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1.
Summary The ultrastructure of fructose-supplemented and unsupplemented nitrogen-fixing (fix +) and nonfixing (fix –)Plectonema boryanum UTEX 581 cells was examined by transmission electron microscopy. The most prominent structural differences included the arrangement and morphology of the thylakoids and alterations in the appearance of the interthylakoidal spaces. These ultrastructural differences, together with other observations such as glycogen content and presence of nitrogenase (using acetylene reduction assay and immunogold localization), readily distinguished nonfixingP. boryanum from nitrogen-fixing cells. 相似文献
2.
Summary The amino acid sequence of the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) small subunit (SSU) from Euglena has been established by alignment of the sequence of peptides obtained by cleavage with chymotrypsin, trypsin, Staphylococcus aureus protease or formic acid. The Euglena SSU has 138 amino acids and thus represents longest SSU sequence described so far. Homology is only 41% with cyanobacteria SSU and about 51% with higher plant SSU, whereas it is around 75% between higher plants. The largest homologous portion between all the known SSU sequences is localized in the second half and covers about 20 amino acids. The phylogenetic tree based on known SSU sequences has been established and the rate of amino acid substitution for SSU is estimated to be about 1.35×10-9 per year and per site. Despite heterogeneity in amino acid sequence, we found that the overall secondary structure is fairly well conserved.Abbreviations DABITC
Dimethyl amino azobenzene isothiocyanate
- HPLC
high pressure liquid chromatography
- Kd
Kilo daltons
- LSU
large subunit
- PITC
phenyl isothiocyanate
- RuBisCO
ribulose-1,5-bisphosphate carboxylase/oxygenase
- SDS
sodium dodecyl sulfate
- SSU
small subunit
- TFA
trifluoric acetic acid 相似文献
3.
Acquisition of the dark heterotrophic growth capacity on glucose in Plectonema boryanum involves both adaptation and enrichment of a fast-growing genotype. The adaptation includes induction of functions involved in glucose incorporation and increase in glucose-6-phosphate dehydrogenase activity. Photosynthetic products are implicated in the control of both systems. Efficient energy conversion in the dark, as measured by cyanophage multiplication, correlates in time with the increase in potential for glucose incorporation while heterotrophic growth capacity correlates with the increase in glucose-6-phosphate dehydrogenase activity. The lower efficiency of heterotrophic growth compared to photoautotrophic growth is discussed in light of the conservation of the photosynthetic potency in the heterotrophic cells.Abbreviations DCMU
3-(3,4-dichlorophenyl)-1,1-dimethylurea
- DTT
dithiothreitol
- G6P
glucose-6-phosphate
- NADP
nicotinamide adenine dinucleotide phosphate
- NTG
N-methyl-N-nitro-N-nitrosoguanidine
- RUDP
ribulose-1,5-diphosphate
- TCA
trichloroacetic acid
Dedicated to Prof. R. Y. Stanier on the occasion of his 60th birthday 相似文献
4.
Iwaki T Haranoh K Inoue N Kojima K Satoh R Nishino T Wada S Ihara H Tsuyama S Kobayashi H Wadano A 《Photosynthesis research》2006,88(3):287-297
A reporter gene assay revealed that promoters derived from Synechococcus PCC7942 (S.7942) psbAI and Synechocystis PCC6803 (S.6803) psbAII were suitable for the expression of foreign ribulose-bisphosphate carboxylase (RuBisCO; EC 4.1.1.39) in S.7942 cells. Transformational vectors with a promoter and a foreign RuBisCO gene, cvrbc originated from Allochromatium vinosum, were constructed on a binary vector, pUC303, and introduced to S.7942 cells. When the cvrbc was expressed with the S.7942 psbAI promoter, the total RuBisCO activity increased 2.5- to 4-fold than that of the wild type cell. The S.6803 psbAII promoter increased the activity of the transformant 1.5–2 times of that of wild type cell. There was a significant increase in the rate of photosynthesis depending on the increase of RuBisCO activity. The maximum rate of photosynthesis of the transformant cell was 1.63 times higher than that of the wild type under the illumination of 400 μmol m−2 s−1, at 20 mM bicarbonate and at 30 °C. Although the photosynthesis of the higher plant is limited by the ability of photosystems under high irradiance and the high CO2 concentration, that of the S.7942 cell is limited by the RuBisCO activity, even at high CO2 concentrations and under high irradiance. 相似文献
5.
Strains of filamentous, non-heterocystous cyanobacteria from the Pasteur Culture Collection (PCC), able to synthesize nitrogenase under anaerobic test conditions, were tested for growth with N2 as sole nitrogen source at low O2 partial pressure (less than 0.05%).
Plectonema boryanum (PCC 73110) exhibited exponential growth under these conditions. This capacity was restricted to light intensities not exceeding 500 lux. Growth rates were 0.014/h at 200 and 0.023 at 500 lux and similar to those of anaerobic and aerobic control cultures with nitrate as N-source. For N2-fixing cultures incubated at 200 and 500 lux, acetylene reduction rates were 4–8 and 5–14 nmol C2H4 per mg protein per min, respectively. The ratio of phycocyanine to chlorophyll was higher (200 lux) or slightly reduced (500 lux) in N2-fixing cultures as compared to control cultures with nitrate as N-source. On the basis of epifluorescence microscopy and microfluorimetry, no differences in pigment contents were found between individual cells or filaments of N2-fixing cultures. Also no noteworthy differences were observed between the pycobiliprotein composition of individual cells in N2 fixing cultures as compared to nitrate-grown controls. Thus the observed exponential growth of P. boryanum at low light intensities implies simultaneous nitrogen fixation and oxygenic photosynthesis. Additional continuous culture experiments showed that N2-fixing exponential growth was dependent on O2 partial pressures lower than 0.2–0.4%.The other strains tested (PCC 6412, 6602, 7403, 7104) did not grow under such conditions.Abbreviations Chl
chlorophyll
- PBP
phycobiliproteins
- PC
phycocyanin
- PCC
Pasteur Culture Collection
- OD
optical density 相似文献
6.
7.
Eugene L. Barsky Michael V. Gusev Kamilla A. Nikitina Vitaly D. Samuilov 《Archives of microbiology》1981,129(1):105-108
Light-induced fluorescence changes of 9-aminoacridine, an indicator of proton gradient in energy-transducing membranes, were
studied in Plectonema boryanum and other cyanobacteria. The fluorescence changes observed in cell suspensions resulted from a superposition of fluorescence
quenching and enhancement as the analysis of the kinetic data shows. Both components of the fluorescence changes are abolished
by 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) and m-chlorocarbonylcyanide phenylhydrazone. The inhibitory effect of DCMU is removed by 2,3,5,6- or N,N,N′,N′-tetramethyl-p-phenylenediamine.
The fluorescence quenching sensitive to substrates and uncouplers of the photophosphorylation is only observed in membrane
vesicles obtained by osmotic shock of P. boryanum spheroplasts. Presumably, light-induced quenching of the dye fluorescence in the cells of cyanobacteria is due to the proton
transport from the cytoplasm in the inner space of thylakoids while fluorescence enhancement is due to the proton efflux from
the cytoplasm into the incubation medium. 相似文献
8.
9.
Ribulose 1,5-bisphosphate (RuBP) carboxylase (EC 4.1.1.39) activity was approximately equally distributed between supernatant and pellet fractions produced by differential centrifugation of disrupted cells of Chlorogloeopsis fritschii. Low ionic strength buffer favoured the recovery of particulate RuBP carboxylase. Density gradient centrifugation of resuspended cell-free particulate material produced a single band of RuBP carboxylase activity, which was associated with the polyhedral body fraction, rather than with the thylakoids or other observable particles. Isolated polyhedral body stability was improved by density gradient centrifugation through gradients of Percoll plus sucrose in buffer, which yielded apparently intact polyhedral bodies. These were 100 to 150 nm in diameter and contained ring-shaped, 12 nm diameter particles. It is inferred that the C. fritschii polyhedral bodies are carboxysomes. Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis of SDS-dissociated polyhedral bodies revealed 8 major polypeptides. The most abundant, with molecular weights of 52,000 and 13,000, correspond with the large and small subunits, respectively, of RuBP carboxylase.Abbreviations RuBP
ribulose 1,5-bisphosphate
- Ru5P
ribulose 5-phosphate
- SDS
sodium dodecyl sulphate
- PAGE
polyacrylamide gel electrophoresis
- EDTA
ethylenediamine tetraacetic acid
- Tris
tris (hydroxymethyl) methylamine
- IB
isolation buffer
- TCA
trichloroacetic acid 相似文献
10.
11.
Dark inhibition of ribulose-1,5-bisphosphate carboxylase/oxygenase in legumes: A biosystematic study
2-carboxyarabinitol 1-phosphate (CA 1-P) is a naturally occurring inhibitor of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Members of the Fabaceae exhibit a particularly wide range in the extent of CA 1-P accumulation during darkness and include Phaseolus vulgaris, whose dark/light regulation of Rubisco activity is principally achieved by synthesis/degradation of CA 1-P. An extensive survey of the degree of dark inhibition of Rubisco was undertaken for the subfamily Papilionoideae to elucidate evolutionary patterns in the occurrence of this regulatory mechanism. Seventy-five species from 21 tribes were examined. Dark inhibition of Rubisco was found in ancestral tribes such as the Sophoreae, but was substantially reduced or absent in representative species of three more recently evolved tribes, Cicereae, Hedysareae and Vicieae. We conclude that regulation of Rubisco by CA 1-P is neither of recent origin nor of restricted distribution among the Papilionoideae. On the contrary, it becomes lost or less pronounced only in a minority of the more evolutionarily advanced species in this important subfamily. 相似文献
12.
The functions of His291, His295 and His324 at the active-site of recombinant A. nidulans ribulose-1,5-bisphosphate carboxylase/ oxygenase have been explored by site-directed mutagenesis. Replacement of His291 by K or R resulted in unassembled proteins, while its replacement by E, Q or N resulted in assembled but inactive proteins. These results are in accord with a metal ion-binding role of this residue in the activated ternary complex by analogy to x-ray crystallographic analyses of tobacco and spinach enzymes.His324 (H327 in spinach), which is located within bonding distance of the 5-phosphate of bound bi-substrate analog 2-carboxyarabinitol 1,5-bisphosphate in the crystal structures, has been substituted by A, K, R, Q and N. Again with the exception of the H324K and R variants, these changes resulted in detectable assembled protein. The mutant H324A protein exhibited no detectable carboxylase activity, whereas the H324Q and H324N changes resulted in purifiable holoenzyme with 2.0 and 0.1% of the recombinant wild-type specific carboxylase activity, respectively. These results are consistent with a phosphate binding role for this residue.The replacement of His295, which has been suggested to aid in phosphate binding, with Ala in the A. nidulans enzyme leads to a mutant with 5.8% of the recombinant wild-type carboxylase activity. All other mutations at this position resulted in unassembled proteins. Purified H295A and H324Q enzymes had elevated Km(RuBP) values and unchanged CO2/O2 specificity factors compared to recombinant wild-type.Abbreviations CABP
D-2-carboxyarabinitol 1,5 bisphosphate
- IPTG
isopropyl-b-d-thiogalactopyranoside
- L
large subunit of rubisco
- PAGE
polyacrylamide gel electrophoresis
- rubisco
ribulose 1,5-bisphosphate carboxylase/oxygenase
- RuBP
ribulose-P2, ribulose 1,5 bisphosphate
- S
small subunit of rubisco
- SDS
sodium dodecyl sulfate
- X-gal
5-bromo-4-chloro-3-indolyl-b-d-galactoside 相似文献
13.
Dissociation of ribulose-1,5-bisphosphate bound to ribulose-1,5-bisphosphate carboxylase/oxygenase and its enhancement by ribulose-1,5-bisphosphate carboxylase/oxygenase activase-mediated hydrolysis of ATP 总被引:5,自引:2,他引:3
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Ribulose bisphosphate (RuBP), a substrate of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), is an inhibitor of Rubisco activation by carbamylation if bound to the inactive, noncarbamylated form of the enzyme. The effect of Rubisco activase on the dissociation kinetics of RuBP bound to this form of the enzyme was examined and characterized with the use of 3H-labeled RuBP and proteins purified from spinach (Spinacia oleracea L.) In the absence of Rubisco activase and in the presence of a large excess of unlabeled RuBP, the dissociation rate of bound [1-3H]RuBP was much faster after a short (30 second) incubation than after an extended incubation (1 hour). After 1 hour of incubation, the dissociation rate constant (Koff) of the bound RuBP was 4.8 × 10−4 per second, equal to a half-time of about 35 minutes, whereas the rate after only 30 seconds was too fast to be accurately measured. This time-dependent change in the dissociation rate was reflected in the subsequent activation kinetics of Rubisco in the presence of RuBP, CO2, and Mg2+, and in both the absence or presence of Rubisco activase. However, the activation of Rubisco also proceeded relatively rapidly without Rubisco activase if the RuBP level decreased below the estimated catalytic site concentration. High pH (pH 8.5) and the presence of Mg2+ in the medium also enhanced the dissociation of the bound RuBP from Rubisco in the presence of RuBP. In the presence of Rubisco activase, Mg2+, ATP (but not the nonhydrolyzable analog, adenosine-5′-O-[3-thiotriphosphate]), excess RuBP, and an ATP-regenerating system, the dissociation of [1-3H]RuBP from Rubisco was increased in proportion to the amount of Rubisco activase added. This result indicates that Rubisco activase-mediated hydrolysis of ATP is required for promotion of the enhanced dissociation of the bound RuBP from Rubisco. Furthermore, product analysis by ion-exchange chromatography demonstrated that the release of the bound RuBP, in an unchanged form, was considerably faster than the observed increase in Rubisco activity. Thus, RuBP dissociation was experimentally separated from activation and precedes the subsequent formation of active, carbamylated Rubisco during activation of Rubisco by Rubisco activase. 相似文献
14.
15.
Spreitzer RJ 《Archives of biochemistry and biophysics》2003,414(2):141-149
Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of CO2 fixation in photosynthesis, but O2 competes with CO2 for substrate ribulose 1,5-bisphosphate, leading to the loss of fixed carbon. Interest in genetically engineering improvements in carboxylation catalytic efficiency and CO2/O2 specificity has focused on the chloroplast-encoded large subunit because it contains the active site. However, there is another type of subunit in the holoenzyme of plants, which, like the large subunit, is present in eight copies. The role of these nuclear-encoded small subunits in Rubisco structure and function is poorly understood. Small subunits may have originated during evolution to concentrate large-subunit active sites, but the extensive divergence of structures among prokaryotes, algae, and land plants seems to indicate that small subunits have more-specialized functions. Furthermore, plants and green algae contain families of differentially expressed small subunits, raising the possibility that these subunits may regulate the structure or function of Rubisco. Studies of interspecific hybrid enzymes have indicated that small subunits are required for maximal catalysis and, in several cases, contribute to CO2/O2 specificity. Although small-subunit genetic engineering remains difficult in land plants, directed mutagenesis of cyanobacterial and green-algal genes has identified specific structural regions that influence catalytic efficiency and CO2/O2 specificity. It is thus apparent that small subunits will need to be taken into account as strategies are developed for creating better Rubisco enzymes. 相似文献
16.
Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a first key enzyme in the Calvin Circle of plant cell photosynthesis. This paper mainly studied gold immunolocalization of Rubisco of Chlorella spp. 640909, and the Native-PAGE and, SDS-PAGE and Western bloting analysis, as well as the observation to pyrenoid ultra structure. The Native-PAGE result showed a main band, evidenced as the Rubisco band by the Western blot with the antibody against the Rubisco from C. prototothecoides, The special immunoacton of Rubisco from Chlorella spp. 640909 and the antibody to large subunit of Rubisco from C. prothecoides showed the large subunit proteins of Rubisco in the two species of Chlorella shared the high homology. The SDS-PAGE and Western blotting maps showed the molecule weight of the large subunit of Rubisco of Chlorella spp. 640909 was about 55 KD. The shape of pyrenoid ultra structure of the electronic microscope was oblong, and was embedded in starch sheath, with 2 swelling thylakoids through out a center portrait channel of the pyrenoid. There were some connections between pyrenoid and the chloroplast stroma. The distribution of the large subunits and the whole Rubisco in the chloroplast of Chrolella spp. 640909 was studied by immunoelectron microscopy by embedded sections with antibody to large subunit and whole enzyme followed by second antibody, goad anti-rabbit immunoglobulin G conjugated to 10 nm gold particles(Sigma production). The result showed the antibodies against large subunit and whole enzyme heavily labeled the pyrenoid, as well as starch sheath region, whereas the thylakoid region of the plastid was lightly labeled. And the whole Rubisco antibody labeled the pyrenoid surface more heavily than the large subunit antibody did. It is demonstrated the pyrenoid and starch sheath have the photosynthesis function. Rubisco concentrating in pyrenoid and starch sheath is valuable to fix CO2 for photosynthesis in algae. 相似文献
17.
The prochlorophytes, oxygenic photosynthetic prokaryotes having no phycobiliprotein but possessing chlorophylls a and b, have been proposed to have a common ancestry with green chloroplasts, yet this is still controversal. We report here that partial sequence comparisons of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, including sequence data from two prochlorophytes, Prochlorococcus and Prochloron, indicate that Prochlorococcus is more closely related to a photosynthetic bacterium, Chromatium vinosum (-purple bacteria), than to cyanobacteria, while Prochloron is closely related to the prochlorophyte Prochlorothrix and to cyanobacteria. The molecular phylogenetic tree indicates that a common ancestor of Prochlorococcus and -purple bacteria branched off from the land plant lineage earlier than Prochloron, Prochlorothrix, and cyanobacteria.Correspondence to: A. Shimada 相似文献
18.
Hans C. P. Matthijs Jan Maarten Van Steenbergen Ruud Kraayenhof 《Photosynthesis research》1985,7(1):59-67
The fluorescent probe 9-amino-6-chloro-2-methoxy acridine was used to study the energy transduction in the thylakoid and cell membranes of the cyanobacterium Plectonema boryanum. Apart from light-driven electron transfer, the dark endogenous respiration also leads to energization resulting in an ACMA fluorescence response, that is sensitive to the electron flow inhibitor 2, 5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, to the energy transfer inhibitors dicyclohexylcarbodiimide and venturicidine and to the uncoupler 5-chloro-3-t-butyl-2-chloro-4-nitrosalicylanilide.In spheroplasts, in which the cell membranes have lost their capacity to maintain a proton gradient, the respiration-and light-induced ACMA fluorescence changes (quenching) are similar to those in chloroplasts. In intact cells a combination of reversible quenching and enhancement of ACMA fluorescence was found. This dualistic behaviour is supposedly caused by an opposite orientation of the thylakoid and cell membranes. ACMA quenching at the level of the thylakoids was obtained either by respiratory or photosynthetic electron transfer and gave similar responses to those obtained in the spheroplasts. The slower ACMA fluorescence enhancement, only observed in cells with intact cell membranes, also evoked by both respiration and light-induced energization is sensitive to the compounds mentioned above and in addition to KCN.Our results support the view [8] that dark oxidation of substrates by O2 proceeds via the thylakoid membrane and terminates at a CN- sensitive oxidase located in the cell membrane which requires the involvement of a mobile cytoplasmic redox mediator.Abbreviations ACMA
9-amino-6-chloro-2-methoxy acridine
- chl a
chlorophyll a
- DBMIB
2, 5-dibromo-3-methyl-6-isopropyl-p-benzoquinone
- DCCD
dicyclohexylcarbodiimide
- DNP
dinitrophenol
- DNP-INT
dinitrophenyl ether of 2-iodo-4-nitrothymol
- FCCP
carbonylcyanide-p-trifluoro-methoxy phenylhydrazone
- S-13
5-chloro-3-t-butyl-2-chloro-4-nitrosalicylanilide
- tricine
N-2 (2-Hydroxy-1, 1-bis (hydroxymethyl) ethyl)-glycine
- Tris
Tris (hydroxymethyl) amino methane 相似文献
19.
A variety of preparative methods for in situ X-ray energy dispersive analysis were tested to determine their effects on the elemental composition of polyphosphate bodies in P. boryanum. The bodies were found to contain large amounts of P and K and small amounts of Ca and Mg. Air drying, freeze-drying and freeze-drying from a liquid nitrogen slush all gave similar results. Fixation of the cells in glutaraldehyde and/or OsO4 resulted in loss of the K and enhancement of the Ca peak. Magnesium was lost during embedding in epoxy. 相似文献