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An autotrophic bacterium able to gain energy from the oxidation of arsenite was isolated from arsenite-containing acid mine drainage waters. It belongs to the genus Thiomonas as shown by DNA-DNA hybridization experiments, 16S rRNA gene sequence, quinone and fatty acid content analyses. Carboxysomes were observed and the cbbSL genes encoding the ribulose 1,5-bisphosphate carboxylase/oxygenase were detected, confirming that this bacterium is able to fix CO(2). Arsenite oxidation was catalysed by a membrane-bound enzyme, and this activity was detected essentially in cells grown in the presence of arsenite. The genes encoding the two subunits of the arsenite oxidase of the Thiomonas isolate have been sequenced. The small subunit has a characteristic Tat signal sequence and contains the residues binding the [2Fe-2S] Rieske-type cluster. The large subunit has the [3Fe-4S] cluster-binding motif as well as the residues proposed to bind arsenite. In addition, most of the residues interacting with the molybdenum cofactor are conserved. The genes encoding both subunits belong to an operon, likely with a gene encoding a cytochrome c. The expression of this operon is greater in cells grown in the presence than in the absence of arsenite, in agreement with a transcriptional regulation in the presence of this metalloid.

Weathering of the As-rich pyrite-rich tailings of the abandoned mining site of Carnoulès (southeastern France) results in the formation of acid waters heavily loaded with arsenic. Dissolved arsenic present in the seepage waters precipitates within a few meters from the bottom of the tailing dam in the presence of microorganisms. An Acidithiobacillus ferrooxidans strain, referred to as CC1, was isolated from the effluents. This strain was able to remove arsenic from a defined synthetic medium only when grown on ferrous iron. This A. ferrooxidans strain did not oxidize arsenite to arsenate directly or indirectly. Strain CC1 precipitated arsenic unexpectedly as arsenite but not arsenate, with ferric iron produced by its energy metabolism. Furthermore, arsenite was almost not found adsorbed on jarosite but associated with a poorly ordered schwertmannite. Arsenate is known to efficiently precipitate with ferric iron and sulfate in the form of more or less ordered schwertmannite, depending on the sulfur-to-arsenic ratio. Our data demonstrate that the coprecipitation of arsenite with schwertmannite also appears as a potential mechanism of arsenite removal in heavily contaminated acid waters. The removal of arsenite by coprecipitation with ferric iron appears to be a common property of the A. ferrooxidans species, as such a feature was observed with one private and three collection strains, one of which was the type strain.Weathering of sulfide-rich rocks results in the formation of highly acidic and heavy metal-laden effluents. At the abandoned Pb-Zn mining site of Carnoulès (southeastern France), the pyrite-rich tailings are subject to bioleaching. Consequently, the Reigous spring, which collects the seepage waters from the waste materials, is acid (pH 3) and contains high levels of solubilized metals (Fe, Zn, and Pb) but also extremely high arsenic (As) concentrations (250 mg liter Ϫ1 on average) (26, 27). The extremely high contents of this metalloid are lowered by 2 to 3 orders of magnitude between the bottom of the tailing dam and few hundred meters downstream. The presence in the sediments of bacteria coated with Fe-As-rich material suggested that arsenic attenuation was due to precipitation mechanisms mediated by microorganisms (24,25,26,27). Among them, Acidithiobacillus ferrooxidans was proposed to be involved in the removal of soluble arsenic (9,26,27,35). The role of microorganisms in arsenic attenuation in this ecosystem was evaluated by two different approaches: (i) an A. ferrooxidans strain was isolated from the effluent and its role in arsenic oxidation and/or precipitation was determined (this paper); (ii) arsenite oxidizing bacteria, one of which belongs to the Thiomonas genus, were isolated and characterized (K. Duquesne, A. Yarzabal, J. Ratouchniak, D. Muller, D. Lièvre-mont, M-C. Lett, and V. Bonnefoy, unpublished results).A. ferrooxidans is an acidophilic chemolithoautotrophic gram-negative bacterium commonly encountered in acid mine drainage. This bacterium is resistant to heavy metals and metallo...

SummaryThe outer membrane of Gram-negative bacteria protects the cell against bactericidal substances. Passage of nutrients and waste is assured by outer membrane porins, beta-barrel transmembrane channels. While atomic structures of several porins have been solved, so far little is known on the supramolecular structure of the outer membrane. Here we present the first high-resolution view of a bacterial outer membrane gently purified maintaining remnants of peptidoglycan on the perisplasmic surface. Atomic force microscope images of outer membrane fragments of the size of~50% of the bacterial envelope revealed that outer membrane porins are by far more densely packed than previously assumed. Indeed the outer membrane is a molecular sieve rather than a membrane. Porins cover~70% of the membrane surface and form locally regular lattices. The potential role of exposed aromatic residues in the formation of the supramolecular assembly is discussed. Finally, we present first structural data of the outer membrane porin from the marine Gramnegative bacteria Roseobacter denitrificans, and we perform a sequence alignment with porins of known structure.

Baeyer–Villiger monooxygenases (BVMOs) catalyze the oxidation of ketones to lactones under very mild reaction conditions. This enzymatic route is hindered by the requirement of a stoichiometric supply of auxiliary substrates for cofactor recycling and difficulties with supplying the necessary oxygen. The recombinant production of BVMO in cyanobacteria allows the substitution of auxiliary organic cosubstrates with water as an electron donor and the utilization of oxygen generated by photosynthetic water splitting. Herein, we report the identification of a BVMO from Burkholderia xenovorans (BVMO Xeno ) that exhibits higher reaction rates in comparison to currently identified BVMOs. We report a 10-fold increase in specific activity in comparison to cyclohexanone monooxygenase (CHMO Acineto ) in Synechocystis sp. PCC 6803 (25 vs 2.3 U g DCW –1 at an optical density of OD 750 = 10) and an initial rate of 3.7 ± 0.2 mM h –1 . While the cells containing CHMO Acineto showed a considerable reduction of cyclohexanone to cyclohexanol, this unwanted side reaction was almost completely suppressed for BVMO Xeno , which was attributed to the much faster lactone formation and a 10-fold lower K M value of BVMO Xeno toward cyclohexanone. Furthermore, the whole-cell catalyst showed outstanding stereoselectivity. These results show that, despite the self-shading of the cells, high specific activities can be obtained at elevated cell densities and even further increased through manipulation of the photosynthetic electron transport chain (PETC). The obtained rates of up to 3.7 mM h –1 underline the usefulness of oxygenic cyanobacteria as a chassis for enzymatic oxidation reactions. The photosynthetic oxygen evolution can contribute to alleviating the highly problematic oxygen mass-transfer limitation of oxygen-dependent enzymatic processes.

Interaction forces of membrane protein subunits are of importance in their structure, assembly, membrane insertion, and function. In biological membranes, and in the photosynthetic apparatus as a paradigm, membrane proteins fulfill their function by ensemble actions integrating a tight assembly of several proteins. In the bacterial photosynthetic apparatus light-harvesting complexes 2 (LH2) transfer light energy to neighboring tightly associated core complexes, constituted of light-harvesting complexes 1 (LH1) and reaction centers (RC). While the architecture of the photosynthetic unit has been described, the forces and energies assuring the structural and functional integrity of LH2, the assembly of LH2 complexes, and how LH2 interact with the other proteins in the supramolecular architecture are still unknown. Here we investigate the molecular forces of the bacterial LH2 within the native photosynthetic membrane using atomic force microscopy single-molecule imaging and force measurement in combination. The binding between LH2 subunits is fairly weak, of the order of k B T , indicating the importance of LH2 ring architecture. In contrast LH2 subunits are solid with a free energy difference of 90 k B T between folded and unfolded states. Subunit α-helices unfold either in one-step, α-and β-polypeptides unfold together, or sequentially. The unfolding force of transmembrane helices is approximately 150 pN. In the two-step unfolding process, the β-polypeptide is stabilized by the molecular environment in the membrane. Hence, intermolecular forces influence the structural and functional integrity of LH2.atomic force microscopy | photosynthesis | protein unfolding | membrane protein assembly M embrane protein function is intimately linked and influenced by its structural integrity and molecular environment (1-3). Photosynthesis is a paradigm of such orchestrated molecular action, for which the implicated proteins have evolved to form supramolecular assemblies (4). Knowledge of supramolecular architecture and interaction forces and energies are indispensable for a complete understanding of a membrane protein structure and function.The atomic force microscope (AFM) (5) can be used as a high-resolution imaging (6) and as a force-measurement tool (7). Most elegantly the two applications were combined allowing the attribution of structural changes to force events (8). More recently, the AFM has proven the unique tool for studying the supramolecular assembly of the bacterial photosynthetic unit (9-12) and has provided a solid basis for the understanding of the ensemble function of the photosynthetic proteins (4, 13-15).The light-harvesting process in photosynthetic bacteria starts with photon capture at the peripheral light-harvesting complex 2 (LH2) that transfers the absorbed excitation energy to the light-harvesting complex 1 (LH1) associated with the reaction center (RC). The LH2 structure is well described by X-ray crystallography (16, 17) and AFM in native membranes (12,(18)(19)(20). Typically, the LH2 complexes appe...

We have investigated the adaptation of the light-harvesting system of the photosynthetic bacterium Phaeospirillum molischianum (DSM120) to very low light conditions. This strain is able to respond to changing light conditions by differentially modulating the expression of a family of puc operons that encode for peripheral light-harvesting complex (LH2) polypeptides. This modulation can result in a complete shift between the production of LH2 complexes absorbing maximally near 850 nm to those absorbing near 820 nm. In contradiction to prevailing wisdom, analysis of the LH2 rings found in the photosynthetic membranes during light adaptation are shown to have intermediate spectral and electrostatic properties. By chemical cross-linking and mass-spectrometry we show that individual LH2 rings and subunits can contain a mixture of polypeptides derived from the different operons. These observations show that polypeptide synthesis and insertion into the membrane are not strongly coupled to LH2 assembly. We show that the light-harvesting complexes resulting from this mixing could be important in maintaining photosynthetic efficiency during adaptation.chromatic adaptation | membrane protein | photosynthetic bacteria | Rhodospirillum molischianum P hotosynthetic bacteria are able to efficiently convert incident light into chemical potential energy via a light-driven cyclic electron transfer pathway (1). This process depends on the efficient absorption of incident light energy and the transfer of this energy to the reaction center. Measurements of the quantum efficiency of the light-harvesting apparatus indicate that very little energy is lost during the migration of the exciton from the point of initial absorption to the reaction center (1). The light-harvesting system of many bacteria contains two different pigment-protein complexes. The core complex contains one or two reaction centers in close association with a light-harvesting system (LH1) and a smaller peripheral complex (LH2) that transfers the absorbed excitation energy to the core complex. Both types of light-harvesting complex, LH1 and LH2, are constructed from oligomers of a similar basic subunit containing 2 polypeptides (α and β) with associated bacteriochlorophyll and carotenoid pigments. Their structures are known from x-ray crystallography (2-4), electron crystallography (5), and more recently from atomic force microscopy (6-11).The LH2 complexes are circular oligomers of typically 9 αβ subunits (3), in which 18 long wavelength-absorbing bacteriochlorophyll molecules are sandwiched between the inner ring of α-polypeptides and an outer-ring of β-polypeptides. A second series of 9 bacteriochlorohpyll molecules, closer to the cytoplasmic membrane surface, occupies gaps between the β-polypeptides. This general architecture is slightly variable, with in particular the number of monomeric units forming the circular architecture depending on the species and environmental conditions (2,7,12,13). In the species studied in this work, Phaeospirillum (Ph.) molischianum...

A critical step in any in vitro analysis of membrane proteins is the solubilization of the membrane to extract the protein of interest in an active form to obtain an aqueous solution containing the membrane protein complexed with detergents and lipids in a form suitable for purification and further analysis. This process is particularly delicate as the aim is to maximally disrupt the lipid components of the membrane while putting the protein components in an un-natural detergent environment without perturbing them. Looked at this way, it is remarkable that it ever works. Although the process is difficult and hard to master, an increasing number of membrane proteins have been successfully solubilized in active forms, allowing some general principles to be established that we illustrate in the method developed in this chapter.