The LH2 Complexes (B800-850 and B800-830) Are Assembled in the Cells of the Sulfur Bacterium Thiorhodospira sibirica Strain Kir-3 without Carotenoids.

The results of assembling the light-harvesting complexes in the cells of the purple sulfur bacterium Thiorhodospira (T.) sibirica strain Kir-3 while suppressing the biosynthesis of carotenoids with diphenylamine (DPA) were studied. LH2 complexes (B800-850 and B800-830) with different carotenoid composition were isolated from the cells obtained. Maximum inhibition of carotenoid biosynthesis (~90% of the control) was reached at an inhibitor concentration of 53.25 µM (9 mg/L). It was established that changes in the qualitative and quantitative composition of carotenoids do not affect the assembly of B800-830 and B800-850 complexes. It is assumed that, in the population of DPA-LH2 complexes from T. sibirica strain Kir-3, both the carotenoidless complexes and the complexes containing one or two carotenoid molecules can be assembled. These results support the hypothesis that carotenoids are not required for assembling B800-850 and B800-830 complexes.

Bacteriochlorophyll Interaction with Singlet Oxygen in Membranes of Purple Photosynthetic Bacteria: Does the Protective Function of Carotenoids Exist?

The direct action of singlet oxygen on the bacteriochlorophyll (BChl) of light-harvesting complexes in the membranes of four species of purple non-sulfur and sulfur photosynthesizing bacteria with and without carotenoids was studied. It was found that BChl in carotenoidless samples is generally more resistant to the action of singlet oxygen compared to the control. It is assumed that carotenoids are not required to protect BChl of bacterial light-harvesting complexes from singlet oxygen, and in the classic work by Griffith et al. [1] the apoptosis process in carotenoidless mutant cells, which involves the destruction of complexes, the appearance of monomeric BChl, and the generation of singlet oxygen caused by BChl, followed by BChl oxidation, was mistakenly attributed to the protective function of carotenoids.

Singlet-triplet Fission of Carotenoid Excitation in the Purple Phototrophic Bacteria Thermochromatium tepidum.

Singlet fission of carotenoid excitation is studied in purple phototrophic bacterium Thermochromatium tepidum. Using time-resolved EPR and magnetic field-induced modulation of fluorescence yield it is shown that the concept of intramolecular excitation fission developed in a number of publications is not supported by the experimental results. The obtained data favor intermolecular fission mechanism involving two carotenoid molecules.

The Influence of the Number of Conjugated Double Bonds in Carotenoid Molecules on the Energy Transfer Efficiency to Bacteriochlorophyll in Light-Harvesting Complexes LH2 from Allochromatium vinosum Strain MSU.

Seven different carotenoids with the number of conjugated double bonds (N) from 5 to 11 were incorporated in vitro into carotenoidless complexes LH2 of the sulfur bacterium Allochromatium vinosum strain MSU. The efficiency of their incorporation varied from 4 to 99%. The influence of N in the carotenoid molecules on the energy transfer efficiency from these pigments to bacteriochlorophyll (BChl) in the modified LH2 complexes was studied for the first time. The highest level of energy transfer was recorded for rhodopin (N = 11) and neurosporene (N = 7) (37 and 51%, respectively). In the other carotenoids, this parameter ranged from 11 to 33%. In the LH2 complexes studied, we found no direct correlation between the decrease in N in carotenoids and increase in the energy transfer efficiency from these pigments to BChl.

Embedding carotenoids of spheroidene-branch biosynthesis into antenna complexes of sulfur photosynthetic bacteria.

The possibility of embedding the carotenoids of spheroidene-branch biosynthesis (spheroidene and spheroidenone) from non-sulfur bacteria into the diphenylamine antenna complexes (DPA-complexes) from the sulfur bacteria Allochromatium minutissimum and Ectothiorhodospira haloalkaliphila with carotenoid synthesis inhibited by diphenylamine (DPA) was studied for the first time. It was found that spheroidene was embedded into the DPA-complexes from these bacteria at a level of 75-87%, with spheroidene embedding efficiency being 41-68% for the LH1-RC DPA-complexes and 71-89% for the LH2 DPA-complexes. The energy transfer efficiency from carotenoids to bacteriochlorophyll was shown to depend not only on the type of carotenoid but also on the very structure on the antenna complex.

3-Acetyl-Chlorophyll Formation in Light-Harvesting Complexes of Purple Bacteria by Chemical Oxidation.

Dedicated to the memory of Yuriy Nikolayevich Kozlov Oxidation of bacteriochlorophyll (BChl) with potassium ferricyanide in membranes and LH2 complexes (carotenoid-less and control samples) from the purple bacteria Allochromatium minutissimum and Rhodobacter sphaeroides as well as BChl photobleaching in a model system have been studied. The oxidation of BChl depended on the type of bacteria. BChl850 was rapidly oxidized in samples from Alc. minutissimum, and BChl800 and BChl850 were slowly oxidized in samples from Rb. sphaeroides. The carotenoids were not involved in protecting BChl from chemical oxidation in the light-harvesting complexes. The appearance of BChl oxidation product was registered in the absorption spectra (absorption maximum about 700 nm) and by HPLC analysis. The oxidized BChl was identified as 3-acetyl-chlorophyll. It differed from BChl by the presence of a double bond in pyrrole ring II at the 7-8 position. The extinction coefficient of 3-acetyl-chlorophyll was about 10 times less than that of BChl850 in the LH2 complex from Alc. minutissimum. In the BChl → 3-acetyl-chlorophyll transition, the binding constant of the latter with LH2 complex as compared with that of BChl did not change dramatically, as indicated by: (i) preserved electrophoretic mobility of the complex; (ii) the presence of 3-acetyl-chlorophyll in the complex after separation; (iii) the presence of a 3-acetyl-chlorophyll CD signal that was proportional to its absorption spectrum.

Distribution of rhodopin and spirilloxanthin between LH1 and LH2 complexes when incorporating carotenoid mixture into the membrane of purple sulfur bacterium Allochromatium minutissimum 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.

Effect of Illumination Intensity and Inhibition of Carotenoid Biosynthesis on Assembly of Peripheral Light-Gathering Complexes in Purple Sulfur Bacteria C Allochromatium vinosum 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 didehydrorhodovibrin) was observed in the B800-820 complex. At 1200 lx, the cells grew slowly at diphe- nylamine (DPA) concentrations not exceeding 53 .iM, while at illumination intensity decreased to 500 Ix they could grow at 71 jiM DPA (DPA cells). Independent on illumination level, the inhibitor is supposed to impair the functioning of phytoine synthetase (resulting in a decrease in the total carotenoid content) and of phyto- ine desturase, which results in formation of neurosporene hydroxy derivatives and ;-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 carotenoid-free 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.

Peripheral Light-Harvesting LH2 Complex Can Be Assembled in Cells of Nonsulfur Purple Bacterium Rhodoblastus acidophilus without Carotenoids.

The effect of carotenoids on the assembly of LH2 complex in cells of the purple nonsulfur bacterium Rhodoblastus acidophilus was investigated. For this purpose, the bacterial culture was cultivated with an inhibitor of carotenoid biosynthesis - 71 µM diphenylamine (DPA). The inhibitor decreased the level of biosynthesis of the colored carotenoids in membranes by ~58%. It was found that a large amount of phytoene was accumulated in them. This carotenoid precursor was bound nonspecifically to LH2 complex and did not stabilize its structure. Thermostability testing of the isolated LH2 complex together with analysis of carotenoid composition revealed that the population of this complex was heterogeneous with respect to carotenoid composition. One fraction of the LH2 complex with carotenoid content around 90% remains stable and was not destroyed under heating for 15 min at 50°C. The other fraction of LH2 complex containing on average less than one molecule of carotenoid per complex was destroyed under heating, forming a zone of free pigments (and polypeptides). The data suggest that a certain part of the LH2 complexes is assembled without carotenoids in cells of the nonsulfur bacterium Rbl. acidophilus grown with DPA. These data contradict the fact that the LH2 complex from nonsulfur bacteria cannot be assembled without carotenoids, but on the other hand, they are in good agreement with the results demonstrated in our earlier studies of the sulfur bacteria Allochromatium minutissimum and Ectothiorhodospira haloalkaliphila. Carotenoidless LH2 complex was obtained from these bacteria with the use of DPA (Moskalenko, A. A., and Makhneva, Z. K. (2012) J. Photochem. Photobiol., 108, 1-7; Ashikhmin, A., et al. (2014) Photosynth. Res., 119, 291-303).

The spirilloxanthine-pathway carotenoid incorporation into light-harvesting complexes of Ectothiorhodospira haloalkaliphila in vitro.

Carotenoidless light-harvesting complexes (DPA-complexes) LH1-RC and LH2 were isolated from the purple sulfur bacterium Ectothiorhodospira haloalkaliphila in which carotenoid biosynthesis was suppressed with diphenylamine (DPA). Carotenoids of the spirilloxanthine series, which were isolated from the same bacterium, were incorporated into the DPA-complexes in vitro with an efficiency of 95-100%. The comparison of characteristics of the complexes with the incorporated carotenoids and the control complexes showed that the LH2 complexes with the incorporated carotenoids restored their absorption spectra, circular dichroism signals, and energy transfer from carotenoids to bacteriochlorophyll, which indicates that carotenoids were correctly incorporated into the structure of this complex.

Singlet-triplet fission of carotenoid excitation in light-harvesting LH2 complexes of purple phototrophic bacteria.

The current generally accepted structure of light-harvesting LH2 complexes from purple phototrophic bacteria conflicts with the observation of singlet-triplet carotenoid excitation fission in these complexes. In LH2 complexes from the purple bacterium Allochromatium minutissimum, a drop in the efficiency of carotenoid triplet generation is demonstrated, which correlates with the extent of selective photooxidation of bacteriochlorophylls absorbing at ~850 nm. We conclude that singlet-triplet fission of carotenoid excitation proceeds with participation of these excitonically coupled bacteriochlorophylls. In the framework of the proposed mechanism, the contradiction between LH2 structure and photophysical properties of carotenoids is eliminated. The possibility of singlet-triplet excitation fission involving a third mediator molecule was not considered earlier.

[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

Light-harvesting complexes from purple sulfur bacteria Allochromatium minutissimum assembled without carotenoids.

Effect of carotenoid (Car) biosynthesis inhibitor diphenylamine (DPA) on purple sulfur bacteria Allochromatium (Alc) minutissimum cell growth has been investigated. Cell growth in the presence of maximum concentration of DPA results in practically complete suppression (∼99%) of carotenoids (Cars) according to the spectrophotometric, HPLC and CD data. Phytoene does not replace the colored carotenoids in these cells. Also Phytoene does not accumulate in large amounts in the cells treated with DPA. A new method for calculating the content of Cars in the complexes from the cells with inhibited Car synthesis including the number of empty Car's "pockets" has been used. Our results together with published data devoted to DPA action on the cell growth of purple bacteria revealed that Phytoene was not accumulated in the cells treated with DPA. We have concluded that (i) DPA completely inhibits or strongly reduces synthesis of the colored Cars in the cells of purple bacteria, (ii) Phytoene is the main one among the trace amounts of the other Cars in the case of significant inhibition of Car biosynthesis (80-90% or higher). The amount of the LH2 complexes presented in the membranes of Alc minutissimum was found to be little dependent on DPA. From DPA-grown cultures it was possible to isolate Car-less both the LH1 (as LH1-RC complex) and the LH2 complexes. Electronic absorption properties of BChl's were very similar to those isolated from the control cells. It is shown by HPLC data that the 100 LH2 complexes from cells of Alc minutissimum, in which the synthesis of Car was depressed, contained ∼9 Car molecules and 5 Phytoene molecules. Thus, only nine (with 1 Car molecule per a complex) or less (if more than one Car molecule per a complex) of the 100 LH2 complexes contain molecules of Cars. It means that 90 or more LH2 complexes from each 100 ones are assembled without any Cars. This is in strong contrast with the previous results obtained with purple non-sulfur bacterium Rhodobacter sphaeroides, where the amount of LH2 presented in the membrane was directly correlated to the amount of the carotenoids synthesized (H.P. Lang, C.N. Hunter, The relationship between carotenoid biosynthesis and the assembly of the light harvesting LH2 complex in Rhodobacter sphaeroides, Biochem. J. 298 (1994) 197-205). Our results show that although the presence of Car molecules is important for the stability of the LH2 complexes the overall native structure can be maintained without any Cars at least in the case of purple sulfur bacteria.

[Roseococcus suduntuyensis sp. nov., a new aerobic bacteriochlorophyll A-containing bacterium isolated from a low-mineralized soda lake of Eastern Siberia].

A novel strain, SHET, of aerobic bacteriochlorophyll a-containing bacteria was isolated from the surface layer of bottom sediments from the soda lake Shuluutai-Ekhe-Torom (Chita oblast, Eastern Siberia, Russia). The lake water has a total mineralization of 3.0 g/l and a pH of 9.2. The cells of strain SHET are cocci or short rods, which reproduce by uniform division. The cells are motile by means of flagella. The cell wall structure is of the gram-negative type. Sparse intracytoplasmic membrane vesicles are located close to the cell wall. The new isolate is an obligate aerobe and facultative alkaliphile which grows in a pH range of 7.5-9.5 (with an optimum at pH 8.5-9.0). The best growth of strain SHET occurred at 2.0 g/l NaCl and 23-28 degrees C. Photosynthetic pigments are represented by bacteriochlorophyll a, with the maximum absorption at 865 nm in the in vivo spectrum, and carotenoids (spirilloxanthin derivatives). Analysis of the 16S rRNA gene sequences demonstrated that strain SHET is closely related to Roseococcus thiosulfatophilus of the alpha-1 subclass of Proteobacteria (98.6 % similarity). The DNA G + C base content is 69.1 mol %. Unlike Rsc. thiosulfatophilus, strain SHET grows well on sugars and glycerol and is not capable of utilizing thiosulfate as an energy source. The new isolate is a facultative alkaliphile and reduces nitrates to nitrites. On the basis of its phenotypic and genetic characteristics, strain SHET was described as a new species of the genus Roseococcus, Rsc. suduntuyensis sp. nov.

[Assembly of LH2 light-harvesting complexes in Rhodopseudomonas palustris cells illuminated by blue and red light].

We investigated the formation of the B800-850 complex in cells of the bacterium Rhodopseudomonas palustris AB illuminated by red and blue light under anaerobic growth conditions. Under red illumination, the B800-850 complex was assembled with a reduced absorption band at 850 nm. The results of re-electrophoresis of the B800-850 complex and oxidation in the presence of potassium iridate suggest its heterogeneity. It may be a mixture of two complexes (B800 and B800-850). The B800-850 complex lacks the capacity for conformational transitions if assembled under blue illumination. Accordingly, the light-harvesting complex assembled in the blue light contains polypeptides that are not synthesized under normal conditions or at increased or decreased light intensities. The mechanism of regulation of the synthesis of the polypeptides of light-harvesting B800-850 complex and its dependence on the spectral composition of the light is discussed.

[Rhodobaca barguzinensis sp. nov., a new alkaliphilic purple nonsulfur bacterium isolated from a soda lake of the Barguzin Valley (Buryat Republic, eastern Siberia)].

A novel strain, alga-05, of alkaliphilic purple nonsulfur bacteria was isolated from sediments of a small saline (60 g/l) soda lake near Lake Algin (Barguzin Valley, Buryat Republic, Russia). These bacteria contain bacteriochlorophyll a and carotenoids of the alternative spirilloxanthin group with predominating demethylspheroidenone. They are facultative anaerobes; their photosynthetic structures are of the vesicular type and arranged along the cell periphery. Growth of this strain is possible in a salinity range of 5-80 g/l NaCl, with an optimum at 20 g/l NaCl. Best growth occurred at 20-35 degrees C. Analysis of the 16S rRNA gene sequences demonstrated that the studied isolate is closely related to the alkaliphilic purple nonsulfur bacterium Rhodobaca bogoriensis (99% similarity) isolated from soda lakes of the African Rift Zone. According to the results of DNA-DNA hybridization, strain alga-05 has a 52% similarity with the type species of the genus Rhodobaca. On the basis of the obtained genotypic data and some phenotypic properties (dwelling in a hypersaline soda lake of Siberia, moderate halophily, ability to grow at relatively low temperatures, etc.), the isolated strain of purple bacteria was described as a new species of the genus Rhodobaca, Rca. barguzinensis sp. nov.