1. Microbial Genetics Lectures
Kelly Doran
Assistant Professor
UCSD School of Medicine
Department of Pediatric Infectious Diseases

2. Microbial Genetics Lectures
Bacterial viruses (pg )
Classification
Reproduction
Transduction
Recombinant DNA Technology (pg )
Recombinant DNA
Vectors and Cloning
Applications

3. Bacteriophages (Phages)
Viruses that infect bacteria
Bacteriophages cannot reproduce and survive on their own, must take over host cell
Fundamentally important microbes
Controlling bacteria populations and energy cycling
Gene transfer and shuffling in the environment
Tools for molecular biology and recombinant DNA technology

5. Applications of Phage Biology
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

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

7. Morphology: T4
Head / Capsid
Base plate
Sheath (Tail)
Tail fibres
DNA

8. dsDNA Phage Life Cycle Vast majority of phages are dsDNA
Two life styles
Lytic (e.g. T4)
Lysogenic (e.g. Lambda)

9. Lytic Life Cycle - 1 Adsorption to the host cell and penetration
Specificity of phage infection
~10 phages for every type of bacteria
Phage attach to specific receptor sites on bacterial surface
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

10. Phage DNA
Bacterial DNA

11. 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 (restriction endonucleases) 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 particles assembled (15 min)

13. 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)

15. 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 phages) are released without lysing the host cell – secreted instead

16. T4 phage
22 min
300 particles

17. Lytic Life Cycle

18. Single-Stranded DNA phages X-174, M13
ssDNA is converted to double-stranded form by host DNA polymerase (ds replicative intermediate)
Double-stranded form directs phage protein synthesis
Two different strategies for lysis
Similar to T4
Secreted from the host cell (filamentous phages, M13)

19. RNA Phages Single-stranded RNA phages (MS2)
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

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

22. 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.

23. 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

26. Lysogeny

27. Prophage Integration Phage lambda attP E. coli genome attB Integration
(Int, IHF)
Excision
(Int, Xis, IHF)
attL
attR

29. Gene Expression in l phage
3 distinct phases
First phase- very early: synthesis of proteins that will take over the host cell
Second phase-early: replication of the bacteriophage genome
Third phase-late lytic or late lysogenic: assembly and packaging of mature phage capsids OR integration into the bacterial host chromosome

30. 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

31. Lysogeny cI
Lytic
Cycle
cro


33. 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

34. 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.

35. Generalized Transduction
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.
Transducing particle- the phage which injects bacterial DNA into a new recipient.

36. Generalized Transduction

37. Specialized Transduction
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

38. Specialized Transduction

39. Recombinant DNA Technology (pg 312-333)
Applications
Vectors and Cloning

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

41. Construction of a Recombinant DNA Molecule
Isolate gene of interest
For example, create many copies of a gene by PCR
Digest the ends of the gene with restriction enzymes
Use DNA ligase to link the gene to a cloning vector
Propagate cloning vector and proceed with applications with cloned gene
Cloning vector – genetic element used to propagate and express genes of interest in bacteria
Plasmids, phages, cosmids, artificial chromosomes

43. 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
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)

44. PCR

46. Bacteria use them to destroy foreign DNA
Restriction enzymes
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
G A A T T C
C T T A A G G
C T T A A
A A T T C G
Eco RI overhang
Eco RI overhang

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

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

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

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

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

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

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

56. 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?

57. Group B Streptococcus (GBS)
Streptococcus agalactiae

58. GBS Disease

59. GBS Invasion of Blood-Brain Barrier

60. Goal: Identify GBS Factors Involved in
BBB Invasion

GBS Mutant

61. Transposon Mutagenesis
Tn917
pTV1OK
At low frequency transposon
hops into bacterial chromosome
Antibiotic selection 30oC
plasmid maintained

62. Select for Transposition Events
Ermr
37oC, Non-permissive
for plasmid replication
Under antibiotic selection
cells with an integrated Tn917
survive

63. Gene Identification orfB iagA Confirm by targeted mutagenesis Tn917
45 bp
457 bp
orfB
iagA
Tn917
Confirm by targeted mutagenesis

64. Thank you!

65. 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