A comparative study of<Emphasis Type="Italic"> bchG</Emphasis> from green photosynthetic bacteria

The gene bchG, coding for bacteriochlorophyll a synthase from a variety of green sulfur bacteria and the filamentous anoxygenic phototrophic bacteria, Chloroflexus aurantiacus, Chloronema sp., and Roseiflexus castenholzii HL08, was partially sequenced and compared. The deduced amino acid consensus sequences for green sulfur bacteria and green filamentous anoxygenic phototrophic bacteria were found to belong to the UbiA enzyme family of polyprenyltransferases with the most similar sequences being those of photosynthetic organisms. All deduced amino acid sequences showed a highly conserved region, which includes the motif DRXXD, characteristic of polyprenyltransferases, which was extended to DREVDAINEP for green sulfur bacteria. Neighbor-joining analysis of a protein similitude matrix displayed a relatively high distance between green sulfur bacteria and the other groups. Sequences from green sulfur bacteria were more closely related to those of purple bacteria than to those of filamentous anoxygenic phototrophic bacteria. In addition, internal grouping within green sulfur bacteria was congruent regarding taxonomic features including cell shape, presence of gas vacuoles and NaCl requirement. In addition to bchlG, another gene encoding for a second chlorophyll synthetase, previously tentatively identified as chlG, was also found in Chlorobium tepidum, showing the highest similarities with polyprenyltransferases from chlorophyll- a-containing organisms.     

Comparative genomics provides evidence for the 3-hydroxypropionate autotrophic pathway in filamentous anoxygenic phototrophic bacteria and in hot spring microbial mats

Stable carbon isotope signatures of diagnostic lipid biomarkers have suggested that Roseiflexus spp., the dominant filamentous anoxygenic phototrophic bacteria inhabiting microbial mats of alkaline siliceous hot springs, may be capable of fixing bicarbonate via the 3-hydroxypropionate pathway, which has been characterized in their distant relative, Chloroflexus aurantiacus. The genomes of three filamentous anoxygenic phototrophic Chloroflexi isolates (Roseiflexus sp. RS-1, Roseiflexus castenholzii and Chloroflexus aggregans), but not that of a non-photosynthetic Chloroflexi isolate (Herpetosiphon aurantiacus), were found to contain open reading frames that show a high degree of sequence similarity to genes encoding enzymes in the C. aurantiacus pathway. Metagenomic DNA sequences from the microbial mats of alkaline siliceous hot springs also contain homologues of these genes that are highly similar to genes in both Roseiflexus spp. and Chloroflexus spp. Thus, Roseiflexus spp. appear to have the genetic capacity for carbon dioxide reduction via the 3-hydroxypropionate pathway. This may contribute to heavier carbon isotopic signatures of the cell components of native Roseiflexus populations in mats compared with the signatures of cyanobacterial cell components, as a similar isotopic signature would be expected if Roseiflexus spp. were participating in photoheterotrophic uptake of cyanobacterial photosynthate produced by the reductive pentose phosphate cycle.     

New class of bacterial membrane oxidoreductases

A new class of bacterial multisubunit membrane-bound electron-transfer complexes has been identified based on biochemical and bioinformatic data. It contains subunits homologous to the three-subunit molybdopterin oxidoreductases and four additional subunits, two of which are c-type cytochromes. The complex was purified from the filamentous anoxygenic phototrophic bacterium Chloroflexus aurantiacus, and putative operons for similar complexes were identified in a wide range of bacteria. In most cases, the presence of the new complex is anticorrelated with the cytochrome bc or bf electron-transfer complex, suggesting that it replaces it functionally. This appears to be a widespread yet previously unrecognized protein complex involved in energy metabolism in bacteria.     

Alkane-1,2-diol-based glycosides and fatty glycosides and wax esters in Roseiflexus castenholzii and hot spring microbial mats

The lipid composition of Roseiflexus castenholzii, a thermophilic filamentous phototrophic bacterium related to uncultivated filamentous phototrophic bacteria that predominate in hot spring microbial mats, is reported. R. castenholzii lipid extracts were dominated by components characterized by alkane-1-ol-2-alkanoate moieties glycosidically bonded to a C(6) sugar. Similar fatty glycosides, with an additional fatty acid esterified, were detected by HPLC-MS. R. castenholzii also produces a suite of wax esters ranging from 37 to 40 carbon atoms in length. In lipid extracts from two nonsulfidic hot spring microbial mats, similar alkane-1,2-diol-based lipids were detected in minor amounts. R. castenholzii lipids are compared to lipids of mats and other thermophilic mat isolates.     

Excitation energy transfer and trapping dynamics in the core complex of the filamentous photosynthetic bacterium Roseiflexus castenholzii

The light-harvesting core complex of the thermophilic filamentous anoxygenic phototrophic bacterium Roseiflexus castenholzii is intrinsic to the cytoplasmic membrane and intimately bound to the reaction center (RC). Using ultrafast transient absorption and time-resolved fluorescence spectroscopy with selective excitation, energy transfer, and trapping dynamics in the core complex have been investigated at room temperature in both open and closed RCs. Results presented in this report revealed that the excited energy transfer from the BChl 800 to the BChl 880 band of the antenna takes about 2?ps independent of the trapping by the RC. The time constants for excitation quenching in the core antenna BChl 880 by open and closed RCs were found to be 60 and 210?ps, respectively. Assuming that the light harvesting complex is generally similar to LH1 of purple bacteria, the possible structural and functional aspects of this unique antenna complex are discussed. The results show that the core complex of Roseiflexus castenholzii contains characteristics of both purple bacteria and Chloroflexus aurantiacus.     

Kinetics and energetics of electron transfer in reaction centers of the photosynthetic bacterium Roseiflexus castenholzii

The kinetics and thermodynamics of the photochemical reactions of the purified reaction center (RC)-cytochrome (Cyt) complex from the chlorosome-lacking, filamentous anoxygenic phototroph, Roseiflexus castenholzii are presented. The RC consists of L- and M-polypeptides containing three bacteriochlorophyll (BChl), three bacteriopheophytin (BPh) and two quinones (Q(A) and Q(B)), and the Cyt is a tetraheme subunit. Two of the BChls form a dimer P that is the primary electron donor. At 285K, the lifetimes of the excited singlet state, P*, and the charge-separated state P(+)H(A)(-) (where H(A) is the photoactive BPh) were found to be 3.2±0.3 ps and 200±20 ps, respectively. Overall charge separation P*→→ P(+)Q(A)(-) occurred with ≥90% yield at 285K. At 77K, the P* lifetime was somewhat shorter and the P(+)H(A)(-) lifetime was essentially unchanged. Poteniometric titrations gave a P(865)/P(865)(+) midpoint potential of +390mV vs. SHE. For the tetraheme Cyt two distinct midpoint potentials of +85 and +265mV were measured, likely reflecting a pair of low-potential hemes and a pair of high-potential hemes, respectively. The time course of electron transfer from reduced Cyt to P(+) suggests an arrangement where the highest potential heme is not located immediately adjacent to P. Comparisons of these and other properties of isolated Roseiflexus castenholzii RCs to those from its close relative Chloroflexus aurantiacus and to RCs from the purple bacteria are made.     

Characterization of the Photosynthetic Apparatus and Proteome of Roseobacter denitrificans

The phototrophic capacity of aerobic anoxygenic phototrophic bacteria endows them with a selective advantage over other heterotrophic bacteria in the oligotrophic ocean. Here, we reported the phototrophic features and proteome of an aerobic phototrophic bacterium Roseobacter denitrificans under starvation stress. The fluorescence induction and relaxation measurements suggested that the photosynthetic capacity in R. denitrificans was preserved but was lower than in the photoautotrophic bacterium Rhodobacter sphaeroides. The existence of light-harvesting complexes (LH1 and LH2) and the reaction center (RC) in the native membrane were demonstrated through atomic force microscopy image analysis as direct evidence of their phototrophy. The homology-based LH1–RC complex structure was proposed in which RC was the Rb. sphaeroides homolog structure surrounded by the LH1. Moreover, the protein expression profiles of cells in the stationary phase under heterotrophic and mixotrophic conditions show that light enhanced or activated some proteins such as carbon monoxide dehydrogenase and NifU to cope with the low levels of amino acids and carbon sources under starvation conditions.     

A new cytochrome subunit bound to the photosynthetic reaction center in the purple bacterium, Rhodovulum sulfidophilum

The nucleotide sequence of the puf operon, which contains the genes encoding the B870 light-harvesting protein and the reaction center complex of the purple photosynthetic bacterium, Rhodovulum sulfidophilum, was determined. The operon, which consisted of six genes, pufQ, pufB, pufA, pufL, pufM, and pufC, is a new variety in photosynthetic bacteria in the sense that pufQ and pufC coexist. The amino acid sequence of the cytochrome subunit of the reaction center deduced from the pufC sequence revealed that this cytochrome contains only three possible heme-binding motifs; the heme-1-binding motif of the corresponding tetraheme cytochrome subunits was not present. This is the first exception of the "tetraheme" cytochrome family in purple bacteria and green filamentous bacteria. The pufC sequence also revealed that the sixth axial ligands to heme-1 and heme-2 irons were not present in the cytochrome either. This cytochrome was actually detected in membrane preparation as a 43-kDa protein and shown to associate functionally with the photosynthetic reaction center as the immediate electron donor to the photo-oxidized special pair of bacteriochlorophyll. This new cytochrome should be useful for studies on the role of each heme in the cytochrome subunit of the bacterial reaction center and the evolution of proteins in photosynthetic electron transfer systems.     

Structure of the puf operon of the obligately aerobic, bacteriochlorophyll alpha-containing bacterium Roseobacter denitrificans OCh114 and its expression in a Rhodobacter capsulatus puf puc deletion mutant.

Roseobacter denitrificans (Erythrobacter species strain OCh114) synthesizes bacteriochlorophyll a (BChl) and the photosynthetic apparatus only in the presence of oxygen and is unable to carry out primary photosynthetic reactions and to grow photosynthetically under anoxic conditions. The puf operon of R. denitrificans has the same five genes in the same order as in many photosynthetic bacteria, i.e., pufBALMC. PufC, the tetraheme subunit of the reaction center (RC), consists of 352 amino acids (Mr, 39,043); 20 and 34% of the total amino acids are identical to those of PufC of Chloroflexus aurantiacus and Rubrivivax gelatinosus, respectively. The N-terminal hydrophobic domain is probably responsible for anchoring the subunit in the membrane. Four heme-binding domains are homologous to those of PufC in several purple bacteria. Sequences similar to pufQ and pufX of Rhodobacter capsulatus were not detected on the chromosome of R. denitrificans. The puf operon of R. denitrificans was expressed in trans in Escherichia coli, and all gene products were synthesized. The Roseobacter puf operon was also expressed in R. capsulatus CK11, a puf puc double-deletion mutant. For the first time, an RC/light-harvesting complex I core complex was heterologously synthesized. The strongest expression of the R. denitrificans puf operon was observed under the control of the R. capsulatus puf promoter, in the presence of pufQ and pufX and in the absence of pufC. Charge recombination between the primary donor P+ and the primary ubiquinone Q(A)- was observed in the transconjugant, showing that the M and L subunits of the RC were correctly assembled. The transconjugants did not grow photosynthetically under anoxic conditions.     

Isolation and characterization of the B798 light-harvesting baseplate from the chlorosomes of Chloroflexus aurantiacus

The B798 light-harvesting baseplate of the chlorosome antenna complex of the thermophilic, filamentous anoxygenic phototrophic bacterium Chloroflexus aurantiacus has been isolated and characterized. Isolation was performed by using a hexanol-detergent treatment of freeze-thawed chlorosomes. The isolated baseplate consists of Bchl a, beta-carotene, and the 5.7 kDa CsmA protein with a ratio of 1.0 CsmA protein/1.6 Bchl a/4.2 beta-carotenes. The baseplate has characteristic absorbance at 798 nm as well as carotenoid absorbance maxima at 519, 489, and 462 nm. The energy transfer efficiency from the carotenoids to the Bchl a is 30% as measured by steady-state and ultrafast time-resolved fluorescence and absorption spectroscopies. Energy equilibration within the Bchl a absorbing regions exhibits ultrafast kinetics. Circular dichroism spectroscopy shows no evidence for excitonically coupled Bchl a pools within the 798 nm region.     

Anoxygenic phototrophic bacteria from microbial communities of Goryachinsk thermal spring (Baikal Area,Russia)

Species composition of anoxygenic phototrophic bacteria in microbial mats of the Goryachinsk thermal spring was investigated along the temperature gradient. The spring belonging to nitrogenous alkaline hydrotherms is located at the shore of Lake Baikal 188 km north-east from Ulan-Ude. The water is of the sulfate-sodium type, contains trace amounts of sulfide, and salinity does not exceed 0.64 g/L, pH 9.5. The temperature at the outlet of the spring may reach 54°C. The cultures of filamentous anoxygenic phototrophic bacteria, nonsulfur and sulfur purple bacteria, and aerobic anoxygenic phototrophic bacteria were identified using the pufLM molecular marker. The fmoA marker was used for identification of green sulfur bacteria. Filamentous cyanobacteria predominated in the mats, with anoxygenic phototrophs comprising a minor component of the phototrophic communities. Thermophilic bacteria Chloroflexus aurantiacus were detected in the samples from both the thermophilic and mesophilic mats. Cultures of nonsulfur purple bacteria similar to Blastochloris sulfoviridis and Rhodomicrobium vannielii were isolated from the mats developed at high (50.6–49.4°C) and low temperatures (45–20°C). Purple sulfur bacteria Allochromatium sp. and Thiocapsa sp., as well as green sulfur bacteria Chlorobium sp., were revealed in low-temperature mats. Truly thermophilic purple and green sulfur bacteria were not found in the spring. Anoxygenic phototrophic bacteria found in the spring were typical of the sulfur communities, for which the sulfur cycle is mandatory. The presence of aerobic bacteriochlorophyll a-containing bacteria identified as Agrobacterium (Rhizobium) tumifaciens in the mesophilic (20°C) mat is of interest.     

Comparison of cultivation-dependent and molecular methods for studying the diversity of anoxygenic purple phototrophs in sediments of an eutrophic brackish lagoon

Phototrophic anoxygenic purple bacteria play a key role in many aquatic ecosystems by oxidizing sulfur compounds and low-molecular-weight organic compounds using light as energy source. In this study, molecular methods based upon pufM gene (photosynthetic unit forming gene) were compared with culture-dependent methods to investigate anoxygenic purple phototrophic communities in sediments of an eutrophic brackish lagoon. Thirteen strains, belonging to eight different genera of purple phototrophic bacteria were isolated with a large dominance of the metabolically versatile purple non-sulfur bacteria (eight strains), some purple sulfur bacteria (three strains) and two strains belonging to the Roseobacter clade (aerobic phototrophs). The pufM genes amplified from the isolated strains were not detected by the molecular methods [terminal-restriction fragment length polymorphism (T-RFLP)] applied on in situ communities. An environmental clone library of the pufM gene was thus constructed from sediment samples. The results showed that most of the clones probably corresponded to aerobic phototrophic bacteria. Our results demonstrate that the culture-dependent techniques remain the best experimental approach for determining the diversity of phototrophic purple non-sulfur bacteria whereas the molecular approach clearly illustrated the abundance of organisms related to the Roseobacter clade in these eutrophic sediments.     

Benthic phototrophic community from Kiran soda lake,south-eastern Siberia

Phototrophic bacterial mats from Kiran soda lake (south-eastern Siberia) were studied using integrated approach including analysis of the ion composition of water, pigments composition, bacterial diversity and the vertical distribution of phototrophic microorganisms in the mats. Bacterial diversity was investigated using microscopic examination, 16S rRNA gene Illumina sequencing and culturing methods. The mats were formed as a result of decomposition of sedimented planktonic microorganisms, among which cyanobacteria of the genus Arthrospira predominated. Cyanobacteria were the largest part of phototrophs in the mats, but anoxygenic phototrophs were significant fraction. The prevailing species of the anoxygenic phototrophic bacteria are typical for soda lakes. The mats harbored aerobic anoxygenic phototrophic bacteria, purple sulfur and non-sulfur bacteria, as well as new filamentous phototrophic Chloroflexi. New strains of Thiocapsa sp. Kir-1, Ectothiorhodospira sp. Kir-2 and Kir-4, Thiorhodospira sp. Kir-3 and novel phototrophic Chloroflexi bacterium Kir15-3F were isolated and identified.

     

Study of the structure of the photosynthetic reaction center of the green thermophilic bacteria Chloroflexus aurantiacus

Analysis of the Chloroflexus aurantiacus reaction centre (RC) using both protein and recombinant DNA techniques resulted in determination of its polypeptide composition and the primary structures of its two subunits. A model of the polypeptide chains' folding in the membrane is suggested based on: i) homology between L- and M-subunits of Chloroflexus aurantiacus RC and their counterparts in purple bacteria; ii) comparison of their hydropathy plots, and iii) data on the tertiary structures of purple bacteria RCs. The role of a number of functionally important amino acid residues in the RC electron transport activity is discussed. Limited proteolysis of the RC under non-denaturing conditions was used to determine the contribution of the N-terminal regions to its thermal stability.     

The nucleotide sequence of the puf operon from the purple photosynthetic bacterium, Rhodospirillum molischianum: Comparative analyses of light-harvesting proteins and the cytochrome subunits associated with the reaction centers

The nucleotide sequence of the puf operon of the purple bacterium, Rhodospirillum molischianum, was determined. The operon includes genes coding for the and subunits of the light-harvesting 1 (LH1) complex and the L, M, and cytochrome subunits of the reaction center complex. As in other purple bacteria, the genes are arranged within the operon in this order. As in Rubrivivax gelatinosus, the deduced amino acid sequence of the cytochrome subunit in Rsp. molischianum contains significant deletions at the attachment site to the M subunit compared with that of Rhodopseudomonas viridis. This suggests that the interaction between the cytochrome subunit and the LM core in Rsp. molischianum and Rvi. gelatinosus is different from that in Rps. viridis. Phylogenetic analysis of the light-harvesting proteins indicated that the LH1 and subunits of Rsp. molischianum are included in the lineage of LH1 polypeptides of the purple bacteria, while the LH2 and subunits are positioned apart from LH2 polypeptides of the other purple bacteria together with those of Chromatium vinosum. Based on these phylogenetic analyses, the classification of the light-harvesting proteins in purple bacteria is discussed.     

Putative novel photosynthetic reaction centre organizations in marine aerobic anoxygenic photosynthetic bacteria: insights from metagenomics and environmental genomics

Photosynthetic core complexes of anoxygenic bacteria consist of reaction centres (RCs) surrounded by light-harvesting complexes (LHC). The structural proteins of the RC-LHC1 complex are encoded by the puf-operon. We find diverse operon organizations of puf-operons that reflect structural differences of the core complex in marine aerobic anoxygenic photosynthetic bacteria (AAnP). By analysis of environmental DNA records coming from AAnP bacteria we find several unknown proteins downstream to the pufM, which were assigned as novel PufX proteins. As all known pufX genes belong to Rhodobacter strains which carry out anaerobic photosynthesis, this may be the first observation of a PufX-containing RCs in aerobic anoxygenic photosynthetic bacteria. Phylogenetic analyses of PufM proteins from cultured as well as from uncultured bacteria show that PufM from operons containing putative novel pufX genes are grouped with Rhodobacter and not with Roseobacter strains.     

SANS investigation of the photosynthetic machinery of Chloroflexus aurantiacus

Green photosynthetic bacteria harvest light and perform photosynthesis in low-light environments, and contain specialized antenna complexes to adapt to this condition. We performed small-angle neutron scattering (SANS) studies to obtain structural information about the photosynthetic apparatus, including the peripheral light-harvesting chlorosome complex, the integral membrane light-harvesting B808-866 complex, and the reaction center (RC) in the thermophilic green phototrophic bacterium Chloroflexus aurantiacus. Using contrast variation in SANS measurements, we found that the B808-866 complex is wrapped around the RC in Cfx. aurantiacus, and the overall size and conformation of the B808-866 complex of Cfx. aurantiacus is roughly comparable to the LH1 antenna complex of the purple bacteria. A similar size of the isolated B808-866 complex was suggested by dynamic light scattering measurements, and a smaller size of the RC of Cfx. aurantiacus compared to the RC of the purple bacteria was observed. Further, our SANS measurements indicate that the chlorosome is a lipid body with a rod-like shape, and that the self-assembly of bacteriochlorophylls, the major component of the chlorosome, is lipid-like. Finally, two populations of chlorosome particles are suggested in our SANS measurements.     

Early colonization of thermal niches in a silica-depositing hot spring in central Tibet

Thermophilic microbial mats dominated by the anoxygenic phototroph Roseiflexus castenholzii commonly develop around sinter-depositing geysers in the Daggyai Tso geothermal field of central Tibet. In this study we used morphological and molecular genetic techniques to reveal a diverse pioneer biofilm community including both archaea and bacteria involved in early colonization of such thermal niches at temperatures ranging from 46 to 77 °C. Sinter precipitation and biomineralization were evident at all locations, but the latter was selective between taxa and most evident on filamentous cells. Evidence for possible indirect biosignatures from biofilms overwhelmed by sinter deposition was found. Succession to a mature community appeared to relate to the growth rate for key taxa outpacing that of silicification within an optimum temperature range of 54–61 °C. The thin surface layer of silicification-resistant cyanobacteria that developed on the surface of mature mats may play a role in preventing biomineralization of the susceptible R. castenholzii beneath within these communities.     

不产氧光合细菌色素蛋白复合体研究进展

摘要: 色素蛋白复合体是光合生物进行光合作用维持生命活动最重要结构基础。目前不产氧光合细菌色素蛋白复合体仍是最具前沿研究领域。本文概述了不产氧光合细菌各种属色素蛋白复合体研究现状,着重对光反应中心色素蛋白复合体（reaction center,RC）和捕光色素蛋白复合体（light-harvesting complex,LH）,尤其是新型捕光色素蛋白复合体LH3和LH4的组成、精细结构、蛋白同源性和功能进行了述评,并就研究中存在的问题和发展趋势进行了讨论。     

Pigment organization in the photosynthetic apparatus of Roseiflexus castenholzii

The light-harvesting-reaction center (LHRC) complex from the chlorosome-lacking filamentous anoxygenic phototroph (FAP), Roseiflexus castenholzii (R. castenholzii) was purified and characterized for overall pigment organization. The LHRC is a single complex that is comprised of light harvesting (LH) and reaction center (RC) polypeptides as well as an attached c-type cytochrome. The dominant carotenoid found in the LHRC is keto-γ-carotene, which transfers excitation to the long wavelength antenna band with 35% efficiency. Linear dichroism and fluorescence polarization measurements indicate that the long wavelength antenna pigments absorbing around 880 nm are perpendicular to the membrane plane, with the corresponding Q_y transition dipoles in the plane of the membrane. The antenna pigments absorbing around 800 nm, as well as the bound carotenoid, are oriented at a large angle with respect to the membrane. The antenna pigments spectroscopically resemble the well-studied LH2 complex from purple bacteria, however the close association with the RC makes the light harvesting component of this complex functionally more like LH1.