Microbial Genetics

1. Microbial Genetics MICB404, Spring 2008 Lecture #16 Bacteriophage Genetics

2. Announcements - Quiz 5 key posted - Quiz 6 posted • Today’s lecture • Bacteriophage • Phage Biology • Phage T4 genetics • Phage λ genetics

3. Phage genetics • Bacteriophage T4 • Life cycle & replication • Genetic analysis

4. T4 • Seymour Benzer, 1950’s • Fine-structure mapping of rII (rapid lysis mutants type II) locus • rII will complete life cycle in E. coli B but not on K12λ • but can infect K12λ • r mutants distinguished based on plaque morphology

5. T4 • Complementation • rII has 2 complementation groups or genes • Recombination • genes can be sub-divided • frequency can map locations of mutations within genes • Deletion • Non-reverting mutations lack genetic information for recombination

6. Complementation Co-infect K12 with 2 rII mutant phage: A a1 a a2 b B B B Defects in different genes: infection has functional copy of each gene: complementation. Massive lysis with progeny mostly of parental Ab or aB genotypes Overlapping defects in same gene: no complementation.No lysis, no progeny phage

7. Recombination Co-infect E. coli B with 2 rII mutant phage: a1 a1 A a2 a2 Collect phage lysate B B B B Non-overlapping defects in same gene: recombination.Low degree of K12 lysis, progeny are almost entirely AB, wild-type

8. Recombination frequency • The closer two sequence regions are to one another, the less room for, and less likelihood of, crossover. • Frequency of recombinants is therefore a measure of how far apart mutations are in the sequences.

9. Recombination Co-infect E. coli B with 2 rII mutant phage: a1 a1 A a2 a2 B B B B Mapping Infect both B and K12 λ B: plaques represent total number of progeny phage K12 : wildtype are ½ of total recombinants Map distance: 2 x f(wild-type progeny) x 100

10. Example Five new mutations in the rII gene of T4 were isolated.  Pair-wise crosses between these mutations were performed with the following results: %(wt):  per cent wild-type phage progeny Derive a genetic map of these mutations 2 x f(wild-type progeny) x 100

11. 2 x f(wild-type progeny) x 100 Example 1 3 2 4 0 .02 .04 .06 .08 .10 .12 .14 Map Units 5    Note that mutation 5 maps to many different places; this is because mutation 5 is a deletion

12. Deletions • Of 2000 mutations, most revert • frequency of 10-2 to 10-8 • 140 mutations did not revert: deletions Deletion 3 Deletion 2 Deletion 1 T4 chromosome Pairwise crosses in K12 strain: 1 X 2 no wildtype results 1 X 3 wildtype recombinant arises (lysis) 2 X 3 no wildtype results

13. Deletions Deletion 2 Deletion 1 Pairwise crosses in K12 strain: 1 X 2 no wildtype results Deletion 1 Deletion 2

14. Deletions Deletion 3 Deletion 1 Pairwise crosses in K12 strain: 1 X 3 wildtype recombinant arises (lysis) Deletion 3 Deletion 1

15. rII deletions

16. Deletions • Deletion mapping is a rapid way to localize new mutations to a specific region of a gene (or chromosome) • New mutations may be crossed to a set of overlapping deletion mutations • If no wild-type recombinants are recovered in a cross, then the mutation maps to the region of the gene covered by the deletion

17. Deletions Cross mutants A, B, C with deletion phage 1 and 2 Deletion 1 Deletion 2 A B C

18. 0.3 * A 0.8 * B 0.1 0.5 C 1 3 2 0 0.6 1.0 Map Units (numbers in the table are percent wild-type recombinants)

19. Major conclusions • A large number of mutable sites occur within genes. • Genetic maps are linear • Most mutations are point mutations and subject to reversion • Deletions • Recombination • Mutation hot spots

20. Temperate phage • Phage T4: virulent • Infection leads to lysis • Phage λ: temperate • Infection leads to • lysis or • lysogeny • phage genome integrates into bacterial genome • bacteria is lysogen; phage is prophage

21. Prophage • Virulence factors or toxins - dysentary, diphtheria, cholera, butulism, tetanus, etc • Transfer of pathogenicity islands • Defective prophages

22. Temperate phage • λ Lysogens display immunity to super-infection by another λ phage • Lysogens can be induced to initiate a lytic cycle

23. λ Life Cycle UV irradiation

24. Infection • Lambda DNA is injected into cell after adsorptionof phage particle to LamB receptor • Genome circularizes • cos, cohesive sequence • ligation

25. Lambda DNA Replication

26. λ Life Cycle • Early, middle, late lytic and late lysogenic phases • Regulatory proteins - cI, cII (in complex with cIII) and cro transcription repressors/activators - N and Q antiterminator proteins that cause RNA polymerase to read through potential termination sequences

27. λ Genome qut, PR’ cos recombination replication Q lysis tail head PL PR cIII N cI cro cII OL OR nutL nutR

28. Immediate Early Gene Expression • N and Cro proteins expressed from PL and PR • N: antiterminator protein • acts on host cell RNA polymerase to permit extensive transcription from PL and PR • Cro: 66 amino acid protein • prevents synthesis of cI repressor • turns off early genes N PL PR tL tR cIII N cI cro cII Q OL OR Cro

29. Delayed Early Gene Expression • Q: antiterminator, permits extended trans-cription from PR (late genes) • cII and cIII: activator proteins • enhance transcription of cI gene • O, P, Gam: DNA replication • Red: DNA recombination cIII PL PR replication N N cIII N cI cro cII O P Q Red Gam OL OR replication recombination Cro cII Q

30. Monday Lambda Reading, chapter 8