Excitation transfer connectivity in different purple bacteria: a theoretical and experimental study.

Photosynthetic membranes accommodate densely packed light-harvesting complexes which absorb light and convey excitation to the reaction center (RC). The relationship between the fluorescence yield (phi) and the fraction (x) of closed RCs is informative about the probability for an excitation reaching a closed RC to be redirected to another RC. In this work, we have examined in this respect membranes from various bacteria and searched for a correlation with the arrangement of the light-harvesting complexes as known from atomic force or electron microscopies. A first part of the paper is devoted to a theoretical study analyzing the phi(x) relationship in various models: monomeric or dimeric RC-LH1 core complexes, with or without the peripheral LH2 complexes. We show that the simple "homogeneous" kinetic treatment used here agrees well with more detailed master equation calculations. We also discuss the agreement between information derived from the present technique and from singlet annihilation experiments. The experimental results show that the enhancement of the cross section of open RCs due to excitation transfer from closed units varies from 1.5 to 3 depending on species. The ratio of the core to core transfer rate (including the indirect pathway via LH2) to the rate of trapping in open units is in the range of 0.5 to 4. It is about 1 in Rhodobacter sphaeroides and does not increase significantly in mutants lacking LH2-despite the more numerous contacts between the dimeric core complexes expected in this case. The connectivity in this bacterium is due in good part to the fast transfer between the two partners of the dimeric (RC-LH1-PufX)(2) complex. The connectivity is however increased in the carotenoidless and LH2-less strain R26, which we ascribe to an anomalous LH1. A relatively high connectivity was found in Rhodospirillum photometricum, although not as high as predicted in the calculations of Fassioli et al. (2010). This illustrates a more general discrepancy between the measured efficiency of core to core excitation transfer and theoretical estimates. We argue that the limited core to core connectivity found in purple bacteria may reflect a trade-off between light-harvesting efficiency and the hindrance to quinone diffusion that would result from too tightly packed LH complexes.

Dual role for a bacteriophytochrome in the bioenergetic control of Rhodopseudomonas palustris: enhancement of photosystem synthesis and limitation of respiration.

In the purple photosynthetic bacterium Rhodopseudomonas palustris, far-red illumination induces photosystem synthesis via the action of the bacteriophytochrome RpBphP1. This bacteriophytochrome antagonizes the repressive effect of the transcriptional regulator PpsR2 under aerobic condition. We show here that, in addition to photosystem synthesis, far-red light induces a significant growth rate limitation, compared to cells grown in the dark, linked to a decrease in the respiratory activity. The phenotypes of mutants inactivated in RpBphP1 and PpsR2 show their involvement in this regulation. Based on enzymatic and transcriptional studies, a 30% decrease in the expression of the alpha-ketoglutarate dehydrogenase complex, a central enzyme of the Krebs cycle, is observed under far-red light. We propose that this decrease is responsible for the down-regulation of respiration in this condition. This regulation mechanism at the Krebs cycle level still allows the formation of the photosynthetic apparatus via the synthesis of key biosynthesis precursors but lowers the production of NADH, i.e. the respiratory activity. Overall, the dual action of RpBphP1 on the regulation of both the photosynthesis genes and the Krebs cycle allows a fine adaptation of bacteria to environmental conditions by enhancement of the most favorable bioenergetic process in the light, photosynthesis versus respiration.

Control of peripheral light-harvesting complex synthesis by a bacteriophytochrome in the aerobic photosynthetic bacterium Bradyrhizobium strain BTAi1.

The recent sequence analysis of the photosynthetic and plant-symbiotic Bradyrhizobium sp. strain BTAi1 revealed the unexpected presence of a pucBA operon encoding the apoproteins of peripheral light-harvesting (LH) complexes. This pucBA operon is found close to a bacteriophytochrome gene (BphP3(B BTAi1)) and a two-component transcriptional regulator gene (TF(BTAi1) gene). In this study, we show that BphP3(B BTAi1) acts as a bona fide bacteriophytochrome and controls, according to light conditions, the expression of the pucBA operon found in its vicinity. This light regulatory pathway is very similar to the one previously described for chromo-BphP4(Rp) in Rhodopseudomonas palustris and conducts the synthesis of a peripheral LH complex. This LH complex presents a single absorption band at low temperature, centered at 803 nm. Fluorescence emission analysis of intact cells indicates that this peripheral LH complex does not act as an efficient light antenna. One putative function of this LH complex could be to evacuate excess light energy in order to protect Bradyrhizobium strain BTAi1, an aerobic anoxygenic photosynthetic bacterium, against photooxidative damage during photosynthesis.

A singular bacteriophytochrome acquired by lateral gene transfer.

Bacteriophytochromes are phytochrome-like proteins that mediate photosensory responses in various bacteria according to their light environment. The genome of the photosynthetic and plant-symbiotic Bradyrhizobium sp. strain ORS278 revealed the presence of a genomic island acquired by lateral transfer harboring a bacteriophytochrome gene, BrBphP3.ORS278, and genes involved in the synthesis of phycocyanobilin and gas vesicles. The corresponding protein BrBphP3.ORS278 is phylogenetically distant from the other (bacterio)phytochromes described thus far and displays a series of unusual properties. It binds phycocyanobilin as a chromophore, a unique feature for a bacteriophytochrome. Moreover, its C-terminal region is short and displays no homology with any known functional domain. Its dark-adapted state absorbs maximally around 610 nm, an unusually short wavelength for (bacterio)phytochromes. This form is designated as Po for orange-absorbing form. Upon illumination, a photo-reversible switch occurs between the Po form and a red (670 nm)-absorbing form (Pr), which rapidly backreacts in the dark. Because of this instability, illumination results in a mixture of the Po and Pr states in proportions that depend on the intensity. These uncommon features suggest that BrBphP3.ORS278 could be fitted to measure light intensity rather than color.

Electrochemically induced FTIR difference spectroscopy in the mid- to far infrared (200 microm) domain: a new setup for the analysis of metal-ligand interactions in redox proteins.

We report the setup of an electrochemical cell with chemical-vapor deposition diamond windows and the use of a Bruker 66 SX FTIR spectrometer equipped with DTGS and Si-bolometer detectors and KBr and mylar beam splitters, to record on the same sample, FTIR difference spectra corresponding to the structural changes associated with the change in redox state of active sites in proteins in the whole 1800-50 cm(-1) region. With cytochrome c we show that reliable reduced-minus-oxidized FTIR difference spectra are obtained, which correspond to single molecular vibrations. Redox-sensitive IR modes of the cytochrome c are detected until 140 cm(-1) with a good signal to noise. This new setup is promising to analyze the infrared spectral region where metal-ligand vibrations are expected to contribute and to extend the analysis of vibrational properties to metal sites or redox states not accessible to (resonance) Raman spectroscopy.

Identification of a Cd2+- and Zn2+-binding site in cytochrome c using FTIR coupled to an ATR microdialysis setup and NMR spectroscopy.

Fourier transform infrared (FTIR) difference spectroscopy allows the study of molecular changes occurring at active sites in proteins with high sensitivity. Reactions are triggered by light, potential, or temperature steps and more recently by the diffusion of buffers containing effectors above membrane proteins deposited as films on ATR crystals. We have adapted a microdialysis system to an ATR, to study metal sites in soluble proteins. In this study, we identified a Cd(2+)- or Zn(2+)-binding site in cytochrome c with dissociation constants of 17 and 42 microM, respectively, which affects the oxidation rate of ferrocytochrome c by hydrogen peroxide. Using the microdialysis ATR-FTIR setup, we determined that a histidine and the carboxylate group of a glutamate are involved in Zn(2+) binding. The implication of His 33 and Glu 104 in the binding site was deduced from the comparison of FTIR data recorded with horse heart and the variant tuna cytochrome c lacking these two amino acids. A two-dimensional NMR analysis of the Zn(2+)-binding site in horse heart cytochrome c confirmed that His 33 and residues close to the C terminus are sensitive to Zn(2+) binding. This study demonstrates that the microdialysis ATR-FTIR setup is promising for the analysis of metal sites in proteins. From H(2)O/(2)H(2)O exchange experiments, we concluded that the impact of Zn(2+) and Cd(2+) binding on the oxidation kinetics of ferrocytochrome c by H(2)O(2) is associated to the perturbation of a hydrogen-bonding network involving His 33 that is sensitive to the redox state of cytochrome c.

A new type of bacteriophytochrome acts in tandem with a classical bacteriophytochrome to control the antennae synthesis in Rhodopseudomonas palustris.

Phytochromes are chromoproteins found in plants and bacteria that switch between two photointerconvertible forms via the photoisomerization of their chromophore. These two forms, Pr and Pfr, absorb red and far-red light, respectively. We have characterized the biophysical and biochemical properties of two bacteriophytochromes, RpBphP2 and RpBphP3, from the photosynthetic bacterium Rhodopseudomonas palustris. Their genes are contiguous and localized near the pucBAd genes encoding the polypeptides of the light harvesting complexes LH4, whose synthesis depends on the light intensity. At variance with all (bacterio)phytochromes studied so far, the light-induced isomerization of the chromophore of RpBphP3 converts the Pr form to a form absorbing at shorter wavelength around 645 nm, designated as Pnr for near red. The quantum yield for the transformation of Pr into Pnr is about 6-fold smaller than for the reverse reaction. Both RpBphP2 and RpBphP3 autophosphorylate in their dark-adapted Pr forms and transfer their phosphate to a common response regulator Rpa3017. Under semiaerobic conditions, LH4 complexes replace specifically the LH2 complexes in wild-type cells illuminated by wavelengths comprised between 680 and 730 nm. In contrast, mutants deleted in each of these two bacteriophytochromes display no variation in the composition of their light harvesting complexes whatever the light intensity. From both the peculiar properties of these bacteriophytochromes and the phenotypes of their deletion mutants, we propose that they operate in tandem to control the synthesis of LH4 complexes by measuring the relative intensities of 645 and 710 nm lights.

Bacteriophytochrome controls photosystem synthesis in anoxygenic bacteria.

Plants use a set of light sensors to control their growth and development in response to changes in ambient light. In particular, phytochromes exert their regulatory activity by switching between a biologically inactive red-light-absorbing form (Pr) and an active far-red-light absorbing form (Pfr). Recently, biochemical and genetic studies have demonstrated the occurrence of phytochrome-like proteins in photosynthetic and non-photosynthetic bacteria--but little is known about their functions. Here we report the discovery of a bacteriophytochrome located downstream from the photosynthesis gene cluster in a Bradyrhizobium strain symbiont of Aeschynomene. The synthesis of the complete photosynthetic apparatus is totally under the control of this bacteriophytochrome. A similar behaviour is observed for the closely related species Rhodopseudomonas palustris, but not for the more distant anoxygenic photosynthetic bacteria of the genus Rhodobacter, Rubrivivax or Rhodospirillum. Unlike other (bacterio)phytochromes, the carboxy-terminal domain of this bacteriophytochrome contains no histidine kinase features. This suggests a light signalling pathway involving direct protein-protein interaction with no phosphorelay cascade. This specific mechanism of regulation may represent an important ecological adaptation to optimize the plant-bacteria interaction.

Bacteriophytochrome and regulation of the synthesis of the photosynthetic apparatus in Rhodopseudomonas palustris: pitfalls of using laboratory strains.

The synthesis of the photosynthetic apparatus of different strains of Rhodopseudomonas palustris has been studied as a function of the oxygen concentration and far-red light. For strain CEA001, only a small amount of photosynthetic apparatus is synthesized in the dark for oxygen concentration higher than 8% whereas synthesis is strongly enhanced by far-red light illumination. This enhancement is due to the action of a bacteriophytochrome (ORF2127/ORF2128), which antagonizes the repressor PpsR. On the contrary, a large fraction of photosystem is synthesized in the dark and far-red illumination induces no enhancement in strain CGA009. This difference in phenotype of strain CGA009 is explained by a single point-mutation R428C in the helix-turn-helix DNA binding motif of PpsR, rendering it inactive. In addition, a frame-shift mutation had occurred in the gene encoding bacteriophytochrome (ORF2127/ORF2128), conducting to a truncated inactive sensor. We propose that these mutations occurred in culture. Bacteria have developed a sophisticated regulatory process to synthesize their photosynthetic apparatus when light is available. This process is a critical advantage for the bacteria under natural conditions since they optimize their development depending on the available energy resources. On the contrary, under laboratory growth conditions where there is no substrate limitation, there is no crucial need for such a regulation and deleterious mutations affecting this process are of no importance.