Ultrastructure of the obligate halophilic bacterium Halobacterium halobium

The fine structure of Halobacterium halobium was studied by means of a modified double-fixation technique. The cell envelope is shown to consist of both a “wall” and a plasma membrane. Some electron-dense strands were seen inside the cytoplasm running parallel to the cell envelope. An unusual organelle (or organelles) appeared inside the cytoplasm in the form of parallel striated strands.     

Structure of the Purple Membrane

IN the preceding article¹ it has been shown that a cell membrane fragment, the purple membrane, isolated from Halobacterium halobium² contains retinal bound in a mole ratio of 1 : 1 to a protein of molecular weight 26,000 which is the only protein present. We now describe the structure of the purple membrane and its localization in the bacterial cell envelope.     

THE CELL ENVELOPES OF TWO EXTREMELY HALOPHILIC BACTERIA

The cell envelope of Halobacterium halobium was seen in thin sections of permanganate-fixed cells to consist of one membrane. This membrane appeared mostly as a unit membrane but in a few preparations it resembled a 5-layered compound membrane. The cell envelope of Halobacterium salinarium at high resolution was always seen as a 5-layered structure different in appearance from the apparent compound membrane of H. halobium. The "envelopes" which were isolated in 12.5 per cent NaCl from each organism were indistinguishable from each other in the electron microscope and comprised, in each case, a single unit membrane with an over-all thickness of about 110 A. Some chemical analyses were made of isolated membranes after freeing them from salt by precipitating and washing with trichloroacetic acid. Such precipitated membranes consisted predominantly of protein, with little carbohydrate and no peptido-aminopolysaccharide (mucopeptide). Sectioned whole cells of H. halobium contained intracellular electron-opaque structures of unknown function.     

The cell envelope of Thermoproteus tenax: three-dimensional structure of the surface layer and its role in shape maintenance

The sulphur-dependent archaebacterium Thermoproteus tenax has a cylindrical cell shape variable in length, but constant in diameter. Its whole surface is covered by a regular protein layer (S-layer). The lattice has p6 symmetry and a lattice constant of 32.8 nm. The three-dimensional reconstruction from a tilt series of isolated and negatively stained S-layer shows a complex mass distribution of the protein: a prominent, pillar-shaped protrusion is located at the 6-fold crystallographic axis with radiating arms connecting neighbouring hexamers in the vicinity of the 3-fold axis. The base vectors of the S-layer lattice have a preferred orientation with respect to the longitudinal axis of the cell. The layer can be seen as a helical structure consisting of a right-handed, two-stranded helix, with the individual chains running parallel. Supposing that new S-layer protein is inserted at lattice faults (wedge disclinations) near the poles, growing of the layer would then proceed by moving a disclination at the end of the helix. The constant shape of the cell, as well as the particular structure of the layer, strongly suggest that this S-layer has a shape-maintaining function.     

The cell surface glycoprotein layer of the extreme halophile Halobacterium salinarum and its relation to Haloferax volcanii: cryo-electron tomography of freeze-substituted cells and projection studies of negatively stained envelopes

We have studied the surface layer (S-layer) of Halobacterium salinarum (formerly Halobacterium halobium), an extreme halophile requiring high concentrations of sodium, by electron microscopy of (a) isolated, negatively stained, flattened envelopes and (b) cryo-fixation of intact cells in their high-salt growth medium followed by freeze substitution and tomography of thin sections. From the negatively stained isolated envelopes we have calculated a two-dimensional, projection map that is strikingly similar to that of Haloferax volcanii, an extreme halophile requiring high concentrations of magnesium; both projection maps show the hexagonal arrangement of the morphological units with an identical center-to-center spacing of 150 A; each of the morphological units of the two species has six subunits with a similar density distribution and apparent domain organization. In contrast to the two-dimensional map, the tomographic reconstruction of Halob. salinarum does not agree in a straightforward way with the three-dimensional, electron crystallographic map of negatively stained Halof. volcanii envelopes, although the main features of the lattice and the morphological units are evident. The tomographic reconstruction of sections from epoxy-embedded material suffers from directional compression due to sectioning stress and continuous dimensional changes and mass loss due to electron irradiation. This communication consists, therefore, of three parts: (a) a comparison of the projection maps of negatively stained envelopes of Halof. volcanii and Halob. salinarum; (b) a comparison of the three-dimensional maps obtained by electron crystallography (Halof. volcanii) and low-dose cryo-tomography (Halob. salinarum); and (c) a methodological study of mass loss and dimensional changes of plastic-embedded material under low-dose conditions at room and liquid nitrogen temperatures.     

Cell surface display of chimeric glycoproteins via the S-layer of Paenibacillus alvei

The Gram-positive, mesophilic bacterium Paenibacillus alvei CCM 2051^T possesses a two-dimensional crystalline protein surface layer (S-layer) with oblique lattice symmetry composed of a single type of O-glycoprotein species. Herein, we describe a strategy for nanopatterned in vivo cell surface co-display of peptide and glycan epitopes based on this S-layer glycoprotein self-assembly system. The open reading frame of the corresponding structural gene spaA codes for a protein of 983 amino acids, including a signal peptide of 24 amino acids. The mature S-layer protein has a theoretical molecular mass of 105.95 kDa and a calculated pI of 5.83. It contains three S-layer homology domains at the N-terminus that are involved in anchoring of the glycoprotein via a non-classical, pyruvylated secondary cell wall polymer to the peptidoglycan layer of the cell wall. For this polymer, several putative biosynthesis enzymes were identified upstream of the spaA gene. For in vivo cell surface display, the hexahistidine tag and the enhanced green fluorescent protein, respectively, were translationally fused to the C-terminus of SpaA. Immunoblot analysis, immunofluorescence staining, and fluorescence microscopy revealed that the fused epitopes were efficiently expressed and successfully displayed via the S-layer glycoprotein matrix on the surface of P. alvei CCM 2051^T cells. In contrast, exclusively non-glycosylated chimeric SpaA proteins were displayed, when the S-layer of the glycosylation-deficient wsfP mutant was used as a display matrix.     

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.     

Bacterial and Archaeal S-Layers as a Subject of Nanobiotechnology

Many bacteria and archaea have a crystalline surface layer (S-layer), which overlies the cell envelope. S-layers each consist of one protein or glycoprotein species. Protein subunits of the S-layer noncovalently interact with each other and with the underlying cell-envelope component. On average, the S-layer lattice has pores of 2–6 nm and is 5–10 nm high. Isolated S-layer proteins recrystallize to form two-dimensional crystalline structures in solution, on a solid support, and on planar lipid membranes. Owing to this unique property, S-layers have a broad range of applications. This review focuses on the structural features and applications of S-layers and their proteins, with special emphasis on their use in nanobiotechnology.     

Lipid-Induced Modulation of the Protein Packing in Two-Dimensional Crystals of Bacteriorhodopsin

The mechanism by which bacteriorhodopsin (BR), the light-driven proton pump from the purple membrane (PM) of Halobacterium halobium, arranges in a 2D hexagonal array has been studied by reconstitution of BR in complexes of two types of bilayer made either with PM-derived lipids or with PM lipids and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). The unit cell dimensions of the 2D protein crystals, determined by correlation averaging analysis of freeze-fracture electron micrographs, were compared with the lattice constant of the PM. In complexes made with delipidated BR and with the polar lipids extracted from H. halobium cells (HHPL), BR trimers are arranged in a hexagonal lattice with the same lattice constant of 5.9 ± 0.2 nm as found in the PM. In BR-containing complexes made with PM-derived lipids and DMPC at several protein:lipid mole ratios, BR trimers are also arranged in a hexagonal lattice, but with a unit cell dimension of 9.2 ± 0.2 nm, which is about one-third larger compared to that measured in PM (Michel et al. , 1980). In a subclass of this type of complexes, orthogonal BR arrays were observed with a lattice constant of 5.9 × 9.9 ± 0.2 nm. It appears that insertion of DMPC into the BP/PM-derived lipid complexes increases the center-to-center distances in both array types by a discrete amount.     

Identification and characterization of fusion and processing domains of the human immunodeficiency virus type 2 envelope glycoprotein.

The envelope glycoprotein of the human immunodeficiency virus type 2 (HIV-2) is synthesized as a polyprotein precursor which is proteolytically processed to produce the mature surface and transmembrane envelope glycoproteins. The processed envelope glycoprotein species are responsible for the fusion between the viral envelope and the host cell membrane during the infection process. The envelope glycoprotein also induces syncytium formation between envelope-expressing cells and receptor-bearing cells. To characterize domains of the HIV-2 envelope glycoprotein involved in membrane fusion and in proteolytic processing, we introduced single amino acid mutations into the region of the HIV-2 surface glycoprotein corresponding to the principal neutralizing determinant (the V3 loop) of HIV-1, the putative HIV-2 envelope precursor-processing sequence, and the hydrophobic amino terminus of the HIV-2 transmembrane envelope glycoprotein. The effects of these mutations on syncytium formation, virus infectivity, envelope expression, envelope processing, and CD4 binding were analyzed. Our results suggest that the V3-like region of the HIV-2 surface glycoprotein and the hydrophobic amino terminus of the transmembrane glycoprotein are HIV-2 fusion domains and characterize the effects of mutations in the HIV-2 envelope glycoprotein precursor-processing sequence.     

BamA is a pivotal protein in cell envelope synthesis and cell division in Deinococcus radiodurans

The beta-barrel assembly machinery (BAM) is an indispensable complex for protein transportation located at the outer membrane of bacteria. BAM is composed of five subunits (BamA-E) in the model bacterium Escherichia coli. DR_0379 is a BamA homolog in Deinococcus radiodurans, but the other subunits have not been detected in this species. In the present study, deletion of bamA resulted in decreased growth rate and altered morphology of D. radiodurans. ΔbamA cells underwent abnormal cell division, leading to aggregated bacteria of diverse size and shape, and the cell envelope was detached from the cell surface, resulting in reduced resistance to high ionic strength. Oxidative stress resistance was significantly enhanced in the mutant, which may be attributed to increased manganese ion concentration and Mn/Fe ratio. Numerous proteins were released into the medium from ΔbamA cells, including surface layer (S-layer) proteins and various transporters located in the periplasm and outer membrane. These results indicate that BamA affects the synthesis and assembly of the outer membrane and S-layer, and thereby influences material transport and cell division. The findings highlight the special functions of BamA in D. radiodurans, and promote our understanding of the multi-layer structure of the D. radiodurans cell envelope.     

The quenching effect of blue light on halorhodopsin

A mutant of Halobacterium halobium which contains halorhodopsin was isolated from strain S₉. An absorbance change at 380 nm caused by steady orange light illumination (λ ?530 nm) was observed. This change depended upon the intensity of the actinic light. The bleached envelope vesicles and vesicles derived from nicotine-grown cells showed a small or no absorbance change at 380 nm, suggesting that the change stemmed from the photochemical intermediate of halorhodopsin (referred to as P-380). When blue light was superimposed on orange background illumination, the membrane potential (Δψ) of the envelope vesicles decreased. Δψ was determined from the tetraphenylphosphonium cation (TPP⁺) distribution by means of a TPP⁺ electrode. When blue light intensity was increased, both Δψ and the amount of P-380 were decreased. An equation was derived which showed that Δψ is proportional to the concentration of P-380 formed by illumination under the assumption that the ionic composition is not significantly changed upon illumination. This equation was checked experimentally from the following three points: The blue light effect, the relationship between Δψ and light intensity, and the effect of gramicidin. The data obtained accorded well with the theoretical relationship.     

Bacterial and archaeal S-layers as object of bionanotechnology

Many species of Bacteria and Archaea posses a regularly structured surface layers (S-layers) as outermost cell envelope component. S-layers composed of a single protein or glycoprotein species. The individual subunits of S-layers interact with each other and with the supporting bacterial envelope component through non-covalent forces. Pores in the crystalline protein network are with mean diameter of 2-6 nm, the thickness of S-layer is 5-10 nm. The isolated S-layer subunits reassemble into two-dimensional crystalline arrays in solution, on solid supports, on planar lipid films. These unique features of S-layers have led to a broad spectrum applications. This review focuses on the structural properties S-layers and S-proteins and their applications with accent to using this structures in nanobiotechnology.     

Frequency and structure of spinae on Chlorobium spp.

Several Chlorobium species have been observed to possess spinae. Spinae are non-prosthecate, helically wound, rigid structures that extend from the outer bacterial cell surface into the external environment. Spinae length was variable within and between Chlorobium species. Spinae width was fairly consistent within species but varied between species (39.4 ± 2.6 nm to 82.6 ± 8.0 nm). The number of spinae per cell varied. The spinae did not penetrate the bacterial cell envelope and were randomly located on the cell surface. Spinae were not geographically restricted. The observation of spinae on pure cultures of Chlorobium spp. maintained for 25–30 years suggests that spinae may be of significant use to the cell.     

Role of the S layer in morphogenesis and cell division of the archaebacterium Methanocorpusculum sinense.

Thin sections, freeze-etched, and negatively stained preparations of Methanocorpusculum sinense cells reveal a highly lobed cell structure with a hexagonally arranged surface layer (S layer). Digital image processing of negatively stained envelope fragments show that the S layer forms a porous but strongly interconnected network. Since the S layer is the exclusive cell envelope component outside the cytoplasmic membrane it must have a cell shape determining and maintaining function. Although lattice faults such as disclinations and dislocations are a geometrical necessity on the surface of a closed protein crystal, our data indicate that they also play important roles as sites for the incorporation of new morphological units, in the formation of the lobed cell structure, and in the cell division process. In freeze-etched preparations of intact cells numerous positive and negative 60 degree wedge disclinations can be detected which form pentagons and heptagons in the hexagonal array. Complementary pairs of pentagons and heptagons are the termination points of edge dislocations. They can be expected to function both as sites for incorporation of new morphological units into the lattice and as initiation points for the cell division process. The latter is determined by the ratio between the increase of protoplast volume and the increase in actual S-layer surface area during cell growth. We postulate that this mode of cell fission represents a common feature in lobed archaebacteria which possess an S layer as the exclusive wall component.     

Distribution of nuclear pores and perinuclear dense substances in spermatocytes I of some oedionychina fleabeetles

The nuclear envelope of growing postpachytene spermatocyte I differs notably in structure between the fleabeetles Omophoita cyanipennis and Oedionychus bicolor. The former species shows a more conventional structure with an even and probably random distribution of nuclear pores, and a strongly electron-opaque layer of fibrogranular material (FM) separated from the outer nuclear membrane by an intermediate layer of about 40 nm thickness. Peripherally from the FM layer, a continuous corona of granular dense material (GM) is accumulated around the nucleus. In Oedionychus, all nuclear pores are clustered in “nuclear sieves”, i.e., shallow, cup-like indentations of the nuclear envelope. The sieves are filled with an electron-opaque substance resembling the FM of the Omophoita spermatocytes. This substance is kept at a distance of about 40 nm from the outer nuclear membrane. GM is produced only at the sieves, and is thus discontinuous. The sieves with their contents are called nuclear sieve complexes (NSC).     

Three-dimensional structure of a cell wall glycoprotein

Electron microscopy and computer image analysis have been used to determine the three-dimensional structure of the crystalline glycoprotein cell wall layer of the alga Lobomonas piriformis. Images of negatively stained specimens, tilted through a range of angles up to 70 °, were combined to give a map of the molecular envelope to a resolution of 2.0 nm. The cell wall layer consists of crystalline plates the centres and edges of which display distinctly different but isomorphous structures. A comparison of three-dimensional reconstructions of the two areas shows the difference probably to be due to a conformational change of one of the glycoprotein subunits. The structure consists of two sets of dimers composed of rod-shaped subunits which lie with their long axes approximately in the plane of the crystal. The centre-edge transition may have significance in the pathway of accretion of new subunits during cell wall growth.     

Structural properties of the outer membrane and the regular surface protein of Comamonas acidovorans

The outer membrane of Comamonas acidovorans, formerly Pseudomonas acidovorans, contains a regularly arrayed surface protein. The tetragonal lattice (p4 symmetry, unit cell dimensions a = B = 10.5 nm is composed of a single type of polypeptide. It forms dimeric morphological complexes as revealed by means of electron microscopy in conjunction with image processing, STEM mass determination, and IR analysis. The surface protein has tightly associated carbohydrates and behaves like a glycoprotein in electrophoresis and IR spectroscopy. The outer membrane proteins Omp21 and Omp32 are not regularly arrayed. Omp32 has the characteristic attributes of an intrinsic outer membrane protein, such as moderate hydrophobicity, a high β-structure content, and a typical solubilization behavior. It forms channels in black lipid membranes and it, therefore, represents the major porin of C. acidovorans.     

Reconstitution of an ordered structure from major outer membrane constituents and the lipoprotein-bearing peptidoglycan sacculus of Escherichia coli.

An ordered hexagonal lattice structure with a lattice constant of about 7 nm was reconstituted on the entire surface of the lipoprotein-bearing peptidoglycan from outer membrane protein O-8 and lipopolysaccharide. The lattice structure resembled that observed in the cell envelope which had been treated with sodium dodecyl sulfate (Steven et al., J. Cell Biol. 72:292-301, 1977). The omission of either O-8 or lipopolysaccharide resulted in the failure of formation of the lattice structure. No ordered lattice was formed on the peptidoglycan lacking the bound form of the lipoprotein. In the absence of the lipoprotein-bearing peptidoglycan, O-8 and lipopolysaccharide assembled into vesicles with an ordered hexagonal lattice, the lattice constant of which was also about 7 nm. A preliminary experiment indicated that protein O-9 gave the same result as did O-8. These results strongly indicate that O-8 and/or O-9 and lipopolysaccharide provide the ordered framework of the outer membrane and that the bound form of the lipoprotein plays a role in the holding of the framework on the peptidoglycan layer.     

The crystalline glycoprotein cell wall of the green alga Chlorogonium elongatum: a structural analysis.

Members of the Chlamydomonaceae, mostly single-celled green algae, have been shown to contain a crystalline glycoprotein cell wall component. Most of the species examined fall into a class of algae whose walls have an identical crystalline unit cell. Chlorogonium elongatum has been chosen as a representative of this class in order to investigate in more detail its cell wall structure. The alga has a spindleshaped cell wall which retains its asymmetric shape on isolation. Sections from walls fized in the presence of tannic acid clearly reveal a regular subunit monolayer, about 20 nm thick, within the wall. Sodium dodecylsulphate (SDS) polyacrylamide gel electrophoresis shows the presence of at least 2 major glycoprotein species in the wall. Negatively stained purified cell walls demonstrate the crystalline nature of the cell wall. Optical diffraction of bright-field images and direct electron diffraction both give clear diffraction patterns whose spacings extend out to 3 nm and fall on a reciprocal lattice whose vectors describe a 2-dimensional unit cell within the wall 21.5 nm X 7.0 nm and an included angle of 80 degrees. Lattice defects within the cell wall are revealed by both negative staining and surface replication. Through-focal series were used to choose images with the optimal degree of underfocus for image processing. Linear integration and optical filtering of such images gave essentially the same result. A similar image was also obtained by computing the autocorrelation function of the amplitudes in the electron-diffraction pattern and the optical-diffraction pattern of the in-focus image. On the basis of these data a 2-dimensional model of the crystalline cell wall layer is presented.