Download The F plasmid and conjugation

Chapter 14
The Prokaryotic
Chromosome: Genetic
Analysis in Bacteria
Outline of Chapter 14
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General overview of bacteria
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The bacterial genome
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Structure
Organization
Transcription
Replication
Evolution of large, circular chromosomes
Structure and function of small circular plasmids
Gene transfer in bacteria
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Range of sizes
Metabolic activity
How to grow them for study
Transformation
Conjugation
Transduction
A comprehensive example
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Genetic tools to dissect bacterial chemotaxis
General overview of bacteria
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One of the three major lineages of life
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Eukaryotes – organisms whose cells have encased nuclei
Prokaryotes – lack a nuclear membrane
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Archea
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Bacteria
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1996 complete genome of Methanococcus jannaschii sequenced
More than 50% of genes completely different than bacteria and eukaryotes
Of those that are similar, genes for replication, transcription, and
translation are same as eukaryotes
Genes for survival in unusual habitats similar to some bacteria
Similar genome structure, morphology, and mechanisms of gene transfer to
archea
Evolutionary biologist believe earliest single celled organism,
probably prokaryote existed 3.5 billion years ago
A family tree of living organisms
Fig. 14.1
Diversity of bacteria
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Outnumber all other organisms on Earth
10,000 species identified
Smallest – 200 nanometers in diameter
 Largest – 500 micrometers in length (10 billion
times larger than the smallest bacteria)
 Habitats range from land, aquatic, to parasitic
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Remarkable metabolic diversity allows
them to live almost anywhere
Common features of bacteria
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Lack defined nuclear membrane
Lack membrane bound organelles
Chromosomes fold to form a nucleoid body
Membrane encloses cells with mesosome which
serves as a source of new membranes during cell
division
Most have a cell wall
Mucus like coating called a capsule
Many move by flagella
Power of bacterial genetics is the
potential to study rare events
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Bacteria multiply rapidly
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Liquid media – E. coli grow to concentration of 109 cells per
milliliter within a day
Agar media – single bacteria will multiply to 107 – 108 cells in less
than a day
Most studies focus on E. coli
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Inhabitant of intestines in warm blooded animals
Grows without oxygen
Strains in laboratory are not pathogenic
Prototrphic – makes all the enzymes it needs for amino acid and
nucleotide synthesis
Grows on minimal media containing glucose as the only carbon
source
Divides about once every hour in minimal media and every 20
minutes in enriched media
Rapid multiplication make it possible to observe very rare genetic
events
The bacterial genome is composed of
one circular chromosome
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4-5 Mb long
Condenses by supercoiling and looping into a
densely packed nucleoid body
Chromosomes replicate inside cell and cell divides
by binary fission
Fig. 14.4 b
E. coli lysed to release chromosome
Fig. 14.4 a
How to find mutations in bacterial
genes
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Mutations affecting colony morphology
Mutations conferring resistance to antibiotics or
bacteriophages
Mutations that create auxotrophs
Mutations affecting the ability of cells to break
down and use complicated chemicals in the
environment
Mutations in essential genes whose protein
products are required under all conditions of
growth
How to identify mutations by a
genetic screen
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Genetic screens provide a way to observe
mutations that occur very rarely such as
spontaneous mutations (1 in 106 to 1 in 108 cells)
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Replica plating – simultaneous transfer of thousands of
colonies from one plate to another
Treatments with mutagens – increase frequency of
mutations
Enrichment procedures – increase the proportion of
mutant cells by killing wild-type cells
Testing for visible mutants on a petri plate
Bacteria nomenclature
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wild-type – ‘+’
mutant gene – ‘-’
three lower case, italicized letters – a gene
(e.g., leu+ is wild type leucine gene)
The phenotype for a bacteria at a specific gene
is written with a capital letter and no italics
(e.g., Leu+ is a bacteria with that does not
need leucine to grow, and Leu- is a bacteria
that does need leucine to grow.)
Structure and organization of E. coli
chromosome
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4.6 million base pairs
open reading frames (ORFs)
90% of genome encodes protein (compare that to humans!)
4288 genes, 40% of which we do not know what they do.
almost no repeated DNA
427 genes have a transport function, other classes also
identified
bacteriophage sequences found in 8 places (must have been
invaded by viruses at least 8 times during history.
Insertion sequences dot the E. coli
chromosome
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Transposable elements place DNA sequences at
various locations in the genome.
Geneticists use transposable elements to insert DNA
at various locations in bacterial genomes.
If you were to insert a piece of DNA into a bacterial
genome using a transposable element, can you think
of a molecular method that you could use to find out
which gene you inserted the DNA into?
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Transposable
elements in
bacteria
Fig. 14.6
Transcription in bacteria
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Transcription machinery moves clockwise
Different strands code for different genes
Several genes may be transcribed in one
segment
RNA polymerase may transcribe adjacent
genes at the same time in a
counterclockwise direction
Highly transcribed genes generally oriented
in direction of replication fork movement
DNA replication in E. coli
Fig. 14.7
Plasmids: smaller circles of DNA
that do not carry essential genes
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Plasmids vary in size ranging from 1kb – 3 Mb.
Plasmids can carry genes that confer resistance to
antibiotics and toxic substances.
Plasmids are not needed for reproduction or normal
growth, but they can be beneficial.
Plasmids can carry genes from one bacteria to
another. Bacteria can thus become resistant to a drug,
put the resistance gene in the plasmid, and transfer it
to other bacteria. This transfer of plasmid DNA can
even occur across species.
Some plasmids contain multiple
antibiotic resistance genes
Gene Transfer in Bacteria
Fig. 14.9
Transformation
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Fragments of donor DNA enter the recipient
and alter its genotype
Natural transformation – recipient cell has
enzymatic machinery for DNA import
 Artificial transformation – damage to recipient
cell walls allows donor DNA to enter cells
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Treat cells by suspending in calcium at cold
temperatures
 Electroporation – mix donor DNA with recipient
bacteria and subject to very brief high-voltage shock
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Mechanism of
natural
transformation
Fig. 14.10
Conjugation – A type of gene
transfer requiring cell-to-cell contact
Fig. 14.11
The F plasmid and conjugation
Fig. 14.12 a
The process of conjugation
The F plasmid occasionally integrates into the
E. coli chromosome
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Fig. 14.13
Hfr cells have
integrated part of
chromosome
Episomes – plasmids
that can integrate into
host chromosome
Exconjugate –
recipient cell with
integrated DNA
Integrated plasmid
can initiate DNA
transfer by
conjugation, but may
take some of bacterial
chromosome as well
Gene transfer
in a mating
between Hfr
donor and F
recipient
Fig. 14.14
Mapping genes in Hfr and F- crosses
by interrupted mating experiments
Interrupted mating studies confirm
bacterial chromosome is a circle
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Cross between Hfr
and FThe F plasmid
integrates into
different locations in
different
orientations into the
circular donor
chromosome
Fig. 14.16 a, b
Partial genetic map of the E. coli
chromosome
Fig. 14.16 c
Recombination analysis improves
accuracy of map
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Interupted mating experiments accurate to only 2
minutes
Frequency of recombination between genes is
more accurate
Start by considering only exconjugates that have
all of the genes to be mapped (select for the last
gene transferred)
Living cells must have even number of crossovers
Consider as a three-point cross
Mapping genes using a three-point cross
Fig. 14.17
Different classes of crossovers: quadruple
crossover is least frequent
Fig. 14.17 c
F’ plasmids can be used for
complementation studies
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F’ plasmids replicate as discrete circles of DNA
inside host cells.
Transferred in same manner as F plasmids
A few chromosomal genes will always be
transferred as part of the F’ plasmid
Can create partial diploids
Merozygotes – partial diploids in which two gene
copies are identical
Heterogenotes – partial dipoids carrying different
alleles of the same gene
F’ plasmid
formation and
transfer
Fig. 14.18 a, b
Complementation testing using F’ plasmids
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Fig. 14.18 c
Creation of a
heterogenote
Phenotype of
partial diploid
establishes
whether
mutations
complement each
other or not
Transduction: Gene transfer via
bactgeriophages
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Bacteriophages
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Widely distributed in nature
Infect, multiply, and kill bacterial host cells
Transduction - may incorporate some of bacterial
chromosome into its own chromosome and transfer it to
other cells
Bacteriophage particles are produced by the lytic
cycle
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Phage inject DNA into cell
Phage DNA expresses its genes in host cell and replicate
Reassemble into 100-200 new phage particles
Cells lyse and phage infect other cells
Lysate is population of phage after lytic cyle is complete
Generalized
transduction
Fig. 14.19
Mapping genes by generalized transduction
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Frequency of recombination between genes
P1 bacteriophage often used for mapping
90kb can be constransduced corresponding
to about 2% recombination or 2 minutes
First find approximate location of gene by
mating mutant strain to different Hfr
strains
P1 transduction then used to map to specific
location
Fig. 14.20
Temperate phage can integrate into bacterial genome
through lysogenic cycle creating a prophage
Fig. 14.21
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Fig. 14.22 b
Recombination between att sites on the
phage and bacterial chromosomes allows
integration of the prophage
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Errors in prophage excision produce
specialized transducing phage
Adjacent genes are included in circular
phage DNA that forms after excision
Fig. 14.22 c