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Genetics of Bacteria Help (page 2)

By — McGraw-Hill Professional
Updated on Aug 23, 2011

Culture Techniques

The elements required for most life on this planet include carbon, hydrogen, nitrogen, oxygen, phosphorus and sulfur, plus some mineral elements, such as magnesium and zinc. The elements are used by cells to form the basic molecular building blocks (e.g., proteins, carbohydrates, nucleic acids, lipids) of the cell. Cells also require an energy source, such as sugars or light from the sun, to help them generate the needed power for cellular reactions. Bacteria grown in the laboratory in an aqueous solution of nutrients and energy sources are referred to as a bacterial culture. Bacteria can also be grown in a broth or liquid culture medium or on a medium that is made solid by the addition of agar. Agar is a complex carbohydrate isolated from algae that forms a solid matrix similar to gelatin. In addition, and unlike gelatin, most bacteria cannot digest agar. Solid media of this type are often poured into flat, circular containers called Petri dishes or into test tubes to make a slant. The inoculation of bacteria onto an agar surface is called plating. When a dilute sample from a culture is plated, each bacterial cell reproduces itself into a cluster of thousands of cells called a bacterial colony or clone that is visible to the naked eye. Barring mutation, all members of a colony are genetically identical and are thus, clones. The number of cells in a culture can be estimated by plating.

EXAMPLE 10.1 Suppose that 0.1 ml of a 106-fold dilution of a bacterial culture is plated on nutrient agar and 200 colonies develop. If 0.1 ml produces 200 colonies, l ml should produce 10 times as many colonies. Furthermore, since the original culture was diluted 106, it must contain 106 more bacteria than the diluted sample. Thus, the cell density of the original culture is estimated to be 200 × 10 × 106 =2 × 109 cells per milliliter.

When an undiluted sample of a dense culture is plated, the colonies are so numerous that they form a lawn of solid bacterial growth over the entire surface of the agar. Rare mutants can be easily isolated from such a lawn by several techniques.

Bacterial Phenotypes and Genotypes

Bacteria exist in a number of morphological forms: bacilli (rod-shaped), cocci (spherical), spirilla (spiral), spirochetes (helical), and branched. Because they are so small, individual bacterial cells are rarely studied in genetics. However, bacterial colonies are large enough to examine macroscopically and often exhibit variations in size, shape or growth habit, texture, color, and response to nutrients, dyes, drugs, antibodies, and viral pathogens, such as bacterial viruses, called bacteriophage or phage. Some bacteria can grow on minimal media containing a carbon and energy source (e.g., glucose), a nitrogen source, a source of sulfur, a few inorganic salts, and water. Bacteria that can grow on such minimal, "unsupplemented" medium are said to be prototrophic. If any other organic substance must be added to minimal medium to obtain growth, the bacteria are said to be auxotrophic. A medium that contains all the organic nutrients (amino acids, nucleotides, etc.) that could be required by any auxotrophic cell is called complete medium. In laboratory practice, prototrophic bacterial strains are typically "wild type" and auxotrophic strains contain mutations in genes that normally encode enzymes in essential biochemical pathways. When one or more of these critical enzyme genes are mutated, the bacterial cell containing the mutation cannot complete the pathway, and thus, cannot survive.

Five major types of phenotypic changes are commonly produced by bacterial mutations:

  1. A change from prototrophy to auxotrophy or vice versa; i.e., the loss or recovery of the ability to produce products of biosynthetic pathways. For example, amutation that produces a defect in the gene that specifies the enzyme that converts glutamic acid to glutamine would cause the cell to be dependent on the environment for glutamine.
  2. The loss or recovery of the ability to use alternative nutrients. For example, a mutation in the gene for the enzyme that converts the sugar lactose into glucose and galactose renders the cell incapable of growing in a medium where lactose is the only carbon source. These kinds of mutations that are involved in catabolic (degradative) reactions are independent of prototrophy or auxotrophy.
  3. A change from drug sensitivity to drug resistance or vice versa. For example, most bacteria are sensitive to the antibiotic streptomycin, but resistant strains can be produced by mutation.
  4. A change from phage sensitivity to phage resistance or vice versa. For example, a mutation in the bacterial receptor for the phage would render the cell resistant to infection.
  5. The loss or recovery of structural components of the cell surface. For example, one pneumococcus strain may possess a polysaccharide capsule, whereas another strain may not have a capsule.
EXAMPLE 10.2 If the cell can synthesize its own leucine, its phenotype is symbolized Leu+ and its genotype, leu+. The substance that characterizes the phenotype in this case (leucine) is symbolized Leu. The genotype that is auxotrophic for leucine is leu or leu, and the phenotype in this case is Leu (unable to grow without leucine supplementation). If more than one gene is required to produce the substance, the three-letter symbol would be followed by an italicized letter, such as leuA, leuB, etc. The genotype for resistance to the antibiotic drug penicillin is penr or pen-r; Penr or Pen-r is the corresponding phenotype. In partial diploids, the two haploid sets are separated by a diagonal line; thus, leu+/leuA.

Genetically different members of the same bacterial species are sometimes recognized as different strains if the differences are small, or as different varieties if the differences are substantial.

EXAMPLE 10.3   One of the most thoroughly studied bacterial species is Escherichia coli, or E. coli. Strains are designated by adding an unitalicized capital letter or number after the species name, thus E. coli B, E. coli S, etc. The three most commonly used strains of E. coli are E. coli B (host for phages of the T series), E. coli C (host for the single-stranded DNA phage 174), and E. coli K12 (harboring the lambda pro-phage). Note that variants within a strain are indicated by adding a number after the strain letter.
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