Interaction of Mg-2+ with peptidoglycan and its relation to the prevention of lysis of a marine pseudomonad.

Intact cells of marine pseudomonad B-16 (ATCC 19855) which have been washed with a solution of NaCl require only 0.001 M MgSO4 and 100 to 300 times this concentration of NaCl or KCl to prevent lysis. Conversion of intact cells to mureinoplasts, a process involving removal of the outer double-track layer (outer membrane) and the periplasmic space layer of the cell wall, approximately doubled the requirement for the three salts to prevent lysis. The formation of protoplasts from mureinoplasts by removing the peptidoglycan layer again doubled the requirement for Na+ and K+ salts but increased the requirement for the Mg-2+ salt 200- to 300-fold. Cells of the marine pseudomonad suspended in solutions containing Mg-2+ salts failed to lyse on subsequent repeated suspension in distilled water, whereas cells presuspended in NaCl lysed immediately. Isolated envelope layers including the peptidoglycan layer, when dialyzed against solutiions containing Mg-2+ salts, retained Mg-2+ after subsequent suspension in distilled water. Envelope layers exposed to solutions of Na+ or K+ salts failed to retain these ions after exposure to distilled water. Na+ displaced Mg-2+ from the cell envelope layers. The results obtained indicate that the capacity of Mg-2+ salts at very low concentration to prevent lysis of intact cells and mureinoplasts of this organism is due primarily to the interaction of Mg-2+ with the peptidoglycan layer of the cell wall. Ion interaction with the layers lying outside of the peptidoglycan layer contributes only a small amount to the mechanical strength of the wall.     

Electron Microscopic Observations on the Structure of the Envelopes of Mature Elementary Bodies and Developmental Reticulate Forms of Chlamydia psittaci

Purified suspensions of Chlamydia psittaci were prepared from L cells. Thin sections of intact elementary bodies and intact developmental reticulate bodies and of their purified envelopes were observed by electron microscopy. In both intact organisms and partially purified envelopes, two membranous structures, each appearing in electron micrographs as two darkly stained layers, were observed. In the elementary body sections, the outer membrane was round, apparently rigid, and was not soluble in 0.5% sodium dodecyl sulfate. The inner layer was irregular in shape and was completely removed by detergent treatment. We interpret these results to indicate that the outer rigid layer of the envelope is the cell wall and the inner layer is the cytoplasmic membrane. When the fragile reticulate body envelopes were similarly studied, the outer cell wall was clearly visible, and some evidence of an inner membrane was seen. After treatment with nucleases and detergent, all evidence of inner or cytoplasmic membrane was removed, but the outer cell wall remained. Thus, it appears that the cell wall of this organism is continuous throughout the growth cycle and that the fragility and lack of rigidity of the reticulate body cell is due to changes in chemical composition or structure of the cell wall.     

Separation and localization of cell wall layers of a gram-negative bacterium

The cell envelope of a marine pseudomonad as seen in thin section by electron microscopy has the double-membrane structure typical of other gram-negative bacteria. Cells washed with a solution containing Na(+), K(+), and Mg(++) at their concentrations in the growth medium, when suspended briefly in 0.5 m sucrose, lost 13% of their hexosamine in a form nonsedimentable by centrifugation at 73,000 x g. Since the resulting cells in thin section appeared unchanged, it was concluded that the material released was derived from a nonstaining, loosely bound outer layer. This same layer could be removed from the cells by washing with 0.5 m NaCl. A second nonsedimentable fraction was released after successive suspension of the cells in 0.5 m sucrose. Since this material was released only when the outer double-track structure had broken, it was concluded that it arose from a layer immediately underlying the latter layer. The three layers differed in their content of hexosamine and protein. None of the layers released contained muramic or diaminopimelic acid. The cell form remaining was rod shaped and appeared in thin section to be bounded only by its cytoplasmic membrane. This form contained all the muramic and diaminopimelic acid in the cell. Treatment with lysozyme released the muramic and diaminopimelic acid and converted the rod form to a protoplast, indicating that in the rod form (mureinoplast) a thin layer of peptidoglycan is located on the outside surface of the cytoplasmic membrane. Thus, five separate layers have been detected in the cell envelope of this marine pseudomonad.     

Biochemical Localization of Alkaline Phosphatase in the Cell Wall of a Marine Pseudomonad

The various layers of the cell envelope of marine pseudomonad B-16 (ATCC 19855) have been separated from the cells and assayed directly for alkaline phosphatase activity under conditions established previously to be optimum for maintenance of the activity of the enzyme. Under conditions known to lead to the release of the contents of the periplasmic space from the cells, over 90% of the alkaline phosphatase was released into the medium. Neither the loosely bound outer layer nor the outer double-track layer (cell wall membrane) showed significant activity. A small amount of the alkaline phosphatase activity of the cells remained associated with the mureinoplasts when the outer layers of the cell wall were removed. Upon treatment of the mureinoplasts with lysozyme, some alkaline phosphatase was released into the medium and some remained with the protoplasts formed. Cells washed and suspended in 0.5 M NaCl were lysed by treatment with 2% toluene, and 95% of the alkaline phosphatase in the cells was released into the medium. Cells washed and suspended in complete salts solution (0.3 M NaCl, 0.05 M MgSO(4), and 0.01 M KCl) or 0.05 M MgSO(4) appeared intact after treatment with toluene but lost 50 and 10%, respectively, of their alkaline phosphatase. The results suggest that the presence of Mg(2+) in the cell wall is necessary to prevent disruption of the cells by toluene and may also be required to prevent the release of alkaline phosphatase by toluene when disruption of the cells by toluene does not take place.     

Native cell wall organization shown by cryo-electron microscopy confirms the existence of a periplasmic space in Staphylococcus aureus

The current perception of the ultrastructure of gram-positive cell envelopes relies mainly on electron microscopy of thin sections and on sample preparation. Freezing of cells into a matrix of amorphous ice (i.e., vitrification) results in optimal specimen preservation and allows the observation of cell envelope boundary layers in their (frozen) hydrated state. In this report, cryo-transmission electron microscopy of frozen-hydrated sections of Staphylococcus aureus D2C was used to examine cell envelope organization. A bipartite wall was positioned above the plasma membrane and consisted of a 16-nm low-density inner wall zone (IWZ), followed by a 19-nm high-density outer wall zone (OWZ). Observation of plasmolyzed cells, which were used to artificially separate the membrane from the wall, showed membrane vesicles within the space associated with the IWZ in native cells and a large gap between the membrane and OWZ, suggesting that the IWZ was devoid of a cross-linked polymeric cell wall network. Isolated wall fragments possessed only one zone of high density, with a constant level of density throughout their thickness, as was previously seen with the OWZs of intact cells. These results strongly indicate that the IWZ represents a periplasmic space, composed mostly of soluble low-density constituents confined between the plasma membrane and OWZ, and that the OWZ represents the peptidoglycan-teichoic acid cell wall network with its associated proteins. Cell wall differentiation was also seen at the septum of dividing cells. Here, two high-density zones were sandwiched between three low-density zones. It appeared that the septum consisted of an extension of the IWZ and OWZ from the outside peripheral wall, plus a low-density middle zone that separated adjacent septal cross walls, which could contribute to cell separation during division.     

Distribution of lipopolysaccharide and the detection of a new subfraction in the cell envelope of a marine pseudomonad.

The three outer layers of the cell envelope of marine pseudomonad B-16, the loosely bound outer layer, the outer membrane, and the periplasmic space layer, are the only ones containing appreciable amounts of both lipid and carbohydrate. These layers and a fraction released into the medium during growth of the cells were examined for the presence of common antigens by double immunodiffusion using anti-whole serum. Each of the layers, the medium fraction, and lipopolysaccharide (LPS) isolated from the organism were shown to contain two or more diffusible components showing reactions of identity. Thus LPS is found in each of the three outer layers of the cell envelope of this gram-negative bacterium. The periplasmic space layer was found to contain a fraction accounting for 20% of the dry weight of the layer, which was sedimentable at 30,000 x g and contained lipid, protein, and carbohydrate. Double-immunodiffusion tests indicated that the fraction contained at least one of the two antigens present in isolated LPS. A particulate material was released by the cells during growth which gave a positive test for 2-keto-3-deoxyoctulosonic acid and cross-reacted serologically with LPS.     

Characterization of the protein import apparatus in isolated outer envelopes of chloroplasts

Isolated outer envelope membrane from pea (Pisum sativum L.) chloroplasts can be used in vitro to study binding and partial translocation of precursor proteins destined for the inside of the organelle. Efficient binding to a receptor protein on the outside of the membrane vesicle and generation of a translocation intermediate depends strictly on the presence of ATP. Protease treatment of the translocation intermediate demonstrates its insertion into the membrane. The membrane-inserted precursor protein cannot be extracted by 1 M NaCl and is also NaOH resistant to a large extent. Mild solubilization of outer envelope membranes by detergent resulted in the isolation of a complex which still contained the precursor protein. We have identified a constitutively expressed homologue hsc 70 as part of this membrane complex. Antibodies against hsp 70 (inducible heat shock protein 70) were able to immuno-precipitate the complex bound precursor protein. A second protein of 86 kDa molecular weight (OEP 86) from the outer envelope membrane was also identified as a major component of this complex.     

Chemical analysis of the outer membrane and other layers of the cell envelope of Acinetobacter sp

Chemical analysis of fractions of the cell envelope of Acinetobacter sp. strain MJT/F5/199A, prepared by breakage in the French press and removal of plasma membranes, followed by sequential treatment with lysozyme and with papain, confirmed the existence of layers previously identified by electron microscopy. Outside the plasma membrane and periplasmic space, the envelope is composed of (i) a peptidoglycan-containing dense layer, (ii) an intermediate layer, (iii) a lipopolysaccharide-containing outer membrane, and (iv) an ordered array of protein subunits. A small amount of carbohydrate (3%) is found associated with protein in the fraction containing both the surface subunits and the intermediate layer. The papain-treated outer membranes contain 67% protein, 24% lipid, together with 11% lipopolysaccharide, and about 6% of non-lipopolysaccharide hexosamine. Lipid is located only in the papain-treated outer-membrane and is mainly phospholipid: 29% phosphatidyl glycerol, 30% phosphatidyl ethanolamine, and 40% cardiolipin. The principal fatty acid is C(18:1). Significant amounts of alcohols(16:1) and alcohols(18:1), which are found in Acinetobacter waxes, were recovered from the outer membrane.     

Relationship of a Wall-Associated Enzyme with Specific Layers of the Cell Wall of a Gram-Negative Bacterium

In untreated cells of the marine pseudomonad studied here, alkaline phosphatase was found to be located in the periplasmic space, at the cell surface, and in the medium into which it had been shed during growth. Washing in 0.5 M NaCl, which removed the loosely bound outer layer, caused a shift of periplasmic enzyme to the outer aspect of the double-track layer and released some of the cell surface-associated enzyme. When the double-track layer of the cell wall was partially deranged, large amounts of this cell wall-associated enzyme were released, and, when the double-track was removed from the cells to produce mureinoplasts, alkaline phosphatase was released into the menstruum. There was no significant association of the enzyme with the peptidoglycan layer of the cell wall, which is the outermost structure of the mureinoplast, and no association of the enzyme with the cytoplasmic membrane of these modified cells. This study has shown that alkaline phosphatase is specifically associated with the outer layers of the cell walls of cells of this organism and is retained within the cell wall by virtue of this association.     

ELECTRON MICROSCOPE STUDY OF MYCOBACTERIUM LEPRAE AND ITS ENVIRONMENT IN A VESICULAR LEPROUS LESION.

Imaeda, Tamotsu (Instituto Venezolano de Investigaciones Cientificas, Caracas, Venezuela) and Jacinto Convit. Electron microscope study of Mycobacterium leprae and its environment in a vesicular leprous lesion. J. Bacteriol. 83:43-52. 1962.-Biopsied specimens of a borderline leprosy lesion were observed with the electron microscope. In this lesion, the majority of Mycobacterium leprae were laden with cytoplasmic components. The bacilli were separated from the cytoplasm of host cells by an enclosing membrane, thus differing from the environment of well-developed lepra cells in lepromatous lesions.The cell wall is composed of a moderately dense layer. A diffuse layer is discernible outside the cell wall, separated from it by a low density space. It is suggested that the cell wall is further coated by a low density layer, although the nature of the outermost diffuse layer has not yet been determined.The plasma membrane consists of a double layer, i.e., dense inner and outer layers separated by a low density space. The outer layer is closely adjacent to the cell wall. In the region where the outer layer of the plasma membrane enters the cytoplasm and is transformed into a complex membranous structure, the inner layer encloses this membranous configuration. Together they form the intracytoplasmic membrane system.In the bacterial cytoplasm, moderately dense, presumably polyphosphate bodies are apparent. As neither these bodies nor the intracytoplasmic membrane system are visible in the degenerating bacilli, it seems probable that these two components represent indicators of the state of bacillary activity.     

Components of the regular surface array of Aquaspirillum serpens MW5 and their assembly in vitro.

The two-layered regular surface array of Aquaspirillum serpens MW5 was removed from cell envelopes and dissociated into subunits by treatment with 6 M urea. The surface components reassembled onto an outer membrane surface and self-assembled into planar sheets in vitro in the presence of Ca2+ or Sr2+. The two layers were removed sequentially from cell envelopes by a two-step extraction procedure involving initial treatment with a high-pH buffer to remove the outermost surface layer and subsequent treatment with 6 M urea to remove the innermost layer. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the outer and inner layers of the array were composed of two proteins with molecular weights of 125,000 and 150,000, respectively. The two layers assembled sequentially; the 150,000-molecular-weight protein formed an array on an outer membrane surface, and the 125,000-molecular-weight protein required that array as a template for its in vitro assembly.     

Morphological alterations of cell wall concomitant with protein release in a protein-producing bacterium, Bacillus brevis 47.

Bacillus brevis 47 secreted vast amounts of protein into the medium and had a characteristic three-layered cell wall. The three layers are designated, from the outermost to the innermost layer, as the outer wall (4.2 nm), the middle wall(8.5 nm), and the inner wall (2.1-3.7 nm). The inner wall might be a peptidoglycan layer. The fine cell wall structure was morphologically altered to various extents, depending on the growth period. At the early stationary phase of growth, cells began to shed the outer two layers of a limited area of the surface. This shedding was complete after further cell growth. The morphological alterations in the cell wall occurred concomitantly with a prominent increase in protein excretion. When protein secretion was severely inhibited by growing cells with Mg2+, morphological alterations in the cell wall were not observed, even at the late stationary phase of growth. This was also the case with a nonprotein-producing mutant, strain 47-5-25. When cells were incubated in buffers, the outer two layers of the cell wall were specifically removed, leaving cells surrounded only by the inner wall layer. The layers removed by incubation were recovered by high-speed centrifugation. This fraction consisted of two layers resembling the outer and middle wall layers. Protein secreted by B. brevis 47-5 consisted mainly of two proteins with approximate molecular weights of 150,000 and 130,000. Proteins released by incubating cells in buffers and proteins in the outer- and middle-wall-enriched fraction were also composed mainly of two proteins with the same molecular weights as those secreted into the medium. Therefore, we conclude that B. brevis 47 secretes proteins derived from the outer two layers of cell wall and these components are synthesized even after the shedding of the outer two layers.     

Comparative studies of the cell structures of Mycobacterium leprae and M. tuberculosis using the electron microscopy freeze-substitution technique

The cell envelope and cytoplasmic architecture of the Mycobacterium leprae Thai-53 strain were examined using the freeze-substitution technique of electron microscopy and compared with those of the M. tuberculosis H37Rv strain. Both strains had similarly multilayered envelope architectures composed of an electron-translucent layer, a peptidoglycan layer and the plasma membrane, from outside to inside. A comparison of the structures of these two mycobacteria revealed that the M. leprae cell was smaller in size and had a thinner peptidoglycan layer than the M. tuberculosis cell. The cell widths measured on electron micrographs were 0.44 microm for M. tuberculosis and 0.38 microm for M. leprae. The peptidoglycan layer of M. leprae was 4-5 nm, while the corresponding layer of M. tuberculosis was 10-15 nm.     

NUCLEAR ENVELOPE-CHLOROPLAST RELATIONSHIPS IN ALGAE

In Ochromonas danica and two related species (Chrysophyceae) and in Rhodomonas lens and Cryptomonas sp. (Cryptophyceae), the chloroplast is surrounded by an outer double-membraned envelope which lies outside the usual double-membraned chloroplast envelope. At the borders of the area where the chloroplast lies adjacent to the nucleus, this outer envelope is continuous with the outer membrane of the nuclear envelope as a double-membraned outfolding, so that the entire chloroplast in these species lies within a double-membraned sac, one wall of which is the nuclear envelope. In Olisthodiscus sp. (Chrysophyceae ?), each of the small peripheral chloroplasts is surrounded by a similar double-membraned outer envelope, but in this species no connections with the nuclear envelope were observed. In the Ochromonadaceae, a characteristic array of tubules is present within the sac in the narrow space which separates the chloroplast from the nucleus. In the other species studied, tubules are present at places between the chloroplast envelope and the outer envelope. In the Cryptophyceae, the starch grains lie outside the chloroplast envelope, but within the outer double-membraned sac. A double-membraned outer envelope appears to be present outside the chloroplasts of the Phaeophyta and Euglenophyta, but seems to be absent in the other groups of algae.     

Heterocysts with reduced cell walls in populations of cycad cyanobionts

The ultrastructure of the cyanobionts of the greenhouse-grown cycads Cycads circinalis, Ceratozamia mexicana, and Encephalartos villosus was studied. In addition to heterocysts with the typical ultrastructure, the cyanobiont microcolonies also contained altered heterocysts with reduced cell walls, which might dominate in all regions of the coralloid roots. The altered heterocysts represented a protoplast enclosed in a heterocyst-specific envelope with additional layers. Some heterocysts contained an additional reticular protoplast-enclosing sheath below the heterocyst-specific envelope, whereas the other heterocysts contained an additional electron-opaque outer layer. The substance of the inner sheath of the former heterocysts resembled the polysaccharides of mucilage, which fills the intercellular space of plant tissues, whereas the electron-opaque outer layer of the latter heterocysts probably had a protein nature. The substances that constitute the sheath and the outer layer are likely to be synthesized intracellularly and then released with the aid of membrane-bounded vesicles or by channels in the cytoplasmic membrane.     

Morphological and ultrastructural study of the cell envelope of thermophilic and acidophilic microorganisms as compared to Thiobacillus ferrooxidans

The cell envelope of a Sulfolobus-like microorganism has an arrayed hexagonal subunit structure, a double-layered cytoplasmic membrane, and a hollow periplasmic space between the plasma membrane and the outermost arrayed layer. A dense peptidoglycan layer outside the plasma membrane found in the case of Thiobacillus ferrooxidans was not seen. The cell envelope of a thermophile isolated from a leaching environment has a well-defined envelope with two well-stained layers distinclty seen. While the peptidoglycan layer is also not seen in this thermophile, a long flagellum similar to that in the case of T. ferrooxidans is present. The presence of pili in the Sulfolobus-like organism and its arrayed subunit cell envelope structure could account for the organism's selective attachment to sulfide phases in the leaching of low-grade ores. The observations of a well-defined cell envelope in the two thermophiles is consistent with the structure-function relationship previously established for T. ferrooxidans.     

Capacity of the outer membrane of a gram-negative marine bacterium in the presence of cations to prevent lysis by Triton X-100.

Cells of marine pseudomonad B-16 (ATCC 19855) washed with a solution containing 0.3 M NaCl, 50 mM MgCl2, and 10 mM KCl (complete salts) could be protected from lysis in a hypotonic environment if the suspending medium contained either 20 mM Mg2+, 40 mM Na+, or 300 mM K+. When the outer double-track layer (the outer membrane) of the cell envelope was removed to yield mureinoplasts, the Mg2+, Na+ or K+, requirements to prevent lysis were raised to 80, 210, and 400 mM, respectively. In the presence of 0.1% Triton X-100, 220, 320, and 360 mM Mg2+, Na+ or K+, respectively, prevented lysis of the normal cells. Mureinoplasts and protoplasts, however, lysed instantly in the presence of the detergent at all concentrations of Mg2+, Na+, or K+ tested up to 1.2 M. Thus, the structure of the outer membrane appears to be maintained by appropriate concentrations of Mg2+ or Na+ in a form preventing the penetration of Triton X-100 and thereby protecting the cytoplasmic membrane from dissolution by the detergent. K+ was effective in this capacity with cells washed with complete salts solution but not with cells washed with a solution of NaCl, suggesting that bound Mg2+ was required in the cell wall membrane for K+ to be effective in preventing lysis by the detergent. At high concentrations (1 M) K+ and Mg2+, but not Na+, appeared to destabilize the structure of the outer membrane in the presence of Triton X-100.     

Isolation and characterization of the outer membrane of Neisseria gonorrhoeae

The cell envelope of Neisseria gonorrhoeae strain 2686, colonial type 4, was isolated from spheroplasts formed by the action of ethylenediaminetetraacetic acid and lysozyme. Isopycnic centrifugation of osmotically ruptured spheroplasts resolved the cell envelope into two main membrane fractions. Chemical and enzymatic analyses were used to characterize these isolated membranes. Succinic dehydrogenase, reduced nicotinamide adenine dinucleotide oxidase, and d-lactate dehydrogenase were localized in the membrane fraction of buoyant density, rho degrees = 1.141 g/cm(3). Lipopolysaccharide and over half of the cell envelope protein were associated with the membrane that banded in sucrose at rho degrees = 1.219 g/cm(3). These fractions were consequently designated cytoplasmic and outer or L-membrane, respectively. Sodium dodecyl sulfate-polyacrylamide electrophoresis of isolated membranes demonstrated the relative simplicity of the protein spectrum of the outer membrane. The majority of the protein in this membrane could be accounted for by proteins of molecular weights 34,500, 22,000, and 11,500. The protein of molecular weight 34,500 accounted for 66% of the total protein of the L-membrane. Isoelectric precipitation at pH 4.6 with 10% acetic acid selectively removed this protein from a 150 mM NaCl in 10 mM tris(hydroxymethyl)aminomethane-hydrochloride, pH 7.4, extract of purified outer membrane. At pH 4.0, the other proteins of the L-membrane were precipitated. It was concluded that the membrane components of the cell envelope of N. gonorrhoeae were similar to those of other gram-negative bacteria. The cell envelope fractions described here, in particular the outer membrane, are sufficiently well defined to provide a valuable tool for future biochemical and immunological studies on N. gonorrhoeae.     

Tegumentary glands of the supra-anal pit of Scutigerellidae (Symphala, Myriapoda)]

Tegumentary glands of the 'supra-anal pit' in the genus Scutigerella are ductule-associated glandular cells. The invaginated cavity consists of two distinct parts, the inner bearing microvilli collector. The efferent ductule penetrates into the upper part of the cavity by means of a receiving tubule, the wall of which is perforated and composed of two layers having different electron densities. The glandular cell cytoplasm is packed with smooth endoplasmic reticulum which arises from rough endoplasmic reticulum and by blebbing of the outer membrane of the nuclear envelope, blebs immediately losing their ribosomes. Secretion granules are released into the extracellular invaginated cavity between the microvilli and form an amorphous layer that covers the cuticular invagination of the 'supra-anal pit'.