Periplasmic Bodies Research Articles

Ultrastructure of periplasmic bodies in gram-negative bacteria

Perhaps the data about periplasmic enzymes are obtained through biochemical methods but lack of morphological description. We have proved the existence of periplasmic bodies by electron microscope and described their ultrastructures. We hope this report may draw the attention of biochemists and mrophologists to collaborate on researches in periplasmic enzymes or periplasmic bodies with each other.One or more independent bodies may be seen in the periplasmic space between outer and inner membranes of Gram-negative bacteria, which we called periplasmic bodies. The periplasmic bodies have been found in seven species of bacteria at least, including the Pseudomonas aeroginosa. Shigella flexneri, Echerichia coli. Yersinia pestis, Campylobacter jejuni, Proteus mirabilis, Clostridium tetani. Vibrio cholerae and Brucella canis.

Scanning electron microscopy of cells and protoplasts ofSaccharomyces rouxii

Cells ofSaccharomyces rouxii from a normal broth culture were subjected to a high osmotic pressure (2 M KCl), fixed in 3% glutaraldehyde fortified with 2 M KCl, and then processed routinely for examination in a scanning electron microscope. Micrographs revealed birth and bud scars typical for the genus and an apparently undamaged surface topography. Protoplasts were prepared from the same material by digestion of cell walls with snail gut enzymes in the presence of 2 M KCl. Naked protoplasts were obtained and these exhibited surface invaginations. In addition, spheroidal protrusions were noted and these structures were equated with the periplasmic bodies previously described by transmission electron microscopy. The propensity for periplasmic body formation inSaccharomyces rouxii is contrasted with otherSaccharomyces species and the circumstantial evidence that relates periplasmic bodies to cryptic β-fructofuranosidase inS. rouxii is briefly discussed.

Isolation and Characterization of Protoplasts from Saccharomyces rouxii

Cells of the osmotolerant yeast Saccharomyces rouxii were transformed to protoplasts in good yield (85%) by digesting cell walls with snail-gut enzyme in the presence of 10 mM dithioerythritol, 0.1 M sodium phosphate buffer (pH 6.8), and 2.0 M KCl. The requirement for 2.0 M KCl compares with that for S. bisporus var. mellis (another osmotolerant species) and contrasts with the 0.3 to 0.8 M KCl concentrations used in the preparation of most yeast protoplasts. Short digestions (60 min or less) produced mostly spheroplasts; longer incubations (90 min or more) yielded mostly protoplasts as judged by electron micrographs. These protoplasts could be transferred to 1.0 M KCl or 2.0 M sorbitol without lysing, but lysis was pronounced in 0.5 M KCl or 1.0 M mannitol and complete in 0.02 M KCl. Protoplasts were separated from isolated cell wall remnants and debris by centrifugation on a linear gradient of Ficoll 400 (35 to 17.5%, wt/vol) containing 2.0 M KCl. Both crude and fractionated protoplast preparations contained vesicles which were identified with the periplasmic bodies of whole cells. Some of the periplasmic bodies were connected to protoplasts by fine pedicels; others appeared free. Independent degeneracy of periplasmic bodies was occasionally observed. beta-Fructofuranosidase (EC 3.2.1.26) activity is cryptic (physically) in cells of S. rouxii in contrast to the expressed enzyme (periplasmic space) of other Saccharomyces species. This enzyme remains cryptic in protoplast preparations of S. rouxii but is expressed upon lysis. The same specific activities were found per unit cell or protoplast. The possible association of the cryptic enzyme with periplasmic bodies is discussed.

Periplasmic structure in Saccharomyces rouxii (Boutroux), an osmophil.

Electron micrographs of ultrathin sections of S. rouxii displayed electrondense, membrane-circumscribed structures between the protoplasmic membrane and the cell wall. These periplasmic bodies were numerous in cells from a 3-day culture and absent or rare in older cells. Periplasmic bodies were fewer and smaller (flattened) in specimens grown in a medium fortified with 10% sucrose; they were not detected in cells grown in 20% sucrose. A brief treatment with ethyl acetate caused the periplasmic bodies of young cells to become electron light. Periplasmic bodies were most prevalent in the regions of the bud scars and were often accommodated within large invaginations in the protoplasmic membrane. In general, conditions which favor the prevalence and electron density of periplasmic bodies are those which also mask the activity of beta-fructofuranosidase in this species.

Periplasmic Structure in Saccharomyces rouxii (Boutroux), an Osmophil

Electron micrographs of ultrathin sections of S. rouxii displayed electrondense, membrane-circumscribed structures between the protoplasmic membrane and the cell wall. These periplasmic bodies were numerous in cells from a 3-day culture and absent or rare in older cells. Periplasmic bodies were fewer and smaller (flattened) in specimens grown in a medium fortified with 10% sucrose; they were not detected in cells grown in 20% sucrose. A brief treatment with ethyl acetate caused the periplasmic bodies of young cells to become electron light. Periplasmic bodies were most prevalent in the regions of the bud scars and were often accommodated within large invaginations in the protoplasmic membrane. In general, conditions which favor the prevalence and electron density of periplasmic bodies are those which also mask the activity of beta-fructofuranosidase in this species.

Microanatomy of the Periplasm of Bacillus Licheniformis

Periplasm of bacteria is the space outside the permeability barrier of plasma membrane but enclosed by the cell wall. The contents of this special milieu exterior could be regulated by the plasma membrane from the internal, and by the cell wall from the external environment of the cell. Unlike the gram-negative organism, the presence of this space in gram-positive bacteria is still controversial because it cannot be clearly demonstrated. We have shown the importance of some periplasmic bodies in the secretion of penicillinase from Bacillus licheniformis.In negatively stained specimens prepared by a modified technique (Figs. 1 and 2), periplasmic space (PS) contained two kinds of structures: (i) fibrils (F, 100 Å) running perpendicular to the cell wall from the protoplast and (ii) an array of vesicles of various sizes (V), which seem to have evaginated from the protoplast.

Periplasmic structure of frozen-etched and negatively stained cells of Bacillus licheniformis as correlated with penicillinase formation.

Bacillus licheniformis strain 749/C (constitutive for penicillinase formation) and uninduced cells of strain 749 (penicillinase-inducible) were examined after freezeetching. In the early stationary phase, strain 749/C organisms had clusters of vesicles (30 to 40 nm in diameter) on the outer surface of the plasma membrane. These are randomly distributed on the membrane, including the region of septum formation. The vesicles are not intimately associated with the plasma membrane, and their inner and outer surfaces are devoid of particles. Periplasmic vesicles were not detected by freeze-etching in strain 749 (uninduced) or in young cells of 749/C; however, the membrane of mid-logarithmic phase 749/C cells had a corrugated appearance. Negatively stained 749/C cells (logarithmic phase) also showed many vesicular and tubular bodies in the periplasm as well as septal and cytoplasmic mesosomes of typical morphology. The periplasmic structures appear to be formed either by evagination of plasma membrane or by migration of vesicular bodies from the membranous pockets of the cytoplasm. Stationary phase cells of 749/C still have many periplasmic vesicular bodies; however, the mesosomes are greatly reduced both in number and size. In sharp contrast, strain 749 organisms have very few structures similar to the periplasmic bodies of strain 749/C. These findings support our previous view that penicillinase-producing cells of 749/C have periplasmic membranous structures that are rare in the uninduced strain 749, though there is some lack of correspondence between freeze-etching, negative staining, and thin section data. These structures may be important for the retention or storage of penicillinase in the cell.