Bacterial Genetics

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 • 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 • 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 Thymine dimer repair • 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

16. Repair of Damaged DNA • Repair of modified bases • 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. Transformation • 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

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 • 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

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

32. Conjugation - Hfr • 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