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.

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.

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.

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.

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.'

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.

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.

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.

Inorganic pyrophosphate: formation in bacterial photophosphorylation.

Inorganic pyrophosphate is identified as the major product of photophosphorylation by isolated chromatophores from Rhodospirillum rubrum in the absence of added nucleotides.

Carotenoid biosynthesis in Rhodospirillum rubrum: effect of pteridine inhibitor.

A known inhibitor of pteridine utilization (4-phenoxy,2,6-diamino pyridine) blocks the synthesis of colored carotenoids in the photosynthetic bacterium Rhodospirillum rubrum. In many ways the effect is similar to the inhibition of the synthesis of colored carotenoids by diphenylamine. This inhibition is probably independent of other effects of pteridine on photosynthetic electron transport since it is not as readily reversible as the total inhibition of photosynthetic activity by pteridine analogs.

Watching the components of photosynthetic bacterial membranes and their in situ organisation by atomic force microscopy.

The atomic force microscope has developed into a powerful tool in structural biology allowing information to be acquired at submolecular resolution on the protruding structures of membrane proteins. It is now a complementary technique to X-ray crystallography and electron microscopy for structure determination of individual membrane proteins after extraction, purification and reconstitution into lipid bilayers. Moving on from the structures of individual components of biological membranes, atomic force microscopy has recently been demonstrated to be a unique tool to identify in situ the individual components of multi-protein assemblies and to study the supramolecular architecture of these components allowing the efficient performance of a complex biological function. Here, recent atomic force microscopy studies of native membranes of different photosynthetic bacteria with different polypeptide contents are reviewed. Technology, advantages, feasibilities, restrictions and limits of atomic force microscopy for the acquisition of highly resolved images of up to 10 A lateral resolution under native conditions are discussed. From a biological point of view, the new insights contributed by the images are analysed and discussed in the context of the strongly debated organisation of the interconnected network of membrane-associated chlorophyll-protein complexes composing the photosynthetic apparatus in different species of purple bacteria.

Investigation of the effects of different carotenoids on the absorption and CD signals of light harvesting 1 complexes.

Absorption and circular dichroism (CD) spectra of light-harvesting (LH)1 complexes from the purple bacteria Rhodobacter (Rba.) sphaeroides and Rhodospirillum (Rsp.) rubrum are presented. The complexes exhibit very low intensity, highly nonconservative, near-infrared (NIR) CD spectra. Absorption and CD spectra from several mutant and reconstituted LH1 complexes, with the carotenoid neurosporene and the precursor phytoene replacing the wild-type (WT) carotenoids, are also examined. The experiments show that the position of the carotenoid bands as well as the bacteriochlorophyll (BChl)/carotenoid ratio affect the NIR CD spectra: bluer bands and larger ratios make the NIR CD signal more conservative. Modeling results that support this finding are presented. This study, combined with the theoretical approach of the companion paper, where modeling of such complexes is presented and discussed in detail, provide a complete explanation of the origin of the nonconservative NIR CD spectra of LH1 and B820.

Labeling of chromatophore membranes and reaction centers from the photosynthetic bacterium Rhodospirillum rubrum with the hydrophobic marker 5-[125I]iodonaphthyl-1-azide..

Chromatophores of the photosynthetic bacterium Rhodospirillum rubrum and isolated reaction centers were labeled with the lipophilic membrane marker 5-[125I]iodonaphthyl-1-azide. The two smaller reaction center proteins L and M bind more label than the larger subunit H, a fact supporting the proposed localisation of the 3 subunits obtained with hydrophilic labels. Besides these integral proteins the lipids, among them mainly the pigments and the quinones, are highly labeled suggesting a hydrophobic environment around these molecules and a preferred reactivity to iodonaphthylazide. Such a hydrophobic environment may be of great importance for the function of the photosynthetic reaction centers especially for the charge separation and the primary reactions in electron transport.

Comparative studies on nicotinamide nucleotide transhydrogenase from different sources.

(1) Nicotinamide nucleotide transhydrogenases in submitochondrial particles from beef heart mitochondria, chromatophores from Rhodospirillum rubrum and membrane preparations from Escherichia coli and Pseudomonas aeruginosa have been compared with respect to the following properties: stereospecificity for the 4-hydrogen of NADH, reactivity with 3'-NADP, inhibition by palmityl-CoA, sensitivity tot rypsin, and effects of Ca2+ and 2'-AMP on the reaction rates. (2) Transhydrogenases from submitochondrial particles, R. rubrum chromatophores and E. coli membrane preparations have A-side stereospecificity for NADH, do not react with 3'-NADP, are inhibited by palmityl-CoA and are extremely sensitive to trypsin treatment. No effects of Ca2+ or 2'-AMP on the reaction rates were observed. In R. rubrum chromatophores trypsin-sensitive sites are present both in the soluble transhydrogenase factor and in the membrane preparation devoid of transhydrogenase factor. (3) In contrast, P. aeruginosa transhydrogenase is allosterically regulated by Ca2+ and 2'-AMP, is reactive with 3'-NADP, has B-side stereospecificity for NADH and is insensitive to palmityl-CoA or trypsin treatment. (4) It is concluded that the properties characterizing the transhydrogenase in E. coli, R. rubrum chromatophores and submitochondrial particles are closely connected with the interaction of the enzyme with an energy-conserving membrane system.

Role of water in processes of energy transduction: Ca2+-transport ATPase and inorganic pyrophosphatase.

After the proposal of the chemiosmotic theory by Mitchell (1966, 1979) it has been recognized that different membrane-bound enzymes are able to use the energy derived from ionic gradients for the synthesis of ATP. These include the F1-ATPases of mitochondria and chloroplasts, the Ca2+-dependent ATPase of sarcoplasmic reticulum and the (Na+,K+)-ATPase of plasma membrane. In these systems the process of energy transduction is fully reversible. The enzyme can use the energy derived from the hydrolysis of ATP to build up a concentration gradient of ions across the membrane and, in the reverse process, use the energy derived from the gradient to synthesize ATP. Another interesting system in which these forms of energy are interconverted is found in photosynthetic bacteria. In chromatophores of Rhodospirillum rubrum there is a membrane-bound pyrophosphatase that, like the transport ATPases, catalyses the synthesis of pyrophosphate from Pi when a light-dependent proton gradient is formed across the chromatophore membrane. Like F1-ATPase, this enzyme is also able to generate an electrochemical potential gradient of protons at the expense of pyrophosphate hydrolysis. The mechanism by which the energy derived from a gradient is used by membrane-bound enzymes to catalyse the synthesis of high-energy phosphate compounds is still far from understood. Among the different enzymes studied, Ca2+-dependent ATPase is probably the system in which most is known about the mechanism of energy transduction. We now know of experimental conditions which allow us to move the different intermediary steps of the catalytic cycle of the enzyme in the direction of ATP synthesis. Thus, ATP synthesis can be attained after a single catalytic cycle in the absence of a transmembrane Ca2+ gradient. The net synthesis of ATP can be promoted by a variety of perturbations, including Ca2+, pH and water activity. These experiments indicate that during the catalytic cycle different forms of energy are interconverted by the Ca2+-dependent ATPase. The ultimate step of the cycle seems to be a change of water activity within the catalytic site of the ATPase. A common feature of all membrane-bound enzymes mentioned above is that during the catalytic cycle there are steps in which the hydrolysis of a phosphate compound (ATP, pyrophosphate or an acyl phosphate residue) is accompanied by only a small change in free energy. In conditions similar to those found in the cytosol, the hydrolysis of these phosphate compounds is accompanied by a much larger change in free energy.(ABSTRACT TRUNCATED AT 400 WORDS)

Protein phosphorylation and control of excitation energy transfer in photosynthetic purple bacteria and cyanobacteria.

The function of phosphorylation of light-harvesting polypeptides is well characterised in chloroplasts of green plants, but the prokaryotic cyanobacteria and purple photosynthetic bacteria have quite different light-harvesting polypeptides whose structure and function cannot be controlled in precisely the same way. Nevertheless, cyanobacteria show light-dependent phosphorylation of membrane polypeptides associated with photosystem II and with the light-harvesting phycobilisome, and purple bacteria show light-dependent phosphorylation of low molecular-weight chromatophore membrane polypeptides. In both cases membrane protein phosphorylation is associated with functional changes observed by chlorophyll fluorescence spectroscopy or chlorophyll fluorescence induction kinetics. Here we report on our recent protein sequence and other data concerning the identities of these phosphoproteins. We also discuss the significance of these findings for regulation by protein phosphorylation of photosynthesis in prokaryotes.

Amino acid sequences of cytochromes c2 and c' from the moderately halophilic purple phototrophic bacterium Rhodospirillum salexigens.

Rhodospirillum salexigens is a moderately halophilic purple phototrophic bacterium which grows optimally in 8% NaCl. The amino acid sequences of the two principal soluble cytochromes c have been determined. One of these is a cytochrome c2, similar in size to mitochondrial cytochrome c. While clearly of the same sequence class as mitochondrial cytochrome c and the proteins from several other Gram-negative bacteria, it does not show particular affinity to any already known sequence in terms of the percentage sequence identity. The other protein is a cytochrome c', but is also a divergent member of this widespread group. The lack of appreciable sequence identity to other species is probably due to a limit of divergence which has been reached for the majority of purple bacterial species. However, the numbers of insertions and deletions and their locations in cytochromes c2 and c' suggest that R salexigens may be related to Rhodospirillum molischianum. Like other electron transport proteins from halophiles, both of these cytochromes are notable for their high content of arginine as compared with lysine and both are acidic. However, they do not show any particular sequence homology to electron transport proteins that have been characterized from the extremely halophilic phototrophes of the genus Ectothiorhodospira. Thus, it appears that adaptation to halophilic habitats has independently occurred more than once in purple bacteria.

X-ray diffraction studies on chromatophore membrane from photosynthetic bacteria. II. Comparison of diffraction patterns of photosynthetic units from various purple bacteria.

Comparative X-ray diffraction studies, in conjunction with infrared absorption spectroscopy, were performed on chromatophores isolated from various purple photosynthetic bacteria in order to achieve a better understanding of the molecular structure of the photosynthetic unit. Purple non-sulfur bacteria used were Rhodospirillum rubrum, Rhodospirillum molischianum, Rhodopseudomonas sphaeroides, and Rhodopseudomonas palustris. Chromatophores of Chromatium vinosum, as a typical example of purple sulfur bacteria, were also investigated. The results were as follows. Distinct equatorial X-ray diffraction patterns were obtained from chromatophores of all the bacteria examined. They showed diffuse, continuous diffraction patterns having several maxima, and the patterns are evidently distinguished from those of either crystalline or amorphous material. The pattern indicates that the photosynthetic unit in the chromatophore has a highly organized molecular structure in the plane of the membrane. Bacteria whose major photosynthetic pigment is bacteriochlorophyll alpha can be categorized in three groups from the viewpoint of near infrared absorption spectra. X-ray diffraction patterns are also grouped accordingly, although the differences are minimal and the patterns display common features. In other words, the bacteriochlorophyll forms, which are bacteriochlorophyll-protein complexes exhibiting different near-infrared absorption spectra, show different X-ray patterns: the molecular structure of photosynthetic units is closely related to the state of pigment in each complex, although the "X-ray" molecular structure is mainly concerned with the arrangement of constituent protein molecules at the present resolution, whereas the "spectroscopic" structure reflects the local environment of pigment.(ABSTRACT TRUNCATED AT 250 WORDS)