Genetics of Bacteria Help (page 3)
Characteristics of Bacteria
Each cellular organism is classified as either prokaryote or eukaryote. A prokaryote is generally a single-celled organism whose DNA is not confined within a true nucleus. A eukaryote can be single-celled or multicelled and has its genetic material isolated from the rest of the cell by a nuclear membrane. All bacteria are prokaryotes. There are two main prokaryotic groups—the Eubacteria (true bacteria) or Bacteria and the Archaebacteria (ancient bacteria) or Archaea. These two groups are distinguished based on their cellular structures and DNA sequences. The Eubacteria contains most of the organisms we typically identify as bacteria, such as the common gut microbe Escherichia coli, and the causative agent of strep throat, Streptococcus pyogenes. The Archaea contains organisms that have been hypothesized to be evolutionarily older, or at least distinct, from the Eubacteria, such as the methanogens (bacteria that produce methane). All other forms of life (fungi, plants, animals) are eukaryotes in the Eukarya group. Bacteria are part of the larger category of organisms called microorganisms. This category includes bacteria as well as some smaller eukaryotes, such as the fungi, protozoans, and algae.
Most bacterial cells have a wall surrounding their plasma membrane. The wall contains a unique chemical called peptidoglycan (also called murein). There are two main types of bacteria based on the structure of their walls, and, in part, on the amount of peptidoglycan in their walls. The Gram-positive bacteria have a thick peptidoglycan layer, while the Gram-negative bacteria have a thinner peptidoglycan layer with an extra outer membrane. The antibiotic penicillin is effective against bacteria because it disrupts the synthesis of peptidoglycan.
Bacteria do not reproduce sexually (i.e., by formation of haploid gametes produced by meiosis, and fusion of gametes to form diploid zygotes) and do not use mitosis as a mechanism for cellular division. The bacterial chromosome does not condense, it has no centromere, and no spindle develops. Instead, the circular bacterial chromosome is replicated and, as the cell elongates, new cell wall material is laid down and the chromosome copies move apart in a process called binary fission. Bacteria can divide much more rapidly than eukaryotes (once every 20 min under ideal conditions, in contrast to 24–48 h or longer for many eukaryotic cells). Bacteria are typically about 1 μm wide and 1–5 μm long. Some bacteria can be as wide as 50 μm and several millimeters long, but this is rare. As mentioned earlier, bacteria are primarily single-celled organisms; however, there are exceptions. The Actinomycetes contain bacterial species that form filamentous, multicellular structures and, more recently, many different types of bacteria previously known only as single-celled organisms have been observed to form multicellular, slimy structures called biofilms. Biofilms are difficult to destroy due to difficulty in penetrating their multilayer, complex structure. They have been observed to accumulate in pipes used for food processing, medical catheters, or in patient tissues—resulting in difficult-to-treat diseases. In fact, symptoms of cystic fibrosis are thought to be due, in part, to biofilm accumulation in the patient's lungs.
Most of the genetic information of a bacterial cell resides in a single, circular, double-strandedDNAmolecule, commonly referred to as the bacterial chromosome, in a region of the cell called the nucleoid. Some bacterial DNA is complexed with basically charged proteins to form a kind of bacterial chromatin analogous to the association of histones and other chromosomal proteins with DNA in eukaryotic chromatin. Some bacteria may also contain small, self-replicating DNA circles called plasmids. There seldom are any membrane-bound organelles in bacterial cells.
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:
- 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.
- 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.
- 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.
- 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.
- 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.
Isolation of Bacterial Mutants
There are several different mutant bacterial phenotypes that can be used to identify mutations affecting various functional pathways. When the mutant phenotype has an advantage over the wild type under a specified set of conditions, a selection scheme is used to isolate the mutant. For example, it is relatively easy to isolate phage- or drug-resistant mutant strains by plating the bacteria on a medium that contains a selective agent, such as a phage or drug. Only resistant cells will form colonies on such plates. Prototrophic mutants can be isolated from an auxotrophic culture by plating on minimal medium; only prototrophic colonies would grow on such a plate. Isolation of auxotropic mutants is more difficult because they do not have a growth advantage over wild type. Generally, either an enrichment or a screening scheme must be used in this case. There are at least four methods for isolating auxotrophic mutants from prototrophic cultures, each of which is enhanced by first treating the culture with a mutagenic agent to increase the mutation rate.
- In the delayed enrichment method, a diluted culture is plated on minimal medium and then covered with an agar layer of the same medium. The plate is incubated and the locations of prototrophic colonies are marked on the plate.Alayer of nutrient medium is then added, and the nutrients from this agar are allowed to diffuse through the minimal agar. After another incubation period, the appearance of any new colonies may represent auxotrophs that could only grow after supplementation.
- The limited enrichment method is a simplification of the delayed enrichment method. Bacteria are plated on minimal medium containing a very small amount of nutrient supplementation. Under such conditions, auxotrophic bacteria will undergo limited growth until the supply of nutrients is exhausted, and hence will form small colonies. Prototrophic bacteria will continue to grow and produce large colonies.
- Penicillin interferes with the development of the bacterial cell wall only in growing cells, causing them to rupture. In the penicillin enrichment technique, the bacterial culture is exposed to penicillin in minimal medium. Growing prototrophic cells die, whereas auxotrophic cells cannot grow and therefore are not killed. The culture is then plated on nutrient medium without penicillin. Only auxotrophic colonies should form on the plate.
- In the replica plating technique (Fig. 10-1), the bacteria are first plated on nutrient agar and allowed to form colonies. A sterile velvet pad is then pressed onto the surface of this "master plate." The nap of the velvet picks up representatives from each colony on the plate. The pad is then pressed onto the sterile surface of one or more "replica plates" containing minimal medium. Only prototrophic colonies grow on the replica plate. Colonies on the master plate that are not represented on the replica plate may be auxotrophs. These colonies can be picked from the master plate to form a "pure" auxotrophic culture.
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