Genetics Lecture Lytic Cycle of a Bacteriophage
Prefinal Notes
CHAPTER 7. Genetics of Bacteria and Viruses
The Genetic Organization of Bacteria and Viruses
Bacterial cell
❖ Nucleoid - a region that contains the genetic material
which lacks clear boundaries
1. A phage particle attaches to a host bacterium.
2. It injects its nucleic acid into the bacterial cell.
Escherichia coli
❖ in lower intestines of warm-blooded animals
❖ produces vitamin K2 Three types of useful bacterial mutants:
- reduces risk of cancer & heart disease 1. Antibiotic-resistant Mutants
- supports immune system & bone health ❖ Grow in streptomycin (Str) and tetracycline (Tet)
❖ Wildtype (prototroph)
Viruses ❖ Str-r = resistant ; Str-s = sensitive
❖ Inert particles if no host
❖ Capsid (protective coat) - encloses a viral genetic
material
❖ "living" only when their genetic material directs their own
multiplication
Bacteriophages
❖ Viruses infecting bacterial cells
❖ = phages Non-resistant bacteria multiply, and upon drug treatment, the bacteria
die. Drug resistant bacteria multiply as well, but upon drug
treatment, the bacteria continue to spread.
2. Nutritional Mutants
❖ Unable to utilize nutrients unless the required
nutrient is supplied in the medium
❖ Arginine auxotroph (Arg-)
- only grows in a medium with Arg
❖ (+) wildtype ; (-) mutant
Three types of genetic transfer in bacteria
❖ Adenine prototrophs (Ad+)
1. Bacterial Transformation
- DNA molecule is taken up from an external
environment and incorporated into the genome.
2. Conjugation
- donor DNA is transferred by direct contact from one
bacterial cell to another.
3. Carbon-source Mutants
❖ Cannot utilize sources of carbon (energy) 3. Transduction
❖ lac- cannot utilize lactose for growth - DNA is transferred from one bacterial cell to
another by a bacterial virus.
Bacterial Transformation
Phenotype in Bacteria
❖ 3 letters, first is capitalized ❖ Free DNA can become available in the soil by lysis
❖ (+) : the presence of a designated character (spontaneous breakage) of donor cells.
❖ (-) : absence
❖ s : sensitivity
❖ r : resistance
Ex:
Lac+ (can use lactose for growth)
Tet-r (can grow on medium with tetracycline)
Genotype in Bacteria
❖ 3 letters, lowercase, italicized
Ex:
lac+, tet-r
Transformation
What is the frequency of transformation?
❖ 1 transformed cell per 103 cells = 10-3
❖ 2 genes far in the chromosome (contained in 2 DNA
fragments)
• 10-3 x 10-3 = 1 transformant per 106 cells
❖ 2 genes near each other (contained in 1 DNA fragment)
• 10-3 = 1 cotransformant per 103 cells
- Cotransformation of two genes at a frequency substantially
greater than the product of the single-gene transformation
frequencies implies that the two genes are close together in the
bacterial chromosome.
Conjugation (Bacterial Mating)
❖ Plasmids - non-essential, DNA that exists inside bacteria
• replicate independent of bacterial genome
• 1 to 50 copies / host cell
• few kb to few 100 kb
• rely on the enzymes for DNA replication of their host
cell for reproduction
• their genes control the initiation of host's replication
❖ F Plasmid
- mediates conjugation between E. coli
• F+ : donor with F (“fertility”) factor
• F- : recipient; no F factor Conjugation begins with physical contact between a donor cell
• F ("fertility") factor: 100 kb and a recipient cell. The F plasmid DNA moves through a pore
- exists as free in a cell or integrated in the in the membrane from the donor to the recipient. The transfer
chromosome (episome) is always accompanied by replication of the plasmid. Contact
• 1-2 copies / cell between an F1 and an F2 cell initiates rolling-circle replication
• sex pilus (p. pili) - starts the physical contact that results in the transfer of a single-stranded linear branch
of the rolling circle to the recipient cell.
❖ Joshua Lederberg (1951)
- microbiologist
- discovered conjugation in E. coli
Hfr x F- mating
1. Coming together between donor (Hfr) and F- cells.
2. Rolling-circle replication in donor cell follows.
❖ Transfer of 1 strand of F factor to F- cell.
3. Transfer of ss-DNA into the recipient
❖ DNA is synthesized in both F+ & F- cells.
Part of F factor → Chromosomal genes → final part
❖ Circularization of new DNA strand in F- cell.
of F factor
❖ Connecting bridge breaks apart.
4. ss-DNA is converted into ds-DNA in the recipient.
❖ Both cells contain F factor and can act as donors.
5. The conjugating cells break apart long before the
entire bacterial chromosome is transferred. The last
High frequency of recombination (Hfr) Cells
part of the F factor is not transferred.
6. Recombination between Hfr fragment & the F-
chromosome follows.
Interrupted-mating Technique
❖ In an Hfr x F- cross, a technique by which donor &
recipient cells are broken apart at specific times, allowing
only a particular length of DNA to be transferred.
❖ How? By violent agitation of the suspension of mating
cells.
❖ When it is performed with Hfr x F- matings, the number of
recombinants of any particular allele increases with the
time during which the cells are in contact.
❖ Mixing of cells
❖ Agitating of samples violently
❖ Plating on a series of media with streptomycin & different
❖ Integration of F factor by recombination between a combinations of the 5 substances A through E.
nucleotide sequence in F plasmid & a homologous
sequence in the bacterial chromosome.
1. The number of recombinants in each curve increases with
length of time of mating.
2. For each marker, there is a time (the time of entry) before
which no recombinants are detected.
3. Each curve has a linear region that can be extrapolated
back to the time axis, defining the time of entry of each
gene a+, b+, . . . , e+.
4. The number of recombinants of each type reaches a
maximum, the value of which decreases with successive
times of entry.
Circular genetic map of E. coli
Time-of-Entry Mapping
Map distances are given in minutes; the total map length is 100 minutes.
For some of the loci that encode functionally related gene products, the
map order of the clustered genes is shown, along with the direction of
transcription and length of transcript (black arrows). The purple
arrowheads show the origin and direction of transfer of a number of
Hfr strains. For example, HfrH transfers thr very early, followed by leu
another genes in a clockwise direction.
❖ Due to circularity of the E. coli chromosome in F- cells and
the many points of integration of the F plasmid
❖ Both the DNA molecule and the genetic map are circular.
❖ The entire chromosome requires 100 minutes to be
transferred (it usually breaks first), so the total map length
is 100 minutes.
(A) Time-of-entry curves for one Hfr strain. (B) The linear map Generalized Transduction
derived from the data in part A. (C) A linear map obtained with the
same Hfr but with a different F2 strain containing the alleles b- e- f- g- ❖ bacterial DNA is transferred from one bacterial cell to
h-. (D) A composite map formed from the maps in parts B and C. (E) A
another by a phage particle (transducing phage) containing
linear map from another Hfr strain. (F) The circular map (gold)
the DNA.
obtained by combining the two (green and blue) maps of parts D and
E.
Cotransduction depends on how close the genes are
(A) A P1 transducing particle carrying the leu1 allele can convert a leu2
gal2 cell into a leu1 gal2 genotype (but cannot produce a leu1 gal1
genotype). (B) The transductants that could be formed by three possible
types of transducing particles—one carrying gal1, one carrying bio1, and
one carrying the linked alleles gal1 bio1. The third type results in
Phage P1 infects a leu1 donor, yielding predominantly normal P1 phages cotransduction. For clarity, the distance between gal and bio, relative to
with an occasional one carrying bacterial DNA instead of phage DNA. If that between leu and gal, is greatly exaggerated, and the size of the
the phage population infects a bacterial culture, then the normal phages DNA fragment in a transducing particle, relative to the size of the
produce progeny phages, whereas the transducing particle yields a bacterial chromosome, is not drawn to scale.
transductant. Note that the recombination step requires two
crossovers. For clarity, doublestranded phage DNA is drawn as a single
line.
Two types of transducing phages:
Step 1. E. coli phage P1 infects a bacterium 1. Specialized transducing phages – can transduce only certain
bacterial genes
Step 2. P1 DNA directs the host cell to make more DNA. Ex: lambda phage bio
Step 3. P1 phage makes nuclease that cuts the bacterial lambda phage gal
2. Generalized transducing phages - transduce any gene
DNA into fragments.
Step 4. New P1 phages are formed with phage DNA.
Step 5. Occasional phage carries bacterial DNA (arg+). Formation of an F' lac Plasmid (merodiploids / partial diploids)
Step 6. Transducing phage infects new host cell & injects the
bacterial DNA (arg+) into a recipient bacterium (arg-).
Step 7. Recombination occurs & yields a transductant
bacterium (arg+). Arg- fragment is digested by nucleases.
leu+ Single Transduction
Formation of an F9 lac plasmid by aberrant excision of F from an Hfr
chromosome. Breakage and reunion are between nonhomologous
regions.
The role of F' plasmids Transposable Elements
❖ DNA sequences capable of moving from one location to
❖ They render any recipient bacteria diploid for the region another in a genome
of the chromosome they carry. ❖ Transposon - a TE that has bacterial genes,
ex: antibiotic resistance genes.
Importance of diploid regions:
❖ Allow dominance tests
❖ Allow studies on gene-dosage tests
Lysogenic Life Cycle of E. coli phage λ
Transposable Elements (TEs) in Bacteria
❖ Transposons (bacterial TEs) can insert randomly at
potential target sites in the genome.
❖ Many transposons code for their own transposase.
❖ TEs have 1 or > genes for antibiotic resistance
that serve as genetic markers for selection.
Insertion Sequence (IS 50) = 1-3 kb
❖ Codes for transposase - for transposition
❖ Codes for repressor - regulates transposition
Transposon 5 (Tn5)
❖ with transposase and repressor
❖ genes for resistance to antibiotics: neomycin, bleomycin &
streptomycin
(A) Insertion sequence IS50. The element is terminated by short, nearly perfect
inverted-repeat sequences, the terminal nine base pairs of which are indicated.
IS50 contains a region that codes for the transposase and for a repressor of
transposition. The coding regions are identical in the region of overlap, but the
repressor is somewhat shorter because it begins at a different place. (B)
Composite transposon Tn5. The central sequence contains genes for resistance
to neomycin, neo-r; bleomycin, ble-r; and streptomycin, str-r. It is flanked by two
copies of IS50 in inverted orientation. The left-hand element (IS50L) contains
mutations and is nonfunctional, so the transposase and repressor are made by
1. A phage injects its nucleic acid into a bacterium.
the right-hand element (IS50R).
2. The ends of A ss-DNA are cohesive & complementary in
sequence so they can pair forming a circular molecule.
Methods of Transposition:
3. The circular A DNA & the E. coli DNA undergo site- specific
recombination.
4. Integrase catalyzes the recombination event.
5. Integration of the A DNA (prophage) into the bacterial
DNA (lysogen).
Lytic and Lysogenic Phages
❖ All phage species can undergo lytic cycle.
❖ Temperate phages - undergo lysogenic cycle
❖ Virulent phages - undergo lytic cycle only
CHAPTER 12. Molecular Mechanisms of Mutation and DNA
Repair
Mutation
- any heritable change in the genetic material.
❖ causes pyrimidine dimers
❖ brings 2 thymines closer
❖ distorts the double helix
❖ blocks transcription.& DNA replicatio transiently
b) Ionizing Radiation (X-ray; <100 nm)
❖ 1898 - status symbol in NY
- "no danger"
❖ Mid-1930s - x-ray deaths increased due to cancer
❖ 1960s - full hazard was exposed, mutagenic effects
1. Origin c) Nitrous Acid (HNO2)
❖ Spontaneous - random; 10% occurrence
❖ Induced- occurs due to a mutagen
❖ A potentially favorable mutation does not arise
because the organism has a need for it.
Mutagens:
a) UV Light (100-400 nm)
❖ alters the H-bonding specificity of the bases
(deamination)
The UVC Led light runs within a specific wavelength of 260- 1. A becomes hypoxanthine, pairs with C
270nm, which is the perfect wavelength to kill germs and 2. C becomes U, pairs with A
bacteria.
d) Ethyl Methanesulfonate (EMS) 2. Mutations based on Cell Type
❖ Somatic
- causes cancer in the body
- can have normal kids
- shortens one's life
❖ Germ-line
- occurs in reproductive cells
- can affect kids
- can have longer life
Which is worse? Somatic or germ-line?
Depends on the lifestyle
3. Molecular Change
❖ Insertion - 1 or more extra nucleotide present
❖ Deletion - 1 or more extra nucleotide missing
❖ alters base pairing
❖ distorts DNA structurally; creates bulky side of DNA
❖ reacts more to T & G
e) Nitrogen Mustard
❖ Base substitution (also called Point Mutation)
- a base pair in DNA is replaced with a different
nucleotide
- example 3’ – TC – 5’
from: 5’ – AG – 3’ 5’ – AG – 3’
to: 5’ – CG – 3’ 5’ – CG – 3’
❖ gas causing pain & lung damage when enhaled
❖ a warfare in WWI in Europe
❖ causes bulky side of DNA; more to T & G
f) Acridine
❖ Transition
- pyrimidine → pyrimidine
Ex: C → T or T → C
- purine > purine
Ex: A → G or G → A
❖ Transversion
- pyrimidine → purine: T → A or G
C → A or G
- purine → pyrimidine: A → C or T
G → C or T
❖ from coal tar
❖ raw material for dye production & some drugs ❖ The ratio of transitions to transversions among
❖ nicks DNA causing deletion or addition of a base or few spontaneous base substitution is 2:1.