1. 1 Bacterial Genetics Part II

2. 2 Review of the Lac Operon Repressors turn off gene –Lac repressor Inducers bind to and inactivate repressors –Allolactose Activators turn on genes –CRP (cAMP receptor protein) binds to cAMP for it to be activator (remember high glucose-low cAMP low or no glucose- high cAMP)

3. 3 CRP Polycistronic mRNA (No cAMP) Repressor doesn’t bind because of allolactose

4. 4 Tryptophan Operon Escherichia coli operon Five genes involved in the synthesis of tryptophan –Trp A, B,C,D & E Regulatory elements – Promoter – Operator – Repressor – Attenuator – Corepressor textbookofbacteriology.net

5. 5 [Trp]  RNA pol binds Transcription of 5 genes in operon [Trp]  Repressor protein binds to operator Prevents binding of RNA polymerase

6. 6 Trp Operon textbookofbacteriology.net R = repressor O = operator P = promoter Attenuator DNA sequence between the operator and the structural genes RNA polymerase must cross the attenuator to transcribe the structural genes  [Trp] RNApolymerase molecules dissociate from the DNA  [Trp] RNApolymerase navigates the attenuator sequence and transcribe the trp genes Trp L regulatory gene Codes for the repressor protein

7. 7  [Trp] RNAp navigates the attenuator sequence and transcribe the trp genes textbookofbacteriology.net

8. 8  [Trp] RNApolymerase dissociates from the DNA textbookofbacteriology.net Repressor protein is “inactive” until tryptophan binds.

9. 9 Two Types of Regulation (Promoters) Constitutive –Allows continuous transcription of its genes lacI trpL “House keeping” genes involved in basic metabolism –Glycolysis –RNA polymerases –DNA repair enzymes –Ribosomal proteins Inducible –Transcription is linked to a special circumstance Presence of a sugar Concentration of a metabolite Stress

10. 10 Transcription and Translation are Coupled in Prokaryotes No compartmentalization in prokaryotic cells Transcription and translation occur in same place Ribosomes can associate with transcript while it is still being made Results in coupled transcription/translation chromosome RNA polymerase transcript chromosome RNA polymerase transcript ribosome protein

11. 11 Multiple Ribosomes can Associate with the Growing Transcript Highly expressed genes require high levels of translation Multiple ribosomes associate with growing transcripts to accomplish this Resulting structure is called a poly-some Allows prokaryotes to make a lot of protein very quickly.

12. 12 Experimental Bacterial Genetics Generate mutations to determine gene function Wild-type: normal or non-mutant form of a species or gene Mutant: aberrant form of a species or gene There is a change in DNA sequences Mutation A randomly or intentionally-produced, heritable change in the DNA sequence

13. 13 One Gene – One Enzyme 1941 Neurospora, a red orange bread mold Minimal medium –Sucrose –Minerals –Vitamins Induced mutations using X-rays Screened for auxotrophs Beadle & Tatum Wild-type strain can synthesize all its own amino acids

14. 14 Auxotroph A mutant that requires a nutrient for growth X Each mutant lacked a different enzyme along a pathway Genes code for enzymes One gene for one enzyme X X

15. 15 Wild-type Missense mutation Nonsense mutation Frameshift mutation Silent mutation Alters a base but does not change the amino acid

16. 16 Loss of function mutations Reduce or eliminate the activity of a gene Gain of function mutations Might increase the activity of a gene “overexpressed gene” but might be active at inappropriate circumstances Extremely rare, but sometimes confers a new function to gene… produces a protein that oes something new that might be advantageous

17. 17 Positive Selection for Mutants mutagen present Solid media containing penicillin Wish to generate a mutant that is resistant to penicillin Grow normal bacteria in presence of mutagen Chemical / physical agent/ irradiation that induces changes in DNA sequence Plate on solid media that contains penicillin or penicillin analog Only bacteria that have acquired a mutation that confers resistance to penicillin will survive Termed positive selection for desired mutation VERY powerful experiment penicillin resistant colonies Gain of function mutation New function = resistance to penicillin Mutagens discussed on page 277, Table 9.4

18. 18 Positive selection is not always possible Positive selection cannot identify loss of function mutations Wish to identify a mutant that cannot synthesize histidine No positive selection Alternate Strategy Grow bacteria in presence of mutagen Plate on rich media Transfer colonies using velvet Plate on media containing and lacking histidine Minimal medium Strategy employed by Beadle and Tatum

19. 19 His no His Auxotrophic mutant minimal medium

20. 20 Loss of Function Mutant Hunt Hypothesis –Capsule production by Streptococcus pneumonia is a virulence factor

21. 21 Mutagenize culture of a smooth, virulent strain of S. pneumonia Likelihood of mutation should be equal for all genes. Plate out mutants on blood agar plates Identify bacterial colonies that DO NOT produce a capsule. Loss of function Grow each “rough” mutant Inoculate mice Screen for virulence

22. 22 Are the “rough” mutants avirulent? Living mice is consistent with the hypothesis that capsule is a necessary virulence factor. Dead mice are not consistent with the hypothesis. Dead mice indicate that there may be other factors (gene products) involved in virulence. Studies of this type generally result in living and a smaller percentage of dead mice. This experiment needs a control. Inoculating mice with a wild-type culture of smooth, virulent S. pneumonia is an appropriate control. What would be the expected outcome?

23. 23 CONTROL GROUP Wildtype, smooth S. pneumonia Capsule present EXPERIMENTAL GROUP Mutant “rough” strains No visible capsule Inoculate mice Analyze Results Conclusion Rough mutants have greatly reduced virulence as compared to the wildtype strain Capsule production by Streptococcus pneumonia is a virulence factor Perform Experiment 48 of 50 mice (96.0%) die 10 of 50 (20%) mice die Significant difference

24. 24 Interruption of capsule production in Streptococcus pneumonia serotype 3 by insertion of transposon Tn916. D. A. Watson and D. M. Musher Infect Immun. 1990 September; 58(9): 3135–3138. LD 50 of the wildtype was 1 CFU LD 50 of 3 selected rough mutants was 1 x 1.3 10 5, 1.4 x 10 6 CFU and 1.3 x 10 5 CFU “…capsule was the principle virulence factor…” Transposons page 285

25. 25 Perform additional experiments to identify the gene controlling capsule production Locate the gene from the wild-type genome that will restore or “complement” the mutation.

26. 26 Isolate DNA from the wild-type strain Cut up genomic DNA with a restriction enzyme. Page 290 Ligate fragments of DNA into specialized plasmids. Page 290 Transform a “rough” mutant strain with the different plasmids. Screen transformants for capsule production. Red area represents the gene(s) controlling capsule production.

27. 27 Identify the transformants that produce a capsule. Gene(s) on plasmid “complements” the mutation on the chromosome. Inoculate mice with the complemented mutants. This experiment needs a control. Use rough mutants, a wild-type strain and water. Poor drawing of capsule production by transformants.

28. 28 Percentage of Mice that Die after Inoculation Transformants (capsule) Rough mutants (no capsule) Wild-type (capsule) Water 45/50 = 90.0%10/50 = 20.0% 49/50 = 98.0% 0/50 = 0% Conclusion