1. Bacterial Genetics
Chapter 8

2. Diversity in Bacteria
Bacteria use three different mechanism to adapt to changing environments
Mutation
Gene transfer
Regulation of gene expression

3. Spontaneous Mutation
Spontaneous mutations occur in natural environment
Occur infrequently and randomly
Rate of mutation
probability that a mutation will be observed in a given gene each time the cell divides
Rate is generally between 1 in 10,000 and 1 in a trillion
Low rate is due to cellular repair mechanisms

4. Spontaneous Mutation
Mutations are stable heritable changes in the base sequence of DNA
Can occur from
Base substitutions
Removal or addition of nucleotides
Transposable elements

5. Spontaneous Mutation Base substitutions Most common type of mutation
Results from mistakes during DNA synthesis
Incorrect base is incorporated into DNA
Point mutations
Occur when one base pair is changes
Missense mutation
Mutation resulting from amino acid substitution is called
Nonsense mutation
Mutation that changes a amino acid codon to a stop codon

6. Spontaneous Mutation Removal and addition of nucleotides
Shifts the translational reading frame
Shifts the codons
Mutation called frameshift mutation
Affects all amino acids downstream from addition or deletion
Mutations frequently result in premature stop codons

7. Spontaneous Mutation Transposable elements
Special segments of DNA that move spontaneously from gene to gene
Elements called transposons
Transposons disrupt proper function of gene
Gene or gene product generally non functional

8. Induced Mutations Mutations are essential for understanding genetics
Mutations can be intentionally produced to demonstrate function of particular gene or set of genes
These mutations are termed induced
Mutations can be induced via
Chemical mutagens
Transposition
Radiation

9. Induced Mutations Chemical mutagens Nitrous acid Alkylating agents
Converts amino group to a keto group
Changes cytosine to uracil
Uracil binds to adenine while cytosine binds to guanine
Alkylating agents
Largest group of chemical mutagens
Alters hydrogen bonding of bases
Nitrosoguanine is common alkylating agent

10. Induced Mutations Chemical mutagens Base analogs
Chemicals that are structurally similar to the nitrogenous bases but have slightly altered base pairing properties
Base analogs include:
2-aminopurine which incorporates in the place of adenine but binds with cytosine
5-bromouracil which incorporates in the place of thymine but binds with guanine

11. Induced Mutations Chemical mutagens Intercalating agents
Molecules that insert themselves between adjacent bases
Creates space between bases
Extra base is often added to fill space
Ethidium bromide is common intercalating agent
Potential carcinogen

12. Induced Mutations Transposition
Common procedure used to induce mutation in laboratory
Gene that receives transposon will undergo a knockout mutation
Termed insertion mutation

13. Induced Mutations Radiation Two types Ultraviolet light X rays
Causes covalent bonding between adjacent thymine bases
Forms thymine dimers which distorts DNA
X rays
Cause breaks and alterations in DNA
Breaks that occur on both strand are often lethal

14. Repair of Damaged DNA Repair of base substitution
Cells develop two methods of repair
Proofreading
DNA polymerase has proofreading function
Able to excise incorrect base and replace with correct one
Mismatch repair
Endonuclease enzyme removes short stretch of nucleotide
DNA polymerase fills gap
DNA ligase joins ends
Mismatch repair

15. Repair of Damaged DNA Repair of thymine dimers Two mechanisms
Light repair
A.k.a photoreactivation
Enzyme uses visible light to break covalent bonds between bases
Dark repair
A.k.a excision repair
Endonuclease excises damages section
New section replicated and joined to original strand
Thymine dimer repair

16. Repair of Damaged DNA Repair of modified bases SOS repair
Enzyme cuts DNA backbone and removes base
DNA polymerase incorporates new base
SOS repair
Last ditch effort to bypass damage
Damage induces SOS system
Produces new DNA polymerase
Highly error prone
Mutations can arise from synthesis with new enzyme

17. Mutations and Their Consequences
Mutation provide organism way to respond to changing environments
Environment selects for cells suited to survive
Environment does not cause mutation

18. Mutation Selection
Major problem in induced mutation is identifying bacteria with desired mutation
Techniques used include
Direct selection
Involves inoculating population of bacteria on medium on which only mutants will grow
Used to select antimicrobial resistant organisms
Indirect selection
Required to isolate organisms that require growth factor that parent strain does not
Replica plating
Replica Plating

19. Mutation Selection Testing for cancer causing chemicals
Many mutagens are also carcinogens
Cancer causing agents
Microbes used to test potential carcinogenic activity
Tests are based on effect chemical has on microbial DNA
Ames test common chemical carcinogen test
Test rate of reversion of Salmonella auxotroph
Also test potential lethality

20. Mechanisms of Gene Transfer
Genes are naturally transferred between bacteria using three mechanisms
DNA-mediated transformation
Transduction
Conjugation

21. Mechanisms of Gene Transfer
Gene exchange in bacteria
Two event occur in gene exchange
1. Donor DNA is transferred and accepted by the recipient cell
Donor DNA is transferred one of three ways
Transformation
Transduction
Conjugation
2. Donor DNA is integrated onto the recipient cell’s chromosome

22. Mechanisms of Gene Transfer
DNA-mediated transformation
Definition: the transfer of naked DNA from one bacterium to another
Discovered by Fredrick Griffith in 1928 while working with Streptococcus pneumoniae

23. Mechanisms of Gene Transfer
Griffith realized S. pneumonic existed in two forms
Encapsulated, virulent form
Smooth in appearance
Nonencapsulated, avirulent form
Rough in appearance
Griffith wondered if injections with the smooth strain could be used as a vaccine against pneumonia
He injected mice with the different strains and recorded his results

24. Griffith Experiment

25. Mechanisms of Gene Transfer
DNA-mediated transformation
Involves the transfer of naked DNA from the environment to the recipient cell
Cells rupture during the stationary and death phase
The chromosome breaks into small pieces and explode through the ruptured cell wall
Recipient cell picks up piece of the naked DNA
The naked DNA is integrated onto the recipient chromosome
Replaces the homologous gene on the chromosome of the recipient cell

26. Mechanisms of Gene Transfer
DNA-mediated transformation
Natural transformation occurs when bacterial cells are “competent”
Competence is a condition in which bacterial cells are capable of taking up and integrating larger fragments of DNA
Competence occurs during the late log, early stationary phase

27. Natural transformation occurs in four stages Entry of the DNA
Only single strands enter, double strands are degraded
Integration of the donor DNA
Donor DNA is integrated by hydrogen bonding
Enzymes cleave recipient DNA
Donor DNA is put in place
Mismatch repair
Repair mechanism remove either donor or recipient DNA that doesn’t match
Repairs with correct nucleotides
Cell multiplication
Transformed cells multiply under selective conditions in which non-transformed cell will not grow
Transformation

28. Mechanisms of Gene Transfer
Transduction
Bacterial DNA that is transferred from donor to recipient via a bacterial virus (bacteriophage)
Two types of transduction
Generalized
Any gene of donor can be transferred
Specialized
Only specific genes can be transferred

29. Transduction is a mis-packaging of DNA during viral replication
The mis-packaged phage infects a new bacterial cell and insert the donor DNA into the recipient cell
The donor DNA is integrated and mismatched pairs are repaired
Transduction

30. Mechanisms of Gene Transfer
Conjugation – only form of gene exchange in which donor survives
Conjugation is mediated by a plasmid
Plasmid is self replicating extrachromosomal piece of DNA
Can code for traits that give bacteria advantage
Conjugation requires direct contact between cells
Cells must be of opposite mating types
Donor cells carry a plasmid that codes for fertility factor or “F factor”
This cell is designated F+
Recipient cell does not carry a plasmid
This cell is designated F-

31. During conjugation, the plasmid is replicated in the donor cell and is transferred to the recipient
After plasmid is transferred, F- cell becomes F+
Conjugation

32. In some F+ cells, the F factor integrates onto the host chromosome
Converts F+ to Hfr
Hfr= High frequency of recombination
Conjugation between Hfr and F- cell results in only a portion of the F factor being replicated and transferred
F- remain F-
F- has new information but may not have the F factor gene
Conjugation - Hfr