Ice-minus bacteria

Last updated

Ice-minus bacteria is a common name 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 P. syringae. The "ice-plus" protein (INA protein, "Ice nucleation-active" protein) found on the outer bacterial cell wall acts as the nucleating centers for ice crystals. [1] 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 ice-minus strain to be created from the ice-plus strain in the lab.

Contents

The ice nucleating nature of P. syringae 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 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 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:

  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

Icy lingonberry Icy LingonBerry.jpg
Icy lingonberry

In the United States alone, it has been estimated that frost accounts for approximately $1 billion in crop damage each year.[ citation needed ] 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 – 0 °C (32 °F). 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 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. [2] 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 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, 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. [3]

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. [4] In 1987, the ice-minus strain of P. syringae became the first genetically modified organism (GMO) to be released into the environment [5] when a strawberry field in California 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. [6]

Controversy

At the time of Lindow's work on ice-minus P. syringae, genetic engineering was considered to be very controversial. Jeremy Rifkin 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. [4] [7] 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". [5] The BBC quoted Andy Caffrey from Earth First!: "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." [5]

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, which continues to govern US regulatory decisions. [4]

The controversy drove many biotech companies away from use of genetically engineering microorganisms in agriculture. [8]

See also

Related Research Articles

<span class="mw-page-title-main">Bacterial conjugation</span> Method of bacterial gene transfer

Bacterial conjugation is the transfer of genetic material between bacterial cells by direct cell-to-cell contact or by a bridge-like connection between two cells. This takes place through a pilus. It is a parasexual mode of reproduction in bacteria.

<span class="mw-page-title-main">Frost</span> Coating or deposit of ice

Frost is a thin layer of ice on a solid surface, which forms from water vapor that deposits onto a freezing surface. Frost forms when the air contains more water vapor than it can normally hold at a specific temperature. The process is similar to the formation of dew, except it occurs below the freezing point of water typically without crossing through a liquid state.

<span class="mw-page-title-main">Genetic engineering</span> Manipulation of an organisms genome

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 the transfer of genes within and across species boundaries to produce improved or novel organisms. New DNA is obtained by either isolating and copying the genetic material of interest using recombinant DNA methods or by artificially synthesising the DNA. A construct is usually created and used to insert this DNA into the host organism. The first recombinant DNA molecule was made by Paul Berg in 1972 by combining DNA from the monkey virus SV40 with the lambda virus. As well as inserting genes, the process can be used to remove, or "knock out", genes. The new DNA can be inserted randomly, or targeted to a specific part of the genome.

<i>Escherichia coli</i> Enteric, rod-shaped, gram-negative bacterium

Escherichia coli ( ESH-ə-RIK-ee-ə KOH-ly) is a gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus Escherichia that is commonly found in the lower intestine of warm-blooded organisms. Most E. coli strains are harmless, but some serotypes such as EPEC, and ETEC are pathogenic and can cause serious food poisoning in their hosts, and are occasionally responsible for food contamination incidents that prompt product recalls. Most strains are part of the normal microbiota of the gut and are harmless or even beneficial to humans (although these strains tend to be less studied than the pathogenic ones). For example, some strains of E. coli benefit their hosts by producing vitamin K2 or by preventing the colonization of the intestine by pathogenic bacteria. These mutually beneficial relationships between E. coli and humans are a type of mutualistic biological relationship — where both the humans and the E. coli are benefitting each other. E. coli is expelled into the environment within fecal matter. The bacterium grows massively in fresh fecal matter under aerobic conditions for three days, but its numbers decline slowly afterwards.

<span class="mw-page-title-main">Freezing</span> Phase transition in which a liquid turns into a solid due to a decrease in thermal energy

Freezing is a phase transition where a liquid turns into a solid when its temperature is lowered below its freezing point. In accordance with the internationally established definition, freezing means the solidification phase change of a liquid or the liquid content of a substance, usually due to cooling.

Virulence is a pathogen's or microorganism's ability to cause damage to a host.

<span class="mw-page-title-main">Transformation (genetics)</span> Genetic alteration of a cell by uptake of genetic material from the environment

In molecular biology and genetics, transformation is the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material from its surroundings through the cell membrane(s). For transformation to take place, the recipient bacterium must be in a state of competence, which might occur in nature as a time-limited response to environmental conditions such as starvation and cell density, and may also be induced in a laboratory.

<i>Agrobacterium tumefaciens</i> Bacterium, genetic engineering tool

Agrobacterium radiobacter is the causal agent of crown gall disease in over 140 species of eudicots. It is a rod-shaped, Gram-negative soil bacterium. Symptoms are caused by the insertion of a small segment of DNA, from a plasmid into the plant cell, which is incorporated at a semi-random location into the plant genome. Plant genomes can be engineered by use of Agrobacterium for the delivery of sequences hosted in T-DNA binary vectors.

<span class="mw-page-title-main">Recombinant DNA</span> DNA molecules formed by human agency at a molecular level generating novel DNA sequences

Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination that bring together genetic material from multiple sources, creating sequences that would not otherwise be found in the genome.

The F-plasmid allows genes to be transferred from one bacterium carrying the factor to another bacterium lacking the factor by conjugation. The F factor was the first plasmid to be discovered. Unlike other plasmids, F factor is constitutive for transfer proteins due to a mutation in the gene finO. The F plasmid belongs to F-like plasmids, a class of conjugative plasmids that control sexual functions of bacteria with a fertility inhibition (Fin) system.

<span class="mw-page-title-main">Blue–white screen</span> DNA screening technique

The blue–white screen is a screening technique that allows for the rapid and convenient detection of recombinant bacteria in vector-based molecular cloning experiments. This method of screening is usually performed using a suitable bacterial strain, but other organisms such as yeast may also be used. DNA of transformation is ligated into a vector. The vector is then inserted into a competent host cell viable for transformation, which are then grown in the presence of X-gal. Cells transformed with vectors containing recombinant DNA will produce white colonies; cells transformed with non-recombinant plasmids grow into blue colonies.

<i>Pseudomonas syringae</i> Species of bacterium

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 international culture collections such as the NCPPB, ICMP, and others.

Bioprecipitation is the concept of rain-making bacteria and was proposed by David Sands from Montana State University in the 1970s. The formation of ice in clouds is required for snow and most rainfall. Dust and soot particles can serve as ice nuclei, but biological ice nuclei are capable of catalyzing freezing at much warmer temperatures. The ice-nucleating bacteria currently known are mostly plant pathogens. Recent research suggests that bacteria may be present in clouds as part of an evolved process of dispersal.

Transposons are semi-parasitic DNA sequences which can replicate and spread through the host's genome. They can be harnessed as a genetic tool for analysis of gene and protein function. The use of transposons is well-developed in Drosophila and in Thale cress and bacteria such as Escherichia coli.

<span class="mw-page-title-main">Genetically modified bacteria</span> First organisms to be modified in the laboratory

Genetically modified bacteria were the first organisms to be modified in the laboratory, due to their simple genetics. These organisms are now used for several purposes, and are particularly important in producing large amounts of pure human proteins for use in medicine.

Bacterial ice-nucleation proteins is a family of proteins that enable Gram-negative bacteria to promote nucleation of ice at relatively high temperatures. These proteins are localised at the outer membrane surface and can cause frost damage to many plants. The primary structure of the proteins contains a highly repetitive domain that dominates the sequence. The domain comprises a number of 48-residue repeats, which themselves contain 3 blocks of 16 residues, the first 8 of which are identical. It is thought that the repetitive domain may be responsible for aligning water molecules in the seed crystal.

 [.........48.residues.repeated.domain..........]  / / | | \ \  AGYGSTxTagxxssli AGYGSTxTagxxsxlt AGYGSTxTaqxxsxlt  [16.residues...] [16.residues...] [16.residues...]
<span class="mw-page-title-main">Molecular cloning</span> Set of methods in molecular biology


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 method involves the replication of one molecule to produce a population of cells with identical DNA molecules. Molecular cloning generally uses DNA sequences from two different organisms: the species that is the source of the DNA to be cloned, and the species that will serve as the living host for replication of the recombinant DNA. Molecular cloning methods are central to many contemporary areas of modern biology and medicine.

<i>Escherichia coli</i> in molecular biology Gram-negative gammaproteobacterium

Escherichia coli is a Gram-negative gammaproteobacterium commonly found in the lower intestine of warm-blooded organisms (endotherms). The descendants of two isolates, K-12 and B strain, are used routinely in molecular biology as both a tool and a model organism.

<span class="mw-page-title-main">History of genetic engineering</span>

Genetic engineering is the science of manipulating genetic material of an organism. The first artificial genetic modification accomplished using biotechnology was transgenesis, the process of transferring genes from one organism to another, first accomplished by Herbert Boyer and Stanley Cohen in 1973. It was the result of a series of advancements in techniques that allowed the direct modification of the genome. Important advances included the discovery of restriction enzymes and DNA ligases, the ability to design plasmids and technologies like polymerase chain reaction and sequencing. Transformation of the DNA into a host organism was accomplished with the invention of biolistics, Agrobacterium-mediated recombination and microinjection. The first genetically modified animal was a mouse created in 1974 by Rudolf Jaenisch. In 1976 the technology was commercialised, with the advent of genetically modified bacteria that produced somatostatin, followed by insulin in 1978. In 1983 an antibiotic resistant gene was inserted into tobacco, leading to the first genetically engineered plant. Advances followed that allowed scientists to manipulate and add genes to a variety of different organisms and induce a range of different effects. Plants were first commercialized with virus resistant tobacco released in China in 1992. The first genetically modified food was the Flavr Savr tomato marketed in 1994. By 2010, 29 countries had planted commercialized biotech crops. In 2000 a paper published in Science introduced golden rice, the first food developed with increased nutrient value.

Steven Earl Lindow is an American plant pathologist. He has researched the application of transgenic bacteria to prevent frost damage in crops as well as using bacteria to prevent russeting of fruit. He is a member of several scientific societies, including the National Academy of Sciences, and has been co-editor of the Annual Review of Phytopathology with Jan E. Leach since 2015.

References

  1. ↑ Love, J.; Lesser, W. (April 1989). "The Potential Impact of Ice-Minus Bacteria as a Frost Protestant in New York Tree Fruit Production" (PDF). Northeastern Journal of Agricultural and Resource Economics. 18 (1): 26–34. doi:10.1017/S0899367X00000234. S2CID   53310881.
  2. ↑ Parrott, Carolyn C. (1993). "Recombinant DNA to Protect Crops". Archived from the original on September 18, 2012. Retrieved February 11, 2007.
  3. ↑ Hynes, Patricia H. (1989). "Biotechnology in agriculture: an analysis of selected technologies and policy in the United States" (PDF). Reproductive and Genetic Engineering. 2 (1): 39–49. Archived from the original (PDF) on December 4, 2014.
  4. 1 2 3 Bratspies, Rebecca (2007). "Some Thoughts on the American Approach to Regulating Genetically Modified Organisms" (PDF). Kansas Journal of Law and Public Policy. 16 (3): 393. SSRN   1017832.[ dead link ]
  5. 1 2 3 "GM crops: A bitter harvest?". BBC News. June 14, 2002. Retrieved April 4, 2016.
  6. ↑ Thomas H. Maugh II (June 9, 1987). "Altered Bacterium Does Its Job : Frost Failed to Damage Sprayed Test Crop, Company Says". Los Angeles Times. Retrieved April 4, 2016.
  7. ↑ Maykuth, Andrew (January 10, 1986). "Genetic wonders to come: Some see boon, others calamity". The Philadelphia Inquirer. Retrieved February 11, 2007.
  8. ↑ Baskin, Yvonne (1987). "Testing The Future". Alicia Patterson Foundation. Retrieved February 11, 2007.
This page is based on this Wikipedia article
Text is available under the CC BY-SA 4.0 license; additional terms may apply.
Images, videos and audio are available under their respective licenses.