Visualization of Bacterial Microcompartment Facet Assembly Using High-Speed Atomic Force Microscopy

Bacterial microcompartments (BMCs) are proteinaceous organelles widespread among bacterial phyla. They compartmentalize enzymes within a selectively permeable shell and play important roles in CO2 fixation, pathogenesis, and microbial ecology. Here, we combine X-ray crystallography and high-speed atomic force microscopy to characterize, at molecular resolution, the structure and dynamics of BMC shell facet assembly. Our results show that preformed hexamers assemble into uniformly oriented shell layers, a single hexamer thick. We also observe the dynamic process of shell facet assembly. Shell hexamers can dissociate from and incorporate into assembled sheets, indicating a flexible intermolecular interaction. Furthermore, we demonstrate that the self-assembly and dynamics of shell proteins are governed by specific contacts at the interfaces of shell proteins. Our study provides novel insights into the formation, interactions, and dynamics of BMC shell facets, which are essential for the design and engineering of self-assembled biological nanoreactors and scaffolds based on BMC architectures

Discovery of anaerobic lithoheterotrophic haloarchaea, ubiquitous in hypersaline habitats

Hypersaline anoxic habitats harbour numerous novel uncultured archaea whose metabolic and ecological roles remain to be elucidated. Until recently, it was believed that energy generation via dissimilatory reduction of sulfur compounds is not functional at salt saturation conditions. Recent discovery of the strictly anaerobic acetotrophic Halanaeroarchaeum compels to change both this assumption and the traditional view on haloarchaea as aerobic heterotrophs. Here we report on isolation and characterization of a novel group of strictly anaerobic lithoheterotrophic haloarchaea, which we propose to classify as a new genus Halodesulfurarchaeum. Members of this previously unknown physiological group are capable of utilising formate or hydrogen as electron donors and elemental sulfur, thiosulfate or dimethylsulfoxide as electron acceptors. Using genome-wide proteomic analysis we have detected the full set of enzymes required for anaerobic respiration and analysed their substrate-specific expression. Such advanced metabolic plasticity and type of respiration, never seen before in haloarchaea, empower the wide distribution of Halodesulfurarchaeum in hypersaline inland lakes, solar salterns, lagoons and deep submarine anoxic brines. The discovery of this novel functional group of sulfur-respiring haloarchaea strengthens the evidence of their possible role in biogeochemical sulfur cycling linked to the terminal anaerobic carbon mineralisation in so far overlooked hypersaline anoxic habitats.</p

Bacterial microcompartments

Bacterial microcompartments (BMCs) are self-assembling organelles that consist of an enzymatic core that is encapsulated by a selectively permeable protein shell. The potential to form BMCs is widespread and found across the kingdom Bacteria. BMCs have crucial roles in carbon dioxide fixation in autotrophs and the catabolism of organic substrates in heterotrophs. They contribute to the metabolic versatility of bacteria, providing a competitive advantage in specific environmental niches. Although BMCs were first visualized more than 60 years ago, it is mainly in the past decade that progress has been made in understanding their metabolic diversity and the structural basis of their assembly and function. This progress has not only heightened our understanding of their role in microbial metabolism but is also beginning to enable their use in a variety of applications in synthetic biology. In this Review, we focus on recent insights into the structure, assembly, diversity and function of BMCs

Visualization of Bacterial Microcompartment Facet Assembly Using High-Speed Atomic Force Microscopy.

Bacterial microcompartments (BMCs) are proteinaceous organelles widespread among bacterial phyla. They compartmentalize enzymes within a selectively permeable shell and play important roles in CO2 fixation, pathogenesis, and microbial ecology. Here, we combine X-ray crystallography and high-speed atomic force microscopy to characterize, at molecular resolution, the structure and dynamics of BMC shell facet assembly. Our results show that preformed hexamers assemble into uniformly oriented shell layers, a single hexamer thick. We also observe the dynamic process of shell facet assembly. Shell hexamers can dissociate from and incorporate into assembled sheets, indicating a flexible intermolecular interaction. Furthermore, we demonstrate that the self-assembly and dynamics of shell proteins are governed by specific contacts at the interfaces of shell proteins. Our study provides novel insights into the formation, interactions, and dynamics of BMC shell facets, which are essential for the design and engineering of self-assembled biological nanoreactors and scaffolds based on BMC architectures

Desulfovibrio piezophilus sp nov., a piezophilic, sulfate-reducing bacterium isolated from wood falls in the Mediterranean Sea

A novel sulfate-reducing bacterium, designated C1TLV30(T), was isolated from wood falls at a depth of 1693 m in the Mediterranean Sea. Cells were motile vibrios (2-4x0.5 mu m). Strain C1TLV30(T) grew at temperatures between 15 and 45 degrees C (optimum 30 degrees C) and at pH 5.4-8.6 (optimum 7.3). It required NaCl for growth (optimum at 25 g NaCl l(-1)) and tolerated up to 80 g NaCl l(-1). Strain C1TLV30(T) used as energy sources: lactate, fumarate, formate, malate, pyruvate and ethanol. The end products from lactate oxidation were acetate, H(2)S and CO(2) in the presence of sulfate as terminal electron acceptor. Besides sulfate, thiosulfate and sulfite were also used as terminal electron acceptors, but not elemental sulfur, fumarate, nitrate or nitrite. Strain C1TLV30(T) possessed desulfoviridin and was piezophilic, growing optimally at 10 MPa (range 0-30 MPa). The membrane lipid composition of this strain was examined to reveal an increase in fatty acid chain lengths at high hydrostatic pressures. The G+C content of the genomic DNA was 49.6% and the genome size was estimated at 3.5 +/- 0.5 Mb. Phylogenetic analysis of the SSU rRNA gene sequence indicated that strain C1TLV30(T) was affiliated to the genus Desulfovibrio with Desulfovibrio pro fundus being its closest phylogenetic relative (similarity of 96.4%). On the basis of SSU rRNA gene sequence comparisons and physiological characteristics, strain C1TLV30(T) (=DSM 21447(T) =JCM 1548(T)) is proposed to be assigned to a novel species of the genus Desulfovibrio, Desulfovibrio piezophilus sp. nov

Structure and Function of a Bacterial Microcompartment Shell Protein Engineered to Bind a [4Fe-4S] Cluster

Bacterial microcompartments (BMCs) are self-assembling organelles composed of a selectively permeable protein shell and encapsulated enzymes. They are considered promising templates for the engineering of designed bionanoreactors for biotechnology. In particular, encapsulation of oxidoreductive reactions requiring electron transfer between the lumen of the BMC and the cytosol relies on the ability to conduct electrons across the shell. We determined the crystal structure of a component protein of a synthetic BMC shell, which informed the rational design of a [4Fe-4S] cluster-binding site in its pore. We also solved the structure of the [4Fe-4S] cluster-bound, engineered protein to 1.8 Å resolution, providing the first structure of a BMC shell protein containing a metal center. The [4Fe-4S] cluster was characterized by optical and EPR spectroscopies; it has a reduction potential of −370 mV vs the standard hydrogen electrode (SHE) and is stable through redox cycling. This remarkable stability may be attributable to the hydrogen-bonding network provided by the main chain of the protein scaffold. The properties of the [4Fe-4S] cluster resemble those in low-potential bacterial ferredoxins, while its ligation to three cysteine residues is reminiscent of enzymes such as aconitase and radical <i>S</i>-adenosymethionine (SAM) enzymes. This engineered shell protein provides the foundation for conferring electron-transfer functionality to BMC shells

Structure and Function of a Bacterial Microcompartment Shell Protein Engineered to Bind a [4Fe-4S] Cluster

Bacterial microcompartments (BMCs) are self-assembling organelles composed of a selectively permeable protein shell and encapsulated enzymes. They are considered promising templates for the engineering of designed bionanoreactors for biotechnology. In particular, encapsulation of oxidoreductive reactions requiring electron transfer between the lumen of the BMC and the cytosol relies on the ability to conduct electrons across the shell. We determined the crystal structure of a component protein of a synthetic BMC shell, which informed the rational design of a [4Fe-4S] cluster-binding site in its pore. We also solved the structure of the [4Fe-4S] cluster-bound, engineered protein to 1.8 Å resolution, providing the first structure of a BMC shell protein containing a metal center. The [4Fe-4S] cluster was characterized by optical and EPR spectroscopies; it has a reduction potential of −370 mV vs the standard hydrogen electrode (SHE) and is stable through redox cycling. This remarkable stability may be attributable to the hydrogen-bonding network provided by the main chain of the protein scaffold. The properties of the [4Fe-4S] cluster resemble those in low-potential bacterial ferredoxins, while its ligation to three cysteine residues is reminiscent of enzymes such as aconitase and radical <i>S</i>-adenosymethionine (SAM) enzymes. This engineered shell protein provides the foundation for conferring electron-transfer functionality to BMC shells