The size of whole Rhodobacter sphaeroides prevents 3D visualization of centermost chromatophores in their native environment. This study combines cryo-focused ion beam milling with cryo-electron tomography to probe vesicle architecture both in situ and in 3D. Developing chromatophores are membrane-bound buds that remain in topological continuity with the cytoplasmic membrane and detach into vesicles when mature. Mature chromatophores closest to the cell wall are typically isolated vesicles, whereas centermost chromatophores are either linked to neighboring chromatophores or contain smaller, budding structures. Isolated chromatophores comprised a minority of centermost chromatophores. Connections between vesicles in growing bacteria are through ~10 nm-long, ~5 nm-wide linkers, and are thus physical rather than functional in terms of converting photons to ATP. In cells in the stationary phase, chromatophores fuse with neighboring vesicles, lose their spherical structure, and greatly increase in volume. The fusion and morphological changes seen in older bacteria are likely a consequence of the aging process, and are not representative of connectivity in healthy R. sphaeroides. Our results suggest that chromatophores can adopt either isolated or connected morphologies within a single bacterium. Revealing the organization of chromatophore vesicles throughout the cell is an important step in understanding the photosynthetic mechanisms in R. sphaeroides.

Omp21, a minor outer membrane protein of the soil bacteriumComamonas acidovorans, was purified from a spontaneous mutant lacking a surface layer and long-chain lipopolysaccharide. Omp21 synthesis is enhanced by oxygen depletion, and the protein has a variable electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis due to its heat-modifiable behavior. The structural gene omp21 encodes a precursor of 204 amino acids with a putative signal peptide of 21 amino acids. Mature Omp21 is a typical outer membrane protein with a high content of β structure as determined by infrared spectroscopy. Sequence comparisons show that it belongs to a new outer membrane protein family, characterized by eight amphipathic β strands, which includes virulence proteins, such as the neisserial opacity proteins,Salmonella typhimurium Rck, and Yersinia enterocolitica Ail, as well as the major outer membrane proteins OmpA from Escherichia coli and OprF fromPseudomonas aeruginosa.

Photosynthesis by plants, algae, and photosynthetic bacteria is a significant source of the global energy supply. Photosynthetic vesicles, such as the chromatophore in Rhodobacter sphaeroides, are fascinating both fundamentally and technologically. Each chromatophore is capable of photosynthesis by absorbing sunlight as electronic excitation and converting it into chemical energy in form of ATP. Previous scientific inquiries prioritized characterizing the protein-pigment complexes on a chromatophore surface, and how they inform our understanding of photosynthesis by eukaryotic organelles, like chloroplasts. More recent work demonstrates new approaches to engineering biorenewable devices with chromatophore complexes as the active components, renewing interest in Rba. sphaeroides. Despite significant advances in understanding how proteins interact on the surface of a single vesicle, decadeslong deliberation on how chromatophores in Rba. sphaeroides interact with neighboring vesicles continues today. As the reticulated nature of chromatophores may have a significant impact on the photosynthetic mechanism in Rba. sphaeroides and biorenewable technologies, it is imperative to investigate the extent of connectivity between vesicles throughout the bacteria both in situ and in 3D.Understanding chromatophore connectivity is hampered by limitations of techniques typically used to assess their structure. For instance, atomic force microscopy has been used to image the arrangement of proteins on the surface of chromatophores, but isolation of vesicles from the bacteria is required and can disturb their network. Transmission electron microscopy (TEM) provides visualization of peripheral structure in Rba. sphaeroides with nanoscale detail, but resolving the internal structure is often hampered by cellular thickness. The conventional thinning technique is sectioning by cryoultramicrotomy, which can introduce compression artifacts. Most of these experimental limitations can be avoided with alternative techniques such as cryo-focused ion beam milling (cryo-FIB) and cryoelectron tomography (cryo-ET). Sample fixation by vitrification preserves the sample in a near-native, liquid-hydrated state avoiding staining artifacts. Cryo-FIB milling thins a specimen with a collimated beam of Ga + ions, providing site-specific, ultra-thin sections without the artifacts associated with cryoultramicrotomy [1-2]. Furthermore, thinning occurs directly on the TEM grid where specimens have been previously vitrified, reducing the risk of contamination. Cryo-FIB is a robust technique that has been used with cryo-ET to visualize both bacteria and photosynthetic organelles in 3D [3][4].In this study, we use an on-grid cryo-FIB preparation of fully-hydrated, vitrified Rba. sphaeroides to gain an unprecedented view of the internal structure of the bacteria in its native state (Fig. 1 B,C). We describe the macromolecular landscape of Rba. sphaeroides, including vesicular biogenesis, development, organization, and connectivity ( Fig. 2 A,B). This study pro...

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