Z. K. Makhneva

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.

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 presence of two interacting carotenoid molecules is made.

Reconstitution of Okenone into light harvesting complexes from Allochromatium minutissimum.

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).

Some Spectral Characteristics of Pigment–Protein Complexes and Their Interaction in Membranes of Thiorhodospira sibirica

The structure of the photosynthetic apparatus of purple photosynthetic bacteria is much simpler than in other photosynthesizing organisms. Usually, the photosynthetic apparatus of purple bacteria consists of two light-harvesting complexes (B800-850 and B880 or LH2 and LH1, respectively) and a reaction center (RC) [1–3]. The absence of the LH2 complex in carotenoidless strains of nonsulfur purple bacteria studied thus far is a specific feature of these bacteria [1, 4, 5]. It was shown in the preceding works [2, 6] that the complete set of antenna complexes is synthesized in the sulfur photosynthesizing bacterium Chromatium minutissimum even in the presence of agents causing a 95–99% inhibition of carotenoid biosynthesis. The results of studies of antenna complexes of this bacterium were inconsistent with the existing views on the mechanisms of inhibition of carotenoid biosynthesis and the role of carotenoids in bacterial photosynthesis. The following results should be emphasized in this context: a relatively low content of phytoine in membranes treated with inhibitor, formation of LH2 complexes even against the background of a 99% inhibition of carotenoid biosynthesis, absence of the blue shift of the long-wave band of the absorption spectrum of LH1, and direct evidence for carotenoid-mediated stabilization of the structure of antenna complexes [2, 3, 6].

Formation of Bacteriochlorophyll Form B820 in Light Harvesting 2 Complexes from Purple Sulfur Bacteria Treated with Dioxane

Treatment of some sulfur bacteria (Allochromatium minutissimum, Thiorhodospira sibirica, and Ectothiorhodospira halovacuolata WN22) with dioxane results in formation of the bacteriochlorophyll form B820 in the light harvesting complex LH2. This form characterized by absorption maximum at 820 nm has the same absorption spectrum as B820 subcomplex from LH1 complex. Appearance of the B820 form was accompanied by a sharp decrease in absorption in the carotenoid region. This phenomenon observed in all LH2 complexes investigated may be attributed to formation of colorless carotenoid aggregates. This is very similar to the previously reported dissociation of the LH1 complex with carotenoids into B820 subcomplexes. Although the B820 form corresponded the bacteriochlorophyll dimer, its circular dichroism spectrum showed that pigment molecules in this dimer exhibit different interaction than those in the B820 subcomplex. The dioxane treatment of LH2 complexes isolated from Rhodopseudomonas palustris bacteria grown under normal or low intensity illumination did not result in formation of such dimers. It is suggested that bacteriochlorophyll B820 formation is related to unique structure of LH2 complexes from the sulfur bacteria.

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 the B800-850 complex and its dependence on the spectral composition of the light is discussed.

Carotenoids are not Required to Provide Protection of Bacteriochlorophyll Clusters against Photooxidation in Light-Harvesting Complexes of Photosynthetic Bacteria

According to the generally accepted view, carotenoids play several important roles in photosynthesis, including light-harvesting, protective, and structural functions [1, 2]. The goal of this work was to analyze the protective function of carotenoids in more detail. For the first time, the possible protective role of carotenoids in photosynthesis was suggested in [3]. It was found [3] that the growth of a carotenoidless mutant of Rhodopseudomonas sphaeroides was inhibited, the concentration of bacteriochlorophyll in mutant cells gradually decreased, and exposure to low-intensity light in the presence of oxygen caused cell death. In contrast, wild-type cells containing carotenoids were tolerant to the presence of oxygen. It was suggested [3] on the basis of these results that carotenoids were able to protect bacteriochlorophyll against photooxidation, thereby preventing cell death. A series of experimental works was performed later using pigment‐protein complexes and model systems exposed to light of extreme intensity [1, 2]. A hypothetical mechanism of the protective effect of carotenoids was suggested on the basis of these studies. This mechanism can be illustrated by the following diagram: