Objectives
- To obtain plasmid cured bacteria.
- To determine the effect of Acridine orange as plasmid curing agent.
Theory
Plasmids are defined as extrachromasomally replicating molecules of DNA. They are different from the chromosomal DNA and are present in bacteria. Bacteria are a large group of single-celled microorganisms, many of which cause infections and disease in animals and humans. For example, the Enterobacteriaceae are a large family of bacteria, which occur both commensally and pathogenically in the intestines, causing wide –spread disease. Most of the essential genes in a bacterium that are required for survival under normal or optimum conditions are contained in the chromosome. However, many bacteria also carry a variety of Mobile Genetic Elements (MGE) that can contribute significantly to their diversity and adaptability. These MGEs do not carry anything essential for the survival under non-stressful circumstances, but may be important for specialized functions, such as the ability of the bacterium to form biofilms or to be resistant to antibiotics. Plasmids are the most easily identified MGE, because they are physically separate from the chromosome and can be the visualized by lysis and electrophoretic separation of the released DNA molecules. It is expected, therefore, that in any growing population of plasmid carrying bacteria, plasmidless segregants will occasionally be produced as the result of an error in the process of plasmid replication or partitioning to daughter bacteria. The survival of such bacteria indicated that the plasmid lost does not encode functions vital for growth under the prevailing environmental conditions. Furthermore the bacteria can only regain the lost functions by acquiring, the necessary genes from an external source. Thus the instability of bacterial property, either a common or uncommon feature of that species, can be an indicator of plasmid involvement.
Bacterial plasmids are known to harbor genes for: (i) resistances to antibiotics and metals, (ii) catabolic pathways such as lactose utilization and degradation of hydrocarbons (iii) biosynthesis of certain antibiotics, etc. In many cases the characteristics of the host organism conferred by the plasmids remain elusive, and such cryptic plasmids are abundant in nature. Curing of this cryptic plasmid from a bacterial strain is a method to substantiate the relationship between a genetic trait and carriage of that specific trait in the plasmid. Various methods involving chemical and physical agents have been developed to eliminate plasmids. Protocols for curing plasmids consist frequently of exposure of a culture to sub-inhibitory concentrations of some chemical agents, e.g. acridine orange, acriflavine, and sodium dodecyl sulfate or to a super-optimal temperature followed by selection of cured derivatives.
In the instances where the plasmid is stable or the loss of property difficult to determine, the bacteria can be treated with curing agents. These include chemical and physical agents, some of which can mutate DNA, interfere specifically with its replication, or affect particular structural components or enzymes of the bacterial cell. Protocols for curing plasmids consist frequently of exposure of a culture to sub-inhibitory concentrations of some chemical agents, e.g. acridine orange, acriflavine, and sodium dodecyl sulfate or to a super-optimal temperature followed by selection of cured derivatives. The DNA intercalating agents such as Acridine orange and ethidium bromide are the most commonly used because they are found to be effective against plasmids in a wide variety of genera. Although all of these agents have been used to enhance the recovery of plasmid less derivatives of various bacteria, they are individually effective only against some plasmids and their likely response is unpredictable. The elimination of a plasmid (curing) from a bacterial culture is the best method to substantiate the relationship between a genetic trait and carriage of specific plasmid by the culture as the phenotypic characters which are associated with the plasmid are not expressed in cured derivatives but on the re-introduction of the plasmid in to the cured strain the lost phenotype is re appeared. The efficiency of curing can also vary widely depending on the plasmid and the particular bacterial host carrying it. In most instances, the underlying mechanism of curing is not known. The agent may interfere directly with plasmid replication as occurs with the heat induced curing of certain temperature sensitive palsmid or curing of them by acridines or ethidium bromide. Alternatively, curing may interfere with the growth of plasmid carrying bacteria thereby allowing spontaneously arising plasmidless segregants to become predominant. This occurs in certain instances of curing by acridines, sodium dodecyl sulphate and urea. Curing by MitomycinC is also effective. Curing experiments are usually performed under conditions similar to those used for the routine culture of the bacteria unless the limitation of an essential component is specifically intended. When treatment is to be for a continuous period, the agent is used at concentration just less than that required to inhibit growth, and the number of bacteria initially is small. If acridine is used, the culture is maintained at pH 7.6 and incubated in the dark.
