Purified Cell Walls Research Articles

Ultrastructure of the cell wall of Escherichia coli and chemical nature of its constituent layers

The chemical nature of the layers constituting the wall of E. coli was studied by examining the modifications induced in the ultrastructure of the wall by several enzymatic and chemical treatments. The structure of the wall visible in thin sections is composed of an outer triple-layered (L) membrane and a backing component (G) (ref. 8), the latter consisting of an outer transparent layer (g1) and an inner dense layer (g2). A transparent space separates this structure from the cytoplasmic membrane. Lysozyme digests the g2 layer. Proteolytic enzymes detach g2 from the L membrane, apparently digesting the material present in g1. The L membrane is left substantially unaffected by proteolytic enzymes, but is completely removed by phenol (and sometimes by sodium dodecylsulfate). Empty walls and purified cell wall components, i.e., lipopolysaccharides and “murein sacculi” (mucopeptides), were isolated and their characteristics compared with those of the various layers of the wall of intact and treated cells. No evidence for the existence of unstained material lying outside the L membrane was found in cells of “rough” colonies of E. coli B, in which the cells come often in close contact, or in aggregated isolated walls, which frequently show closely apposed L membranes. A model of the wall of E. coli, which could account for the available electron microscopic and chemical data, is discussed.

Electron microscopy of the lysis of Staphylococcus aureus cell walls by Aeromonas lytic factor.

Isolated and purified cell walls of Staphylococcus aureus were treated with a purified fraction of the culture supernatant fluid of a species of Aeromonas. The course of lysis of the cell walls was followed over a period of time by examination of samples under an electron microscope. The undifferentiated cell wall was rapidly digested, but the equatorial rings were more resistant. The undifferentiated cell wall became a very thin sheet before completely dissolving, leaving a series of equatorial rings of various widths. As digestion proceeded, solubilization of the entire cell wall occurred. Analogous findings were obtained with purified S. aureus mucopeptide. It is concluded that the Aeromonas lytic principle is an enzyme, and that susceptible bonds are more concentrated in the undifferentiated cell wall mucopeptide.

Cell wall studies ofHistoplasma capsulatum

Purified cell walls, prepared from the filamentous and parasitic phases of Histoplasma capsulatum, were analyzed chemically. Analysis of alkali extractable and alkali resistant materials revealed only quantitative differences in polysaccharide content. Glucose was the only neutral hexose associated with these fractions. Hydrolysis of the alkali resistant cell wall material and subsequent amino acid determinations revealed quantitative and qualitative differences. The alkali resistant material of the filamentous phase contains more amino acid material than the yeast phase. The purified cell walls were immunogenic in rabbits. Purificadas paredes celulares, preparadas de la fase filamentosa y parasitica del Histoplasma capsulatum, fueron analizadas quimicamente. Análisis de extractos alcalinos y materiales alcalino resistentes revelaron solamente cuantitative diferencia en su contenido polisacárido. La glucosa fue la única exosa neutra asociada con estas fracciones. La hidrolisis del material alcalino resistente de la pared celular, y subsecuentes determinaciones de amino ácidos, revelaron cuantitiva y cualitativa diferencia. El material alcalino resistente de la fase filamentosa contenia más material amino ácido que el de la fase parasitica. Las parades celulares purificadas fueron “immunogenic” en conejos.

Chemical composition of the cell wall of Caryophanon latum.

THE “giant micro-organism” Caryophanon latum was first described by Peshkoff as the “missing link” between the blue-green algae and the bacteria. He isolated the microorganism from cow manure near Moscow, noting its unusual morphology including “its long rod forms containing a varying number of nuclei”1. Others have since isolated this organism in England and in the United States2,3. Pringsheim and Robinow have described this micro-organism. Caryophanon latum Peshkoff as “a very large Gram negative, peritrichously flagellated bacterium of unusual structural complexity”2. On the other hand, Provost and Doetsch reported the organism to be a Gram positive bacterium, susceptible to egg-white lysozyme and able to form protoplasts readily4. The work described here was designed to obtain further evidence as to the bacterial nature of the micro-organism by analysing the isolated and purified cell walls of Caryophanon latum for its constituent amino sugars, amino acids and monosaccharides.

Elevated cell wall serine in pleiotropic staphylococcal mutants.

Korman, Ruth Z. (Cornell University, Ithaca, N.Y.). Elevated cell wall serine in pleiotropic staphylococcal mutants. J. Bacteriol. 92:762-768. 1966.-Physically purified cell walls were prepared from two staphylococcal strains and from pleiotropic variants derived from them. The quantitative amino acid and amino sugar content of these walls is reported. The pleiotypes, which are identified culturally by their failure to elaborate coagulase, their resistance to bacteriophage, and their sensitivity to mannitol, have altered molar ratios of amino acids and amino sugars in their cell walls. In comparison with lysine content, the serine content of the mutant wall is elevated and the glycine content is reduced. The glucosamine content is reduced also. It is postulated that the pleiotropic mutants possess an altered cell wall biosynthetic pathway.

Antigenic properties of the cell wall and other fractions of the yeast form of Histoplasma capsulatum.

Pine, Leo (Communicable Disease Center, Atlanta, Ga.), Clarence J. Boone, and Dave McLaughlin. Antigenic properties of the cell wall and other fractions of the yeast form of Histoplasma capsulatum. J. Bacteriol. 91:2158-2168. 1966.-Yeast-form cells of Histoplasma capsulatum were fractionated in an attempt to obtain complement-fixing antigens with greater specificity than those of the currently used whole yeast form. No specific fraction was isolated. Rupture of the whole cell produced soluble and insoluble highly antigenic fractions. The soluble fractions rapidly lost antigenicity. Marked reduction of activity of some particulate fractions was also observed. The purified cell wall, stripped of its protein components, was antigenic, relatively stable, and demonstrated low cross-reactivity with heterologous sera. All fractions cross-reacted with sera from cases of North American blastomycosis to a greater degree than did the whole yeast-form antigen of H. capsulatum. The production of certain cellular fractions varied with the strains used. In complement-fixation tests, insignificant differences in specificity and antigenicity were found with whole-cell antigens from 13 H. capsulatum strains as well as with purified cell wall fractions prepared from 2 of these strains.

Structure of purified cell walls of dense forms of meningopneumonitis organisms.

Manire, G. P. (Institute for Virus Research, Kyoto University, Kyoto, Japan). Structure of purified cell walls of dense forms of meningopneumonitis organisms. J. Bacteriol. 91:409-413. 1966.-Purified suspensions of the dense form of the meningopneumonitis organism were prepared by differential centrifugation, sonic treatment, enzyme treatment, and sucrose gradient centrifugation. When mixed with glass beads and sonically treated at 10 kc/sec, the particles were completely disrupted in 15 min when 10-ml volumes were used, and in 5 min when 2-ml volumes were used. Purified cell walls were prepared by enzyme treatment and sucrose gradient centrifugation. When shadow-cast in high vacuum with platinum-palladium alloy, the cell walls appeared to be composed of an inner layer of hexagonally packed macromolecular structures approximately 100 A in diameter and an outer layer of unknown material.

DIFFERENTIAL STAINING OF YEAST FOR PURIFIED CELL WALLS, BROKEN CELLS, AND WHOLE CELLS.

Intact yeast cells are Gram positive but broken or disrupted cells are Gram negative. A counterstain with methyl green provides differential staining between cell wall and cytoplasm. The cells and cell fragments are dried on a slide and stained by a standard Gram stain. The preparation is then treated for 5 min with 1% phosphomolybdic acid, washed, and stained 0.5 min with 1% aqueous methyl green (unpurified by CHCl3 extraction). Under these conditions whole, intact cells are dark purple or black, walls of broken cells and purified walls are light green, and the exposed cytoplasm stains light purple. All fractions can be easily differentiated.

Localization of Antigens in Mechanically Disrupted Cells of Certain Species of the Genera Candida and Torulopsis

Summary The antigenic structure of mechanically disrupted cells of seven species of Candida and of T. glabrata was studied by means of agar gel diffusion methods. Two purified cell wall components of C. albicans, both glucomannanproteins (GMP I and GMP II), were compared with cell wall fractions of the seven species studied and found to be components common to all of them as well as to T. glabrata. Use of antisera to C. albicans, portions of which had been rendered monospecific for various antigens present in the genus by adsorption with cells of heterologous species, permitted an identification of antigens 1 to 3 as cell wall components, and of antigen 5 as a cytoplasmic component. Unique thermolabile antigens present in C. pseudotropicalis, C. krusei and C. parakrusei were determined to be cytoplasmic. Other antigens found were present in both cytoplasmic and cell wall components.

Localization of the site of fixation of the inducer, penicillin, in Bacillus cereus

1. 1. The rupture of [ 35S]penicillin treated Bacillus cereus in a pressure cell solubilized the majority of the specifically bound radioactivity. Upon conversion of [ 35S]penicillin treated cells to protoplasts approx. 95% of the label was found in the lysozyme digest supernatant and not associated with the protoplasts. In either case the bound label was still associated with a large molecular weight component. 2. 2. The bound label was not found to be associated with the ribosomes, messenger RNA, purified cell wall, or purified cytoplasmic membrane. Localization of the bound penicillin to a component closely associated with the cytoplasmic membrane-cell wall area is strengthened by the finding that a combination of lipase (EC 3.1.1.3) and trypsin (EC 3.4.4.4) caused a major portion of the bound radioactivity to become dialyzable. This latter result indicates that the cell structure to which penicillin binds is a lipoprotein. 3. 3. The release of the penicillin-receptor complex during protoplast formation explains the results that protoplasts do not fix [ 35S]penicillin similar to whole cells and that protoplasts cannot be induced to a higher rate of penicillinase (penicillin amidohydrolase, EC 3.5.2.6) formation by addition of penicillin. A 30 to 60 min induction of the inducible strain with penicillin is required for the subsequently formed protoplasts to synthesize penicillinase at comparable rates to induced whole cells.

Mycobacterial cell walls. I. Methods of preparation and treatment with various chemicals.

Takeya, Kenji (Kyushu University, Fukuoka, Japan) and Kazuhito Hisatsune. Mycobacterial cell walls. I. Methods of preparation and treatment with various chemicals. J. Bacteriol. 85:16-23. 1963.-Several methods of preparation of mycobacterial cell walls were examined, and the grinding method with glass powder, using Dry Ice, was found to give fairly good cell-wall preparations. "Paired fibrous structures" were clearly seen on the purified cell wall. The appearance of the cell wall as revealed by the electron microscope was not altered by digestion with trypsin, pronase, or pronase in 5% alcoholic solution, nor by treatment with 95% alcohol, acetone-alcohol mixture, or ether-alcohol mixture. By treatment with alcoholic KOH solution, the fibrous structure was removed. The remaining thin layer of the cell wall was tentatively designated the "basal layer" of the mycobacterial cell wall. The fibers appeared also to be removed by chloroform treatment. Nagarse digestion seemed to solubilize some constituents of the cell wall. The cell wall lost its shape and rigidity after lysozyme digestion.

Use of pressure cell to prepare cell walls from Mycobacteria.

Use of a pressure cell for preparation of purified cell walls of mycobacteria is described. This technic eliminated difficulties encountered earlier with the Mickle procedure and the yields were considerably increased.

Safety glass specifications : (American Standards Association.)

Cleistothecia and associated mycelia of Sphaerotheca mors-uvae (Schw.) Berk. at different stages of development and overwintering have been examined by chemical analysis, decomposition by micro-organisms and enzymes, and by electron microscopy.Chemical analysis of purified cell walls showed that immature and mature stages contain 35 and 26% hexosan and 31 and 22% glucan respectively. Galactose and mannose were the only other monosaccharides found in hydrolysates by GLC. Hydrolysates of walls contained about 10% hexosamine, most of which was N-acetylglucosamine. Melanin, some of which was probably associated with glucosamine and protein, comprised 18–20% of the heavily pigmented mature wall.Chitin-degrading micro-organisms isolated from soil and leaves grew on media containing cell walls from immature and mature samples but only cleared media from the former. Chitinase and glucanase released the expected amounts of N-acetylglucosamine and glucose from immature cell walls but unless they were homogenized by ultrasound, mature walls yielded reduced amounts of N-acetylglucosamine. Filtrates of cell-wall media after growth of Penicillium javanicum Szilvinyi or Streptomyces sp. isolated from over-wintered cleistothecia contained chitinase and β-1,3- and β-1,6-glucanases. Only Streptomyces sp. caused complete lysis of pigmented and non-pigmented mycelial cell walls but mature cleistothecial walls were not solubilized by either organism in 13 days.Cell walls of mature cleistothecia were very electron dense and fungi (including an asco-mycete) and bacteria occurred in cell lumina, even at an early stage. Bacteria were also present within the cell walls in mature cleistothecia and sometimes signs of wall lysis occurred around them.