The anatomy of the bacterial surface.

Publisher Summary This chapter describes physical and chemical methods for disintegrating microbes for preparing biologically active fractions. Direct measurement of cell disintegration can be sometimes dispensed with and the criteria of yield of the desired enzyme activity be relied on, especially when one of the chemical methods of extraction is used. In any comparative work and where control or location is studied by means of a physical method of disruption or with spheroplasts, some method of assessing disintegration is essential. Estimation of cell disintegration by measuring the release of typical cell components can be made quantitatively more precise than direct counting. There is some difficulty in interpretation and in obtaining a normal zero time and 100% standards. For instance, the measurement of the release of soluble protein is often used. This depends partly on the degree of disruption, but also on the method used to separate soluble protein from insoluble debris.

Chapter I The Disintegration of Micro-organisms

Mohan, Raam R. (Warner-Lambert Research Institute, Morris Plains, N.J.), Donald P. Kronish, Roland S. Pianotti, Ray L. Epstein, and Benjamin S. Schwartz. Autolytic mechanism for spheroplast formation in Bacillus cereus and Escherichia coli. J. Bacteriol. 90:1355–1364. 1965.—Spheroplasts of Bacillus cereus strain T and Escherichia coli B were prepared by incubating early log-phase cells in appropriate buffers and stabilizers for 3 hr at 30 and 37 C, respectively. Upon incubation in 0.05 m tris(hydroxymethyl)aminomethane buffer osmotically stabilized with 16% polyethylene glycol at pH 7.5, 99% of the B. cereus cells formed spheroplasts; 90% of the E. coli cells were converted to spheroplasts in 0.4 m sodium acetate buffer osmotically stabilized with 1.6 m sucrose at pH 6.0. The extent of spheroplast formation was determined by phase-contrast microscopic examination, by measuring the rate of fall of optical density in the reaction mixture when subjected to osmotic shock, and by viable intact cell counts. The effect of a selected group of metabolic inhibitors on the autolytic system of B. cereus and E. coli has been examined. B. cereus and E. coli wall components comprising 26% of the dry weight of the original cellular material were recovered from dialyzed fractions by precipitation in 70% ethyl alcohol. Chemical and chromatographic analysis of cell-wall hydrolysates from B. cereus and E. coli indicated the presence of glucosamine, alanine, lysine, glycine, aspartic acid, diaminopimelic acid, glutamic acid, and muramic acid.

Autolytic Mechanism for Spheroplast Formation in Bacillus cereus and Escherichia coli

Publisher Summary This chapter discusses the system of calcium homeostasis in birds. The importance of Ca 2+ in controlling a variety of metabolic functions in the body, ranging from muscle contraction to blood coagulation is reviewed. The Ca 2+ participates together with inositol phosphates in the mediation of hormone action in the target cell, including the regulation of secretion of several hormones. Although over 99% of body calcium is contained in the skeleton, the metabolic priority of the organism is the maintenance of a constant concentration of calcium in plasma and extracellular fluids, at the expense of bone calcification and in extreme conditions by the net breakdown of skeletal material. Thus, the relatively insoluble calcium phosphates of bone serve as a calcium reservoir to be utilized during need. This function of bone is particularly prominent in female birds during reproduction. The regulation of Ca 2+ concentration in body fluids is achieved through the action of a complex feedback control system which includes several subsystems and regulating hormones.

Calcium homeostasis in birds.

Cells of Clostridium botulinum type A strain 190 harvested at logarithmic growth phase rapidly autolysed in phosphate buffer and most of the cells were converted autolytically into spheroplasts in 0.5 M sucrose-phosphate buffer within 2–3 hr at 37 C. Electron microscope observations on the process of autolysis and spheroplast formation revealed that lysis of the cell wall commenced at one end of the cell and the cytoplasmic contents were released through such lesion. The rod cell was thusly transformed into a fragile spherical form in the hypertonic sucrose-buffer. The lysis of the cell wall proceeded centripetally and finally morphological integrity of the cell wall was completely lost. From these findings it is suggested that the autolysis of the organism is preceded by autodigestion of the cell wall at one end of the cell. A crude cell wall fraction isolated from log-phase cultures by sonication and fractionation rapidly autolysed in phosphate buffer. Reducing sugars and amino sugars of the wall were released from the autolysing wall fraction. Electron microscopy of the residues obtained from wall-autolysates demonstrated that the rigid structure of the wall completely disappeared and only fragile membranous or amorphous fragments remained after autolysis of the crude wall fraction. Heated wall preparations digested with trypsin and nagarse were dissolved by a soluble wall-autolysate, but not by a soluble cytoplasmic fraction. It seems likely that autolytic enzyme(s) may exist at or near the cell wall.

Autolytic Formation of Spheroplasts and Autolysis of Cell Walls in Clostridium botulinum Type A

WHEN certain bacteria are suspended in sucrose solution of suitable concentration and incubated with lysozyme, the rigid cell-walls are dissolved away, leaving relatively stable spherical protoplasts1 Lytic enzymes which dissolve the isolated cell-walls of vegetative Bacillus cereus have been found in extracts of mechanically disintegrated resting spores2 and partial autolysates of sporulating cells3 of this organism When heat-treated, intact vegetative cells were treated with preparations of these enzymes, the walls were dissolved, leaving the coagulated cell-contents apparently unchanged It has now been found that when viable organisms are suspended in sucrose solution and treated with these enzymes, relatively stable protoplasts are obtained in good yield

F. A. Dark

Papers

Bacterial protoplasts from Bacillus species by the action of autolytic enzymes.