Ice-minus bacteria

Ice-minus bacteria is a nickname given to a variant of the common bacterium Pseudomonas syringae (P. syringae). This strain of P. syringae lacks the ability to produce a certain surface protein, usually found on wild-type "ice-plus" P. syringae. This protein found on the outer bacterial cell wall acts as the nucleating centers for ice crystals. This facilitates ice formation, hence the designation "ice-plus." The ice-minus variant of P. syringae is a mutant, lacking the gene responsible for ice-nucleating surface protein production. This lack of surface protein provides a less favorable environment for ice formation. Both strains of P. syringae occur naturally, but recombinant DNA technology has allowed for the synthetic removal or alteration of specific genes, enabling the creation of the ice-minus strain.

Production
To systematically create the ice-minus strain of P. syringae, its ice-forming gene must be isolated, amplified, deactivated and reintroduced into P. syringae bacterium. The following steps are often used to isolate and generate ice-minus strains of P. syringae:


 * 1)  Digest P. syringae's DNA with restriction enzymes.
 * 2)  Insert the individual DNA pieces into a plasmid.  Pieces will insert randomly, allowing for different variations of recombinant DNA to be produced.
 * 3)  Transform the bacterium Escherichia coli (E.coli) with the recombinant plasmid.  The plasmid will be taken in by the bacteria, rendering it part of the organism's DNA.
 * 4)  Identify the ice-gene from the numerous newly developed E. coli recombinants.  Recombinant E. coli with the ice-gene will possess the ice-nucleating phenotype, these will be "ice-plus."
 * 5)  With the ice nucleating recombinant identified, amplify the ice gene with techniques such as polymerase chain reactions (PCR).
 * 6)  Create mutant clones of the ice gene through the introduction of mutagenic agents such as UV radiation to inactivate the ice gene, creating the "ice-minus" gene.
 * 7)  Repeat previous steps (insert gene into plasmid, transform E. coli, identify recombinants) with the newly created mutant clones to identify the bacteria with the ice-minus gene.  They will possess the desired ice-minus phenotype.
 * 8)  Insert the ice-minus gene into normal, ice-plus P. syringae bacterium.
 * 9)  Allow recombination to take place, rendering both ice-minus and ice-plus strains of P. syringae.

Economic importance


The success of the agricultural world is heavily dependent on the weather. Cold weather conditions are directly responsible for the appearance of frost on plants and most importantly, crops. In the United States alone, it has been estimated that frost accounts for approximately $1 billion in crop damage each year. As P. syringae commonly inhabits plant surfaces, its ice nucleating nature incites frost development, freezing the buds of the plant and destroying the occurring crop. The introduction of an ice-minus strain of P. syringae to the surface of plants would incur competition between the strains. Should the ice-minus strain win out, the ice nucleate provided by P. syringae would no longer be present, lowering the level of frost development on plant surfaces at normal water freezing temperature (0oC). Even if the ice-minus strain does not win out, the amount of ice nucleate present from ice-plus P. syringae would be reduced due to competition. Decreased levels of frost generation at normal water freezing temperature would translate into a lowered quantity of crops lost due to frost damage, rendering higher crop yields overall.

Historical perspective
Dr. Hall Hoppe of the U.S. Department of Agriculture was the first to notice a connection between bacteria and frost damage. In 1961, Dr. Hoppe studied a corn fungus by grinding up infected leaves each season, then applying the powder to test corn for the following season to track the disease. A surprise frost occurred that year, leaving peculiar results. Only plants infected with the diseased powder incurred frost damage, leaving healthy plants unfrozen. This phenomenon would baffle scientists until graduate student Stephen Lindow of the University of Wisconsin-Madison found a bacterium in the dried leaf powder in the early 1970s. Dr. Lindow, now a plant pathologist at the University of California-Berkeley, found that when this particular bacterium was introduced to plants where it is originally absent, the plants became very vulnerable to frost damage. He would go on to identify the bacterium as P. syringae, investigate P. syringae's role in ice nucleation and in 1977, discover the mutant ice-minus strain. He was later successful at developing the ice-minus strain of P. syringae through recombinant DNA technology as well.

In 1983, Advanced Genetic Sciences (AGS) obtained U.S. government authorization to perform field tests with the ice-minus strain of P. syringae, but environmental groups and protestors delayed the field tests for four years with legal challenges. In 1987, the ice-minus strain of P. syringae became the first genetically modified organism (GMO) to be released into the environment. A strawberry field in California was spayed with the ice-minus strain of P. syringae just before a frost in 1987. The results were promising, showing lowered frost damage to the treated plants, but the data was in suspect as environment activists destroyed some of the plants. Dr. Lindow also conducted an experiment on a crop of potato seedlings sprayed with ice-minus P. syringae. He was successful in protecting the potato crop from frost damage with a strain of ice-minus P. syringae.

Controversy
At the time of Dr. Lindow's work on ice-minus P. syringae, genetic engineering was considered to be very controversial. The controversy primarily revolved around fears of introducing new organisms that may permanently disrupt the ecosystem. The fear was that the introduction of ice-minus bacteria to the environment would eliminate bacterial and plant varieties. This was true in the case of the gypsy moth's accidental introduction into the U.S. Without a predator in the U.S., the gypsy moth is still causing overwhelming destruction to the hardwood forests of northeastern U.S.