Comparison of the Energy-Transfer Rates in Structural and Spectral Variants of the B800-850 Complex from Purple Bacteria.

Photosynthetic light harvesting can occur with a remarkable near-unity quantum efficiency. The B800-850 complex, also known as light-harvesting complex 2 (LH2), is the primary light-harvesting complex in purple bacteria and has been extensively studied as a model system. The bacteriochlorophylls of the B800-850 complex are organized into two concentric rings, known as the B800 and B850 rings. However, depending on the species and growth conditions, the number of constituent subunits, the pigment geometry, and the absorption energies vary. While the dynamics of some B800-850 variants have been exhaustively characterized, others have not been measured. Furthermore, a direct and simultaneous comparison of how both structural and spectral differences between variants affect these dynamics has not been performed. In this work, we utilize ultrafast transient absorption measurements to compare the B800 to B850 energy-transfer rates in the B800-850 complex as a function of the number of subunits, geometry, and absorption energies. The nonameric B800-850 complex from Rhodobacter (Rb.) sphaeroides is 40% faster than the octameric B800-850 complex from Rhodospirillum (Rs.) molischianum, consistent with structure-based predictions. In contrast, the blue-shifted B800-820 complex from Rs. molischianum is only 20% faster than the B800-850 complex from Rs. molischianum despite an increase in the spectral overlap between the rings that would be expected to produce a larger increase in the energy-transfer rate. These measurements support current models that contain dark, higher-lying excitonic states to bridge the energy gap between rings, thereby maintaining similar energy-transfer dynamics. Overall, these results demonstrate that energy-transfer dynamics in the B800-850 complex are robust to the spectral and structural variations between species used to optimize energy capture and flow in purple bacteria.

The Effect of Zn(II) Ions and Reactive Oxygen on the Uptake of Zinc and Production of Carotenoids by Selected Red Yeasts.

Three strains of red yeast Rhodosporidium kratochvilovae, Rhodotorula glutinis and Sporidiobolus salmonicolor were studied for their responses to the presence metal stress, oxidative stress and a combination of these stress factors. For all yeast strains, the production of ß-carotene increased in stress conditions. The combination of H2 O2 and Zn2+ significantly activated the pathways for the production of torularhodin in the strain R. glutinis (from 250 to 470 µg g-1 DCW) as well as ß-carotene (from 360 to 1100 µg g-1 DCW) and torulene (from 100 to 360 µg g-1 DCW) in Sp. salmonicolor. Strains of R. glutinis and Rh. kratochvilovae bound the majority of Zn(II) ions to the fibrillar part of the cell walls, whereas the strain Sp. salmonicolor bound them to both extracellular polymers and the fibrillar part of the cell walls. A decrease in the ability of yeasts to tolerate higher concentrations of Zn(II) in the presence of free radicals (hydrogen peroxide) was also found.

Directed -in vitro- evolution of Precambrian and extant Rubiscos.

Rubisco is an ancient, catalytically conserved yet slow enzyme, which plays a central role in the biosphere's carbon cycle. The design of Rubiscos to increase agricultural productivity has hitherto relied on the use of in vivo selection systems, precluding the exploration of biochemical traits that are not wired to cell survival. We present a directed -in vitro- evolution platform that extracts the enzyme from its biological context to provide a new avenue for Rubisco engineering. Precambrian and extant form II Rubiscos were subjected to an ensemble of directed evolution strategies aimed at improving thermostability. The most recent ancestor of proteobacteria -dating back 2.4 billion years- was uniquely tolerant to mutagenic loading. Adaptive evolution, focused evolution and genetic drift revealed a panel of thermostable mutants, some deviating from the characteristic trade-offs in CO2-fixing speed and specificity. Our findings provide a novel approach for identifying Rubisco variants with improved catalytic evolution potential.

Anti-Obesity Effect of Red Radish Coral Sprout Extract by Inhibited Triglyceride Accumulation in a Microbial Evaluation System and in High-Fat Diet-Induced Obese Mice.

Rhodosporidium toruloides, an oleaginous yeast, can be used as a fast and reliable evaluation tool to screen new natural lipid-lowering agents. Herein, we showed that triglyceride (TG) accumulation was inhibited by 42.6% in 0.1% red radish coral sprout extract (RRSE)-treated R. toruloides. We also evaluated the anti-obesity effect of the RRSE in a mouse model. The body weight gain of mice fed a high-fat diet (HFD) with 0.1% RRSE (HFD-RRSE) was significantly decreased by 60% compared with that mice fed the HFD alone after the 8-week experimental period. Body fat of the HFD-RRSE-fed group was dramatically reduced by 38.3% compared with that of the HFD-fed group.

Rhodospirillum rubruml-asparaginase targets tumor growth by a dual mechanism involving telomerase inhibition.

Rhodospirillum rubruml-asparaginase mutant RrA E149R, V150P, F151T (RrA) was previously identified to down-regulate telomerase activity along with catalyzing the hydrolysis of l-asparagine. The aim of this study was to define the effect of prolonged RrA exposure on telomerase activity, maintenance of telomeres and proliferation of cancer cells in vitro and in vivo. RrA could inhibit telomerase activity in SCOV-3, SkBr-3 and A549 human cancer cell lines due to its ability to down-regulate the expression of telomerase catalytic subunit hTERT. Telomerase activity in treated cells did not exceeded 29.63 ± 12.3% of control cells. Continuous RrA exposure of these cells resulted in shortening of telomeres followed by cell death in vitro. Using real time PCR we showed that length of telomeres in SCOV-3 cells has been gradually decreasing from 10105 ± 2530 b.p. to 1233 ± 636 b.p. after 35 days of cultivation. RrA treatment of xenograft models in vivo showed slight inhibition of tumor growth accompanied with 49.5-53.3% of decrease in hTERT expression in the all tumors. However down-regulation of hTERT expression, inhibition of telomerase activity and the loss of telomeres was significant in response to RrA administration in xenograft models. These results should facilitate further investigations of RrA as a potent therapeutic protein.

Identification and characterization of a novel bifunctional Δ(12)/Δ(15)-fatty acid desaturase gene from Rhodosporidium kratochvilovae.

OBJECTIVES: To elucidate the biosynthesis pathway of linoleic acid and α-linolenic acid in Rhodosporidium kratochvilovae YM25235 and investigate the correlation of polyunsaturated fatty acids with its cold adaptation. RESULTS: A 1341 bp cDNA sequence, designated as RKD12, putatively encoding a Δ(12)-desaturase was isolated from YM25235. Sequence analysis indicated that this sequence comprised a complete ORF encoding 446 amino acids of 50.6 kDa. The encoded amino acid sequence shared higher similarity to known fungal Δ(12)-desaturases that are characteristic of three conserved histidine-rich motifs. RKD12 was further transformed into Saccharomyces cerevisiae INVScl for functional characterization. Fatty acid analysis showed the yeast transformants accumulated two new fatty acids: linoleic acid and α-linolenic acid. Furthermore, mRNA expression level of RKD12 and the content of linoleic acid and α-linolenic acid were increased significantly with the culture temperature downshift from 30 to 15 °C, which might be helpful for the cold adaptation of YM25235. CONCLUSION: RKD12 is a novel bifunctional ∆(12)/∆(15)-desaturase gene, and the increased RKD12 mRNA expression level and PUFAs content at low temperature might be helpful for the cold adaptation of YM25235.

Chemoenzymatic synthesis of (S)-duloxetine using carbonyl reductase from Rhodosporidium toruloides.

A chemoenzymatic strategy was developed for (S)-duloxetine production employing carbonyl reductases from newly isolated Rhodosporidium toruloides into the enantiodetermining step. Amongst the ten most permissive enzymes identified, cloned, and overexpressed in Escherichia coli, RtSCR9 exhibited excellent activity and enantioselectivity. Using co-expressed E. coli harboring both RtSCR9 and glucose dehydrogenase, (S)-3-(dimethylamino)-1-(2-thienyl)-1-propanol 3a was fabricated with so far the highest substrate loading (1000mM) in a space-time yield per gram of biomass (DCW) of 22.9mmolL(-1)h(-1)gDCW(-1) at a 200-g scale. The subsequent synthetic steps from RtSCR9-catalyzed (S)-3a were further performed, affording (S)-duloxetine with 60.2% overall yield from 2-acethylthiophene in >98.5% ee.

Light harvesting by lamellar chromatophores in Rhodospirillum photometricum.

Purple photosynthetic bacteria harvest light using pigment-protein complexes which are often arranged in pseudo-organelles called chromatophores. A model of a chromatophore from Rhodospirillum photometricum was constructed based on atomic force microscopy data. Molecular-dynamics simulations and quantum-dynamics calculations were performed to characterize the intercomplex excitation transfer network and explore the interplay between close-packing and light-harvesting efficiency.

Reclassification of Rhodospirillum photometricum Molisch 1907, Rhodospirillum sulfurexigens Anil Kumar et al. 2008 and Rhodospirillum oryzae Lakshmi et al. 2013 in a new genus, Pararhodospirillum gen. nov., as Pararhodospirillum photometricum comb. nov., Pararhodospirillum sulfurexigens comb. nov. and Pararhodospirillum oryzae comb. nov., respectively, and emended description of the genus Rhodospirillum.

The genus Rhodospirillum is represented by four species, with three of them showing phylogenetic divergence compared to the type species, Rhodospirillum rubrum. Differences in the major diagnostic properties such as internal photosynthetic membranes, quinones, fatty acids, carotenoid composition and a few other phenotypic properties warrant the reclassification of members of this genus. Resultantly, a new genus, Pararhodospirillum gen. nov., is proposed based on the analysis of nine strains to accommodate Rhodospirillum photometricum, Rhodospirillum sulfurexigens and Rhodospirillum oryzae as Pararhodospirillum photometricum comb. nov., Pararhodospirillum sulfurexigens comb. nov. and Pararhodospirillum oryzae comb. nov., respectively. The type species of the genus is Pararhodospirillum photometricum comb. nov. An emended description of the genus Rhodospirillum is also proposed.

Lateral organization of biological membranes: role of long-range interactions.

The lateral organization of biological membranes is of great importance in many biological processes, both for the formation of specific structures such as super-complexes and for function as observed in signal transduction systems. Over the last years, AFM studies, particularly of bacterial photosynthetic membranes, have revealed that certain proteins are able to segregate into functional domains with a specific organization. Furthermore, the extended non-random nature of the organization has been suggested to be important for the energy and redox transport properties of these specialized membranes. In the work reported here, using a coarse-grained Monte Carlo approach, we have investigated the nature of interaction potentials able to drive the formation and segregation of specialized membrane domains from the rest of the membrane and furthermore how the internal organization of the segregated domains can be modulated by the interaction potentials. These simulations show that long-range interactions are necessary to allow formation of membrane domains of realistic structure. We suggest that such possibly non-specific interactions may be of great importance in the lateral organization of biological membranes in general and in photosynthetic systems in particular. Finally, we consider the possible molecular origins of such interactions and suggest a fundamental role for lipid-mediated interactions in driving the formation of specialized photosynthetic membrane domains. We call these lipid-mediated interactions a 'lipophobic effect.'

Spectra and dynamics in the B800 antenna: comparing hierarchical equations, Redfield and Förster theories.

We model the spectra (absorption and circular dichroism) and excitation dynamics in the B800 ring of the LH2 antenna complex from Rs. molischianum using different theoretical approaches, i.e., Förster theory, standard and modified versions of the Redfield theory, and the more versatile nonperturbative approach based on hierarchically coupled equations for the reduced density operator. We demonstrate that, although excitations in the B800 ring are localized due to disorder, thermal effects, and phonons, there are still sizable excitonic effects producing shift, narrowing, and asymmetry of the spectra. Moreover, the excitation dynamics reveals the presence of long-lived (up to 1 ps) non-oscillatory coherences between the exciton states maintained due to nonsecular population-to-coherence transfers. The sub-ps decay of the coherences is followed by slow motion of the excitation around the ring, producing equilibration of the site populations with a time constant of about 3-4 ps, which is slower than the B800 → B850 transfer. The exact solution obtained with the hierarchical equations is compared with other approaches, thus illustrating limitations of the Förster and Redfield pictures.

Rhodospirillum oryzae sp. nov., a phototrophic bacterium isolated from rhizosphere soil of paddy.

A reddish-brown bacterium, designated strain JA318(T), was purified from a photoheterotrophic enrichment culture obtained from the rhizosphere soil of paddy. Cells of strain JA318(T) are spiral shaped, Gram-stain-negative and motile by means of amphitrichous flagella. Strain JA318(T) has no NaCl requirement for growth but can tolerate up to 1.5 % (w/v) NaCl. Internal photosynthetic membranes are present as lamellar stacks. Photoorganoheterotrophy is the only growth mode observed. Strain JA318(T) contains bacteriochlorophyll a, lycopene and rhodopin as major carotenoids. Thiamine, niacin and para-aminobenzoic acid (PABA) are required as growth factors. Major fatty acids are C18 : 1ω7c and C16 : 0. Ubiquinone-8 and rhodoquinone-8 are the observed quinones. Diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an unidentified aminolipid are the major polar lipids in strain JA318(T). Phylogenetic analysis based on 16S rRNA gene sequences showed that the strain JA318(T) clustered with species of the genus Rhodospirillum which belongs to the class Alphaproteobacteria. The highest sequence similarity of strain JA318(T) was found with Rhodospirillum sulfurexigens JA143(T) (99.9 %). The DNA-DNA reassociation values of strain JA318(T) with Rsp. sulfurexigens JA143(T) and Rhodospirillum photometricum DSM 122(T) were 52 ± 2 % and 45 ± 1 %, respectively. The genomic DNA G+C content of strain JA318(T) was 60.2 mol%. Based on the morphological, physiological, chemotaxonomical and molecular evidence, strain JA318(T) is significantly different from the type strains of species of the genus Rhodospirillum, of the family Rhodospirillaceae, and it is proposed that the strain be classified as a representative of a novel species for which the name Rhodospirillum oryzae sp. nov. is proposed. The type strain is JA318(T) (= KCTC 5960(T) = NBRC 107573(T)).

Shotgun genome sequence of the large purple photosynthetic bacterium Rhodospirillum photometricum DSM122.

Here, we present the shotgun genome sequence of the purple photosynthetic bacterium Rhodospirillum photometricum DSM122. The photosynthetic apparatus of this bacterium has been particularly well studied by microscopy. The knowledge of the genome of this oversize bacterium will allow us to compare it with the other purple bacterial organisms to follow the evolution of the photosynthetic apparatus.

Forces guiding assembly of light-harvesting complex 2 in native membranes.

Interaction forces of membrane protein subunits are of importance in their structure, assembly, membrane insertion, and function. In biological membranes, and in the photosynthetic apparatus as a paradigm, membrane proteins fulfill their function by ensemble actions integrating a tight assembly of several proteins. In the bacterial photosynthetic apparatus light-harvesting complexes 2 (LH2) transfer light energy to neighboring tightly associated core complexes, constituted of light-harvesting complexes 1 (LH1) and reaction centers (RC). While the architecture of the photosynthetic unit has been described, the forces and energies assuring the structural and functional integrity of LH2, the assembly of LH2 complexes, and how LH2 interact with the other proteins in the supramolecular architecture are still unknown. Here we investigate the molecular forces of the bacterial LH2 within the native photosynthetic membrane using atomic force microscopy single-molecule imaging and force measurement in combination. The binding between LH2 subunits is fairly weak, of the order of k(B)T, indicating the importance of LH2 ring architecture. In contrast LH2 subunits are solid with a free energy difference of 90 k(B)T between folded and unfolded states. Subunit α-helices unfold either in one-step, α- and ß-polypeptides unfold together, or sequentially. The unfolding force of transmembrane helices is approximately 150 pN. In the two-step unfolding process, the ß-polypeptide is stabilized by the molecular environment in the membrane. Hence, intermolecular forces influence the structural and functional integrity of LH2.

In silico predictions of LH2 ring sizes from the crystal structure of a single subunit using molecular dynamics simulations.

Most of the currently known light-harvesting complexes 2 (LH2) rings are formed by 8 or 9 subunits. As of now, questions like "what factors govern the LH2 ring size?" and "are there other ring sizes possible?" remain largely unanswered. Here, we investigate by means of molecular dynamics (MD) simulations and stochastic modeling the possibility of predicting the size of an LH2 ring from the sole knowledge of the high resolution crystal structure of a single subunit. Starting with single subunits of two LH2 rings with known size, that is, an 8-ring from Rs. moliscianum (MOLI) and a 9-ring from Rps. acidophila (ACI), and one with unknown size (referred to as X), we build atomic models of subunit dimers corresponding to assumed 8-, 9-, and 10-ring geometries. After inserting each of the dimers into a lipid-water environment, we determine the preferred angle between the corresponding subunits by three methods: (1) energy minimization, (2) free MD simulations, and (3) potential of mean force calculations. We find that the results from all three methods are consistent with each other, and when taken together, it allows one to predict with reasonable level of confidence the sizes of the corresponding ring structures. One finds that X and ACI very likely form a 9-ring, while MOLI is more likely to form an 8-ring than a 9-ring. Finally, we discuss both the merits and limitations of all three prediction methods.

The photosensor protein Ppr of Rhodocista centenaria is linked to the chemotaxis signalling pathway.

BACKGROUND: Rhodocista centenaria is a phototrophic α-proteobacterium exhibiting a phototactic behaviour visible as colony movement on agar plates directed to red light. As many phototrophic purple bacteria R. centenaria possesses a soluble photoactive yellow protein (Pyp). It exists as a long fusion protein, designated Ppr, consisting of three domains, the Pyp domain, a putative bilin binding domain (Bbd) and a histidine kinase domain (Pph). The Ppr protein is involved in the regulation of polyketide synthesis but it is still unclear, how this is connected to phototaxis and chemotaxis. RESULTS: To elucidate the possible role of Ppr and Pph in the chemotactic network we studied the interaction with chemotactic proteins in vitro as well as in vivo. Matrix-assisted coelution experiments were performed to study the possible communication of the different putative binding partners. The kinase domain of the Ppr protein was found to interact with the chemotactic linker protein CheW. The formation of this complex was clearly ATP-dependent. Further results indicated that the Pph histidine kinase domain and CheW may form a complex with the chemotactic kinase CheAY suggesting a role of Ppr in the chemotaxis signalling pathway. In addition, when Ppr or Pph were expressed in Escherichia coli, the chemotactic response of the cells was dramatically affected. CONCLUSIONS: The Ppr protein of Rhodocista centenaria directly interacts with the chemotactic protein CheW. This suggests a role of the Ppr protein in the regulation of the chemotactic response in addition to its role in chalcone synthesis.

Time-dependent atomistic view on the electronic relaxation in light-harvesting system II.

Aiming at a better understanding of the molecular details in light absorption during photosynthesis, spatial and temporal correlation functions as well as spectral densities have been determined. At the focus of the present study are the light-harvesting II complexes of the purple bacterium Rhodospirillum molischianum. The calculations are based on a time-dependent combination of molecular dynamics simulations and quantum chemistry methods. Using a 12 ps long trajectory, different quantum chemical methods have been compared to each other. Furthermore, several approaches to determine the couplings between the individual chromophores have been tested. Correlations between energy gap fluctuations of different individual pigments are analyzed but found to be negligible. From the energy gap fluctuations, spectral densities are extracted which serve as input for calculations of optical properties and exciton dynamics. To this end, the spectral densities are tested by determining the linear absorption of the complete two-ring system. One important difference from earlier studies is given by the severely extended length of the trajectory along which the quantum chemical calculations have been performed. Due to this extension, more accurate and reliable data have been obtained in the low frequency regime which is important in the dynamics of electronic relaxation.

Light-harvesting mechanism of bacteria exploits a critical interplay between the dynamics of transport and trapping.

Light-harvesting bacteria Rhodospirillum photometricum were recently found to adopt strikingly different architectures depending on illumination conditions. We present analytic and numerical calculations which explain this observation by quantifying a dynamical interplay between excitation transfer kinetics and reaction center cycling. High light-intensity membranes exploit dissipation as a photoprotective mechanism, thereby safeguarding a steady supply of chemical energy, while low light-intensity membranes efficiently process unused illumination intensity by channeling it to open reaction centers. More generally, our analysis elucidates and quantifies the trade-offs in natural network design for solar energy conversion.

Revealing linear aggregates of light harvesting antenna proteins in photosynthetic membranes.

How light energy is harvested in a natural photosynthetic membrane through energy transfer is closely related to the stoichiometry and arrangement of light harvesting antenna proteins in the membrane. The specific photosynthetic architecture facilitates a rapid and efficient energy transfer among the light harvesting proteins (LH2 and LH1) and to the reaction center. Here we report the identification of linear aggregates of light harvesting proteins, LH2, in the photosynthetic membranes under ambient conditions by using atomic force microscopy (AFM) imaging and spectroscopic analysis. Our results suggest that the light harvesting protein, LH2, can exist as linear aggregates of 4 +/- 2 proteins in the photosynthetic membranes and that the protein distributions are highly heterogeneous. In the photosynthetic membranes examined in our measurements, the ratio of the aggregated to the nonaggregated LH2 proteins is about 3:1 to 5:1 depending on the intensity of the illumination used during sample incubation and on the bacterial species. AFM images further identify that the LH2 proteins in the linear aggregates are monotonically tilted at an angle 4 +/- 2 degrees from the plane of the photosynthetic membranes. The aggregates result in red-shifted absorption and emission spectra that are measured using various mutant membranes, including an LH2 knockout, LH1 knockout, and LH2 at different population densities. Measuring the fluorescence lifetimes of purified LH2 and LH2 in membranes, we have observed that the LH2 proteins in membranes exhibit biexponential lifetime decays whereas the purified LH2 proteins gave single exponential lifetime decays. We attribute that the two lifetime components originate from the existence of both aggregated and nonaggregated LH2 proteins in the photosynthetic membranes.

Energy transfer in light-adapted photosynthetic membranes: from active to saturated photosynthesis.

In bacterial photosynthesis light-harvesting complexes, LH2 and LH1 absorb sunlight energy and deliver it to reaction centers (RCs) with extraordinarily high efficiency. Submolecular resolution images have revealed that both the LH2:LH1 ratio, and the architecture of the photosynthetic membrane itself, adapt to light intensity. We investigate the functional implications of structural adaptations in the energy transfer performance in natural in vivo low- and high-light-adapted membrane architectures of Rhodospirillum photometricum. A model is presented to describe excitation migration across the full range of light intensities that cover states from active photosynthesis, where all RCs are available for charge separation, to saturated photosynthesis where all RCs are unavailable. Our study outlines three key findings. First, there is a critical light-energy density, below which the low-light adapted membrane is more efficient at absorbing photons and generating a charge separation at RCs, than the high-light-adapted membrane. Second, connectivity of core complexes is similar in both membranes, suggesting that, despite different growth conditions, a preferred transfer pathway is through core-core contacts. Third, there may be minimal subareas on the membrane which, containing the same LH2:LH1 ratio, behave as minimal functional units as far as excitation transfer efficiency is concerned.