Nutrition and Metabolism of Marine Bacteria XVI. Formation of Protoplasts, Spheroplasts, and Related Forms from a Gram-negative Marine Bacterium

Abstract

When cells of a marine pseudomonad were washed and suspended in 0.5 M sucrose, they retained their rod shape, but thin sections, when examined in an electron microscope, revealed that the outer layer of the cell wall had separated a considerable distance from the cytoplasmic membrane. Treatment of such cells with lysozyme alone produced no obvious change, but treatment with ethylenediaminetetraacetic acid (EDTA) alone caused the outer wall to disappear. A combination of EDTA and lysozyme resulted in the rapid formation of spheres essentially free from hexosamine and indistinguishable from protoplasts of gram-positive bacteria. When cells were washed with 0.5 M NaCl and then suspended in 0.5 m sucrose, they also retained their rod shape, but in this case the outer layer separated from the cells completely and could be recovered from the suspending medium. Such cells were converted to protoplasts by the action of lysozyme alone. Cells washed and finally suspended in 0.5 M NaCl, when treated with EDTA and lysozyme, slowly became spherical. Thin sections revealed typical spheroplasts of gram-negative bacteria in which the outer wall remained intact. Protoplasts took up a-aminoisobutyric acid by a Na+-dependent process. When certain species of gram-positive bacteria are treated with lysozyme, the entire outer wall of the cell is removed and the resulting osmotically sensitive structure is bounded only by its cytoplasmic membrane. Such a structure is referred to as a protoplast (3). Gram-negative bacteria are more resistant to the action of lysozyme and are converted to osmotically sensitive forms by this enzyme only if auxiliary agents or procedures are employed. The latter include heat treatment of the cells (L. E. Myerholtz and S. E. Hartsell, Bacteriol. Proc., p. 34, 1952), freezing and thawing (Ii), the addition of ethylenediaminetetraacetic acid (EDTA) in tris(hydroxymethyl)aminomethane (Tris) buffer (17), and plasmolysis (2). Such auxiliary treatments appear to increase the penetrability of the outer layers of the cell wall, permitting lysozyme to reach andattack the underlying mucopeptide 1 lssued as Macdonald College Journal Series No.

Biochemical Localization of Alkaline Phosphatase in the Cell Wall of a Marine Pseudomonad

“…The appearance of hexosamine-containing material at this point indicated that the expected release of outer double-track layer and the underlying (periplasmic space) layer of the cell wall had occurred (9). The cell form which arises when the outer three layers of the wall have been removed is referred to as a mureinoplast (6). Traces of alkaline phosphatase activity remained associated with the mureinoplasts formed.…”

“…Neither the loosely bound outer layer nor the outer double-track layer (cell wall membrane) showed evidence of more than traces of alkaline phosphatase activity. The cell form left after release of the loosely bound outer layer, the outer double-track layer, and underlying (periplasmic space) layer is a mureinoplast, a cell form bounded only by its cytoplasmic membrane and a layer of peptidoglycan (6). A small amount of the total alkaline phosphatase activity in the cells remained attached to this cell form and was, in part, released on treatment of the mureinoplasts with lysozyme.…”

“…The possibility must be considered that the enzyme is normally associated with the surface of the mureinoplast or with the inside surface of the outer double-track layer of the cell wall of the organism and is released from these structures under the conditions leading to the release of the contents of the periplasmic space. This possibility is rendered less likely by the observation that the enzyme shows no tendency to reassociate with either of these structures on incubation with Mg2+, since Mg2`is believed to be primarily responsible for holding the various components of the cell wall together (6,9).…”

“…More probably, the discrepancy arises from the difficulty of applying the cytochemical technique to the marine pseudomonad. Unlike most bacteria of terrestrial origin, the marine pseudomonad has special requirements for inorganic ions to maintain the various layers of the cell wall intact (2,6). Cells of the organism are suspended in complete salts solution to maintain them under as nearly normal physiological conditions as possible.…”

Localization of Alkaline Phosphatase in Three Gram-Negative Rumen Bacteria

“…All experiments described herein used rumen fluid medium, unless otherwise stated. At given times of incubation, cultures were harvested by centrifugation in a Sorvall RC-2B refrigerated centrifuge for 10 min at 15,000 x g. The optical density of the culture was measured at 660 nm. The optical densities of cultures of B. ruminicola, B. succinogenes, and M. elsdenii were correlated with the dry weight of the cells by means of a calibration curve following drying to a constant weight at 90 C. From the curve it was determined that an optical density of Cell extracts and enzyme assays.…”