Structure of bacterial L forms and their parent bacteria

Weibull, Claes (Rocky Mountain Laboratory, Hamilton, Mont.). Structure of bacterial L forms and their parent bacteria. J. Bacteriol. 90:1467-1480. 1965.-Light and electron microscopic studies were done on normal cells and L forms of Proteus mirabilis, Staphylococcus aureus, and Corynebacterium sp. grown in liquid media. Under the prevailing growth conditions, the L forms studied were morphologically indistinguishable from one another. They appeared as approximately spherical elements occurring singly or more often connected with each other by thinner portions of cell material. In sections of large L forms, the following structures were seen: a peripheral, triple-layered ("unit") membrane, a granular cytoplasm, nuclear regions, and vacuoles limited by membranes. Small bodies often were present inside the vacuoles. These bodies also contained a peripheral membrane and a granular cytoplasm but usually no nuclear regions. The normal bacteria from which the L forms were derived differed markedly in structure from one another, especially in the surface layers of the cells.     

Morphology and Reproductive Processes of the L Forms of Bacteria II. Comparative Study of L Forms and Mycoplasma with the Electron Microscope

Representative electron micrographs, from the study of eight strains of L forms and one strain of Mycoplasma, are presented. A- and B-type L forms were derived from two strains of Proteus, two other L forms were derived from a diphtheroid and from a staphylococcus strain, and two strains (designated as LX) were isolated from L forms derived from a group A beta-hemolytic streptococcus and from a staphylococcus. The Mycoplasma strain was isolated from goats. Sections were made of young colonies grown within agar and from parts of surface colonies embedded in the agar. B-type L colonies of Proteus were produced by inoculation of bacteria into media containing penicillin. The large bodies developing from the bacteria and the organisms in B-type L colonies of Proteus, like the parent bacteria, had a cell wall consisting of a plasma membrane and an outer cell wall. The loss of rigidity in the cell wall indicated an alteration in its structure. The A-type L cultures of Proteus consisted of irregular branching masses extending in several directions, of small dense organisms corresponding to the elementary corpuscles present in cultures of Mycoplasma, and of intermediary forms. In contrast to the B-type, all organisms in the A-type colonies were surrounded by a single unit membrane corresponding to the plasma membrane of bacteria. The structures inside the cell membrane, both in the A- and B-type, seemed to correspond to the structure of the parent bacteria, which contained ribosomes and threads of DNA. The elementary corpuscles formed chains and filaments, and, apparently, these corpuscles took part in the multiplication by gradual enlargement. The organisms seen in the cultures of all L forms and Mycoplasma studied, except in the B-type L forms of Proteus, corresponded in size, shape, and structure, as well as in the development of elementary corpuscles, to the organisms in the A-type L form of Proteus. In contrast to the spherical organisms usually seen in broth cultures, the organisms in young cultures of Mycoplasma, which were grown within the agar, were similar in morphology, as well as in the discernible structure of the organisms, to L forms. Significant morphological and structural differences were not apparent between the L forms and Mycoplasma (in cultures grown within agar media) under the conditions of this investigation.     

Reconstitution of model membranes from phospholipid and outer membrane proteins of Proteus mirabilis. Role of proteins in the formation of hydrophilic pores and protection of membranes against detergents.

Outer membrane proteins extracted from isolated cell walls of Proteus mirabilis were able to combine the cell wall phospholipids in a model membrane system. The presence of outer membrane proteins in vesicular model membranes mediated the release of previously entrapped [14C]sucrose while [3H]inulin was retained. Incorporation of lipopolysaccharide from the same cell walls was not required for the formation of such selectively permeable membranes. Three major outer membrane proteins of apparent molecular weights 39000, 36000 and 17000 were isolated using acetic acid and sodium deoxycholate solution as solvents and avoiding the strongly denaturing sodium dodecyl sulfate. The isolated proteins were assayed for their ability to form hydrophilic pores in reconstituted membranes. The trypsin-sensitive 39000-Mr protein and the peptidoglycan-associated 36000-Mr protein were equally effective in this function whereas the 17000-Mr protein mediated little penetration of low molecular weight solute. The 39000-Mr and 36000-Mr proteins also protected reconstituted membrane vesicles from disruption by detergent while 17000-Mr protein was ineffective in this regard.     

Identification of L Forms by Polyacrylamide-Gel Electrophoresis

Crude membranes were obtained from L forms by allowing the cells to lyse in distilled water. The crude membranes were washed several times in distilled water, lyophilized, and extracted with phenol-acetic acid-water. The membrane proteins were separated electrophoretically in polyacrylamide gels run at pH 4.5. Electrophoretic patterns and densitometric tracings of the gels showed distinct, reproducible intergeneric differences among L forms of Proteus, Streptobacillus, Staphylococcus, and Streptococcus. Differences within a genus could not be detected except between the group A streptococcal L forms and the one group D F-24 L form. This electrophoretic method offers possibilities for ready identification of L forms through the use of standard reference strains.     

Localization of crystals in diseased oats treated with uranyl acetate

Uranyl acetate, a suppressor of victorin-induced electrolyte leakage in oat leaves when applied together with, or before, victorin, also suppressed victorin-induced changes in ultra-structure. Uranyl crystallized in cell walls and near the plasmalemma of vascular cells, but was excluded from the protoplasm. Fewer crystals occurred near the plasmalemma when leaves were allowed to take up uranyl and victorin simultaneously than when uranyl alone was absorbed, but deposition in cell walls was similar in the two treatments. No differences in crystal distribution were found in uranyl-treated leaves which subsequently took up either water or victorin. The most striking effect of prolonged exposure to uranyl was increased vesicular activity in the protoplasm, formation of complex concentric membranes, and tonoplast damage. Following victorin treatment, uranyl post-treatment was ineffective in suppressing electrolyte leakage or preserving normal cellular ultrastructure. More severe ultrastructural damage was found in victorintreated leaves after uranyl post-treatments than after post-treatment with water, a result of victorin-induced damage which facilitates uranyl entry into the protoplasm.     

Polyacrylamide Gel Identification of Bacterial L-Forms and Mycoplasma Species of Human Origin

Polyacrylamide gel electrophoretic patterns of acidified phenol extracts prepared from whole cells can be used for the identification of bacterial L-forms and Mycoplasma species of human origin. Ten human Mycoplasma serotypes and eight L-forms belonging to five different genera were studied. The gel patterns were sufficiently distinct and reproducible that it was possible not only to identify L-forms at the genus level (group with streptococci) and different Mycoplasma serotypes but also to differentiate between the two of them. The parentage of L-forms of Streptobacillus moniliformis L1, Listeria monocytogenes, Streptococcus MG, and Staphylococcus aureus Smith strain was established by relating their gel patterns directly to parent bacteria. It was found that an L-form designated S. moniliformis An (ATCC 14220) was actually an L-form of Proteus. In addition, it was shown electrophoretically that no relationship existed between the Streptococcus MG L-form and M. pneumoniae. The applicability of this method as a diagnostic and taxonomic tool for the differentiation of L-forms and mycoplasmas is discussed.     

Preparation of endotoxin-free bacteriophages

Bacteriophages (phages) are bacterial viruses that interact with bacterial walls and invade bacterial cells. Moreover, they disturb bacterial metabolism and lead to bacteria lysis. In the case of Gram-negative bacteria crude phage cultures, apart from the phages themselves, the bacterial debris, bacterial proteins and nucleic acids contain endotoxins. These endotoxins (lipopolysaccharides) posses a high degree of toxicity in vitro and in vivo, and their removal is essential for safety in antibacterial bacteriophage therapy. An effective, scaleable purification of bacteriophages from endotoxins was accomplished by sequential ultrafiltration through polysulfone membrane (30 nm) followed by chromatography on sepharose 4B and Matrex Cellulofine Sulfate. The phage fraction after gel filtration chromatography routinely contained endotoxins in the 150-2500 EU/ml range. The procedure yielded bacteriophages contaminated with as little as 0.4-7 EU/ml (Limulus assay). This value lies within the permitted level for intravenous applications (5 EU/kg/h by European Pharmacopoeia, 1997).     

The lipid content of cell walls obtained from juvenile,yeast-like and filamentous cells ofCandida albicans

Purified cell walls ofCandida albicans obtained from juvenile cells, mature yeast-like cells and filamentous cells were analyzed for their lipid components. Chloroform: methanol (2:1 v v) extraction of the acetone-treated dried cell walls indicated the total lipid content to be 2.1% of the dry weight of the juvenile cell walls, 1.8% of the mature yeast-like cell walls and 4.5% of the filamentous cell walls. Separation of the chloroform: methanol extractable fraction through a silicie acid column and quantitative determination of the fractions showed significant amounts of sterol esters, triglycerides, sterols, free fatty acids, and phospholipids in these extracts. Following acetone extraction sterols were shown to constitute a greater percentage of the cell wall of juvenile cells than mature cells. Thin-layer chromatography separated the acetone-extractable lipids into at least four components. Diethyl ether extracts of the cell walls indicated the presence of small amounts of glycerol phospholipids in the cell walls of juvenile and mature yeast cells. Boiling 95% ethanol also removed a small lipid fraction from the cell walls of both juvenile and mature yeast which could include sphingosine phosphatides or glycosides.     

Occult Endotoxin in Bacterial Protoplasm

Protoplasm separated from disrupted cells of gram-negative bacteria was extracted with hot phenol-water or was precipitated with ethyl alcohol after digestion with Pronase. These methods recovered about 10 times more endotoxin than was detectable in the untreated protoplasm. Inactivation of endotoxin by protoplasm also occurred in vitro when the endotoxin was first dissociated into subunits before reaction with protoplasm. Despite this increased yield from another source, the major proportion of endotoxin was still found in the cell walls.     

Structural and immunological studies on the protoplast membrane of the yeast Candida utilis

Cell membranes of the yeast Candida utilis isolated by lysis of protoplasts have been shown to be lipoprotein in nature. Electron microscopy shows that Mg⁺⁺ is responsible for maintaining the integrity of the membrane. A close serological relationship was found between membranes and cell walls isolated from the yeast. This relationship was exhibited not only by membranes obtained by strepzyme treatment but also by those obtained from the action of helicase enzyme. No such relationship existed between membranes and whole cells. Related data have been obtained by treatment of yeasts with different digestive enzymes. All of the results suggest that the protoplast membrane possesses traces of structural cell wall material. This material is detectable by serological tests, but not by electron microscopy.     

Synthesis of plasmalemmal glycoproteins in intestinal epithelial cells. Separation of Golgi membranes from villus and crypt cell surface membranes; glycosyltransferase activity of surface membrane

The relationship between Golgi and cell surface membranes of intestinal cells was studied. These membranes were isolated from intestinal crypt cells and villus cells. The villus cell membranes consisted of microvillus membrane, a Golgi-rich fraction, and two membrane fractions interpreted as representing lateral-basal membranes. The villus cell microvillus membrane was purified by previously published techniques while the other membranes were obtained from isolated cells by differential centrifugation and density gradient velocity sedimentation. The two membrane fractions obtained from villus cells and considered to be lateral-basal membranes were enriched for Na+,K+-ATPase activity, but one also showed enrichment in glycosyltransferase activity. The Golgi membrane fraction was enriched for glycosyltransferase activity and had low to absent Na+,K+-ATPase activity. Adenylate cyclase activity was present in all membrane fractions except the microvillus membrane but co-purified with Golgi rather than lateral-basal membranes. Electron microscopy showed that the Golgi fraction consisted of variably sized vesicles and cisternalike structures. The two lateral-basal membrane fractions showed only vesicles of smaller, more uniform size. After 125I labeling of isolated intact cells, radioactivity was found associated with the lateral-basal and microvillus membrane fractions and not with the Golgi fraction. Antibody prepared against lateral-basal membrane fractions reacted with the surface membrane of isolated villus cells. The membrane fractions from isolated crypt cells demonstrated that all had high glycosyltransferase activity. The data show that glycosyltransferase activity, in addition to its Golgi location, may be a significant property of the lateral-basal portion of the intestinal villus cell plasma membrane. Data obtained with crypt cells support earlier data and show that the crypt cell surface membrane possesses glycosyltransferase activity.     

Microextraction of bacterial lipid A: easy and rapid method for mass spectrometric characterization

Endotoxins (lipopolysaccharides) are the main components of Gram-negative bacterial outer membranes. A quick and simple way to isolate their lipid region (lipid A) directly from whole bacterial cells was devised. This method using hot ammonium-isobutyrate solvent was applied to small quantities of cells and proved to be indispensable when a rapid characterization of lipid A structure by mass spectrometry was required. Biological activities of endotoxins are directly related to the lipid A structures, which vary greatly with cell growth conditions. This method is suitable for rough- and smooth-type bacteria and very efficient for screening variations in lipid A structures. Data are acquired in a few hours and avoid the use of phenol in extraction.     

Electron Microscopy and Viability of Lysostaphin-induced Staphylococcal Spheroplasts, Protoplast-like Bodies, and Protoplasts

The cell walls of a selected isolate of Staphylococcus aureus FDA 209P were observed undergoing progressive disintegration when exposed to lysostaphin (1 unit/ml) in 24% NaCl solution. Electron micrographs of ultrathin sections of test cells after exposure to lysostaphin for 2 min showed only superficial evidence of lytic damage. However, an average of 89% of these cells were osmotically fragile, and 21% were damaged beyond their capacity to regenerate cell walls and to grow as normal staphylococci. The 68% (average) of the osmotically fragile cells which retained the capacity to revert to normal staphylococci were designated spheroplasts. Neither perforations of the cell walls nor separation of the cell walls from the plasma membranes were observed in the micrographs of these 2-min spheroplasts. Thus, it appears that the osmotic fragility of these and possibly all lysostaphin-induced staphylococcal spheroplasts results from the hydrolysis of a critical number of the pentapeptide cross-linkages of the murein of the cell wall. Electron micrographs of cells exposed to lysostaphin for 5 to 10 min showed perforations and more extensive damage, including the separation of walls from the plasma membranes and the disintegration of large sections of the walls. Smaller numbers of spheroplasts (21 and 8%) were recovered from these 5- and 10-min preparations; those recovered probably represent cells which were attacked more slowly than the majority by the lytic enzyme. The nonrevertible, osmotically fragile cells that retained segments of cell wall were designated protoplast-like bodies. After 20-min exposure to lysostaphin, all of the cell wall was digested away from most of the cells, and true staphylococcal protoplasts were produced. These lysostaphin-induced, osmotically fragile forms appear to have different osmotic properties from the staphylococcal "protoplasts" reported by other investigators and should serve as the basis for a variety of fundamental investigations.     

Localization of enzymes and endotoxin in proteus L-forms and in their parent bacteria

By comparing three methods for disruption of normal bacteria and bacterial L-forms (process ing in the Ribi-Sorvall Cell Fractionator or in the X-press of Edebo, and osmotic shock), it was shown that the outcome of fractionation experiments with disintegrated bacteria is highly influenced by the disintegration method employed. The localization of seven enzymes in normal Proteus bacteria and Proteus L-forms was studied. The experimental results indicate that the succinic dehydrogenase and NADH oxidase are located in the membranous part of both types of organisms, the glucose-6-phosphate and isocitric dehydrogenase, the fumarase, the catalase, and the acid phosphatase in the “soluble” protoplasm.     

Isolation of Outer Membranes with an Ordered Array of Surface Subunits from Acinetobacter

A method has been developed for the isolation of outer membranes from Acinetobacter sp. strain MJT/F5/199A. Washed cells were broken in a French press and, after deoxyribonuclease and ribonuclease treatment, removal of intact cells, and four washes in 20 mosmol phosphate buffer, pH 7.4, with centrifugation at 25,000 x g for 10 min, preparations of cell wall fragments from which almost all pieces of plasma membrane had been removed resulted. Treatment of the cell walls with lysozyme and further washing, in the presence of 20 mM MgCl(2), yielded preparations of outer membranes. Electron microscopy of freeze-etched preparations shows that a regular pattern of subunits is present on the outer surfaces of intact cells. After negative staining, these subunits are visible on isolated walls and outer membranes; they can be removed by brief treatment with papain. In section, the cell wall structure is that typical of gram-negative bacteria, but the subunits are not detectable on the surface of the outer membrane. The outer membrane retains the appearance of a "unit membrane" in the cell wall, isolated outer membrane, and papain-treated outer membrane fractions. Both cell walls and outer membranes contain a high percentage of protein (76 and 84%, respectively) and not more than 5% carbohydrate, of which glucose and galactose are constitutents. The outer membranes of this Acinetobacter thus differ in structure and composition from those of bacteria in the Enterobacteriaceae.     

Composition of the outer membrane of Proteus mirabilis in relation to serum sensitivity in progressive stages of cell form defectiveness.

A serum-resistant strain of Proteus mirabilis was used to determine whether changes in the composition of surface components could be detected following induction of progressive stages of cell form defectiveness by beta-lactam antibiotics. The critical stage was the conversion from filaments to the spheroplast form, which was accompanied by increased susceptibility to the bactericidal action of human serum. Inner and outer membranes of the bacterium, its filament form and its spheroplast form were separated by sucrose density-gradient centrifugation after digestion of peptidoglycan, followed by osmotic lysis of the cells. Outer membranes of the bacterial and the filament forms sedimented at the same density, whilst the outer membrane fraction of the spheroplast form sedimented in a region of lesser density. In addition, the amounts of two major outer-membrane proteins as well as the O-polysaccharide content of the lipopolysaccharide were reduced in the spheroplast form. These results indicate a general disorganization in structure and assembly of components in regard to their interactions with one another in the outer membrane of the spheroplast form.     

Basal structure and attachment of flagella in cells of Proteus vulgaris

Abram, Dinah (Purdue University, Lafayette, Ind.), Henry Koffler, and A. E. Vatter. Basal structure and attachment of flagella in cells of Proteus vulgaris. J. Bacteriol. 90:1337-1354. 1965.-The attachment of flagella to cells of Proteus vulgaris was studied electron microscopically with negatively stained and shadow-cast preparations of ghosts from standard cultures and from special cultures that produced "long forms." The flagellum, the basal portion of which is hooked, arises within the cell from a nearly spherical structure, 110 to 140 A in diameter. This structure appears to be associated with the cytoplasmic membrane; it may be a part of the membrane or a separate entity that lies just beneath the membrane. Flagella associated with cell walls free from cytoplasmic membrane frequently have larger bodies, 200 to 700 A in diameter, associated with their base. These structures probably consist at least partly of fragments of the cytoplasmic membrane, a portion of which folds around a smaller structure. Flagella in various stages of development were observed in long forms of P. vulgaris cells grown at low temperature. The basal structure of these flagella was similar to that of the long or "mature" flagella. Strands connecting the basal structures were observed in ghosts of long forms; these strands appear to be derived from the cytoplasmic membrane. Flagella were found to be attached to fragments of cell wall and to cytoplasmic membrane in a similar manner as they are attached to ghosts. In isolates of flagella that have been separated from the cells mechanically, the organelles often terminate in hooks which almost always appear naked, but have a different fine structure than the flagellum proper.     

Alteration in protein kinase C activity and subcellular distribution in sickle erythrocytes

In agreement with previous data, membrane protein phosphorylation was found to be altered in intact sickle cells (SS) relative to intact normal erythrocytes (AA). Similar changes were observed in their isolated membranes. The involvement of protein kinase C (PKC) in this process was investigated. The membrane PKC content in SS cells, measured by [3H]phorbol ester binding, was about 6-times higher than in AA cells. In addition, the activity of the enzyme, measured by histone phosphorylation was also found to be increased in SS cell membranes but decreased in their cytosol compared to the activity in AA cell membranes and cytosol. The increase in membrane PKC activity was observed mostly in the light fraction of SS cells, fractionated by density gradient, whereas the decrease in cytosolic activity was only observed in the dense fraction. PKC activity, measured in cells from the blood of reticulocyte-rich patients, exhibited an increase in both membranes and cytosol, thus explaining some of the effects observed in the SS cell light fraction, which is enriched in reticulocytes. The increase in PKC activity in the membranes of SS cells is partly explained by their young age but the loss of PKC activity in their cytosol, particularly in that of the dense fraction, seems to be specific to SS erythrocytes. The relative decrease in membrane PKC activity between the dense and the light fractions of SS cells might be related to oxidative inactivation of the enzyme.     

Babesia bovis: bovine helper T cell lines reactive with soluble and membrane antigens of merozoites.

Babesia bovis-specific T cell lines were established from cattle infected with either tick-derived or cultured parasites by stimulation of peripheral blood mononuclear cells with a crude parasite membrane fraction. Induction and enrichment of CD4+ T cells occurred over time. All cell lines responded vigorously and in a dose-dependent, MHC-restricted manner to intact merozoites, and to soluble and membrane fractions derived from merozoites by homogenization and high-speed centrifugation. Solubilization of the membrane fraction with nondenaturing zwitterionic or nonionic detergents yielded antigenic extracts which also stimulated the T cells. However, a differential response was observed, in that cell lines from one animal proliferated vigorously to the detergent extracts of the membrane fraction, whereas cell lines from a second animal proliferated only weakly to these extracts. SDS-PAGE analysis revealed common protein bands of 90 and 22 kDa in the various immunogenic fractions. Cell lines from the animal infected with cultured parasites also responded to parasite culture supernatant "exoantigens" and to the related parasite, Babesia bigemina. We conclude that antigens present in merozoite membranes and soluble parasite extracts preferentially stimulate CD4+ T cells from cattle immune to Babesia bovis. The differential pattern of response of T cell lines from different cattle suggests that more than one protein or epitope is immunodominant for T cells.     

Non‐pathogenic association of L‐form bacteria (pseudomonas syringae pv. phaseolicola) with bean plants (Phaseolus vulgaris L.) and its potential for biocontrol of halo blight disease

L‐forms of the halo blight pathogen, Pseudomonas syringae phaseolicola, were maintained in a medium which suppressed cell wall synthesis. These L‐forms, unlike revertants (walled forms derived from unstable L‐forms) and cell walled (parent) organisms, did not elicit a hypersensitive response in tobacco leaves. Association of L‐forms with Phaseolus vulgaris was established by seed imbibition in L‐form suspensions compared with appropriate control treatments (5% mannitol or heat‐killed cells). Seedling emergence and plant growth was not affected by L‐form imbibition. The association was detected by agglutination assays using polyclonal antibody. The L‐form association was localized to the lower shoot tissue and was progressively lost with age of plants. Plants with associated L‐forms had vigour and shoot weights equivalent to controls and showed no disease symptoms. The cell walled form could not be isolated from plants showing positive agglutination. On challenge with the pathogen, plants associated with L‐forms showed significantly less disease symptoms than controls. Stem extracts, from associated plants, were inhibitory to in vitro cultures of both L‐forms and parent forms of Ps. syr. phaseolicola. These results indicate that L‐form associations confer induced systemic resistance to bean plants and might be developed as novel biocontrol systems.