Extracellular phosphomannan as a phosphate reserve in the yeast Kuraishia capsulata

We have found that extracellular phosphomannan is the main phosphate reserve in the yeast Kuraishia capsulata, in contrast to other yeast species effectively absorbing P_i. Under nitrogen starvation, K. capsulata absorbed essentially all P_i from the medium containing 240 mM glucose, 2.5 mM MgSO₄, and 11 mM KH₂PO₄. Inorganic polyphosphate level in the cells was about 14% of the P_i absorbed. Most of the P_i (～60%) was found in the fraction of extracellular phosphomannan that can be used as a carbon and phosphorus source by this yeast in deficient media.     

Properties and regulation of synthesis of two ferredoxins from Rhodopseudomonas capsulata

Two ferredoxins from nitrogen-fixing cells of the phototrophic bacterium Rhodopseudomonas capsulata, strain B10, are purified to a homogeneous state and characterized. The molecular mass of ferredoxin I is about 12 kDa and that of ferredoxin II, 18 kDa. Ferredoxin I contains 8 Fe²⁺ and 8 S^2?; ferredoxin II has 4 Fe²⁺ and 4 S^2? per molecule. The redox potential of ferredoxin I is about ?270 mV and that of ferredoxin II ?419 mV. Ferredoxin I is more labile to the action of O₂, O^?₂, H₂O₂ and heating. The ferredoxins are also different in their absorption and EPR spectra, amino acid composition and electron-transfer activity to Rps. capsulata nitrogenase: both C₂H₂ reduction and H₂ evolution by Rps. capsulata nitrogenase proceed faster in the presence of ferredoxin I than in case of ferredoxin II. Synthesis of ferredoxin I takes place only in Rps. capsulata nitrogen-fixing cells grown in light under anaerobic conditions whereas ferredoxin II formation does not depend on the source of nitrogen or the growth medium, though the amount of ferredoxin II varies with the growth conditions. Its highest level has been found in the cells grown in lactate-limited medium in the presence of CO₂ and light or in the presence of glutamate in darkness under anaerobic conditions.     

Structure of D-glyceraldehyde-3-phosphate dehydrogenase fromPalinurus versicolor in a tetragonal crystal form

D-glyceraldehyde-3-phosphate dehydrogenase (holo-GAPDH) from Palinurus versicolor was crystallized in a novel crystal form by the method of sitting-drop vapor diffusion. The crystals have space group P4212, cell parameters a=15.49 nm, c=8.03 nm and two subunits per asymmetric unit. The crystal structure at 0.34 nm was determined by the molecular replacement method. The final model has crystallographic Rfree and R factors of 0.274 and 0.262, and r.m.s. deviations of 0.002 nm for bond lengths and 2.33?for bond angles. The two subunits in asymmetric unit are similar to each other not only in the three-dimensional structure, but also in average temperature factors. This result demonstrates that the obvious difference in average temperature factors for the different subunits in C2 crystal form reported previously may be attributed to the different crystallographic environments of the subunits. This further supports that holo-GAPDH has a good 222 molecular symmetry.     

Asymmetry of an energy transducing membrane. The location of cytochrome c2 in Rhodopseudomonas spheroides and Rhodopseudomonas capsulata

Monospecific antibodies have been prepared against cytochrome c₂ from Rhodopseudomonas spheroides and Rhodopseudomonas capsulata, and against cytochrome c′ from Rps. capsulata. These antibodies precipitated their respective antigens, but did not cross react with a wide range of procaryotic or eucaryotic cytochromes, or with other bacterial proteins. The cytochromes produced during aerobic growth were immunologically indistinguishable from those produced during photosynthetic growth.Cytochrome c₂ is located in vivo in the periplasmic space between the cell wall and the cell membrane, and when chromatophores are prepared from whole cells the cytochrome becomes trapped inside these vesicles. The implications of these results to energy coupling in the photosynthetic bacteria are discussed.     

Photoproduction of H2 from Cellulose by an Anaerobic Bacterial Coculture

Cellulomonas sp. strain ATCC 21399 is a facultatively anaerobic, cellulose-degrading microorganism that does not evolve hydrogen but produces organic acids during cellulose fermentation. Rhodopseudomonas capsulata cannot utilize cellulose, but grows photoheterotrophically under anaerobic conditions on organic acids or sugars. This report describes an anaerobic coculture of the Cellulomonas strain with wild-type R. capsulata or a mutant strain lacking uptake hydrogenase, which photoevolves molecular hydrogen by the nitrogenase system of R. capsulata with cellulose as the sole carbon source. In coculture, the hydrogenase-negative mutant produced 4.6 to 6.2 mol of H₂ per mol of glucose equivalent, compared with 1.2 to 4.3 mol for the wild type.     

A preliminary crystallographic study of β-glucuronidase from rat preputial gland

Large single crystals of rat preputial gland β-glucuronidase have been grown by dialysis against 2-methyl-2,4-pentanediol. The crystals are tetragonal, space group P4₁2₁2 or P4₃2₁2 with cell dimensions $\text{a = 103.5}\text{A}\text{?}\text{and}\text{c = 279.8}\text{A}\text{?}$. Each asymmetric unit contains one half of a tetramer composed of subunits of 69,000 molecular weight.     

Crystallization and preliminary crystallographic data of rabbit uteroglobin

Uteroglobin is a steroid-binding protein of 15,800 molecular weight, composed of two chemically identical subunits which, in the oxidized form, are covalently linked by disulphide bridges. Large crystals have been grown from ammonium sulphate solutions by vapour diffusion as well as by equilibrium dialysis. The crystals are very stable under X-rays, diffract to at least 2.2 Å resolution and belong to space group P2₁. The unit cell, with dimensions $\text{a = 43.3}\text{A}\text{?}\text{, b = 31.1}\text{A}\text{?}\text{, c = 34.5}\text{A}\text{?}\text{, and}\text{β = 90.7 °}$, contains two dimeric molecules. The crystals exhibit a prominent pseudo symmetry corresponding to space group P2₁2₁2 which indicates that the two subunits should be structurally nearly identical.     

Manganese superoxide dismutases from Escherichia coli and from yeast mitochondria: Preliminary X-ray crystallographic studies

The Mn superoxide dismutase from Escherichia coli has been obtained in three crystal forms: (I) from 68% saturated (NH₄)₂SO₄, space group P222 or P222₁, $\text{a = 47}\text{A}\text{?}\text{, b = 103}\text{A}\text{?}\text{, c = 47.5}\text{A}\text{?}$, with one subunit per asymmetric unit; (II) from 50% polyethylene glycol 6000, space group C222₁ (with approx. P4₁2₁2 symmetry), $\text{a = 101}\text{A}\text{?}\text{, b = 108}\text{A}\text{?}\text{, c = 180}\text{A}\text{?}$, with four subunits (2 molecules) per asymmetric unit; (III) from 52% polyethylene glycol with a different method of preparing the enzyme solution, space group P2₁2₁2, $\text{a = 47}\text{A}\text{?}\text{, b = 51}\text{A}\text{?}\text{, c = 188}\text{A}\text{?}$, with two subunits per asymmetric unit.The yeast mitochondrial Mn superoxide dismutase has yielded the same crystal form both from 30% 2-methyl-2,4-pentane diol and from 23% polyethylene glycol 6000: space group P2₁2₁2₁, $\text{a = 63}\text{A}\text{?}\text{, b = 115}\text{A}\text{?}\text{, c = 125}\text{A}\text{?}$, with four subunits (one molecule) per asymmetric unit.A full X-ray crystallographic study of at least one of these enzymes is planned.     

Energy transduction in photosynthetic bacteria. VIII. Activation of the energy-transducing ATPase by inorganic phosphate

ATPase activity and ATP-induced energization of photosynthetic membranes from Rhodopseudomonas capsulata are stimulated by phosphate; the maximum stimulatory effect occurs at a concentration between 1 and 2 mM.The sensitivity of the ATPase to oligomycin increases in the presence of phosphate since all the P_i-stimulated activity is inhibited by this antibiotic. Aurovertin, which has no effect on ATPase in the absence of phosphate, inhibits completely the activity elicited by this anion.The addition of P_i induces a substantial increase in the V of ATPase activity without changing the affinity of the enzyme for ATP or ADP.Arsenate, at the same concentrations, produces effects very similar to those of phosphate. The stimulation by arsenate of the transfer of energy from ATP to the membrane suggests a non-hydrolytic role of this anion as a modifier of the ATPase activity.     

Unit cell and space group of catalase from Micrococcus luteus.

Octahedral shaped crystals of about 1 mm were grown from catalase of Micrococcus luteus. The space group was determined as P4₂2₁2₁ with $\text{a = b = 106.6}\text{A}\text{?}\text{and}\text{c = 106.3}\text{A}\text{?}$. In contrast to mammalian catalase, the bacterial catalase contains only one subunit per asymmetric unit. This proves that the four subunits of bacterial catalase are identical.     

Characterization of the gene transfer agent made by an overproducer mutant of Rhodopseudomonas capsulata.

Most wild-type isolates of the photosynthetic bacterium Rhodopseudomonas capsulata spontaneously produce nucleoprotein particles that act as vectors of genetic exchange in this species. The low yield of these particles, termed gene transfer agents, has made their characterization difficult. We have utilized a new screening technique to select a mutant, Y262, which produces about three orders of magnitude more GTA² per culture. The GTA produced by Y262 seem to be identical to those of wild type by immunological, genetic and sedimentation analyses, and they have been further characterized by a variety of techniques. GTA resemble tailed bacteriophage, although GTA with a head diameter of 30 nm are smaller than most morphologically similar viruses. DNA isolated from GTA is in the form of linear duplex molecules of about 3 × 10⁶ molecular weight. There is no phage-like DNA in GTA particles as judged by C_ot and restriction endonuclease analyses. Instead, GTA DNA shows the same kinetic complexity and restriction endonuclease digestion pattern as the R. capsulata genome. The GTA of ten independently isolated wild-type strains of R. capsulata show a high degree of immunological cross-reactivity; thus this genetic exchange system, while not a species-wide trait, does seem to be the product of a conservative evolutionary process.     

Crystal structure of a bacterial protein proteinase inhibitor (Streptomyces subtilisin inhibitor) at 2.6 A resolution

The crystal structure of a bacterial protein proteinase inhibitor (Streptomyces subtilisin inhibitor) was solved at 2·6 Å resolution. Each subunit of the dimeric inhibitor has a five-stranded antiparallel β-sheet and two short α-helices. The subunit-subunit interface formed by a stack of two β-sheets provided by the two subunits resembles the dimer-dimer interface of concanavalin A. Conformation of the reactive site around the scissible bond Met73-Val74 seems very rigid. Between bovine pancreatic trypsin inhibitor (Kunitz) and the Streptomyces inhibitor, the reactive site conformations are almost identical with each other from the P₂ to P₂′ residues, while between the soybean trypsin inhibitor (Kunitz) and the Streptomyces inhibitor they are similar from the P₂ to P₁′ residues. There are overall similarities in conformation extending from the P₃ to P₂′ residues between the Streptomyces inhibitor and a hypothetical substrate presumed (Robertus et al., 1972b) to be bound to subtilisin BPN′ in a productive binding mode. Apart from the reactive site, there seems to be no structural relationship among the Streptomyces, bovine pancreatic and soybean inhibitors, suggesting their convergent evolution from separate ancestral proteins.     

Assessment of the metal removal capability of two capsulated cyanobacteria, Cyanospira capsulata and Nostoc PCC7936

Two capsulated, exopolysaccharide-producing cyanobacteria, Cyanospira capsulata and Nostoc PCC7936, were tested with regard to their metal removal capability by using copper as model metal. The experiments, carried out with the sole cyanobacterial biomass suspended in distilled water and confined into small dialysis tubings, showed that C. capsulata biomass is characterized by the best efficiency in metal removal, with a q_max (maximum amount of copper removed per biomass unit) of 96 ± 2 mg Cu(II) removed per g of protein in comparison with the value of 79 ± 3 of Nostoc PCC7936 biomass. The experimental data obtained with both cyanobacterial biomass best fit the Langmuir sorption isotherm. The sorption of copper started from the first minutes of contact with the metal and attained the equilibrium state, when no more copper removal was evident, after 5 and 6 hours, for C. capsulata and Nostoc PCC7936, respectively. The best efficiency in Cu(II) removal was obtained at pH 6.1–6.2, while the presence of Mg²⁺ or Ca²⁺ reduced copper removal capability of both species to 60–70% of their q_max. The results showed that the biomass of C. capsulata and Nostoc PCC7936 possesses a high affinity and a high specific uptake for copper, comparable with the best performances shown by other microbial biomass, and suggest the possibility to use the capsulated trichomes of the two cyanobacteria for the bioremoval of heavy metals from polluted water bodies.     

Light-induced oxygen reduction as a probe of electron transport between respiratory and photosynthetic components in membranes of Rhodopseudomonas capsulata

Membrane vesicles prepared from photosynthetically grown cells of Rhodopseudomonas capsulata strain M7 are able to perform light-induced oxygen uptake. In contrast, chromatophores from mutant strain M6, lacking the cytochrome b₂₆₀-containing pathway of oxygen reduction, are completely devoid of oxygen uptake induced by light. Therefore, cytochrome b₂₆₀ (cyt b₂₆₀), previously demonstrated to be the alternative oxidase in photosynthetic and aerobic membranes of R. capsulata (La Monica, R. F., and Marrs, B. L. (1976) Biochim. Biophys. Acta449, 431–439; Zannoni, D., Melandri, B. A., and Baccarini-Melandri, A. (1976) Biochim. Biophys. Acta449, 386–400), also appears to be involved in light-induced oxygen uptake. Light-driven oxygen reduction activity is inhibited by high concentrations of cyanide (5 × 10^?3m) and by carbon monoxide in accord with the sensitivity of cyt b₂₆₀ to these inhibitors reported in aerobic membranes (Zannoni, D., Melandri, B. A., and Baccarini-Melandri, A. (1976) Biochim. Biophys. Acta423, 413–430). Further evidence for a direct involvement of the cyt b₂₆₀-containing pathway in light-induced oxygen uptake has been obtained by comparing membranes from semiaerobically grown cells to those from photosynthetic cells of R. capsulata M7. The substrate-dependent dark oxidase activity associated with the cyt b₂₆₀-containing pathway is 4.6 times higher in semiaerobic than in photosynthetic membranes, and a parallel enhancement of light-induced oxygen uptake is observed. The data presented are in agreement with and extend previous results on the composition and function of the respiratory and photosynthetic apparatus, supporting an association of cytochrome b₂₆₀ with both the aerobic and photosynthetic systems present in membranes of R. capsulata. These findings clearly demonstrate that respiratory electron carriers have access to electrons flowing from the photosynthetic reaction center, i.e., the two systems are “electrically connected” in membrane fragments from this organism.     

Crystallization of cowpea chlorotic mottle virus

Cowpea chlorotic mottle virus belongs to the brome mosaic virus group of the small spherical plant viruses. It contains 180 protein subunits, which are arranged on a T = 3 icosahedral surface lattice. The virus crystallizes in orthorhombic space group P2₁2₁2 ($\text{a = 394}\text{A}\text{?}\text{, b = 382}\text{A}\text{?}\text{,}\text{and}\text{c = 397}\text{A}\text{?}$). The unit cell contains four virus particles, while the crystallographic asymmetric unit consists of one complete virion. X-ray diffraction data from the crystals extend to nearly 3.0 Å resolution.     

Structural role of bacterioruberin in the trimeric structure of archaerhodopsin-2

Archaerhodopsin-2 (aR2), a retinal protein-carotenoid complex found in the claret membrane of Halorubrum sp. aus-2, functions as a light-driven proton pump. In this study, the membrane fusion method was utilized to prepare trigonal P321 crystals (a = b = 98.2 Å, c = 56.2 Å) and hexagonal P6₃ crystals (a = b = 108.8 Å, c = 220.7 Å). The trigonal crystal is made up of stacked membranes in which the aR2 trimers are arranged on a honeycomb lattice. Similar membranous structures are found in the hexagonal crystal, but four membrane layers with different orientations are contained in the unit cell. In these crystals, the carotenoid bacterioruberin [5,32-bis(2-hydroxypropan-2-yl)-2,8,12,16,21,25,29,35-octamethylhexatriaconta-6,8,10,12,14,16,18,20,22,24,26,28,30-tridecaene-2,35-diol] binds to crevices between the subunits of the trimer. Its polyene chain is inclined from the membrane normal by an angle of about 20° and, on the cytoplasmic side, it is surrounded by helices AB and DE of neighbouring subunits. This peculiar binding mode suggests that bacterioruberin plays a striking structural role for the trimerization of aR2. When compared with the aR2 structure in another crystal form containing no bacterioruberin, the proton release channel takes a more closed conformation in the P321 or P6₃ crystal; i.e., the native conformation of protein is stabilized in the trimeric protein-bacterioruberin complex. Interestingly, most residues participating in the trimerization are not conserved in bacteriorhodopsin, a homologous protein capable of forming a trimeric structure in the absence of bacterioruberin. Despite a large alteration in the amino acid sequence, the shape of the intratrimer hydrophobic space filled by lipids is highly conserved between aR2 and bacteriorhodopsin. Since a transmembrane helix facing this space undergoes a large conformational change during the proton pumping cycle, it is feasible that trimerization is an important strategy to capture special lipid components that are relevant to the protein activity.     

Light-induced electron transport pathways in membrane preparations from Rhodopseudomonas capsulata

The photosynthetic electron transport chain in Rhodopseudomonas capsulata cells was investigated by studying light-induced noncyclic electron transport from external donors to O₂. Two membrane preparations with opposite membrane polarity, heavy chromatophores and regular chromatophores, were used to characterize this electron transport. It was shown that with lipophylic electron donors such as dichloroindophenol, diaminobenzidine, and phenazine methosulfate the electron transport activities were similar in both types of chromatophores, whereas horse heart cytochrome c, K₄Fe(CN)₆, 3-sulfonic acid phenazine methosulfate, and ascorbate, which cannot penetrate the membrane, were more active in the heavy chromatophores than in the regular chromatophores. Partial depletion of cytochrome c₂ from the heavy chromatophores caused a decrease in the light-induced O₂ uptake from reduced dichloroindophenol or ascorbate. The activity could be restored with higher concentrations of dichloroindophenol or with purified cytochrome c₂ from Rps. capsulata. It is assumed that in the heavy chromatophores the artificial electron donors are oxidized on the cytochrome c₂ level which faces the outside medium. However, cytochrome c₂ is not exposed to the outside medium in the regular chromatophores. Therefore, only lipophylic donors would interact with cytochrome c₂ in this system, while hydrophylic donors would be oxidized by another component of the electron transport chain which is exposed to the external medium. Studies with inhibitors of photophosphorylation show that antimycin A enhances the light-dependent electron transport to O₂ whereas 1:10 phenanthroline inhibited the reaction, but dibromothymoquinone did not affect it. It is assumed that a nonheme iron protein is taking part in this electron transport but not a dibromothymoquinone-sensitive quinone. The terminal oxidase of the light-dependent pathway is different from the two oxidases of the respiratory chain. The ratio between electrons entering the system and molecules of O₂ consumed is 4, which means that the end product of O₂ reduction is H₂O.     

Complementarity of regulation for the two glutamine synthetases from Bacillus caldolyticus, an extreme thermophile

Chromatophores from Rhodopseudomonas capsulata cells grown semiaerobically in the dark oxidize NADH, succinate, and dichlorophenolindophenol. In the presence of N₃^? these activities are inhibited, but light induces oxidation of dichlorophenolindophenol with O₂ as a terminal electron acceptor. Cyanide also inhibits electron transport but much higher concentrations are required to inhibit the photooxidation than the dark oxidation. The photooxidation was studied in a mutant strain of Rhodopseudomonas capsulata (YIV) which cannot grow anaerobically in the light, but similarly to the wild type, grows in the presence of oxygen. Chromatophores from YIV mutant catalyze photophosphorylation and dark oxidation activities with the same properties as those of the wild type. However, the rate of photooxidation in the mutant is only one-third that of the wild type. Based on the differential inhibitor sensitivity and on the mutation it is suggested that the photooxidase is different from the two respiratory oxidases and that this photooxidation activity might be essential for growth of the cells under anaerobic conditions in the light.     

Crystallization of tobacco ringspot virus

Tobacco ringspot virus belongs to the nepovirus group of the small spherical plant viruses. It has been suggested that the protein capsid, which displays icosahedral symmetry, consists of 240 protein subunits which are arranged on a T = 4 surface lattice. The virus crystallizes in orthorhombic space group P2₁2₁2₁ ($\text{a = 388}\text{A}\text{?}\text{, b = 396}\text{A}\text{?}\text{,}\text{and}\text{c = 405}\text{A}\text{?}$). The unit cell contains four virus particles while the crystallographic asymmetric unit consists of one complete virion. X-ray diffraction data from the crystals extend to at least 3.3 Å resolution.     

Characterization and preliminary crystallographic data on the VL-related fragment of the human kI Bence Jones protein Wat

A “naturally occurring” human κI V_L dimer, designated Wat, has been isolated and crystallized. Protein Wat consists of two non-covalently bound monomers, each having a molecular weight of ~ 11,500. The monomer subunit is composed of an entire variable region light chain (V_L) domain closely homologous to that of the κI Bence Jones protein Roy (Hilschmann &; Craig, 1965) as evidenced from amino acid composition, tryptic peptide map, and sequence analysis. Immunochemical studies substantiated that protein Wat is of the κ chain subgroup κI and lacks the isotypic and allotypic antigenic determinants associated with the κ constant region light chain domain. Two types of crystals of V_L dimer Wat were obtained from ammonium sulfate or polyethylene glycol solutions. The type I crystals have unit cell dimensions of $\text{a = b = 82.6}\text{A}\text{?}\text{, c = 60.3}\text{A}\text{?}$, and the space group is hexagonal P6₂ or P6₄. The asymmetric unit consists of one V_L dimer; the fractional volume of unit cell occupied by solvent is 0.51. The unit cell dimensions of the type II crystals are $\text{a = b = 1,08.3}\text{A}\text{?}\text{, c = 108.8}\text{A}\text{?}$; the space group is hexagonal P6₁22 or P6₅22. Three variable domains constitute the asymmetric unit of the type II crystals; the fractional value of the solvent (0.52) is compatible with the value obtained for the type I crystals.