FliL ring enhances the function of periplasmic flagella.

SignificanceHow flagella sense complex environments and control bacterial motility remain fascinating questions. Here, we deploy cryo-electron tomography to determine in situ structures of the flagellar motor in wild-type and mutant cells of Borrelia burgdorferi, revealing that three flagellar proteins (FliL, MotA, and MotB) form a unique supramolecular complex in situ. Importantly, FliL not only enhances motor function by forming a ring around the stator complex MotA/MotB in its extended, active conformation but also facilitates assembly of the stator complex around the motor. Our in situ data provide insights into how cooperative remodeling of the FliL-stator supramolecular complex helps regulate the collective ion flux and establishes the optimal function of the flagellar motor to guide bacterial motility in various environments.

Structural basis of MsbA-mediated lipopolysaccharide transport.

Lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria is critical for the assembly of their cell envelopes. LPS synthesized in the cytoplasmic leaflet of the inner membrane is flipped to the periplasmic leaflet by MsbA, an ATP-binding cassette transporter. Despite substantial efforts, the structural mechanisms underlying MsbA-driven LPS flipping remain elusive. Here we use single-particle cryo-electron microscopy to elucidate the structures of lipid-nanodisc-embedded MsbA in three functional states. The 4.2 Å-resolution structure of the transmembrane domains of nucleotide-free MsbA reveals that LPS binds deep inside MsbA at the height of the periplasmic leaflet, establishing extensive hydrophilic and hydrophobic interactions with MsbA. Two sub-nanometre-resolution structures of MsbA with ADP-vanadate and ADP reveal an unprecedented closed and an inward-facing conformation, respectively. Our study uncovers the structural basis for LPS recognition, delineates the conformational transitions of MsbA to flip LPS, and paves the way for structural characterization of other lipid flippases.

BB0326 is responsible for the formation of periplasmic flagellar collar and assembly of the stator complex in Borrelia burgdorferi.

Borrelia burgdorferi is a highly motile spirochete due to its periplasmic flagella. Unlike flagella of other bacteria, spirochetes' periplasmic flagella possess a complex structure called the collar, about which little is known in terms of function and composition. Using various approaches, we have identified a novel protein, BB0326, as a key component of the collar. We show that a peripheral portion of the collar is diminished in the Δbb0326 mutant and restored in the complemented bb0326+ cells, leading us to rename BB0326 as periplasmic flagellar collar protein A or FlcA. The ΔflcA mutant cells produced fewer, abnormally tilted and shorter flagella, as well as diminished stators, suggesting that FlcA is crucial for flagellar and stator assemblies. We provide further evidence that FlcA interacts with the stator and that this collar-stator interaction is essential for the high torque needed to power the spirochete's periplasmic flagellar motors. These observations suggest that the collar provides various important functions to the spirochete's periplasmic flagellar assembly and rotation.

Macromolecular Crowding In Vitro, In Vivo, and In Between.

Biochemical processes take place in heterogeneous and highly volume-occupied or crowded environments that can considerably influence the reactivity and distribution of participating macromolecules. We summarize here the thermodynamic consequences of excluded-volume and long-range nonspecific intermolecular interactions for macromolecular reactions in volume-occupied media. In addition, we summarize and compare the information content of studies of crowding in vitro and in vivo. We emphasize the importance of characterizing the behavior not only of labeled tracer macromolecules but also the composition and behavior of unlabeled macromolecules in the immediate vicinity of the tracer. Finally, we propose strategies for extending quantitative analyses of crowding in simple model systems to increasingly complex media up to and including intact cells.

From Chaperones to the Membrane with a BAM!

Outer membrane proteins (OMPs) play a central role in the integrity of the outer membrane of Gram-negative bacteria. Unfolded OMPs (uOMPs) transit across the periplasm, and subsequent folding and assembly are crucial for biogenesis. Chaperones and the essential ß-barrel assembly machinery (BAM) complex facilitate these processes. In vitro studies suggest that some chaperones sequester uOMPs in internal cavities during their periplasmic transit to prevent deleterious aggregation. Upon reaching the outer membrane, the BAM complex acts catalytically to accelerate uOMP folding. Complementary in vivo experiments have revealed the localization and activity of the BAM complex in living cells. Completing an understanding of OMP biogenesis will require a holistic view of the interplay among the individual components discussed here.

Icosahedral bacteriophage ΦX174 forms a tail for DNA transport during infection.

Prokaryotic viruses have evolved various mechanisms to transport their genomes across bacterial cell walls. Many bacteriophages use a tail to perform this function, whereas tail-less phages rely on host organelles. However, the tail-less, icosahedral, single-stranded DNA ΦX174-like coliphages do not fall into these well-defined infection processes. For these phages, DNA delivery requires a DNA pilot protein. Here we show that the ΦX174 pilot protein H oligomerizes to form a tube whose function is most probably to deliver the DNA genome across the host's periplasmic space to the cytoplasm. The 2.4 Å resolution crystal structure of the in vitro assembled H protein's central domain consists of a 170 Å-long α-helical barrel. The tube is constructed of ten α-helices with their amino termini arrayed in a right-handed super-helical coiled-coil and their carboxy termini arrayed in a left-handed super-helical coiled-coil. Genetic and biochemical studies demonstrate that the tube is essential for infectivity but does not affect in vivo virus assembly. Cryo-electron tomograms show that tubes span the periplasmic space and are present while the genome is being delivered into the host cell's cytoplasm. Both ends of the H protein contain transmembrane domains, which anchor the assembled tubes into the inner and outer cell membranes. The central channel of the H-protein tube is lined with amide and guanidinium side chains. This may be a general property of viral DNA conduits and is likely to be critical for efficient genome translocation into the host.

BamA ß16C strand and periplasmic turns are critical for outer membrane protein insertion and assembly.

Outer membrane (OM) ß-barrel proteins play important roles in importing nutrients, exporting wastes and conducting signals in Gram-negative bacteria, mitochondria and chloroplasts. The outer membrane proteins (OMPs) are inserted and assembled into the OM by OMP85 family proteins. In Escherichia coli, the ß-barrel assembly machinery (BAM) contains four lipoproteins such as BamB, BamC, BamD and BamE, and one OMP BamA, forming a 'top hat'-like structure. Structural and functional studies of the E. coli BAM machinery have revealed that the rotation of periplasmic ring may trigger the barrel ß1C-ß6C scissor-like movement that promote the unfolded OMP insertion without using ATP. Here, we report the BamA C-terminal barrel structure of Salmonella enterica Typhimurium str. LT2 and functional assays, which reveal that the BamA's C-terminal residue Trp, the ß16C strand of the barrel and the periplasmic turns are critical for the functionality of BamA. These findings indicate that the unique ß16C strand and the periplasmic turns of BamA are important for the outer membrane insertion and assembly. The periplasmic turns might mediate the rotation of the periplasmic ring to the scissor-like movement of BamA ß1C-ß6C, triggering the OMP insertion. These results are important for understanding the OMP insertion in Gram-negative bacteria, as well as in mitochondria and chloroplasts.

The Bacteroid Periplasm in Soybean Nodules Is an Interkingdom Symbiotic Space.

The functional role of the periplasm of nitrogen-fixing bacteroids has not been determined. Proteins were isolated from the periplasm and cytoplasm of Bradyrhizobium diazoefficiens bacteroids and were analyzed using liquid chromatography tandem mass spectrometry proteomics. Identification of bacteroid periplasmic proteins was aided by periplasm prediction programs. Approximately 40% of all the proteins identified as periplasmic in the B. diazoefficiens genome were found expressed in the bacteroid form of the bacteria, indicating the periplasm is a metabolically active symbiotic space. The bacteroid periplasm possesses many fatty acid metabolic enzymes, which was in contrast to the bacteroid cytoplasm. Amino acid analysis of the periplasm revealed an abundance of phosphoserine, phosphoethanolamine, and glycine, which are metabolites of phospholipid metabolism. These results suggest the periplasm is a unique space and not a continuum with the peribacteroid space. A number of plant proteins were found in the periplasm fraction, which suggested contamination. However, antibodies to two of the identified plant proteins, histone H2A and lipoxygenase, yielded immunogold labeling that demonstrated the plant proteins were specifically targeted to the bacteroids. This suggests that the periplasm is an interkingdom symbiotic space containing proteins from both the bacteroid and the plant.

Leptospiral structure, physiology, and metabolism.

Members of the family Leptospiraceae are thin, spiral, highly motile bacteria that are best visualized by darkfield microscopy. These characteristics are shared with other members of the Order Spirochaetales, but few additional parallels exist among spirochetes. This chapter describes basal features of Leptospira Leptospira that are central to survival and, in the case of pathogenic leptospiral species, intimately linked with pathogenesis, including its morphology, characteristic motility, and unusual metabolism. This chapter also describes the general methodology and critical requirements for in vitro cultivation and storage of Leptospira within a laboratory setting.

[EFFECT OF PULSE-PERIODIC CORONA DISCHARGE ON VIABILITY OF ESCHERICHIA COLI M17 CELLS IN BIOFILMS].

AIM: Detection of bactericidal effect of pulse-periodic corona discharge (PPCD) on cells and biofilms of Escherichia coli M17. MATERIALS AND METHODS: A gas-discharge device was created based on PPCD in air with power supply parameters: amplitude values of voltage of 30 - 60 kV, pulse repetition rate of 250 - 400 kHz. Ultrastructure changes in cells and biofilms of E. coli M17, affected by PPCD, generated in air, were studied by typical methods of transmission electron microscopy. RESULTS: Disturbances of integrity of surface and abyssal structures of biofilms, as well as changes of morphological properties of E. coli M17 cells, characteristic for sub-lethal heat impact, were detected. Destructive changes of bacterial cells were developed by formation of focal disturbance of cytoplasmic membrane, extension of periplasmic space, formation of globular structures, characteristic for heat effect, and destruction of cytoplasm. CONCLUSION: Bactericidal effect of PPCD on E. coli M17 cells as part of biofilms was shown. Destructive morphological changes in cells and biofilms of E. coli M17 after the effect of PPCD were detected for the first time on electron-microscopic level.

Porphyromonas gingivalis outer membrane vesicles exclusively contain outer membrane and periplasmic proteins and carry a cargo enriched with virulence factors.

Porphyromonas gingivalis, a keystone pathogen associated with chronic periodontitis, produces outer membrane vesicles (OMVs) that carry a cargo of virulence factors. In this study, the proteome of OMVs was determined by LC-MS/MS analyses of SDS-PAGE fractions, and a total of 151 OMV proteins were identified, with all but one likely to have originated from either the outer membrane or periplasm. Of these, 30 exhibited a C-terminal secretion signal known as the CTD that localizes them to the cell/vesicle surface, 79 and 27 were localized to the vesicle membrane and lumen respectively while 15 were of uncertain location. All of the CTD proteins along with other virulence factors were found to be considerably enriched in the OMVs, while proteins exhibiting the OmpA peptidoglycan-binding motif and TonB-dependent receptors were preferentially retained on the outer membrane of the cell. Cryo-transmission electron microscopy analysis revealed that an electron dense surface layer known to comprise CTD proteins accounted for a large proportion of the OMVs' volume providing an explanation for the enrichment of CTD proteins. Together the results show that P. gingivalis is able to specifically concentrate and release a large number of its virulence factors into the environment in the form of OMVs.

Ultrastructure and histochemistry of the periplasm in oocytes of sturgeons during egg envelope formation.

In the cytoplasm of oocytes (ooplasm) located in ovarian follicles with diameters 2000 microm and 2150 microm in Acipenser gueldenstaedtii, and 2000 microm and 2350 microm in A. baerii, periplasm containing a basophilic compartment and endoplasm containing reserve materials was formed. Vesicles involved in polyspermy blocking and in the formation of the embryo were located in the periplasm. These included compact (cCGs), low-electron-dense cortical granules (lCGs), and lamellar bodies. The cCGs were bounded by a membrane, comprised fibrillar material, fibrils and rod-shaped components. The lCGs were membrane-bounded and contained fibrillar material and granular inclusions. Endoplasmic reticulum (ER) and Golgi complexes were involved in the formation of cCG and lCG. The basophilic compartment, ER and Golgi vesicles participated in the formation of lamellar bodies. They comprised numerous membranes and fibrillar material. It is assumed that they transfer membranes and their precursors to the growing furrow during cleavage and release their content to organize the extracellular matrix. The location of compounds in the developing egg envelope of A. gueldenstaedtii was presented and discussed. Ovaries of both investigated species represented the first pubertal stages of development. Such fish should not be used for reproduction.

Reward for Bdellovibrio bacteriovorus for preying on a polyhydroxyalkanoate producer.

Bdellovibrio bacteriovorus HD100 is an obligate predator that invades and grows within the periplasm of Gram-negative bacteria, including mcl-polyhydroxyalkanoate (PHA) producers such as Pseudomonas putida. We investigated the impact of prey PHA content on the predator fitness and the potential advantages for preying on a PHA producer. Using a new procedure to control P. putida KT2442 cell size we demonstrated that the number of Bdellovibrio progeny depends on the prey biomass and not on the viable prey cell number or PHA content. The presence of mcl-PHA hydrolysed products in the culture supernatant after predation on P. putida KT42Z, a PHA producing strain lacking PhaZ depolymerase, confirmed the ability of Bdellovibrio to degrade the prey's PHA. Predator motility was higher when growing on PHA accumulating prey. External addition of PHA polymer (latex suspension) to Bdellovibrio preying on the PHA minus mutant P. putida KT42C1 restored predator movement, suggesting that PHA is a key prey component to sustain predator swimming speed. High velocities observed in Bdellovibrio preying on the PHA producing strain were correlated to high intracellular ATP levels of the predator. These effects brought Bdellovibrio fitness benefits as predation on PHA producers was more efficient than predation on non-producing bacteria.

Desulfovibrio magneticus RS-1 contains an iron- and phosphorus-rich organelle distinct from its bullet-shaped magnetosomes.

Intracellular magnetite crystal formation by magnetotactic bacteria has emerged as a powerful model for investigating the cellular and molecular mechanisms of biomineralization, a process common to all branches of life. Although magnetotactic bacteria are phylogenetically diverse and their crystals morphologically diverse, studies to date have focused on a few, closely related species with similar crystal habits. Here, we investigate the process of magnetite biomineralization in Desulfovibrio magneticus sp. RS-1, the only reported species of cultured magnetotactic bacteria that is outside of the alpha-Proteobacteria and that forms bullet-shaped crystals. Using a variety of high-resolution imaging and analytical tools, we show that RS-1 cells form amorphous, noncrystalline granules containing iron and phosphorus before forming magnetite crystals. Using NanoSIMS (dynamic secondary ion mass spectroscopy), we show that the iron-phosphorus granules and the magnetite crystals are likely formed through separate cellular processes. Analysis of the cellular ultrastructure of RS-1 using cryo-ultramicrotomy, cryo-electron tomography, and tomography of ultrathin sections reveals that the magnetite crystals are not surrounded by membranes but that the iron-phosphorus granules are surrounded by membranous compartments. The varied cellular paths for the formation of these two minerals lead us to suggest that the iron-phosphorus granules constitute a distinct bacterial organelle.

Electron transfer at the cell-uranium interface in Geobacter spp.

The in situ stimulation of Fe(III) oxide reduction in the subsurface stimulates the growth of Geobacter spp. and the precipitation of U(VI) from groundwater. As with Fe(III) oxide reduction, the reduction of uranium by Geobacter spp. requires the expression of their conductive pili. The pili bind the soluble uranium and catalyse its extracellular reductive precipitation along the pili filaments as a mononuclear U(IV) complexed by carbon-containing ligands. Although most of the uranium is immobilized by the pili, some uranium deposits are also observed in discreet regions of the outer membrane, consistent with the participation of redox-active foci, presumably c-type cytochromes, in the extracellular reduction of uranium. It is unlikely that cytochromes released from the outer membrane could associate with the pili and contribute to the catalysis, because scanning tunnelling microscopy spectroscopy did not reveal any haem-specific electronic features in the pili, but, rather, showed topographic and electronic features intrinsic to the pilus shaft. Pili not only enhance the rate and extent of uranium reduction per cell, but also prevent the uranium from traversing the outer membrane and mineralizing the cell envelope. As a result, pili expression preserves the essential respiratory activities of the cell envelope and the cell's viability. Hence the results support a model in which the conductive pili function as the primary mechanism for the reduction of uranium and cellular protection in Geobacter spp.

Ultrastructural changes of sparkling wine lees during long-term aging in real enological conditions.

Ultrastructural changes of lees of three series of sparkling wines produced using the traditional method during long-term aging (4 years) were assessed by high-pressure freezing in combination with transmission electron microscopy. The stratified structure of the cell wall disappeared throughout aging. After 18 months, the microfibrous material of the cell wall appeared more diffuse and the amorphous midzone of the inner wall layer was progressively degraded. From 30 months onward, the cell wall consisted of a tangled structure of fibers. In spite of these changes, the cell wall of yeasts remained unbroken at 48 months of wine aging. Cell membrane breakage was observed for the first time in lees of Saccharomyces cerevisiae. An increase in the thickness of the periplasmic space owing to plasmolysis and of the number of cells with less cytoplasmic content was observed during aging. Morphological evidence of microautophagy was detected for the first time in S. cerevisiae in enological conditions.

Microscopic studies on Thermosipho globiformans implicate a role of the large periplasm of Thermotogales.

Thermosipho globiformans is a member of Thermotogales, which contains rod-shaped, Gram-negative, anaerobic (hyper)thermophiles. These bacteria are characterized by an outer sheath-like envelope, the toga, which includes the outer membrane and an amorphous layer, and forms large periplasm at the poles of each rod. The cytoplasmic membrane and its contents are called "cell", and the toga and its contents "rod", to distinguish between them. Optical cells were constructed to observe binary fission of T. globiformans. High-temperature microscopy of rods adhering to optical cells' coverslips showed that the large periplasm forms between newly divided cells in a rod, followed by rod fission at the middle of the periplasm, which was accompanied by a sideward motion of the newly generated rod pole(s). Electron microscopic observations revealed that sessile rods grown on a glass plate have nanotubes adhered to the glass, and these may be involved in the sideward motion. Epifluorescence microscopy with a membrane-staining dye suggested that formation of the septal outer membrane is distinct from cytokinesis. Transmission electron microscopy indicated that the amorphous layer forms in the periplasm between already-divided cells. These findings suggest that the large periplasm is the structure in which the septal toga forms, an event separate from cytokinesis.

[Ultrastructural changes in cells of mycobacteria affected by flurenizid derivatives].

In cells of micobacteria of all investigated samples the following ultrastructural changes were observed: disorganization of a nucleoid and citoplasm, formation of the citoplasmic vacuoles and endocellular lipide-like inclusions, and also change of the cells form into spheroid and formeless mass, disappearance of periplasmatic space, occurrence of cells of small-size and short incompletely divided cells in the samples. They were observed more often after 72 hours of exposition of the cultures with antibacterial substances, than after 24 hours of exposition. The highest concentration of these substances being used, the ultrastructural changes were more essential. No significant difference between the nature of changes in the structure of cells studied using antibacterial substances has been found.

High-resolution architecture of the outer membrane of the Gram-negative bacteria Roseobacter denitrificans.

The 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 approximately 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 approximately 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 Gram-negative bacteria Roseobacter denitrificans, and we perform a sequence alignment with porins of known structure.

Comparative cryo-electron tomography of pathogenic Lyme disease spirochetes.

Spirochetes of the Borrelia burgdorferi sensu lato group, the causative agents of Lyme borreliosis, exhibit a complex biology evolved in its zoonotic cycle. Cryo-electron tomography was used to investigate structural features of three species, B. burgdorferi, B. garinii and B. afzelii, known to cause different clinical manifestations in humans. All three organisms revealed an overall similar architecture and showed different numbers of periplasmic flagellar filaments, polar periplasmic void regions, vesicles budding from the outer membrane sheath, which was covered by an amorphous slime layer. The latter was shown to be distinct in its density when comparing the three human-pathogenic Lyme disease spirochetes and Borrelia hermsii, a species causing relapsing fever. Tomograms of dividing bacteria revealed vesicles near the site of division and new basal bodies that were attached at each end of newly establishing cytoplasmic cylinder poles, while periplasmic flagellar filaments still passed the impending site of division. Two different kinds of cytoplasmic filaments showed similarities to MreB or FtsZ filaments of other bacteria. The similar and distinct structural features of Borrelia and the previously investigated pathogenic and non-pathogenic Treponema species emphasize the importance of further studying phylogenetically distant spirochetes.