Safe Food_ Bacteria, Biotechnology, and Bioterrorism - Marion Nestle [178]
Agrobacteria can infect a variety of plants that have been scratched, torn, or “wounded” in some way. At the wound site, the bacteria induce the plant to form swellings—crown galls—a form of plant cancer. The bacteria do not actually penetrate into the plant’s tissues. Instead, they attach to the wound site and transfer a special piece of their DNA into the plant. This piece, called transfer-DNA (T-DNA), contains genes and DNA base sequences that enable it to enter the plant cells, find the plant’s DNA, integrate into it, and specify the production of proteins that cause plant cells to make crown galls. Why might Agrobacterium do this? The most likely explanation is that the T-DNA also contains genes that cause crown galls to produce unusual amino acid derivatives called opines. Opines are not normally made by plants and do nothing for them. Instead, they serve as a preferential food for Agrobacteria, giving them a competitive edge in the ecological world of soil bacteria.
What makes Agrobacterium tumifaciens uniquely qualified to transfer genes from other organisms to plants is that the T-DNA is not really part of its own DNA. Instead, the T-DNA is carried on a small, entirely separate, circular piece of DNA called a plasmid. Most bacteria contain plasmids (but without T-DNA). Plasmids are self-replicating, which means that they contain genes that specify their own reproductive functions; they multiply independently of the bacterial chromosome—the structure that contains the bacteria’s DNA.
Typically, plasmids carry genes for traits that are useful—but not essential—for bacterial growth or reproduction. Agrobacterium tumifaciens plasmids, for example, carry T-DNA and its genes for crown gall. Other bacteria contain plasmids with genes for a variety of functions highly germane to issues discussed in this book: the ability to fix atmospheric nitrogen, synthesize the Bacillus thuringiensis (Bt) toxin, produce pathogenic toxins (E. coli O157:H7 and Bacillus anthracis), resist certain antibiotics, and—most important—infect other bacteria. Plasmid genes for these last two characteristics, for example, are often responsible for the widespread dissemination of resistance to antibiotics within a bacterial species, and from one kind of bacteria to another.
Agrobacterium plasmids are unique in containing T-DNA. On these plasmids, the T-DNA is flanked by DNA base sequences that mark its borders. As the T-DNA enters the plant, any DNA that lies between its border regions will be transferred into the plant’s cells, regardless of where that DNA came from. Agrobacterium plasmids, therefore, solve a major technical problem: how to get desirable genes from bacteria or other foreign sources inserted into the cells of food plants.
Plant biotechnologists select the genes they want from any organism, get rid of unwanted T-DNA genes responsible for crown gall and opines, insert desired genes and regulatory DNA sequences between the T-DNA border regions, and use the Agrobacterium system to inject the newly constructed T-DNA into plant cells. This system does not work efficiently, and only a rare plant accepts the T-DNA. To identify the successful transfers, scientists add marker genes to the T-DNA, usually for resistance to antibiotics. The constructed plasmid—with the original genes for infectivity (but with crown gall functions removed), and the desired genes, regulatory elements, and markers inserted into the T-DNA—is called a transmission vector. When the system works, the bacteria containing the vector attach to the plant and actively transfer the T-DNA to the plant’s cells. Once in the plant, the T-DNA genes and sequences integrate