Protoplasts and spheroplasts

Protoplasts and spheroplasts are altered forms of bacteria or yeast , in which the principal shape-maintaining structure of the bacteria is weakened. Each bacterium forms a sphere, which is the shape that allows the bacterium to withstand the rigors, particularly osmotic, of the fluid in which it resides.

The term protoplast refers to the spherical shape assumed by Gram-positive bacteria. Spheroplast refers to the spherical shape assumed by Gram-negative bacteria. The difference is essentially the presence of a single membrane, in the case of the protoplast, and the two membranes (inner and outer) of the Gram-negative spheroplasts. It is also possible to generate a gram-negative protoplast by the removal of the outer membrane. Thus, in essence, protoplast refers to a bacterial sphere that is bounded by a single membrane and spheroplast refers to a sphere that is bounded by two membranes.

Bacteria are induced to form protoplasts or spheroplasts typically by laboratory manipulation. However, formation of the structures can occur naturally. Such bacteria are referred to as L-forms. Examples of bacterial genera that can produce L-forms include Bacillus, Clostridium, Haemophilus, Pseudomonas, Staphylococcus, and Vibrio.

The peptidoglycan is the main stress-bearing layer of the bacterial cell wall and the peptidoglycan also gives the bacterium its shape. In the laboratory, weakening the peptidoglycan network in the cell wall generates both protoplasts and spheroplasts.

By exposing bacteria to an enzyme called lysozyme, the interconnecting strands of the two particular sugars that form the peptidoglycan can be cut. When this is done, the peptidoglycan loses the ability to serve as a mechanical means of support.

The situation in yeast is slightly different, as other components of the yeast cell wall are degraded in order to form the protoplast.

The process of creating protoplasts and spheroplasts must be done in a solution in which the ionic composition and concentration of the fluid outside of the bacteria is the same as that inside the bacteria. Once the structural support of the peptidoglycan is lost, the bacteria are unable to control their response to differences in the ionic composition between the bacterial interior and exterior. If the inner concentration is greater than the outer ionic concentration, water will flow into the bacterium in an attempt to achieve an ionic balance. The increased volume can be so severe that the bacteria will burst. Conversely, if the inner ionic concentration is less than the exterior, water will exit the bacterium, in an attempt to dilute the surroundings. The bacteria can shrivel to the point of death.

Preservation of ionic balance is required to ensure that bacteria will not be killed during their transformation into either the protoplast or the spheroplast form. Living protoplasts and spheroplasts are valuable research tools. The membrane balls that are the protoplasts or spheroplasts can be induced to fuse more easily with similar structures as well as with eukaryotic cells. This facilitates the transfer of genetic material between the two cells. As well, the sequential manufacture of spheroplasts and protoplasts in Gram-negative bacteria allows for the selective release of the contents of the periplasm . This approach has been popular in the identification of the components of the periplasm, and in the localization of proteins to one or the other of the Gram-negative membranes. For example, if a certain protein is present in a spheroplast population—but is absent from a protoplast population—then the protein is located within the outer membrane.

See also Bacterial ultrastructure; Biotechnology; Transformation