F- mating materials able to generate a mating signal in mating with HfrH dnaB(Ts) cells.

The purified outer membrane from F- (W1-3) cells was shown to inhibit mating effectively, but the purified cytoplasmic (inner) membrane did not. These membranes, heat-treated minicells, and ultraviolet-irradiated minicells were examined for their ability to generate a mating signal at 43 degrees C in mating with HfrH dnaB(Ts) cells. The outer and inner membranes and heat-treated minicells all failed to stimulate incorporation of radioactive thymine; only ultraviolet-irradiated minicells retained the ability to generate a mating signal for the donor to initiate transfer replication.     

Freeze-Etch Study of Pseudomonas aeruginosa: Localization Within the Cell Wall of an Ethylenediaminetetraacetate-Extractable Component

A freeze-etch study of normal cells of Pseudomonas aeruginosa and of cells after incubation with ethylenediaminetetraacetate (EDTA) and tris(hydroxymethyl)aminomethane (Tris) was performed. When cells were freeze-etched without a cryoprotective agent, a smooth outer cell wall layer, which showed a regular array of subunits, and the presence of flagella and pili were observed. These features were not observed in cells freeze-etched after cryoprotection with glycerol. Four fracture surfaces, which resulted from splitting down the center of the outer wall membrane and of the inner cytoplasmic membrane, were revealed in freeze-etched glycerol-protected cells. The murein layer was seen in profile between the outer cell wall membrane and the cytoplasmic membrane. Spherical units and small rods composed of the spherical units were observed in the inner layer of the outer cell wall membrane. These spherical units appeared to be attached to, or embedded in, the inner face of the outer layer of the outer cell wall membrane. These spherical units were removed from cells on exposure to EDTA-Tris, resulting in cells that were osmotically fragile. The spherical units were detected via electron microscopy of negatively stained preparations in the supernatant fluid of cellular suspensions treated with EDTA-Tris. Upon addition of Mg(2+), the spherical units were reaggregated into the inner layer of the outer cell wall membrane and the cells were restored to osmotic stability. The spherical units were shown to consist primarily of protein. These data are thought to represent the first ultrastructural demonstration of reaggregation of cell wall components within a living cell system.     

Fine Structure of Selected Marine Pseudomonads and Achromobacters

The fine structure of more than 20 marine pseudomonads and more than 15 achromobacters was examined. Under the conditions extant, clear differences between members of these two groups were seen. The pseudomonads displayed the characteristic gram-negative morphology: the cell wall was irregularly undulant and the cytoplasmic membrane more nearly planar, ribonucleoprotein (RNP) particles were loosely packed throughout the periphery of the cytoplasm, and the deoxyribonucleic acid (DNA) was axially disposed. Cell division appeared to be by constriction. Some strains characteristically produced evaginations or blebs of the cell wall. Occasionally, thick, densely stained ring structures were seen which are possibly analogous to mesosomes. In contrast, the achromobacters demonstrated a regularly undulant outer cell wall element and a planar inner wall. The cytoplasmic membrane was thin and not readily observed. RNP particles were densely stained and tightly packed in the cytoplasm; the DNA was most often lobate in disposition. Cellular division was mediated by the formation of a septum which consisted of the cytoplasmic membrane and the inner element of the cell wall. Mesosomes were observed in all of the strains examined. Dense inclusion bodies were also seen in many strains.     

Electron microscopic studies on protoplast release from I.C.A.-1.65 line (I.F.C.-1.65 line) of Bacillus subtilis cells.

Under lysozyme action a minicell-forming line (I.C.A.-1.65) of B. subtilis releases protoplasts. The main cytologic events which proceed protoplast releasing are described. Different areas of the cell wall prove a remarkable difference in their sensitivity to enzymatic lysozyme action. Central areas of the cell wall are most sensitive and the polar areas are most resistant. Mesosomal vesicles and tubules are extruded and released together with other cytoplasmic extrusion during the protoplasting process. The cell wall of minicells does not prove resistance particularities to lysozyme action. The minicells release protoplasts.     

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.     

Proteomic screening and identification of differentially distributed membrane proteins in Escherichia coli

Bacteria show asymmetric subcellular distribution of many proteins involved in diverse cellular processes such as chemotaxis, motility, actin polymerization, chromosome partitioning and cell division. In many cases, the specific subcellular localization of these proteins is critical for proper regulation and function. Although cellular organization of the bacterial cell clearly plays an important role in cell physiology, systematic studies to uncover asymmetrically distributed proteins have not been reported previously. In this study, we undertook a proteomics approach to uncover polar membrane proteins in Escherichia coli. We identified membrane proteins enriched in E. coli minicells using a combination of two-dimensional electrophoresis and mass spectrometry. Among a total of 173 membrane protein spots that were consistently detected, 36 spots were enriched in minicell membranes, whereas 15 spots were more abundant in rod cell membranes. The minicell-enriched proteins included the inner membrane proteins MCPs, AtpA, AtpB, YiaF and AcrA, the membrane-associated FtsZ protein and the outer membrane proteins YbhC, OmpW, Tsx, Pal, FadL, OmpT and BtuB. We immunolocalized two of the minicell-enriched proteins, OmpW and YiaF, and showed that OmpW is a bona fide polar protein whereas YiaF displays a patchy membrane distribution with a polar and septal bias.     

Identification and characterization of the TolC protein, an outer membrane protein from Escherichia coli

We used the cloned tolC gene to identify, locate, and purify its gene product. Strains carrying pPR13 or pPR42 overproduced a cell envelope protein (molecular weight, 52,000). A protein of the same molecular weight was identified in radioactively labeled minicells carrying pPR13; this protein was absent in pPR11-carrying minicells. This protein was the tolC gene product, since pPR11 differed from pPR13 in having a Tn10 insertion in the tolC gene. The protein seen in cell envelopes of whole cells (TolC protein) was found to exist in an aggregated state in the outer membrane; under conditions in which OmpC and OmpF were peptidoglycan associated, TolC protein was not likewise associated. Using these properties, we purified the TolC protein and determined the sequence of twelve amino acids from the amino-terminal end. The location of the TolC protein in the outer membrane was consistent with the proposed function for the tolC gene product as a processing protein in the outer membrane.     

Relationship between Cell Wall, Cytoplasmic Membrane, and Bacterial Motility

High-resolution electron microscopy of polarly flagellated bacteria revealed that their flagella originate at a circular, differentiated portion of the cytoplasmic membrane approximately 25 nm in diameter. The flagella also have discs attaching them to the cell wall. These attachment discs are extremely resistant to lytic damage and are firmly bound to the flagella. The cytoplasm beneath the flagellum contains a granulated basal body about 60 nm in diameter, and a specialized polar membrane. The existence of membrane-bound basal bodies is shown to be an artifact arising from adherence of cell wall and cytoplasmic membrane fragments to flagella in lysed preparations. Based on structures observed, a mechanism to explain bacterial flagellar movement is proposed. Flagella are considered to be anchored to the cell wall and activated by displacement of underlying cytoplasmic membrane to which they are also firmly attached. An explanation for the membrane displacement is given.     

Organization of K88ac-encoded polypeptides in the Escherichia coli cell envelope: use of minicells and outer membrane protein mutants for studying assembly of pili

Escherichia coli K-12 minicells, harboring recombinant plasmids encoding polypeptides involved in the expression of K88ac adhesion pili on the bacterial cell surface, were labeled with [35S]methionine and fractionated by a variety of techniques. A 70,000-dalton polypeptide, the product of the K88ac adhesion cistron adhA, was primarily located in the outer membrane of minicells, although it was less clearly associated with this membrane than the classical outer membrane proteins OmpA and matrix protein. Two polypeptides of molecular weights 26,000 and 17,000 (the products of adhB and adhC, respectively) were located in significant amounts in the periplasmic space. The 29,000-dalton polypeptide was shown to be processed in E. coli minicells. The 23.500-dalton K88ac pilus subunit (the product of adhD) was detected in both inner and outer membrane fractions. E. coli mutants defective in the synthesis of murein lipoprotein or the major outer membrane polypeptide OmpA were found to express normal amounts of K88ac antigen on the cell surface, whereas expression of the K88ac antigen was greatly reduced in perA mutants. The possible functions of the adh cistron products are discussed.     

Ultrastructural study of polymyxin-resistant isolates of Pseudomonas aeruginosa.

Upon exposure to 6,000 U of polymyxin B sulfate per ml, cells of the polymyxin-sensitive PAO 1 strain of Pseudomonas aeruginosa displayed in thin sections long projections arising from the outer membrane of the cell wall and extensive cytoplasmic degradation with accumulation of cytoplasmic membrane infoldings. Polymyxin-resistant isolates derived from the PAO 1 strain, however, grew well in the presence of 6,000 U of polymyxin per ml and exhibited none of these effects, having instead the appearance of a typically healthy cell. Freeze-etching of cells of the sensitive strain grown in basal medium without polymyxin revealed a concave cell wall layer studded with numerous particles. Freeze-etching of cells of the resistant isolates grown in basal medium containing 6,000 U of polymyxin per ml revealed a concave cell wall layer (i.e., the outer half of the outer membrane) in which most of these particles were absent. Thus, acquisition of resistance to polymyxin was correlated with an alteration in the architecture of the outer membrane. When the resistant isolates were grown in the basal medium lacking polymyxin and then freeze-etched, the particle distribution in the concave cell wall layer resembled that of the sensitive parent strain. The cells had regained sensitivity to polymyxin upon suspension in medium containing 6,000 U/ml as determined by their failure to grow and by internal damages seen in thin sections. These cells also had acquired increased sensitivity to ethylenediaminetetraacetate, whereas the polymyxin-resistant cells grown in the presence of polymyxin were resistant to lysis by ethylenediaminetetraacetate. The polymyxin-resistant isolates were not stable mutants but instead represented an adaptive response to the presence of polymyxin in the medium.     

Electron microscope studies on Leucothrix mucor

Summary Electron microscopic studies of thin sections of filaments, knots, resettes, gonidia, and gonidial-forming filaments of Leucothrix mucor were carried out. The cell wall is typical of gram-negative bacteria, with a double outer layer of variable thickness, a single thin middle layer which is probably peptidoglycan, and a double inner layer which is the cell membrane. The transverse septa of these filaments show two peptidoglycan layers, and no clearly demarked outer layer. During gonidial formation, there is a gradual rounding up of the cells, and the transverse septa become part of the gonidial wall. The cell membrane contains many invaginations, both along the outer wall and along the transverse septa. Thin sections through rosettes show the holdfast as material which is a heavily-staining amorphous material peripheral to the outer wall layer. Sections through knots show highly contorted cell walls, closely appressed. Fibrillar nuclear material, ribosomes, and storage granules can be seen within the cytoplasmic matrix.     

Characterization of the cell wall of the unicellular cyanobacterium Synechocystis PCC 6714

Cell walls free of cytoplasmic- and thylakoid membranes were isolated from Synechocystis PCC 6714 by sucrose density gradient centrifugation and extraction with Triton X-100. The Triton-insoluble cell wall fraction retained the multilayered fine structure. Peptidoglycan, proteins, polysaccharides, lipopolysaccharides, lipids and carotenoids were found as constituents of the cell wall. Polypeptide and lipid patterns of cell walls were completely different from that of the cytoplasmic/thylakoid membrane fraction. The purified cell walls contained about twelve outer membrane proteins. The two major polypeptides (M_r 67,000 and 61,000) were found to be associated with the peptidoglycan by ionic interactions.Myxoxanthophyll (major carotenoid), related carotenoid-glycosides and zeaxanthin were the predominating carotenoids of the cell wall of Synechocystis PCC 6714 over echinenone and -carotene. A polar unknown carotenoid was observed, the absorption spectrum of which resembled that of myxoxanthophyll. It was exclusively found in cell walls, but not in the cytoplasmic/thylakoid membrane fraction.Abbreviations Hep heptose - DGDG digalactosyldiglyceride - MGDG monogalactosyldiglyceride - SL sulfolipid - PC phosphatidylcholin - PG phosphatidylglyceride Dedicated to Prof. Dr. G. Drews on the occasion of his 60th birthday     

Cross wall synthesis and the arrangement of the wall polymers in the cell wall of Staphylococcus spp

The growing process and the fine structure of the cross wall of Staphylococcus were investigated by electron microscopy. Examination of the tangentially sectioned cross wall revealed that it was initially synthesized as a thin cell wall layer by an invaginated cytoplasmic membrane. The wall thickness soon increased by additional synthesis of the wall from the cytoplasmic membrane located at the side region of the cross wall. Scanning electron microscopic observation of sodium dodecyl sulfate-treated and mechanically separated cross walls revealed that the outer surface of the cross wall exhibits regular circular structures and the inner surface showed has an irregular surface. This indicates that cell wall materials were arranged in a regular circular manner in the initially synthesized thin layer. It is conceivable that in Staphylococcus spp. two cell wall synthesizing systems are present: wall-elongation synthesis in which wall materials are arranged in a regular circular manner and wall-thickening synthesis in which wall materials are arranged in an irregular manner.     

Cell Envelope Morphology of Rumen Bacteria

The cell walls of three species of rumen bacteria (Bacteroides ruminicola, Bacteroides succinogenes, and Megasphaera elsdenii) were studied by a variety of morphological methods. Although all the cells studied were gram-negative and had typical cytoplasmic membranes and outer membranes, great variation was observed in the thickness of their peptidoglycan layers. Megasphaera elsdenii evidenced a phenomenally thick peptidoglycan layer whose participation in septum formation was very clearly seen. All species studied have cell wall "coats" external to the outer membrane. The coat of Bacteroides ruminicola is composed of large (approximately 20 nm) globules that resemble the protein coats of other organisms, whereas the coat of Bacteroides succinogenes is a thin and irregular carbohydrate coat structure. Megasphaera elsdenii displays a very thick fibrillar carbohydrate coat that varies in thickness with the age of the cells. Because of the universality of extracellular coats among rumen bacteria we conclude that the production of these structures is a protective adaptation to life in this particular, highly competitive, environment.     

Electron Microscope Study of Septum Formation in Escherichia coli Strains B and B/r During Synchronous Growth

The formation of cell wall septa was monitored in Escherichia coli B and B/r during synchronous growth in glucose media at 37 C by means of electron microscopy. The visible events of septation comprised the following sequence, starting at about 30 min of incubation: (a) bleb formation of the outer membrane; (b) invagination of mucopeptide and cytoplasmic membrane (with associated mesosomes); the outer membrane is excluded from the septum; (c) formation of a cross-wall; (d) ingrowth of the outer membrane during cell separation. The septum is composed of a fold of cytoplasmic membrane plus mucopeptide, and the latter is a double structure, composed of two opposed lamellae separated by an electron-transparent gap. Experiments with chloramphenicol and nalidixic acid suggested that division could occur in the presence of these inhibitors once a round of deoxyribonucleic acid replication is completed. The initial stages of septation, as estimated by the potential of the cells to produce bulges in the presence of ampicillin, may involve the modification of mucopeptide by hydrolases at the end of the C period. Assembly of the septum may occur during the first half of the D period by means of precursors synthesized during the preceding C period.     

ELECTRON MICROSCOPIC STUDIES ON THE DEVELOPMENT OF VESICULAR STOMATITIS VIRUS IN KB CELLS

The development of vesicular stomatitis virus in KB cells was studied by electron microscopy. Sections of infected cells were made 1, 4, 7, 10, and 20 hours after inoculation of the cell cultures, and at the same intervals the supernatant fluid was assayed for virus titer by the plaque test in chick embryo cells. At 10, 14, and 20 hours after inoculation, virus rods were observed attached to cytoplasmic membranes, inside cytoplasmic vacuoles, and attached to the membranes delimiting these vacuoles; they were also found on the surface membrane of the cells. Besides the rods, spherical particles of different sizes and shapes were seen. The possibility that these structures are related to the development of virus rods is discussed. A similarity was noted between the site of maturation of vesicular stomatitis virus rods and that of some other arbor viruses.     

Isolation of the carotenoid-containing cell wall of three unicellular cyanobacteria.

A carotenoid-containing membrane fraction devoid of chlorophyll and phycobiliproteins was isolated from three unicellular cyanobacteria, Synechococcus sp., Synechococcus leopoliensis UTEX 625, and Anacystis nidulans R-2, by aqueous-phase separation, hydrophobic chromatography, and differential centrifugation. The presence of 2-keto-3-deoxyoctonate, muramic acid, and diaminopimelic acid suggests that the preparation is highly enriched in cell wall. Electron micrographs of thin sections of this material showed C-shaped membrane profiles similar to those seen in other gram-negative cell wall preparations. The inactivation of cyanophage AS-1 by this fraction confirmed its identity as cell wall. The cell wall contained approximately equal weights of total carbohydrate and protein. Absorption maxima at 434, 452, and 488 nm indicated the presence of carotenoids. These were in the outer membrane and were not due to contaminating cytoplasmic or thylakoid membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the preparations showed a broad band of approximately 50,000 molecular weight which contained 35% of the total outer membrane protein. This band was resolved into at least two components running at approximately 50,000 and 52,000 molecular weight. The smaller of these polypeptides was a glycoprotein. The polypeptide components were unaffected by protease or detergent treatment in either whole cells or isolated cell wall preparations, indicating that the polypeptide components were not exposed to the surface or easily removed from the hydrophobic environment.     

Mode of cell wall synthesis in gram-positive bacilli.

Ultrastructural experiments on plasmolyzed cells suggested that the information for the position and orderly synthesis of septa is not determined by the attachment of cell membrane to previously formed wall. These experiments, in conjunction with others on cells disrupted by the freeze-fracture technique, are most consistent with wall growth over the entire surface of the rods, with wall material gradually moving from a position next to the cell membrane to a position at the outer surface of the cell.     

Structural aspects of bulge formation during root hair initiation

Using light and electron microscopy, the early stages of root hair initiation were investigated under control conditions and in a situation where F-actin polymerization was effectively inhibited by latrunculin B. Trichoblasts in their early stage of bulge formation possessed large vacuole traversed by cytoplasmic strands and enclosed within a narrow peripheral layer of cytoplasm. The nucleus was settled at the inner periclinal cell wall, typically opposite the site of bulge formation. Within the bulging area, dense cytoplasm and numerous ER elements, and other organelles were accumulated, together with pleiomorphic membrane-bound structures, the identity and nature of which will require further studies. These unusual structures, which were associated with the outer cell wall, contained material similar to that of the cell wall. Similar cell wall-like bodies were observed also in the cytoplasm and sometimes within vacuoles. The possible role of these novel organelles of plant cells in cell wall thinning/degradation or in the turgor pressure maintenance are discussed. Latrunculin B treatment allowed bulge formation but prevented the switch from the slow and diffuse expansion of bulge into the rapid tip-growth characteristic of the emerging root hair. Moreover, the cytoplasm of the bulging domain became extensively vacuolated and lacked abundant ER elements and other organelles including the membrane-bound structures. These results indicate important roles of F-actin in the switch from diffuse to highly polarized tip growth.