Reversible Changes in the Structural Features of Photosynthetic Light-Harvesting Complex 2 by Removal and Reconstitution of B800 Bacteriochlorophyll a Pigments.

Light-harvesting complex 2 (LH2) is an integral membrane protein in purple photosynthetic bacteria. This protein possesses two types of bacteriochlorophyll (BChl) a, termed B800 and B850, which exhibit lowest-energy absorption bands (Qy bands) around 800 and 850 nm. These BChl a pigments in the LH2 protein play crucial roles not only in photosynthetic functions but also in folding and maintaining its protein structure. We report herein the reversible structural changes in the LH2 protein derived from a purple photosynthetic bacterium, Rhodoblastus acidophilus, induced by the removal of B800 BChl a (denoted as B800-free LH2) and the reconstitution of exogenous BChl a. Atomic force microscopy observation clearly visualized the nonameric ring structure of the B800-free LH2 with almost the same diameter as the native LH2. Size exclusion chromatography measurements indicated a considerable decrease in the size of the protein induced by the removal of B800 BChl a. The protein size was almost recovered by the insertion of BChl a pigments into the B800 binding sites. The decrease in the LH2 size would mainly originate from the shrinkage of the B800 binding sites perpendicular to the macrocycle of B800 BChl a without deformation of the circular arrangement. The reversible changes in the LH2 structure induced by the removal and reconstitution of B800 BChl a will be helpful for understanding the structural principle and the folding mechanism of photosynthetic pigment-protein complexes.

Skin Anti-Aging Activities of Bacteriochlorophyll a from Photosynthetic Bacteria, Rhodobacter sphaeroides.

In this work, the anti-aging skin effects of bacteriochlorophyll a isolated from Rhodobacter sphaeroides are first reported, with notably low cytotoxicity in the range of 1% to 14% in adding 0.00078 (% (w/w)) of the extracts, compared with the normal growth of both human dermal fibroblast and keratinocyte cells without any treatment as a control. The highest production of procollagen from human fibroblast cells (CCD-986sk) was observed as 221.7 ng/ml with 0.001 (% (w/w)) of bacteriochlorophyll a, whereas 150 and 200 ng/ml of procollagen production resulted from addition of 0.001 (% (w/w)) of the photosynthetic bacteria. The bacteriochlorophylla- induced TNF-α production increased to 63.8%, which was lower secretion from HaCaT cells than that from addition of 0.00005 (% (w/w)) of bacteriochlorophyll a. Additionally, bacteriochlorophyll a upregulated the expression of genes related to skin anti-aging (i.e., keratin 10, involucrin, transglutaminase-1, and MMPs), by up to 4-15 times those of the control. However, crude extracts from R. sphaeroides did not enhance the expression level of these genes. Bacteriochlorophyll a showed higher antioxidant activity of 63.8% in DPPH free radical scavenging than those of water, ethanol, and 70% ethanol extracts (14.0%, 57.2%, and 12.6%, respectively). It was also shown that the high antioxidant activity could be attributed to the skin anti-aging effect of bacteriochlorophyll a, although R. sphaeroides itself would not exhibit significant anti-aging activities.

Marked seasonality of aerobic anoxygenic phototrophic bacteria in the coastal NW Mediterranean Sea as revealed by cell abundance, pigment concentration and pyrosequencing of pufM gene.

The abundance and diversity of aerobic anoxygenic phototrophs (AAPs) were studied for a year cycle at the Blanes Bay Microbial Observatory (NW Mediterranean) and their potential links to an array of environmental variables were explored. Cell numbers were low in winter and peaked in summer, showing a marked seasonality that positively correlated with day length and light at the surface. Bacteriochlorophyll a concentration, their light-harvesting pigment, was only detected between April and October, and pigment cell quota showed large variations during this period. Pyrosequencing analysis of the pufM gene revealed that the most abundant operational taxonomic units (OTUs) were affiliated to phylogroup K (Gammaproteobacteria) and uncultured phylogroup C, although they were outnumbered by alphaproteobacterial OTUs in spring. Overall, richness was higher in winter than in summer, showing an opposite trend to abundance and day length. Clustering of samples by multivariate analyses showed a clear seasonality that suggests a succession of different AAP subpopulations over time. Temperature, chlorophyll a and day length were the environmental drivers that best explained the distribution of AAP assemblages. These results indicate that AAP bacteria are highly dynamic and undergo seasonal variations in diversity and abundance mostly dictated by environmental conditions as exemplified by light availability.

[Fingerprinting analysis of photopigments in purple bacteria].

OBJECTIVE: Photopigments, including carotenoid and bacteriochlorophyll a, are the most important functional units of photosynthesis in purple bacteria. We developed rapid qualitative and quantitative methods to determine photopigments. METHODS: Using Rhodopesudomonas palustris CQV97 as a reference, we used image gray intensity analysis, absorption spectrophotometry, thin layer chromatography (TLC), HPLC and mass spectrometry (MS) for photopigment analysis. RESULTS: The total amount of photopigments increased by 13.5% by using modified acetone-methanol extraction. We developed two types of photopigment fingerprintings by TLC and HPLC, estimated the apparent relative content of each photopigment of fingerprintings, and determined the corresponding relationships between R(f) value of each photopigment on TLC fingerprinting and retention time of each photopigment elution in HPLC fingerprinting. Based on the data from the absorption spectra, MS and related photopigment biosynthetic pathway analysis, we identified 11 photopigments in CQV97 strain. Using this strain as a standard, we analyzed photopigments of the tested samples by TLC or HPLC. It was shown that (1) the relative standard deviation (RSD) of the two methods was less than 5%; (2) the compositions and contents of the theory sample were consistent with that of the standard sample; (3) the photopigment compositions of the real sample was the same as the standard sample, but the photopigment content was different. CONCLUSION: Both of TLC and HPLC analyses for photopigment determination have good stability and repeatability. The fingerprintings analyses are suitable for rapid determination of photopigments of purple bacteria and have important application in control of regulation mechanism for photopigment synthesis.

[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.

Description of Labrenzia alexandrii gen. nov., sp. nov., a novel alphaproteobacterium containing bacteriochlorophyll a, and a proposal for reclassification of Stappia aggregata as Labrenzia aggregata comb. nov., of Stappia marina as Labrenzia marina comb. nov. and of Stappia alba as Labrenzia alba comb. nov., and emended descriptions of the genera Pannonibacter, Stappia and Roseibium, and of the species Roseibium denhamense and Roseibium hamelinense.

A slightly pink-coloured strain, strain DFL-11(T), was isolated from single cells of the marine dinoflagellate Alexandrium lusitanicum and was found to contain the genes encoding two proteins of the photosynthetic reaction centre, pufL and pufM. 16S rRNA gene sequence analysis revealed that the novel strain belonged to the alpha-2 subgroup of the Proteobacteria and was most closely related to Stappia aggregata (97.7 % similarity), Stappia alba (98.0 %) and Stappia marina (98.0 %). Dark-grown cells of strain DFL-11(T) contained small amounts of bacteriochlorophyll a (bchl a) and a carotenoid. Cells of strain DFL-11(T) were rods, 0.5-0.7 x 0.9-3.0 microm in size and motile by means of a single, subpolarly inserted flagellum. The novel strain was strictly aerobic and utilized a wide range of organic carbon sources, including fatty acids, tricarboxylic acid cycle intermediates and sugars. Biotin and thiamine were required as growth factors. Growth was obtained at sea salt concentrations of between 1 and 10 % (w/v), at a pH between 6 and 9.2 and at a temperature of up to 33 degrees C (optimum, 26 degrees C). Nitrate was not reduced and indole was not produced from tryptophan. Strain DFL11(T) was resistant to potassium tellurite and transformed it to elemental tellurium. The major respiratory lipoquinone was ubiquinone 10 (Q10). The polar lipids comprised phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylcholine, an unidentified aminolipid and the glycolipid sulphoquinovosyldiacylglyceride. The fatty acids comprised 16 : 1 omega7c, 16 : 0, 18 : 1 omega7c, 18 : 0, 11-methyl 18 : 1 omega6t, 11-methyl 20 : 1 omega6t, 20 : 1 omega7c, 22 : 0, 22 : 1 and the hydroxy fatty acids 3-OH 14 : 0, 3-OH 16 : 0 (ester-linked), 3-OH 18 : 0, 3-OH 20 : 1 and 3-OH 20 : 0, all of which are amide-linked. The DNA G+C value was 56 mol%. Comparative analysis of alpha-2 subgroup 16S rRNA gene sequences showed that the type species of the genus Stappia, Stappia stellulata, is only distantly related to S. aggregata (95.3 % sequence similarity). Based on the combination of the 16S rRNA gene sequence data, a detailed chemotaxonomic study and the biochemical and physiological properties of members of the genera Stappia, Pannonibacter and Roseibium, it is proposed that S. aggregata, S. alba, S. marina are transferred to a new genus, Labrenzia gen. nov., as Labrenzia aggregata comb. nov., Labrenzia alba comb. nov. and Labrenzia marina comb. nov. The type species of the new genus is Labrenzia alexandrii sp. nov., with strain DFL-11(T) (=DSM 17067(T)=NCIMB 14079(T)) as the type strain. The pufLM genes of the photosynthesis reaction centre were shown to be present in some, but not all, species of the new genus Labrenzia and they were identified for the first time in S. stellulata. In accordance with the new data collected in this study, emended descriptions are provided for the genera Pannonibacter, Roseibium and Stappia.

Dinoroseobacter shibae gen. nov., sp. nov., a new aerobic phototrophic bacterium isolated from dinoflagellates.

A novel group of aerobic anoxygenic phototrophic bacteria was isolated from marine dinoflagellates, and two strains were characterized in detail. Cells were Gram-negative cocci or ovoid rods and were motile by means of a single, polarly inserted flagellum. They were obligate aerobes requiring 1-7 % salinity. The optimal pH range for growth was 6.5-9.0 and the temperature optimum was 33 degrees C. The bacteria contained bacteriochlorophyll a and spheroidenone as the only carotenoid. The in vivo absorption spectrum displayed two maxima in the infrared region at 804 and 868 nm. The distinct 804 nm band indicates the presence of light-harvesting system 2. Various organic carbon sources were assimilated, including many carboxylic acids, glucose and glycerol, but not butyrate, ethanol or methanol. Dissimilatory nitrate reduction was found for both strains. The physiological characteristics of the new strains resembled those of Roseobacter denitrificans, but there were differences in the lipid composition. Based on 16S rRNA gene sequence analysis the new strains are relatively distant from other recognized species, with the closest relatives Jannaschia helgolandensis, Ruegeria atlantica and Rhodobacter veldkampii showing 94.1-93.4 % similarity. Similarity to Roseobacter denitrificans was only 92.2 %, in line with numerous other species of the Roseobacter group. Therefore, it is proposed to classify the strains in a new genus and species within the Roseobacter clade, Dinoroseobacter shibae gen. nov., sp. nov. The type strain is DFL 12(T) (=DSM 16493(T)=NCIMB 14021(T)).

Porphyrobacter donghaensis sp. nov., isolated from sea water of the East Sea in Korea.

Two Gram-negative, motile, non-spore-forming, bacteriochlorophyll a-containing slightly halophilic strains, SW-132(T) and SW-158, were isolated from sea water of the East Sea in Korea, and subjected to a polyphasic taxonomic study. The two isolates were characterized chemotaxonomically as having Q-10 as the predominant respiratory lipoquinone and major amounts of unsaturated fatty acids C(18 : 1)omega7c and C(17 : 1)omega6c. The DNA G+C contents of the two strains were in the range 66.8-65.9 mol%. The 16S rRNA gene sequences of strains SW-132(T) and SW-158 were 99.9 % (1 nt difference) similar and their mean level of DNA-DNA relatedness was 86 %. The 16S rRNA gene sequence analysis showed that strains SW-132(T) and SW-158 are phylogenetically closely related to Porphyrobacter species and Erythromicrobium ramosum. Similarities between the 16S rRNA gene sequences of strains SW-132(T) and SW-158 and the type strains of Porphyrobacter species and E. ramosum ranged from 97.8 to 99.0 %. DNA-DNA relatedness data indicated that strains SW-132(T) and SW-158 are members of a genomic species that is separate from the four Porphyrobacter species. On the basis of phenotypic and phylogenetic data and genetic distinctiveness, strains SW-132(T) (=KCTC 12229(T)=DSM 16220(T)) and SW-158 (=KCTC 12230) are classified as a novel Porphyrobacter species, for which the name Porphyrobacter donghaensis sp. nov. is proposed.

Synthetic analogues of the histidine-chlorophyll complex: a NMR study to mimic structural features of the photosynthetic reaction center and the light-harvesting complex.

Mg(II)-porphyrin-ligand and (bacterio)chlorophyl-ligand coordination interactions have been studied by solution and solid-state MAS NMR spectroscopy. (1)H, (13)C and (15)N coordination shifts due to ring currents, electronic perturbations and structural effects are resolved for imidazole (Im) and 1-methylimidazole (1-MeIm) coordinated axially to Mg(II)-OEP and (B)Chl a. As a consequence of a single axial coordination of Im or 1-MeIm to the Mg(II) ion, 0.9-5.2 ppm (1)H, 0.2-5.5 ppm (13)C and 2.1-27.2 ppm (15)N coordination shifts were measured for selectively labeled [1,3-(15)N]-Im, [1,3-(15)N,2-(13)C]-Im and [1,3-(15)N,1,2-(13)C]-1-MeIm. The coordination shifts depend on the distance of the nuclei to the porphyrin plane and the perturbation of the electronic structure. The signal intensities in the (1)H NMR spectrum reveal a five-coordinated complex, and the isotropic chemical shift analysis shows a close analogy with the electronic structure of the BChl a-histidine in natural light harvesting 2 complexes. The line broadening of the ligand responses support the complementary IR data and provide evidence for a dynamic coordination bond in the complex.

Distribution of chloropigments in suspended particulate matter and benthic microbial mat of a meromictic lake, Lake Kaiike, Japan.

We investigated the distribution of chloropigments in a small meromictic lake, Lake Kaiike, south-west Japan. In the water-column, concentrations of Chl a related to cyanobacteria, BChl a related to purple sulphur bacteria, and three types of BChl e homologues (BChls e1, e2 and e3) related to brown-coloured green sulphur bacteria, were maximal at the redox boundary. Below the redox boundary, absolute concentrations of Chl a and BChl a gradually decreased with depth, whereas BChls e remained rather constant. Suspended particulate matter (SPM) at the deeper region of the anoxic water-column was enriched in highly alkylated BChl e homologues compared with SPM at the redox boundary. The shift in the relative content of highly alkylated BChl e homologues beneath the boundary was associated with community related adaptation of brown-coloured green sulphur bacteria to changes in light quality/quantity, resulting from the optical absorption and reflectance of SPMs in the overlying water-column. Benthic microbial mats were characterized by high abundances of BChls e, in which highly alkylated homologues were substantially abundant. This suggests that the BChls e in the microbial mat may be derived from the low-light adapted brown-coloured green sulphur bacteria forming the bacterial mat.

Structure of the Rhodobacter sphaeroides light-harvesting 1 beta subunit in detergent micelles.

The light harvesting 1 antenna (LH1) complex from Rhodobacter sphaeroides funnels excitation energy to the photosynthetic reaction center. Our ultimate goal is to build up the structure of LH1 from structures of its individual subunits, much as the antenna can self-assemble from its components in membrane-mimicking detergent micelles. The beta subunit adopts a nativelike conformation in Zwittergent 3:12 micelles as demonstrated by its ability to take the first step of assembly, binding BChl a. Multidimensional NMR spectroscopy shows that the beta subunit folds as a helix((L12-S25))-hinge((G26-W28))-helix((L29-W44)) structure with the helical regions for the 10 lowest-energy structures having backbone rmsds of 0.26 and 0.24 A, respectively. Mn(2+) relaxation data and the protein-detergent NOE pattern show the C-terminal helix embedded in the micelle and the N-terminal helix lying along the detergent micelle surface with a 60 degrees angle between their long axes. (15)N relaxation data for residues L12-W44 are typical of a well-ordered protein with a correlation time of 8.25 +/- 2.1 ns. The presence of the hinge region placing the N-terminal helix along the membrane surface may be the structural feature responsible for the functional differences observed between the LH1 and LH2 beta subunits.