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A very high rate for cyclic electron flow (CEF) around PSI (~180 ssup-/sup1 or 210 ssup-1/supin minimum medium or in the presence of a carbon source respectively) is measured in the presence of methyl viologen (MV) in intact cells of Chlamydomonas reinhardtii under anaerobic conditions. The observation of an efficient CEF in the presence of methyl viologen is in agreement with the previous results reports of Asada et al. in broken chloroplasts (Plant Cell Physiol. 31(4) (1990) 557-564). From the analysis of the P700 and PC absorbance changes, we propose that a confinement between 2 PC molecules, 1 PSI and 1 cytbsub6/subf corresponding to a functional supercomplex is responsible for these high rates of CEF. Supercomplex formation is also observed in the absence of methyl viologen, but with lower maximal CEF rate (about 100 ssup-1/sup) suggesting that this compound facilitates the mediation of electron transfer from PSI acceptors to the stromal side of cytbsub6/subf. Further analysis of CEF in mutants of Chlamydomonas defective in state transitions shows the requirement of a kinase-driven transition to state 2 to establish this functional supercomplex configuration. However, a movement of the LHCII antennae is not involved in this process. We discuss the possible involvement of auxiliary proteins, among which is a small cytbsub6/subf-associated polypeptide, the PETO protein, which is one of the targets of the STT7 kinase.

Deletion of two of the major electron carriers, the reaction center-bound tetrahemic cytochrome and the HiPIP, involved in the light-induced cyclic electron transfer pathway of the purple photosynthetic bacterium, Rubrivivax gelatinosus, significantly impairs its anaerobic photosynthetic growth. Analysis on the light-induced absorption changes of the intact cells of the mutants shows, however, a relatively efficient photo-induced cyclic electron transfer. For the single mutant lacking the reaction center-bound cytochrome, we present evidence that the electron carrier connecting the reaction center and the cytochrome bc1 complex is the High Potential Iron–sulfur Protein. In the double mutant lacking both the reaction center-bound cytochrome and the High Potential Iron–sulfur Protein, this connection is achieved by the high potential cytochrome c8. Under anaerobic conditions, the halftime of re-reduction of the photo-oxidized primary donor by these electron donors is 3 to 4 times faster than the back reaction between P+ and the reduced primary quinone acceptor. This explains the photosynthetic growth of these two mutants. The results are discussed in terms of evolution of the type II RCs and their secondary electron donors.

The purple photosynthetic bacterium Rubrivivax gelatinosus has, at least, four periplasmic electron carriers, i.e., HiPIP, two cytochromes c 8 with low- and high-midpoint potentials, and cytochrome c 4 as electron donors to the photochemical reaction center. The quadruple mutant lacking all four electron carrier proteins showed extremely slow photosynthetic growth. During the long-term cultivation of this mutant under photosynthetic conditions, a suppressor strain recovering the wild-type growth level appeared. In the cells of the suppressor strain, we found significant accumulation of a soluble c -type cytochrome that has not been detected in wild-type cells. This cytochrome c has a redox midpoint potential of about + 280 mV and could function as an electron donor to the photochemical reaction center in vitro . The amino acid sequence of this cytochrome c was 65% identical to that of the high-potential cytochrome c 8 of this bacterium. The gene for this cytochrome c was identified as nirM on the basis of its location in the newly identified nir operon, which includes a gene coding cytochrome cd 1 -type nitrite reductase. Phylogenetic analysis and the well-conserved nir operon gene arrangement suggest that the origin of the three cytochromes c 8 in this bacterium is NirM. The two other cytochromes c 8 , of high and low potentials, proposed to be generated by gene duplication from NirM, have evolved to function in distinct pathways.

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.

Since the first discovery of a bacteriophytochrome in Rhodospirillum centenum, numerous bacteriophytochromes have been identified and characterized in other anoxygenic photosynthetic bacteria. This review is focused on the biochemical and biophysical properties of bacteriophytochromes with a special emphasis on their roles in the synthesis of the photosynthetic apparatus.

A new type of membrane-bound cytochrome c was found in a marine purple photosynthetic bacterium, Rhodovulum sulfidophilum. This cytochrome c was significantly accumulated in cells growing under anaerobic photosynthetic conditions and showed an apparent molecular mass of approximately 100 kDa when purified and analyzed by SDS-PAGE. The midpoint potential of this cytochrome c was 369 mV. Flash-induced kinetic measurements showed that this new cytochrome c can work as an electron donor to the photosynthetic reaction center. The gene coding for this cytochrome c was cloned and analyzed. The deduced molecular mass was nearly equal to 50 kDa. Its C-terminal heme-containing region showed the highest sequence identity to the water-soluble cytochrome c(2), although its predicted secondary structure resembles that of cytochrome c(y). Phylogenetic analyses suggested that this new cytochrome c has evolved from cytochrome c(2). We, thus, propose its designation as cytochrome c(2m). Mutants lacking this cytochrome or cytochrome c(2) showed the same growth rate as the wild type. However, a double mutant lacking both cytochrome c(2) and c(2m) showed no growth under photosynthetic conditions. It was concluded that either the membrane-bound cytochrome c(2m) or the water-soluble cytochrome c(2) work as a physiological electron carrier in the photosynthetic electron transfer pathway of Rvu. sulfidophilum.

Bacterial cytochrome bc1-complex encoded by the petABC operon consists of three subunits, the Rieske iron-sulphur protein, the b-type cytochrome, and the c1-type cytochrome. Disruption of the petA gene of Rubrivivax gelatinosus is not lethal under photosynthetic growth conditions. However, deletion of both petA and petB results in a photosynthesis-deficient strain, suggesting the presence of a second gene encoding a Rieske protein and rescuing a functional cytochrome bc1-complex in the PETA1 mutant. The corresponding petA2 gene was identified and the PETA2 mutant could also grow under photosynthetic conditions. The double mutant PETA12, however, was unable to grow photosynthetically. The presence of a photo-induced cyclic electron transfer was tested by monitoring the kinetics of cytochrome photo-oxidation on intact cells; the data confirm the capacity of petA2 to replace petA1 in the bc1-complex to support photosynthesis. Soluble forms of both PetA1 and PetA2 Rieske proteins were purified from Escherichia coli and found to contain correctly inserted [2Fe-2S] clusters. Electron paramagnetic resonance (EPR) spectroscopy and midpoint potential measurements showed typical [2Fe-2S] signals and E(m) values of +275 mV for both Rieske proteins. The high amino acid sequence similarity and the obtained midpoint potential values argue for a functional role of these proteins in the cytochrome bc1-complex. The presence of duplicated Rieske genes is not restricted to R. gelatinosus. Phylogenetic trees of Rieske genes from Rubrivivax and other proteobacteria as well as from cyanobacteria were reconstructed. On the basis of the phylogenetic analyses, differing evolutionary origins of duplicated Rieske genes in proteo- and cyanobacteria are proposed.

The kinetics of reoxidation of the primary acceptor Q(a) has been followed by measuring the changes in the fluorescence yield induced by a series of saturating flashes in intact cells of Rhodobacter sphaeroides in anaerobic conditions. At 0 degrees C, about half of Q(a)(-) is reoxidized in about 200 ms while reoxidation of the remaining fraction is completed in several seconds to minutes. The fast phase is associated with the transfer of ubiquinone formed at site Q(o) of the cytochrome bc(1) complex while the slowest phase is associated with the diffusion of ubiquinone present in the membrane prior to the flash excitation. The biphasic kinetics of Q(a)(-) oxidation is interpreted assuming that the electron chain is organized in supercomplexes that associate two RCs and one cyt bc(1) complex, which allows a fast transfer of quinone formed at the level of cyt bc(1) complex to the RCs. In agreement with this model, the fast phase of Q(a)(-) reoxidation is inhibited by myxothiazol, a specific inhibitor of cyt bc(1). The PufX-deleted mutant displays only the slowest phase of Q(a)(-) oxidation; it is interpreted by the lack of supramolecular organization of the photosynthetic chain that leads to a larger average distance between cyt bc(1) and RCs.

The two closely related bacteria Bradyrhizobium and Rhodopseudomonas palustris show an unusual mechanism of regulation of photosystem formation by light thanks to a bacteriophytochrome that antirepresses the regulator PpsR. In these two bacteria, we found out, unexpectedly, that two ppsR genes are present. We show that the two Bradyrhizobium PpsR proteins exert antagonistic effects in the regulation of photosystem formation with a classical repressor role for PpsR2 and an unexpected activator role for PpsR1. DNase I footprint analysis show that both PpsR bind to the same DNA TGTN12ACA motif that is present in tandem in the bchC promoter and the crtED intergenic region. Interestingly, the cycA and aerR promoter regions that contain only one conserved palindrome are recognized by PpsR2, but not PpsR1. Further biochemical analyses indicate that PpsR1 only is redox sensitive through the formation of an intermolecular disulfide bond, which changes its oligomerization state from a tetramer to an octamer under oxidizing conditions. Moreover, PpsR1 presents a higher DNA affinity under its reduced form in contrast to what has been previously found for PpsR or its homolog CrtJ from the Rhodobacter species. These results suggest that regulation of photosystem synthesis in Bradyrhizobium involves two PpsR competing for the binding to the same photosynthesis genes and this competition might be modulated by two factors: light via the antagonistic action of a bacteriophytochrome on PpsR2 and redox potential via the switch of PpsR1 oligomerization state.

The photosynthetic cyclic electron transfer of the purple bacterium Rubrivivax gelatinosus, involving the cytochrome bc(1) complex and the reaction center, can be carried out via two pathways. A high potential iron-sulfur protein (HiPIP) acts as the in vivo periplasmic electron donor to the reaction center (RC)-bound cytochrome when cells are grown under anaerobic conditions in the light, while cytochrome c is the soluble electron carrier for cells grown under (8)aerobic conditions in the dark. A spontaneous reversion of R. gelatinosus C244, a defective mutant in synthesis of the RC-bound cytochrome by insertion of a Km(r) cassette leading to gene disruption with a slow growth rate, restores the normal photosynthetic growth. This revertant, designated C244-P1, lost the Km(r) cassette but synthesized a RC-bound cytochrome with an external 77-amino acid insertion derived from the cassette. We characterized the RC-bound cytochrome of this mutant by EPR, time-resolved optical spectroscopy, and structural analysis. We also investigated the in vivo electron transfer rates between the two soluble electron donors and this RC-bound cytochrome. Our results demonstrated that the C244-P1 RC-bound cytochrome is still able to receive electrons from HiPIP, but it is no longer reducible by cytochrome c(8). Combining these experimental and theoretical protein-protein docking results, we conclude that cytochrome c(8) and HiPIP bind the RC-bound cytochrome at two distinct but partially overlapping sites.

The cytochrome bound to the photosynthetic reaction center of Rhodovulum sulfidophilum presents two unusual characteristics with respect to the well characterized tetraheme cytochromes. This cytochrome contains only three hemes because it lacks the peptide motif CXXCH, which binds the most distal fourth heme. In addition, we show that the sixth axial ligand of the third heme is a cysteine (Cys-148) instead of the usual methionine ligand. This ligand exchange results in a very low midpoint potential (-160 +/- 10 mV). The influence of the unusual cysteine ligand on the midpoint potential of this distal heme was further investigated by site-directed mutagenesis. The midpoint potential of this heme is upshifted to +310 mV when cysteine 148 is replaced by methionine, in agreement with the typical redox properties of a His/Met coordinated heme. Because of the large increase in the midpoint potential of the distal heme in the mutant, both the native and modified high potential hemes are photooxidized at a redox poise where only the former is photooxidizable in the wild type. The relative orientation of the three hemes, determined by EPR measurements, is shown different from tetraheme cytochromes. The evolutionary basis of the concomitant loss of the fourth heme and the down-conversion of the third heme is discussed in light of phylogenetic relationships of the Rhodovulum species triheme cytochromes to other reaction center-associated tetraheme cytochromes.

High potential iron-sulfur protein (HiPIP), a small soluble redox protein, has been shown to serve in vivo as electron donor to the photosynthetic reaction centre (RC) in Rubrivivax gelatinosus [Biochemistry 34 (1995) 11736]. The results of time-resolved optical spectroscopy on membrane-fragments from this organism indicates that the photooxidized RC is re-reduced by HiPIP even in the absence of the soluble fraction. This implies that a significant fraction of HiPIP can firmly bind to the membrane in a conformation able to interact with the RCs. Salt treatment of the membrane-fragments abolishes these re-reduction kinetics, demonstrating the presence of HiPIP on the membrane due to association with the RC rather than due to simple trapping in hypothetical chromatophores. The existence of such a functional complex in membranes is confirmed and its structure further examined by electron paramagnetic resonance (EPR) performed on membrane-fragments. Orientation-dependent EPR spectra of HiPIP were recorded on partially ordered membranes, oxidized either chemically or photochemically. Whereas hardly any preferential orientation of the HiPIP was seen in the chemically oxidised sample, a subpopulation of HiPIP showing specific orientations could be photooxidised. This fraction arises from the electron transfer complex between HiPIP and the RC.

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)1,2. Recently, biochemical and genetic studies have demonstrated the occurrence of phytochrome-like proteins in photosynthetic and non-photosynthetic bacteria3,4,5,6,7—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.

A mutant of the phototrophic species belonging to the β-proteobacteria, Rubrivivax gelatinosus, lacking the photosynthetic growth ability was constructed by the removal of genes coding for the L, M, and cytochrome subunits of the photosynthetic reaction center complex. The L, M, and cytochrome genes derived from five other species of proteobacteria, Acidiphilium rubrum, Allochromatium vinosum, Blastochloris viridis, Pheospirillum molischianum, and Roseateles depolymerans, and the L and M subunits from two other species, Rhodobacter sphaeroides and Rhodopseudomonas palustris, respectively, have been introduced into this mutant. Introduction of the genes from three of these seven species, Rte. depolymerans, Ach. vinosum, and Psp. molischianum, restored the photosynthetic growth ability of the mutant of Rvi. gelatinosus, although the growth rates were 1.5, 9.4, and 10.7 times slower, respectively, than that of the parent strain. Flash-induced kinetic measurements for the intact cells of these three mutants showed that the photo-oxidized cytochrome c bound to the introduced reaction center complex could be rereduced by electron donor proteins of Rvi. gelatinosus with a t1/2 of less than 10 ms. The reaction center core subunits of photosynthetic proteobacteria appear to be exchangeable if the sequence identities of the LM core subunits between donor and acceptor species are high enough, i.e., 70 % or more.

Some leguminous species of the genus Aeschynomene are specifically stem-nodulated by photosynthetic bradyrhizobia. To study the effect of bacterial photosynthesis during symbiosis, we generated a photosynthesis-negative mutant of the Bradyrhizobium sp. strain ORS278 symbiont of Aeschynomene sensitiva . The presence of a functional photosynthetic unit in bacteroids and the high expression of the photosynthetic genes observed in stem nodules demonstrate that the bacteria are photosynthetically active during stem symbiosis. Stem inoculation by the photosynthetic mutant gave a 50% decrease in stem-nodule number, which reduced nitrogen fixation activity and plant growth in the same proportion. These results indicate an important role of bacterial photosynthesis in the efficiency of stem nodulation.

The organization of chromophores in the cytochrome b 6 f fromChlamydomonas reinhardtii has been studied spectroscopically. Linear dichroism (LD) measurements, performed on the complex co-reconstituted into vesicles with photosynthetic reaction centers as an internal standard, allow the determination of the orientations of the chromophore with respect to the membrane plane. The orientations of the b H- andb L-hemes are comparable to those determined crystallographically on the cytochrome bc 1. The excitonic CD signal, resulting from the interaction betweenb-hemes, is similar to that reported for the cytochromebc 1. LD and CD data are consistent with the differences between the b 6 f andbc 1 leaving the orientation of theb-hemes unaffected. By contrast, the LD data yield a different orientation for the heme f as compared either to the heme c 1 in the crystallographic structures or to the heme f as studied by electron paramagnetic resonance. This difference could either result from incorrect assumptions regarding the orientations of the electronic transitions of the f-heme or may point to the possibility of a redox-dependent movement of cytochrome f. The chlorophyll a was observed in a well defined orientation, further corroborating a specific binding site for it in theb 6 f complex.

The orientation of the chromophores in the cytochrome bc 1 of Rhodospirillum rubrum, Rhodobacter sphaeroides, and beef heart mitochondria is reported. The combination of redox-resolved absorption spectrophotometry and linear dichroism experiments at low temperature allows the determination of the orientation of the three hemes with respect to the membrane plane. The orientations of theb H-and b L-hemes of theR. sphaeroides and beef heart mitochondrial complexes are similar to those determined by crystallographic studies of the mitochondrial cytochrome bc 1. On the other hand the orientations of the b-hemes of the R. rubrum complex lead to the conclusion that theb H-heme is more perpendicular to the membrane plane than the b L-heme. This could be explained by a specific organization of the b-hemes due to subunit composition of the complex or, alternatively, to a different spatial position of the heme transitions with respect to the porphyrin macrocycle compared with the other complexes. Moreover, our results demonstrate a different orientation of the hemec 1 of the three studied complexes in comparison to crystallographic studies. This difference may arise from the above hypothesis on the transitions of the heme or from flexibility of this subunit in function of its redox state.

The three phases of the flash-induced membrane potential rise were measured in Rhodobacter sphaeroides, at temperatures between 16°C and −30°C, in the liquid or the frozen state. We show that phase II, which is myxothiazol-insensitive, includes phase IIa and phase IIb, completed at −8°C in about 2 ms and 10 ms, respectively. Phase IIa is very likely associated with the protonation of the doubly-reduced quinone acceptor QB= (Drachev, L.A., Mamedov, A., Mulkidjanian, A.Y., Semenov, A.Y., Shinkarev, V.P. and Verkhovsky, M.J. (1988) FEBS Lett. 233, 315–318); phase IIb is associated with the oxidation of cytochrome c2 (Jackson, J.B. and Dutton, P.L., 1973, Biochim. Biophys. Acta 325, 102–113). Freezing the sample does not modify the kinetics of phase IIa but slows down phase IIb by a factor of 2. The amplitude of phase III, which is exclusively related to electron and proton transfer within the cytochrome b/c1 complex, is temperature-independent between room temperature and −16°C. In the frozen state, the rate of phase III is mainly limited by the electron transfer from cytochrome c2 to the reaction centers and not by the movement of cytochrome c2 between the two membrane complexes. These results are interpreted assuming that one cytochrome c2 is trapped in a supercomplex formed by the association of two reaction centers and one cytochrome b/c1 complex.

Shewanella oneidensisMR-1 reduces selenite and tellurite preferentially under anaerobic conditions. The Se(0) and Te(0) deposits are located extracellularly and intracellularly, respectively. This difference in localization and the distinct effect of some inhibitors and electron acceptors on these reduction processes are taken as evidence of two independent pathways.

International audience; Tropical aquatic species of the legume genus Aeschynomene are stem- and root-nodulated by bradyrhizobia strains that exhibit atypical features such as photosynthetic capacities or the use of a nod gene-dependent (ND) or a nod gene-independent (NI) pathway to enter into symbiosis with legumes. In this study we used a comparative genomics approach on nine Aeschynomene symbionts representative of their phylogenetic diversity. We produced draft genomes of bradyrhizobial strains representing different phenotypes: five NI photosynthetic strains (STM3809, ORS375, STM3847, STM4509 and STM4523) in addition to the previously sequenced ORS278 and BTAi1 genomes, one photosynthetic strain ORS285 hosting both ND and NI symbiotic systems, and one NI non-photosynthetic strain (STM3843). Comparative genomics allowed us to infer the core, pan and dispensable genomes of Aeschynomene bradyrhizobia, and to detect specific genes and their location in Genomic Islands (GI). Specific gene sets linked to photosynthetic and NI/ND abilities were identified, and are currently being studied in functional analyses.

International audience; Ramlibacter tataouinensis TTB310(T) (strain TTB310), a betaproteobacterium isolated from a semi-arid region of South Tunisia (Tataouine), is characterized by the presence of both spherical and rod-shaped cells in pure culture. Cell division of strain TTB310 occurs by the binary fission of spherical "cyst-like" cells ("cyst-cyst" division). The rod-shaped cells formed at the periphery of a colony (consisting mainly of cysts) are highly motile and colonize a new environment, where they form a new colony by reversion to cyst-like cells. This unique cell cycle of strain TTB310, with desiccation tolerant cyst-like cells capable of division and desiccation sensitive motile rods capable of dissemination, appears to be a novel adaptation for life in a hot and dry desert environment. In order to gain insights into strain TTB310's underlying genetic repertoire and possible mechanisms responsible for its unusual lifestyle, the genome of strain TTB310 was completely sequenced and subsequently annotated. The complete genome consists of a single circular chromosome of 4,070,194 bp with an average G+C content of 70.0%, the highest among the Betaproteobacteria sequenced to date, with total of 3,899 predicted coding sequences covering 92% of the genome. We found that strain TTB310 has developed a highly complex network of two-component systems, which may utilize responses to light and perhaps a rudimentary circadian hourglass to anticipate water availability at the dew time in the middle/end of the desert winter nights and thus direct the growth window to cyclic water availability times. Other interesting features of the strain TTB310 genome that appear to be important for desiccation tolerance, including intermediary metabolism compounds such as trehalose or polyhydroxyalkanoate, and signal transduction pathways, are presented and discussed.

Reaction centers (RCs) with one antenna complex attached (RC/LH1 complexes) were isolated from the alkaliphilic and moderately halophilic, phototrophic purple sulfur bacterium Ectothiorhodospira (E.) mobilis and analyzed with respect to quinone composition and charge recombination kinetics, using time-resolved (low- and room-temperature) spectroscopy and thin-layer chromatography. The RCs contain menaquinone as primary and ubiquinone as secondary quinone electron acceptor, QA and QB, respectively. QB is lost during isolation of the RC/LH1 complexes. P+Q−A charge recombination kinetics, which were shown to be pH independent, were only slightly temperature dependent, indicating that this recombination proceeds via the direct (electron tunnelling) route. At room temperature, its average lifetime was 34.5 ms. These decay kinetics were shown to be monophasic at room temperature and biphasic at low temperature. Addition of an excess of UQ6 to RC/LH1 complexes resulted in retardation of the P+ recovery, due to charge recombination of the state P+QAQ−B. Functional reconstitution of QB was also evident from flash-induced binary oscillations at 450 nm, which could be observed in RC/LH1 complexes in the presence of an excess of UQ6. This reconstitution of QB activity was, however, incomplete and decreased with increasing pH. The P+QAQ−B decay kinetics were pH independent; the average lifetime was about 6 s at room temperature. The apparent equilibrium constant K2 between the states Q−AQB and QAQ−B, and consequently the free energy difference between these states, were relatively large and pH independent. K2 was on average 167, whereas the mean value of the free energy difference between the two states was 130 meV. Finally, a scheme is presented for the kinetics and free energy changes of the electron transfer steps in isolated E. mobilis RCs.

Purple bacteria have thus far been considered to operate light-driven cyclic electron transfer chains containing ubiquinone (UQ) as liposoluble electron and proton carrier. We show that in the purple γ-proteobacterium Halorhodospira halophila , menaquinone-8 (MK-8) is the dominant quinone component and that it operates in the Q B -site of the photosynthetic reaction center (RC). The redox potentials of the photooxidized pigment in the RC and of the Rieske center of the bc 1 complex are significantly lower ( E m = +270 mV and +110 mV, respectively) than those determined in other purple bacteria but resemble those determined for species containing MK as pool quinone. These results demonstrate that the photosynthetic cycle in H. halophila is based on MK and not on UQ. This finding together with the unusual organization of genes coding for the bc 1 complex in H. halophila suggests a specific scenario for the evolutionary transition of bioenergetic chains from the low-potential menaquinones to higher-potential UQ in the proteobacterial phylum, most probably induced by rising levels of dioxygen 2.5 billion years ago. This transition appears to necessarily proceed through bioenergetic ambivalence of the respective organisms, that is, to work both on MK- and on UQ-pools. The establishment of the corresponding low- and high-potential chains was accompanied by duplication and redox optimization of the bc 1 complex or at least of its crucial subunit oxidizing quinols from the pool, the Rieske protein. Evolutionary driving forces rationalizing the empirically observed redox tuning of the chain to the quinone pool are discussed.

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.

Bacteriophytochromes are red/far-red photoreceptors that bacteria use to mediate sensory responses to their light environment. Here, we show that the photosynthetic bacterium Rhodopseudomonas palustris has two distinct types of bacteriophytochrome-related protein (RpBphP4) depending upon the strain considered. The first type binds the chromophore biliverdin and acts as a light-sensitive kinase, thus behaving as a bona fide bacteriophytochrome. However, in most strains, RpBphP4 does not to bind this chromophore. This loss of light sensing is replaced by a redox-sensing ability coupled to kinase activity. Phylogenetic analysis is consistent with an evolutionary scenario, where a bacteriophytochrome ancestor has adapted from light to redox sensing. Both types of RpBphP4 regulate the synthesis of light harvesting (LH2) complexes according to the light or redox conditions, respectively. They modulate the affinity of a transcription factor binding to the promoter regions of LH2 complex genes by controlling its phosphorylation status. This is the first complete description of a bacteriophytochrome signal transduction pathway involving a two-component system.

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.

Leguminous plants (such as peas and soybeans) and rhizobial soil bacteria are symbiotic partners that communicate through molecular signaling pathways, resulting in the formation of nodules on legume roots and occasionally stems that house nitrogen-fixing bacteria. Nodule formation has been assumed to be exclusively initiated by the binding of bacterial, host-specific lipochito-oligosaccharidic Nod factors, encoded by the nodABC genes, to kinase-like receptors of the plant. Here we show by complete genome sequencing of two symbiotic, photosynthetic, Bradyrhizobium strains, BTAi1 and ORS278, that canonical nodABC genes and typical lipochito-oligosaccharidic Nod factors are not required for symbiosis in some legumes. Mutational analyses indicated that these unique rhizobia use an alternative pathway to initiate symbioses, where a purine derivative may play a key role in triggering nodule formation.

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 auto-phosphorylate in their dark-adapted Pr forms and transfer their phosphate to a common response regulator Rpa3017. Under semi-aerobic 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.

Intramolecular electron transfer within proteins is an essential process in bioenergetics. Redox cofactors are embedded in proteins, and this matrix strongly influences their redox potential. Several cofactors are usually found in these complexes, and they are structurally organized in a chain with distances between the electron donor and acceptor short enough to allow rapid electron tunneling. Among the different interactions that contribute to the determination of the redox potential of these cofactors, electrostatic interactions are important but restive to direct experimental characterization. The influence of interaction between cofactors is evidenced here experimentally by means of redox titrations and time-resolved spectroscopy in a chimeric bacterial reaction center (Maki, H., Matsuura, K., Shimada, K., and Nagashima, K. V. P. (2003) J. Biol. Chem. 278, 3921–3928) composed of the core subunits of Rubrivivax gelatinosus and the tetraheme cytochrome of Blastochloris viridis. The absorption spectra and orientations of the various cofactors of this chimeric reaction center are similar to those found in their respective native protein, indicating that their local environment is conserved. However, the redox potentials of both the primary electron donor and its closest heme are changed. The redox potential of the primary electron donor is downshifted in the chimeric reaction center when compared with the wild type, whereas, conversely, that of its closet heme is upshifted. We propose a model in which these reciprocal shifts in the midpoint potentials of two electron transfer partners are explained by an electrostatic interaction between them.

Aerobic photosynthetic bacteria possess the unusual characteristic of producing different classes of carotenoids. In this study, we demonstrate the presence of two distinct crt gene clusters involved in the synthesis of spirilloxanthin and canthaxanthin in a Bradyrhizobium strain. Each cluster contains the genes crtE, crtB, and crtI leading to the common precursor lycopene. We show that spirilloxanthin is associated with the photosynthetic complexes, while canthaxanthin protects the bacteria from oxidative stress. Only the spirilloxanthin crt genes are regulated by light via the control of a bacteriophytochrome. Despite this difference in regulation, the biosyntheses of both carotenoids are strongly interconnected at the level of the common precursors. Phylogenetic analysis suggests that the canthaxanthin crt gene cluster has been acquired by a lateral gene transfer. This acquisition may constitute a major selective advantage for this class of bacteria, which photosynthesize only under conditions where harmful reactive oxygen species are generated.

Technetium (99Tc) is a fission product of 235U formed during the generation of nuclear power. The solubility and mobility of Tc are highly dependent upon its redox state. Under oxic conditions, Tc is present in its most stable form, the pertechnetate anion [Tc(VII)O4−]. This form, which is highly soluble and mobile in the environment, can enter the food chain as a sulfate analogue (2, 20, 34). These properties, coupled with its long half-life (2.13 × 105 years), make contamination by Tc one of the major factors in the long-term impact of the nuclear fuel cycle. One approach to remove 99Tc from aqueous solution is to reduce the pertechnetate form Tc(VII) into the insoluble, low-valence form Tc(IV). This can be achieved by abiotic (5) or biotic (19, 20) processes. Abiotic reduction involves electron transfer between Fe(II)-containing minerals and Tc(VII) (3). The most efficient mineral appears to be magnetite, particularly when it is anodically polarized (5). Biotic precipitation of pertechnetate, probably its reduction into a low-valence, insoluble Tc oxide, has been reported for several species of bacteria during the past few years. These include species such as Geobacter metallireducens (14), Geobacter sulfurreducens (17), Escherichia coli (13), Desulfovibrio desulfuricans (16), Shewanella putrefaciens (33), and Deinococcus radiodurans (9). Both indirect (chemical) and direct (enzymatic) reduction processes have been observed, depending on the bacterial growth conditions. Chemical processes have been clearly demonstrated in the case of the sulfate-reducing bacterium D. desulfuricans and in the case of the metal-reducing bacterium G. sulfurreducens (15, 17). Cultures of D. desulfuricans supplied with sulfate and lactate as electron acceptor and donor, respectively, precipitated Tc extracellularly as an insoluble sulfide. In this case, the Tc sulfide results from the chemical reaction between H2S, formed during reduction of sulfate, and TcO4− (19). A chemical reduction of Tc(VII) by the Fe(II) is also observed during reduction of Fe(III) by G. sulfurreducens (17). In addition, enzymatic reduction of Tc(VII) has been reported for different bacterial species. There are several lines of evidence indicating that this process involves hydrogenase, an enzyme which reversibly catalyzes the splitting of molecular hydrogen into protons and electrons. Indeed, hydrogen is an effective electron donor for Tc(VII) reduction for E. coli, D. desulfuricans, and S. putrefaciens (13, 15, 16, 33). Similarly, in the case of G. sulfurreducens, Tc(VII) reduction has an exclusive requirement for hydrogen as the electron donor, unlike the reduction of Fe(III), which can be coupled to the oxidation of different organic electron donors (17). The most convincing evidence has come from the work of Lloyd et al. (13), who showed that mutants of E. coli defective in the synthesis of transcription factor FNR, of molybdenum cofactor, or of formate dehydrogenase H were unable to reduce Tc(VII), indicating a role for the formate-hydrogenlyase complex in the reduction. Desulfovibrio fructosovorans (24) is a sulfate-reducing bacterium amenable to molecular biological study. Three different hydrogenases have been identified in this bacterium: [NiFe] and [Fe] hydrogenases (1, 10, 27), localized in the periplasm, and a heterotetrameric NADP-reducing [Fe] hydrogenase, localized in the cytoplasm (4, 21, 22). In the present work, we have combined physiological, genetic, and biochemical approaches to determine, at the molecular level, the exact roles of these different hydrogenases in Tc(VII) reduction and precipitation.

The effect of selenite on the growth rate and protein synthesis has been investigated in Rhodobacter sphaeroides . This photosynthetic bacterium efficiently reduces selenite with intracellular accumulation under both dark aerobic and anaerobic photosynthetic conditions. Addition of 1 mM selenite under these two growth conditions does not affect the final cell density, although a marked slowdown in growth rate is observed under aerobic growth. The proteome analysis of selenite response by two-dimensional gel electrophoresis shows an enhanced synthesis of some chaperones, an elongation factor, and enzymes associated to oxidative stress. The induction of these antioxidant proteins confirms that the major toxic effect of selenite is the formation of reactive oxygen species during its metabolism. In addition, we show that one mutant unable to precipitate selenite, selected from a transposon library, is affected in the smoK gene. This encodes a constituent of a putative ABC transporter implicated in the uptake of polyols. This mutant is less sensitive to selenite and does not express stress proteins identified in the wild type in response to selenite. This suggests that the entry of selenite into the cytoplasm is mediated by a polyol transporter in R. sphaeroides .

The cytochrome subunit bound to the photosynthetic reaction center (RC) complex in Rhodovulum sulfidophilum lacks one heme-binding motif (CXXCH) out of four motifs found in other purple bacteria resulting in the absence of the most distal heme from the RC-core complex (S. Masuda et al., J. Biol. Chem. 274 (1999) 10795). Cytochrome c2, which acts as the electron donor to the RC was purified, and its gene was cloned and sequenced. The redox midpoint potential of cytochrome c2 was determined to be Em=357 mV. The photo-oxidation and re-reduction of purified cytochrome c2 were observed in the presence of membrane preparations. Flash-induced photo-oxidation and re-reduction of the RC-bound cytochrome were also observed in intact cells. Despite the unusual nature of the RC-bound cytochrome subunit, the cyclic electron transfer system in Rdv. sulfidophilum was shown to be similar to those in other purple bacteria.

We have cloned the nap locus encoding the periplasmic nitrate reductase in Rhodobacter sphaeroides f. sp. denitrificans IL106. A mutant with this enzyme deleted is unable to grow under denitrifying conditions. Biochemical analysis of this mutant shows that in contrast to the wild-type strain, the level of synthesis of the nitrite and N 2 O reductases is not increased by the addition of nitrate. Growth under denitrifying conditions and induction of N oxide reductase synthesis are both restored by the presence of a plasmid containing the genes encoding the nitrate reductase. This demonstrates that R. sphaeroides f. sp. denitrificans IL106 does not possess an efficient membrane-bound nitrate reductase and that nitrate is not the direct inducer for the nitrite and N 2 O reductases in this species. In contrast, we show that nitrite induces the synthesis of the nitrate reductase.

Native tubular membranes were purified from the purple non-sulfur bacterium Rhodobacter sphaeroides. These tubular structures contain all the membrane components of the photosynthetic apparatus, in the relative ratio of one cytochrome bc1 complex to two reaction centers, and approximately 24 bacteriochlorophyll molecules per reaction center. Electron micrographs of negative-stained membranes diffract up to 25 A and allow the calculation of a projection map at 20 A. The unit cell (a = 198 A, b = 120 A and gamma = 103 degrees) contains an elongated S-shaped supercomplex presenting a pseudo-2-fold symmetry. Comparison with density maps of isolated reaction center and light-harvesting complexes allowed interpretation of the projection map. Each supercomplex is composed of light-harvesting 1 complexes that take the form of two C-shaped structures of approximately 112 A in external diameter, facing each other on the open side and enclosing the two reaction centers. The remaining positive density is tentatively attributed to one cytochrome bc1 complex. These features shed new light on the association of the reaction center and the light-harvesting complexes. In particular, the organization of the light-harvesting complexes in C-shaped structures ensures an efficient exchange of ubihydroquinone/ubiquinone between the reaction center and the cytochrome bc1 complex.

Tellurite and selenate reductase activities were identified in extracts of Escherichia coli. These activities were detected on non-denaturing polyacrylamide gels using an in situ methyl viologen activity-staining technique. The activity bands produced from membrane-protein extracts had the same RF values as those of nitrate reductases (NRs) A and Z. Tellurite and selenate reductase activities were absent from membranes obtained from mutants deleted in NRs A and Z. Further evidence of the tellurite and selenate reductase activities of NR was demonstrated using rocket immunoelectrophoresis analysis, where the tellurite and selenate reductase activities corresponded to the precipitation arc of NR. Additionally, hypersensitivity to potassium tellurite was observed under aerobic growth conditions in nar mutants. The tac promoter expression of NR A resulted in elevated tellurite resistance. The data obtained also imply that a minimal threshold level of NR A is required to increase resistance. Under anaerobic growth conditions additional tellurite reductase activity was identified in the soluble fraction on non-denaturing gels. Nitrate reductase mutants were not hypersensitive under anaerobic conditions, possibly due to the presence of this additional reductase activity.

Seven species of obligately aerobic photosynthetic bacteria of the genera Erythromicrobium, Erythrobacter, and Roseococcus demonstrated high-level resistance to tellurite and accumulation of metallic tellurium crystals. High-level resistance without tellurite reduction was observed for Roseococcus thiosulfatophilus and Erythromicrobium ezovicum grown with certain organic carbon sources, implying that tellurite reduction is not essential to confer tellurite resistance.

In Rhodopseudomonas sphaeroides , light causes the transfer of electrons from bacteriochlorophyll to ubiquinone in the photochemical reaction centers. Electrons from this “primary” ubiquinone move on to a secondary ubiquinone. We have studied the manner in which o -phenanthroline inhibits the transfer of electrons from primary to secondary ubiquinone in chromatophores (intracytoplasmic membrane fragments) and isolated reaction centers of Rp. sphaeroides . The formation of anionic semiquinones, both primary and secondary, is signaled by an absorption band at 450 nm and by band shifts of bacteriopheophytin and bacteriochlorophyll in the near infrared. The pattern of band shifts is different for primary and secondary semiquinone, allowing us to distinguish which quinone has become semireduced. This point was established for isolated reaction centers [Vermeglio, A. & Clayton, R. K. (1977) Biochim. Biophys. Acta 461, 159-165] and is confirmed here for chromatophores. By adding o -phenanthroline at various times during a sequence of actinic light flashes and monitoring the resulting optical absorbance changes, we have found that o -phenanthroline inhibits electron transfer from primary semiquinone to secondary quinone if the latter is in its oxidized form, but not if the latter is semireduced. Our findings can explain the decay kinetics of delayed fluorescence from bacteriochlorophyll in Rp. sphaeroides as measured by R. P. Carithers and W. W. Parson [(1976) Biochim. Biophys. Acta 440, 215-232].

Low temperature absorption and linear dichroism measurements on oriented reaction centers of Rhodopseudomonas viridis at different redox potentials allow the α-bands of the four hemes to be spectrally resolved. The high-potential heme C 556 presents an α-band split into two components absorbing at 555 and 551 nm. The corresponding linear dichroism spectrum shows two transitions of opposite sign and equal amplitudes. Comparison of our experimental results with the linear dichroism values calculated from the atomic coordinates leads us to assign unambiguously the high-potential C 556 cytochrome with heme 2, the third heme away from the primary donor. Taken together with other available spectroscopic observations, this result implies the following sequence for the redox centers in Rhodopseudomonas viridis : C 554 C 556 C 552 C 559 P.

The orientations of high potential cytochromes with respect to photosynthetic membranes was investigated in spinach chloroplasts and in Rhodopseudomonas viridis. The general approach consists in detection with polarized light of photoinduced absorbance changes related to the oxidation of the cytochromes. The orientation of cytochrome c-558 was measured at room temperature in chromatophores and whole cells of Rps. viridis, oriented on glass slides and in a magnetic field, respectively. The orientation of cytochrome b-559 of green plants was detected at 77 K in magnetically oriented chloroplasts. In both cases the dichroic ratio for the alpha band shows that the heme plane makes an angle greater than 35 degrees C with the membrane plane. Moreover, the dichroic ratio is not constant throughout the alpha and beta bands, for both cytochrome c-558 and b-559. Linear dichroism spectra of oriented pure horse heart cytochrome c and cytochrome c2 of Rhodopseudomonas sphaeroides in stretched polyvinyl alcohol films show that the variations of the dichroic ratio in the alpha and beta bands can be explained by the occurrence of x- and y-polarized transitions absorbing at slightly different wavelengths.

The photooxidation of c 559 , c 556 , and c 552 hemes in Rhodopseudomonas viridis cytochrome has been characterized by light-induced FTIR difference spectroscopy. Apart from the common features at 1659 cm −1 and 1561/1551 cm −1 which could arise from one (or possibly two) peptide bond(s), no evidence for major structural rearrangement of the polypeptide backbone was observed. A significant difference with respect to redox-induced FTIR spectra of cytochrome c is the absence of the Tyr marker at 1514/1518 cm −1 in Rps. viridis cytochrome, indicating that the localized shift of a Tyr side chain observed between ferro- and ferri-cytochrome c does not occur in Rps. viridis cytochrome.

introduction During the last years there has been a great advance in the knowledge of the structure of photosynthetic membranes, largely based on studies of purified reaction centers, and on detailed investigations of the asymmetry of the membranes and of the orientation of different components relatively to the membrane plane. In this respect it has been shown that the long wavelength transition moment of WOO (or Chl aI), the trap of Photosystem I, was oriented nearly parallel to the membrane plane [l-3] . In this report we take advantage of the recent observation [4] of an absorption band peaking at 825 nm of the oxidized trap of Photosystem II (named ChliI) for studying the orientation of this trap. We conclude that, like for WOO, the long wavelength transition moment of Chl,, is predominantly parallel to the thylakoid membrane plane. 2. Materials

Preliminary studies showed that the periplasmic nitrate reductase (Nap) ofRhodobacter sphaeroidesand the membrane-bound nitrate reductases ofEscherichia coliare able to reduce selenate and tellurite in vitro with benzyl viologen as an electron donor. In the present study, we found that this is a general feature of denitrifiers. Both the periplasmic and membrane-bound nitrate reductases ofRalstonia eutropha, Paracoccus denitrificans, andParacoccus pantotrophuscan utilize potassium selenate and potassium tellurite as electron acceptors. In order to characterize these reactions, the periplasmic nitrate reductase ofR. sphaeroidesf. sp.denitrificansIL106 was histidine tagged and purified. TheVmaxandKmwere determined for nitrate, tellurite, and selenate. For nitrate, values of 39 μmol · min−1· mg−1and 0.12 mM were obtained forVmaxandKm, respectively, whereas theVmaxvalues for tellurite and selenate were 40- and 140-fold lower, respectively. These low activities can explain the observation that depletion of the nitrate reductase inR. sphaeroidesdoes not modify the MIC of tellurite for this organism.