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1.
Norbert Kaufmann Hendrik Hüdig Gerhart Drews 《Molecular & general genetics : MGG》1984,198(1):153-158
Summary Three plasmids containing the transposon Tn5, i.e. pSUP201::Tn5, pACYC184::Tn5 and pJB4JI were transferred from Escherichia coli to Rhodopseudomonas capsulata in order to mutagenize the genome. Mutants defective in bacteriochlorophyll and carotenoid synthesis and mutants unable to form the photochemical reaction center or one of the light-harvesting complexes were isolated. Of special interest were mutants that could not form the light-harvesting complex B800-850. Two of these mutants synthesized only two of the three polypeptides of this complex whereas the corresponding near infrared absorbance bands were not observed. Complementation analysis with the Rprime plasmid pRPS404, which contains a 50 kb region of the genome of R. capsulata carrying most genes responsible for expression of photosynthetic apparatus, revealed that some genes of the B800-850 light-harvesting complex lie outside this photosynthetic gene cluster.Abbreviations Bchl
Bacteriochlorophyll
- Cm
chloramphenicol
- Km
kanamycin
- Tc
tetracycline
- Ap
ampicillin
- Gm
gentamicin
- Spc
spectinomycin 相似文献
2.
Pak-Lam Yu Barbara Hohn Heinz Falk Gerhart Drews 《Molecular & general genetics : MGG》1982,188(3):392-398
Summary Chromosomal segments of Rhodopseudomonas capsulata carrying the ribosomal operons and cloned with the cosmid vector pHC79 have been identified by cross hybridization with 32P-ATP labeled rRNAs. At least seven rRNA operons are present in the R. capsulata chromosome. By R-loop analyses of DNA-RNA hybrids, two distinct loop structures of sizes 1.50 kb and 2.52 kb corresponding to the 16S and 23S RNA molecules, respectively, were detected. Intact 23S RNA molecules can be isolated from R. capsulata ribosomes by sucrose density centrifugation. However, fragmentation of the 23S RNA molecule into a 16S-like molecule was observed during gel electrophoresis. Restriction mapping and hybridization of a 9 kb PstI fragment that contained one copy of the rRNA operon showed the following sequence of the RNA genes in R. capsulata 16S, 23S, and 5S. A spacer region of 0.91 kb was found between the 16S and the 23S RNA genes. 相似文献
3.
The release of protons from intact cells of Rhodopseudomonas capsulata after either 4μs flashes or during brief periods of continuous illumination has been measured with the indicator, cresol red. The half-time for H+-release after a flash was 35 ms and the extent, 1H+ per 134 bacteriochlorophyll. Myxothiazol completely inhibited the flash-induced H+-release and antimycin A reduced it by 37%. The proton-releasing reaction is discussed with reference to the protonmotive Q-cycle. During continuous illumination the rapid phase of H+ release is followed by a lag and then by another period of acidification, suggesting that other protolytic reactions may be in operation. 相似文献
4.
The amino-terminal sequences have been determined by Edman degradation for the reaction center polypeptides from a carotenoidless mutant of Rhodopseudomonas capsulata. Individual polypeptides were isolated by preparative electrophoresis and electroelution. By comparison with the sequences deduced from the DNA (Youvan, D.C., Alberti, M., Begush, H., Bylina, E.J. and Hearst, J.E. (1984) Proc. Natl. Acad. Sci. USA 81, 189–192) we conclude that the M and L subunits are processed so as to remove the amino-terminal methionine, whereas the H subunit is not processed at the amino-terminus after translation. None of the subunits is synthesized with a significant amino-terminal extension peptide. 相似文献
5.
Absorption and fluorescence emission spectra of Rhodopseudomonas capsulata, strains 37b4 (wild type), A1a+ (blue-green mutant strain), Y5 (phototroph negative, having only B-800–850 bacteriochlorophyll-carotenoid-protein complex) at 4 K, 77 K and 300 K were measured. The fluorescence emission at 890 nm of the B-870 bacteriochlorophyll band dominates the emission of other spectral forms of the strains 37b4 and A1a+, while in strain Y5 a fluorescence emission band at 865 nm of the B-850 bacteriochlorophyll dominates. Very little fluorescence was observed at 805 nm. A linear relation between relative fluorescence intensity and the exciting light intensity was observed. The integrated fluorescence yield increased as the temperature was lowered from 300 K to 4 K. The results are discussed in the light of the arrangement of pigment molecules in the membrane and the process of energy migration within the photosynthetic apparatus. 相似文献
6.
In this paper a number of experiments with the purple bacteria Rhodospirillum rubrum and Rhodopseudomonas capsulata is described in which the total fluorescence yield and/or the total fraction of reaction centers closed after a picosecond laser pulse were measured as a function of the pulse intensity. The conditions were such that the reaction centers were either all in the open or all in the closed state before the pulse arrived. These experiments are analysed using the theoretical formalism discussed in the preceding paper (Den Hollander, W.T.F., Bakker J.G.C., and Van Grondelle, R., Biochim. Biophys. Acta 725, 492–507). From the experimental results the number of connected photosynthetic units, λ, the rate of energy transfer between neighboring antenna molecules, kh, and the rate of trapping by an open reaction center, kot, can be estimated. For R. rubrum it is found that λ = 14−17, kh = (1−2)·1012 s−1 and kot = (4−6)·1011 s−1, for Rps. capsulata λ ≈ 30, kh ≈ 4·1011 s−1 and kot ≈ 3·1011 s−1. The findings are discussed in terms of current models for the structure of the antenna and the kinetic properties of the decay processes occurring in these purple bacteria. 相似文献
7.
8.
Augusto F. Garcí a Giovanni Venturoli Nasser Gad'on Javier G. Fern ndez-Velasco B. Andrea Melandri Gerhart Drews 《BBA》1987,890(3):335-345
(1) Cells of Rhodopseudomonas capsulata (wild-type) were grown photoheterotrophically in a turbidostat under very high and very low light intensity. Membranes were isolated from cells adapted to the respective light conditions and fractionated by sucrose density centrifugation. The molar ratios of ubiquinone and cytochromes c2, c1, b-561 and b-566 per reaction center were 3-fold to 5-fold higher in high-light than in low-light membranes. (2) Most of the Cyt(c1 + c2) and Cyt b-561 detected in dark redox titrations undergoes light-induced redox changes, both in high- and in low-light membranes. (3) The fractions of the total photooxidizable reaction center and Cyt(c1 + c2) oxidized under continuous light in the absence of antimycin are higher in membranes from low-light- than from high-light-grown cells. (4) From these data and results of kinetic studies it is proposed that cyclic electron flow under saturating light intensities is faster in high-light-grown cells. 相似文献
9.
The spectral and functional properties of carotenoids associated with each of the two light-harvesting complexes of the Rhodopseudomonas capsulata photosynthetic antenna system have been distinguished by studying mutants lacking one or the other complex. In mutants containing only the light-harvesting I complex (LH-I), the absorption spectrum of the carotenoids is blue-shifted compared to wild type. Carotenoid absorption in mutants possessing only the light-harvesing II complex (LH-II) complex is red-shifted. The circular dichroism spectrum of carotenoids in each complex is also distinctive. Although carotenoids in each complex function with approximately the same efficiency in harvesting and transmitting light energy for photosynethesis, only the carotenoids associated with LH-II undergo an electrochromic bandshift upon generation of a transmembrane potential. These observations are interpreted to indicate that both the orientation of carotenoid molecules with respect to the plane of the membrane, and the immediate electrochemical environment of these molecules differ in the two light-harvesting complexes. 相似文献
10.
Cells of Rhodopseudomonas capsulata were grown in a turbido-stat and adapted to high (1400 W/m2) or low (40 W/m2) light intensities. In high-light-grown cells the specific BChl content was about 10-times lower, the number of intracytoplasmatic membrane vesicles smaller by a factor of about 20, the photosynthetic unit smaller by a factor of 1.9 and the reaction center content about 5-times lower than in low-light-grown cells. However, the photophosphorylation rate per reaction center under saturating light was higher in high-light-grown cells by a factor of 7.7, apparently compensating the lower amount of reaction centers. Adaptation of the cells to different irradiances not only seems to comprise a variation of the size and composition of the antennae, but also a change in the affinity of the photosynthetic system to light, as concluded from saturation curves obtained from the two adaptation stages of cells. 相似文献
11.
We have isolated from Rhodopseudomonas spheroides a pigment-protein complex of apparent weight 9 kdaltons that bears more than 60% of the light harvesting bacteriochlorophyll. The isolation procedure involved exposure to 1% lauryl dimethyl amine oxide (LDAO). The purified 9-kdalton fraction showed the light harvesting bacteriochlorophyll components B800 and B850, plus carotenoids. The ratio of bacteriochlorophyll to protein was 17%. This protein is probably the same as the “band 15” protein of Fraker and Kaplan. It may exist in vivo as characteristic aggregates of higher molecular weight. LDAO added to Rps. spheroides chromatophores converted the bacteriochlorophyll component B870 to a form absorbing at 770 nm but had little effect on the “B800 + B850” system, causing only a reversible shift of the 850-nm band to 845 nm. Anti-reaction center serum, added to subcellular fractions from Rps. spheroides with 1% LDAO, precipitated reaction center chromoprotein unaccompanied by light harvesting bacteriocholorophyll. Other antisera precipitated light harvesting components and left the reaction center chromophores in solution. A major protein of apparent weight 45 kdaltons was found in relatively nonpigmented fractions from Rps. spheroides, associated with cell wall fragments. The 45-kdalton protein showed considerable interstrain variability, whereas the 9-kdalton and reaction center proteins appeared constant. 相似文献
12.
The absorbance-detected magnetic-resonance technique has been applied to the study of the triplet state of the primary donor in chromatophores of the photosynthetic bacterium Rps. viridis. The results confirm the triplet-minus-singlet absorbance-difference spectrum and its interpretation as previously obtained for isolated reaction centers (Den Blanken, H.J. and Hoff, A.J. (1982) Biochim. Biophys. Acta 681, 365–374). Our present results affirm that the primary donor is a bacteriochlorophyll b dimer, and that there is no blue exciton band at 850 nm. We show that the reaction centers are not identical, but have a small heterogeneity in their properties. In chromatophores and sometimes in isolated reaction centers a shoulder is observed in the long-wavelength absorbance-difference band of the primary donor. This shoulder is possibly caused by charge transfer interaction of the donor with an adjacent chromophore (Vermeglio, A. and Paillotin. G. (1982) Biochim. Biophys. Acta 681, 32–40; Maslov, V.G., Klevanik, A.V., Ismailov, M.A. and Shuvalov, V.A. (1983) Doklady Akad. Nauk. SSSR 269, 1217–1221) or it reflects a slight heterogeneity in the reaction-center geometry, which cannot be removed with the selection offered by the magnetic resonance technique. The zero-field triplet-ESR spectrum and the sublevel decay rates of the triplet state of the primary donor are presented, as detected in whole cells at the antenna fluorescence, and in chromatophores and isolated reaction centers at the absorbance-difference band at 838 nm. We do not observe the expected reversal of the sign of the ESR transitions monitored with the two techniques. A tentative explanation is given in terms of energy transfer from unrelaxed excited states of the antenna pigments to the reaction center. 相似文献
13.
Suzanne Sommer Jelena Knezevic Adriana Bailone Raymond Devoret 《Molecular & general genetics : MGG》1993,239(1-2):137-144
The actions of UmuDC and RecA proteins, respectively in SOS mutagenesis are studied here with the following experimental strategy. We used lexAl (Ind–) bacteria to maintain all SOS proteins at their basal concentrations and then selectively increased the concentration of either UmuDC or RecA protein. For this purpose, we isolated operator-constitutive mutations o
c in the umuDC and umuD'C operons and also used the o
98
c
-recA mutation. The o
1
c
-umuDC mutation prevents LexA repressor from binding to the operator and improves the Pribnow box consensus sequence. As a result, 5000 UmuD and 500 UmuC molecules per cell were produced in lexAl bacteria. This concentration is sufficient to restore SOS mutagenesis. The level of RecA protein present in the repressed state promoted full UmuD cleavage. Overproduction of RecA alone did not promote SOS mutagenesis. Increasing the level of RecA in the presence of high concentrations of UmuDC proteins has no further effect on SOS mutgenesis. We conclude that, after DNA damage, umuDC is the only SOS operon that must be induced in Escherichia coli to promote SOS mutagenesis. 相似文献
14.
A photosynthetically-incompetent mutant Rhodopseudomonas spheroides that lacks bacteriochlorophyll was isolated. Spectroscopic evidence from CO difference spectra and cyanide difference spectra suggested that a cytochrome oxidase was present in this mutant that contained two components, corresponding to cytochromes a and a3 of mitochondria. Potentiometric titration at 607 nm also showed the presence of two components with oxidation-reduction mid-point potentials of +375 mV and +200 mV. They were present in a ratio close to unity. No cytochrome of the the c-type corresponding to mitochondrial cytochrome c was detected, but a minor c component (near 10% of the total cytochrome c) with an oxidation-reduction mid-point potential of +120 mV was found
Growth of the mutant in medium with low aeration or lacking added copper diminished the concentration of the a-type cytochrome but not the concentrations of cytochromes of the b and c-type. 相似文献
15.
Chromatophores from various strains of Rhodopseudomonas sphaeroides were excited with laser flashes lasting about 20 ns. Fluorescence from the antenna bacteriochlorophyll of the photosynthetic apparatus was measured both during the laser flash, and during a weak Xe flash following the laser flash. Strong laser flashes caused severe quenching of the fluorescence, which could be correlated with the formation of triplet states of the antenna pigments. Triplet states of both BChl and carotenoids acted as quenchers, but bacteriochlorophyll triplets were the more effective of the two. In the double-flash experiments, the reciprocal of the fluorescence yield was proportional to the concentration of triplet quenchers remaining at the time of the second flash. This relationship indicates that singlet excitations can migrate over large domains in the antenna, rather than being restricted by boundaries separating individual reaction centers. Comparisons of chromatophores from different strains and from cells grown under different conditions showed that excitations are concentrated rapidly in the antenna complexes with the longest wavelength absorption bands (B870), and that the migration of excitations to trapping sites is relatively insensitive to the amount of antenna bacteriochlorophyll absorbing at shorter wavelengths (B800–B850). This suggests that the B870 complexes are organized in the membrane so as to interconnect many reaction centers, and that the B800–B850 complexes are arranged peripherally. 相似文献
16.
The optical spectra of the reaction center (RC) of Rhodopseudomonas viridis including absorption (A), linear dichrosism (LD), circular dichroism (CD), absorption-detected magnetic resonance (ADMR) and its linear dichroism (LD-ADMR) are simulated by an exciton model. It involves the Qy transitions of six prosthetic groups: four (bacteriochlorophyll-b molecules, two bacteriopheophytin-b molecules and the Soret bands By of the special-pair pigments, which couple most strongly with the corresponding Qy transitions. For the ADMR-spectra additional excitations of the special-pair triplet are introduced. While interactions between the Qy transitions and the By transitions are in principle determined from the structural arrangement of the pigments, the interactions to the other states are adjusted to fit the spectral features for the various transitions. The interaction of the newly introduced states are interpreted in terms of a simple model. 相似文献
17.
Centrifugation through a cesium chloride density gradient and agarose gel electrophoresis of the DNA from the purple non-sulfur photosynthetic bacterium Ectothiorhodospira sp. resolved a single extrachromosomal element, plasmid pDG1. Its size was estimated to be 13.2 kilobases by restriction endonuclease mapping. Plasmid pDG1 and two restriction fragments thereof were cloned in Escherichia coli C600 with plasmid pBR327 as a vector to form mixed plasmids pDGBR1, pDGBR2, and pDGBR3. The resistance to streptomycin and mercury found in Ectothiorhodospira sp. was transferred to E. coli C600 after transformation with pDGBR1 but not with pDGBR2 and pDGBR3. The replication origin of pDG1 was estimated to be within a 2-kilobase restriction fragment of pDG1 by monitoring its replication in E. coli HB101, using a kanamycin resistance reporter gene. High stringency molecular hybridization with 32P-labeled pDG1 identified specific fragments of genomic DNA, suggesting the integration of some plasmid sequences. In accordance with the hypothesis that this integration is due to a transposon, we tested the transfer of streptomycin resistance from pDG1 into plasmid pVK 100 used as a target. For this test, we regrouped in the same cells of E. coli HB101, pDGBR1 and mobilizable plasmid pVK100 (tetr, kmr). We used the conjugation capacity of the pVK100/pRK2013 system to rescue the target plasmid pVK100 into nalidixic acid-resistant E. coli DH1. The transfer frequency of streptomycin resistance into pVK100 was 10−5, compatible with a transposition event. In line with the existence of a transposon on pDG1, heteroduplex mapping indicated the presence of inverted repeats approximately 7.5 kb from one another. 相似文献
18.
Mutant strains of Rhodopseudomonas spheroides have been isolated which contain 5–50 times more bacteriochlorophyll and carotenoids than the wild type when grown under highly aerobic conditions in the dark. Their pigment content is similar to the wild type when grown in the light. One of the mutants (TA-R) grew more slowly than its parent strain under aerobic conditions but formed pigments at about 60% of the rate observed under photosynthetic conditions. The other mutants grew at rates similar to the wild type under all conditions. Synthesis of bacteriochlorophyll by suspensions of the mutants began without delay upon transfer from conditions of high to low aeration. In contrast to the wild type, magnesium protoporphyrin-S-adenosylmethionine methyltransferase (EC 2.1.1.11) activity in particulate preparations from the mutants was not repressed by growth under aerobic conditions in the light or dark. Ribulose diphosphate carboxylase (EC 4.1.1.39) activity was repressed by O2 in the mutants as in the wild type. Other enzyme activities were compared in mutant TA-R and its parent strain grown under the same conditions. NADH oxidase activity in particles from aerobically grown TA-R was about one third that found in the parent strain. However, the respiration rates of the intact cells did not differ. Light inhibited the respiration of aerobically grown TA-R, indicating that the bacteriochlorophyll formed under these conditions had photochemical activity. It is concluded that the insensitivity of the mutants to O2 repression is due to defects in the regulatory system which controls formation of the enzymes concerned in pigment synthesis. 相似文献
19.
Michael C. W. Evans 《BBA》1987,894(3):524-533
The oxidation-reduction potential of the iron-quinone electron acceptors in the reaction centre of Rhodopseudomonas viridis has been reinvestigated. In chromatophores treated with o-phenanthroline to remove the secondary acceptor Qb, two steps were observed in the reduction of the primary electron acceptor Qa with Em ≈ − 100 and ≈ − 330 mV. In isolated reaction centres only one step was observed in the reduction of Qa with E ≈ −150 mV. Reconstitution of the reaction centres with additional menaquinone resulted in an increase in the Qa EPR signal and reconstitution of the low-potential step in the oxidation-reduction titration. Reconstitution with ubiquinone resulted in the recovery of the secondary quinone Qb. The addition of ubiquinone did not reconstitute the low-potential step of Qa reduction, or affect the reconstitution of this step by menaquinone. It is concluded that menaquinone can bind to two sites on the reaction centre. Both have properties of the Qa site but with different pK values. It is unlikely that either is the same as the Qb site. 相似文献
20.
(1) Current models for the mechanism of cyclic electron transport in Rhodopseudomonas sphaeroides and Rhodopseudomonas capsulata have been investigated by observing the kinetics of electron transport in the presence of inhibitors, or in photosynthetically incompetent mutant strains. (2) In addition to its well-characterized effect on the Rieske-type iron sulfur center, 5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) inhibits both cytochrome b50 and cytochrome b?90 reduction induced by flash excitation in Rps. sphaeroides and Rps. capsulata. The concentration dependency of the inhibition in the presence of antimycin (approx. 2.7 mol UHDBT/mol reaction center for 50% inhibition of extent) is very similar to that of its inhibition of the antimycin-insensitive phase of ferricytochrome c re-reduction. UHDBT did not inhibit electron transfer between the reduced primary acceptor ubiquinone (Q?I) and the secondary acceptor ubiquinone (QII) of the reaction center acceptor complex. A mutant of Rps. capsulata, strain R126, lacked both the UHDBT and antimycin-sensitive phases of cytochrome c re-reduction, and ferricytochrome b50 reduction on flash excitation. (3) In the presence of antimycin, the initial rate of cytochrome b50 reduction increased about 10-fold as the Eh(7.0) was lowered below 180 mV. A plot of the rate at the fastest point in each trace against redox potential resembles the Nernst plot for a two-electron carrier with Em(7.0) ≈ 125 ± 15 mV. Following flash excitation there was a lag of 100–500 μs before cytochrome b50 reduction began. However, there was a considerably longer lag before significant reduction of cytochrome c by the antimycin-sensitive pathway occurred. (4) The herbicide ametryne inhibited electron transfer between Q?I and QII. It was an effective inhibitor of cytochrome b50 photoreduction at Eh(7.0) 390 mV, but not at Eh(7.0) 100 mV. At the latter Eh, low concentrations of ametryne inhibited turnover after one flash in only half of the photochemical reaction centers. By analogy with the response to o-phenanthroline, it is suggested that ametryne is ineffective at inhibiting electron transfer from Q?I to the secondary acceptor ubiquinone when the latter is reduced to the semiquinone form before excitation. (5) At Eh(7.0) > 200 mV, antimycin had a marked effect on the cytochrome b50 reduction-oxidation kinetics but not on the cytochrome c and reaction center changes or the slow phase III of the electrochromic carotenoid change on a 10-ms time scale. This observation appears to rule out a mechanism in which cytochrome b50 oxidation is obligatorily and kinetically linked to the antimycin-sensitive phase of cytochrome c reduction in a reaction involving transmembrane charge transfer at high Eh values. However, at lower redox potentials, cytochrome b50 oxidation is more rapid, and may be linked to the antimycin-sensitive reduction of cytochrome c. (6) It is concluded that neither a simple linear scheme nor a simple Q-cycle model can account adequately for all the observations. Future models will have to take account of a possible heterogeneity of redox chains resulting from the two-electron gate at the level of the secondary quinone, and of the involvement of cytochrome b?90 in the rapid reactions of the cyclic electron transfer chain 相似文献