Electron Microscopy of the Spherical Body of Oral Spirochetes In Vitro: Further Studies

The surface and inner structure of the spherical bodies (SB) produced by the human oral treponeme strain G7201, similar to Treponema macrodentium, were studied by electron microscopy. Ultrathin sectioning and scanning techniques demonstrated that in the presence of a high concentration of sucrose, the outer envelope of one or both terminal ends of this oral spirochete changed into a swollen structure, the SB. Spirochetal cells adhered firmly to the surface of the resultant body. The membrane of the SB, i.e. the outer envelope, enclosed the coiled protoplasmic cylinder and five axial fibrils which were located between the envelope and the cylinder. Large expanded protoplasmic cylinders were observed, surrounded by a partially disrupted double membrane in some SBs. A number of frizzly fibrous structures, which differed from axial fibrils in number and shape, were also observed within these SBs. Except for abnormal or partially broken cylinders, the protoplasmic cylinders tended to be located close to the inner surface of the SB membrane, resulting in a central vacant space with occasional axial fibrils. These findings suggest that the oral spirochete produces an SB by terminal expansion of the outer envelope in the presence of high concentrations of sucrose. The outer envelope of the SB, which consists of two electron-dense layers, has the property of binding spirochetal cells to its outer layer and the protoplasmic cylinder and axial fibrils to the inner layer. Some protoplasmic cylinders were also observed to be swollen in the presence of high sucrose concentrations.     

Purification, Ultrastructure, and Composition of Axial Filaments from Leptospira

The ultrastructure of three strains of water Leptospira was studied by negative staining, thin sectioning, and freeze-etching. The cells possessed a triple-layered sheath which covered two independent axial filaments, one inserted subterminally in each end of the cell. The protoplasmic cylinder was surrounded by a triple-layered cell wall and possessed ribosomes, lamellar structures, and a typical procaryotic nuclear region. The axial filament was comprised of several component structures. An axial fibril, with a diameter of 20 to 25 nm, consisted of a solid inner core (13 to 16 nm in diameter) surrounded by a coat. A terminal knob (40 to 70 nm in length) was connected to a series of disc insertion structures at the terminal end of the axial fibril. The axial fibril was surrounded by a helical outer coat (35 to 60 nm in diameter) which was composed of a continuously coiled fiber, 3 to 4 nm in diameter, embedded in an electron-dense material. A procedure for the purification of the axial fibrils was presented and their ultrastructural, physical, and chemical properties were determined. Similarities in ultrastructural, physical, and chemical properties were noted between the axial fibrils and bacterial flagella. A schematic model of the leptospiral axial filament is presented, and a mechanism is proposed for its function as a locomotor organelle.     

Variable cellular composition of Chromatium in growing cultures

Summary A strictly anaerobic spirochete was isolated from a sample of marine mud. The organism possessed two axial fibrils entwined with the regularly coiled protoplasmic cylinder. An outer envelope or sheath enclosed both protoplasmic cylinder and axial fibrils. The spirochete grew in chemically defined media containing glucose, amino acids or NH₄Cl, sulfide, NaCl, vitamins, coenzyme A, and in-organic salts. A reducing agent, such as sodium sulfide or l-cysteine, as well as exogenous supplements of biotin, niacin and coenzyme A were required for growth. Pantothenate replaced coenzyme A as an exogenous growth factor, but the resulting cell yields were low. The spirochete grew in media prepared with sea water, but not in fresh water media containing less than 0.05 M NaCl (optimum concentration 0.35 M). Both Na⁺ and Cl^- were required. Carbohydrates served as fermentable substrates. Amino acids, sugar alcohols, tricarboxylic acid cycle intermediates, and other organic acids and alcohols were not fermented. Glucose was fermented to ethyl alcohol, acetate, CO₂, H₂, and small amounts of lactate, formate and pyruvate. The guanine + cytosine content of the DNA of the spirochete was 50.5 moles-% (buoyant density). It is proposed that the marine isolate be considered a new species and that it be named Spirochaeta litoralis.     

Isolation and characterization of an obligately anaerobic,polysaccharolytic, extremely thermophilic member of the genus Spirochaeta

An obligately anaerobic, extremely thermophilic Spirochaeta species was isolated from a thermal spring on the edge of Green Lake on Raoul Island of the Kermadec archipelago. The strain, designated RI 19.B1 (=DSM 6192) displayed the morphological characteristics typical of Spirochaeta species: regularly coiled long thin cells consisting of a crenulated outer sheath surrounding a central coiled protoplasmic cylinder. Between the outer sheath and the protoplasmic cylinder were two longitudinal periplasmic fibrils in a 1-2-1 arrangement, each anchored by an insertion disc near one pole, whereas the other end was not anchored. The strain displayed a strictly anaerobic, chemoorganotrophic, fermentative metabolism and was able to grow on a variety of mono-, di- and polysaccharides, including cellulose. Sugar alcohols, organic and amino acids were not utilized. Growth supplements were not required, but CO₂ was required to produce consistent growth. Strain RI 19.B1 had temperature, pH and salinity optima of 64–66°C, pH 6.95 and 0.4% NaCl respectively. The maximum growth temperature and salinity were 73°C and 2.5% respectively. Glucose was fermented to lactate, acetate, carbon dioxide, and hydrogen. Succinate, ethanol and formate were not detected. The strain displayed the resistance to rifampicin typical of Spirochaeta species. The mol % G+C of DNA from strain RI 19.B1 was 52%.     

Fine-structural and chemical analyses on inner and outer sheath of the cyanobacteriumGloeothece sp. PCC 6909

Cells of the unicellular cyanobacteriumGloeothece sp. PCC 6909 are surrounded by an inner (enclosing 1–2 cells) and an outer (enclosing cell groups) sheath. Using conventional Epon-embedding in combination with ruthenium-red staining, the inner and outer sheaths appeared similar and displayed multiple bands of electron-dense subunits. However, embedding in Nanoplast resin to avoid shrinkage led to the detection of two distinct zones (inner and outer zone) each with several distinct layers. The zone delimited by the electron-dense thick inner sheath layer, and the zone enclosed by the thin electron-dense outer sheath layer, are composed of a homogeneous material of little electron-contrast. Whereas the outer zone appears to be of even contrast, the inner zone is characterized by a distinct electron-transparent layer. Element distribution analysis revealed that the electron-transparent layer contained relatively large amounts of sulfur, carbon, and oxygen but only little nitrogen.Inner and outer sheath fractions were isolated by differential mechanical cell breakage and centrifugation. The outer sheath fraction was less hydrated than the inner one. The two fractions differed little in their contents of uronic acids, carbohydrate and protein, although the outer sheath fraction contained less sulfate. A soluble polysaccharide with a chemical composition similar to that of inner and outer sheath fractions was also obtained from the culture supernatant.     

Electron Microscopy of Immune Disruption of Leptospires: Action of Complement and Lysozyme

Sequential disruption of the sheath of avirulent leptospires of the serotype canicola with antibody and complement was monitored by electron microscopy. Loosening and separation of the sheath from the protoplasmic cylinder was observed as early as 2 min after exposure to complement. Virulent leptospires of this serotype were morphologically intact after 1 hr of exposure to antibody and complement. Similarly, treatment of leptospires of the serotype patoc with normal serum and complement severely damaged the sheath structure. Removal of the sheath of both serotypes permitted lysozyme to act on the wall of the protoplasmic cylinder. Thus, morphological evidence for the location of the mucopeptide-containing structure of these leptospires was obtained. Viable leptospires with intact sheaths were resistant to lysozyme alone. Sections and negatively stained preparations of sheaths of serotypes canicola and patoc revealed three dense layers with two intermediate light zones and an overall thickness of about 110 A. A periodicity of 40 A was observed in sheath fragments produced by complement. The 70 A wallmembrane complex of leptospires of both serotypes consisted of two dense layers with an intermediate light zone. Structures apparent after removal of the outer sheath included membranous bodies or mesosomes, axial filaments attached to terminal knobs at opposite ends of the cell, and electron-dense intracellular bodies.     

The ultrastructure and development of the colonial sheath of Microcystis marginata

The colonial sheath of Microcystis marginata has a definite structure as seen by light and electron microscopy, consisting of a relatively smooth inner surface and densely packed, long fibrils on the outer surface. The sheath initially forms around the single cell and expands by continual deposition of sheath material to accomodate the synchronously dividing cells of the colony.     

The cell envelope of the hyperthermophilic archaebacterium Pyrobaculum organotrophum consists of two regularly arrayed protein layers: three-dimensional structure of the outer layer

The cell envelope of the hyperthermophilic sulphur-reducing archaebacterium Pyrobaculum organotrophum H10 was found to be composed of two distinct hexagonally arranged crystalline protein arrays. Electron microscopic analysis of freeze-etched cells and isolated envelopes in conjunction with image processing showed that the inner layer (lattice centre-to-centre spacing 27.9 nm) is essentially identical to the protein array of Pyrobaculum islandicum GEO3, a complex, rigid structure implicated in the maintenance of cell shape. The outer layer has clear p6 symmetry and a lattice spacing of 20.6 nm. Its three-dimensional structure was reconstructed from a negative stain tilt series of an intact double-layered envelope using Fourier filtration to separate the desired information from the other lattices present. The outer layer is a unique, porous network of blocklike dimers disposed around six-fold axes, and exhibits minimal asymmetry between its inner and outer faces. It appears to be rather loosely associated with the outer surface of the inner layer. In most H10 envelopes, the inner layer is orientated with one base vector exactly perpendicular to the long axis of the cell, so that the cylindrical portion is composed of a series of parallel cell-girdling hoops of hexameric morphological units. All the other known Pyrobaculum strains were found to have a GEO3-type envelope structure, consisting of a single rigid protein array and a fibrous capsule. Although H10 does not possess a capsule, fibrils appear to be sandwiched between the two protein layers.     

Treponema succinifaciens sp. nov., an anaerobic spirochete from the swine intestine

The morphology, the general physiological characteristics, and the energy-yielding metabolism of an obligately anaerobic spirochete isolated from the colon of a swine were studied. Electron microscopy showed that the helical spirochetal cells possessed an outer sheath, a protoplasmic cylinder, and 4 periplasmic fibrils in a 2-4-2 arrangement. The spirochete grew in an atmosphere of N₂ in prereduced media containing a carbohydrate, NaHCO₃, rumen fluid, yeast extract, peptone, l-cysteine, and inorganic salts. The spirochete fermented carbohydrates and required substrate amounts of CO₂ (HCO ₃ ^- ) for growth. Amino acids were not fermented. Major fermentation products of cells growing with glucose as the substrate and in the presence of CO₂ were acetate, formate, succinate, and lactate. Small amounts of 2,3-butanediol, pyruvate, and acetoin were also formed. Determinations of enzymatic activities in cell extracts, and of radioactivity in products formed by growing cells from [1-¹⁴C]glucose, indicated that this sugar was dissimilated to pyruvate via the Embden-Meyerhof pathway. The spirochetes used a coliform-type clastic reaction to metabolize pyruvate. Determinations of radioactivity in products formed from [¹⁴C]NaHCO₃ indicated that CO₂ was assimilated and used in succinate production. The guanine+cytosine content of the DNA was 36 mol%. This study indicates that this intestinal spirochete represents a new species of Treponema. It is proposed that the new species be named Treponema succinifaciens.Abbreviations cpm counts per minute - DTT dithiothreitol - EM Embden-Meyerhof - GC guanine plus cytosine - IgG immunoglobulin G - PC protoplasmic cylinder - PF periplasmic fibrils (axial fibrils) - OS outer sheath     

Electron Microscopic Observations on the Structure of Treponema zuelzerae and Its Axial Filaments

The fine structure of the spirochete Treponema zuelzerae, and particularly of its axial filaments, was investigated by using the electron microscope. The cell consists of a protoplasmic core surrounded by two concentric envelopes, each approximately 12 nm in width. Between these envelopes are two axial filaments, one originating at each pole of the cell, which overlap and lie side by side in the central region of the cell. The diameter of the axial filaments is 18.0 to 18.5 nm. The terminal region of each filament at its proximal end consists of a hook-like structure, very similar in appearance to the proximal end of a bacterial flagellum. The outer envelope of the cell is readily disrupted with distilled water, and this treatment often results in the release of the filaments from their axial position. A sheath is seen surrounding the filaments when cells are treated with distilled water for no more than 1 min and fixed immediately with osmium tetroxide or glutaraldehyde. This sheath has a striated fine structure and a diameter of 46 nm.     

Ultrastructure of Treponema microdentium and Borrelia vincentii

Bladen, Howard A. (National Institute of Dental Health, Bethesda, Md.), and Edward G. Hampp. Ultrastructure of Treponema microdentium and Borrelia vincentii. J. Bacteriol. 87:1180-1191.-A small oral Treponema (FM) and Borrelia vincentii (N9) were harvested after 3 to 7 days of incubation and either embedded in Vestopal W or negatively stained with phosphotungstate. The protoplasmic cylinders of both strains were identical except for size, and had a triple-structured cell wall as well as intracellular concentric laminations. Protoplasmic cylinders of both strains were enclosed in a cell envelope which appeared amorphous in negatively stained preparations, but which had a triple-structured wall when viewed in thin sections. The cell envelope of strain FM also acted as an envelope for the terminal filament; no filament envelope was evident in strain N9. Large structures which contained variable numbers of organisms and which were representative of spirochetal granules were observed. Protoplasmic cylinders contained within such granules frequently were devoid of cell envelopes. The axial filament consisted of several individual fibers which usually terminated in small end knobs. Occasionally, a fiber of the axial filament became a fiber of the terminal filament. Fibers of the terminal filament originated in end knobs similar to, but separate from, those to which the axial filament was attached. A periodicity of 60 A was occasionally observed in the terminal filament envelope of strain FM. A microperiodicity of approximately 20 A was also observed. The fibers of the terminal filament of strain N9 were composed of a large number of fibrils approximately 15 A wide. The periodicity and fibrillar structure of the terminal filament is discussed with reference to proposed models of bacterial flagella suggested by X-ray diffraction data.     

Analysis of sheath and core structures of the axial filament of Treponema pallidum

Electron microscopy and SDS-PAGE have been used to analyse the polypeptide and antigenic composition of the sheath and core components of the axial filament of Treponema pallidum. The sheath contains a major 37 kDa polypeptide which was solubilized by a combination of trypsin and urea treatments with concurrent loss of binding of anti-37 kDa monoclonal antibody. These studies also indicated some antigenic heterogeneity within the axial filament population. Trypsin treatment alone removed a number of antigenic determinants from the axial filament but left others intact, suggesting differences in their location within the sheath structure. A second 31.5 kDa polypeptide may also be associated with the sheath. The axial filament core comprises at least two components, an antigenically dominant 33.5 kDa polypeptide and a second of 34 kDa. The structure of the axial filament in T. pallidum and Treponema phagedenis biotype Reiterii was similar, but antigenic cross-reactivity of sheath and core components was incomplete.     

Novel ultrastructures of Treponema primitia and their implications for motility

Members of the bacterial phylum Spirochaetes are generally helical cells propelled by periplasmic flagella. The spirochete Treponema primitia is interesting because of its mutualistic role in the termite gut, where it is believed to cooperate with protozoa that break down cellulose and produce H₂ as a by-product. Here we report the ultrastructure of T. primitia as obtained by electron cryotomography of intact, frozen-hydrated cells. Several previously unrecognized external structures were revealed, including bowl-like objects decorating the outer membrane, arcades of hook-shaped proteins winding along the exterior and tufts of fibrils extending from the cell tips. Inside the periplasm, cone-like structures were found at each pole. Instead of the single peptidoglycan layer typical of other Gram-negative bacteria, two distinct periplasmic layers were observed. These layers formed a central open space that contained two flagella situated adjacent to each other. In some areas, the inner membrane formed flattened invaginations that protruded into the cytoplasm. High-speed light microscopic images of swimming T. primitia cells showed that cell bodies remained rigid and moved in a helical rather than planar motion. Together, these findings support the 'rolling cylinder' model for T. primitia motility that posits rotation of the protoplasmic cylinder within the outer sheath.     

The notochordal sheath in amphioxus--an ultrastructural and histochemical study

The notochord and notochordal sheath of 10 adult amphioxus were investigated ultrastructurally and histochemically. The notochord in amphioxus consists of parallel notochordal cells (plates) and each plate consists of parallel thicker and thinner fibrils and numerous profiles of smooth endoplasmic reticulum situated just beneath the cell membrane. Histochemical staining shows that the notochordal plates resemble neither the connective tissue notochordal sheath nor the typical muscular structure myotomes. The notochordal sheath has a complex three-layered organization with the outer, middle and inner layer The outer and middle layer are composed of collagen fibers of different thickness and course, that correspond to collagen type I and collagen type III in vertebrates, respectively, and the inner layer is amorphous, resembles basal lamina, and is closely attached to the notochord by hemidesmosome junctions. These results confirm the presence of collagen fibers and absence of elastic fibers in amphioxus.     

Spherical body formation in the spirochaete Brachyspira hyodysenteriae

When cultures of Brachyspira hyodysenteriae were grown under a wide range of in vitro conditions, at least 1% of the cells formed spherical bodies different to the normal helical form. This percentage increased considerably in aging cultures or following their incubation in caramelized media. Spherical body formation was initiated from a terminal localized swelling of the outer sheath followed by a retraction of the protoplasmic cylinder into the resulting swollen vesicle. As this occurred, the periplasmic flagella seemed to unwind from the protoplasmic cylinder. Once retracted, the protoplasmic cylinder was found to be wrapped in an organized manner around the inner surface of the membrane of the swollen vesicle. Although most were 2-3 microm in diameter, some much larger spherical bodies (6-12 microm diameter) were occasionally seen, with a corresponding increase in the visible number of peripheral protoplasmic cylinder cross-sections. Spherical bodies from older cultures did not contain protoplasmic cylinders arranged around the periphery, but instead were characterized by the presence of a centrally located, electron-dense body c. 0.5-0.8 mum in diameter. Brachyspira hyodysenteriae spherical bodies differ in both their structural organization and probable method of formation from similar structures described in other spirochaete genera.     

MORPHOLOGY AND FINE STRUCTURE OF FISCHERELLA AMBIGUA1

Fischerella ambigua is a branching blue-green alga, the filamentous nature of which is maintained almost entirely by sheath material. Cell division in this organism most closely resembles the septal division found in most unicellular organisms. In all filamentous blue-green algae previously examined with the electron microscope, cell division has resulted from the imagination of the plasma membrane and inner wall layer only; both the middle wall and the outer wall layers remain continuous throughout the length of the filament. In Fischerella, by contrast, the plasma membrane and the inner wall layer invaginate to produce initially 2 cells. However, the middle wall layer, outer wall layer, and sheath also invaginate to separate the daughter cells. The sheath alone remains continuous throughout the length of the filament.     

The fine structure ofCristispira from the lamellibranchCryptomya californica conrad

The fine structure ofCristispira from the lamellibranchCryptomya californica Conrad was examined by Nomarski differential interference, Hoffman modulation contrast, phase contrast, polarizing, and darkfield optics, which were useful for observing the motility, morphology, and organelles of livingCristispira within the crystalline style. Transmission electron microscopy studies of double-stained sections and negatively stained whole cells revealed details of the outer sheath, axial filament, and the protoplasmic cylinder. Hundreds of periplasmic flagella were observed, as well as membrane-bound vesicles and lipid accumulations in the protoplasm, which may help to explain the mutualistic relationship which seems to exist betweenCristispira spp. and their molluscan hosts. Also described are a dialysis method for producing concentrated populations of viableCristispira from the clam, and a minimal-salts medium for their short-term maintenance.     

Fibrillar Array in the Cell Wall of a Gliding Filamentous Cyanobacterium

The cell walls of a number of filamentous, gliding cyanobacteria of the genus Oscillatoria were examined by transmission electron microscopy of ultrathin sections, of freeze-etched replicas, and of whole cells crushed between glass slides and negatively stained. All three techniques revealed the presence of a highly ordered array of parallel fibrils, seen in transverse sections to be situated between the peptidoglycan and the outer membrane. Approximately 200 individual fibrils, each 25 to 30 nm in width, form a parallel, helical array that completely surrounds each cyanobacterial filament, running at an angle of 25 to 30° to its long axis. This highly regular arrangement of the fibrillar layer may imply some underlying symmetry responsible for its organization. A possible source of such symmetry would be the peptidoglycan, and some form of interaction between this layer and the fibrils might provide the necessary scaffolding for the fibrillar array. In crushed, negatively stained samples of fresh cells, individual fibrils were seen outside the filament, released from the cell wall. These released fibrils were of the same width as those observed in situ but were in short lengths, mostly of 100 to 200 nm, and were invariably bent, sometimes even into U shapes, implying great flexibility. Negative staining of released fibrils showed no evidence that they were hollow tubes but did give some indication of a substructure, implying that they were composed of many subunits. The function of this fibrillar array is unknown, although its position in the cell wall, as well as the correspondence between the angle of the fibrils with respect to the long axis of the filament and the rotation of the filament during gliding, may imply an involvement in gliding motility.     

Treponema bryantii sp. nov., a rumen spirochete that interacts with cellulolytic bacteria

A saccharolytic spirochete that associated and interacted with cellulolytic bacteria was isolated from bovine rumen fluid. Isolation was accomplished by means of a procedure involving serial dilution of a sample of rumen fluid into a cellulose-containing agar medium. Clear zones appeared within the medium as a result of cellulose hydrolysis by rumen bacteria. The saccharolytic spirochete and a cellulolytic bacterium later identified as a strain of Bacteroides succinogenes were isolated from the clear zones. The spirochete did not utilize cellulose, but grew in coculture with the cellulolytic bacterium in cellulose-containing media. When cocultured in these media the spirochete used, as fermentable substrates, soluble sugars released from cellulose by the cellulolytic bacterium. In cellulosecontaining agar medium the spirochete enhanced cellulose breakdown by the B. succinogenes strain. Electron microscopy showed that the helical spirochete cells possessed an outer sheath, a protoplasmic cylinder, and two periplasmic fibrils. Under a CO₂ atmosphere, in a reduced medium containing inorganic salts, rumen fluid, glucose, and NaHCO₃, the spirochete grew to a final density of 1.9×10⁹ cells/ml. Succinate, acetate, and formate were products of the fermentation of glucose by growing cells. CO₂ (HCO₃ ^-), branched short-chain fatty acids, folic acid, biotin, niacinamide, thiamine, pyridoxal, and a carbohydrate were required for growth of the spirochete. The results of this study indicated that the rumen spirochete represents a new species of Treponema. It is proposed that the new species be named Treponema bryantii.Abbreviations cpm counts per minute - GC guanine plus cytosine - T_m melting temperature - PC protoplasmic cylinder - PF pertplasmic fibrils (axial fibrils) - OS outer sheath - ID insertion disk     

Ultrastructure and chemical composition of the sheath of Leptothrix discophora SP-6.

Light microscopy and transmission electron microscopy of thin sections and metal-shadowed specimens showed that the sheath of Leptothrix discophora SP-6 (ATCC 51168) is a tube-like extracellular polymeric structure consisting of a condensed fabric of 6.5-nm-diameter fibrils underlying a more diffuse outer capsular layer. In thin sections, outer membrane bridges seen to contact the inner sheath layer suggested that the sheath fabric was attached to the outer layer of the gram-negative cell wall. The capsular polymers showed an affinity for cationic colloidal iron and polycationic ferritin, indicating that they carry a negative charge. Cell-free sheaths were isolated by treatment with a mixture of lysozyme, EDTA, and N-lauroylsarcosine (Sarkosyl) or sodium dodecyl sulfate (SDS). Both Sarkosyl- and SDS-isolated sheaths were indistinguishable in microscopic appearance. However, the Mn-oxidizing activity of Sarkosyl-isolated sheaths was more stable than that of SDS-isolated sheaths. The Sarkosyl-isolated sheaths also contained more 2-keto-3-deoxyoctanoic acid and more outer membrane protein than SDS-isolated sheaths. The oven-dried mass of detergent-isolated sheaths represented approximately 9% of the total oven-dried biomass of SP-6 cultures; the oven-dried sheaths contained 38% C, 6.9% N, 6% H, and 2.1% S and approximately 34 to 35% carbohydrate (polysaccharide), 23 to 25% protein, 8% lipid, and 4% inorganic ash. Gas-liquid chromatography showed that the polysaccharide was an approximately 1:1 mixture of uronic acids (glucuronic, galacturonic, and mannuronic acids and at least one other unidentified uronic acid) and an amino sugar (galactosamine). Neutral sugars were not detected. Amino acid analysis showed that sheath proteins were enriched in cysteine (6 mol%). The cysteine residues in the sheath proteins probably provide sulfhydryls for disulfide bonds that play an important role in maintaining the structural integrity of the sheath (D. Emerson and W.C. Ghiorse, J. Bacteriol. 175:7819-7827, 1993).