Genetics of Bacteria and their viruses (Chapter 9) 9th Ed. Chap8
nonconjugative plasmid
bacterial genome
X homologous
recombination
cointegrate site specific recombinase
homologous recombination
Bacteriophage P1 has loxP sites and Cre enzyme
Conditional Knockout using LoxP and Flp
Integrons
and cassettes
Integron contains promoter Cassette contains antibiotic resistance gene.
attI: integron attachment site attC: cassette attachment site need integrase protein
cassette
excission
WT prototroph
minimal medium
(glucose+inorganic salts)
auxotroph cannot grow
1. conjugation
2. transformation 3. transduction
Leu-
Horizontal gene transfer
Conjugation
Conjugation of F plasmid from F+ to F- through pilus
Not all plasmids are F plasmids. Need 20 genes to make pilus F plasmid low copy number 100 kb
relaxosome binds to the F-factor origin of transfer, oriT.
relaxase, encoded in the F-factor gene TraI, nicks the F-factor DNA at a , unique site called nic, binds covalently to the 5ʹ end of the nicked strand, and unwinds about 200 base pairs of DNA
protein-capped single strand of DNA that is transferred through the pore into the F− cell
rolling circle replication
Replicative transfer
do NOT confuse with origin of replication!
transformation
transformation frequency 1/10^3
cotransformation
1/10^6 if genes distant
if genes close more like 1/10^3
info about gene order need very high
concentration of extracellular DNA
transformation frequency 1/10 million
“crossing” bacteria
thi-
Cell Contact is necessary: Conjugation
no transformation
HfR high frequency of recombination cells
episome: either free or integrated into chromosome (different than a plasmid!)
not drawn to scale!
Hfr × F− conjugation process
Hfr × F− conjugation process
Hfr × F− conjugation process
Note that F episome did not incorporate oriT is where transfer initiates
the F- cell does NOT become Hfr
because complete F episome is not transferred!
genome size 100 kb for F 4600 kb for E. coli K12
transfer time: 2min vs 100min
How do you know that the bacteria growing is Hfr or F- after conjugation?
Hfr leu+str-s x F− leu−str-r cells, grow on streptomycin.
F− leu− parent cannot grow
recombinant F− leu+ cells can grow
Hfr Str-s cells die under streptomycin selection
leu selection marker
strep counterselection marker
here there is no blender: free mating
Selection versus counter selection
Time-of-Entry Mapping
interrupted-mating technique : kitchen blender again
4 mins to enter
Hfr a + b + c + d + X F - a - b - c - d -
minus B medium
minus A medium
minus E medium
minus C medium
- D
Transfer always begins at oriT of F
Not all cells conjugate at the same time: why number increases why plateau??
E. coli genome
F can integrate at numerous sites in the chromosome (both directions)
purple arrow: different Hfr strain
(different direction) Black direction of gene!
genes can be on either strand clockwise
anti-clockwise
F′ Plasmids
strain that has F’ plasmid contains bacterial chromosome derived gene.
If transferred to F- bacteria, it will generate partial diploid strain.
Used for dominance tests and copy number effects
Transduction
The Phage Lytic Cycle
P1 transducing particle contains 100–115 kb of bacterial DNA (about 50 genes)
Generalized transduction: phage infection helps gene transfer.
random digestion
1 leu − cell in 10
6becomes leu+
Dead end infection No more phage
Transduction
The Phage Lytic Cycle
P1 phage infects Leu+Gal+Bio+ strain then infects leu-gal-bio-
you get transformants leu+
gal+
bio+
gal+bio+ (genes are close-linked)
plaque formation on bacterial lawns
allows the determination of
phage concentration
Phage co-infection: r− (rapid lysis) allele results in large plaques, and the h− (host range) allele results in clear plaques r-h+ (large turbid plaque) X r+h- (small clear)
Genetic Recombination in the Lytic Cycle
Seymour Benzer Phage recombination
Wild-type T4 bacteriophage is able to multiply in E. coli strains B and K12(λ) and gives small ragged plaques K12(λ) signifies a K12 strain that is lysogenic for bacteriophage λ
Mutations in the rIIgene of T4 result in large round plaques on strain B but completely prevent T4 from propagating in strain K12(λ)
Bacteriopahge lambda
attP
attB
