1. Microbial Genetics Lectures John Buchanan –Research Scientist School of Medicine Department of Pediatric Infectious Diseases –Research Projects The genetics of bacterial virulence Alternatives to antibiotics to treat bacterial infections

2. Microbial Genetics Lectures Lecture 2 –Bacterial viruses (372-386) Classification Reproduction Transduction –Recombinant Technology (312-333) Recombinant DNA Vectors and Cloning Applications

3. –Bacterial viruses (372-386) Classification Reproduction Transduction

4. Bacteriophages (Phages) Viruses that infect bacteria Bacteriophages cannot reproduce and survive on their own, must take over host cell Fundamentaly important microbes –Ecologically- 10 31 total phages 10 8 - 10 9 / ml in water –Controlling bacteria populations and energy cycling –Gene shuffling in the environment –Tools for molecular biology and recombinant technology

6. Morphology Head / Capsid Base plate Sheath (Tail)Tail fibres DNA

7. Classification of Bacteriophages The most important criteria used for classification are phage morphology and nucleic acid properties dsDNA –Contractile tails –Noncontractile tails –Tailless –Filamentous –Head shape ssDNA ssRNA dsRNA

8. Classification of Bacteriophages The most important criteria used for classification are nucleic acid properties and phage morphology dsDNA –Contractile tails –Noncontractile tails –Tailless –Filamentous –Head shape ssDNA ssRNA dsRNA Contractile tail (T4) Flexible tail (lambda) Filamentous (fd) Tailless (SSV-1)

9. dsDNA Phage Life Cycle Vast majority of phages Two life styles –Lytic (T4) –Lysogenic (Lambda)

10. Lytic Life Cycle - 1 Adsorption to the host cell and penetration –Specificity of phage infection ~10 phages for every type of bacteria –Viruses attach to specific receptor sites Proteins Lipopolysaccharides Teichoic acids and cell wall components Carbohydrates Sex pilus –Phages then inject DNA into the cell Tail contraction (T4) Injection (PRD1) Unknown mechanisms

11. Phage DNA Bacterial DNA

12. Lytic Life Cycle - 2 Synthesis of phage nucleic acids and proteins –mRNA molecules transcribed early in the infection are synthesized using host RNA polymerase (1 min) Make viral enzymes required to take over the host cell –Degradation of host DNA (3 min) –Transcription of viral genes (5-9 min) Phage DNA is replicated (5 min) –Phage DNA sometimes modified protect the phage DNA from host enzymes that would degrade the viral DNA The assembly of phage particles –Phage mRNA directs the synthesis of capsid proteins and other proteins involved in assembly and release of the virus (12 min) –DNA packaged into the head (13 min) –Phage pieces assembled (15 min)

14. Lytic Life Cycle - 3 Release of phage particles (22 min – 300 new phage particles) –Many phages lyse their host by damaging the cell membrane and cell wall Holin – enzyme which destabilizes the host cell membrane (pokes holes) Lysin – phage enzyme which breaks host cell wall (lyses host bacteria)

16. Lytic Life Cycle - Summary Adsorption to the host cell and penetration –Viruses attach to specific receptor sites (proteins, lipopolysaccharides, teichoic acids, etc.) on the host cell –Many viruses inject DNA into the host cell, leaving an empty capsid outside Synthesis of phage nucleic acids and proteins –mRNA molecules transcribed early in the infection (early mRNA) are synthesized using host RNA polymerase; make viral enzymes required to take over the host cell –Transcription of viral genes follows Phage DNA is replicated –Phage DNA sometimes modified protect the phage DNA from host enzymes that would degrade the viral DNA The assembly of phage particles –Phage mRNA directs the synthesis of capsid proteins and other proteins involved in assembly and release of the virus –Phage pieces assembled –DNA packaged into the head Release of phage particles –Many phages lyse their host by damaging the cell wall or the cytoplasmic membrane –A few phages (e.g., filamentous fd phages) are released without lysing the host cell – secreted instead

17. T4 phage 22 min 300 particles

18. Single-Stranded DNA phages ssDNA is converted to double-stranded form by host DNA polymerase Double-stranded form directs phage protein synthesis Two different strategies for lysis –Similar to T4 –Secreted from the host cell (filamentous phages) Parasitic relationship

19. RNA Phages Single-stranded RNA phages –Codes for RNA replicase (enzyme for replicating the RNA genome) The RNA genome can usually act as mRNA to direct the synthesis of the replicase RNA is then converted to dsRNA dsRNA is then used as a template for production of multiple copies of the genomic RNA –Capsid proteins are made, and ssRNA is packaged into new virions –Very small genomes Lyses host through inhibition of cell wall formation Only one dsRNA phage has so far been discovered (f6); it infects Pseudomonas phaseolicola and possesses a membranous envelope

20. Measuring Phage Number – Plaque Assays Plaque assay – method for enumerating the number of phage particles in a sample; results are giving in plaque forming units (PFU) Mix phage and bacteria agar plate (1.5%)

21. Measuring Phage Number – Plaque Assays Plaque assay – method for enumerating the number of phage particles in a sample; results are giving in plaque forming units (PFU) Mix phage and bacteria agar plate (1.5%)

22. Applications of Phage Biology

23. Need for alternative therapies for treating bacterial infections –Resistance exists to every antibiotic we have Phages are potent antibacterials Self-replicating (smart drugs?) Narrow specificity so don’t damage the normal flora Resistance not as significant Resurgent interest in the application of phages to agriculture and human health Used for years in Eastern Europe and Russia

24. dsDNA Phage Life Cycle Vast majority of phages Two life styles –Lytic (T4) – lyses host cell –Lysogenic (Lambda) - Instead of destroying host to produce virus progeny, the viral genome remains within the host cell and replicates with the bacterial chromosome.

25. Temperate Bacteriophages and Lysogeny Temperate phages are capable of lysogeny, a nonlytic relationship with their hosts (virulent phages lyse their hosts - lytic) –Temperate = lysogenic –Virulent = lytic –In lysogeny, the viral genome (called a prophage) remains in the host (usually integrated into the host chromosome) but does not kill (lyse) the host cell; It may switch to the lytic cycle at some later time –The switching to a lytic cycle is called induction Lambda phages

27. Establishment of lysogeny DNA is double stranded with cohesive ends (cos sites) which are ss stretches of DNA that are complementary to each other –Circularizes immediately after injection into the host Once a closed circle is formed transcription by host RNA polymerase is initiated The BIG Decision: Lytic or Lysogenic life cycle? Battle between two repressors, cI or cro which compete for the same binding sites (operators) on phage DNA –If cI binds, represses synthesis of all genes = Lysogenic –If cro binds, represses synthesis of cI = Lytic If cI repressor wins the circular DNA is inserted into the chromosome via a process called integration and is maintained there –At this stage it is called a prophage If cI levels drop, cro takes over and the phage becomes lytic Environmental factors, such as UV light or chemical mutagens, that damage host DNA causes a host protein, recA, to act as a protease and cleave the cI repressor Decrease in cI stops repression of phage genes and balance shifts to cro and the lytic cycle

29. Notes For lambda and most temperate phages the viral genome integrates into the host chromosome; however, some temperate phages can establish lysogeny without integration Most bacteriophages are temperate indicating that this life strategy is advantageous –1 T4 phage = 300 new phages (may exterminate hosts) –1 lambda phage infects one host Host produces 1000 daughter cells (can live with hosts) Lambda emerges with 100 phages per cell = 100,000 new phages

30. Lysogenic conversion Lysogenic conversion is a change that is induced in the host phenotype by the presence of a prophage –Not directly related to the completion of the viral life cycle –Expression of additional genes from prophage –Production of diphtheria toxin only by lysogenized strains of Corynebacterium diphtheriae –Toxins that make Vibrio cholerae pathogenic are carried on a phage

31. Transduction Transduction is the transfer of bacterial genes by phages. –Bacterial genes are incorporated into a phage capsid due to errors made during the virus life cycle. –The virus containing these genes then injects them into another bacteria Mistakes in bacteriophage replication – can generate diversity at the genomic level and shuffle the genes of bacteria into novel combinations Most common mechanism for gene exchange and recombination in bacteria.

32. Transducing particle- the phage which injects bacterial DNA into a new recipient. Generalized transduction – Transfer of random portions of host genomic DNA by bacteriophages during the lytic cycle of virulent or temperate phages –Any part of the bacterial genome can be transferred –The phage degrades host chromosome into randomly sized fragments –During assembly, fragments of host DNA can be mistakenly packaged into a phage head –When the next host is infected, the bacterial genes are injected –Preservation of the transferred genes requires their integration into the host chromosome Specialized transduction - transfer of only specific portions of the bacterial genome by temperate phages that have integrated their DNA into the host chromosome –The prophage is sometimes excised incorrectly and contains portions of the bacterial DNA that was adjacent to the phageís integration site on the chromosome –The excised phage genome is defective because some of its own genes have been replaced by bacterial genes; therefore, the bacteriophage cannot reproduce –When the next host is infected, the donor bacterial genes are still injected and can become incorporated

34. –Recombinant Technology (312-333)\ Recombinant DNA Vectors and Cloning Applications

35. Genetic engineering - the deliberate modification of an organism's genetic information by directly changing its nucleic acid Recombinant DNA technology - the collection of methods used to accomplish genetic engineering Recombinant DNA - DNA with a new sequence formed by joining fragments from different sources

36. The Polymerase Chain Reaction (PCR) PCR is used to synthesize large quantities of a specific DNA fragment in vitro (in a test tube) Synthetic DNA molecules with sequences identical the target sequence are created during the reaction Made possible by bacteria - Replication is carried out in successive heating-cooling cycles using a heat- stable DNA polymerase from a thermophilic bacteria PCR has proven valuable in molecular biology, medicine (e.g., PCR-based diagnostic tests) and in biotechnology (e.g., use of DNA fingerprinting in forensic science)

37. Restriction enzymes (endonucleases) - bacterial enzymes that recognize and cleave specific sequences of DNA (4-8 bp long) –Bacteria use them to destroy foreign DNA – Valuable molecular biology tools –Enzyme EcoR1 (Restriction enzyme R1 from E. coli) Cuts at GAATTC (palindrome) –Leaves a cleaved DNA molecule with specific ends Restriction enzymes G A A T T C C T T A A G G C T T A A Eco RI overhang A A T T C G Eco RI overhang

38. G C T T A A Eco RI overhang A A T T C G Eco RI overhang + DNA Ligase G A A T T C C T T A A G

39. CONSTRUCTION OF A RECOMBINANT DNA MOLECULE 1.Isolate gene of interest For example, create many copies of a gene by PCR 2.Digest the ends of the gene with restriction enzymes 3.Use DNA ligase to link the gene to a cloning vector 4.Progate cloning vector and proceed with applications with cloned gene Cloning vector – genetic element used to propogate and express genes of interest in bacteria Plasmids, phages, cosmids, artificial chromosomes

40. Gene of Interest – Green Fluorescent Protein EcoRI G A A T T C C T T A A G G A A T T C C T T A A G Digest with EcoR1 Green Fluorescent Protein G C T T A A A A T T C G

41. Origin of replication Ampicillin resistance gene EcoRI BamH1 HindIII Digest with EcoR1 A A T T C G C T T A A Cloning Vector

42. Green Fluorescent Protein G C T T A A A A T T C G Add DNA Ligase A A T T C G C T T A A

43. Green Fluorescent Protein G C T T A A A A T T C G Add DNA Ligase A A T T C G C T T A A Origin of replication Ampicillin resistance gene BamH1 HindIII GFP

44. Origin of replication Ampicillin resistance gene EcoRI BamH1 HindIII Cloning Vector GFP EcoRI

45. Ampicillin resistance gene Origin of replication BamH1 HindIII GFP Origin of replication Ampicillin resistance gene EcoRI BamH1 HindIII Cloning Vector GFP GFP Expression Plasmid EcoRI

46. Selection for Bacteria with Gene of Interest Only bacteria containing the resistance gene grow Medium contains Ampicillin TRANSFORMATION SELECTION FOR BACTERIA WITH PLASMID

48. Applications of Recombinant Technology Use similar techniques for bacterial expression of medically important proteins –Insulin –Interleukins –Growth hormone Industrial and agricultural application Use recombinant technology to understand the genetics of organisms Recombinant technology is the alteration of DNA –Genetically modified organisms –Increased efficiency and economic value –Risks and social concerns?

49. Any questions?

51. Cloning Vectors –Cloning vector - small, well-characterized DNA molecule that contains at least one replication origin, can be replicated within the appropriate bacterial host, and code for a phenotype that is easily detected Antibiotic resistance, color change –Plasmids vectors Easy to isolate and purify Can be introduced into bacteria by transformation Often bear antibiotic resistance genes that can be used to select recombinants –Phage vectors Are more conveniently stored for long periods Contain insertion sites that do not interfere with replication when foreign DNA is inserted Recombinant phage DNA can be packaged into viral capsids and used to infect a host cell –Cosmids - plasmids with lambda phage cos sites; Cosmids can be packaged into lambda capsids and then manipulated as a phage Can also exist in the cell like a plasmid Can be used to clone very large pieces of DNA –Artificial chromosomes - can be yeast or bacterial; have all of the elements necessary to propagate as a chromosome; they can be used to clone DNA fragments from 100kb to 2000kb in length

52. G C T T A A EcoRI A A T T C G EcoRI Ligate with DNA ligase G A A T T C C T T A A G Eco RI G C T T A A EcoRI A A T T C G EcoRI Ligate with DNA ligase G A A T T C C T T A A G Eco RI