Producing fast and active Rubisco in tobacco to enhance photosynthesis.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) performs most of the carbon fixation on Earth. However, plant Rubisco is an intrinsically inefficient enzyme given its low carboxylation rate, representing a major limitation to photosynthesis. Replacing endogenous plant Rubisco with a faster Rubisco is anticipated to enhance crop photosynthesis and productivity. However, the requirement of chaperones for Rubisco expression and assembly has obstructed the efficient production of functional foreign Rubisco in chloroplasts. Here, we report the engineering of a Form 1A Rubisco from the proteobacterium Halothiobacillus neapolitanus in Escherichia coli and tobacco (Nicotiana tabacum) chloroplasts without any cognate chaperones. The native tobacco gene encoding Rubisco large subunit was genetically replaced with H. neapolitanus Rubisco (HnRubisco) large and small subunit genes. We show that HnRubisco subunits can form functional L8S8 hexadecamers in tobacco chloroplasts at high efficiency, accounting for ∼40% of the wild-type tobacco Rubisco content. The chloroplast-expressed HnRubisco displayed a ∼2-fold greater carboxylation rate and supported a similar autotrophic growth rate of transgenic plants to that of wild-type in air supplemented with 1% CO2. This study represents a step toward the engineering of a fast and highly active Rubisco in chloroplasts to improve crop photosynthesis and growth.

Structural basis and evolution of the photosystem I-light-harvesting supercomplex of cryptophyte algae.

Cryptophyte plastids originated from a red algal ancestor through secondary endosymbiosis. Cryptophyte photosystem I (PSI) associates with transmembrane alloxanthin-chlorophyll a/c proteins (ACPIs) as light-harvesting complexes (LHCs). Here, we report the structure of the photosynthetic PSI-ACPI supercomplex from the cryptophyte Chroomonas placoidea at 2.7-Å resolution obtained by crygenic electron microscopy. Cryptophyte PSI-ACPI represents a unique PSI-LHCI intermediate in the evolution from red algal to diatom PSI-LHCI. The PSI-ACPI supercomplex is composed of a monomeric PSI core containing 14 subunits, 12 of which originated in red algae, 1 diatom PsaR homolog, and an additional peptide. The PSI core is surrounded by 14 ACPI subunits that form 2 antenna layers: an inner layer with 11 ACPIs surrounding the PSI core and an outer layer containing 3 ACPIs. A pigment-binding subunit that is not present in any other previously characterized PSI-LHCI complexes, ACPI-S, mediates the association and energy transfer between the outer and inner ACPIs. The extensive pigment network of PSI-ACPI ensures efficient light harvesting, energy transfer, and dissipation. Overall, the PSI-LHCI structure identified in this study provides a framework for delineating the mechanisms of energy transfer in cryptophyte PSI-LHCI and for understanding the evolution of photosynthesis in the red lineage, which occurred via secondary endosymbiosis.

Molecular interactions of the chaperone CcmS and carboxysome shell protein CcmK1 that mediate ß-carboxysome assembly.

The carboxysome is a natural proteinaceous organelle for carbon fixation in cyanobacteria and chemoautotrophs. It comprises hundreds of protein homologs that self-assemble to form a polyhedral shell structure to sequester cargo enzymes, ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and carbonic anhydrases. How these protein components assemble to construct a functional carboxysome is a central question in not only understanding carboxysome structure and function but also synthetic engineering of carboxysomes for biotechnological applications. Here, we determined the structure of the chaperone protein CcmS, which has recently been identified to be involved in ß-carboxysome assembly, and its interactions with ß-carboxysome proteins. The crystal structure at 1.99 Å resolution reveals CcmS from Nostoc sp. PCC 7120 forms a homodimer, and each CcmS monomer consists of five α-helices and four ß-sheets. Biochemical assays indicate that CcmS specifically interacts with the C-terminal extension of the carboxysome shell protein CcmK1, but not the shell protein homolog CcmK2 or the carboxysome scaffolding protein CcmM. Moreover, we solved the structure of a stable complex of CcmS and the C-terminus of CcmK1 at 1.67 Å resolution and unveiled how the CcmS dimer interacts with the C-terminus of CcmK1. These findings allowed us to propose a model to illustrate CcmS-mediated ß-carboxysome assembly by interacting with CcmK1 at the outer shell surface. Collectively, our study provides detailed insights into the accessory factors that drive and regulate carboxysome assembly, thereby improving our knowledge of carboxysome structure, function, and bioengineering.

Membrane Dynamics in Phototrophic Bacteria.

Photosynthetic membranes are typically densely packed with proteins, and this is crucial for their function in efficient trapping of light energy. Despite being crowded with protein, the membranes are fluid systems in which proteins and smaller molecules can diffuse. Fluidity is also crucial for photosynthetic function, as it is essential for biogenesis, electron transport, and protein redistribution for functional regulation. All photosynthetic membranes seem to maintain a delicate balance between crowding, order, and fluidity. How does this work in phototrophic bacteria? In this review, we focus on two types of intensively studied bacterial photosynthetic membranes: the chromatophore membranes of purple bacteria and the thylakoid membranes of cyanobacteria. Both systems are distinct from the plasma membrane, and both have a distinctive protein composition that reflects their specialized roles. Chromatophores are formed from plasma membrane invaginations, while thylakoid membranes appear to be an independent intracellular membrane system. We discuss the techniques that can be applied to study the organization and dynamics of these membrane systems, including electron microscopy techniques, atomic force microscopy, and many variants of fluorescence microscopy. We go on to discuss the insights that havebeen acquired from these techniques, and the role of membrane dynamics in the physiology of photosynthetic membranes. Membrane dynamics on multiple timescales are crucial for membrane function, from electron transport on timescales of microseconds to milliseconds to regulation and biogenesis on timescales of minutes to hours. We emphasize the open questions that remain in the field.

Molecular mechanism underlying transport and allosteric inhibition of bicarbonate transporter SbtA.

SbtA is a high-affinity, sodium-dependent bicarbonate transporter found in the cyanobacterial CO2-concentrating mechanism (CCM). SbtA forms a complex with SbtB, while SbtB allosterically regulates the transport activity of SbtA by binding with adenyl nucleotides. The underlying mechanism of transport and regulation of SbtA is largely unknown. In this study, we report the three-dimensional structures of the cyanobacterial Synechocystis sp. PCC 6803 SbtA-SbtB complex in both the presence and absence of HCO3- and/or AMP at 2.7 Å and 3.2 Å resolution. An analysis of the inward-facing state of the SbtA structure reveals the HCO3-/Na+ binding site, providing evidence for the functional unit as a trimer. A structural comparison found that SbtA adopts an elevator mechanism for bicarbonate transport. A structure-based analysis revealed that the allosteric inhibition of SbtA by SbtB occurs mainly through the T-loop of SbtB, which binds to both the core domain and the scaffold domain of SbtA and locks it in an inward-facing state. T-loop conformation is stabilized by the AMP molecules binding at the SbtB trimer interfaces and may be adjusted by other adenyl nucleotides. The unique regulatory mechanism of SbtA by SbtB makes it important to study inorganic carbon uptake systems in CCM, which can be used to modify photosynthesis in crops.

Native architecture and acclimation of photosynthetic membranes in a fast-growing cyanobacterium.

Efficient solar energy conversion is ensured by the organization, physical association, and physiological coordination of various protein complexes in photosynthetic membranes. Here, we visualize the native architecture and interactions of photosynthetic complexes within the thylakoid membranes from a fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 (Syn2973) using high-resolution atomic force microscopy. In the Syn2973 thylakoid membranes, both photosystem I (PSI)-enriched domains and crystalline photosystem II (PSII) dimer arrays were observed, providing favorable membrane environments for photosynthetic electron transport. The high light (HL)-adapted thylakoid membranes accommodated a large amount of PSI complexes, without the incorporation of iron-stress-induced protein A (IsiA) assemblies and formation of IsiA-PSI supercomplexes. In the iron deficiency (Fe-)-treated thylakoid membranes, in contrast, IsiA proteins densely associated with PSI, forming the IsiA-PSI supercomplexes with varying assembly structures. Moreover, type-I NADH dehydrogenase-like complexes (NDH-1) were upregulated under the HL and Fe- conditions and established close association with PSI complexes to facilitate cyclic electron transport. Our study provides insight into the structural heterogeneity and plasticity of the photosynthetic apparatus in the context of their native membranes in Syn2973 under environmental stress. Advanced understanding of the photosynthetic membrane organization and adaptation will provide a framework for uncovering the molecular mechanisms of efficient light harvesting and energy conversion.

Rubisco accumulation factor 1 (Raf1) plays essential roles in mediating Rubisco assembly and carboxysome biogenesis.

Carboxysomes are membrane-free organelles for carbon assimilation in cyanobacteria. The carboxysome consists of a proteinaceous shell that structurally resembles virus capsids and internal enzymes including ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), the primary carbon-fixing enzyme in photosynthesis. The formation of carboxysomes requires hierarchical self-assembly of thousands of protein subunits, initiated from Rubisco assembly and packaging to shell encapsulation. Here we study the role of Rubisco assembly factor 1 (Raf1) in Rubisco assembly and carboxysome formation in a model cyanobacterium, Synechococcus elongatus PCC7942 (Syn7942). Cryo-electron microscopy reveals that Raf1 facilitates Rubisco assembly by mediating RbcL dimer formation and dimer-dimer interactions. Syn7942 cells lacking Raf1 are unable to form canonical intact carboxysomes but generate a large number of intermediate assemblies comprising Rubisco, CcaA, CcmM, and CcmN without shell encapsulation and a low abundance of carboxysome-like structures with reduced dimensions and irregular shell shapes and internal organization. As a consequence, the Raf1-depleted cells exhibit reduced Rubisco content, CO2-fixing activity, and cell growth. Our results provide mechanistic insight into the chaperone-assisted Rubisco assembly and biogenesis of carboxysomes. Advanced understanding of the biogenesis and stepwise formation process of the biogeochemically important organelle may inform strategies for heterologous engineering of functional CO2-fixing modules to improve photosynthesis.

Single-Organelle Quantification Reveals Stoichiometric and Structural Variability of Carboxysomes Dependent on the Environment.

The carboxysome is a complex, proteinaceous organelle that plays essential roles in carbon assimilation in cyanobacteria and chemoautotrophs. It comprises hundreds of protein homologs that self-assemble in space to form an icosahedral structure. Despite its significance in enhancing CO2 fixation and potentials in bioengineering applications, the formation of carboxysomes and their structural composition, stoichiometry, and adaptation to cope with environmental changes remain unclear. Here we use live-cell single-molecule fluorescence microscopy, coupled with confocal and electron microscopy, to decipher the absolute protein stoichiometry and organizational variability of single ß-carboxysomes in the model cyanobacterium Synechococcus elongatus PCC7942. We determine the physiological abundance of individual building blocks within the icosahedral carboxysome. We further find that the protein stoichiometry, diameter, localization, and mobility patterns of carboxysomes in cells depend sensitively on the microenvironmental levels of CO2 and light intensity during cell growth, revealing cellular strategies of dynamic regulation. These findings, also applicable to other bacterial microcompartments and macromolecular self-assembling systems, advance our knowledge of the principles that mediate carboxysome formation and structural modulation. It will empower rational design and construction of entire functional metabolic factories in heterologous organisms, for example crop plants, to boost photosynthesis and agricultural productivity.

Probing the Internal pH and Permeability of a Carboxysome Shell.

The carboxysome is a protein-based nanoscale organelle in cyanobacteria and many proteobacteria, which encapsulates the key CO2-fixing enzymes ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and carbonic anhydrase (CA) within a polyhedral protein shell. The intrinsic self-assembly and architectural features of carboxysomes and the semipermeability of the protein shell provide the foundation for the accumulation of CO2 within carboxysomes and enhanced carboxylation. Here, we develop an approach to determine the interior pH conditions and inorganic carbon accumulation within an α-carboxysome shell derived from a chemoautotrophic proteobacterium Halothiobacillus neapolitanus and evaluate the shell permeability. By incorporating a pH reporter, pHluorin2, within empty α-carboxysome shells produced in Escherichia coli, we probe the interior pH of the protein shells with and without CA. Our in vivo and in vitro results demonstrate a lower interior pH of α-carboxysome shells than the cytoplasmic pH and buffer pH, as well as the modulation of the interior pH in response to changes in external environments, indicating the shell permeability to bicarbonate ions and protons. We further determine the saturated HCO3- concentration of 15 mM within α-carboxysome shells and show the CA-mediated increase in the interior CO2 level. Uncovering the interior physiochemical microenvironment of carboxysomes is crucial for understanding the mechanisms underlying carboxysomal shell permeability and enhancement of Rubisco carboxylation within carboxysomes. Such fundamental knowledge may inform reprogramming carboxysomes to improve metabolism and recruit foreign enzymes for enhanced catalytical performance.

Construction of à la carte QconCAT protein standards for multiplexed quantification of user-specified target proteins.

BACKGROUND: QconCATs are quantitative concatamers for proteomic applications that yield stoichiometric quantities of sets of stable isotope-labelled internal standards. However, changing a QconCAT design, for example, to replace poorly performing peptide standards has been a protracted process. RESULTS: We report a new approach to the assembly and construction of QconCATs, based on synthetic biology precepts of biobricks, making use of loop assembly to construct larger entities from individual biobricks. The basic building block (a Qbrick) is a segment of DNA that encodes two or more quantification peptides for a single protein, readily held in a repository as a library resource. These Qbricks are then assembled in a one tube ligation reaction that enforces the order of assembly, to yield short QconCATs that are useable for small quantification products. However, the DNA context of the short construct also allows a second cycle of loop assembly such that five different short QconCATs can be assembled into a longer QconCAT in a second, single tube ligation. From a library of Qbricks, a bespoke QconCAT can be assembled quickly and efficiently in a form suitable for expression and labelling in vivo or in vitro. CONCLUSIONS: We refer to this approach as the ALACAT strategy as it permits à la carte design of quantification standards. ALACAT methodology is a major gain in flexibility of QconCAT implementation as it supports rapid editing and improvement of QconCATs and permits, for example, substitution of one peptide by another.

Structural Visualization of Septum Formation in Staphylococcus warneri Using Atomic Force Microscopy.

Cell division of Staphylococcus adopts a "popping" mechanism that mediates extremely rapid separation of the septum. Elucidating the structure of the septum is crucial for understanding this exceptional bacterial cell division mechanism. Here, the septum structure of Staphylococcus warneri was extensively characterized using high-speed time-lapse confocal microscopy, atomic force microscopy, and electron microscopy. The cells of S. warneri divide in a fast popping manner on a millisecond timescale. Our results show that the septum is composed of two separable layers, providing a structural basis for the ultrafast daughter cell separation. The septum is formed progressively toward the center with nonuniform thickness of the septal disk in radial directions. The peptidoglycan on the inner surface of double-layered septa is organized into concentric rings, which are generated along with septum formation. Moreover, this study signifies the importance of new septum formation in initiating new cell cycles. This work unravels the structural basis underlying the popping mechanism that drives S. warneri cell division and reveals a generic structure of the bacterial cell.IMPORTANCE This work shows that the septum of Staphylococcus warneri is composed of two layers and that the peptidoglycan on the inner surface of the double-layered septum is organized into concentric rings. Moreover, new cell cycles of S. warneri can be initiated before the previous cell cycle is complete. This work advances our knowledge about a basic structure of bacterial cell and provides information on the double-layered structure of the septum for bacteria that divide with the "popping" mechanism.

Roles of RbcX in Carboxysome Biosynthesis in the Cyanobacterium Synechococcus elongatus PCC7942.

Rubisco is the essential enzyme mediating the fixation of atmospheric CO2 during photosynthesis. In cyanobacteria, Rubisco enzymes are densely packed and encapsulated in a specialized organelle known as the carboxysome. Well-defined Rubisco assembly and carboxysome formation are pivotal for efficient CO2 fixation. Numerous chaperone proteins, including RbcX, are essential for proper protein folding and Rubisco assembly. In this study, we investigated the in vivo function of RbcX in the cyanobacterium Synechococcus elongatus PCC 7942 (Syn7942) using molecular, biochemical, and live-cell fluorescence imaging approaches. Our results show that genetic deletion of the rbcX gene affects Rubisco abundance, as well as carboxysome formation and spatial distribution. Moreover, RbcX appears as one component of the carboxysome and shows a dynamic interaction with Rubisco enzymes. These in vivo observations provide insight into the role of RbcX from Syn7942 in mediating carboxysome assembly. Understanding the molecular mechanism underlying Rubisco assembly and carboxysome biogenesis will provide essential information required for engineering functional CO2-fixing complexes in heterogeneous organisms, especially plants, with the aim of boosting photosynthesis and agricultural productivity.

Insights into the Origin of Distinct Medin Fibril Morphologies Induced by Incubation Conditions and Seeding.

Incubation conditions are an important factor to consider when studying protein aggregation in vitro. Here, we employed biophysical methods and atomic force microscopy to show that agitation dramatically alters the morphology of medin, an amyloid protein deposited in the aorta. Agitation reduces the lag time for fibrillation by ~18-fold, suggesting that the rate of fibril formation plays a key role in directing the protein packing arrangement within fibrils. Utilising preformed sonicated fibrils as seeds, we probed the role of seeding on medin fibrillation and revealed three distinct fibril morphologies, with biophysical modelling explaining the salient features of experimental observations. We showed that nucleation pathways to distinct fibril morphologies may be switched on and off depending on the properties of the seeding fibrils and growth conditions. These findings may impact on the development of amyloid-based biomaterials and enhance understanding of seeding as a pathological mechanism.

Distribution and dynamics of electron transport complexes in cyanobacterial thylakoid membranes.

The cyanobacterial thylakoid membrane represents a system that can carry out both oxygenic photosynthesis and respiration simultaneously. The organization, interactions and mobility of components of these two electron transport pathways are indispensable to the biosynthesis of thylakoid membrane modules and the optimization of bioenergetic electron flow in response to environmental changes. These are of fundamental importance to the metabolic robustness and plasticity of cyanobacteria. This review summarizes our current knowledge about the distribution and dynamics of electron transport components in cyanobacterial thylakoid membranes. Global understanding of the principles that govern the dynamic regulation of electron transport pathways in nature will provide a framework for the design and synthetic engineering of new bioenergetic machinery to improve photosynthesis and biofuel production. This article is part of a Special Issue entitled: Organization and dynamics of bioenergetic systems in bacteria, edited by Conrad Mullineaux.

Supramolecular architecture of photosynthetic membrane in red algae in response to nitrogen starvation.

The availability of nitrogen is one of the most important determinants that can limit the growth of photosynthetic organisms including plants and algae; however, direct observations on the supramolecular architecture of photosynthetic membranes in response to nitrogen stress are still lacking. Red algae are an important evolutionary group of algae which contain phycobilisomes (PBSs) on their thylakoid membranes, as do cyanobacteria. PBSs function not only as light-harvesting antennae but also as nitrogen storage. In this report, alterations of the supramolecular architecture of thylakoid membranes from red alga Porphyridium cruentum during nitrogen starvation were characterized. The morphology of the intact thylakoid membrane was observed to be round vesicles. Thylakoid membranes were reduced in content and PBSs were degraded during nitrogen starvation. The size and density of PBSs were both found to be reduced. PBS size decreased by less than one-half after 20days of nitrogen starvation, but their hemispherical morphology was retained. The density of PBSs on thylakoid membranes was more seriously affected as time proceeded. Upon re-addition of nitrogen led to increasing of PBSs on thylakoid membranes. This work reports the first direct observation on alterations in the supramolecular architecture of thylakoid membranes from a photosynthetic organism in response to nitrogen stress.

Light Modulates the Biosynthesis and Organization of Cyanobacterial Carbon Fixation Machinery through Photosynthetic Electron Flow.

Cyanobacteria have evolved effective adaptive mechanisms to improve photosynthesis and CO2 fixation. The central CO2-fixing machinery is the carboxysome, which is composed of an icosahedral proteinaceous shell encapsulating the key carbon fixation enzyme, Rubisco, in the interior. Controlled biosynthesis and ordered organization of carboxysomes are vital to the CO2-fixing activity of cyanobacterial cells. However, little is known about how carboxysome biosynthesis and spatial positioning are physiologically regulated to adjust to dynamic changes in the environment. Here, we used fluorescence tagging and live-cell confocal fluorescence imaging to explore the biosynthesis and subcellular localization of ß-carboxysomes within a model cyanobacterium, Synechococcus elongatus PCC7942, in response to light variation. We demonstrated that ß-carboxysome biosynthesis is accelerated in response to increasing light intensity, thereby enhancing the carbon fixation activity of the cell. Inhibition of photosynthetic electron flow impairs the accumulation of carboxysomes, indicating a close coordination between ß-carboxysome biogenesis and photosynthetic electron transport. Likewise, the spatial organization of carboxysomes in the cell correlates with the redox state of photosynthetic electron transport chain. This study provides essential knowledge for us to modulate the ß-carboxysome biosynthesis and function in cyanobacteria. In translational terms, the knowledge is instrumental for design and synthetic engineering of functional carboxysomes into higher plants to improve photosynthesis performance and CO2 fixation.

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.

Oxidative Stress Alters the Morphology and Toxicity of Aortic Medial Amyloid.

The aggregation and fibril deposition of amyloid proteins have been implicated in a range of neurodegenerative and vascular diseases, and yet the underlying molecular mechanisms are poorly understood. Here, we use a combination of cell-based assays, biophysical analysis, and atomic force microscopy to investigate the potential involvement of oxidative stress in aortic medial amyloid (AMA) pathogenesis and deposition. We show that medin, the main constituent of AMA, can induce an environment rich in oxidative species, increasing superoxide and reducing bioavailable nitric oxide in human cells. We investigate the role that this oxidative environment may play in altering the aggregation process of medin and identify potential posttranslational modification sites where site-specific modification and interaction can be unambiguously demonstrated. In an oxidizing environment, medin is nitrated at tyrosine and tryptophan residues, with resultant effects on morphology that lead to longer fibrils with increased toxicity. This provides further motivation to investigate the role of oxidative stress in AMA pathogenicity.

The architecture of Rhodobacter sphaeroides chromatophores.

The chromatophores of Rhodobacter (Rb.) sphaeroides represent a minimal bio-energetic system, which efficiently converts light energy into usable chemical energy. Despite extensive studies, several issues pertaining to the morphology and molecular architecture of this elemental energy conversion system remain controversial or unknown. To tackle these issues, we combined electron microscope tomography, immuno-electron microscopy and atomic force microscopy. We found that the intracellular Rb. sphaeroides chromatophores form a continuous reticulum rather than existing as discrete vesicles. We also found that the cytochrome bc1 complex localizes to fragile chromatophore regions, which most likely constitute the tubular structures that interconnect the vesicles in the reticulum. In contrast, the peripheral light-harvesting complex 2 (LH2) is preferentially hexagonally packed within the convex vesicular regions of the membrane network. Based on these observations, we propose that the bc1 complexes are in the inter-vesicular regions and surrounded by reaction center (RC) core complexes, which in turn are bounded by arrays of peripheral antenna complexes. This arrangement affords rapid cycling of electrons between the core and bc1 complexes while maintaining efficient excitation energy transfer from LH2 domains to the RCs.

Single-molecule in vivo imaging of bacterial respiratory complexes indicates delocalized oxidative phosphorylation.

Chemiosmotic energy coupling through oxidative phosphorylation (OXPHOS) is crucial to life, requiring coordinated enzymes whose membrane organization and dynamics are poorly understood. We quantitatively explore localization, stoichiometry, and dynamics of key OXPHOS complexes, functionally fluorescent protein-tagged, in Escherichia coli using low-angle fluorescence and superresolution microscopy, applying single-molecule analysis and novel nanoscale co-localization measurements. Mobile 100-200nm membrane domains containing tens to hundreds of complexes are indicated. Central to our results is that domains of different functional OXPHOS complexes do not co-localize, but ubiquinone diffusion in the membrane is rapid and long-range, consistent with a mobile carrier shuttling electrons between islands of different complexes. Our results categorically demonstrate that electron transport and proton circuitry in this model bacterium are spatially delocalized over the cell membrane, in stark contrast to mitochondrial bioenergetic supercomplexes. Different organisms use radically different strategies for OXPHOS membrane organization, likely depending on the stability of their environment.