Escherichia coli

Genetic "Vital Statistics"

Genome size:

4.6 Mb

Chromosomes:

1, circular

Number of genes:

4000

Percentage with human homologs:

8%

Average gene size:

1 kb, no introns

Transposons:

Strain specific, ~ 60 copies per genome

Genome sequenced in:

1997

Escherichia coli

Key organism for studying:

  • Transcription, translation, replication, recombination

  • Mutation

  • Gene regulation

  • Recombinant DNA technology

The unicellular bacterium Escherichia coli is widely known as a disease-causing pathogen, a source of food poisoning and intestinal disease. However, this negative reputation is undeserved. Although some strains of E. coli are harmful, others are natural and essential residents of the human gut. As model organisms, strains of E. coli play an indispensable role in genetic analyses. In the 1940s, several groups began investigating the genetics of E. coli. The need was for a simple organism that could be cultured inexpensively to produce large numbers of individual bacteria to be able to find and analyze rare genetic events. Because E. coli can be obtained from the human gut and is small and easy to culture, it was a natural choice. Work on E. coli defined the beginning of “black box” reasoning in genetics: through the selection and analysis of mutants, the workings of cellular processes could be deduced even though an individual cell was too small to be seen.

E. coli genome. Electron micrograph of the genome of the bacterium E. coli, released from the cell by osmotic shock.
[G. Murti/Science Source.]

Special features

Much of E. coli’s success as a model organism can be attributed to two statistics: its 1-μm cell size and a 20-minute generation time. (Replication of the chromosome takes 40 minutes, but multiple replication forks allow the cell to divide in 20 minutes.) Consequently, this prokaryote can be grown in staggering numbers—a feature that allows geneticists to identify mutations and other rare genetic events such as intragenic recombinants. E. coli is also remarkably easy to culture. When cells are spread on plates of nutrient medium, each cell divides in situ and forms a visible colony. Alternatively, batches of cells can be grown in liquid shake culture. Phenotypes such as colony size, drug resistance, ability to obtain energy from particular carbon sources, and colored dye production take the place of the morphological phenotypes of eukaryotic genetics.

Bacterial colonies.
[Biophoto Associates/Science Source.]

Life Cycle

Escherichia coli reproduces asexually by simple cell fission; its haploid genome replicates and partitions with the dividing cell. In the 1940s, Joshua Lederberg and Edward Tatum discovered that E. coli also has a type of sexual cycle in which cells of genetically differentiated “sexes” fuse and exchange some or all of their genomes, sometimes leading to recombination (see Chapter 5). “Males” can convert “females” into males by the transmission of a particular plasmid. This circular extragenomic 100-kb DNA plasmid, called F, determines a type of “maleness.” F+ cells acting as male “donors” transmit a copy of the F plasmid to a recipient cell. The F plasmid can integrate into the chromosome to form an Hfr cell type, which transmits the chromosome linearly into F recipients. Other plasmids are found in E. coli in nature. Some carry genes whose functions equip the cell for life in specific environments; R plasmids that carry drug-resistance genes are examples.

Length of life cycle: 20 minutes

795

Geneticists have also taken advantage of some unique genetic elements associated with E. coli. Bacterial plasmids and phages are used as vectors to clone the genes of other organisms within E. coli. Transposable elements from E. coli are harnessed to disrupt genes in cloned eukaryotic DNA. Such bacterial elements are key players in recombinant DNA technology.

Genetic analysis

Spontaneous E. coli mutants show a variety of DNA changes, ranging from simple base substitutions to the insertion of transposable elements. The study of rare spontaneous mutations in E. coli is feasible because large populations can be screened. However, mutagens also are used to increase mutation frequencies.

To obtain specific mutant phenotypes that might represent defects in a process under study, screens or selections must be designed. For example, nutritional mutations and mutations conferring resistance to drugs or phages can be obtained on plates supplemented with specific chemicals, drugs, or phages. Null mutations of any essential gene will result in no growth; these mutations can be selected by adding penicillin (an antibacterial drug isolated from a fungus), which kills dividing cells but not the nongrowing mutants. For conditional lethal mutations, replica plating can be used: mutated colonies on a master plate are transferred by a felt pad to other plates that are then subjected to some toxic environment. Mutations affecting the expression of a specific gene of interest can be screened by fusing it to a reporter gene such as the lacZ gene, whose protein product can make a blue dye, or the GFP gene, whose product fluoresces when exposed to light of a particular wavelength.

After a set of mutants affecting the process of interest have been obtained, the mutations are sorted into their genes by recombination and complementation. These genes are cloned and sequenced to obtain clues to function. Targeted mutagenesis can be used to tailor mutational changes at specific protein positions.

In E. coli, crosses are used to map mutations and to produce specific cell genotypes (see Chapter 5). Recombinants are made by mixing Hfr cells (having an integrated F plasmid) and F cells. Generally an Hfr donor transmits part of the bacterial genome, forming a temporary merozygote in which recombination takes place. Hfr crosses can be used to perform mapping by time-of-marker entry or by recombinant frequency. By transfer of F′ derivatives carrying donor genes to F, it is possible to make stable partial diploids to study gene interaction or dominance.

Techniques of Genetic Modification

Standard mutagenesis:

Chemicals and radiation

Random somatic mutations

Transposons

Random somatic insertions

 

Transgenesis:

On plasmid vector

Free or integrated

On phage vector

Free or integrated

Transformation

Integrated

 

Targeted gene knockouts:

Null allele on vector

Gene replacement by recombination

Engineered allele on vector

Site-directed mutagenesis by gene replacement

Genetic engineering

Transgenesis. E. coli plays a key role in introducing transgenes to other organisms (see Chapter 10). It is the standard organism used for cloning genes of any organism. E. coli plasmids or bacteriophages are used as vectors, carrying the DNA sequence to be cloned. These vectors are introduced into a bacterial cell by transformation, if a plasmid, or by transduction, if a phage, where they replicate in the cytoplasm. Vectors are specially modified to include unique cloning sites that can be cut by a variety of restriction enzymes. Other “shuttle” vectors are designed to move DNA fragments from yeast (“the eukaryotic E. coli”) into E. coli, for its greater ease of genetic manipulation, and then back into yeast for phenotypic assessment.

A plasmid designed as a vector for DNA cloning. Successful insertion of a foreign gene into the plasmid is detected by inactivation of either drug-resistance gene (tetR or ampR). Restriction sites are identified.

Targeted gene knockouts. A complete set of gene knockouts is being accumulated. In one procedure, a kanamycin-resistance transposon is introduced into a cloned gene in vitro (by using a transposase). The construct is transformed in, and resistant colonies are knockouts produced by homologous recombination.

Main contributions

Pioneering studies for genetics as a whole were carried out in E. coli. Perhaps the greatest triumph was the elucidation of the universal 64-codon genetic code, but this achievement is far from alone on the list of accomplishments attributable to this organism. Other fundamentals of genetics that were first demonstrated in E. coli include the spontaneous nature of mutation (the fluctuation test), the various types of base changes that cause mutations, and the semiconservative replication of DNA (the Meselson and Stahl experiment). This bacterium helped open up whole new areas of genetics, such as gene regulation (the lac operon.) and DNA transposition (IS elements). Last but not least, recombinant DNA technology was invented in E. coli, and the organism still plays a central role in this technology today.

Other areas of contribution

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