration. Two of the cytochromes; flavocytochrome c-554 and cytochrome c, were similar to cytochromes found in anoxygenic photosynthetic bacteria. Flavocytochrome c-554 had an apparent molecular weight of 21,000, an isoelectric focusing point at pH 4.4, contained FMN as the flavin component and had absorption maxima at 410, 450 and 470 nm in the oxidized form and at 417, 523 and 554 nm in the dithionite-reduced from. Cytochrome c was also an acidic protein with a pI of 4.8 and an apparent molecular weight of 18,000. The absorption spectra maxima were at 400, 490 and 635 nm in the oxidized form, at 424 and 550 nm in the dithione-reduced form and at 415 and 555 nm in the dithionite-reduced plus CO form. The third cytochrome characterized, cytochrome c-553 had an apparent molecular weight of 13,000, an isoelectric point at pH 4.4 and showed absorption maxima at 411 nm in the oxidized form and at 418, 523 and 553 nm in the dithionite-reduced form. Cytochrome c-553 was also isolated as a complex with a non-heme protein with a molecular weight of 16,000. The non-heme protein altered the absorption spectra and isoelectric point of cytochrome c-553.Abbreviations IEF isoelectric focusing - M _r molecular weight - pI isoelectric point     

An isolated reaction center complex from the green sulfur bacterium Chlorobium vibrioforme can photoreduce ferredoxin at high rates

Chlorosome-depleted membranes and a reaction center complex with well-defined subunit composition were prepared from the green sulfur bacterium Chlorobium vibrioforme under anaerobic conditions. The reaction center complex contains a 15-kDa polypeptide with the N-terminal amino acid sequence MEPQLSRPETASNQVR/. This sequence is nearly identical to the N-terminus of the pscD gene product from Chlorobium limicola (Hager-Braun et al. (1995) Biochemistry 34: 9617–9624). In the presence of ferredoxin and ferredoxin:NADP⁺ oxidoreductase, the membranes and the isolated reaction center complex photoreduced NADP⁺ at rates of 333 and 110 mol (mg bacteriochlorophyll a)^–1 h^–1, respectively. This shows that the isolated reaction center complex contains all the components essential for steady state electron transport. Midpoint potentials at pH 7.0 of 160 mV for cytochrome c ₅₅₁ and of 245 mV for P840 were determined by redox titration. Antibodies against cytochrome c ₅₅₁ inhibit NADP⁺ reduction while antibodies against the bacteriochlorophyll a-binding Fenna-Matthews-Olson protein do not.Abbreviations FMO protein Fenna-Matthews-Olson protein - TMBZ 3,3,5,5-tetramethylbenzidine     

Vectorial electron transport in Degulfovibrio vulgaris (Marburg) growing on hydrogen plus sulfate as sole energy source

Desulfovibrio vulgaris (Marburg) was grown on hydrogen plus sulfate as sole energy source in a medium containing excess iron. The topography of electron transport components was investigated. The bacterium contained per mg cells (dry weight) 30U hydrogenase (1U=1 mol/min), 35 g desulfoviridin (= bisulfite reductase), 0.6 U adenosine phosphosulfate reductase, 30 mU thiosulfate reductase, 0.3 nmol cytochrome c ₃ (M _r=13,000), 0.04 nmol cytochrome b, 0.85 nmol menaquinone, and 0.4 nmol ferredoxin. Hydrogenase (>95%) and cytochrome c ₃ (82%) were localized on the periplasmic side and desulfoviridin (95%), adenosine phosphosulfate reductase (87%), thiosulfate reductase (74%), and ferredoxin (71%) on the cytoplasmic side of the cytoplasmic membrane; menaquinone and cytochrome b were exlusively found in the membrane fraction. The location of the oxidoreductases indicate that in D. vulgaris (Marburg) H₂ oxidation and sulfate reduction take place on opposite sides of the cytoplasmic membrane rather than on the same side, as has recently been proposed.     

The enzymatic system thiosulfate: Cytochrome c oxidoreductase from photolithoautotrophically grown Rhodopseudomonas palustris

Rhodopseudomonas palustris cells, characterized by a lamellar type intracytoplasmic chromatophore membrane system after phototrophic growth, yielded a crude supernatant cell-free fraction (S-144) after ultracentrifugation which retained the contents of both the cell compartments. After thiosulfate-dependent growth, a protein system was isolated from S-144 which catalyzed the thiosulfate-linked reduction of an endogenous c-type cytochrome. — The colorless oxidoreductase protein, after purification to homogeneity, revealed a molecular weight of 93,000 and, after SDS treatment, a particle weight of 48,000. It was focused at an average pI of 5.45. Apparent K _m values for several substrates were in the M range. The electron acceptor for thiosulfate oxidation was found to be a cytochrome c from S-144. The homogeneous acceptor protein, at liquid nitrogen temperature, exhibited absorption maxima at 549.0, 518.5 and 418.0 nm, and shoulders at 525.5, 512.0 and 508.0 nm. Its molecular weight was found to be 17,000 (gel filtration) and 16,000 (SDS gel electrophoresis). It was characterized by a pI of 10.0. Its midpoint redox potential of E _m,7.0=+228 mV was determined by redox titrations and the value of +205 mV by spectrophotometric calculations.Abbreviations BSA bovine serum albumin - HiPIP high potential nonheme iron protein - IEF isoclectric focusing - SDS dodecylsulfate, sodium salt - Temed N,N,N,N-tetramethylethylenediamine     

Identification of two RAPD markers tightly linked with the Nicotiana debneyi gene for resistance to black root rot of tobacco

Linkage of randomly amplified polymorphic DNA (RAPD) markers with a single dominant gene for resistance to black root rot (Chalara elegans Nag Raj and Kendrick; Syn. Thielaviopsis basicola [Berk. and Broome] Ferraris) of tobacco (Nicotiana tabacum L.), which was transferred from N. debneyi Domin, was investigated in this study. There were 2594 repeatable RAPD fragments generated by 441 primers on DNAs of Delgold tobacco, a BC₅F₈ near isogenic line (NIL) carrying the resistance gene in a Delgold background, and PB19, the donor parent of the resistance gene. Only 7 of these primers produced eight RAPD markers polymorphic between Delgold and PB19, indicating there are few RAPD polymorphisms between them despite relatively dissimilar pedigrees. Five of the eight RAPD markers were not polymorphic between Delgold and the NIL. All of these markers proved to be unlinked with the resistance gene in F₂ linkage tests. Of the remaining three RAPD markers polymorphic between Delgold and the NIL, two were shown to be strongly linked with the resistance gene; one in coupling and the other in repulsion. Application of the two RAPDs in the elimination of linkage drag associated with the N. debneyi resistance gene and marker-assisted selection for the breeding of new tobacco cultivars with the resistance gene is discussed.