Regulation of Gene Expression
Advances in Microbiology
Use of Reporter Genes to Study Gene Regulation
The ability to quantitate gene expression under different growth conditions or after alterations have been made to the regulatory elements of the gene of interest has led to a greater understanding of the mechanisms of gene regulation. Direct quantitation of gene expression is not always possible. In many cases, the gene of interest encodes a protein that either does not have a biochemical activity or has an activity that cannot be easily assayed. In these cases, the study of gene regulation has been greatly facilitated by the use of reporter genes. Reporter genes encode protein products that can be easily and quantifiably assayed. The most useful reporter genes encode proteins with activities that can be easily measured in liquid assays and that can also be visualized as colonies grown on agar plates. Reporter genes are placed under the control of the regulatory region of the gene of interest; thus, the expression of the reporter gene should mimic that of the gene of interest.
A number of reporter genes are commonly used in bacteria, with lacZ being the most predominant. This gene encodes beta-galactosidase, an enzyme that hydrolyzes lactose into galactose and glucose. This enzymatic activity can be easily quantitated in liquid assays, and the presence of this enzyme in colonies growing on plates is detected by the presence of X-gal, a substrate that can be cleaved by beta-galactosidase to produce a blue product. Cells producing high levels of this enzyme turn a dark blue color on plates containing X-gal, while colonies that do not produce this enzyme do not change color at all. Other commonly used reporter genes are xylE and lux. xylE encodes a catechol dioxygenase that cleaves catechol to produce a bright yellow product that can be seen on agar plates or measured in liquid assays. lux encodes luciferase, which catalyzes a light-emitting reaction.
A gene which has proven very useful in the study of in vivo gene expression is the gfp gene encoding green fluorescent protein (GFP) from Aquorea victorea (jelly fish). Although GFP has some limitations, there is one aspect that has made it the reporter of choice for the study of bacterial pathogenesis. The advantage of using GFP is that it allows the use of a fluorescence-activated cell sorter (FACS) (also known as flow cytometry). Cells that fluoresce are diverted from a stream of cells passing through the machine to separate them from the non-fluorescent cells.
Salmonella is a causative agent of food poisoning and also serves as a model for human typhoid fever. GFP and FACS have been used to identify Salmonella typhimurium genes preferentially expressed within host macrophages. Salmonella cells carrying plasmids with random fragments of chromosomal DNA cloned upstream of a promoterless gfp gene were used to infect a macrophage cell line. Since the gfp gene does not have its own promoter, its expression is dependent upon a promoter present in the Salmonella DNA cloned upstream. Macrophages harboring bacteria that fluoresce were separated by FACS. Bacteria that did not fluoresce or exhibited only weak fluorescence on normal growth medium were studied further, as these bacteria must harbor plasmids in which gfp expression was regulated by intracellularly expressed gene fusions.
It is expected that the study of the genes identified by this method will elucidate what genes are required for intracellular survival of human pathogens. This methodology has been applied to other pathogenic bacteria such as Mycobacterium species, and these studies may help identify targets for treatment of infections by these organisms.