Escherichia coli is a single-celled bacterium. It is a natural occupant of the animal gut and is typically harmless, although unusual toxic varieties exist. E. coli is small (microscopic) and reproduces rapidly by fission to form clonal colonies on agar plates, with tens of millions of cells produced in a day. It can also be grown in liquid media to produce astounding numbers of progeny—an important characteristic for studies of rare genetic events. E. coli contains a single circular chromosome, and studying mutants is much easier in a haploid organism than in a diploid organism, as there are no dominant genes to mask a mutation. Common mutant phenotypes are resistance to bacteriophages, ability to grow on certain carbon sources, antibiotic resistance, and colony size and shape. E. coli also offers a plentiful, uniform source of starting material for biochemical studies. Thus, the study of E. coli combines the power of genetics with the power of biochemistry to understand life processes at the molecular level.

E. coli is not a eukaryote and thus has limited homology to humans. This restricts its usefulness in studying all but the most basic cellular processes. For example, E. coli lacks nucleosomes, and most of its proteins are unmodified. Eukaryotic protein expression in transformed E. coli sometimes does not work, because it requires posttranslational modifications that the bacterium cannot carry out.

Early Studies of E. coli as a Model Organism

The marriage of biochemistry and genetics made E. coli a rich source for the discovery of new information. The famous “fluctuation analysis” by Salvadore Luria and Max Delbruck in 1943 demonstrated that E. coli mutates spontaneously to become resistant to bacteriophage and passes the phage-resistance trait to new progeny—thus establishing E. coli as a model system for understanding the nature and function of genes. The discovery by Joshua Lederberg and Edward Tatum that E. coli undergoes a type of mating and crossing over (DNA conjugation mediated by the F plasmid) made possible DNA exchange and complementation tests, firmly rootingE. coli as a model for genetics. In 1952, Alfred Hershey and Martha Chase used E. coli to identify DNA as the genetic material of T2 phage (see the How We Know section at the end of Chapter 2). In 1958, Matthew Meselson and Franklin Stahl selected E. coli for their studies to demonstrate that DNA replication occurs by a semiconservative mechanism (see Figure 11-1). In the same year, Arthur Kornberg and his colleagues published their results on the purification and characterization of the first-discovered DNA polymerase (see the How We Know section at the end of Chapter 11).

Francis Crick and Sydney Brenner used E. coli genetics to establish the triplet nature of the genetic code in 1961. By 1966, the genetic code had been cracked by Marshall Nirenberg, Heinrich Matthaei, Gobind Khorana, and their colleagues, using synthetic oligonucleotides and E. coli extracts (see Chapter 17). A completely new era of gene regulation research was opened up by the studies of François Jacob and Jacques Monod on the detailed workings of the lac operon (see Chapter 20 and the How We Know section at the end of Chapter 5). The identification of plasmids in E. coli, and methods to handle them, ushered in the era of recombinant DNA technology. Plasmids continue to be wonderful biotech tools, valuable beyond compare (see Chapter 7).

Life Cycle

Like most bacteria, E. coli reproduces by binary fission (Figure A-1). As the cell grows, it senses when it has reached the correct size to begin replication. The duplicate chromosomes partition to opposite poles of the cell. Cytokinesis splits the cell in half to produce two daughter cells, each containing an identical chromosome. The entire process requires only about 20 minutes at 37°C, the average mammalian body temperature. Thus, a large, visible colony of millions of identical bacteria form within only 24 hours on a Petri plate of rich medium. Huge quantities of E. coli can also be grown in liquid culture within a day.

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Genetic Techniques

For a complete description of many of these techniques, see Chapter 7.

Plasmids E. coli has several natural plasmids that can harbor foreign DNA in the cell. Researchers use these plasmids for protein expression, as shuttle vectors, or for building and sequencing large genomes.

Introduction of DNA E. coli can be made to accept foreign DNA by chemical transformation or electroporation. Cells transformed with plasmid DNA containing an antibiotic-resistance gene can be selected and maintained by adding that antibiotic to the growth medium.

Gene Knockouts Plasmids without a replication origin but containing an antibiotic-resistance gene can be selected for integration into the bacterial chromosome. Integration is typically achieved by homologous recombination, convenient for gene deletion, or knockout.

Complementation This technique is often used to prove that a mutation is in a particular gene by adding a wild-type gene to restore function. Complementation analysis of mutant cells can be performed with plasmids that contain a wild-type copy of the gene of interest. Complementation analysis of unknown mutations often makes use of a genomic plasmid library. Positive clones (cells that survive under conditions in which the mutant cannot live) can then be sequenced to identify the complementing gene.

Recombinant Protein Expression Rapid and inexpensive growth of E. coli makes it the most widely used vehicle for the expression and purification of foreign proteins. Foreign proteins are typically expressed from genes located on high-copy-number plasmids. Expression is induced using regulatory elements derived from well-studied E. coli and phage promoters.

E. coli as a Model Organism Today

Multiprotein Machines Biochemical and structural studies are now illuminating the once unknown atomic details of structures and processes of information transfer. These include the structure and function of the DNA replication apparatus and the DNA repair and recombination proteins, regulation of RNA polymerase, and the structure and chemistry of the ribosome. These multiprotein structures and processes occur in bacteria and eukaryotes alike, but they are streamlined in the relatively simple E. coli cell.

Cytologic Studies Processes can be visualized in living E. coli cells through the use of fluorescent fusion proteins. For example, recent studies of this sort have allowed detection of the proteins involved in chromosomal replication and their movements along the chromosome during replication.

Recombinant DNA Technology E. coli holds the central position in recombinant DNA technology. Its plasmids are widely used as shuttle vectors to amplify and maintain the DNA libraries of other organisms. E. coli is also still one of the most widely used vehicles for the expression and purification of foreign proteins.

Systems Biology Its relatively small genome makes E. coli an attractive system in which to pioneer approaches to systems biology. DNA chips have been constructed to examine the response of every transcript to a wide variety of conditions. The functions of about 35% of E. coli genes are still unknown; a genome-wide collection of E. coli gene knockouts is helping to assign function to these genes and to identify new gene interaction networks.

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