Gel electrophoresis makes it possible to identify unknown bacteria by comparing the protein subfractions digested from their cells and cell walls with those of known samples. This molecular separation technology, termed DNA EXPRESSION FINGERPRINTING, (DEF), directly compares individual polypeptides, the products of gene expression. It is a technology that soon will replace gram-staining and biochemical identification methods routinely in use today.
DEF is accomplished by growing the bacteria culture to a known density and then lysing the cells by first digesting the cell walls with lysozyme and then dissolving the resultant protoplasts with a solubilization buffer containing SDS and a reducing agent such as 2-mercaptoethanol (BME). Lysozyme is an enzyme that cleaves the polysaccharide component of the bacterial cell wall causing it to fractionate. SDS solubilizes hydrophilic and hydrophobic proteins. In addition, SDS confers a net negative charge to the polypeptide in proportion to its length. When a protein is treated with SDS and a reducing agent, the polypeptides become rods of negative charges with equal charge per unit length. This treatment allows the negatively charged protein rods to separate on SDS gels on the basis of their molecular weight. Treatment of proteins with a reducing agent, such as BME, breaks the disulfide bonds that form between some polypeptides as they are processed into higher levels of protein structure. This allows us to compare individual polypeptides, each representing the product of gene expression.
Bacteria come in several shapes, each of which is determined by the configuration of certain macromolecules in the bacterial cell wall. There are two distinct classes of bacterial cell envelopes, known as gram-positive and gram-negative based on the gram-reaction. These two forms are distinguished by a universally-applied test that determines the ability of the bacterial cell wall to retain a crystal violet-iodine stain (gram-stain) when treated with alcohol. Bacteria that retain the stain are gram-positive and stain purple; those that do not are gram-negative and stain pink. Although the result of the gram-staining test is conditioned to some extent by the physiological state of the cell, the test detects the major differences in the biochemical composition of bacterial cell walls.
The cell walls of all prokaryotic cells (except Tenericulates and archebacteria) have a common structural component called peptidoglycan layer. It is present as a thick outer layer consisting of both sugar and peptide units. The peptidoglycan forms a saclike macromolecule that encloses the entire inner membrane and cytoplasm of the bacterium, providing the support needed for the cell to maintain its structure. In cell walls of gram-negative bacteria, peptidoglycan occurs as a single layer, and in small quantity (1-10%). In gram-positive bacteria, there are many layers and a much greater quantity (60-100%) of peptidoglycan. In addition to the peptidoglycan layer, each bacterial cell wall contains a unique assortment of other macromolecules such as phospholipids, teichoic acids and polysaccharides that are specific to it alone.
Figure 1 illustrates the differences of macromolecular complexity in cell walls of gram-negative and gram-positive bacteria.
Using polyacrylamide gel electrophoresis (PAGE), the protein subfractions of the bacterial cell wall digest and other cell proteins are separated according to their molecular weight. The banding pattern in each gel lane makes up the DEF code for a particular bacterial species or strain. To determine a perfect bacterial identification match, the two DEF codes must be identical. If the DEF codes are similar and only differ in several bands, then the two strains probably are closely related. The DEF code of distantly related or unrelated bacteria would be radically different.
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