BACTERIAL GENETICS

1. BACTERIAL GENETICS Pneumococcal reversion Mutations Regulation of gene expression Gene exchange Genetic engineering

2. Live virulent smooth cocci with capsule Heat-killed smooth pneumococci (Streptococcus pneumoniae) with capsule Live non-virulent cocci with no capsule Frederick Griffith 1928 Live smooth cocci isolated from dead mouse

4. 5’ end 3’ end 3’ end

5. DNA Replication • Semi-conservative • old strand-template • new strand-complementary • Replication fork • multiple enzymes • DNA unwinds • exposes nucleotides • synthesize new strand • one direction: 5’ to 3’ Ingraham & Ingraham

6. DNA Replication • Complementary nucleotides match (A=T; G=C) • DNA polymerase III binds nucleotides, releasing pyrophosphate Ingraham & Ingraham

7. Bacterial chromosomes • Replication of circular chromosome • Origin of replication • bubble forms • DNA unwinds • Replication occurs in both directions • Two replication forks • Continues until replication forks meet • Strands separate Ingraham & Ingraham

8. MUTATIONS • Mutagenicity • Ames test • Silent mutations: Amino acid sequence unchanged • Missense mutations: Different amino acid insertion • Nonsense mutations: Stop protein synthesis • Null mutations: Impair gene function

9. REGULATION OF GENE EXPRESSION • Inducible genes: Beta galactosidase induction in • the presence of lactose • Repressible genes: Histidine in the medium turns • off histidine biosynthesis pathway

10. When glucose is exhausted, growth stops temporarily, but begins again at a slower rate using lactose as an energy source

11. Transcription and Translation • Simultaneous transcription and translation • mRNA chain is transcribed • translation begins • multiple ribosomes on single mRNA • polysome Ingraham & Ingraham

12. Transcriptional regulation lac operon • lacZ • lacY • lacA • regulated by lacI • Lactose absent • repressor binds • stops transcription Ingraham & Ingraham

13. Transcriptional regulation • Lactose present • repressor bound by product of lactose • allolactose • transcription occurs • gene products of all genes are made Ingraham & Ingraham

14. GENE EXCHANGE Transformation Transduction Conjugation

15. GENE EXCHANGE

16. GENE EXCHANGE

17. TRANSFORMATION • Addition of foreign DNA in the laboratory • Requires calcium chloride or • electroporation • In cultures of Neisseria, Streptococcus, • Staphylococcus, and Bacillus

18. TRANSDUCTION Transfer of bacterial DNA from one cell to another via bacteriophage infection Replication of the bacterial chromosome disrupted Components of the phage produced and assembled The bacterium lyses, phage particles are released

20. TRANSDUCTION Lysogeny: Infection by a temperate bacteriophage does not cause lysis The genome of the phage integrates into the bacterial chromosome Reversion to a virulent state leads to the production of phage particles and bacterial lysis The new phage may carry with it a part of the bacterial chromosome

21. TRANSDUCTION The "tox" gene that codes for diphtheria exotoxin is introduced into Corynebacterium diphtheriae by a lysogenic bacteriophage (“B-corynephage”) Antibiotic resistance or ß-galactosidase production may be transduced

22. CONJUGATION Bacterial conjugation: The direct transfer of the DNA of the donor into the recipient bacterium Donor bacteria: Fertility factor positive (F+) Recipient bacteria: Fertility factor negative (F-) F: On a plasmid transferred with high frequency

23. E. coli with F pili, made visible by phages binding to it

24. CONJUGATION “High-frequency recombination" (Hfr): Strain of bacteria in which the F+ plasmid is integrated into the chromosome Only one strand of DNA is transferred to the recipient, while the other remains in the donor

25. PLASMIDS • Circular DNA consisting of 1-30 kb • (E. coli chromosome: 4,000 kb) • Code for • Resistance to toxins and antibiotics (R plasmids) • Fertility factor • Adhesiveness or protective coating • Bacteriocins (e.g. colicins in E. coli)

26. TRANSPOSONS • Segments of DNA that can move: • from one position to another in the genome • from chromosomal DNA to a plasmid • from a plasmid to chromosomal DNA • e.g. R plasmid

27. GENETIC ENGINEERING Manipulation of genetic material to alter the characteristics of an organism A particular gene can be inserted into a cloning vector which replicates normally in bacteria Recombinant DNA technology Restriction endonuclease --> Incorporation of donor DNA via a ligase --> Introduction into E. coli --> Growth, lysis and isolation of plasmid

28. Cloning vector Selective marker Multiple cloning site Origin of replication

29. Cloning a specific gene University of Illinois at Chicago

31. Gene cloning: Cutting DNA • Restriction endonucleases (RE; e.g. EcoRI) • natural products of bacteria • protect from foreign viral DNA • cut DNA at specific sequences • select RE that cuts outside the gene of interest • also cut the vector (plasmid) with same RE 5’ G A A T T C 3’ C T T A A G 3’ 5’ EcoRI G A A T T C 5’ 3’ 5’ 3’ C T T A A G 3’ 5’ 3’ 5’

34. Transformed cells are plated on selective medium Only the cells with a plasmid will survive

35. How do we find the clone with the gene we are looking for? Phenotypic screening Colony has different color or morphology Screening with antibodies

36. DNA probe Using DNA hybridization

40. Cloning eukaryoticgenes • Eukaryotic DNA has noncoding regions • introns • After mRNA is transcribed, the introns are removed • Reverse transcribe • make DNA copy of the mRNA • cDNA

41. cDNA cloning mRNA isolated from cell mRNA reverse transcriptase and dNTPs cDNA DNA polymerase and dNTPs dsDNA ligate into plasmid vector

42. Gene cloning: Gel electrophoresis • Electrophoresis • agarose gel • apply electrical current • DNA separates by size (length) • negative charge on DNA • moves toward positive electrode • small move faster than larger • isolate DNA containing gene of interest (after probe hybridization) • cut out of gel and remove from agarose

43. GENETIC ENGINEERING Complementary DNA (cDNA) obtained by revese transcription of mRNA The vector and cDNA cleaved by a restriction enzyme, producing "sticky ends" DNA ligase to produce a recombinant DNA vector Genomic library: All the recombinant vectors obtained when chromosomal DNA is cloned

44. GENETIC ENGINEERING The recombinant DNA is introduced into a bacterial host by transformation Bacterial colonies are screened by transferring them onto membranes and probing with labelled DNA or antibodies