Ice-minus bacteria
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Ice-minus bacteria is a common name given to a variant of the common
bacterium Bacteria (; singular: bacterium) are ubiquitous, mostly free-living organisms often consisting of one biological cell. They constitute a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria were among ...
''
Pseudomonas syringae ''Pseudomonas syringae'' is a rod-shaped, Gram-negative bacterium with polar flagella. As a plant pathogen, it can infect a wide range of species, and exists as over 50 different pathovars, all of which are available to researchers from internat ...
'' (''P. syringae''). This strain of ''P. syringae'' lacks the ability to produce a certain surface
protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
, usually found on wild-type ''P. syringae''. The "ice-plus" protein (INA protein, "Ice nucleation-active" protein) found on the outer bacterial
cell wall A cell wall is a structural layer surrounding some types of cells, just outside the cell membrane. It can be tough, flexible, and sometimes rigid. It provides the cell with both structural support and protection, and also acts as a filtering mech ...
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 In biology, and especially in genetics, a mutant is an organism or a new genetic character arising or resulting from an instance of mutation, which is generally an alteration of the DNA sequence of the genome or chromosome of an organism. It ...
, lacking the
gene In biology, the word gene (from , ; "...Wilhelm Johannsen coined the word gene to describe the Mendelian units of heredity..." meaning ''generation'' or ''birth'' or ''gender'') can have several different meanings. The Mendelian gene is a ba ...
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 Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word ''cloning'' refers to the fact that the metho ...
has allowed for the synthetic removal or alteration of specific genes, enabling the ice-minus strain to be created from the ice-plus strain in the lab. The ice nucleating nature of ''P. syringae'' incites frost development, freezing the
bud In botany, a bud is an undeveloped or embryonic shoot and normally occurs in the axil of a leaf or at the tip of a stem. Once formed, a bud may remain for some time in a dormant condition, or it may form a shoot immediately. Buds may be spec ...
s of the plant and destroying the occurring crop. The introduction of an ice-minus strain of ''P. syringae'' to the surface of plants would reduce the amount of ice nucleate present, rendering higher crop yields. The recombinant form was developed as a commercial product known as Frostban. Field-testing of Frostban in 1987 was the first release of a
genetically modified organism A genetically modified organism (GMO) is any organism whose genetic material has been altered using genetic engineering techniques. The exact definition of a genetically modified organism and what constitutes genetic engineering varies, with ...
into the environment. The testing was very controversial and drove the formation of US biotechnology policy. Frostban was never marketed.


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'': # Digest ''P. syringae''s DNA with
restriction enzymes A restriction enzyme, restriction endonuclease, REase, ENase or'' restrictase '' is an enzyme that cleaves DNA into fragments at or near specific recognition sites within molecules known as restriction sites. Restriction enzymes are one class o ...
. # Insert the individual DNA pieces into a
plasmid A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; how ...
. Pieces will insert randomly, allowing for different variations of recombinant DNA to be produced. # Transform the bacterium ''
Escherichia coli ''Escherichia coli'' (),Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. also known as ''E. coli'' (), is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus ''Escher ...
'' (''E.coli'') with the recombinant plasmid. The plasmid will be taken in by the bacteria, rendering it part of the organism's DNA. # 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 In genetics, the phenotype () is the set of observable characteristics or traits of an organism. The term covers the organism's morphology or physical form and structure, its developmental processes, its biochemical and physiological proper ...
, these will be "ice-plus". # With the ice nucleating recombinant identified, amplify the ice gene with techniques such as
polymerase chain reaction The polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete or partial) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of it) t ...
s (PCR). # Create mutant clones of the ice gene through the introduction of mutagenic agents such as
UV radiation Ultraviolet (UV) is a form of electromagnetic radiation with wavelength from 10 nm (with a corresponding frequency around 30  PHz) to 400 nm (750  THz), shorter than that of visible light, but longer than X-rays. UV radiation i ...
to inactivate the ice gene, creating the "ice-minus" gene. # 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. # Insert the ice-minus gene into normal, ice-plus ''P. syringae'' bacterium. # Allow recombination to take place, rendering both ice-minus and ice-plus strains of ''P. syringae''.


Economic importance

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
bud In botany, a bud is an undeveloped or embryonic shoot and normally occurs in the axil of a leaf or at the tip of a stem. Once formed, a bud may remain for some time in a dormant condition, or it may form a shoot immediately. Buds may be spec ...
s 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 – . 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

In 1961, Paul Hoppe of the
U.S. Department of Agriculture The United States Department of Agriculture (USDA) is the federal executive department responsible for developing and executing federal laws related to farming, forestry, rural economic development, and food. It aims to meet the needs of com ...
studied a corn
fungus A fungus ( : fungi or funguses) is any member of the group of eukaryotic organisms that includes microorganisms such as yeasts and molds, as well as the more familiar mushrooms. These organisms are classified as a kingdom, separately from th ...
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 A university () is an educational institution, institution of higher education, higher (or Tertiary education, tertiary) education and research which awards academic degrees in several Discipline (academia), academic disciplines. Universities ty ...
with D.C. Arny and C. Upper found a bacterium in the dried leaf powder in the early 1970s. Lindow, now a plant pathologist at the
University of California-Berkeley The University of California, Berkeley (UC Berkeley, Berkeley, Cal, or California) is a public land-grant research university in Berkeley, California. Established in 1868 as the University of California, it is the state's first land-grant univ ...
, 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), a biotech company, applied for 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 A genetically modified organism (GMO) is any organism whose genetic material has been altered using genetic engineering techniques. The exact definition of a genetically modified organism and what constitutes genetic engineering varies, with ...
(GMO) to be released into the environment when a strawberry field in
California California is a U.S. state, state in the Western United States, located along the West Coast of the United States, Pacific Coast. With nearly 39.2million residents across a total area of approximately , it is the List of states and territori ...
was sprayed with the ice-minus strain of ''P. syringae''. The results were promising, showing lowered frost damage to the treated plants. 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 Lindow's work on ice-minus ''P. syringae'',
genetic engineering Genetic engineering, also called genetic modification or genetic manipulation, is the modification and manipulation of an organism's genes using technology. It is a set of technologies used to change the genetic makeup of cells, including t ...
was considered to be very controversial.
Jeremy Rifkin Jeremy Rifkin (born January 26, 1945) is an American economic and social theorist, writer, public speaker, political advisor, and activist. Rifkin is the author of 23 books about the impact of scientific and technological changes on the economy, ...
and his Foundation on Economic Trends (FET) sued the NIH in federal court to delay the field trials, arguing that NIH had failed to conduct an Environmental Impact Assessment and had failed to explore the possible effects "Ice-minus" bacteria might have on ecosystems and even global weather patterns. Once approval was granted, both test fields were attacked by activist groups the night before the tests occurred: "The world's first trial site attracted the world's first field trasher". The BBC quoted Andy Caffrey from
Earth First! Earth First! is a radical environmental advocacy group that originated in the Southwestern United States. It was founded in 1980 by Dave Foreman, Mike Roselle, Howie Wolke, Bart Koehler, and Ron Kezar. Today there are Earth First! groups around ...
: "When I first heard that a company in Berkley was planning to release these bacteria Frostban in my community, I literally felt a knife go into me. Here once again, for a buck, science, technology and corporations were going to invade my body with new bacteria that hadn't existed on the planet before. It had already been invaded by smog, by radiation, by toxic chemicals in my food, and I just wasn't going to take it anymore." Rifkin's successful legal challenge forced the Reagan Administration to more quickly develop an overarching regulatory policy to guide federal decision-making about agricultural biotechnology. In 1986, the Office of Science and Technology Policy issued the
Coordinated Framework for Regulation of Biotechnology The Coordinated Framework for Regulation of Biotechnology, proposed in 1984 by the White House Office of Science and Technology Policy and finalized in 1986, spells out the basic federal policy for regulating the development and introduction of p ...
, which continues to govern US regulatory decisions. The controversy drove many biotech companies away from use of genetically engineering microorganisms in agriculture.


See also

*
Bacterial ice-nucleation proteins Bacterial ice-nucleation proteins is a family of proteins that enable Gram-negative bacteria to promote nucleation of ice at relatively high temperatures (above -5C). These proteins are localised at the outer membrane surface and can cause frost ...


References


External links


''P. syringae'' genomic information from Cornell University's Pseudomonas-Plant Interaction Project
{{Genetic engineering Pseudomonadales Genetically modified organisms Genetically modified organisms in agriculture