Chapter 2 Introduction: The bar gene
Members of the genus Streptomyces (Actinobacteria: Actinomycetales) produce hundreds of antibiotics, one of which is bialaphos (also known as bilanafos or PTT). The chemical structure of bialaphos can be seen below. It consists of a glutamic acid analogue moiety, called phosphinothricin [PTC or glufosinate = 2-amino-4-(hydroxymethylphosphinyl) butanoate] and two alanine residues.
Bialaphos is an inhibitor of the key enzyme in the nitrogen assimilation pathway, glutamine synthetase (GS). It becomes active after removal of the alanine residues by intracellullar peptidases. The remaining glufosinate compound inhibits GS and as a result, leads to accumulation of toxic levels of ammonia in both bacteria and plant cells. The biochemical and toxicological characteristics of glufosinate have made it a popular, nonselective herbicide, which has been commercialized under the names Basta®, Buster® and Liberty® by Bayer Crop Science (formerly Aventis). Meiji Seika, Japan has also commercialized it as Bilanafos.
Some microorganisms can detoxify glufosinate by producing an enzyme that causes acetylation of the amino group. Genes encoding the acetylating enzyme were isolated from Streptomyces hygroscopicus (Thompson et al., 1987) and from S. viridochromogenes (Wohlleben et al., 1988). A bar (bialaphos resistance) gene encodes a phosphinothricin acetyl transferase (PAT) enzyme (in this paper we will use bar when referring to the gene and PAT when referring to the enzyme).
A bar gene has also been isolated from Alcaligenes faecalis (Proteobacteria: Beta subdivision). In multiple ongoing genome annotation projects, homologous (i.e. sharing common ancestry) genes have also been identified in the genomic sequences of many other microorganisms, including archaebacteria. The Alcaligenes, Streptomyces and archaebacterial PAT enzymes are ca. 30% identical at the amino acid level (ca. 50% using conservative replacements).
Treatment of genetically modified plants carrying a bar gene with glufosinate or bialaphos provides a very efficient means of selection in genetic transformation protocols. Although this use of the bar gene was popular in many laboratories, according to some, the patent owners expressed concern about indiscriminate use of the gene in fields, expecting that it could lead to cross-pollination and possible tolerance buildup in weeds, thereby undermining commercial applications of glufosinate. This led to enforcement of restrictions on the use of the bar gene as a selectable marker gene (personal communication).
Commercial use so far has been restricted to a few major crops, and seed distribution is mainly in the hands of the patent owner, nowadays Bayer Crop Science, and very few other companies.
In countries with commercial transgenic crops, those carrying the bar gene include sugar beet, canola, soybean, rice and maize. A few resistant hybrid maize lines are registered to Dekalb (which was acquired by Monsanto), Mycogen (now controlled by Dow) and Syngenta. A male sterile chicory variety carrying the gene is registered to Bejo Zaden, NL (data obtained from www.agbios.com).