Spirilloxanthin incorporation into the LH2 and LH1-RC pigment-protein complexes from a purple sulfur bacterium <Emphasis Type="Italic">Allochromatium minutissimum</Emphasis>

Incorporation of spirilloxanthin into carotenoidless LH2 and LH1-RC complexes from a purple sulfur bacterium Allochromatium (Alc.) minutissimum was studied. Carotenoidless cells of Alc. minutissimum were obtained using diphenylamine, a carotenoid biosynthesis inhibitor. In the course of incorporation of the carotenoid mixture, the composition of which corresponded to that of Alc. minutissimum control photosynthetic membranes, no selective incorporation of spirilloxanthin into the LH1-RC complex was detected. It is assumed that in vivo carotenoids are not incorporated into the LH2 and LH1-RC complexes from a common pool. Pure spirilloxanthin destroys both the LH2 and LH1-RC complexes. Within the concentration range of spirilloxanthin in the incorporated mixture from 27% to 52%, it was found to be incorporated into the LH2 and LH1-RC complexes with the efficiency of 13% and 33%, respectively. The possible existence of different sites of assembly for the LH2 and LH1-RC complexes is discussed, as well as of two fractions of LH2 complexes, in one of which rhodopin may be integrated, and in the other (minor) one, spirilloxanthin.     

Effects of growth conditions on formation of cytochrome system of a denitrifying bacterium, Pseudomonas stutzeri

Cytochrome systems in cells of a denitrifying bacterium, Pseudomonasstutzeri (VAN NIEL strain), grown under different atmosphericconditions were compared with reference to the effects of nitrateand nitrite on cytochrome synthesis. When a culture was sufficiently aerated (aerobic conditions),synthesis of all cytochrome components was repressed, regardlessof the presence or absence of nitrate and nitrite. When aeratedmoderately (semi-aerobic conditions), both soluble and paniculatecytochromes c-552 and cytochrome b-558 contents markedly increasedeven in the absence of nitrate and nitrite. Under anaerobic or semi-aerobic conditions, nitrite inducedcytochrome a₂–c synthesis. This inductive effect of nitritewas counteracted by nitrate. Nitrate also repressed particulatecytochrome c-552 synthesis to some extent but nitrite did not. ¹Present address: Department of Biochemistry, Hiroshima UniversitySchool of Dentistry, Hiroshima, Japan (Received June 24, 1969; )     

Distribution of rhodopin and spirilloxanthin between LH1 and LH2 complexes when incorporating carotenoid mixture into the membrane of purple sulfur bacterium <Emphasis Type="Italic">Allochromatium minutissimum</Emphasis> in vitro

Carotenoid mixture enriched by rhodopin and spirilloxanthin was incorporated in LH2 and LH1 complexes from Allochromatium (Alc.) minutissimum in vitro. The maximum incorporating level was ~95%. Rhodopin (56.4%) and spirilloxanthin (13.8%) were incorporated into the LH1 complex, in contrast to the control complex, which contained primarily spirilloxanthin (66.8%). After incorporating, the LH2 complex contained rhodopin (66.7%) and didehydrorhodopin (14.6%), which was close to their content in the control (67.4 and 20.5%, respectively). Thus, it was shown that carotenoids from the total pool are not selectively incorporated into LH2 and LH1 complexes in vitro in the proportion corresponding to the carotenoid content in the complexes in vivo.     

Identification and Subcellular Distribution of Carotenoids in the Aerobic Photosynthetic Bacterium, Pseudomonas radiora Strain MD-1

The carotenoid compositions in the aerobic methylotrophic photosyntheticbacterium, Pseudomonas radiora (Methylobacterium radiotolerans)strain MD-1, were investigated. Carotenoids bound to the BChl-proteincomplex were identified as spirilloxanthin, anhydrorhodovibrinand rhodovibrin, which have been generally found in purple photosyntheticbacteria. The ratio of BChl a to carotenoid in the BChl-proteincomplex was about 2 to 1. The transfer of excitation energyfrom carotenoids to BChl in the BChl-protein complex was observedalthough its efficiency was as low as that in the purple bacterialcomplexes which have spirilloxanthin as the major carotenoidcomponent. By contrast, carotenoic acids were found as majorforms of carotenoids in the cells, and most of them were notbound to the BChl-protein complex. These carotenoic acids werealso found as the major carotenoids in other methylotrophs thathave been characterized as aerobic photosynthetic bacteria. (Received November 28, 1994; Accepted April 21, 1995)     

The isolation and preliminary characterization of a halophilic photosynthetic bacterium

Summary The isolation of a photosynthetic bacterium which is apparently an obligate halophile is reported. The organism (strain SL-1) grows in a medium with either thiosulfate of sulfide and bicarbonate containing 22% NaCl but not in one containing 4% NaCl. The morphology is abnormal in media with 8% NaCl. Strain SL-1 is an obligate anaerobe. The major carotenoid pigment is spirilloxanthin. The spectrum of the chlorophyllous pigment is very similar to that of bacteriochlorophyll; however, its identity with bacteriochlorophyll has not been demonstrated.Strain SL-1 is a spirillum. Cells possess a single, polar, sheathed flagellum. The ultrastructure of the cells is depicted.Dedicated to Professor C. B. van Niel on the occasion of his 70th birthday.Supported in part by a grant from the National Science Foundation.     

Carotenoid Profiles in Pigment-Protein Complexes of Rhodospirillum rubrum

Young cells of Rhodospirillum rubrum contain a set of carotenoidsfrom lycopene to spirilloxanthin. During growth, intermediatesare almost completely converted to spirilloxan-thin. The ratioof the different carotenoid precursors vs. spirilloxanthin foundin material of a certain age is the same in cells, chromatophores,light-harvesting complexes and reaction centers. Independentof the carotenoid composition and the age of the cells, thesame detergent treatment can be used for isolation of pigment-proteincomplexes. Light-harvesting complexes of young cells containingmainly precursors of spirilloxanthin, as well as those of oldcells in which spirilloxanthin dominates, both have their absorptionmaximum at 880 nm. It is thus assumed that all carotenoids ofthe spirilloxanthin series interact with bacteriochlorophylla similarly to spirilloxanthin itself. From these results it is concluded that the micro-environmentof these membrane-complexes is not influenced by the type ofcarotenoid present and that the assembly of the pigment-proteincomplexes in a growing membrane takes place before carotenoidbiosynthesis has lead to the final product. (Received October 26, 1988; Accepted March 6, 1989)     

Effects of Carotenoid Inhibition on the Photosynthetic RC–LH1 Complex in Purple Sulphur Bacterium Thiorhodospira sibirica

Core complexes (LH1–RC) were isolated using preparative gel electrophoresis from photosynthetic membranes of the purple bacterium, Thiorhodospira sibirica, grown in the absence or presence of the carotenoid biosynthesis inhibitor, diphenylamine. The biosynthesis of carotenoids is affected by diphenylamine both quantitavely and qualitatively: after inhibition, the level of carotenoids in core complexes reaches only 10% of the normal content, as analyzed by HPLC and absorption spectroscopy. The normally grown bacterium biosynthesizes spirilloxanthin, rhodopin, anhydrorhodovibrin and lycopene, whereas after inhibition only neurosporene, ζ-carotene and their derivatives are found in the complexes. There is no concomitant accumulation of appreciable amounts of colorless carotenoid precursors. Interestingly, the main absorption band of the core light harvesting complex isolated from carotenoid-inhibited cells, shows a red shift to 889 nm, instead of a blue shift observed in many carotenoid-deficient species of purple photosynthetic bacteria. The stability of isolated core complexes against n-octyl-β-D-glucopyranoside clearly depends on the presence of carotenoids. Subcomplexes resulting from the detergent treatment, were characterized by non-denaturating gel electrophoresis combined with in situ absorption spectroscopy. Core complexes with the native carotenoid complement dissociate into three subcomplexes: (a) LH1 complexes partially depleted of carotenoids, with an unusual spectrum in the NIR region (λ_max = 791, 818, 847 and 875 nm), (b) reaction centers associated with fragments of LH1, (c) small amounts of a carotenoidless B820 subcomplex. The core complex from the carotenoid-deficient bacterium is much less stable and yields only the two sub-complexes (b) and (c). We conclude that carotenoids contribute critically to stability and interactions of the core complexes with detergents.     

Polarization angle dependence of stark absorption spectra of spirilloxanthin bound to the reconstituted LH1 complexes using LH1-subunits isolated from the purple photosynthetic bacterium Rhodospirillum rubrum

Reconstituted LH1 complexes were prepared using the LH1 subunit-type complexes, isolated from the purple photosynthetic bacterium Rhodospirillum (Rs.) rubrum, and purified all-trans spirilloxanthin. Stark absorption spectra of spirilloxanthin bound to both the native and reconstituted LH1 complexes were compared in different polarization angles (χ) against the external electric field. From the polarization angle dependence of the Stark absorption spectra, two angles were determined in reference to the direction of transition dipole moment (m) of spirilloxanthin: one is the change in polarizability upon photoexcitation (Δα), θ(Δα) and the other is the change in static dipole moment upon photoexcitation (Δμ), θ(Δμ). Despite the symmetric molecular structure of all-trans spirilloxanthin, its Stark absorption spectra show pronounced values of Δμ. This large Δμ values essentially caused by the effect of induced dipole moment through Δα both in the cases for native and reconstituted LH1 complexes. However, slightly different values of θ(Δα) and θ(Δμ) observed for the native LH1 complex suggest that spirilloxanthin is asymmetrically distorted when bound to the native LH1 complex and gives rise to intrinsic Δμ value.     

Characterisation of the LH2 spectral variants produced by the photosynthetic purple sulphur bacterium Allochromatium vinosum

This study systematically investigated the different types of LH2 produced by Allochromatium (Alc.) vinosum, a photosynthetic purple sulphur bacterium, in response to variations in growth conditions. Three different spectral forms of LH2 were isolated and purified, the B800-820, B800-840 and B800-850 LH2 types, all of which exhibit an unusual split 800 peak in their low temperature absorption spectra. However, it is likely that more forms are also present. Relatively more B800-820 and B800-840 are produced under low light conditions, while relatively more B800-850 is produced under high light conditions. Polypeptide compositions of the three different LH2 types were determined by a combination of HPLC and TOF/MS. The B800-820, B800-840 and B800-850 LH2 types all have a heterogeneous polypeptide composition, containing multiple types of both α and β polypeptides, and differ in their precise polypeptide composition. They all have a mixed carotenoid composition, containing carotenoids of the spirilloxanthin series. In all cases the most abundant carotenoid is rhodopin; however, there is a shift towards carotenoids with a higher conjugation number in LH2 complexes produced under low light conditions. CD spectroscopy, together with the polypeptide analysis, demonstrates that these Alc. vinosum LH2 complexes are more closely related to the LH2 complex from Phs. molischianum than they are to the LH2 complexes from Rps. acidophila.     

Photodestruction of Pigments in Higher Plants by Herbicide Action: I. THE EFFECT OF DCMU (DIURON) ON ISOLATED CHLOROPLASTS

3-(3',4'-Dichlorophenyl)-1',1'-dimethyl urea (DCMU) inducedthe photobleaching of chlorophylls and carotenoids in isolatedchloroplasts of Hordeum vulgare. In chloroplasts illuminatedin both the absence and presence of DCMU (5.0 mmol m^–3),the destruction of carotenoid preceded that of the chlorophylls.The rate of photodestruction was accelerated by the presenceof DCMU. After only 2 h illumination the rates of loss of ß-caroteneand of the epoxyxanthophylls, neoxanthin and violaxanthin, weresimilar (approximately 40–50% loss in the presence of5–0 mmol m^–3 DCMU) but weremuch greater than thatof lutein (25% loss). Analysis of the individual pigment-proteincomplexes, isolated from chloroplasts following such treatment,showed that whilst pigment destruction had occurred in all complexes,the relative content of the LHCP2/CPa complexes (containingthe PSII core) had fallen to the greatest extent. Further illuminationof the chloroplasts, for up to 22 h, resulted in far greaterbleaching but showed a similar pattern of pigment loss, withDCMU again accelerating the rate at which this loss occurred.ß-Carotene-5,6-epoxide was identified as a productof such photo-oxidative conditions. Key words: DCMU, carotenoids, chlorophylls, photobleaching, ß-carotene-5,6-expoxide     

Accumulation of Pigments during the Greening of Etiolated Seedlings of Hordeum vulgare L.

The pigment changes that occur during transformation of etioplaststo fully developed chloroplasts have been studied in seedlingsof barley (Hordeum vulgare L.) by greening with white lightof low (15–25 µmol m^–2 s^–1) and medium(150 µmol m^–2 s^–1) intensity. At least 24h longer was required in the low light regime for the same concentrationof pigment to be accumulated in the seedlings. The increasein pigment content was mainly due to the synthesis of chlorophyllsa and b. Of the carotenoids present, the increases in the levelsof neoxanthin and, especially, ß-carotene were muchgreater than those observed for the other carotenoids. Levelsof lutein also increased but this change was small by comparisonto those observed for neoxanthin and ß-carotene. Inthe long-term the concentration of violaxanthin remained unalteredalthough significant transient changes were recorded. The levelsof antheraxanthin and zeaxanthin were markedly reduced duringgreening. The rate of pigment synthesis decreased with increasingcell age, i.e. from the base to the tip of the primary leaf.Overall, carotenoid levels increased by approximately 100% atthe base of the seedling but hardly at all at the tip. Key words: Hordeum vulgare, carotenoids, violaxanthin-cycle, etiolation     

COMPARATIVE STUDIES ON GROWTH, RESPIRATION, PHOTOSYNTHESIS AND PIGMENT CONTENT IN RHODOPSEUDOMONAS PALUSTRIS

1. Comparative studies were performed on growth, photosyntheticand respiratory activities, and pigment content in Rhodopseudomonaspalustris.
2. The growth of the organism, as influenced by variousculturalconditions such as light, aerobiosis, anaerobiosisand nutritionalfactors was investigated.
3. The respiratoryactivity of the bacterium was found to be higherin dark-growncells than in cells grown in the light. The photosyntheticactivitydid not significantly depend on the growth conditionsof theculture. Cells of younger cultures were found to be moreactivethan those of older cultures, with respect both to respirationand photosynthesis.
4. The pigment content was found to be higherin the light-growncells than in the dark-grown ones. The ratiophotosyntheticactivity/bacteriochlorophyll was significantlyhigher in thelatter than in the former.
5. Light, as well asvarious nutritional factors, was found toexert a marked accelerationon pigment formation, although ithas not yet been possibleto culture cells completely lackingin photosynthetic pigmentsand accordingly in photosyntheticactivity.

¹ Present address: Division of Dermatology and Urology, TokyoMetropolitan Hiroo Hospital, Tokyo. ² Present address: Department of Biology, Saitama University,Urawa. ³ Present address: Department of Biochemistry, School of Medicine,Yokohama University, Yokohama. ⁴ Present address: Department of Biophysics and Biochemistry,Faculty of Science, University of Tokyo, Tokyo. (Received July 23, 1961; )     

Effect of illumination intensity and inhibition of carotenoid biosynthesis on assembly of peripheral light-harvesting complexes in purple sulfur bacteria <Emphasis Type="Italic">Allochromatium vinosum</Emphasis> ATCC 17899

Effect of illumination intensity and inhibition of carotenoid biosynthesis on assemblage of different spectral types of LH2 complexes in a purple sulfur bacterium Allochromatium (Alc.) vinosum ATCC 17899 was studied. Under illumination of 1200 and 500 lx, the complexes B800-850 and B800-840 and B800-820 were assembled. While rhodopine was the major carotenoid in all spectral types of the LH2 complex, a certain increase in the content of carotenoids with higher numbers of conjugated double bonds (anhydrorhodovibrin and didehydrorhodopin) was observed in the B800-820 complex. At 1200 lx, the cells grew slowly at diphenylamine (DPA) concentrations not exceeding 53 μM, while at illumination intensity decreased to 500 lx they could grow at 71 μM DPA (DPA cells). Independent on illumination level, the inhibitor is supposed to impair the functioning of phytoene synthetase (resulting in a decrease in the total carotenoid content) and of phytoene desaturase, which results in formation of neurosporene hydroxy derivatives and ζ-carotene. In the cells grown at 500 lx, small amounts of spheroidene and OH-spheroidene were detected. These carotenoids were originally found under conditions of carotenoid synthesis inhibition in bacteria with spirilloxanthin as the major carotenoid. Carotenoid content in the LH2 complexes isolated from the DPA cells was ~15% of the control (without inhibition) for the B800-850 and ~20% of the control for the B800-820 and B800-840 DPA complexes. Compared to the DPA pigment-containing membranes, the DPA complexes were enriched with carotenoids due to disintegration of some carotenoidless complexes in the course of isolation. These results support the supposition that some of the B800-820, B800-840, and B800-850 complexes may be assembled in the cells of Alc. vinosum ATCC 17899 without carotenoids. Comparison of the characteristics obtained for Alc. vinosum ATCC 17899 and the literature data on strain D of the same bacteria shows that they belong to two different strains, rather than to one as was previously supposed.     

The Female-specific Protein (Vitellogenic Protein) in Crustacea with Particular Reference to Orchestic gammarella (Amphipoda)

Comparative electrophoretic studies of male and female hemolymphin Crustacea have led to the discovery of a lipoglycoproteinfraction present in females and absent from males. The female-specificprotein fraction also contains a pigment which has been identifiedin a small number of species and appears to be a carotenoid.Further observations indicated that the presence of this fractionis coincident with the presence of maturing ovocytes in theovary. The major protein component of the vitellus is also alipoglycocarotenoprotein complex. Comparative analyses haveshown that the female-specific protein fraction present in hemolymphand the major protein component of the vitellus are electrophoreticallyand immunochemically identical. Moreover, in the Amphipod Orchestiagammarella both components appear to have the same molecularweight, estimated as approximately 3 x 10⁵ by Sephadex G 200gel filtration. Although there is ample evidence to supportthe idea that the female-specific protein is synthesized externallyto the ovary, the site of synthesis still remains unknown. Experimentallyinduced sex reversal in O. gammarella indicates that the synthesisof the female specific protein is under ovarian control.     

The fate of the carotenoid pigment fucoxanthin during passage through the copepod gut: pigment recovery as a function of copepod species, season and food concentration

The amount of fucoxanthin, a taxonomically diagnostic carotenoid,recovered after passage through the guts of the copepods Acartiacalifomiensis and Calanus pacificus, was determined after thecopepods had fed on low (50 µg Cl^–1) and high (350µg C1^–1 for Acartia; 500 ug C H for Calanus) concentrationsof the diatom Thalassiosira weissftogii, during spring (May)and winter (December). Changes in pigment concentrations andcell abundances were assessed in experimental (with copepods)and control (without copepods) samples by standard incubationexperiments. Pigment recovery was assessed by (i) comparingthe amount of ingested pigment recovered in the experimentalgroups with that predicted to have been ingested from cell countdata and (ii) comparing fuco-xanthin/cell ratios in controland experimental samples. Both techniques suggested that pigmentloss is substantial (usually 60–100%), regardless of species,food availability or season. Patterns of pigment conservationdiffered between species, although pigment recovery was alwayshigher at high, than at low, food concentrations. Pigment recoveryin Acartia was higher (9.4–28.0%) in the spring than duringthe winter (0 recovery), regardless of food concentration. InCalanus, however, pigment recovery was always higher at high(34.9–67.8%) than at low (0 recovery) food concentrations,regardless of season.     

Excitation energy transfer and carotenoid radical cation formation in light harvesting complexes — A theoretical perspective

Light harvesting complexes have been identified in all chlorophyll-based photosynthetic organisms. Their major function is the absorption of light and its transport to the reaction centers, however, they are also involved in excess energy quenching, the so-called non-photochemical quenching (NPQ). In particular, electron transfer and the resulting formation of carotenoid radical cations have recently been discovered to play an important role during NPQ in green plants. Here, the results of our theoretical investigations of carotenoid radical cation formation in the major light harvesting complex LHC-II of green plants are reported. The carotenoids violaxanthin, zeaxanthin and lutein are considered as potential quenchers. In agreement with experimental results, it is shown that zeaxanthin cannot quench isolated LHC-II complexes. Furthermore, subtle structural differences in the two lutein binding pockets lead to substantial differences in the excited state properties of the two luteins. In addition, the formation mechanism of carotenoid radical cations in light harvesting complexes LH2 and LH1 of purple bacteria is studied. Here, the energetic position of the S₁ state of the involved carotenoids neurosporene, spheroidene, spheroidenone and spirilloxanthin seems to determine the occurrence of radical cations in these LHCs upon photo-excitation. An elaborate pump-deplete-probe experiment is suggested to challenge the proposed mechanism.     

Reconstitution of Okenone into Light Harvesting Complexes from <Emphasis Type="Italic">Allochromatium minutissimum</Emphasis>

Okenone was reconstituted into light harvesting (LH) complexes of the purple photosynthetic bacterium Allochromatium minutissimum possessing the spirilloxanthin pathway for carotenoid biosynthesis. Suppression of this pathway by diphenylamine, an inhibitor of carotenogenesis, yielded nearly carotenoidless complexes preserving their native spectral properties. Using a previously developed technique, okenone was readily reconstituted into LH1 complex (>90%) whereas its reconstitution into LH2 complex was of low efficacy (10-20%). The absorption band of the reconstituted okenone was shifted to shorter wavelength compared with its position in vivo. This is typical for other reconstituted carotenoids. The reconstitution of okenone was confirmed by Li-DS electrophoresis (in contrast to free okenone the reconstituted okenone migrated with complexes), circular dichroism spectra (reconstituted okenone exhibited optical activity), and fluorescence excitation spectrum (energy transfer from okenone to bacteriochlorophyll was at the control level).     

Singlet-triplet excitation fission in light-harvesting complexes of photosynthetic bacteria and in isolated carotenoids

Time-resolved electron paramagnetic resonance was used to study the properties of carotenoid triplet states populated in LH2 light-harvesting complexes of phototrophic bacteria Allochromatium minutissimum, Rhodopseudomonas palustris, and in carotenoid films free of bacteriochlorophyll. The study was performed on purified LH2 preparations not contaminated by reaction centers, and under selective pigment excitation. The obtained results enable a conclusion that the carotenoid triplet states, both in LH2 complexes and films, are populated in the process of homofission of singlet excitation into two triplets, which involves only carotenoid molecules. It is observed that the fission process in magnetic field leads to predominant population of the T₀ spin sublevel of the triplet. One molecular spin sublevel of the triplet is demonstrated to possess an increased probability of intersystem crossing to the ground state, independent of the carotenoid configuration. Pigment composition of the LH2 protein heterodimers is discussed, and a conclusion of the possible presence of two interacting carotenoid molecules is made.     

Mechanism of Photoregulated Carotenogenesis in Rhodotorula minuta I. Photocontrol of Carotenoid Production

Rhodotorula minuta cells, which have only traces of carotenoidswhen grown in the dark, started carotenoid production with theonset of illumination and the amount increased almost linearlyuntil 70 hr then remained constant thereafter when incubationwas continued under illumination, with the number of cells continuingto increase. The rate of carotenoid production [Vc (µgg^–1 hr^–1)] depended on the intensity of light [I(ergcm^–2 sec^–1)], with the relationship of Vc=0.74 logI–1.46. The final carotenoid content [C(µg g^–1)]of cells incubated under continuous light was also controlledby the light intensity [I], with the relationship of C=52 logI–81. Control of carotenoid production by light occursas a two-phase process consisting of a temperatureindependentphotochemical reaction and light-independent biochemical reactions. (Received September 12, 1981; Accepted February 20, 1982)