Download F factor

Bacterial Genomes
• Remember no nucleus!!
• Bacterial chromosome
- Large ds circular DNA molecule = haploid
- E. coli has about 4,300 genes (~4.2 Mb)
• 100x more DNA than the average virus
• 1000x less DNA than eukaryotic cell
- Chromosome is tightly coiled into dense
body = nucleoid
1
Prok Genome Size
Bacteria
Escherichia coli
Bacillus subtilis
Streptococcus pyrogenes
Mycobacterium genitalium
Archaea
Methanococcus jannaschii
Sulfolobus solfactaricus
Pyrococcus furiosus
Mega = 106
Size (Mbp)
4.64
4.20
1.85
0.58
Size (Mbp)
1.66
2.25
1.75
2
Euk Genome Size
Organism
Mbp
Homo sapiens
Drosophilia melanogaster
Plasmodium falciparum
Saccharomyces cerevisiae
3,000
165
23
12.07
Eukaryotes also have
Mitochondrial DNA
Chloroplast DNA
3
- Bacteria divide by simple
division = binary fission
- Division proceeded by
chromosome replication
from single origin of
replication
- E. coli cells can divide every
20 min under optimal conditions
- DNA molecules are
identical except for
mutations
- Mutation rate ~1 mutation/chromosome/generation
- With short generation time = lots of mutations
~ 107-108 mutations/12 hours
4
Extrachromosomal DNA
- Many bacteria have extrachromosomal
molecules of DNA = plasmids
- Plasmids contain an average of ~10-50 genes
- Cells can contain 1-100 plasmids
Resistance (R) plasmids
- Usually carry genes that detoxify antibiotics
- Allows bacteria to be resistant (R) to drugs
that would normally kill them
- Also often contain genes for sex pilus = can be
transferred by conjugation (F plasmids)
5
Recombination in Bacteria
- Bacteria are haploid, have only 1 copy of each
gene on circular chromosome
- There are mechanisms to introduce pieces of DNA
from one cell to another to produce a partial
diploid
- Partial diploids, because usually only small pieces
of DNA with only a few genes are transferred
- The foreign DNA in a partial diploid can replace
endogenous DNA in the chromosome by
homologous recombination
6
Genetic recombination - exchange of genes between two related
chromosomes, forms new combinations of genes
Involves crossover event between chromosomes
Results in hybrid chromosomes
Each now has properties of both
original chromosomes
In eukaryotes, occurs during meiosis
Increases genetic diversity
7
8
Can generate partial diploids in 3 different ways
Transformation
- Bacteria take up naked foreign DNA from the
environment
- Consequences can be that mutant alleles are
replaced with wildtype alleles or vice versa
by homologous recombination=crossing over
- Not all bacteria can be “naturally” transformed
- Competence
- Can create “competent cells” in the lab
9
Types of Transfer of Genetic Material
Genes can be passed from parental cell to progeny cell –
vertical gene transfer
Only method of transfer in higher eukaryotes (yeasts may
be an exception)
Also used by bacteria
Bacteria can also undergo horizontal gene transfer
Transfer of genetic material from one cell to another
Can result in a recombination event
Generates recombinant bacteria
10
Transforming Principle Experiment 1928
Fred Griffith
Streptococcus pneumoniae
Rough
Avirulent
Smooth
Virulent
α-hemolysis
of RBCs
No capsule!
Blood Agar Plate
11
12
The Transforming Principle
• R cells were “transformed”
• Something in the S cells transformed the R
cells
• The standard assumption was that proteins
were responsible
13
the Bacteria
Rough How
colonieswere
lacked functional
gene for capsule production
Transformed?
DNA containing functional gene from
heat killed smooth bacteria taken up
by rough bacteria
Recombination event replaced
defective capsule production gene
with functional gene
Once rough bacteria can now make
capsule and are transformed to
smooth colony virulent phenotype
14
Conjugation
General features
- Transfer of genetic material between 2 bacteria
that are temporally joined
- The donor cell transfer DNA to the recipient cell
- A sex pilus from the male initially joins the 2
cells via cytoplasmic bridge
- “Maleness” is the ability to form a
sex pilus and donate DNA
- Maleness requires an F factor
found either on the bacterial
chromosome or on a plasmid
15
Conjugation
Bacteria can
exchange genetic
information through
conjugation
Requires presence of fertility plasmid (F plasmid)
Contains genes required for production of sex pilus
Can connect two bacteria with pilus, one
with plasmid (F+) one without (F-)
F plasmid transferred
Converts F- to F+
16
How conjugation works
- F factor is an episome = can exist as an
autonomous or integrated (into bacterial
chromosome) plasmid
- The F factor contains ~25 genes mostly used to
make the sex pilus
- Cells with the F factor = F+ = conjugation donors
- Cells without the F factor = F- = conjugation recipients
- When F+ and F- meet, F+ donates the F factor to
F- cell and converts it to F+
17
F factor on plasmid
- the plasmid is only transferred during mating
F factor integrated into the bacteria chromosome
- occurs at a specific site
- the resulting cell is Hfr (High frequency of
recombination).
18
19
Transduction
- Occurs when phage picks up piece of degraded
bacterial chromosome by mistake
- The bacterial DNA is transferred from one host to
another by the phage during infection
20
More on phage during the virus lectures
21
Regulation of Genes in Prokaryotes
In general, prokaryotic genes are organized (and
Expressed) as operons
An operon consists of:
Several genes that encode enzymes under the control
of a single promoter
-
usually all enzymes needed for a specific activity
all transcribed as one long mRNA
polycistronic mRNA
mRNA that contains that codes for more than one
gene within the same mRNA transcript
22
promoter region
- site where RNA polymerase binds
- binding to promoter is necessary for
transcription of the mRNA that encodes
the enzymes
operator region
- binding site between the promoter and
first structural gene
- acts as an “on-off” switch
repressor protein
- binds to the operator region
- prevents transcription
inducer molecule
- binds to repressor & allows transcription 23
Transcriptional Control in
Prokaryotes
Lac Operon
Escherichia coli
24
Genes in the lac operon are designed to breakdown
and import lactose
Disaccharide
b galactosidase
Monosaccharide
25
Three lactose metabolizing enzymes are under
the control of one promoter
26
Repressor protein binds
to operator
No transcription
Repressor is produced
constitutively
No need to make b-gal & other enzymes
when lactose is not present
Negative Control
27
A small amount is
converted to allolactose
allolactose
28
Positive Control of the Lac Operon
- Activator protein
CRP
cAMP Receptor Protein
- Concentration of glucose is low
- cAMP accumulates
- CRP/cAMP binds to the promoter
- There’s a special sequence / binding location
- Maximal rates of transcription occur
- Synthesize a lot of b-gal & other enzymes
29
30
Positive Control – High Glucose
- There is little cAMP
- CRP can not be activated
- E. coli prefers glucose
If there’s plenty of glucose
No reason for produce b-gal
The lac operon is shut down in the presence of glucose
31
[glucose] 
[cAMP] 
Reduced transcription
32