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International audience; Cyanobacteria have massively contributed to carbonate deposition over the geological 27 history. They are traditionally thought to biomineralize CaCO 3 extracellularly as an 28 indirect byproduct of photosynthesis. However, the recent discovery of freshwater 29 cyanobacteria forming intracellular amorphous calcium carbonates (iACC) challenges 30 this view. Despite the geochemical interest of such a biomineralization process, its 31 molecular mechanisms and evolutionary history remain elusive. Here, using comparative 32 genomics, we identify a new gene (ccyA) and protein family (calcyanin) possibly associated 33 with cyanobacterial iACC biomineralization. Proteins of the calcyanin family are 34 composed of a conserved C-terminal domain, which likely adopts an original fold, and a 35 variable N-terminal domain whose structure allows differentiating 4 major types among 36 the 35 known calcyanin homologs. Calcyanin lacks detectable full-length homologs with 37 known function. The overexpression of ccyA in iACC-lacking cyanobacteria resulted in 38 an increased intracellular Ca content. Moreover, ccyA presence was correlated and/or co-39 localized with genes involved in Ca or HCO 3transport and homeostasis, supporting the 40 hypothesis of a functional role of calcyanin in iACC biomineralization. Whatever its 41 function, ccyA appears as diagnostic of intracellular calcification in cyanobacteria. By 42 searching for ccyA in publicly available genomes, we identified 13 additional 43 cyanobacterial strains forming iACC, as confirmed by microscopy. This extends our 44 knowledge about the phylogenetic and environmental distribution of cyanobacterial 45 iACC biomineralization, especially with the detection of multicellular genera as well as a 46 marine species. Moreover, ccyA was probably present in ancient cyanobacteria, with 47 independent losses in various lineages that resulted in a broad but patchy distribution 48 across modern cyanobacteria.

International audience; There is an emerging scientific need for reliable tools for monitoring membrane protein transport. We present a methodology leading to the reconstitution of efflux pumps from the Gram negative bacteria Pseudomonas aeruginosa in a biomimetic environment that allows for an accurate investigation of their activity of transport. Three prerequisites are fulfilled: compartmentation in a lipidic environment, use of a relevant index for transport and generation of a proton gradient. The membrane protein transporter is reconstituted into liposomes together with bacteriorhodopsin, a light-activated proton pump that generates a proton gradient that is robust as well as reversible and tunable. The activity of the protein is deduced from the pH variations occurring within the liposome, using pyranin, a pH-dependent fluorescent probe. We describe a step-by-step procedure where membrane protein purification, liposome formation, protein reconstitution and transport analysis are addressed. Although they were specifically designed for an RND transporter, the described methods could potentially be adapted for use with any other membrane protein transporter energized by a proton gradient.

In this thesis we focussed on the characterization of the reaction center (RC) protein purified from the photosynthetic bacterium Rhodobacter sphaeroides. In particular, we discussed the effects of native and artificial environment on the light-induced electron transfer processes. The native environment consist of the inner antenna LH1 complex that copurifies with the RC forming the so called core complex, and the lipid phase tightly associated with it. In parallel, we analyzed the role of saccharidic glassy matrices on the interplay between electron transfer processes and internal protein dynamics. As a different artificial matrix, we incorporated the RC protein in a layer-by-layer structure with a twofold aim: to check the behaviour of the protein in such an unusual environment and to test the response of the system to herbicides. By examining the RC in its native environment, we found that the light-induced charge separated state P+QB - is markedly stabilized (by about 40 meV) in the core complex as compared to the RC-only system over a physiological pH range. We also verified that, as compared to the average composition of the membrane, the core complex copurifies with a tightly bound lipid complement of about 90 phospholipid molecules per RC, which is strongly enriched in cardiolipin. In parallel, a large ubiquinone pool was found in association with the core complex, giving rise to a quinone concentration about ten times larger than the average one in the membrane. Moreover, this quinone pool is fully functional, i.e. it is promptly available at the QB site during multiple turnover excitation of the RC. The latter two observations suggest important heterogeneities and anisotropies in the native membranes which can in principle account for the stabilization of the charge separated state in the core complex. The thermodynamic and kinetic parameters obtained in the RC-LH1 complex are very close to those measured in intact membranes, indicating that the electron transfer properties of the RC in vivo are essentially determined by its local environment. The studies performed by incorporating the RC into saccharidic matrices evidenced the relevance of solvent-protein interactions and dynamical coupling in determining the kinetics of electron transfer processes. The usual approach when studying the interplay between internal motions and protein function consists in freezing the degrees of freedom of the protein at cryogenic temperature. We proved that the “trehalose approach” offers distinct advantages with respect to this traditional methodology. We showed, in fact, that the RC conformational dynamics, coupled to specific electron transfer processes, can be modulated by varying the hydration level of the trehalose matrix at room temperature, thus allowing to disentangle solvent from temperature effects. The comparison between different saccharidic matrices has revealed that the structural and dynamical protein-matrix coupling depends strongly upon the sugar. The analyses performed in RCs embedded in polyelectrolyte multilayers (PEM) structures have shown that the electron transfer from QA - to QB, a conformationally gated process extremely sensitive to the RC environment, can be strongly modulated by the hydration level of the matrix, confirming analogous results obtained for this electron transfer reaction in sugar matrices. We found that PEM-RCs are a very stable system, particularly suitable to study the thermodynamics and kinetics of herbicide binding to the QB site. These features make PEM-RC structures quite promising in the development of herbicide biosensors. The studies discussed in the present thesis have shown that, although the effects on electron transfer induced by the native and artificial environments tested are markedly different, they can be described on the basis of a common kinetic model which takes into account the static conformational heterogeneity of the RC and the interconversion between conformational substates. Interestingly, the same distribution of rate constants (i.e. a Gamma distribution function) can describe charge recombination processes in solutions of purified RC, in RC-LH1 complexes, in wet and dry RC-PEM structures and in glassy saccharidic matrices over a wide range of hydration levels. In conclusion, the results obtained for RCs in different physico-chemical environments emphasize the relevance of the structure/dynamics solvent/protein coupling in determining the energetics and the kinetics of electron transfer processes in a membrane protein complex.

The core complex formed by the reaction center and the light harvesting complex 1 (RC-LH1) was purified from the photosynthetic bacterium Rhodobacter sphaeroides. We analyzed the lipid and ubiquinone (UQ) complements copurifying with the RC-LH1 complex and with the peripheral antenna (LH2). ln RC-LH1 UQ was almost ten times more concentrated than in the LH2 and in the native membranes from which the complexes were extracted. The fractional lipid composition of the RC-LH1 complex also differed from that of lhe intact membranes, exhibiting a marked increase in cardiolipin concentration. We propose that the confinement of cardiolipin and ubiquinone observed within the RC-LH1 complex, plays a role in vivo in lhe stabilization of the light-induced charge separation catalyzed by the RC.

We have recently shown that in the reaction center – light harvesting complex 1 (RC-LH1) purified from the photosynthetic bacterium Rhodobacter sphaeroides, at pH=7.8, flash-induced P+QB- recombines with an average rate constant ( = 0.3 s-1), significantly smaller than that measured in RC deprived of the LH1 ( = 1 s-1). Since the kinetics of P+QA- recombination is unaffected by the presence of the antenna, the P+QB- state appears to be energetically stabilized in core complexes (1). The pH dependence of the P+QB- recombination kinetics has been analyzed in dimeric and monomeric forms of RC-LH1 and compared with that observed in RC deprived of the antenna. At 6.5 < pH < 8.5 recombination is essentially pH independent and significantly slower in all forms of core complexes as compared to LH1-deprived RCs. At increasing pH values, however, where the recombination rate increases, this stabilization effect decreases progressively and vanishes at pH > 10.5, indicating the involvement of protonatable groups. The recombination kinetics, moreover, becomes progressively distributed at pH > 9. The width of the rate constant distribution (sigma = 0.3 s-1 at pH < 9.0) increases by more than one order of magnitude at pH 11.0, suggesting a variety of conformations, possibly differing in the protonation state. The observed pH dependence of sigma could be explained when assuming that such conformations interconvert at a rate which is comparable to the rate of charge recombination at physiological pH values but is considerably lower at high pH values. Under this condition the conformational heterogeneity becomes therefore observable. A similar behaviour was observed in chromatophores of Rhodobacter capsulatus FJ2, a c2- and cyminus strain, in which the kinetics of P+QB- recombination could be accurately studied by avoiding any interference due to exogenous electron donors/acceptors. The lipid complement of the examined RC-LH1 complexes, determined by Inductively Coupled Plasma Emission Spectroscopy of phosphorous, ranges between 200 and 400 phospolipid molecules per RC. A large ubiquinone (UQ) pool, varying from 15 to 30 UQ molecules per RC was systematically found to be associated with the core complexes. When similar determinations are performed in chromatophores, it appears that the effective UQ concentration in the lipid phase of core complexes is at least three times higher than the average UQ concentration in the intact membrane. This finding argues strongly in favour of an in vivo heterogeneity in the distribution of the quinone pool within the chromatophore bilayer.

Dimeric photosynthetic reaction center-light harvesting complex (RC-LH1) has been purified from the photosynthetic bacterium Rhodobacter sphaeroides grown under semiaerobic conditions. Removal of the detergent in the presence of lipids leads to the formation of two-dimensional crystals. Analysis by cryoelectron mycroscopy at a resolution of 26 A ° reveals an ‘‘S’’-shaped dimeric complex where the continuity of the LH1 ring that surrounds the RC is interrupted. The higher density of the projection map at the junction between the two monomers of core complex is attributed to a dimer of the PufX peptide (Scheuring et al., 2004, J. Biol. Chem. 279, 3620). These data confirm the structural role of PufX, a single transmembrane protein required for the photosynthetic phenotype (Farchaus et al., 1992, EMBO J., 11, 2779), responsible for the dimerization of the RC-LH1 complex (Francia et al., 1999, Biochemistry 38, 6834).The functionality of the isolated complex, purified from photosynthetically and semiaerobically grown bacteria, was analyzed by time resolved spectroscopy. Upon excitation of the sample with an actinic laser pulse, the kinetics of charge recombination from the state P +QAQB- to the neutral state PQAQB exibhit a slow phase with an half time of approximately 4 s, at least four times larger than what usually observed in RC complex deprived of the LH1. Stoichiometric determinations of the quinone (Q10) present in the RCLH1 indicate a Q10/RC-LH1 ratio >10. These quinones are functionally coupled to the RC-LH1 complex, as judged from the extent of cytochrome c2 rapidly oxidized under continuous illumination. Charge recombination kinetics have been analyzed on the basis of a model proposed by Shinkarev and Wraight (Shinkarev and Wraight, 1993, in ‘‘The photosynthetic reaction center’’ Vol. 1, 193) that take into account the binding of quinone at the QB site when a quinone pool is present. The slowing down of the recombination reaction experimentally detected cannot be simply explained by a quinone concentration effect. The model predicts a lower limit of the charge recombination rate constant when the quinone concentration is raised to infinity that is well above the one measured in the RC-LH1, indicating that, even in the presence of saturating quinone conditions the recombination reaction cannot be so slow as experimentally observed. These data suggests that a stabilization of the charge separated state P +QAQB-, leading to a slower recombination reaction, is induced by the LH1 antenna complex.