A. A. Moskalenko

Structural Role of Carotenoids in Photosynthetic Membranes

Besides the light-harvesting and protecting role, carotenoids are also instrumental as structural components for the assembly of light-harvesting complexes in purple bacteria and green plants, as well as for the formation of photosystem II complex. Carotenoids stabilize those pigm ent-protein complexes, but have no effect on the form ation of the reaction centers of purple bacteria and photosystem I of plants.

Investigation of the neighbour relationships between photosystem II polypeptides in the two types of isolated reaction centres (D1/D2/cytb559 and CP47/D1/D2/cyt b559 complexes).

The nearest neighbour relationships within the D1/D2/cyt b 559 complex (PSIIRC) and the CP47/D1/D2/cyt b 559 complex (RC‐CP47) were investgated by using different length bifunctional crosslinking agents. The crosslinking products were identified by immunoblotting with polyclonal antibodies and by two‐dimensional gel electrophoresis. Seven products (CP47/D2, D1/D2/α, D1/D2, D2/α, D1/α, α/αa, α/β) have been revealed in both complexes. The crosslinking of both complexes does not increase their photostability. The photocrosslinking products (D1/α and D2/α) appeared under illumination of complexes with light of high intensity.

Fluorescence of native and carotenoid-depleted LH2 from Chromatium minutissimum, originating from simultaneous two-photon absorption in the spectral range of the presumed (optically ‘dark’) S1 state of carotenoids

Native and carotenoid‐depleted peripheral purple bacterial light‐harvesting complex (LH2) were investigated by simultaneous two‐photon excited (between 1300–1500 nm) fluorescence (TPF). TPF results from direct bacteriochlorophyll excitation in both samples. The spectral position of the 2Ag − state of rhodopsin is indicated by a diminuition of the bacteriochlorophyll TPF in native LH2. In conclusion, comparison to carotenoid‐depleted samples is a conditio sine qua non for unambiguous interpretation of similar experiments.

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 30 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°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 α-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.

Rubribacterium polymorphum gen. nov., sp. nov., a novel alkaliphilic nonsulfur purple bacterium from an Eastern Siberian soda lake

An alkaliphilic nonsulfur purple bacterial (NPB) strain “Green” was isolated from sediments of the littoral zone of the soda lake (mineralization 22 g/l, pH 9.5) in the Barguzin River valley (Eastern Siberia). The cells of the new isolate are ovoid or polymorphic at latter stages. The photosynthetic membrane structures are of vesicular type. Bacteriochlorophyll a and carotenoids of both spheroidene and spirilloxanthin type are the photosynthetic pigments. Two light-harvesting systems (LH1 and LH2) are present. The new isolate is a photoheterotroph and a facultative aerobe. It grows well in the dark on organic substrates; anaerobic phototrophic growth is poor. The isolate is alkaliphilic with pH optimum of 8.5–9.5. The most abundant cell growth occurred at 5–40 g/l NaCl (optimum at 10 g/l) and 30 °C. The DNA G+C base content was 69.9 mol %. Analysis of 16S rRNA gene sequences revealed a 10% difference with the most closely related NPB (Rhodobacter species). Rubrimonas cliftoensis, a bacteriochlorophyll a-containing bacterium, is the closest relative (93.3% similarity). It is proposed that strain “Green” should be placed in the new genus and new species Rubribacterium polymorphum gen. nov., sp. nov. GenBank accession number: 16S rRNA-EU857676.

Reconstitution of Carotenoids into the Light-Harvesting Complex B800–850 of Chromatium minutissimum

Chromatophores and peripheral light-harvesting complexes B800–850 with a trace of carotenoids were isolated from Chromatium minutissimum cells in which carotenoid biosynthesis was inhibited by diphenylamine. Three methods previously used for the reconstitution of carotenoids into either the light-harvesting (LH1) type complexes or reaction centers (RC) of carotenoidless mutants were examined for the possibility of carotenoid reconstitution into the carotenoid depleted chromatophores. All these methods were found to be unsuitable because carotenoid depleted complex B800–850 from Chr. minutissimum is characterized by high lability. We have developed a novel method maintaining the native structure of the complexes and allowing reconstitution of up to 80% of the carotenoids as compared to the control. The reconstituted complex has a similar CD spectrum in the carotenoid region as the control, and its structure restores its stability. These data give direct proof for the structural role of carotenoids in bacterial photosynthesis.

The development of carotenoid-deficient membranes in plastids of barley seedlings treated with norflurazon☆

The effect of carotenoid (Car) deficiency on the formation of thylakoid membranes in barley seedlings grown with norflurazon (NF) was investigated. To exclude photodestruction during the growth of Car-deficient seedlings, the etiolated seedlings were illuminated for 24 h with 2.5 ms flashes every 12 min. The morphometric analysis of seven main ultrastructural parameters of plastids control and NF-treated seedlings was carried out. The length of one partition and the number of partitions per plastids section, which characterize the initial stages of stacking, showed no difference in etiolated NF-treated and control seedlings. Compared with the control, the number of partitions was considerably lower in those plastids of Car-deficient seedlings which, after flash illumination, were kept for 1.5 h in continuous light of low intensity. The photobleaching of chlorophyll (Chl) in post-illuminated seedlings provides an indication of the photodestructive processes in Car-deficient seedlings exposed to low-intensity light. Chl bleaching did not appear in Car-deficient seedlings illuminated only with flashes; however, the stacking of the membranes was disturbed: the partition lengths and number of partitions per plastid section were higher in flashed, non-treated seedlings than in Car-deficient seedlings. Although the flashed control and NF-treated seedlings had an equal amount of Chl and the same polypeptide composition, no activity of photosystem II (PSII) and no PSII reaction centre complex were found in NF-treated seedlings. Car-deficient seedlings mainly contained photosystem I (PSI) with a ratio of Chl to P700 of 60 compared with 150 in the control. It is therefore suggested that Car deficiency in flashed leaves, which may accumulate only a small amount of light-harvesting Chl ab protein, prevents the assembly of the PSII complex and membrane stacking.

Investigation of light-harvesting complex Rhodopseudomonas Sphaeroides

The investigation of LH complexes is of great interest since the main bulk of bacteriochlorophyll in purple bacteria is associated with them. Similar complexes have been isolated from different types of bacteria [l-5 ] . Two kinds of polypeptides have been found in the LH complexes from Chr. minutissimum [6] , Rh. palustris, T. roseopersicina, Ect. shaposhnikovii [ 71 and R. rubrum [ 8,9] ; however, only one has been discovered in the LH complex from Rh. sphaeroides [5] . This discrepancy may result from the use of two different electrophoretic systems [lo] . In the present study the separation of two polypeptides from the LH complexes in two different electrophoretic systems containing SDS is compared. In contrast with [5] , the LH complex from the wild strain Rh. sphaeroides is now shown to contain two polypeptides (9000 and 12 000 daltons). These polypeptides were not separated by electrophoresis in 10% polyacrylamide gel with SDS as in iI31 *

Investigation of spatial relationships and energy transfer between complexes B800-850 and B890-RC from Chromatium minutissimum reconstituted into liposomes

Spatial relationships between difterent pigment‐protein complexes in the membranes of the purple photosynthetic bacterium, Chromatium minutissimum, have been studied. The possibility of restoring the function of efficient excitation energy transfer from bacteriochlorephyll molecules to the reaction centers in the system of soybean liposomes, reconstituted with pigment‐protein complexes B800‐850 and B890‐RC from C. minutissimum, has been explored. The chemical cross‐linking method, together with stationary and picosecond spectrally resolved fluoresence measurements were employed. It has been shown that after the incorporation of the complexes into the liposame membranes conditions for directed excitation energy transfer from the light‐harvesting pigments to the reaction centers are created, which are less optimal, however, than those in the native state. Possible reasons are considered.

The isolation of the B812 subcomplex of the B880 core complex and the B800–850 complex from membranes of Chromatium minutissimum with extracted carotenoids; the structural role of carotenoids

Abstract The isolation of the B800–850 complex and the B812 subcomplex of the B880 core complex from membranes of Chromatium minutissimum with partially extracted carotenoids is presented. This method, based on that developed to isolate a similar subcomplex (B820) from the B880 core complex of Rhodospirillum rubrum (Miller et al., Biochemistry, 26 (1987) 5055–5062) with some modification, requires the treatment of chromatophores with octylglucoside followed by electrophoresis in polyacrylamide gel. It separates the B880-RC assembly enriched with reaction centres (RCs) and carotenoids but depleted of a significant amount of the B880 (core) complex, the carotenoidless B812 subcomplex of the B880 core complex and the B800–850 complex with a reduced level of carotenoids. The spectral and biochemical characteristics of the B812 subcomplex are the same as those of similar complexes from non-sulphur bacteria. The B800–850 complex exhibits stronger reversible conformational changes (hypsochromic shift of the band centred at 855 nm on treatment with detergent) compared with the control. The role of carotenoids in the structure of these complexes is discussed.