Three-Dimensional Imaging of the Highly Bent Architecture of Bdellovibrio bacteriovorus by Using Cryo-Electron Tomography

Abstract

Bdellovibrio bacteriovorus cells are small deltaproteobacterial cells that feed on other gram-negative bacteria, including human pathogens. Using cryo-electron tomography, we demonstrated that B. bacteriovorus cells are capable of substantial flexibility and local deformation of the outer and inner membranes without loss of cell integrity. These shape changes can occur in less than 2 min, and analysis of the internal architecture of highly bent cells showed that the overall distribution of molecular machines and the nucleoid is similar to that in moderately bent cells. B. bacteriovorus cells appear to contain an extensive internal network of short and long filamentous structures. We propose that rearrangements of these structures, in combination with the unique properties of the cell envelope, may underlie the remarkable ability of B. bacteriovorus cells to find and enter bacterial prey.Bacteria of the genus Bdellovibrio are highly motile predators that reside in diverse aquatic and terrestrial environments, as well as in the mammalian digestive tract (28; for a review, see reference 33). They prey on a variety of gram-negative bacteria, including several human pathogens, and could become an important tool in the probiotic treatment of disease (32). Within this genus, Bdellovibrio bacteriovorus is the species that has been best characterized biochemically and genetically (27). Investigations over the last four decades (20, 27, 33) have helped elucidate several aspects of the complex life cycle of this organism, which includes a free-swimming or attack phase and a prey-bound phase. Attack-phase B. bacteriovorus is propelled by a single long sheathed flagellum and is capable of attaining speeds up to 160 m/s. The predator swims and turns in apparently random directions until it finds and attaches to a prey cell. It then digests a region of the host cell outer membrane to make a small entry pore, penetrates into the host periplasm, and forms a growth chamber by reshaping and resealing the host outer membrane. The established parasite depletes the host cytoplasm and undergoes growth to form a single elongated spiral cell and segmented division to generate multiple progeny, and it develops a flagellum to gain motility. Ultimately, the chamber ruptures, releasing the nascent bacteria to reinitiate the life cycle.Conventional electron microscopic (EM) studies have provided valuable insights into morphological changes that occur during the life cycle of B. bacteriovorus (1, 2, 6, 7). However, room temperature EM imaging typically requires specimen preparation steps that include chemical fixation, removal of the aqueous phase by treatment with organic solvents, and embedding in plastic resins. These procedures can introduce artifacts, such as distortion of the cell shape, deterioration of membrane structures, and aggregation of soluble multiprotein complexes. Moreover, the contrast in the images originates primarily from the stain and not from the intrinsic density of target structures, limiting the resolution attainable...

Three-Dimensional Structure of the Ultraoligotrophic Marine Bacterium “Candidatus Pelagibacter ubique”

“…3F). This is in contrast to the centrally located nucleoid and peripherally located ribosomes observed in many other bacterial species (18,19,32). Three-dimensional modeling of nucleoids and ribosome-like particles indicated that these subcellular structures were compartmentalized into two distinct zones, each taking up roughly one-half of the volume enclosed by the IM (Fig.…”

“…In addition, rapid freezing (which occurs within ϳ1 ms) preserves the cells in a nearly native state without chemical fixation or staining, which allows accurate measurement and examination of the cell dimensions and internal structures. In the past few years, cryo-ET has yielded great insights into the internal organization of many commonly studied Gramnegative bacteria (18)(19)(20), archaebacteria (21), cyanobacteria (22), mycobacteria and mycoplasmae (23), viruses (24,25), and bacterial phages (26) and, more recently, the cellular architecture of eukaryotes (27,28).…”

Proteome-Based Comparative Analyses of Growth Stages Reveal New Cell Cycle-Dependent Functions in the Predatory Bacterium Bdellovibrio bacteriovorus

“…1), strongly suggesting that it does not stem from physical constraints in the bdelloplast but rather results from a regulated cell plan. A recent study has shown that the Bdellovibrio cell is unusually flexible and contains numerous filamentous structures (8). Notably, MreB, an actin-like homolog, is present as two copies in B. bacteriovorus.…”

Spiral Architecture of the Nucleoid in Bdellovibrio bacteriovorus

“…The host-dependent B. bacteriovorus strains used in this study were the following: wild-type predatory HD100; HD100mreB2-mtfp, a strain containing integrated plasmid pAKF40a, a kanamycin resistance gene replacement vector that replaces wild-type mreB2 with an mreB2 that is fused in frame at its C terminus with the coding sequence of monomeric bright teal fluorescent protein, mTFP; and the control strain HD100Bd2345::Km r , a cell line that has an integrated pK18mobsacB kanamycin resistance vector to disrupt the ABC transporter gene to act as a plasmid control for the pK18 backbone of pAKF40a (17). HD100 was cocultured at 22 to 25°C with E. coli strain RP3098, a mutant with all flagellar and chemotaxis proteins deleted and grown as described previously (3). The other two strains were cocultured at 29°C on YPSC (yeast extract, peptone, sodium acetate, and Lcysteine) overlay plates with E. coli strain S17-1 that contains plasmid pZMR100 to confer kanamycin resistance (17).…”