Stabilization of the electron in the quinone acceptor part of the <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> reaction centers

The evolution of the light-induced absorption difference spectrum (380–500 nm) of the reaction centers from photosynthetic purple bacteria Rhodobacter sphaeroides has been examined over 200 μs. The observed changes are interpreted as the effects of proton movement along the H-bond between the primary quinone acceptor and its protein surroundings. A theoretical analysis of the spectral evolution, considering the proton tunneling kinetics, corroborates this interpretation. The electronic state of the primary quinone is stabilized within tens of microseconds; the process is retarded upon deuteration of the reaction center as well as in 90% glycerol, and accelerated upon nondestructive heating to 40°C.     

The structure and function of the cytochrome <Emphasis Type="Italic">c</Emphasis><Subscript>2</Subscript>: reaction center electron transfer complex from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

In the photosynthetic bacterium, Rhodobacter sphaeroides, the mobile electron carrier, cytochrome c₂ (cyt c₂) transfers an electron from reduced heme to the photooxidized bacteriochlorophyll dimer in the membrane bound reaction center (RC) as part of the light induced cyclic electron transfer chain. A complex between these two proteins that is active in electron transfer has been crystallized and its structure determined by X-ray diffraction. The structure of the cyt:RC complex shows the cyt c₂ (cyt c₂) positioned at the center of the periplasmic surface of the RC. The exposed heme edge from cyt c₂ is in close tunneling contact with the electron acceptor through an intervening bridging residue, Tyr L162 located on the RC surface directly above the bacteriochlorophyll dimer. The binding interface between the two proteins can be divided into two regions: a short-range interaction domain and a long-range interaction domain. The short-range domain includes residues immediately surrounding the tunneling contact region around the heme and Tyr L162 that display close intermolecular contacts optimized for electron transfer. These include a small number of hydrophobic interactions, hydrogen bonds and a pi-cation interaction. The long-range interaction domain consists of solvated complementary charged residues; positively charged residues from the cyt and negatively charged residues from the RC that provide long range electrostatic interactions that can steer the two proteins into position for rapid association.     

Femtosecond relaxation processes in <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> reaction centers

Energy relaxation was studied with difference femtosecond spectroscopy in reaction centers of the YM210L mutant of the purple photosynthetic bacterium Rhodobacter sphaeroides at low temperature (90 K). A dynamical long-wavelength shift of stimulated emission of the excited state of the bacteriochlorophyll dimer P was found, which starts simultaneously with P* formation and is accompanied by a change in the spectral shape of this emission. The characteristic value of this shift was about 30 nm, and the characteristic time about 200 fs. Difference kinetics ΔA measured at fixed wavelengths demonstrate the femtosecond shift of the P* stimulated emission appearing as a dependence of these kinetics on wavelength. We found that the reported long-wavelength shift can be explained in terms of electron-vibrational relaxation of the P* excited state with time constants of vibrational and electronic relaxation of 100 and 50 fs, respectively. Alternative mechanisms of the dynamical shift of the P* stimulated emission spectrum are also discussed in terms of energy redistribution between vibrational modes or coherent excitation of the modes.     

Singlet oxygen generation in the reaction centers of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

Singlet oxygen ((1)O(2)) generation in the reaction centers (RCs) of Rhodobacter sphaeroides wild type was characterized by luminescent emission in the near infrared region (time resolved transients and emission spectra) and quantified to have quantum yield of 0.03 +/- 0.005. (1)O(2) emission was measured as a function of temperature, ascorbate, urea and potassium ferricyanide concentrations and as a function of incubation time in H(2)O:D(2)O mixtures. (1)O(2 )was shown to be affected by the RC dynamics and to originate from the reaction of molecular oxygen with two sources of triplets: photoactive dimer formed by singlet-triplet mixing and bacteriopheophytin formed by direct photoexcitation and intersystem crossing.     

Light-Dependent Assimilation of <Emphasis Type="Italic">trans</Emphasis>-Cinnamate by <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> OU5

We present a novel light-dependent metabolism of an aromatic compound (trans-cinnamate) that is assimilatory rather than dissimilatory. Light-dependent assimilation of trans-cinnamate was observed by both growing and resting cells of Rhodobacter sphaeroides OU5. Trans-cinnamate assimilation could be correlated with simultaneous formation of both phenylalanine and tyrosine at near-stoichiometric ratios. Trans-cinnamate assimilation was promoted by carbon source and electron donors, such as glucose, pyruvate, or α-ketoglutarate, whereas malate, succinate, fumarate, and acetate were inhibitory.     

Absorbance changes accompanying the fast fluorescence induction in the purple bacterium <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

The authors present a study of the fluorescence and absorbance transients occurring in whole cells of purple nonsulfur bacterium Rhodobacter sphaeroides on the millisecond timescale under pulsed actinic illumination. The fluorescence induction curve is interpreted in terms of combination of effects of redox changes in the reaction center and the membrane potential. The results of this study support the view that the membrane potential act predominantly to increase the fluorescence yield. Advantages of the pulsed actinic illumination for study of the operation of the electron transport chain in vivo are discussed.     

Femtosecond charge separation in dry films of reaction centers of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> and <Emphasis Type="Italic">Chloroflexus aurantiacus</Emphasis>

In this work, the influence of the crystallographic water on electron transfer between primary donor P and acceptor B_A was studied in reaction centers (RCs) of the purple bacterium Rhodobacter sphaeroides and the green bacterium Chloroflexus aurantiacus. For this purpose, time constants and oscillations of charge separation kinetics are compared between dry film RCs and RCs in glycerol-water buffer at 90 K. A common result of the drying of Rba. sphaeroides and Cfx. aurantiacus RCs is slowing of the charge separation process, decrease in amplitude of the oscillatory components of the kinetics, and the depletion of its spectrum. Thus, the major time constant of stimulated emission decay of P* bacteriochlorophyll dimer at 940 nm is increased from 1.1 psec for water-containing Rba. sphaeroides RCs to 1.9 psec for dry films of Rba. sphaeroides RCs. An analogous increase from 3.5 to 4.2 psec takes place in Cfx. aurantiacus RCs. In dry films of Rba. sphaeroides RCs, the amplitude of coherent oscillations of the absorption band of monomeric bacteriochlorophyll B_A⁻ at 1020 nm is 1.8 times less for the 130-cm⁻¹ component and 2.3 times less for the 32-cm⁻¹ component than the analogous amplitudes for water-containing RCs. Measurements in the analogous band of Cfx. aurantiacus RCs show that strong decrease (∼5-10 times) of the B_A⁻ absorption band and strong slowing (from ∼0.8 to ∼3 psec) of B_A⁻ accumulation together with ∼3-fold decrease in oscillation amplitude occurs on drying of these RCs. The overtones of the 32-cm⁻¹ component disappeared from the oscillations of the kinetics at 940 and 1020–1028 nm after drying of the Rba. sphaeroides and Cfx. aurantiacus RCs. The results are in agreement with the results for GM203L mutant of Rba. sphaeroides, in which the HOH55 water molecule is sterically removed, and with the results for dry films of pheophytin-modified RCs of Rba. sphaeroides R-26 and for YM210W and YM210L Rba. sphaeroides mutant RCs. The data are discussed in terms of the influence (or participation) of the HOH55 water molecule on electron transfer along the chain of polar atomic groups N-Mg(P_B)-N-C-N(HisM202)-HOH55-O=(B_A) connecting P_B and B_A in Rba. sphaeroides RCs.     

Mutant reaction centers of <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> I(L177)H with strongly bound bacteriochlorophyll <Emphasis Type="Italic">a</Emphasis>: Structural properties and pigment-protein interactions

Methods of photoinduced Fourier transform infrared (FTIR) difference spectroscopy and circular dichroism were employed for studying features of pigment-protein interactions caused by replacement of isoleucine L177 by histidine in the reaction center (RC) of the site-directed mutant I(L177)H of Rhodobacter sphaeroides. A functional state of pigments in the photochemically active cofactor branch was evaluated with the method of photo-accumulation of reduced bacteriopheophytin H _A ^? . The results are compared with those obtained for wild-type RCs. It was shown that the dimeric nature of the radical cation of the primary electron donor P was preserved in the mutant RCs, with an asymmetric charge distribution between the bacteriochlorophylls P_A and P_B in the P⁺ state. However, the dimers P in the wild-type and mutant RCs are not structurally identical due probably to molecular rearrangements of the P_A and P_B macrocycles and/or alterations in their nearest amino acid environment induced by the mutation. Analysis of the electronic absorption and FTIR difference P⁺Q^?/PQ spectra suggests the 17³-ester group of the bacteriochlorophyll P_A to be involved in covalent interaction with the I(L177)H RC protein. Incorporation of histidine into the L177 position does not modify the interaction between the primary electron acceptor bacteriochlorophyll B_A and the bacteriopheophytin H_A. Structural changes are observed in the monomer bacteriochlorophyll B_B binding site in the inactive chromophore branch of the mutant RCs.     

Excitation relaxation in the chemically modified photosynthetic reaction center from <Emphasis Type="Italic">Rhodobacter</Emphasis><Emphasis Type="Italic">sphaeroides</Emphasis> 601

The ultrafast excitation relaxation in the sodium borohydride-treated reaction center of Rhodobacter sphaeroides 601 was investigated with selective excitation. From the femtosecond pump-probe measurement at 790 nm, the excitation relaxation demonstrates a biexponential decay with time constants of about 200 fs and 1.4 ps. By comparison with the result from sodium ascorbate-pretreated modified RS601, it could be concluded that the dynamical trace at 790 nm mainly originates from the contribution of accessory bacteriochlorophyll in the active side, and the electrochromic shift arising from the induced positive charge on the special pair primarily affects the absorption band in the red region of the accessory bacteriochlorophyll in RS601. With direct excitation of the special pair, the charge separation and subsequent electron transfer were observed in borohydride-modified RS601. The 2.8 ps component was ascribed to the charge separation and electron transfer from P* to H(A). From the dynamical traces at 790, 800 and 818 nm, the ultrafast energy relaxation from the excited accessory bacteriochlorophyll in the active side is consistent with a two-step energy transfer mechanism. This dynamical observation in modified RS601 is of significance in understanding the physical mechanism of excitation relaxation and energy transfer in the photosynthetic primary process.     

High-precision determination of quantum yields of photoelectric excitation conversion in reaction centers of purple bacteria <Emphasis Type="Italic">Rhodospirillum rubrum</Emphasis> and <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> R-26

Using an original model of primary events in bacterial photosynthetic reaction centers (RCs), a complete set of kinetic and energy characteristics, and temperature dependences of nanosecond luminescence component(s) emitted by the RCs, upper limits to the quantum yield of primary transmembrane charge separation in RCs of the purple bacterium Rhodospirillum rubrum and the carotenoidless mutant Rhodobacter sphaeroides R-26 have been estimated. The calculations have given the values of 0.915 ± 0.045 and 0.978 ± 0.006, respectively. In the case of Rb. sphaeroides R-26, the quantum yield partly overlapped with that reported earlier (1.02 ± 0.04) by Wraight and Clayton (BBA, 1974. vol. 333, pp. 246–260).     

Synthesis of bacteriochlorophyll <Emphasis Type="Italic">a</Emphasis> by the purple nonsulfur bacterium <Emphasis Type="Italic">Rhodobacter capsulatus</Emphasis>

The ability of purple nonsulfur bacteria Rhodobacter capsulatus B10 to synthesize bacteriochlorophyll under phototrophic and dark conditions was studied. The modes for cultivation in the dark with oxygen limitation in a continuous culture at D = 0.1 h^?1 were selected. The yield of biomass reached 20 g/l; the bacteriochlorophyll a output of the process amounted to 16.6 mg/l h^?1.     

Incidence of the endosymbionts <Emphasis Type="Italic">Wolbachia</Emphasis>, <Emphasis Type="Italic">Cardinium</Emphasis> and <Emphasis Type="Italic">Spiroplasma</Emphasis> in phytoseiid mites and associated prey

Endosymbiotic bacteria that potentially influence reproduction and other fitness-related traits of their hosts are widespread in insects and mites and their appeal to researchers’ interest is still increasing. We screened 20 strains of 12 agriculturally relevant herbivorous and predatory mite species for infection with Wolbachia, Cardinium and Spiroplasma by the use of PCR. The majority of specimens originated from Austria and were field collected or mass-reared. Eight out of 20 strains (40%) tested, representing seven of 12 mite species (58%), carried at least one of the three bacteria. We found Wolbachia in the herbivorous spider mites Tetranychus urticae and Bryobia rubrioculus, with the former also carrying Spiroplasma and the latter also carrying Cardinium. Cardinium was furthermore found in two populations of the predatory mite Euseius finlandicus and the spider mite Eotetranychus uncatus. Spiroplasma was detected in the predatory mite Neoseiulus californicus. All bacteria positive PCR products were sequenced, submitted to GenBank and analyzed in BLAST queries. We found high similarities to complete identity with bacteria found in the same and different mite species but also with bacteria found in insect species like ladybirds, butterflies and minute pirate bugs, Orius. We discuss the significance of potential (multiple) infections with the investigated bacteria for biological control.     

Heterologous synthesis and assembly of functional LHII antenna complexes from <Emphasis Type="Italic">Rhodovulum sulfidophilum</Emphasis> in <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> mutant

The light harvesting complexes, including LHII and LHI, are the important components of photosynthetic apparatus. Rhodovulum (Rdv.) sulfidophilum and Rhodobacter (R.) sphaeroides belong to two genera of photosynthetic bacteria, and they are very different in some physiological characteristics and light harvesting complexes structure. The LHII structural genes (pucBsAs) from Rdv. sulfidophilum and the LHI structural genes (pufBA) from R. sphaeroides were amplified, and cloned into an expression vector controlled by puc promoter from R. sphaeroides, which was then introduced into LHI and LHII-minus R. sphaeroides mutants; the transconjugant strains synthesized heterologous LHII and native LHI complexes, which played normal roles in R. sphaeroides. The Rdv. sulfidophilum LHII complex from pucBsAs had near-infrared absorption bands at ~801–853 nm in R. sphaeroides, and was able to transfer energy efficiently to the native LHI complex. The results show that the pucBsAs genes from Rdv. sulfidophilum could be expressed in R. sphaeroides, and the functional foreign LHII and native LHI were assembled into the membrane of R. sphaeroides.     

Influence of chemical chaperones on the properties of lysozyme and the reaction center protein from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

The influence of three chemical chaperones: glycerol, 4-hexylresorcinol, and 5-methylresorcinol on the structure, equilibrium fluctuations, and functional activity of the hydrophilic enzyme lysozyme and the transmembrane reaction center (RC) protein from Rb. sphaeroides in a broad range of concentrations has been studied. The chosen chemical chaperones differ strongly in their structure and action on hydrophilic and membrane proteins. The influence of the chemical chaperones (except methylresorcinol) on the structure, dynamics, and functional properties of lysozyme and RC protein are well described in the framework of extended models of preferential hydration and preferential interaction of protein with a chemical chaperone. A molecule of hexylresorcinol consists of a hydrophobic (alkyl radical) and a hydrophilic (aromatic core) moieties; this provides for additional regulation of the functional activity of lysozyme and RC by hexylresorcinol. The influence of methylresorcinol on proteins differs from that of glycerol and hexylresorcinol. Methylresorcinol interacts with the surface of lysozyme directly, not via water hydrogen bonds. This leads to a decrease in the denaturation temperature and an increase in the amplitude of equilibrium fluctuations, allowing it to be a powerful activator. Methylresorcinol interacts with the membrane RC protein only by the condensation of hydration water, which is negligible in this case. Therefore, methylresorcinol does not affect the functional properties of the RC protein. It is concluded that different chaperones at the same concentration as well as one and the same chaperone at different concentrations produce protein 3D structures differing in dynamic and functional characteristics.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Capsicum chinense</Emphasis> Jacq.

An efficient micropropagation protocol was established for Capsicum chinense Jacq. cv. Umorok, a pungent chilli cultivar. Shoot-tip explants were cultured on Murashige and Skoog (MS) medium containing cytokinins (22.2–88.8 μM 6-benzylaminopurine, BAP, 23.2–93.0 μM kinetin, Kin, or 22.8–91.2 μM zeatin, Z) alone or in combination with 5.7 μM indole-3-acetic acid (IAA). Maximum number of shoots were induced on medium containing 91.2 μM Z or 31.1 μM BAP with 4.7 μM Kin. The separated shoots rooted and elongated on medium containing 2.5 or 4.9 μM indole-3-butyric acid (IBA). Axillary shoots were induced from in vitro raised plantlets by decapitating them. The axillary shoot-tip explants were used for further multiple shoot buds induction. A maximum of about 150 plantlets were obtained from a single seedling. Hardened and acclimatized plantlets were successfully established in the soil.