Microbiology Lecture 14

Front 1

U.V. radiation and what it does to bacteria

Back 1

When bacterial cells are exposed to U.V. radiation adjacent thymines are unnaturally bonded to create thymine-thymine dimers (or just thymine dimers). To combat the effect of U.V. light, many species possess an enzyme induced by visible light which will cleave or break the covalent bonds joining the dimers. IN addition to this method of repair, a dark repair mechanism exists. In this process an enzyme first excises the dimer. A DNA polymerase (second enzyme) then inserts new thymines opposite to the existing adenines to reform the naturally bonded DNA. Too much U.V. radiation can prevent repair. U.V. light is most damaging at wavelengths between 260 and 270 nanometer. The above mentioned mutagens will cause an increased rate of mutation for all genes in the genome. These are general mutagens.

Front 2

1. Mutation rates
2. How do genes mutate?

Back 2

1. The mutation rate for different genes can be different. Genes will have different wavelengths and configurations increasing or decreasing their susceptibilities.

2. Genes will mutate independently of each other. The chance that a single cell will have 2 separate mutations is equal to the product of the single rates

Front 3

SELECTING MUTANT CELLS
1. What are the two categories?

Back 3

1. Direct selection
2. Indirect selection

Front 4

1. Direct selection when selecting mutant cells

Back 4

1. Grow organisms in the presence of an antibiotic. Those that survive will have the mutant gene that allows those cells to live in the presence of the antibiotic. The word prototroph refers to the original cells that do not have the mutant gene. The prototroph population is also referred to as the wild type or normal or parent group of cells. The selected cells growing in the presence of the antibiotic would represent the mutation population.

Front 5

1. Indirect selection when selecting mutant cells

Back 5

Indirect selection
1. Prototroph - grows on a minimal media. Growing cells are susceptible to penicillin.

2. Auxotroph - the mutant cells. Auxotrophs are mutants from X-Men that have additional nutrient requirements. The auxotrophs will require additional amino acids for growth on minimal media.

Front 6

Experimental procedure for Auxotroph selection: has 7 steps

Back 6

1. Expose the prototroph population to U.V light (intermediate dose).
a. Kills some
b. Mutates some (shocked survivors)
c. Some unaffected (shocked survivors)

2. Grow the shocked survivors on a nutritionally complete media to allow for recovery.

3. To separate out the shocked survivors, place them on a minimal media that contains penicillin.

4. The prototrophs will grow and then be killed by the penicillin.

5. The auxotrophs cannot grow on a minimal media. They sit on the media and are not killed.

6. Transfer the cells to a complete media. All auxotrophs grow.

7. Use replica plating technique to separate the different auxotrophs.

Front 7

1. What does the Ames test do?

Back 7

1. The Ames test makes use of Salmonella histidine auxotrophs to efficiently screen for chemical carcinogens. Expose these autotrophs to a suspected carcinogen and look for the presence of the original Salmonella prototroph. The prototroph does not need added histidine for growth and will grow on a minimal media.

Front 8

1. Genetic recombination

Back 8

1. The exchange of genes between two DNA molecules to form new combinations of genes on a chromosome. This process creates diversity like mutation and therefore also drives evolution. Mutation however, only involves a single gene. There are 3 general methods of genetic recombination available to bacteria. They are transformation, conjugation and transduction.

Front 9

Transformation: historical perspective

Back 9

1. In 1928 Frederick Griffith (English physician) was doing work on what made pneumococcus (Streptococcus pneumoniae) pathogenic. He found two different types:
1. Type III(S) - virulent (encapsulated), resisted phagocytosis, smooth colonies, killed mice.
2. Type II (R) - avirulent (no capsule), rough colonies, mice survived.

Griffith then set up an experiment which can be said to consist of 10 steps.

Front 10

1. What type of experiment did Griffith do?

Back 10

1. Griffith set up an experiment to see if capsular material killed the mice (Type III (S))

Front 11

Steps 1 through 4 for Transformation

Back 11

1. Type III(S) killed the mice.
2. Type II(R) had no effect on the mice.
3. Heat-killed Type III(S) had no effect on the mice. IF the capsule was toxic, then the heat-killed cells with the capsular material should have caused disease. Conclusion: living cells plus capsules are needed for disease.
4. Mixture of Heat-killed Type III(S) and living Type II(R) - this killed the mice. The living Type II(R) somehow transformed to Type III(S).