No abstract

Freeze-substitution was performed on strains of Escherichia coli, Pasteurella multocida, Campylobacter fetus, Vibrio cholerae, Pseudomonas aeruginosa, Pseudomonas putida, Aeromonas salmonicida, Proteus mirabilis, Haemophilus pleuropneumoniae, Caulobacter crescentus, and Leptothrix discophora with a substitution medium composed of 2% osmium tetroxide and 2% uranyl acetate in anhydrous acetone. A thick periplasmic gel ranging from 10.6 to 14.3 nm in width was displayed in E. coli K-12, K30, and His 1 (a K-12 derivative containing the K30 capsule genes), P. multocida, C. fetus, P. putida, A. salmonicida, H. pleuropneumoniae, and P. mirabilis. The other bacteria possessed translucent periplasms in which a thinner peptidoglycan layer was seen. Capsular polysaccharide, evident as electron-dense fibers radiating outward perpendicular to the cell surface, was observed on E. coli K30 and His 1 and P. mirabilis cells. A more random arrangement of fibers forming a netlike structure was apparent surrounding cells of H. pleuropneumoniae. For the first time a capsule, distinct from the sheath, was observed on L. discophora. In all instances, capsular polysaccharide was visualized in the absence of stabilizing agents such as homologous antisera or ruthenium red. Other distinct envelope structures were observed external to the outer membrane including the sheath of L. discophora and the S layers of A. salmonicida A450 and C. crescentus CB15A. We believe that the freeze-substitution technique presents a more accurate image of the structural organization of these cells and that it has revealed complex ultrastructural relationships between cell envelope constituents previously difficult to visualize by more conventional means of preparation.

Exponential-growth-phase cultures of BaciUus subtilis 168 were probed with polycationized ferritin (PCF) or concanavalin A (localized by the addition of horseradish peroxidase conjugated to colloidal gold) to distinguish surface anionic sites and teichoic acid polymers, respectively. Isolated cell walls, lysozyme-digested cell walls, and cell walls treated with mild alkali to remove teichoic acid were also treated with PCF. After labelling, whole cells and walls were processed for electron microscopy by freeze-substitution. Thin sections of untreated cells showed a triphasic, fibrous wall extending more than 30 nm beyond the cytoplasmic membrane. Measurements of wall thickness indicated that the wall was thicker at locations adjacent to septa and at pole-cylinder junctions (P < 0.001). Labelling studies showed that at saturating concentrations the PCF probe labelled the outermost limit of the cell wall, completely surrounding individual cells. However, at limiting PCF concentrations, labelling was observed at only discrete cell surface locations adjacent to or overlying septa and at the junction between pole and cylinder. Labelling was rarely observed along the cell cylinder or directly over the poles. Cells did not label along the cylindrical wall until there was visible evidence of a developing septum. Identical labelling patterns were observed by using concanavalin A-horseradish peroxidase-colloidal gold. Neither probe appeared to penetrate between the fibers of the wall. We suggest that the fibrous appearance of the wall seen in freeze-substituted cells reflects turnover of the wall matrix, that the specificity of labelling to discrete sites on the cell surface is indicative of regions of extreme hydrolytic activity in which ot-glucose residues of the wall teichoic acids and electronegative sites (contributed by phosphate and carboxyl groups of the teichoic acids and carboxyl groups of the peptidoglycan polymers) are more readily accessible to our probes, and that the wall of exponentially growing B. subtilis cells contains regions of structural differentiation.The cell wall of Bacillus subtilis 168 is a two-polymer structure comprised virtually of teichoic acids (54%) and peptidoglycan (46%) on a per dry weight basis when cultured in Spizizen minimal medium (4,30,31). Despite this chemical simplicity, the exact mechanisms by which the wall grows, divides, and turns over have not been adequately explained (2).Numerous studies indicate that the predominant mode of wall growth involves insertion of newly synthesized wall polymers in an inside-to-outside fashion at many sites randomly distributed along the cytoplasmic membrane (21, 22). According to the surface stress theory of Koch (18), this newly inserted material is cross-linked to the existing wall fabric at the inner wall surface but is unstressed. It assumes tension only after it is pushed outwards by more recently acquired wall material. Select bonds in this "stretched" wall eventually undergo hydrolysis, turning over or releasing polymers from the cell...

Freeze-substitution and more conventional embedding protocols were evaluated for their accurate preservation of eubacterial ultrastructure. Radioisotopes were specifically incorporated into the RNA, DNA, peptidoglycan, and lipopolysaccharide of two isogenic derivatives of Escherichia coli K-12 as representative gram-negative eubacteria and into the RNA and peptidoglycan of Bacillus subtilis strains 168 and W23 as representative gram-positive eubacteria. Radiolabeled bacteria were processed for electron microscopy by conventional methods with glutaraldehyde fixation, osmium tetroxide postfixation, dehydration in either a graded acetone or ethanol series, and infiltration in either Spurr or Epon 812 resin. A second set of cells were simultaneously freeze-substituted by plunge-freezing in liquid propane, substituting in anhydrous acetone containing 2% (wt/vol) osmium tetroxide, and 2% (wt/vol) uranyl acetate, and infiltrating in Epon 812. Extraction of radiolabeled cell components was monitored by liquid scintillation counting at all stages of processing to indicate retention of cell labels. Electron microscopy was also used to visually confirm ultrastructural integrity. Radiolabeled nucleic acids and wail components were extracted by both methods. In conventionally embedded specimens, dehydration was particularly damaging, with ethanol-dehydrated cells losing significantly more radiolabeled material during dehydration and subsequent infiltration than acetonetreated cells. For freeze-substituted specimens, postsubstitution washes in acetone were the most deleterious step for gram-negative cells, while infiltration was more damaging for gram-positive cells. Autoradiographs of specimens collected during freeze-substitution were scanned with an optical densitometer to provide an indication of freezing damage; the majority of label lost from freeze-substituted cells was a result of poor freezing to approximately one-half of the cell population, thus accounting for the relatively high levels of radiolabel detected in the processing fluids. These experiments revealed that gram-positive and gram-negative cells respond differently to freezing; these differences are discussed with reference to wall structure. It was apparent that the cells frozen first (i.e., the first to contact the cryogen) retained the highest percentage of all radioisotopes, and the highest level of cellular infrastructure, indicative of better preservation. The preservation of these select cells was far superior to that obtained by more conventional techniques.Although freeze-substitution yields images which are strikingly different from those obtained by more conventional techniques and which have profoundly affected current views of bacterial ultrastructure and the native distribution of cellular constituents (11), freeze-substitution has been used only infrequently in the study of procaryotic structure. Several factors are responsible for the limited use of freeze-substitution. The technique requires expertise in ultrarapid freezing, the necessary e...

The majority of Pseudomonas aeruginosa strains synthesize two antigenically distinct types of lipopolysaccharide (LPS), namely, a serotype-specific B-band LPS and a common antigen A-band LPS. A-band LPS consists of uncharged poly-D-rhamnan, which does not bind uranyl ions and is difficult to stain for electron microscopy; the highly charged B-band LPS is more easily visualized. We selected two wild-type strains, PAO1(serotype 05) and IATS 06 (serotype 06), generated isogenic mutants from them, and examined the distribution of LPS on the surface ofthese organisms by freeze-substitution and electron microscopy. On PAO1 cells, which express both A-band and B-band LPSs, a 31-to 36-nm-wide fringe extending perpendicularly from the outer membrane was observed. A fine fibrous material was also observed on the surface of serotype 06 (A+ B+) cells, although this material did not form a uniform layer. When the LPS-deficient mutants, strains AK1401 (A+ B-), AK 1012 (A-B-), rd7513 (A-B-), and R5 (an IATS 06-derived rough mutant; A-B-), were examined, no extraneous material was apparent above the bilayer. However, an asymmetrical staining pattern was observed on the outer leaflet of the outer membrane of each of these mutants, presumably conforming to the anionic charge distribution of the core region of the rough LPS. In all cases, expression of the LPS types was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining.When optical densitometry on electron microscopy negatives was used to analyze the outer membrane staining profiles, subtle differences in the degrees of core deficiency among rough mutants were detectable. This is the first time an electron microscopy technique has preserved the infrastructure produced in the outer membrane by its constituent macromolecules. We conclude that freeze-substitution electron microscopy is effective in the visualization of LPS morphotypes.Lipopolysaccharide (LPS) is anchored on the outer leaflets of the outer membranes of gram-negative bacteria via the lipid A moiety, with the core and 0-antigen polysaccharide regions extending outward from the cell. LPS possesses biologically important properties, such as adjuvant activities that are stimulatory to the immune systems of animal hosts and endotoxic effects that are deleterious to the health of the host (31). It can also act as a receptor for phage, as a physical barrier mediating interactions between a bacterium and its environment, and as a defense against hostile host molecules such as complement.In Pseudomonas aeruginosa, LPS is heterogeneous in size because of variations in the amount of 0-side-chain polymer substitution on the core and in the types of side-chain sugars. Several recent reports have indicated that most P. aeruginosa strains can simultaneously synthesize an A-band form and a B-band form of LPS (1,25,32,33). A-band LPS, a common antigen of uncharged sugars, is composed primarily of a1-->2-and al->3-linked D-rhamnose, whereas B-band LPS is a highly charged 0-antigen-containing LPS ...